transforming-growth-factor-beta and Osteosarcoma

In children and young adults, primary malignant bone tumours are mainly composed of osteosarcoma and Ewing's sarcoma. Despite advances in treatments, nearly 40% of patients succumb to these diseases. In particular, the clinical outcome of metastatic osteosarcoma or Ewing's sarcoma remains poor, with less than 30% of patients who develop metastases surviving five years after initial diagnosis. Over the last decade, the cancer research community has shown considerable interest in the processes of protein ubiquitination and deubiquitination. In particular, a growing number of studies show the relevance to target the ubiquitin-specific protease (USP) family in various cancers. This review provides an update on the current knowledge regarding the implication of these USPs in the progression of bone sarcoma: osteosarcoma and Ewing's sarcoma.

Topics: Antineoplastic Agents; Bone Neoplasms; Child; Drug Delivery Systems; Humans; Osteosarcoma; Transforming Growth Factor beta; Ubiquitin-Specific Proteases; Ubiquitination

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-β (TGF-β) superfamily of proteins; they were initially named after their ability to induce ectopic bone formation. Published studies have proved BMPs' role in a variety of biological processes such as embryogenesis and patterning of body axes, and maintaining adult tissue homeostasis. Other studies have focused on BMPs properties, functions and possible involvement in skeletal diseases, including cancer.. A literature search mainly paying attention to the role of BMPs in musculoskeletal tumors was performed in electronic databases.. This article discusses BMPs synthesis and signaling, and summarizes their prominent roles in the skeletal system for the differentiation of osteoblasts, osteocytes and chondrocytes.. The review emphasizes on the role of BMP signaling in the initiation and progression of musculoskeletal cancer.

Topics: Animals; Bone Morphogenetic Proteins; Bone Neoplasms; Humans; Muscle Neoplasms; Osteosarcoma; Signal Transduction; Transforming Growth Factor beta

The formation of bone tissue involves multiple activities of the osteoblast. The combined application of molecular, biochemical, histochemical and ultrastructural approaches has defined stages in the development of the osteoblast phenotype with each subpopulation of cells exhibiting unique morphologic and functional properties in relation to the ordered deposition of the mineralized bone extracellular matrix (ECM). Peak levels of expressed genes reflect a maturational sequence of osteoblast growth and differentiation characterized by three principal periods: proliferation, ECM maturation and mineralization. A plethora of new information in the past several years provides the basis for insight into molecular mechanisms regulating the development and activities of differentiating osteoblasts. These new concepts will be discussed within the context of understanding cellular responses of bone tissue. To be considered are the following: 1) maturational stages of the osteoblast reflected by the selective expression of transcription factors (e.g., oncogenes, cyclins, homeodomain proteins) and phenotypic genes that provide signals for differentiation through the osteoblast lineage; 2) role of the extracellular matrix in mediating osteoblast growth and differentiation; 3) osteoblast stage specific responses to physiologic mediators (e.g., growth factors and hormones); 4) the developmentally regulated expression and selective responses of osteoblast phenotypic genes are supported by cooperative, synergistic and/or antagonistic activities at multiple basal and enhancer or suppressor sequences in gene promoters; and 5) deregulation of these control mechanisms in transformed osteoblasts and osteosarcoma cells.

Topics: Animals; Cell Differentiation; Cell Division; Cell Line; Extracellular Matrix; Gene Expression; Gene Expression Regulation, Neoplastic; Histones; Humans; Osteoblasts; Osteocalcin; Osteosarcoma; Phenotype; Promoter Regions, Genetic; Transforming Growth Factor beta

Topics: Animals; Bone Neoplasms; Cell Cycle; Growth Substances; Humans; Osteosarcoma; Platelet-Derived Growth Factor; Prognosis; Signal Transduction; Somatomedins; Transforming Growth Factor beta

Novel and effective immunotherapies are required for refractory or recurrent sarcomas. Transforming growth factor-beta (TGF-β) is a diverse regulatory and fibrogenic protein expressed in multiple sarcoma tumors that promotes epithelial-mesenchymal transition and excessive deposition of extracellular matrix. This study evaluated the efficacy and safety of the anti-PD-L1/TGF-β antibody TQB2858 in patients with refractory osteosarcoma and alveolar soft part sarcoma (ASPS).. This single-arm phase 1b exploratory study included patients with refractory osteosarcoma or ASPS who had previously undergone at least two lines of systemic therapy. Patients were administered 1200 mg of TQB2858 once every 3 weeks. The primary endpoint was objective response rate (ORR), with null and alternative hypotheses of ORR ≤5% and ≥20%, respectively. Exploratory biomarker analyses using immunohistochemistry (IHC) staining (for PD-L1 and TGF-β) were performed on pre-treatment tumor samples.. Eleven eligible patients were included in this study. TQB2858 did not demonstrate evidence of efficacy as 0/5 osteosarcomas had any objective response, while 2/6 ASPS showed a partial response. The median progression-free survivals were 1.51 (1.38, Not Evaluable) and 2.86 (1.38, Not Evaluable) months for the osteosarcoma and ASPS groups, respectively. None of the administered cycles met the criteria for unacceptable toxicity. Other Grade 3 toxicities included abnormal liver function and elevation of γ-glutamyl transferase. IHC analysis revealed that functional enrichment in the TGF-β pathway or PD-L1 was not associated with treatment outcomes.. The combination of PD-L1 and TQB2858 did not significantly improve the ORR in patients with recurrent osteosarcoma. However, it improved immunogenic responses in ASPS, even after progression upon anti-PD-1/PD-L1 therapy, with an acceptable safety profile. IHC profiling with pathway enrichment analysis may not have any predictive value for survival outcomes.. Prospectively registered in the Ethical Review Committee of Peking University People's Hospital. The trial registration number is 2021PHA105-001 and 2021PHA140-001 and the registration date was March 2, 2022.. gov Identifier CTR20213001 and CTR20220390.

Topics: Antibodies; Antineoplastic Agents, Immunological; Asian People; B7-H1 Antigen; Bone Neoplasms; East Asian People; Humans; Osteosarcoma; Sarcoma, Alveolar Soft Part; Soft Tissue Neoplasms; Transforming Growth Factor beta

Osteosarcoma, the most common primary malignant bone tumor, is defined by the formation of neoplastic osteoid and/or bone. This sarcoma is a highly heterogeneous disease with a wide range of patient outcomes. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein that is highly expressed in various types of malignant tumors. We previously reported that CD109 is expressed in osteoblasts and osteoclasts in normal human tissues and plays a role in bone metabolism in vivo. While CD109 has been shown to promote various carcinomas through the downregulation of TGF-β signaling, the role and mechanism of CD109 in sarcomas remain largely unknown. In this study, we investigated the molecular function of CD109 in sarcomas using osteosarcoma cell lines and tissue. Semi-quantitative immunohistochemical analysis using human osteosarcoma tissue revealed a significantly worse prognosis in the CD109-high group compared with the CD109-low group. We found no association between CD109 expression and TGF-β signaling in osteosarcoma cells. However, enhancement of SMAD1/5/9 phosphorylation was observed in CD109 knockdown cells under bone morphogenetic protein-2 (BMP-2) stimulation. We also performed immunohistochemical analysis for phospho-SMAD1/5/9 using human osteosarcoma tissue and found a negative correlation between CD109 expression and SMAD1/5/9 phosphorylation. In vitro wound healing assay showed that osteosarcoma cell migration was significantly attenuated in CD109-knockdown cells compared with control cells in the presence of BMP. These results suggest that CD109 is a poor prognostic factor in osteosarcoma and affects tumor cell migration via BMP signaling.

Topics: Antigens, CD; Bone Neoplasms; GPI-Linked Proteins; Humans; Neoplasm Proteins; Neoplastic Processes; Osteosarcoma; Transforming Growth Factor beta

The expression pattern, diagnostic value, and association of PD-L1, IFN-γ and TGF-β with bone tumor type, severity, and relapse are determined in this study. 300 human samples from patients with osteosarcoma, Ewing sarcoma, and GCT were enrolled. The PD-L1 gene and protein expression were assessed by qRT-PCR and immunohistochemistry, respectively. ELISA and flow cytometry was used to detect cytokines and CD4/CD8 T cell percentages, respectively. A considerable increase in PD-L1 level was detected in bone tumor tissues at both gene and protein levels that was considerable in osteosarcoma and Ewing sarcoma. A positive correlation was detected regarding the PD-L1 and tumor metastasis and recurrence in osteosarcoma and Ewing sarcoma. The increased IFN-γ level was detected in patients with metastatic, and recurrent osteosarcoma tumors that were in accordance with the level of TGF-β in these samples. The simultaneous elevation of IFN-γ and TGF-β was detected in Ewing sarcoma and GCT, also the CD4 + /CD8 + ratio was decreased significantly in patients with osteosarcoma compared to GCT tumors. The elevated levels of PD-L1, TGF- β, and IFN-γ were associated with bone tumor severity that can provide insights into the possible role of this axis in promoting immune system escape, suppression, and tumor invasion.

Topics: B7-H1 Antigen; Bone Neoplasms; CD8-Positive T-Lymphocytes; Humans; Immunity; Immunologic Factors; Osteosarcoma; Sarcoma, Ewing; Transforming Growth Factor beta

Osteosarcoma is a highly common malignant bone tumor. Its highly metastatic properties are the leading cause of mortality for cancer. Niclosamide, a salicylanilide derivative, is an oral antihelminthic drug of known anticancer potential. However, the effect of niclosamide on osteosarcoma cell migration, invasion and the mechanisms underlying have not been fully clarified. Therefore, this study investigated niclosamide's underlying pathways and antimetastatic effects on osteosarcoma. In this study, U2OS and HOS osteosarcoma cell lines were treated with niclosamide and then subjected to assays for determining cell migration ability. The results indicated that niclosamide, at concentrations of up to 200 nM, inhibited the migration and invasion of human osteosarcoma U2OS and HOS cells and repressed the transforming growth factor beta-induced protein (TGFBI) expression of U2OS cells, without cytotoxicity. After TGFBI knockdown occurred, cellular migration and invasion behaviors of U2OS cells were significantly reduced. Moreover, niclosamide significantly decreased the phosphorylation of ERK1/2 in U2OS cells and the combination treatment of the MEK inhibitor (U0126) and niclosamide resulted in the intensive inhibition of the TGFBI expression and the migratory ability in U2OS cells. Therefore, TGFBI derived from osteosarcoma cells via the ERK pathway contributed to cellular migration and invasion and niclosamide inhibited these processes. These findings indicate that niclosamide may be a powerful preventive agent against the development and metastasis of osteosarcoma.

Topics: Cell Death; Cell Line, Tumor; Cell Movement; Extracellular Matrix Proteins; Gene Knockdown Techniques; Humans; MAP Kinase Signaling System; Neoplasm Invasiveness; Niclosamide; Osteosarcoma; Transforming Growth Factor beta

Since its first identification in prostate cancers and prostate tissues, transient receptor potential melastatin-subfamily member 8 (TRPM8) is subsequently found to be overexpressed in a wide range of cancers and is shown to be implicated in tumorigenesis and tumor progression. Here, we used N-(3-aminopropyl)-2-[(3-methylphenyl) methoxy] -N-(2-thienylmethyl) benzamide hydrochloride (AMTB), a specific TRPM8 antagonist, to explore its antitumoral effect on osteosarcoma. We find that AMTB suppresses osteosarcoma cell proliferation, metastasis and induces cellular apoptosis. Xenograft model in nude mice experiments also define that AMTB can increase the sensitivity of tumor cells to cisplatin, the cytotoxic chemotherapeutic regimens in treating osteosarcoma. Molecularly, AMTB specifically antagonizes TRPM8 which is upregulated in osteosarcoma and its expression level in osteosarcoma tissues is negatively related to patients' prognosis. Finally, RNA sequencing analysis was performed to explore the mechanism underlying the antitumoral effect of AMTB on osteosarcoma cells and the results prove that AMTB suppresses the Transforming Growth Factor β (TGFβ) signaling pathway. Our study provides evidence that TRPM8 could be a potential therapeutic target and AMTB can suppress growth and metastasis of osteosarcoma cells through repressing the TGFβ signaling pathway and increase the sensitivity of tumor cells to cisplatin.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cisplatin; Humans; Male; Membrane Proteins; Mice; Mice, Nude; Osteosarcoma; Signal Transduction; Transforming Growth Factor beta; TRPM Cation Channels

In the last few decades, biological reconstruction techniques have improved greatly for treating high-grade osteosarcoma patients. To conserve the limb, and its function the affected tumor-bearing bones have been treated using liquid nitrogen and irradiation processes that enable the removal of entire tumors from the bone, and these treated autografts can be reconstructed for the patients. Here, we focus on the expressions of the growth factor family proteins from the untreated and treated autografts that play a crucial role in bone union, remodeling, and regeneration. In this proteomic study, we identify several important cytoskeletal, transcriptional, and growth factor family proteins that showed substantially low levels in untreated autografts. Interestingly, these protein expressions were elevated after treating the tumor-bearing bones using liquid nitrogen and irradiation. Therefore, from our preliminary findings, we chose to determine the expressions of BMP2, TGF-Beta, and FGFR proteins by the target proteomics approach. Using a newly recruited validation set, we successfully validate the expressions of the selected proteins. Furthermore, the increased growth factor protein expression after treatment with liquid nitrogen may contribute to bone regeneration healing, assist in faster recovery, and reduce local recurrence and metastatic spread in high-grade sarcoma patients.

Topics: Autografts; Bone Morphogenetic Protein 2; Bone Neoplasms; Bone Transplantation; Humans; Nitrogen; Osteosarcoma; Proteomics; Transforming Growth Factor beta

Topics: Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; JNK Mitogen-Activated Protein Kinases; Osteosarcoma; Pyrazoles; Pyridines; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

In the widespread adoption of chemotherapy, drug resistance has been the major obstacle to tumor elimination in cancer patients. Our aim was to explore the role of TGF-β in osteosarcoma-associated chemoresistance.. We performed a cytotoxicity analysis of methotrexate (MTX) and cisplatin (CIS) in TGF-β-treated osteosarcoma cells. Then, the metabolite profile of the core metabolic energy pathways in Saos-2 and MG-63 cell extracts was analyzed by. The metabolic analysis revealed enhanced succinate production in osteosarcoma cells after TGF-β treatment. We further found a decrease in SDH expression and an increase in HIF1α expression in TGF-β-treated osteosarcoma cells. Consistently, blockade of SDH efficiently enhanced the resistance of Saos-2 and MG-63 cells to MTX and CIS. Additionally, a HIF1α inhibitor significantly strengthened the anticancer efficacy of the chemotherapeutic drugs in mice with osteosarcoma cancer.. Our study demonstrated that TGF-β attenuated the expression of SDH by reducing the transcription factor STAT1. The reduction in SDH then caused the upregulation of HIF1α, thereby rerouting glucose metabolism and aggravating chemoresistance in osteosarcoma cells. Linking tumor cell metabolism to the formation of chemotherapy resistance, our study may guide the development of additional treatments for osteosarcoma.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glucose; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Methotrexate; Mice; Osteosarcoma; Response Evaluation Criteria in Solid Tumors; STAT1 Transcription Factor; Succinate Dehydrogenase; Transforming Growth Factor beta; Up-Regulation; Warburg Effect, Oncologic; Xenograft Model Antitumor Assays

Although the treatment of osteosarcoma has improved, the overall survival rate of this common type of osseous malignancies has not changed for four decades. Thus, new targets for better therapeutic regimens are urgently needed. In this study, we found that high expression of clathrin heavy chain (CLTC) was an independent prognostic factor for tumor-free survival (HzR, 3.049; 95% CI, 1.476-6.301) and overall survival (HzR, 2.469; 95% CI, 1.005-6.067) of patients with osteosarcoma. Down-regulation of CLTC resulted in tumor-suppressive effects in vitro and in vivo. Moreover, we found that CLTC was transcriptionally regulated by a transcription factor-specificity protein 1 (SP1), which binds to the CLTC promoter at the -320 to -314-nt and +167 to +173-nt loci. Mechanistic investigations further revealed that CLTC elicited its pro-tumor effects by directly binding to and stabilizing trafficking from the endoplasmic reticulum to the Golgi regulator (TFG). Importantly, overexpression of TFG rescued both the tumor-suppressive effect and inhibition of the TGF-β and AKT/mTOR pathways caused by CLTC down-regulation, which indicated that the activity of CLTC was TFG-dependent. Immunohistochemistry analysis confirmed that CLTC expression was positively correlated with TFG expression. These findings collectively highlight CLTC as a new prognostic biomarker for patients with osteosarcoma, and the interruption of the SP1/CLTC/TFG axis may serve as a novel therapeutic strategy for osteosarcoma.

Topics: Adult; Animals; Apoptosis; Biomarkers, Tumor; Bone Neoplasms; Cell Cycle; Cell Movement; Cell Proliferation; Clathrin Heavy Chains; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Osteosarcoma; Prognosis; Proteins; Proto-Oncogene Proteins c-akt; Signal Transduction; Survival Rate; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Xenograft Model Antitumor Assays; Young Adult

Since William Coley utilized bacterial immunotherapy to treat sarcomas in the late 19th century, an association between infection and improved survival has been reported for human and canine osteosarcoma patients. One of the reasons for this improved survival is likely a reactivation of the host immune system towards an inflammatory anti-tumour response, and one of the key players is the macrophage. Yet, despite their importance, the response of macrophages to infectious agents in the context of osteosarcoma has not been thoroughly evaluated. The aim of this study was to evaluate how in vitro exposure to a bacterial agent (Staphylococcus aureus) influenced canine and human macrophage differentiation in the presence of osteosarcoma. Our hypothesis was that S. aureus would, in the presence of osteosarcoma, induce a macrophage phenotype with significantly increased inflammatory signatures. Consistent with our hypothesis, human macrophages co-cultured with osteosarcoma and S. aureus exhibited increased IFN-γ, TNF-α and IL-12p70 cytokine secretion, decreased TGF-β cytokine secretion and increased mRNA expression of TNF-α when compared with macrophages co-cultured with osteosarcoma and to macrophages cultured alone. Canine macrophages similarly exhibited increased IFN-γ and TNF-α cytokine secretion, decreased TGF-β cytokine secretion, increased mRNA expression of TNF-α and increased surface receptor expression of CD80 when co-cultured with osteosarcoma and S. aureus. Collectively, the findings of this study suggest that infection upregulates the inflammatory immune response to counteract osteosarcoma-induced immune suppression. This work informs a potential therapeutic strategy to optimize inflammatory stimuli for triggering an anti-osteosarcoma macrophage response.

Topics: Adolescent; Adult; Animals; Bone Neoplasms; Cytokines; Dog Diseases; Dogs; Down-Regulation; Humans; Macrophages; Osteosarcoma; Staphylococcus aureus; Transforming Growth Factor beta; Young Adult

Long noncoding RNAs (lncRNAs) play crucial roles in tumor development of osteosarcoma (OS). LncRNA PCAT6 was involved in the progression of multiple human cancers. However, the biological function of PCAT6 in OS remains largely unknown. We found that PCAT6 was elevated in OS tissues relative to that in their adjacent normal tissues. The upregulation of PCAT6 was positively associated with metastasis status and advanced stages and predicted poor overall and progression-free survivals in patients with OS. Functionally, silencing PCAT6 inhibited the proliferation, migration and invasion abilities of OS cells. Mechanistically, PCAT6, acting as a competitive endogenous RNA, upregulated expression of TGFBR1 and TGFBR2 to activate TGF-β pathway via sponging miR-185-5p. This study uncovers a novel underlying molecular mechanism of PCAT6-miR-185-5p-TGFBR1/2-TGF-β signaling axis in promoting tumor progression in OS, which indicates that PCAT6 may serve as a promising prognostic factor and therapeutic target again OS.

Topics: Disease Progression; Humans; MicroRNAs; Osteosarcoma; Prognosis; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; RNA, Long Noncoding; Transforming Growth Factor beta; Up-Regulation

Leptin signaling influences osteoblastogenesis and modulates the fate of mesenchymal stem cells (MSCs) during bone and cartilage regeneration. Although MSCs abound in the osteosarcoma (OS) microenvironment, and leptin exhibits pro-tumorigenic properties, leptin's influence on OS progression and chemoresistant signaling in MSCs remains unclear. Using cell viability and apoptosis assays, we showed that medium conditioned by leptin-treated human MSCs promotes cisplatin resistance in cultured human OS cells. Moreover, GFP-LC3 expression and chloroquine treatment experiments showed that this effect is mediated by stimulation of autophagy in OS cells. TGF-β expression in MSCs was upregulated by leptin and suppressed by leptin receptor knockdown. Silencing TGF-β in MSCs also abolished OS cell chemoresistance induced by leptin-conditioned medium. Cisplatin resistance was also induced when leptin-conditioned MSCs were co-injected with MG-63 OS cells to generate subcutaneous xenografts in nude mice. Finally, we observed a significant correlation between autophagy-associated gene expression in OS clinical samples and patient prognosis. We conclude that leptin upregulates TGF-β in MSCs, which promotes autophagy-mediated chemoresistance in OS cells.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Carcinogenesis; Chloroquine; Cisplatin; Drug Resistance, Neoplasm; Leptin; Mesenchymal Stem Cells; Mice; Osteosarcoma; Transforming Growth Factor beta; Tumor Microenvironment; Up-Regulation; Xenograft Model Antitumor Assays

Osteosarcoma (OS) is a progressive primary bone tumor that originates from immature stromal spindle cells. After chemotherapy, the serum-related indexes which are related to the prognosis.. The aim of this study is to investigate the correlation between changes in serum cyclooxygenase-2 (COX-2), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGF-β), and tumor-specific growth factor (TSGF) levels and prognosis of patients with osteosarcoma (OS) before and after treatment.. Data of 75 patients with OS (observation group) and 55 healthy controls (control group) were retrospectively analyzed.. Chemotherapy was administered to the observation group. Serum lactate dehydrogenase, alkaline phosphatase, and TSGF levels were measured before and after treatment. The observation group patients were classified as normal or abnormal according to the changes in serum COX-2, bFGF, VEGF, TGF-β, and TSGF levels after chemotherapy. Patients were followed up for 7.5 years, and the survival rate was determined.. Single-factor influencing prognosis was included in the Cox model, and independent factors influencing prognosis were analyzed.. After chemotherapy, the mean serum COX-2, bFGF, VEGF, and TSGF levels decreased significantly in the observation group but were still higher than those in the control group. Furthermore, serum TGF-β levels increased in the observation group but were still lower than those in the control group. The 5-year survival rate of patients with normal serum COX-2, bFGF, VEGF, and TSGF levels was significantly higher in the normal subgroup than in the abnormal subgroup. Cox analysis showed that the Enneking stage and COX-2 level after chemotherapy were independent prognostic factors.. The serum COX-2, bFGF, VEGF, and TSGF levels of patients with OS significantly changed after chemotherapy, and the short-term survival rate of patients with normal levels of these biomarkers after chemotherapy was high.

Topics: Adolescent; Adult; Antigens, Neoplasm; Biomarkers, Tumor; Bone Neoplasms; Case-Control Studies; Child; Cyclooxygenase 2; DNA-(Apurinic or Apyrimidinic Site) Lyase; Female; Fibroblast Growth Factor 2; Humans; Male; Neoplasm Proteins; Osteosarcoma; Prognosis; Retrospective Studies; Survival Rate; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Young Adult

Topics: Adolescent; Adult; Animals; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Child; Disease Progression; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplastic Stem Cells; Osteosarcoma; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Young Adult

To investigate the effect of gamabufotalin (GBT) on metastasis and modulation of stemness features in osteosarcoma, and the molecular mechanisms underlying such effects.. Human osteosarcoma U2OS/MG-63 cell lines were used in this study. Cell proliferation, migration, and invasion were determined by MTT assay, wound healing assay, and cell invasion assay, respectively. The inhibitive effect of GBT on stemness was assessed by flow cytometry and mammosphere formation. The protein levels of related proteins were detected by western blotting analysis. The effect of GBT on tumorigenicity and metastasis was determined by immunofluorescence staining and immunohistochemistry in vivo experiments.. We found that GBT suppressed the viability of U2OS/MG-63 cells in a time- and dose-dependent manner. Notably, GBT had no effect on the viability of human fetal osteoblastic (hFOB) 1.19 cells. Moreover, GBT increased the width of wounds, reduced the number of invasive osteosarcoma cells and reversed the epithelial-mesenchymal transition phenotype. Notably, we found that, compared with hFOB1.19 cells, the levels of transforming growth factor-β (TGF-β), periostin, phosphorylated-AKT (p-AKT), and phosphorylated-PI3K (p-PI3K) were higher in spheroids group than in parent cells. In addition, GBT reduced the ratio of CD133+ cells, the size of spheroids and Nanog, as well as the protein levels of SRY-box transcription factor 2 (SOX2), and octamer-binding protein 3/4 (OCT3/4). Our in vivo experiments showed that GBT consistently reduced lung metastasis lesions, the expression levels of matrix metalloproteinase 2 (MMP2), TGF-β, periostin, p-AKT, and p-PI3K (immunohistochemistry staining), as well as that of CD133 in tumor tissues (immunofluorescence analysis). From a mechanistic point of view, exogenous TGF-β/periostin/PI3K/AKT overexpression neutralized the reduction of GBT-decreased invasion/migration and the suppression of stemness properties.. Collectively, our data demonstrated that GBT inhibited the viability and tumorigenesis capability of osteosarcoma cells by blocking the TGF-β/periostin/PI3K/AKT signaling pathway. Therefore, GBT may represent a promising therapeutic agent for the management of osteosarcoma.

Topics: Animals; Antineoplastic Agents; Bufanolides; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Humans; Mice; Osteosarcoma; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Our previous study has demonstrated that high expression of ALDH1B1 promoted osteosarcoma tumor progression and was correlated with unfavorable prognosis in osteosarcoma patients. In the current study, we investigated the underlying mechanism and regulation of ALDH1B1 in osteosarcoma.. qRT-PCR assay was applied to detect miR-761 expression. CCK-8, colony formation and EdU assays were conducted to explore the functional role of miR-761/ALDH1B1 axis in osteosarcoma. Bioinformatics analysis and luciferase reporter assay was utilized to assess the regulation between miR-761 and ALDH1B1. Mechanism experiments were implemented to investigate the underlying molecular mechanism of miR-761/ALDH1B1 axis.. ALDH1B1 was negatively regulated by microRNA-761 (miR-761). Functionally, miR-761 suppressed cell growth, migration, and invasion in osteosarcoma via targeting ALDH1B1 in vitro. Xenograft tumor model demonstrated that miR-761 inhibited osteosarcoma tumor development in vivo through regulating ALDH1B1. Consistently, we showed that miR-761 expression was decreased in osteosarcoma patients and low expression of miR-761 was correlated with worse prognosis in osteosarcoma patients. Mechanistically, we revealed that high expression of ALDH1B1 was significantly associated with enhanced TGF-β signaling, epithelial-mesenchymal transition (EMT), and cell adhesion. Furthermore, miR-761 regulated TGF-β and EMT/cell adhesion in osteosarcoma via targeting ALDH1B1.. Taken together, our findings suggest that the oncogenic ALDH1B1 is regulated by miR-761 during osteosarcoma development and progression, which might provide a novel prognostic biomarker and therapeutic strategy for osteosarcoma treatment.

Topics: Aldehyde Dehydrogenase 1 Family; Aldehyde Dehydrogenase, Mitochondrial; Animals; Bone Neoplasms; Cell Line, Tumor; Disease Progression; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Osteosarcoma; Prognosis; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Sarcomas are mesenchymal cancers with poor prognosis, representing about 20% of all solid malignancies in children, adolescents, and young adults. Radio- and chemoresistance are common features of sarcomas warranting the search for novel prognostic and predictive markers. GARP/LRRC32 is a TGF-β-activating protein that promotes immune escape and dissemination in various cancers. However, if GARP affects the tumorigenicity and treatment resistance of sarcomas is not known. We show that GARP is expressed by human osteo-, chondro-, and undifferentiated pleomorphic sarcomas and is associated with a significantly worse clinical prognosis. Silencing of GARP in bone sarcoma cell lines blocked their proliferation and induced apoptosis. In contrast, overexpression of GARP promoted their growth in vitro and in vivo and increased their resistance to DNA damage and cell death induced by etoposide, doxorubicin, and irradiation. Our data suggest that GARP could serve as a marker with therapeutic, prognostic, and predictive value in sarcoma. We propose that targeting GARP in bone sarcomas could reduce tumour burden while simultaneously improving the efficacy of chemo- and radiotherapy.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Bone Neoplasms; Cell Line, Tumor; Cell Proliferation; Child; Child, Preschool; Female; Heterografts; Humans; Male; Membrane Proteins; Mice; Mice, Inbred NOD; Mice, SCID; Middle Aged; Osteosarcoma; Prognosis; Transforming Growth Factor beta; Young Adult

Topics: Antineoplastic Agents; Apoptosis; Caspase 9; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Ketoglutaric Acids; MAP Kinase Kinase 4; Osteosarcoma; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Growth and differentiation factor 15 (GDF15), a novel divergent member of the transforming growth factor‑β (TGF‑β) superfamily, was previously reported to be overexpressed in various types of cancers and was shown to be involved in tumor metastasis; however, the role of GDF15 in the development and malignant progression of osteosarcoma remains unclear. In the present study, reverse transcription‑quantitative polymerase chain reaction, western blot and ELISA analyses were performed to detect mRNA and protein expression, including that of GDF15, SMAD2 and SMAD3. Wound‑healing and cell invasion assays were conducted to determine the migratory and invasive abilities of osteosarcoma cells. A luciferase assay was performed to evaluate the transcriptional activity of a TGF‑β/SMAD‑responsive luciferase reporter. The Kaplan‑Meier method was used to generate survival curves, with a log‑rank test use to evaluate differences in survival. The results revealed that GDF15 expression was upregulated in metastatic osteosarcoma tissues compared with non‑metastatic osteosarcoma tissues. Patients with osteosarcoma that possessed high serum GDF15 levels exhibited significantly decreased overall survival (OS) and pulmonary metastasis‑free survival (PMFS) time compared with patients with low GDF15 expression. Furthermore, high serum GDF15 was an independent prognostic parameter for poor OS and short PMFS. Additionally, it was observed that the knockdown of GDF15 attenuated the migration and invasion of osteosarcoma cells. Silencing GDF15 markedly suppressed the TGF‑β signaling pathway. In conclusion, GDF15 may promote osteosarcoma cell metastasis by regulating the TGF‑β signaling pathway, and serum GDF15 levels may be a potential prognostic and pulmonary metastasis‑predictive biomarker in osteosarcoma.

Topics: Adolescent; Adult; Biomarkers, Tumor; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Growth Differentiation Factor 15; Humans; Kaplan-Meier Estimate; Middle Aged; Models, Biological; Neoplasm Staging; Osteosarcoma; Prognosis; Signal Transduction; Transforming Growth Factor beta; Young Adult

Osteosarcoma (OS) is identified as an aggressive malignancy of the skeletal system and normally occurs among young people. It is well accepted that microRNAs are implicated in biological activities of diverse tumors. Although miR-522 has been proved to elicit oncogenic properties in a wide range of human cancers, the physiological function and latent mechanism of miR-522 in OS tumorigenesis remain largely to be probed. In the current study, we certified that miR-522 was highly expressed in OS cells and presented carcinogenic function by contributing to cell proliferation, migration, and EMT progression whereas dampening cell apoptosis. In addition, miR-522 provoked TGF-β/Smad pathway through targeting PPM1A. Finally, the results of mechanism experiments elucidated that miR-522 stimulated TGF-β/Smad pathway to induce the development of OS via targeting PPM1A, which exposed that miR-522 may become a promising curative target for OS patients.

Topics: Carcinogenesis; Cell Line; Cell Line, Tumor; Cell Movement; Cell Survival; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; MicroRNAs; Osteosarcoma; Protein Phosphatase 2C; Real-Time Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Wound Healing

BACKGROUND Osteosarcoma is a primary bone aggressive cancer, affecting adolescents worldwide. Increasing evidence suggests that dysfunction of microRNAs (miRNAs) plays a pivotal role in malignancies. The aim of this study was to evaluate the potential functions of miR-181c and verifying its regulatory effects on SMAD7 in osteosarcoma. MATERIAL AND METHODS The expressions of miR-181c and SMAD7 in osteosarcoma were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, invasion and migration abilities were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and Transwell assay. Bioinformatics analysis and luciferase reporter assay were used to explore the interaction between miR-181c and SMAD7. Western blot was performed to determine the functions of miR-181c on osteosarcoma cell epithelial-to-mesenchymal transition (EMT) and transforming growth factor-ß (TGF-ß) signaling pathway. RESULTS Decreased expression levels of miR-181c and SMAD7 were identified in osteosarcoma using qRT-PCR. The downregulated miR-181c and SMAD7 expressions indicated poor prognosis of osteosarcoma patients. Moreover, miR-181c overexpression prominently repressed osteosarcoma cell proliferation, invasion, and migration abilities via modulating EMT and TGF-ß signaling pathway. SMAD7 functioned as an important target for miR-181c in osteosarcoma cells. Furthermore, upregulation of miR-181c dramatically suppressed osteosarcoma tumorigenesis in vivo. CONCLUSIONS These findings indicated that miR-181c suppressed osteosarcoma progression, providing new insight into the pathogenesis and representing a potential therapeutic target for osteosarcoma.

Topics: Aged; Aged, 80 and over; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Epithelial-Mesenchymal Transition; Female; Humans; Male; MicroRNAs; Middle Aged; Neoplasm Invasiveness; Osteosarcoma; Signal Transduction; Smad7 Protein; Transforming Growth Factor beta; Up-Regulation

The present study aimed to explore the potential anti-tumor effect of ERβ overexpression and investigate its related mechanism in osteosarcoma. Cell cycle and apoptosis rates were measured by flow cytometry. Cell proliferation and formation of autophagosome were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and dansylcadaverine (MDC) staining assay. Cell migration and invasion were detected by wound healing assay and transwell assay. Western blot analysis was designed to detect the protein expressions of surviving, Bax, LC-3 П, Beclin-1, ERβ, TβRⅠ, TβRⅡ, Smad2, Smad3 and Smad7. Real-Time fluorogenic PCR was designed to examine the mRNA expressions of surviving, Bax, ERβ, TβRⅠ, TβRII, Smad2, Smad3 and Smad7. The results showed that ERβ overexpression inhibited cell proliferation, migration and invasion, blocked cell cycle, and induced apoptosis and autophagy. Additionally, ERβ overexpression significantly inhibited the expression of surviving, TβRⅠ, TβRⅡ, Smad2 and Smad3. Meanwhile, the expressions of Bax, LC-3 П, Beclin-1 and Smad7 were dramatically upregulated by ERβ overexpression. In conclusion, ERβ overexpression could inhibit cell proliferation, migration and invasion, block cell cycle, and promote apoptosis and autophagy in OS by downregulating TNG-β signaling pathway.

Topics: Apoptosis; Bone Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Estrogen Receptor beta; Humans; Osteoblasts; Osteosarcoma; Signal Transduction; Transforming Growth Factor beta

Osteosarcoma is one of the most common malignant tumors in adolescent populations and the prognosis remains incompletely understand. Previous reports have demonstrated that microRNA‑124 (miR‑124) has inhibitory effects on various human malignancies and is associated with tumor progression. However, the clinical significance and potential mechanisms of miR‑124 in the progression of osteosarcoma is not clearly understood. In this study, the potential molecular mechanism of miR‑124 in osteosarcoma tumorigenesis, growth and aggressiveness was investigated. The growth, proliferation, apoptosis, migration and invasion of osteosarcoma cells were investigated following miR‑124 transfection were determined by colony formation assay, western blotting, immunofluorescence, migration/invasion assays and reverse transcription‑quantitative polymerase chain reaction. In vivo anti‑cancer effects of miR‑124 were analyzed by a tumor growth assay, immunohistochemistry and survival rate observations. The results demonstrated that miR‑124 transfection significantly decreased integrin expression in osteosarcoma cells, and further inhibited growth, proliferation, migration and invasion of osteosarcoma cells. Flow cytometry assays indicated that miR‑124 transfection attenuated apoptosis resistance of osteosarcoma to tunicamycin, potentially via the downregulation of P53 and Bcl‑2 apoptosis regulator expression. Mechanistic assays demonstrated that miR‑124 transfection suppressed TGF‑β expression in osteosarcoma. An animal study revealed that tumor growth was reduced in tumor cells transfected with miR‑124 compared with control cells, and the survival rate was prolonged in mice with miR‑124 transfected xenografts compared with control tumors. In conclusion, these results indicate that miR‑124 transection inhibits the growth and aggressive of osteosarcoma, potentially via suppression of TGF‑β‑mediated AKT/GSK‑3β/snail family transcriptional repressor 1 (SNAIL‑1) signaling, suggesting miR‑124 may be a potential anti‑cancer agent/target for osteosarcoma therapy.

Topics: Animals; Bone Neoplasms; Female; Glycogen Synthase Kinase 3 beta; Humans; Mice; Mice, Nude; MicroRNAs; Osteosarcoma; Proto-Oncogene Proteins c-akt; RNA, Neoplasm; Signal Transduction; Snail Family Transcription Factors; Transforming Growth Factor beta

Over the last three decades, the prognosis of osteosarcoma has remained unchanged; the prognosis for patients with lung metastasis is still poor, and the development of new treatments is urgently required. We previously showed that aggressive osteosarcoma cells express more tissue factor (TF) and demonstrate enhanced extrinsic pathway capacity. Furthermore, tumor growth can be suppressed with the anticoagulant low molecular weight heparin. However, the molecular mechanisms underlying TF regulation are still unclear. Here, we report that transforming growth factor-β (TGF-β) upregulates TF, which can occur via activated platelets. TF was found to be expressed on osteosarcoma cell surfaces, which mediated the production of Xa and thrombin. TF induction by TGF-β was observed in several osteosarcoma cells, and especially in MG 63 cells. Both TF expression by TGF-β and extrinsic pathway activity through TF were rapidly increased. This reaction was inhibited by a TGF-β type I receptor inhibitor and TGF-β neutralizing antibody. Although TGF-β was found to phosphorylate both Smad2 and Smad3, their roles were markedly disparate. Surprisingly, Smad2 knockdown resulted in no inhibitory effect, whereas Smad3 knockdown completely suppressed TGF-β-induced TF expression. Next, data suggested that platelets were the source of TGF-β. We confirmed that thrombin-activated platelets and osteosarcoma cells could release TGF-β, and that platelet-derived TGF-β could induce TF expression. These processes were also inhibited by a TGF-β type I receptor inhibitor and Smad3 knockdown. Moreover, CD42b, TF, TGF-β, Smad2/3, and p-Smad2/3 were also detected in a biopsy sample from an osteosarcoma patient. Collectively, these finding suggested that the interaction between osteosarcoma cells and platelets, via thrombin and TGF-β, results in a continuous cycle, and that anti-platelet or anti-TGF-β therapy could be a promising tool for disease treatment. © 2018 American Society for Bone and Mineral Research.

Topics: Animals; Blood Platelets; Cell Line, Tumor; Cell Membrane; Humans; Intracellular Space; Mice; Models, Biological; Osteosarcoma; Phosphorylation; Signal Transduction; Smad2 Protein; Smad3 Protein; Thrombin; Thromboplastin; Transforming Growth Factor beta

Osteosarcoma (OSA) is the most common bone cancer in canines. Both transforming growth factor beta (TGFβ) and Hippo pathway mediators have important roles in bone development, stemness, and cancer progression. The role of Hippo signalling effectors TAZ and YAP has never been addressed in canine OSA. Further, the cooperative role of TGFβ and Hippo signalling has yet to be explored in osteosarcoma. To address these gaps, this study investigated the prognostic value of TAZ and YAP alone and in combination with pSmad2 (a marker of active TGFβ signalling), as well as the involvement of a TGFβ-Hippo signalling crosstalk in tumourigenic properties of OSA cells in vitro. An in-house trial tissue microarray (TMA) which contained 16 canine appendicular OSA cases undergoing standard care and accompanying follow-up was used to explore the prognostic role of TAZ, YAP and pSmad2. Published datasets were used to test associations between TAZ and YAP mRNA levels, metastasis, and disease recurrence. Small interfering RNAs specific to TAZ and YAP were utilized in vitro alone or in combination with TGFβ treatment to determine their role in OSA viability, proliferation and migration.. Patients with low levels of both YAP and pSmad2 when evaluated in combination had a significantly longer time to metastasis (log-rank test, p = 0.0058) and a longer overall survival (log rank test, p = 0.0002). No similar associations were found for TAZ and YAP mRNA levels. In vitro, TAZ knockdown significantly decreased cell viability, proliferation, and migration in metastatic cell lines, while YAP knockdown significantly decreased viability in three cell lines, and migration in two cell lines, derived from either primary tumours or their metastases. The impact of TGFβ signaling activation on these effects was cell line-dependent.. YAP and pSmad2 have potential prognostic value in canine appendicular osteosarcoma. Inhibiting YAP and TAZ function could lead to a decrease in viability, proliferation, and migratory capacity of canine OSA cells. Assessment of YAP and pSmad2 in larger patient cohorts in future studies are needed to further elucidate the role of TGFβ-Hippo signalling crosstalk in canine OSA progression.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Disease Progression; Dog Diseases; Dogs; Female; Male; Osteosarcoma; Signal Transduction; Smad2 Protein; Transcription Factors; Transforming Growth Factor beta

Osteosarcoma is the most common type of bone cancer, especially in children and young adults. Recently, long noncoding RNAs (lncRNAs) have emerged as new prognostic markers and gene regulators in several cancers, including osteosarcoma. In this study, we investigated the contributions of the lncRNA MALAT1 in osteosarcoma with a specific focus on its transcriptional regulation and its interaction with EZH2. Our results showed that MALAT1 was significantly increased in osteosarcoma specimens and cell lines. ROC curve analysis showed that MALAT1 had a higher area under the curve than alkaline phosphatase, and Kaplan-Meier survival analysis indicated that patients with high serum levels of MALAT1 showed reduced survival rate. Knockdown of MALAT1 decreased osteosarcoma cell invasion and promoted E-cadherin expression. Mechanistic investigations showed that MALAT1 was transcriptionally activated by TGF-β. Additionally, EZH2 is highly expressed and associated with the 3' end region of lncRNA MALAT1 in osteosarcoma, and this association finally suppressed the expression of E-cadherin. Subsequently, our gain and loss function assay showed that MALAT1 overexpression promoted cell metastasis and decreased E-cadherin level, however, this effect was partially reversed by EZH2 knockdown. In conclusion, our work illuminates that lncRNA MALAT1 is a potential diagnostic and prognostic factor in osteosarcoma and further demonstrates how MALAT1 confers an oncogenic function. Thus, lncRNA MALAT1 may serve as a promising prognostic and therapeutic target for osteosarcoma patients.

Topics: 3' Untranslated Regions; Adult; Bone Neoplasms; Cadherins; Cell Line, Tumor; Cell Movement; Enhancer of Zeste Homolog 2 Protein; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Middle Aged; Neoplasm Grading; Neoplasm Metastasis; Neoplasm Staging; Osteosarcoma; Prognosis; RNA, Long Noncoding; ROC Curve; Transforming Growth Factor beta; Tumor Burden; Young Adult

Osteosarcoma is suggested to be caused by genetic and molecular alterations that disrupt osteoblast differentiation. Recent studies have reported that transmembrane protein 119 (TMEM119) contributes to osteoblast differentiation and bone development. However, the level of TMEM119 expression and its roles in osteosarcoma have not yet been elucidated. In the present study, TMEM119 mRNA and protein expression was found to be up-regulated in osteosarcoma compared with normal bone cyst tissues. The level of TMEM119 protein expression was strongly associated with tumor size, clinical stage, distant metastasis and overall survival time. Moreover, gene set enrichment analysis (GSEA) of the Gene Expression Omnibus (GEO) GSE42352 dataset revealed TMEM119 expression in osteosarcoma tissues to be positively correlated with cell cycle, apoptosis, metastasis and TGF-β signaling. We then knocked down TMEM119 expression in U2OS and MG63 cells using small interfering RNA, which revealed that downregulation of TMEM119 could inhibit the proliferation of osteosarcoma cells by inducing cell cycle arrest in G0/G1 phase and apoptosis. We also found that TMEM119 knockdown significantly inhibited cell migration and invasion, and decreased the expression of TGF-β pathway-related factors (BMP2, BMP7 and TGF-β). TGF-β application rescued the inhibitory effects of TMEM119 knockdown on osteosarcoma cell migration and invasion. Further in vitro experiments with a TGF-β inhibitor (SB431542) or BMP inhibitor (dorsomorphin) suggested that TMEM119 significantly promotes cell migration and invasion, partly through TGF-β/BMP signaling. In conclusion, our data support the notion that TMEM119 contributes to the proliferation, migration and invasion of osteosarcoma cells, and functions as an oncogene in osteosarcoma.

Topics: Adolescent; Animals; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 7; Bone Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Female; HEK293 Cells; Humans; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Osteosarcoma; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Canine osteosarcoma (OSA) is the most common cancer of the appendicular skeleton and is associated with high metastatic rate to the lungs and poor prognosis. Recent studies have shown the impact of malignant-derived exosomes on immune cells and the facilitation of immune evasion. In the current study, we have characterized the proteomic profile of exosomes derived from healthy osteoblasts and osteosarcoma cell lines. We investigated the direct impact of these exosomes on healthy T cells.. Proteomic cargo of the malignant exosomes was markedly different from osteoblastic exosomes and contained immunosuppressive proteins including TGF-β, α fetoprotein and heat shock proteins. OSA exosomes directly attenuated the rate of T cell proliferation, increased a regulatory (FoxP3+) CD4+ phenotype and diminished the expression of the activation marker CD25+ on CD8+ cells. Exosomes of osteoblasts also demonstrated a direct impact on T cells, but to a lesser degree.. Osteosarcoma-derived exosomes compared to normal osteoblasts contain an immunomodulatory cargo, which reduced the rate of T cell proliferation and promoted T regulatory phenotype. Osteoblast-derived exosomes can also reduce T cell activity, but to lesser degree compared to OSA exosomes and without promoting a T regulatory phenotype.

Topics: Animals; Cell Proliferation; Dogs; Exosomes; Flow Cytometry; Lymphocyte Activation; Osteoblasts; Osteosarcoma; Proteomics; T-Lymphocytes; Transforming Growth Factor beta

This study aimed to investigate the role of long non-coding RNA MEG3 (lncRNA MEG3) in osteosarcoma (OS) and further explore the underlying molecular mechanism.. The expression profiles of MEG3 in OS cell lines and normal osteoblast cell line were detected by qRT-PCR. MEG3 was over-expressed in OS cell line by using LV-MEG3. MTT and colony-formation assays were applied for cell proliferation analysis. Cell migration assay was applied to investigate the cell migration ability. In addition, the expression levels of cell growth and metastasis related factors (Notch1, Hes1, TGF-β, N-cadheren and E-cadheren) were determined to illustrate the mechanisms.. We found that compared with normal osteoblast hFOB1.19 cell line, MEG3 was significantly down-regulated in MG63 and U2OS cell lines, particularly in MG-63 cells. MEG3 was significantly up-regulated in MG63 cells by LV-MEG3. Cell proliferation and migration ability were obviously repressed by MEG3 over-expression. In addition, MEG3 over-expression markedly inhibited Notch1, Hes1,TGF-β and N-cadheren expression, and the expression level of E-cadheren was improved.. These results indicated that MEG3 could prevent cell growth and metastasis of OS by repressing Notch and TGF-β signaling pathway, thus providing a potential therapeutic target for OS treatment (Tab. 1, Fig. 4, Ref. 30).

Topics: Antigens, CD; Bone Neoplasms; Cadherins; Cell Line; Cell Line, Tumor; Cell Movement; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Metastasis; Osteoblasts; Osteosarcoma; Receptor, Notch1; RNA, Long Noncoding; Transcription Factor HES-1; Transforming Growth Factor beta; Tumor Stem Cell Assay; Up-Regulation

Osteosarcoma patients often exhibit pulmonary metastasis, which results in high patient mortality. Understanding the mechanisms of advanced metastasis in osteosarcoma cell is important for the targeted treatment and drug development. Our present study revealed that transforming growth factor-β (TGF-β) treatment can significantly promote the in vitro migration and invasion of human osteosarcoma MG-63 and HOS cells. The loss of epithelial characteristics E-cadherin (E-Cad) and up regulation of mesenchymal markers Vimentin (Vim) suggested TGF-β induced epithelial-mesenchymal transition (EMT) of osteosarcoma cells. TGF-β treatment obviously increased the expression of Snail, a key EMT-related transcription factor, in both MG-63 and HOS cells. Silencing of Snail markedly attenuated TGF-β induced down regulation of E-cad and up regulation of Vim. TGF-β treatment also significantly increased the expression and nuclear translocation of estrogen-related receptors α (ERRα), while had no obvious effect on the expression of ERα, ERβ, or ERRγ. Knock down of ERRα or its inhibitor XCT-790 significantly attenuated TFG-β induced EMT and transcription of Snail in osteosarcoma cells. Collectively, our present study revealed that TGF-β treatment can trigger the EMT of osteosarcoma cells via ERRα/Snail pathways. Our data suggested that ERRα/Snail pathways might be potential therapeutic targets of metastasis of osteosarcoma cells.

Topics: Cell Line, Tumor; Cell Movement; Cell Nucleus; Epithelial-Mesenchymal Transition; ERRalpha Estrogen-Related Receptor; Gene Knockdown Techniques; Humans; Osteosarcoma; Protein Transport; Receptors, Estrogen; Snail Family Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta

Chemokine (C-X-C motif) receptor 6 (CXCR6) is up-regulated in many malignancies, indicating that CXCR6 plays an important role in tumor progression. However, the expression and function of CXCR6 in osteosarcoma (OS) remains unclear. This study aimed to explore the expression levels and function of CXCR6 in OS tissues and osteosarcoma cell lines MG-63, HOS and U2OS. The protein expression levels of CXCR6 in OS patient tissues and three osteosarcoma cell lines MG-63, HOS and U2OS were assessed. CXCR6-overexpression MG-63 cell lines were established and then the proliferation, invasion and the epithelial-mesenchymal transition (EMT) in those cells were assessed. CXCR6 mRNA levels in OS tissues were significantly higher than those in normal bone tissues. Consistently, both of the mRNA and protein levels of CXCR6 in OS cell lines MG-63, HOS and U2OS were higher than those in normal bone cells hFOB1.19. CXCR6 overexpression not only promoted cell proliferation, invasion and EMT, but also enhanced the phosphorylation of Akt in MG-63 cells. After inhibition of Akt-phosphorylation by Akt inhibitor, LY2940023, CXCR6-induced cell proliferation and invasion were dramatically attenuated. In conclusion, the present study demonstrated that CXCR6 enhances OS cell proliferation and invasion through the Akt pathway.

Topics: Carcinogenesis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Gene Expression Regulation; Humans; Oncogene Protein v-akt; Osteoblasts; Osteosarcoma; Phosphorylation; Receptors, Chemokine; Receptors, CXCR6; Receptors, Virus; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Antibodies, Monoclonal, Humanized; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Extracellular Vesicles; Female; Gene Expression Regulation, Neoplastic; Humans; Interleukin-6; Lung Neoplasms; Male; Mesenchymal Stem Cells; Mice; Osteosarcoma; Signal Transduction; STAT3 Transcription Factor; Tissue Array Analysis; Transforming Growth Factor beta

Extracellular signal-regulated kinase (ERK) is a downstream component of the evolutionarily conserved mitogen-activated protein kinase-signaling pathway, which controls the expression of a plethora of genes implicated in various physiological processes. This pathway is often hyper-activated by mutations or abnormal extracellular signaling in different types of human cancer, including the most common primary malignant bone tumor osteosarcomas. p16(INK4A) is an important tumor suppressor gene frequently lost in osteosarcomas, and is associated with the progression of these malignancies. We have shown, here, that the ERK1/2 protein kinase is also activated by p16(INK4A) down-regulation in osteosarcoma cells and normal human as well as mouse cells. This inhibitory effect is associated with the suppression of the upstream kinase MEK1/2, and is mediated via the repression of miR-21-5p and the consequent up-regulation of the MEK/ERK antagonist SPRY2 in osteosarcoma cells. Furthermore, we have shown that p16(INK4) inhibits the migration/invasion abilities of these cells through miR-21-5p-dependent inhibition of ERK1/2. In addition, we present clear evidence that p16(INK4) represses the paracrine pro-migratory effect of osteosarcoma cells on stromal fibroblasts through the inhibition of the TGF-β1 expression/secretion. This effect is also ERK1/2-dependent, indicating that in addition to their cell-autonomous actions, p16(INK4) and ERK1/2 have also non-cell-autonomous cancer-related functions. Together, these results indicate that the tumor suppressor p16(INK4) protein represses the carcinogenic process of osteosarcoma cells not only as a cell cycle regulator, but also as a negative regulator of pro-carcinogenic/-metastatic pathways. This indicates that targeting the ERK pathway is of utmost therapeutic value.

Topics: Animals; Bone Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cyclin-Dependent Kinase Inhibitor p16; Gene Expression Regulation, Neoplastic; Humans; MAP Kinase Signaling System; Mice; MicroRNAs; Neoplasm Metastasis; Neoplasm Proteins; Osteosarcoma; Phosphorylation; Transforming Growth Factor beta

Osteosarcoma is the most common primary bone tumor in adolescents associated with skeletal development. The molecular pathogenesis of osteosarcoma has not been completely determined, although many molecular alterations have been found in human osteosarcomas and cell lines.. We questioned whether (1) we could identify gene expression in osteosarcoma specimens that differs from normal osteoblasts and mesenchymal stem cells and (2) this would provide clues to the molecular pathogenesis of osteosarcoma?. The whole-genome transcriptional profiles of osteosarcomas, including two primary biopsy specimens, two cell lines, two xenografts derived from patient specimens, and one from normal osteoblasts and from mesenchymal stem cells, respectively, were quantitatively measured using serial analysis of gene expression. A statistical enrichment was performed, which selects the common genes altered in each of the osteosarcomas compared with each of the normal counterparts independently.. Sixty (92%) of 65 total genes that were at least twofold downregulated in osteosarcoma compared with osteoblasts and mesenchymal stem cells, could be classified in four categories: (1) seven genes in the insulin–like growth factor (IGF) signaling axis, including three of the IGF-binding proteins (IGFBP) and three of the IGFBPrelated proteins (IGFBPrP); (2) eight genes in the transforming growth factor-b (TGF-b)/bone morphogenetic protein (BMP) signaling cascade; (3) 39 genes encoding cytoskeleton and extracellular matrix proteins that are regulated by TGF-b/BMPs; and (4) six genes involved in cell cycle regulation, including tumor suppressors TP63 and p21.. Based on these transcriptional profiles, a coordinated theme of clustered gene deregulation in osteosarcoma has emerged. Cell proliferation driven by the IGF axes during bone growth is unrestrained owing to downregulation of IGFBPs and cell cycle regulators. Tumor cells may be maintained in an undifferentiated state secondary to impaired TGF-b/BMP signaling. This wellpreserved pattern suggests that the alterations in the signaling axes of IGF-1 and TGF-b, in concert with cell cycle regulators, may be an important pathogenic basis of osteosarcoma. CLINIC RELEVANCE: This study provides a possible molecular basis of pathogenesis of osteosarcoma. This may help to develop new therapeutic targets and strategy for this disease. Preclinical and subsequently clinical testing of inhibitors of the IGF-1 and TGF pathways would be warranted.

Topics: Adolescent; Adult; Animals; Biomarkers, Tumor; Biopsy; Bone Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Child; Computational Biology; Databases, Genetic; Female; Gene Expression Profiling; Genome-Wide Association Study; Genotype; Heterografts; Humans; Male; Mesenchymal Stem Cells; Mice; Neoplasm Transplantation; Osteoblasts; Osteosarcoma; Phenotype; Signal Transduction; Somatomedins; Transforming Growth Factor beta

Topics: Animals; Biomarkers, Tumor; Bone Neoplasms; Female; Gene Expression Profiling; Humans; Male; Osteosarcoma; Signal Transduction; Somatomedins; Transforming Growth Factor beta

Primary cancer cell dissemination is a key event during the metastatic cascade, but context-specific determinants of this process remain largely undefined. Multiple reports have suggested that the p53 (TP53) family member p63 (TP63) plays an antimetastatic role through its minor epithelial isoform containing the N-terminal transactivation domain (TAp63). However, the role and contribution of the major p63 isoform lacking this domain, ΔNp63α, remain largely undefined. Here, we report a distinct and TAp63-independent mechanism by which ΔNp63α-expressing cells within a TGFβ-rich microenvironment become positively selected for metastatic dissemination. Orthotopic transplantation of ΔNp63α-expressing human osteosarcoma cells into athymic mice resulted in larger and more frequent lung metastases than transplantation of control cells. Mechanistic investigations revealed that ΔNp63α repressed miR-527 and miR-665, leading to the upregulation of two TGFβ effectors, SMAD4 and TβRII (TGFBR2). Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound-healing response. We show that ΔNp63α-mediated repression of miR-527/665 controls a TGFβ-dependent signaling node that switches off antimigratory miR-198 by suppressing the expression of the regulatory factor, KSRP (KHSRP). Collectively, these findings reveal that a novel miRNA network involved in the regulation of physiologic wound-healing responses is hijacked and suppressed by tumor cells to promote metastatic dissemination. Cancer Res; 76(11); 3236-51. ©2016 AACR.

Topics: Animals; Apoptosis; Blotting, Western; Bone Neoplasms; Cell Movement; Cell Proliferation; Gene Deletion; Gene Expression Regulation, Neoplastic; Humans; Immunoenzyme Techniques; Lung Neoplasms; Mice; MicroRNAs; Osteosarcoma; Protein Isoforms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Proteins; Wound Healing; Xenograft Model Antitumor Assays

microRNAs (miRs) are small non-coding RNAs involved in the fine regulation of several cellular processes by inhibiting their target genes at post-transcriptional level. Osteosarcoma (OS) is a tumor thought to be related to a molecular blockade of the normal process of osteoblast differentiation. The current paper explores temporal transcriptional modifications comparing an osteosarcoma cell line, Saos-2, and clones stably transfected with CD99, a molecule which was found to drive OS cells to terminally differentiate.. Parental cell line and CD99 transfectants were cultured up to 14 days in differentiating medium. In this setting, OS cells were profiled by gene and miRNA expression arrays. Integration of gene and miRNA profiling was performed by both sequence complementarity and expression correlation. Further enrichment and network analyses were carried out to focus on the modulated pathways and on the interactions between transcriptome and miRNome. To track the temporal transcriptional modification, a PCA analysis with differentiated human MSC was performed.. We identified a strong (about 80 %) gene down-modulation where reversion towards the osteoblast-like phenotype matches significant enrichment in TGFbeta signaling players like AKT1 and SMADs. In parallel, we observed the modulation of several cancer-related microRNAs like miR-34a, miR-26b or miR-378. To decipher their impact on the modified transcriptional program in CD99 cells, we correlated gene and microRNA time-series data miR-34a, in particular, was found to regulate a distinct subnetwork of genes with respect to the rest of the other differentially expressed miRs and it appeared to be the main mediator of several TGFbeta signaling genes at initial and middle phases of differentiation. Integration studies further highlighted the involvement of TGFbeta pathway in the differentiation of OS cells towards osteoblasts and its regulation by microRNAs.. These data underline that the expression of miR-34a and down-modulation of TGFbeta signaling emerge as pivotal events to drive CD99-mediated reversal of malignancy and activation of differentiation in OS cells. Our results describe crucial and specific interacting actors providing and supporting their relevance as potential targets for therapeutic differentiative strategies.

Topics: 12E7 Antigen; Antigens, CD; Cell Adhesion Molecules; Cell Differentiation; Cell Line, Tumor; Clone Cells; Down-Regulation; Gene Expression Profiling; Humans; MicroRNAs; Osteoblasts; Osteosarcoma; Phenotype; RNA, Messenger; Signal Transduction; Time Factors; Transfection; Transforming Growth Factor beta

Aquaporin 1 (AQP1), a member of water channel proteins, functions as a water-selective transporting protein in cell membranes. In recent years, AQP1 has been found to be overexpressed in various tumors. However, the molecular mechanism underlying the biological function of AQP1 in osteosarcoma is still unclear. This study was aimed at elucidating the roles of AQP1 in regulating the biological behavior of osteosarcoma cells. In this study, we found that AQP1 mRNA was elevated in osteosarcoma tissue. High level of AQP1 was associated with poor prognosis in osteosarcoma. Then, we found that knockdown of AQP1 in osteosarcoma cells, U2OS or MG63 cells inhibited cell proliferation and significantly increased cells population in G1 phase. Additionally, suppressing AQP1 expression in osteosarcoma cells dramatically induced cell apoptosis. We also found that down-regulation of AQP1 significantly inhibited cell adhesion and invasion. More importantly, AQP1 knockdown inhibited tumor growth in vivo and prolonged the survival time of nude mice. Gene set enrichment analysis (GSEA) showed that transforming growth factor-β (TGF-β) signaling pathway and focal adhesion genes was correlatively with AQP1 expression. In addition, real time PCR and western blot analysis revealed that expression of TGF-β1/TGF-β2, RhoA and laminin β 2 (LAMB2) was remarkably impaired by AQP1 silencing. In conclusion, AQP1 may be a useful diagnosis and prognosis marker for osteosarcoma. AQP1 knockdown can effectively inhibit cell proliferation, adhesion, invasion and tumorigenesis by targeting TGF-β signaling pathway and focal adhesion genes, which may serve a promising therapeutic strategy for osteosarcoma.

Topics: Animals; Aquaporin 1; Bone Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; G1 Phase Cell Cycle Checkpoints; Gene Expression; Humans; Kaplan-Meier Estimate; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Transplantation; Osteosarcoma; RNA Interference; Signal Transduction; Transforming Growth Factor beta

TRIM66 belongs to the family of tripartite motif (TRIM)-containing proteins. Alterations in TRIM proteins have been implicated in several malignancies. This study was aimed at elucidating the expression and biological function of TRIM66 in osteosarcoma. Here, TRIM66 expression level was higher in osteosarcoma tissues than in normal tissues. High TRIM66 expression was correlated with high rate of local recurrence and lung metastasis, and short survival time. Then, we found that knockdown of TRIM66 in two osteosarcoma cell lines, MG63 and HOS, significantly inhibited cell proliferation and induced G1-phase arrest. Moreover, inhibition of TRIM66 in osteosarcoma cells significantly induced cell apoptosis, while remarkably inhibited cell migration, invasion as well as tumorigenicity in nude mice. Gene set enrichment analysis in Gene Expression Omnibus dataset revealed that apoptosis, epithelial-mesenchymal transition (EMT) and transforming growth factor-β (TGF-β) signaling pathway-related genes were enriched in TRIM66 higher expression patients, which was confirmed by western blot analysis in osteosarcoma cells with TRIM66 silenced. In conclusion, TRIM66 may act as an oncogene through suppressing apoptosis pathway and promoting TGF-β signaling in osteosarcoma carcinogenesis. TRIM66 may be a prognostic factor and potential therapeutic target in osteosarcoma.

Topics: Animals; Apoptosis; Carcinogenesis; Caspase 7; Caspase 9; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; G1 Phase Cell Cycle Checkpoints; Humans; Intracellular Signaling Peptides and Proteins; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Recurrence, Local; Oncogenes; Osteosarcoma; Retrospective Studies; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays

Angiogenesis plays an important role in tumor growth and metastasis and has been reported to be inversely correlated with overall survival of osteosarcoma patients. It has been shown that apurinic/apyrimidinic endonuclease 1 (APE1), a dually functional protein possessing both base excision repair and redox activities, is involved in tumor angiogenesis, although these mechanisms are not fully understood. Our previous study showed that the expression of transforming growth factor β (TGFβ) was significantly reduced in APE1-deficient osteosarcoma cells. Transforming growth factor β promotes cancer metastasis through various mechanisms including immunosuppression, angiogenesis, and invasion. In the current study, we initially revealed that APE1, TGFβ, and microvessel density (MVD) have pairwise correlation in osteosarcoma tissue samples, whereas TGFβ, tumor size, and MVD were inversely related to the prognosis of the cohort. We found that knocking down APE1 in osteosarcoma cells resulted in TGFβ downregulation. In addition, APE1-siRNA led to suppression of angiogenesis in vitro based on HUVECs in Transwell and Matrigel tube formation assays. Reduced secretory protein level of TGFβ of culture medium also resulted in decreased phosphorylation of Smad3 of HUVECs. In a mouse xenograft model, siRNA-mediated silencing of APE1 downregulated TGFβ expression, tumor size, and MVD. Collectively, the current evidence indicates that APE1 regulates angiogenesis in osteosarcoma by controlling the TGFβ pathway, suggesting a novel target for anti-angiogenesis therapy in human osteosarcoma.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Movement; DNA-(Apurinic or Apyrimidinic Site) Lyase; Female; Human Umbilical Vein Endothelial Cells; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neovascularization, Pathologic; Osteosarcoma; Phosphorylation; Prognosis; RNA Interference; RNA, Small Interfering; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Xenograft Model Antitumor Assays

Mesenchymal stem cells (MSCs) are among the most important components of the osteosarcoma microenvironment and are reported to promote tumor progression. However, the means by which osteosarcoma cells modulate MSC behavior remains unclear. The aim of this study was to determine the effects of osteosarcoma cells on both the production of pro-tumor cytokines by mesenchymal stem cells (MSCs) and the osteogenic differentiation of MSCs. High level of transforming growth factor-β (TGF-β) was detected in three osteosarcoma cell lines. Conditioned media (CM) from the osteosarcoma cell lines Saos-2 and U2-OS were used to stimulate the cultured MSCs. We found that osteosarcoma cells promoted the production of IL-6 and VEGF in MSCs by inhibiting their osteogenic differentiation. Furthermore, TGF-β in tumor CM was proved to be an important factor. The TGF-β neutralizing antibody antagonized the effects induced by osteosarcoma CM. The inhibition of Smad2/3 by siRNA significantly decreased the production of IL-6 and VEGF in MSCs and induced their osteogenic differentiation. We also found that Smad2/3 enhanced the expression of β-catenin in MSCs by decreasing the level of Dickkopf-1 (DKK1). Although the inhibition of β-catenin did not affect the production of IL-6 or VEGF, or the gene expression of the early osteogenic markers Runx2 and ALP, it did enhance the gene expression of osteocalcin. Taken together, our data indicate that osteosarcoma cells secrete TGF-β to maintain the stemness of MSCs and promote the production of pro-tumor cytokines by these cells.

Topics: Cell Differentiation; Cells, Cultured; Cytokines; Humans; Mesenchymal Stem Cells; Osteogenesis; Osteosarcoma; RNA, Small Interfering; Smad2 Protein; Smad3 Protein; Structure-Activity Relationship; Transforming Growth Factor beta

Osteosarcoma, the most common primary bone tumor in children and young adolescents, is characterized by local invasion and distant metastasis. But the detailed mechanisms of osteosarcoma metastasis are not well known. In the present study, we found that βig-h3 promotes metastatic potential of human osteosarcoma cells in vitro and in vivo. Furthermore, βig-h3 co-localized with integrin α2β1 in osteosarcoma cells. But βig-h3 did not change integrin α2β1 expression in Saos-2 cells. Interaction of βig-h3 with integrin α2β1 mediates metastasis of human osteosarcoma cells. The second FAS1 domain of βig-h3 but not the first FAS1 domain, the third FAS1 domain or the fourth FAS1 domain mediates human osteosarcoma cells metastasis, which is the α2β1 integrin-interacting domain. We further demonstrated that PI3K/AKT signaling pathway is involved in βig-h3-induced human osteosarcoma cells metastasis process. Together, these results reveal βig-h3 enhances the metastasis potentials of human osteosarcoma cells via integrin α2β1-mediated PI3K/AKT signal pathways. The discovery of βig-h3-mediated pathway helps us to understand the mechanism of human osteosarcoma metastasis and provides evidence for the possibility that βig-h3 can be a potential therapeutic target for osteosarcoma treatment.

Topics: Cell Adhesion; Cell Line, Tumor; Cell Movement; Down-Regulation; Enzyme Activation; Extracellular Matrix Proteins; Humans; Immunoprecipitation; Integrin alpha2beta1; Neoplasm Invasiveness; Neoplasm Metastasis; Osteosarcoma; Phosphatidylinositol 3-Kinases; Protein Binding; Protein Structure, Tertiary; Signal Transduction; Transforming Growth Factor beta

Bone metastasis is a multistep process that includes cancer cell dissemination, colonization, and metastatic growth. Furthermore, this process involves complex, reciprocal interactions between cancer cells and the bone microenvironment. Bone resorption is known to be involved in both osteolytic and osteoblastic bone metastasis. However, the precise roles of the bone resorption in the multistep process of osteoblastic bone metastasis remain unidentified. In this study, we show that bone resorption plays important roles in cancer cell colonization during the initial stage of osteoblastic bone metastasis. We applied bioluminescence/X-ray computed tomography multimodal imaging that allows us to spatiotemporally analyze metastasized cancer cells and bone status in osteoblastic bone metastasis models. We found that treatment with receptor activator of factor-κB ligand (RANKL) increased osteoblastic bone metastasis when given at the same time as intracardiac injection of cancer cells, but failed to increase metastasis when given 4 days after cancer cell injection, suggesting that RANKL-induced bone resorption facilitates growth of cancer cells colonized in the bone. We show that insulin-like growth factor-1 released from the bone during bone resorption and hypoxia-inducible factor activity in cancer cells cooperatively promoted survival and proliferation of cancer cells in bone marrow. These results suggest a mechanism that bone resorption and hypoxic stress in the bone microenvironment cooperatively play an important role in establishing osteoblastic metastasis.

Topics: Animals; Bone Marrow Cells; Bone Neoplasms; Bone Resorption; Cell Hypoxia; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Hypoxia-Inducible Factor 1; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Osteosarcoma; RANK Ligand; Somatomedins; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Microenvironment

Interferon consensus sequence-binding protein (ICSBP) is a transcription factor induced by interferon gamma (IFN-γ) and a member of the interferon regulatory factor (IRF) family. ICSBP is predominantly expressed in hematopoietic cells and regulates the immune response and cell growth and differentiation. However, little is known about its function in non-hematopoietic cells. Here we show a novel function for ICSBP in epithelial-to-mesenchymal transition (EMT)-like phenomena (ELP), cell motility, and invasion in human osteosarcoma cell lines, including U2OS cells. IFN-γ treatment induced ICSBP expression and EMT-like morphological change in U2OS cells, which were suppressed by ICSBP knockdown. To further investigate the role of ICSBP in ELP, we established a stable U2OS cell line that overexpresses ICSBP. ICSBP expression caused U2OS cells to have a more elongated shape and an increased vimentin and fibronectin expression. ICSBP expression also promoted adhesiveness, motility, and invasiveness of U2OS cells. ICSBP upregulated transforming growth factor (TGF)-β receptors and activated TGF-β signaling cascades, which were responsible for ELP as well as increased cell motility and invasion. In addition, ICSBP-induced TGF-β receptor activation resulted in the upregulation of Snail. Knockdown of Snail attenuated the ICSBP-induced augmentation of cell motility and invasion. Upregulation of Snail, ELP, and increased invasion by ICSBP expression were also observed in other osteosarcoma cell lines, such as Saos-2 and 143B. Furthermore, ICSBP and TGF-β receptor I were expressed in 45/54 (84%) and 47/54 (87%) of human osteosarcoma tissues, respectively, and showed significant correlation (r=0.47, P=0.0007) with respect to their expression levels. Taken altogether, these data demonstrate a novel function for ICSBP in ELP, cell motility, and invasion through the TGF-β and Snail signaling pathways.

Topics: Antigens, CD; Bone Neoplasms; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Shape; Coculture Techniques; Epithelial-Mesenchymal Transition; Fibronectins; Human Umbilical Vein Endothelial Cells; Humans; Interferon Regulatory Factors; Interferon-gamma; Neoplasm Invasiveness; Osteosarcoma; Promoter Regions, Genetic; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Snail Family Transcription Factors; Time Factors; Transcription Factors; Transfection; Transforming Growth Factor beta; Vimentin

Platelet-rich plasma (PRP) is widely used to promote tissue repair and accelerate osteogenesis, but there is no agreement about its mechanism of action. We characterized the modulatory effect of PRP on the in vitro osteoblast model SaOS-2, by using cell motility/chemoattraction and osteogenesis/mineralization assays, and a series of osteogenic/ osteoclastogenic genomic markers. Scratch wound assay showed that PRP stimulates cell motility, while transwell assay revealed a strong chemoattraction. Alkaline phosphatase (ALP) and alizarin red-S assays showed that PRP induces slight, but significant, stimulations of ALP activity and mineralization. The TGF-β inhibitor SB431542 reversed these effects, showing a main role for TGF-β1 released by PRP. Analyses of gene expression by qRT-PCR, showed the upregulation of osteocalcin, osteopontin, osteoprotegerin, receptor activator of NFκB (RANK), and runt-related transcription factor 2 (RUNX2) genes, with a total reversion by SB431542 for osteoprotegerin and RANK, and a partial reversion for ostecalcin, osteopontin, and RUNX2. The use of PCR array technique revealed the upregulation of the cathepsin K gene. These data show that PRP induces the development of mixed osteogenic/osteoclastogenic traits in the SaOS-2 model. Such a behavior may favour in vivo bone resorption and reconstitution at post-surgery or post-traumatic sites.

Topics: Alkaline Phosphatase; Benzamides; Bone Neoplasms; Bone Resorption; Calcification, Physiologic; Cell Line, Tumor; Cell Movement; Chemotaxis; Dioxoles; Gene Expression; Humans; Osteogenesis; Osteosarcoma; Phenotype; Platelet-Rich Plasma; RNA, Messenger; Transforming Growth Factor beta

Osteosarcoma is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastasis is present at diagnosis. Because transforming growth factor-β (TGFβ) has been shown to promote metastasis in many solid tumors, we investigated the effect of the natural TGFβ/Smad signaling inhibitor Smad7 and the TβRI inhibitor SD-208 on osteosarcoma behavior.. By using a mouse model of osteosarcoma induced by paratibial injection of cells, we assessed the impact of Smad7 overexpression or SD-208 on tumor growth, tumor microenvironment, bone remodeling, and metastasis development.. First, we demonstrated that TGFβ levels are higher in serum samples from patients with osteosarcoma compared with healthy volunteers and that TGFβ/Smad3 signaling pathway is activated in clinical samples. Second, we showed that Smad7 slows the growth of the primary tumor and increases mice survival. We furthermore demonstrated that Smad7 expression does not affect in vitro osteosarcoma cell proliferation but affects the microarchitectural parameters of bone. In addition, Smad7-osteosarcoma bone tumors expressed lower levels of osteolytic factors such as RANKL, suggesting that Smad7 overexpression affects the "vicious cycle" established between tumor cells and bone cells by its ability to decrease osteoclast activity. Finally, we showed that Smad7 overexpression in osteosarcoma cells and the treatment of mice with SD208 inhibit the development of lung metastasis.. Taken together, these results demonstrate that the inhibition of the TGFβ/Smad signaling pathway may be a promising therapeutic strategy against tumor progression of osteosarcoma, specifically against the development of lung metastasis.

Topics: Animals; Bone Neoplasms; Bone Resorption; Cell Line, Tumor; Disease Models, Animal; Female; Gene Expression; Heterografts; Humans; Lung Neoplasms; Mice; Osteoclasts; Osteosarcoma; Pteridines; Signal Transduction; Smad3 Protein; Smad7 Protein; Transforming Growth Factor beta; Tumor Burden

Osteosarcoma and Ewing's sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40 h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-β. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.

Topics: Antibody-Dependent Cell Cytotoxicity; Antigens, Differentiation, T-Lymphocyte; Cell Line, Tumor; Coculture Techniques; Cytotoxicity, Immunologic; Humans; Interleukin-15; Killer Cells, Natural; Lymphocyte Activation; Natural Cytotoxicity Triggering Receptor 3; NK Cell Lectin-Like Receptor Subfamily K; Osteosarcoma; Receptors, IgG; Receptors, Natural Killer Cell; Sarcoma, Ewing; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is known to promote tumor migration and invasion. Bone morphogenetic proteins (BMPs) are members of the TGF-β family expressed in a variety of human carcinoma cell lines. The role of bone morphogenetic protein 9 (BMP9), the most powerful osteogenic factor, in osteosarcoma (OS) progression has not been fully clarified. The expression of BMP9 and its receptors in OS cell lines was analyzed by RT-PCR. We found that BMP9 and its receptors were expressed in OS cell lines. We further investigated the influence of BMP9 on the biological behaviors of OS cells. BMP9 overexpression in the OS cell lines 143B and MG63 inhibited in vitro cell migration and invasion. We further investigated the expression of a panel of cancer-related genes and found that BMP9 overexpression increased the phosphorylation of Smad1/5/8 proteins, increased the expression of ID1, and reduced the expression and activity of matrix metalloproteinase 9 (MMP9) in OS cells. BMP9 silencing induced the opposite effects. We also found that BMP9 may not affect the chemokine (C-X-C motif) ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis to regulate the invasiveness and metastatic capacity of OS cells. Interestingly, CXCR4 was expressed in both 143B and MG63 cells, while CXCL12 was only detected in MG63 cells. Taken together, we hypothesize that BMP9 inhibits the migration and invasiveness of OS cells through a Smad-dependent pathway by downregulating the expression and activity of MMP9.

Topics: Cell Line, Tumor; Cell Movement; Chemokine CXCL12; Down-Regulation; Gene Expression Regulation, Neoplastic; Growth Differentiation Factor 2; Growth Differentiation Factors; HEK293 Cells; Humans; Matrix Metalloproteinase 9; Osteosarcoma; Receptors, CXCR4; Signal Transduction; Smad Proteins, Receptor-Regulated; Transforming Growth Factor beta

Signaling pathways initiated by transforming growth factor-β (TGF-β) and insulin-like growth factors (IGFs) are important in osteosarcoma cell growth. We have investigated a role for endogenous IGF binding protein-3 (IGFBP-3) in mediating cross-talk between TGF-β receptor and type I IGF receptor (IGF1R) signaling pathways in MG-63 osteosarcoma cells. TGF-β1 indirectly activated the Ras/Raf/MAPK pathway and induced the expression of IGFBP-3, an important regulator of IGF1R activity. IGFBP-3 attenuated TGF-β1 activation of ERK1/2 and Akt in MG-63 cells, and inhibited TGF-β1-induced cell cycle progression and proliferation. This effect of IGFBP-3 was blocked by inhibiting IGF1R signaling. TGF-β1 phosphorylated Smad2 on the non-receptor substrate sites (Ser245/250/255). Blocking the TGF-β1-induced expression of IGFBP-3 enhanced pSmad2(Ser245/250/255) and increased its nuclear accumulation. These results suggest an important role for TGF-β1 in osteosarcoma cell growth, with the induction of IGFBP-3 by TGF-β1 serving in a negative-feedback loop to control cell growth by preventing activation of the IGF1R.

Topics: Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; Insulin-Like Growth Factor Binding Protein 3; MAP Kinase Signaling System; Osteosarcoma; Phosphorylation; Phosphoserine; Receptor Cross-Talk; Receptor, IGF Type 1; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Substrate Specificity; Transforming Growth Factor beta

Overexpressed cysteine-rich protein 61 (Cyr61) is believed to enhance osteosarcoma (OS) cell metastasis, but the mechanism of Cyr61 overexpression in OS is not clear so far. In this study 33 OS samples were analyzed by immunostaining and focused on two parts: the correlation between overexpression of Cyr61 and OS metastasis; the mechanism of regulating Cyr61 expression in OS. Twenty-five out of 33 cases (75.76 %) with metastasis showed high expression of Cyr61. Furthermore, Cyr61 expression in Saos-2 cells was reduced by siRNA, and lower expression of Cyr61 in Saos-2 cell resulted in a cell migration deficiency and had no effect on cell proliferation. Particularly, Cyr61 expression was significantly increased in Saos-2 cells in response to different dosages of transforming growth factor beta (TGF-β), indicating that the expression of Cyr61 is TGF-β dependent. A transwell assay showed that Saos-2 cells stimulated with TGF-β had a greater capacity for migration than the control cells. The p38 MAPK-specific inhibitor SB203580 was able to reduce Cyr61 expression and inhibit the migration of Saos-2 cells stimulated with TGF-β. These results obtained provide new evidence that overexpressed Cyr61 plays a key role in the metastasis of OS cells and Cyr61 is a potential target downstream of TGF-β/p38 MAPK to regulate cell migration.

Topics: Adolescent; Adult; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Child; Cysteine-Rich Protein 61; Enzyme Inhibitors; Female; Humans; Imidazoles; Male; Neoplasm Metastasis; Osteosarcoma; p38 Mitogen-Activated Protein Kinases; Pyridines; RNA Interference; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; Young Adult

A right amount of oxygen and nutrients is essential for a tumor to develop. The role of oxygen dependent pathways and their regulators is therefore of utmost importance although little is known about the detailed impact they can have. Recently we have shown that inhibition of the oxygen sensor PHD2 in tumor cells blocks tumor growth due to the anti-proliferative activity of TGFβ. In this study, we refined these results by comparing different shPHD2 sequences in depth in the early phase of tumor growth. Our findings also reveal an intriguing role for MMP2 and MT1MMP in these settings, as these activated proteases display an anti-proliferative characteristic through the activation of downstream TGFβ targets. In conclusion, PHD2 inhibition is essential for the regulation of the anti-tumoral activity in mouse tumor cells and might bring some new insight in our understanding of tumor growth inhibition.

Topics: Animals; Bone Neoplasms; Cell Growth Processes; Cell Line, Tumor; Disease Models, Animal; Female; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Immunohistochemistry; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Mice, Inbred C3H; Osteosarcoma; Procollagen-Proline Dioxygenase; RNA, Small Interfering; Transforming Growth Factor beta

We previously reported the combination of tumor cryotreatment with dendritic cells to promote antitumor immunity. The effect of the combination treatment with dendritic cells and antitransforming growth factor-β (anti-TGF-β) antibody on the elimination of regulatory T cells and the inhibition of tumor growth was investigated.. The effects of the combination treatment with dendritic cells and anti-TGF-β antibody on the enhancement of systemic immune responses and inhibition of metastatic tumor growth were investigated in a murine osteosarcoma (LM8) model.. To evaluate activation of the immune response, we established the following three groups of C3H mice (60 mice total): (1) excision only; (2) tumor excision and administration of anti-TGF-β antibody; and (3) tumor excision and administration of dendritic cells exposed to cryotreated tumor lysates with anti-TGF-β antibody.. The mice that received dendritic cells exposed to cryotreated tumor lysates with anti-TGF-β antibody showed increased numbers of CD8(+) T lymphocytes, reduced regulatory T lymphocytes in the metastatic lesion, and inhibition of metastatic growth. The combined therapy group showed reduced numbers of regulatory T lymphocytes in the spleen and high serum interferon γ level.. The control of the inhibitory condition induced by regulatory T cells is important to improve the suppression of the cytotoxic lymphocytes. Combining dendritic cells with anti-TGF-β antibody enhanced the systemic immune response.. We suggest that our immunotherapy could be developed further to improve the treatment of osteosarcoma.

Topics: Animals; Antibodies, Monoclonal; Bone Neoplasms; Cancer Vaccines; CD8-Positive T-Lymphocytes; Combined Modality Therapy; Dendritic Cells; Female; Mice; Mice, Inbred C3H; Osteosarcoma; Transforming Growth Factor beta; Treatment Outcome; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The tumor microenvironment plays a critical role in modulating malignant behavior and can dramatically influence cancer treatment strategies. We investigated whether statins inhibit the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and transforming growth factor-β (TGF-β) mRNA in the mouse osteosarcoma cell line LM8. We found that statins significantly inhibited mRNA expressions of bFGF, HGF, and TGF-β, and bFGF, HGF, and TGF-β secretions at concentrations that did not have antiproliferative effects on LM8 cells, but had no effect on the mRNA expression and secretion of VEGF. The inhibition of bFGF, HGF, and TGF-β mRNA expression, and bFGF, HGF, TGF-β secretions was reversed when geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway, was used in combination with statins. Furthermore, statins reduced the membrane localization of K-Ras, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated Akt. Our research indicates that statins inhibit GGPP biosynthesis in the mevalonate pathway, and then inhibit signal transduction in the Ras/ERK and Ras/Akt pathways, thereby inhibiting bFGF, HGF, TGF-β expression in LM8 cells. These results suggest that statins are potentially useful as anti-angiogenic agents for the treatment of osteosarcoma.

Topics: Animals; Cell Line, Tumor; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Hepatocyte Growth Factor; Mevalonic Acid; Mice; Mitogen-Activated Protein Kinase Kinases; Neovascularization, Pathologic; Osteosarcoma; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Polyisoprenyl Phosphates; Proto-Oncogene Proteins c-akt; ras Proteins; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Virtually all solid tumors are dependent on a vascular network to provide them with the right amount of nutrients and oxygen. In that sense, low oxygen tension or hypoxia leads to an adaptive response that is transcriptionally regulated by the hypoxia-inducible factors (HIF), which are tightly controlled by the HIF prolyl hydroxylases (PHD). In this study, we show that inhibition of the oxygen sensor PHD2 in tumor cells stimulates vessel formation but paradoxically results in a profound reduction of tumor growth. This effect relies on the antiproliferative nature of the TGFβ signaling pathway, in a largely HIF-independent manner. Moreover, our findings reveal that PHD2 has an essential function in controlling the dual nature of TGFβ during tumorigenesis and may offer an alternative opportunity for anticancer therapy.

Topics: Animals; Bone Neoplasms; Cell Growth Processes; Cell Line, Tumor; Female; Gene Knockdown Techniques; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Melanoma, Experimental; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Neoplasms, Experimental; Osteosarcoma; Procollagen-Proline Dioxygenase; RNA, Small Interfering; Transforming Growth Factor beta

Osteogenic differentiation is coordinated by the exposure of cells to temporal changes in a combination of growth factors and elements within the extracellular matrix (ECM). Many of the key proteins that drive these changes share the property of being dependent on ECM glycosaminoglycans (GAGs) for their activity. Here, we examined whether GAGs isolated from proliferating, differentiating and mineralizing MG-63 osteosarcoma cells differed in their physical properties, and thus in their capacities to coordinate the osteogenic cascade both in human MG-63 osteosarcoma cells and primary human mesenchymal stem cells (hMSCs). Our results show that the size distribution of GAGs, the expression of GAG-carrying proteoglycan cores and the expression of enzymes involved in their modification systematically change as MG-63 cells mature in culture. When dosed back onto cells exogenously in soluble form, GAGs regulated MG-63 survival and growth in a dose-dependent manner, but not differentiation in either cell type. In contrast, hMSCs aggregated into distinct colonies when grown on GAG-coated substrates, while MG-63 cells did not. Heparin-coated substrates improved hMSC viability without inducing aggregation. These results suggest a complex role for GAGs in coordinating the emergence of the osteoblast phenotype, and provide further evidence for the use of heparans in bone tissue repair applications.

Topics: Cell Aggregation; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Shape; Cell Survival; Chondroitin Sulfates; Dermatan Sulfate; Fibroblast Growth Factor 2; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparin; Humans; Mesenchymal Stem Cells; Osteogenesis; Osteosarcoma; Phenotype; Protein Binding; Time Factors; Transforming Growth Factor beta

Transforming growth factor beta-induced protein (TGFBIp), is secreted into the extracellular space. When fragmentation of C-terminal portions is blocked, apoptosis is low, even when the protein is overexpressed. If fragmentation occurs, apoptosis is observed. Whether full-length TGFBIp or integrin-binding fragments released from its C-terminus is necessary for apoptosis remains equivocal. More importantly, the exact portion of the C-terminus that conveys the pro-apoptotic property of TGFBIp is uncertain. It is reportedly within the final 166 amino acids. We sought to determine if this property is dependent upon the final 69 amino acids containing the integrin-binding, EPDIM and RGD, sequences. With MG-63 osteosarcoma cells, transforming growth factor (TGF)-beta1 treatment increased expression of TGFBIp over 72 h (p<0.001). At this time point, apoptosis was significantly increased (p<0.001) and was prevented by an anti-TGFBIp, polyclonal antibody (p<0.05). Overexpression of TGFBIp by transient transfection produced a 2-fold increase in apoptosis (p<0.01). Exogenous purified TGFBIp at concentrations of 37-150 nM produced a dose dependent increase in apoptosis (p<0.001). Mass spectrometry analysis of TGFBIp isolated from conditioned medium of cells treated with TGF-beta1 revealed truncated forms of TGFBIp that lacked integrin-binding sequences in the C-terminus. Recombinant TGFBIp truncated, similarly, at amino acid 614 failed to induce apoptosis. A recombinant fragment encoding the final 69 amino acids of the TGFBIp C-terminus produced significant apoptosis. This apoptosis level was comparable to that induced by TGF-beta1 upregulation of endogenous TGFBIp. Mutation of the integrin-binding sequence EPDIM, but not RGD, blocked apoptosis (p<0.001). These pro-apoptotic actions are dependent on the C-terminus most likely to interact with integrins.

Topics: Amino Acid Sequence; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Extracellular Matrix Proteins; Humans; Integrins; Osteosarcoma; Peptide Fragments; Protein Binding; Transforming Growth Factor beta; Transforming Growth Factor beta1

Bone metastases are common complications of prostate cancer cells. The bone morphogenetic protein-2 (BMP-2) is constitutively secreted by osteoblasts and plays a key role in bone formation. Integrins are the major adhesive molecules in mammalian cells, and has been associated with cancer cells metastasis to bone. The aim of this study was to investigate whether osteoblast-derived BMP-2 is associated with prostate cancer metastasis.. Cancer cells migration activity was examined using the Transwell assay. The ERK and AKT phosphorylation was examined by using Western blot method. The siRNA was used to inhibit the expression of BMP-2. The cell surface expression of integrins was examined by using flow cytometry. A transient transfection protocol was used to examine NF-kappaB activity.. We found that osteoblast conditioned medium (OBCM) increased the migration and cell surface expression of beta1 or beta 3 integrin in human prostate cancer cells. beta1 or beta 3 integrin monoclonal antibodies or siRNA against beta1 or beta 3 integrin inhibited the OBCM-induced increase the migration of prostate cancer cells. BMP-2 siRNA specifically reduced the OBCM-induced migration and integrins upregulation. BMP-2 siRNA also suppressed the OBCM-induced ERK, AKT and NF-kappaB activation.. This study suggest that the osteoblast-derived BMP-2 act through Akt and ERK, which in turn activates IKK alpha/beta and NF-kappaB, resulting in the activations of beta1 and beta 3 integrins and contributing the migration of prostate cancer cells.

Topics: Adenocarcinoma; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Line, Tumor; Cell Movement; Extracellular Signal-Regulated MAP Kinases; Humans; I-kappa B Kinase; Integrin beta1; Integrin beta3; Male; NF-kappa B; Osteoblasts; Osteosarcoma; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Signal Transduction; Transforming Growth Factor beta

Bone cells produce many glycoproteins potentially involved in the maintenance of healthy bone tissues. Two cytokines produced in inflamed joints, tumor necrosis factor (TNF)alpha and transforming growth factor (TGF)beta, have previously been shown to alter cellular glycosylation which may potentially affect the expression and function of glycoproteins. In order to evaluate models to study the glycodynamics of bone cells, we examined primary human osteoblastic cells from osteoarthritis patients, and compared these to human osteosarcoma cells MG63 and SJSA-1. We showed here for the first time that all of the human osteoblastic cells actively synthesize complex N- and O-glycan chains of bone cell glycoproteins, with quantitative differences between cell types. TNFalpha-induced apoptosis or TGFbeta-induced cell differentiation and proliferation had significant effects on both cell surface carbohydrates and glycosyltransferase activities of osteoblasts and osteosarcoma cells. The results indicate that cultured human bone-derived osteoblastic cells are good models to examine the glycodynamics of osteoblasts under conditions of cell growth and cell death. The changes induced by cytokines can result in altered cell surface functions which may be of importance in osteoarthritis, osteoporosis and other bone diseases.

Topics: Apoptosis; Bone and Bones; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Glycosylation; Humans; Osteoarthritis; Osteoblasts; Osteosarcoma; Polysaccharides; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Lung metastasis is the most crucial event affecting the therapeutic outcome of osteosarcoma. The prevention of lung metastasis is therefore important in improving the prognosis of patients with osteosarcoma. Decorin is a major extracellular matrix protein which has become the focus of various cancer studies. The biological role of decorin in osteosarcoma has yet to be clarified. The aim of this study was to examine the potential of decorin as a novel biological target for the treatment of osteosarcoma. In this study, the LM8 murine osteosarcoma cell line (LM8) with high metastatic potential to the lung was used. The two cell lines established were LM8-DCN which stably expressed human decorin (hDCN) and LM8-mock, established as a control. The LM8-DCN cell line was subcutaneously injected into the backs of mice. Significantly fewer pulmonary metastases were observed in mice with LM8-DCN compared to mice inoculated with LM8 and LM8-mock (P<0.001). In addition, the mice in the LM8-DCN inoculated group survived significantly longer than those in the LM8 and LM8-mock inoculated group, based on the Kaplan-Meier survival analysis and log-rank tests (P<0.005). The effect of decorin on the growth rates, motility and invasion ability of LM8 was investigated in vitro. There was no difference in the morphology and growth rates, but the motility and invasion of LM8 were inhibited by decorin. These results suggest that decorin has the therapeutic potential to prevent lung metastasis in osteosarcoma.

Topics: Animals; Blotting, Western; Bone Neoplasms; Cell Movement; Decorin; Extracellular Matrix Proteins; Genetic Vectors; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred C3H; Neoplasm Invasiveness; Osteosarcoma; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Survival Rate; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Regulatory T cells (Treg) are a distinct group of T lymphocytes with immunosuppressive properties that serve normally to prevent harmful autoimmune responses. However, Tregs can also interfere with beneficial immune responses such as anti-tumor and anti-viral immunity in humans and rodents. Given the overall importance of Tregs, it is likely that they play an important role in diseases of dogs as well. However, at present reagents required for identification of Tregs in dogs are not available. Therefore, we investigated whether expression of FoxP3, a transcription factor that is highly expressed in Tregs in humans and rodents could also be used to identify Tregs in dogs. We found that a cross-reactive FoxP3 antibody identified a subset of CD4(+) T cells in blood and lymph nodes of dogs. By flow cytometry the mean percentage of FoxP3(+)CD4(+) T cells in normal dogs was 4.3% in blood and 9.8% in the lymph nodes. In dogs with cancer, there was a significant increase in numbers of Treg in blood (7.5%) and tumor-draining lymph nodes (17.1%) compared to age-matched healthy control dogs. We also found that FoxP3(+)CD4(+) T cells in dogs could be significantly expanded in vitro by TCR activation together with addition of TGF-beta and IL-2. Treated cells also significantly increased expression of TGF-beta and IL-10mRNA. We conclude from these studies that a cross-reactive FoxP3 antibody can be used to identify Tregs in dogs and that this reagent may serve as a useful tool for investigating the role of Treg in a variety of diseases of dogs.

Topics: Animals; Biomarkers, Tumor; Dog Diseases; Dogs; Flow Cytometry; Forkhead Transcription Factors; Interleukin-10; Lymph Nodes; Lymphocyte Activation; Melanoma; Osteosarcoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

To clarify the mechanism of the stimulatory effect of statins on bone formation, we have assessed the effect of simvastatin and atorvastatin on osteoblast activity by analysing cell proliferation, as well as collagen, osteocalcin, and bone morphogenetic protein-2 (BMP2) gene expression in primary human osteoblast (hOB) and MG-63 cell line cultures. Explants of bone from patients without any metabolic disease under orthopedic hip procedures were used to obtain hOB. Cell cultures were established, synchronized, and different concentrations of simvastatin or atorvastatin were added (10(-9) M, 10(-8) M, 10(-7) M, 10(-6) M) during the experiment. Cell proliferation was analyzed after 24 h. Collagen polypeptide alpha1 type 1 (COL1A1) gene expression, osteocalcin, and BMP2 expression levels were quantified by real-time PCR after 24 h incubation with statins. There was a statistically significant decrease in cell proliferation related to simvastatin or atorvastatin addition at all concentrations in primary hOB compared with those not treated. A significant increase in COL1A1, osteocalcin, and BMP2 gene expression was detected when hOB cultures were treated with simvastatin or atorvastatin at different concentrations. Similar but less significant effects were found on MG-63 cells. After statin treatment we observed both an arrest of proliferation in hOB cells and an increase in collagen, osteocalcin, and BMP2 gene expression, consistent with a stimulatory effect towards mature osteoblast differentiation. These findings support the bone-forming effect of statins, probably through the BMP2 pathway.

Topics: Aged; Atorvastatin; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Collagen Type I; Gene Expression Regulation; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Osteoblasts; Osteocalcin; Osteosarcoma; Pyrroles; Simvastatin; Transforming Growth Factor beta

The transcription factor NFkappaB is constitutively activated in various tumor cells where it promotes proliferation and represses apoptosis. The bone morphogenetic proteins (BMPs) delay cell proliferation and promote differentiation and apoptosis of bone cells through activation of Smad downstream effectors and via Smad-independent mechanisms. Thus, NFkappaB and BMP pathways play opposing roles in regulating osteoblastic cell fate. Here, we show that in osteosarcoma Saos2 osteoblasts, NFkappaB regulates the activity of the BMP/Smad signaling. Inhibition of NFkappaB by overexpression of mIkappaB leads to the induction of osteoblast differentiation. Saos2 cells overexpressing mIkappaB (Saos2-mIkappaB) exhibit higher expression of osteoblast phenotypic genes such as alkaline phosphatase, Runx2 and osteocalcin and are more responsive to BMP2 in comparison to wild-type cells (Saos2-wt) or empty vector infected controls (Saos2-EV). Furthermore, BMP-2 signaling and Smad phosphorylation are significantly increased in Saos2-mIkappaB cells in comparison to Saos2-EV cells. Inhibition of NFkappaB signaling in Saos2-mIkappaB cells is associated with decreased expression of the BMP signaling inhibitor Smad7. While gain of Smad7 function in Saos2-mIkappaB cells results in inhibition of BMP signaling, anti-sense knockdown of Smad7 in Saos2-EV cells leads to upregulation of BMP signaling. We therefore conclude that in osteosarcoma Saos2 cells, NFkappaB represses BMP/Smad signaling and BMP2-induced differentiation through Smad7.

Topics: Alkaline Phosphatase; Blotting, Western; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Neoplasms; Cell Differentiation; Core Binding Factor Alpha 1 Subunit; Gene Expression Regulation; Humans; I-kappa B Kinase; Luciferases; NF-kappa B; Osteocalcin; Osteosarcoma; Phosphorylation; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Smad7 Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

The POS-1 murine model of osteolytic osteosarcoma was used to elucidate the molecular and cellular mechanisms involved in the development of primary bone tumors and associated lung metastasis. The POS-1 cell line is derived from an osteosarcoma tumor which develops spontaneously in C3H mice. The POS-1 cell line was characterized in vitro by mineralization capacity and expression of bone markers by semi-quantitative RT-PCR, compared to primary osteoblasts and bone marrow cells. POS-1 cells showed no mineralization capacity and exhibited an undifferentiated phenotype, expressing both osteoblastic and unexpected osteoclastic markers (TRAP, cathepsin K and RANK). Thereby, experiments were performed to determine whether RANK was functional, by studying the biological activity of murine RANKL through the receptor RANK expressed on POS-1 cells. Results revealed a RANKL-induced increase in ERK phosphorylation, as well as BMP-2 induction at the mRNA and protein levels, and a decrease of POS-1 cell proliferation in the presence of 10 ng/ml RANKL. BMP-2 induction is dependent on the ERK 1/2 signal transduction pathway, as its expression is abolished in the presence of UO126, a specific synthetic inhibitor of the ERK 1/2 pathway. Moreover, a 2-fold molar excess of soluble RANK blocks the RANKL-induced BMP-2 expression, demonstrating that the biological effects of RANKL observed in POS-1 cells are mediated by RANK. This is the first report describing a functional RANK expressed on osteosarcoma cells, as shown by its ability to induce signal transduction pathways and biological activity when stimulated by RANKL.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Neoplasms; Carrier Proteins; Cell Proliferation; Gene Expression Profiling; Humans; Lung Neoplasms; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Osteosarcoma is the most common primary bone tumour in young adults. Despite improved prognosis, resistance to chemotherapy remains responsible for failure of osteosarcoma treatment. The identification of signals that promote apoptosis may provide clues to develop new therapeutic strategies for chemoresistant osteosarcoma. Here, we show that lipophilic statins (atorvastatin, simvastatin, cerivastatin) markedly induce caspases-dependent apoptosis in various human osteosarcoma cells, independently of bone morphogenetic protein (BMP)-2 signaling and cell differentiation. Although statins increased BMP-2 expression, the proapoptotic effect of statins was not prevented by the BMP antagonist noggin, and was abolished by mevalonate and geranylgeranylpyrophosphate, suggesting the involvement of defective protein geranylgeranylation. Consistently, lipophilic statins induced membrane RhoA relocalization to the cytosol and inhibited RhoA activity, which resulted in decreased phospho-p42/p44- mitogen-activated protein kinases (MAPKs) and Bcl-2 levels. Constitutively active RhoA rescued phospho-p42/p44-MAPKs and Bcl-2 and abolished statin-induced apoptosis. Thus, lipophilic statins induce caspase-dependent osteosarcoma cell apoptosis by a RhoA-p42/p44 MAPKs-Bcl-2-mediated mechanism, independently of BMP-2 signaling and cell differentiation.

Topics: Anticholesteremic Agents; Apoptosis; Atorvastatin; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Caspases; Cell Differentiation; Cell Survival; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation, Neoplastic; Heptanoic Acids; Humans; Osteosarcoma; Phenotype; Phosphorylation; Polyisoprenyl Phosphates; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Pyridines; Pyrroles; rhoA GTP-Binding Protein; RNA, Messenger; Signal Transduction; Simvastatin; Transforming Growth Factor beta

Versican, a large sized chondroitin-sulphate proteoglycan (PG), and its binding partner, hyaluronan (HA), are extracellular matrix (ECM) components that play an essential role in transformed cell behavior. Expression of certain versican isoforms has been implicated in cell migration and proliferation of cancer cells and, on the other hand, disruption of HA synthesis by inhibiting hyaluronan synthase-2 (HAS2) expression in osteosarcoma cells by suppressing cell proliferation, invasiveness and motility. Considering that growth factors, such as TGF-beta, bFGF and PDGF-BB, are important regulators for the expression of the ECM macromolecules, in this study we examined the effect of these growth factors on the expression of the various versican isoforms, HA synthases as well as HA synthesis by MG-63 osteosarcoma cells and normal human osteoblastic periodontal ligament cells (hPDL). Real-time PCR and metabolic labelling followed by fine HPLC analysis coupled to radiochemical detection were the methods utilized. It was found that, contrary to normal hPDL cells, osteosarcoma MG-63 cells do not constitutively express the versican isoforms V0 and V1. Exogenous addition of TGF-beta2 stimulated the versican transcript levels mainly by forcing osteosarcoma cells to express V1 and V0 isoforms. PDGF-BB and bFGF had only minor effects in these cells. In hPDL cells a strong stimulation of the V3 transcript by all growth factors was observed. TGF-beta2 was also the major stimulator of HAS2 isoform expression as well as hyaluronan synthesis in osteosarcoma cells, while PDGF-BB exerted dominant influence on HAS2 isoform expression and hyaluronan biosynthesis by osteoblasts. The obtained results show for the first time that TGF-beta2 triggers the malignant phenotype pattern of versican and hyaluronan expression in human osteosarcoma cells and indicate that this growth factor may account for the metastatic potential of these cells.

Topics: Becaplermin; Bone Neoplasms; Cell Line, Tumor; Chondroitin Sulfate Proteoglycans; Fibroblast Growth Factors; Gene Expression; Glucuronosyltransferase; Humans; Hyaluronan Synthases; Hyaluronic Acid; Lectins, C-Type; Osteosarcoma; Platelet-Derived Growth Factor; Protein Isoforms; Proto-Oncogene Proteins c-sis; Transforming Growth Factor beta; Transforming Growth Factor beta2; Versicans

It has been shown previously that the suppression of tumor immunosurveillance may be a mechanism by which tumors resist immune detection and elimination. In this study, we evaluated the role of the immunoregulatory natural killer T (NKT) cells in the biology of immunosurveillance of osteosarcoma. The K7M2 mouse osteosarcoma cell line was implanted orthotopically into wild-type and NKT cell-deficient CD1d knockout (KO) BALB/c mice, and mice were monitored for growth of primary tumors. Further, we examined the role of CD4(+) and/or CD8(+) cells by depleting the cells in vivo and measuring CTL activity in vitro. We also asked the role of interleukin (IL)-4 receptor alpha (IL-4Ralpha)-signal transducer and activator of transcription 6 (STAT6) signaling, including IL-13, and transforming growth factor beta (TGF-beta) by using gene-disrupted mice or treating mice with cytokine antagonists. We were surprised to find a high rate of rejection of osteosarcoma primary tumors in 88% (14 of 16) of CD1d KO mice compared with syngeneic wild-type BALB/c mice that showed rejection of tumor in <24% of mice. Further studies suggested that the rejection of tumor in CD1d KO mice was dependent on CD8(+) lymphocytes. Distinct from other murine tumor models, the negative regulation induced by CD1d-restricted NKT cells was not dependent on IL-4Ralpha-STAT6 signaling, including IL-13, or on TGF-beta. These data suggest that a novel CD1d-restricted NKT cell-mediated mechanism for tumor immunosuppression is active in the K7M2 osteosarcoma model and that NKT cells can regulate immunosurveillance through more than one pathway.

Topics: 3T3 Cells; Animals; Antigens, CD1; Antigens, CD1d; Bone Neoplasms; Down-Regulation; Female; Killer Cells, Natural; Mice; Mice, Inbred BALB C; Mice, Knockout; Osteosarcoma; Receptors, Interleukin-4; STAT6 Transcription Factor; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta

Hedgehog (HH) signalling is important for specific developmental processes, and aberrant, increased activity has been described in various tumours. Disturbed HH signalling has also been implicated in the hereditary syndrome, Multiple Osteochondromas. Indian Hedgehog (IHH), together with parathyroid hormone-like hormone (PTHLH), participates in the organization of growth plates in long bones. PTHLH signalling is absent in osteochondromas, benign tumours arising adjacent to the growth plate, but is reactivated when these tumours undergo malignant transformation towards secondary peripheral chondrosarcoma. We describe a gradual decrease in the expression of Patched (PTCH) and glioma-associated oncogene homologue 1 (GLI1) (both transcribed upon IHH activity), and GLI2 with increasing malignancy, suggesting that IHH signalling is inactive and PTHLH signalling is IHH independent in secondary peripheral chondrosarcomas. cDNA expression profiling and immunohistochemical studies suggest that transforming growth factor-beta (TGF-beta)-mediated proliferative signalling is active in high-grade chondrosarcomas since TGF-beta downstream targets were upregulated in these tumours. This is accompanied by downregulation of energy metabolism-related genes and upregulation of the proto-oncogene jun B. Thus, the tight regulation of growth plate organization by IHH signalling is still seen in osteochondroma, but gradually lost during malignant transformation to secondary peripheral chondrosarcoma and subsequent progression. TGF-beta signalling is stimulated during secondary peripheral chondrosarcoma progression and could potentially regulate the retained activity of PTHLH.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; beta Catenin; Chi-Square Distribution; Child; Chondrosarcoma; Disease Progression; Female; Gene Expression Profiling; Growth Plate; Hedgehog Proteins; Humans; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Membrane Proteins; Middle Aged; Neoplasm Proteins; Nerve Tissue Proteins; Oligonucleotide Array Sequence Analysis; Osteosarcoma; Plasminogen Activator Inhibitor 1; Proto-Oncogene Mas; Proto-Oncogene Proteins c-jun; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Wnt1 Protein

Cell migration is essential for both organogenesis and tumor progression. Bone morphogenetic proteins (BMPs) are reported to be critical for not only bone formation but also tumor invasion. Here, we found that treatment with recombinant human BMP-2 (rhBMP-2) enhanced the haptotactic response of murine osteoblastic MC3T3-E1 and osteosarcoma Dunn cells to various extracellular matrix (ECM) components, including fibronectin, type I collagen, and laminin-1. Function-blocking antibody against integrin alpha5beta1 partially inhibited haptotaxis to fibronectin, suggesting that the response was propagated via these integrins. rhBMP-2 slightly increased the expression level of integrin beta1, and enhanced the speed of cell spreading on fibronectin, focal adhesion formation and phosphorylation of focal adhesion kinase (FAK) at Tyr397. By means of sucrose gradient flotation, incorporation of integrin beta1 in fractions of detergent (CHAPS) resistant membrane was increased when the cells were treated with rhBMP-2. Further, treatment with methyl-beta-cyclodextrin to deplete membrane cholesterol abrogated the effect of rhBMP-2 on haptotaxis, and exogenously added cholesterol reversed this inhibitory effect. Collectively, these results provide insights into the mechanism by which BMP signaling enhances cell migration by modulating fibronectin-integrin beta1 signaling via cholesterol enriched membrane microdomains, lipid rafts.

Topics: 3T3 Cells; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Humans; Integrin beta1; Membrane Microdomains; Mice; Osteoblasts; Osteosarcoma; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta

We have previously shown p53 to have a specific role in osteoblast differentiation by its ability to regulate expression of certain bone specific proteins. In this study, we show mineralized matrix formation in vivo to be directly related to the presence of wild type p53 in osteoblastic osteosarcoma cells. In order to further understand the importance of p53 in differentiation, we investigated the relationship between p53 and Bone Morphogenetic Proteins (BMPs) (BMP 1, 2, 3A, 3B (GDF-10), 4, 5, 6, 7, 8A and 8B) during osteoblast differentiation. The expression of several BMPs were tested using RNase Protection Assay in differentiating ROS17/2.8 osteoblastic osteosarcoma cells. The expression of BMPs 1, 2, 3a, 3b and 7 showed time dependent modulation during in vitro differentiation. In order to determine if p53 has a role in this process, we used a murine osteosarcoma cell line stably expressing a temperature sensitive p53. Cells were exposed to ascorbic acid and glycerophosphates to hasten in vitro osteoblast differentiation and maintained either at 32 or 37 degrees C for expression of the wild type or mutant p53 phenotype. The expression of BMP-2, BMP-4 and BMP-7 were modulated in a p53 dependent fashion. We were able to confirm the p53 dependency of BMP-2 independently by RT-PCR. While BMP-2 expression was evident in the presence of both wild type and mutant p53, regulated expression was seen only in cells expressing wild type p53. Transient over expression of wild type p53 did not result in the same BMP-2 response as stable expression showing that the presence of p53 may be important for an orderly development of osteoblast differentiation rather than a direct effect on gene expression. The functional relationship between p53 and these bone specific markers is discussed.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line, Tumor; Osteoblasts; Osteocalcin; Osteosarcoma; Promoter Regions, Genetic; Rats; RNA, Messenger; Transfection; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Both transforming growth factor-beta (TGF-beta)-induced expression of biglycan (BGN) and activation of p38 MAPK have been implicated in cellular adhesion and migration. Here, we analyzed the role of adhesive events and the small GTPase Rac1 in TGF-beta regulation of BGN. TGF-beta1 induction of BGN expression and activation of p38 was abolished or strongly reduced when cells were kept in suspension or exposed to either the actin cytoskeleton-disrupting agent cytochalasin D or a specific chemical Rac1 inhibitor. Ectopic expression of a dominant negative mutant (T17N) of Rac1 abrogated both TGF-beta-induced p38 MAPK activation and BGN up-regulation but did not affect TGF-beta-induced phosphorylation of Smad3 or transcriptional induction of Growth Arrest DNA Damage 45beta, previously shown to be crucial for TGF-beta regulation of BGN. Overexpression of wild type Rac1 greatly enhanced the TGF-beta effect on BGN in adherent cells, whereas ectopic expression of constitutively active Rac1 (Q61L) activated p38 and in the presence of exogenous TGF-beta was able to rescue BGN expression in nonadherent cells. Endogenous Rac1 was activated by TGF-beta treatment in PANC-1 cells in an adhesion-dependent fashion. Like Rac1-T17N, the NADPH oxidase inhibitor diphenylene iodonium and the tyrosine kinase inhibitor herbimycin A blocked TGF-beta-induced p38 activation and BGN expression, suggesting that Rac1 exerts its effect on BGN and p38 through increasing NADPH oxidase activity and subsequent production of reactive oxygen species. These results show that the TGF-beta effect on BGN is dependent on cell adhesion and that activated Rac1, presumably acting through NADPH oxidase(s), is necessary but not sufficient for TGF-beta-induced BGN expression.

Topics: Biglycan; Carcinoma; Cell Adhesion; Cell Line, Tumor; Cytochalasin D; Enzyme Activation; Enzyme Inhibitors; Extracellular Matrix Proteins; Gene Expression Regulation; Humans; Models, Biological; Mutation; NADPH Oxidases; Nucleic Acid Synthesis Inhibitors; Onium Compounds; Osteosarcoma; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Pancreatic Neoplasms; Proteoglycans; rac1 GTP-Binding Protein; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

This article demonstrates that the degree of VEG expression could be used as a clinically signific prognostic factor in osteosarcoma.

Topics: Adolescent; Adult; Biomarkers, Tumor; Bone Neoplasms; Child; Female; Humans; Male; Middle Aged; Osteosarcoma; Prognosis; Protein Isoforms; Retrospective Studies; Survival Analysis; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Degradation of extracellular matrix (ECM) is a hallmark of tumor invasion, metastasis and angiogenesis. Based on the Rath multitargeted approach to cancer using natural substances to control ECM stability and enhancing its strength, we developed a novel formulation (NM) of lysine, proline, ascorbic acid and green tea extract that has shown significant anti-cancer activity against a number of cancer cell lines. The aim of the present study was to determine whether NM exhibits anti-angiogenic and anti-metastatic effects using in vitro and in vivo experimental models. Angiogenesis was measured using a chorioallantoic membrane (CAM) assay in chick embryos and bFGF-induced vessel growth in C57BL/6J female mice. To determine the in vivo effect of NM on the tumor xenograft growth, male nude mice were inoculated with 3 x 10(6) MNNG-HOS cells. Control mice were fed a mouse chow diet, while the test group was fed a mouse chow diet supplemented with 0.5% NM for 4 weeks. In vitro studies on cell proliferation (MTT assay), MMP expression (zymography) and Matrigel invasion were conducted on human osteosarcoma U2OS, maintained in McCoy medium, supplemented with 10% FBS, penicillin and streptomycin in 24-well tissue culture plates and tested with NM at 0, 10, 50, 100, 500, and 1000 microg/ml in triplicate at each dose. NM at 250 microg/ml caused a significant (p<0.05) reduction in bFGF-induced angiogenesis in CAM. NM inhibited tumor growth of osteosarcoma MNNG-HOS cell xenografts in nude mice by 53%; furthermore, tumors in NM-treated mice were less vascular and expressed lower levels of VEGF and MMP-9 immunohistochemically than tumors in the control group. In addition, NM exhibited a dose-dependent inhibition of osteosarcoma U2OS cell proliferation (up to 60% at 1000 microg/ml), MMP-2 and -9 expression (with virtual total inhibition at 500 microg/ml NM), and invasion through Matrigel (with total inhibition at 100 microg/ml NM). Moreover, NM decreased U2OS cell expression of VEGF, angiopoietin-2, bFGF, PDGF and TGFbeta-1. These results together with our earlier findings suggest that NM is a relatively non-toxic formulation, which inhibits growth, invasion, metastasis, and angiogenesis of tumor cells.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Ascorbic Acid; Cell Line, Tumor; Cell Proliferation; Chick Embryo; Chorioallantoic Membrane; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Extracellular Matrix; Fibroblast Growth Factor 2; Formaldehyde; Humans; Immunohistochemistry; In Vitro Techniques; Laminin; Lysine; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Osteosarcoma; Plant Extracts; Platelet-Derived Growth Factor; Proline; Proteoglycans; Tea; Tetrazolium Salts; Thiazoles; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Growth and differentiation factor 5 (GDF-5) belongs to the large TGF-beta superfamily of secreted signalling proteins and plays a pivotal role in skeletal development during embryogenesis. The gene for human GDF-5 was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained that diffracted to 2.2 A resolution. A native data set was acquired, showing that the crystals belong to a trigonal space group, i.e. P3(1)21 or P3(2)21, with unit-cell parameters a = b = 97.1, c = 48.3 A. Initial analysis suggest the presence of only one monomer in the asymmetric unit, resulting in a high solvent content of 72% in the crystal.

Topics: Bone Morphogenetic Proteins; Cell Line, Tumor; Cloning, Molecular; Crystallization; Crystallography, X-Ray; Escherichia coli; Growth Differentiation Factor 5; Humans; Osteosarcoma; Recombinant Proteins; Transforming Growth Factor beta

Chitosan has a variety of biological activities. However, little is known about how chitosan modulates the hard tissue forming cells. When we cultured an osteoblastic cell line in alpha-MEM supplemented with 10% FBS and 0.005% chitooligosaccharide for 3 days, alkaline phosphatase (ALP) activity was significantly high compared with the control culture group (p<0.05). This study was focused on gene expression in osteoblasts cultured with water-soluble chitooligosaccharide. cDNA probes were synthesized from isolated RNA and labeled with fluorescent dye. They were hybridized with Human 1.0((R)) cDNA microarray, and fluorescent signal was analyzed. cDNA microarray analysis revealed that 16 genes were expressed at >/=1.5-fold higher signal ratio levels in the experimental group compared with the control group after 3 days. RT-PCR analysis showed that chitosan oligomer induced an increase in the expression of two genes, CD56 antigen and tissue-type plasminogen activator. Furthermore, the expression of mRNAs for BMP-2 was almost identical in the experimental and control groups after 3 days of culture, but slightly increased after 7 days of culture with chitosan oligomer. These results suggest that a super-low concentration of chitooligosaccharide could modulate the activity of osteoblastic cells through mRNA levels and that the genes concerning cell proliferation and differentiation can be controlled by water-soluble chitosan.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; CD56 Antigen; Cell Culture Techniques; Cell Differentiation; Cell Division; Cell Line, Tumor; Chitin; Chitosan; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Molecular Weight; Oligonucleotide Array Sequence Analysis; Osteoblasts; Osteogenesis; Osteosarcoma; Reverse Transcriptase Polymerase Chain Reaction; Solubility; Tissue Plasminogen Activator; Transforming Growth Factor beta; Water

This study was performed to elucidate the possible mechanism of the disturbance of hemopoiesis by HCMV infection. Saos-2 cells constitutively express mRNA of GM-CSF, and its expression was profoundly decreased by HCMV infection, which required full replication of the virus and was mediated by soluble factors released from the HCMV-infected Saos-2 cells. TGF-beta1 production was statistically and significantly increased from one day after HCMV infection. Expression and production of GM-CSF in Saos-2 cells were restored when a culture supernatant of HCMV-infected Saos-2 cells was reacted with neutralizing anti-TGF-beta antibody. Conclusively, HCMV inhibits GM-CSF expression in Saos-2 cells partly by the increased production of TGF-beta1.

Topics: Cell Line; Cytomegalovirus; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Osteosarcoma; Transforming Growth Factor beta; Tumor Cells, Cultured; Virus Replication

Differential expression of multiple osteogenic factors may be responsible for the different osteoinductivity of osteosarcoma cell lines. We compared in vivo osteoinductivity of human osteosarcoma cell lines (Saos-2 vs. U-2 OS) in nude mice, and their in vitro expression of various osteogenic factors of protein level by quantitative immunocytochemistry and mRNA level by RT-PCR and/or in situ hybridization. Saos-2 cells, but not U-2 OS, were osteoinductive in vivo. Significantly higher expression (independent t-test, all p < 0.005) of osteogenic factors were observed in Saos-2 cells compared with U-2 OS, which included bone morphogenetic proteins (particularly BMPs-2, 3, 4, and 7), transforming growth factor-beta (TGF-beta), BMP receptor (BMPR)-1A, receptor-regulated Smads (R-Smads), Smads 1, 2, and 5, and common-mediator Smad (Co-Smad), Smad 4. In contrast, U-2 OS cells expressed higher levels of inhibitory Smad 6 (I-Smad) protein than Saos-2 cells (p < 0.001). These results suggest that a combination of osteogenic factors (BMPs, TGF-beta, BMPRs, and R/Co-Smads) against I-Smad may play important roles in the Saos-2 cell osteoinductivity. This may have a clinical implication in selecting key osteogenic factors for combined therapy for bone defect diseases. The characterized cell lines can be used as positive and negative controls for the assessments of both in vitro and in vivo bone formation capabilities of designed tissues or biomaterials.

Topics: Animals; Bone Morphogenetic Proteins; Cell Line, Tumor; Diffusion Chambers, Culture; Female; Growth Substances; Humans; Immunohistochemistry; In Situ Hybridization; Mice; Mice, Inbred BALB C; Mice, Nude; Ossification, Heterotopic; Osteogenesis; Osteosarcoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

To evaluate the effects of transforming growth factor beta1 (TGFbeta1) autocrine blockage on proliferation activity and drug sensitivity of osteosarcoma. METHODS; Northern blot, MTT determination, and 3H thymidine incorporation were used to investigate the effects of antisense TGF beta1 gene on osteosarcoma.. The proliferation of osteosarcoma cells transfected by antisense TGF beta1 gene was suppressed markedly, and adriamycin sensitivity was significantly increased.. Blockage of osteosarcoma cells TGF beta1 autocrine loop inhibits cell proliferation and enhances chemotherapy sensitivity.

Topics: Antineoplastic Agents; Antisense Elements (Genetics); Autocrine Communication; Bone Neoplasms; Cell Division; Cell Line, Tumor; Doxorubicin; Humans; Osteosarcoma; RNA, Messenger; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

Parathyroid hormone (PTH) exerts potent and diverse effects in bone and cartilage through activation of type 1 PTH receptors (PTH1R) capable of coupling to protein kinase A (PKA) and PKC. We have used macroarrays to identify zinc finger protein butyrate response factor-1 (BRF1) as a novel PTH regulated gene in clonal and normal osteoblasts of human and rodent origin. We further demonstrate that in human osteoblast-like OHS cells, biologically active hPTH(1-84) and hPTH(1-34) stimulate BRF1 mRNA expression in a dose- and time-dependent manner, while the amino-terminally truncated hPTH(3-84) which does not activate PTH1R has no effect. Moreover, using specific stimulators or inhibitors of PKA and PKC activity, the PTH-elicited BRF1 mRNA expression is mediated through the PKA signaling pathway. In mouse calvarial osteoblasts, BRF1 mRNA levels are upregulated by PTH(1-84) and reduced in response to bone morphogenetic protein 2 (BMP-2). Hence, our data showing that BRF1 is expressed in osteoblastic cells and regulated by PTH and BMP-2, suggest an important role for BRF1 in osteoblasts within the molecular network of PTH-dependent bone remodeling.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Butyrate Response Factor 1; Cell Line, Tumor; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; DNA-Binding Proteins; Gene Expression Regulation; Hedgehog Proteins; Humans; Immediate-Early Proteins; Mice; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Peptide Fragments; Rats; Receptor, Parathyroid Hormone, Type 1; Receptors, Parathyroid Hormone; Trans-Activators; Transforming Growth Factor beta; Zinc Fingers

Bone morphogenetic proteins (BMPs) are thought to be responsible for bone formation; they cause bone to form in soft tissues and are clinically used in helping fracture union or tumor reconstructions. Skeletal metastases from epithelial tumors may be either bone-forming (blastic) or non-bone-forming (lytic).. We studied the expression of BMPs in a variety of primary and secondary lesions of bone (both bone-forming and non-bone-forming) to determine if there was a consistent relationship between bone formation and BMP expression.. We compared a bone-forming lesion (fibrous dysplasia) with a non-bone-forming lesion (desmoid tumor), using reverse transcription-polymerase chain reaction, Northern blot analysis, and immunohistochemistry to detect BMPs. We also studied a number of non-bone-forming secondary lesions (carcinomas that formed lytic metastases to the skeleton) and found BMP production in most of these tumors.. We found that BMPs were expressed in both bone-forming and non-bone-forming benign musculoskeletal lesions. In the first part of the study, BMPs were found in both fibrous dysplasia and desmoid tumors. Bone morphogenetic proteins were also expressed by several tumors. In the next part of the study (paraffin-embedded tissue), BMPs were expressed by a variety of tumors, irrespective of the radiological nature (blastic or lytic) of their metastases.. We conclude that BMP production alone cannot explain bone formation, and other factors either alone or in combination may be responsible for blastic metastases to the skeleton and for bone formation by primary bone lesions, such as fibrous dysplasia.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Proteins; Bone Neoplasms; Carcinoma; Cell Line; Cell Line, Tumor; Female; Fibroblasts; Fibromatosis, Aggressive; Fibrous Dysplasia, Monostotic; Fibrous Dysplasia, Polyostotic; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Osteosarcoma; Protein Serine-Threonine Kinases; Receptors, Growth Factor; RNA; RNA, Neoplasm; Transforming Growth Factor beta

The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been characterized. Recently, the phenotype of Sp8 null mice has been described, being tailless and having severe truncation of both fore and hind limbs. They also have malformed brains with defective closure of the anterior and posterior neuropore during brain development.. The human Sp8 gene is a three-exon gene that maps to 7p21.3, close to the related Sp4 gene. From an osteosarcoma cell line we cloned two transcript variants that use two different first exons and have a common second exon. One clone encodes a 508-residue protein, Sp8L (isoform 1) and the other a shorter 490-residue protein, Sp8S (isoform 2). These two isoforms are conserved being found also in mice and zebrafish. Analysis of the Sp8L protein sequence reveals an amino-terminal hydrophobic Sp-motif that is disrupted in Sp8S, a buttonhead box and three C2H2 zinc-fingers. Sp8 mRNA expression was detected in a wide range of tissues at a low level, with the highest levels being found in brain. Treatment of the murine pluripotent cell line C3H10T1/2 with 100 ng/mL BMP-2 induced Sp8 mRNA after 24 hours.. There is conservation of the two Sp8 protein isoforms between primates, rodents and fish, suggesting that the isoforms have differing roles in gene regulation. Sp8 may play a role in chondrogenic/osteoblastic differentiation in addition to its role in brain and limb development.

Topics: Amino Acid Sequence; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Line; Cell Line, Tumor; Chromosome Mapping; Chromosomes, Human, Pair 7; Cloning, Molecular; Conserved Sequence; DNA-Binding Proteins; DNA, Complementary; Exons; Gene Expression Regulation; Humans; Hydrophobic and Hydrophilic Interactions; Mice; Mice, Inbred C3H; Molecular Sequence Data; Organ Specificity; Osteoblasts; Osteosarcoma; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Zebrafish Proteins; Zinc Fingers

Notch signaling releases the Notch receptor intracellular domain (ICD), which complexes with CBF1 and Mastermind (MAM) to activate responsive genes. We previously reported that MAM interacts with CBP/p300 and promotes hyperphosphorylation and degradation of the Notch ICD in vivo. Here we show that CycC:CDK8 and CycT1:CDK9/P-TEFb are recruited with Notch and associated coactivators (MAM, SKIP) to the HES1 promoter in signaling cells. MAM interacts directly with CDK8 and can cause it to localize to subnuclear foci. Purified recombinant CycC:CDK8 phosphorylates the Notch ICD within the TAD and PEST domains, and expression of CycC:CDK8 strongly enhances Notch ICD hyperphosphorylation and PEST-dependent degradation by the Fbw7/Sel10 ubiquitin ligase in vivo. Point mutations affecting conserved Ser residues within the ICD PEST motif prevent hyperphosphorylation by CycC:CDK8 and stabilize the ICD in vivo. These findings suggest a role for MAM and CycC:CDK8 in the turnover of the Notch enhancer complex at target genes.

Topics: Adaptor Proteins, Signal Transducing; Animals; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Cell Line, Tumor; Chromatin; Coculture Techniques; Cyclin-Dependent Kinase 8; Cyclin-Dependent Kinase 9; Cyclin-Dependent Kinases; DNA-Binding Proteins; Drosophila Proteins; Enhancer Elements, Genetic; Genes, Reporter; HeLa Cells; Homeodomain Proteins; Humans; L Cells; Luciferases; Membrane Proteins; Mice; Nuclear Proteins; Osteosarcoma; Phosphorylation; Point Mutation; Precipitin Tests; Promoter Regions, Genetic; Protein Structure, Tertiary; Receptors, Notch; Signal Transduction; Trans-Activators; Transcription Factor HES-1; Transcription Factors; Transcriptional Activation; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

To investigate the effect of BMP-2 on (the) gene expression of rat osteosarcoma cells (ROS17/2.8) under rotating clinostat simulated weightlessness.. ROS17/2.8 cells were cultivated in 1 G control and rotating clinostat simulated weightlessness with 500 ng/ml BMP-2 in the culture medium. Total RNA in cells was isolated after 24, 48 and 72 h. Reverse transcription PCR analysis was made to examine the gene expression of alpha 1 chain of type I collagen (collagen I alpha 1) and alkaline phosphatase (ALP).. The expression of COL-I alpha 1 mRNA induced by BMP-2 was much more than that without BMP-2 in 24 h and 48 h group (P<0.01). The level of ALP mRNA induced by BMP-2 was significantly high in 48 h or 72 h group (respectively P<0.01, P<0.05). The level of BMP-2 induced expression of COL-I alpha 1 mRNA was significantly lower under 48 h of simulated weightlessness than in 1 G condition (P<0.01). The level of ALP mRNA was significantly lower under simulated weightlessness for 48 h or 72 h (P<0.01).. BMP-2 can stimulate the differentiation of ROS17/2.8 cells in 1 G condition and this process is reduced under simulated weightlessness.

Topics: Alkaline Phosphatase; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cells, Cultured; Collagen Type I; Gene Expression; Osteosarcoma; Rats; RNA, Messenger; Rotation; Time Factors; Transforming Growth Factor beta; Weightlessness Simulation

The metalloproteinase ADAMTS-2 has procollagen I N-proteinase activity capable of cleaving procollagens I and II N-propeptides in vitro, whereas mutations in the ADAMTS-2 gene in dermatosparaxis and Ehlers-Danlos syndrome VIIC show this enzyme to be responsible in vivo for most biosynthetic processing of procollagen I N-propeptides in skin. Yet despite its important role in the regulation of collagen deposition, information regarding regulation and substrate specificity of ADAMTS-2 has remained sparse. Here we demonstrate that ADAMTS-2 can, like the procollagen C-proteinases, be regulated by transforming growth factor-beta 1 (TGF-beta 1), with implications for mechanisms whereby this growth factor effects net increases in formation of extracellular matrix. TGF-beta 1 induced ADAMTS-2 mRNA approximately 8-fold in MG-63 osteosarcoma cells in a dose- and time-dependent, cycloheximide-inhibitable manner, which appeared to operate at the transcriptional level. Secreted ADAMTS-2 protein induced by TGF-beta 1 was 132 kDa and was identical in size to the fully processed, active form of the protease. Biosynthetic processing of ADAMTS-2 to yield the 132-kDa form is shown to be a two-step process involving sequential cleavage by furin-like convertases at two sites. Surprisingly, purified recombinant ADAMTS-2 is shown to cleave procollagen III N-propeptides as effectively as those of procollagens I and II, whereas processing of procollagen III is shown to be decreased in Ehlers-Danlos VIIC. Thus, the dogma that procollagen I and procollagen III N-proteinase activities are provided by separate enzymes appears to be false, whereas the phenotypes of dermatosparaxis and Ehlers-Danlos VIIC may arise from defects in both type I and type III collagen biosynthesis.

Topics: ADAM Proteins; ADAMTS Proteins; ADAMTS4 Protein; Amino Acid Sequence; Blotting, Western; Collagen; Collagen Type I; Ehlers-Danlos Syndrome; Electrophoresis, Polyacrylamide Gel; Fibroblasts; Gene Expression Regulation; Glycosylation; Humans; Keratinocytes; Kinetics; Molecular Sequence Data; Mutation; Osteosarcoma; Procollagen; Procollagen N-Endopeptidase; Protein Precursors; Recombinant Proteins; RNA, Messenger; Skin; Substrate Specificity; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Chitosan is a natural bioactive material. Although it has been reported that chitosan promotes osteogenesis in bone lesions, little is known about how chitosan modulates this process. The present study was designed to investigate the effect of water-soluble chitosan relative to initiation of biologic mineralization, especially in the matrix-vesicles-(MVs) mediated process in vitro. A human osteoblastic cell line (NOS-1) was used. After 3 days of incubation, the number of cells and alkaline phosphatase (ALP) activity increased significantly in the chitosan group. RT-PCR analysis revealed that chitosan induced an increase in the expression of bone morphogenetic protein-2 mRNA after 7 days of incubation. MVs were isolated from NOS-1 cells using a collagenase digestion and ultracentrifugation method. ALP activity of MVs isolated from chitosan-supplemented cells was significantly higher than that of the control group. Furthermore, isolated MVs were incubated in medium supplemented with Na-beta-glycerophosphate without fetal bovine serum. Needle-like crystals were observed in association with MVs after 24 h of incubation. These needle-like crystals were densely accumulated in the chitosan group. The present findings suggest that water-soluble chitosan would promote osteoblast proliferation and differentiation and may be useful for the acceleration of initial biologic mineralization.

Topics: Alkaline Phosphatase; Base Sequence; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcification, Physiologic; Cell Line, Tumor; Chitin; Chitosan; Culture Media; DNA Primers; Humans; Osteosarcoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Solubility; Transforming Growth Factor beta; Water

To construct the antisense transforming growth factor beta 1 (TGF beta 1) gene and investigate the effect of TGF beta 1 autocrine loop blockage on the proliferation of osteosarcoma cells. TGF beta 1 cDNA was cloned by RT-PCR from human osteosarcoma cells (MG-63) and inserted into pcDNA3 to construct an antisense expression vector, which was dubbed pcDNA3-TGF beta 1(-). MTT was used to detect the proliferation of osteosarcoma cells transfected by antisense TGF beta 1 gene. Our results showed that the proliferation of the transfected osteosarcoma cells was suppressed markedly. It is concluded that TGF beta 1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation, which might be helpful for gene therapy of osteosarcoma.

Topics: Antisense Elements (Genetics); Bone Neoplasms; Cell Division; Humans; Osteosarcoma; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Bone-forming osteoblasts differentiate from pluripotent mesenchymal stem cells (MSCs) in a multistage process that can be modeled in vitro using MSCs isolated from adult human trabecular bone or bone marrow. To identify new genes involved in osteoblast differentiation, we have performed large-scale gene expression profiling using high-density cDNA microarrays in primary human MSCs treated with the known osteogenic agent bone morphogenetic protein 2 (BMP-2). The vascular endothelial growth factor (VEGF) family member placental growth factor (PlGF) was found as an early regulated gene whose induction was already detected after 2 h treatment with BMP-2. Tissue distribution analysis of PlGF mRNA expression using microarrays revealed a very restricted expression of PlGF only in BMP-2-treated MSCs and in placenta as expected. Ribonuclease protection assay (RPA) confirmed the induction of PlGF and showed preferential expression of the PlGF-1 isoform over PLGF-2 in MSCs and MG63 cells. BMP-2 stimulated PlGF expression in MG63 cells with an EC50 of about 50 ng/ml and mRNA levels peaked between 24 and 32 h after stimulation. Furthermore, induction of PlGF by BMP-2 appeared specific, as other osteogenic agents including vitamin D3, transforming growth factor beta, and basic fibroblast growth factor were inactive. BMP-2 stimulated PlGF secretion from MG63 and MSC cells, but PlGF had no effect on MSC proliferation and osteoblastic differentiation. Based on the known function of PlGF in the recruitment of endothelial and hematopoietic stem cells, these results suggest a paracrine role for MSC-derived PlGF in the angiogenesis and hematopoiesis that accompany BMP-2-induced bone formation.

Topics: Amino Acid Sequence; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line, Tumor; Cells, Cultured; Gene Expression; Humans; Mesoderm; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Osteoblasts; Osteosarcoma; Paracrine Communication; Placenta Growth Factor; Pregnancy Proteins; RNA, Messenger; Stem Cells; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) has growth-stimulating effects on mesenchymal cells and several tumor cell lines. The signaling pathway for this effect is, however, not well understood. We examined how TGF-beta stimulates proliferation of MG63 human osteosarcoma cells. Two distinct type I receptors for TGF-beta, ALK-1 and ALK-5, were expressed and functional in MG63 cells. Of these two receptors, ALK-5 appears to be responsible for the growth stimulation because expression of constitutively active ALK-5, but not ALK-1, stimulated proliferation of MG63 cells. SB-431542 (0.3 microM), a novel inhibitor of ALK4/5/7 kinase, suppressed TGF-beta-induced growth stimulation. DNA microarray analysis as well as quantitative real-time PCR analysis of RNAs from TGF-beta-treated cells demonstrated that several growth factors, including platelet-derived growth factor AA, were induced in response to TGF-beta in MG63 cells. Gleevec (1 microM) as well as AG1296 (5 microM) inhibited TGF-beta-induced growth stimulation of MG63 cells, suggesting that platelet-derived growth factor AA was mainly responsible for the growth-stimulatory effect of TGF-beta. We also examined the mechanisms of perturbation of growth-suppressing signaling in MG63 cells. We found that expression of c-Myc, which is down-regulated by TGF-beta in many other cells, was up-regulated in MG63 cells, suggesting that up-regulation of c-Myc expression may be the mechanism canceling growth-suppressing signaling of TGF-beta in MG63 cells.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; Animals; Antineoplastic Agents; Benzamides; Bone Neoplasms; Cell Division; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Dioxoles; Drug Interactions; Gene Expression Regulation, Neoplastic; Humans; Imatinib Mesylate; Mice; NIH 3T3 Cells; Oligonucleotide Array Sequence Analysis; Osteosarcoma; Piperazines; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Pyrimidines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

To observe the regulative effects of 17 beta-estradiol (17 beta-E(2)) and ICI 182780 on the expression of osteoprotegerin (OPG), the ligand of osteoprotegerin (OPGL) and the related cytokines [macrophage colony-stimulating factor (M-CSF), TRAIL and transforming growth factor beta(TGF beta)] in MG63 cells.. The expression of OPG, OPGL, M-CSF, TRAIL and TGF beta mRNA was examined by reverse transcriptase (RT)-PCR. The expression of OPG and OPGL protein was measured with Western blot.. (1) 17 beta-E(2) up-regulated the expression of OPG and TGF beta but down-regulated OPGL, M-CSF and TRAIL expression in MG63 cells. (2) ICI 182780 showed varied transactivating capability when regulating the expression of OPG, OPGL, TRAIL, M-CSF and TGF beta genes in MG63 cells.. (1) One of the key pathogenetic factors of postmenopausal osteoporosis is that estrogen deficiency leads to the decreasing expression of OPG and TGF beta but the increasing expression of some bone-resorbing cytokines such as OPGL, M-CSF and TRAIL in osteoblasts, then stimulation of osteoclast differentiation and activity which potentiates bone resorption and bone loss; (2) ICI 182780 shows partial transactivating activity in osteoblasts, so it should belong to a kind of selective estrogen receptor modulators.

Topics: Apoptosis Regulatory Proteins; Blotting, Western; Carrier Proteins; Cell Line, Tumor; Cytokines; Estradiol; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Macrophage Colony-Stimulating Factor; Membrane Glycoproteins; Osteoprotegerin; Osteosarcoma; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Reverse Transcriptase Polymerase Chain Reaction; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Cytokines are considered to play an important role in tumor pathogenesis and progression, and recent studies have demonstrated that a variety of forms, including interleukins (ILs) and transforming growth factor-beta(s) (TGF-beta(s)), may regulate tumors. In the present study, the expression of TGF-beta isoforms and ILs was investigated in cell lines from a rat osteosarcoma and a malignant fibrous histiocytoma (MFH), both established from transplantable tumors induced by 4-(hydroxyamino) quinoline 1-oxide (4-HAQO) in syngeneic F344 male rats. The results of a multiprobe RNase protection assay showed TGF-beta1 expression to be remarkably elevated, with no TGF-beta2 and beta3 detectable in MFH cells, while TGF-beta1 and -beta2 were found to be moderately and TGF-beta3 weakly expressed in osteosarcoma lines. All cell lines of osteosarcomas and MFHs expressed macrophage migration inhibitory factor at similar levels. In contrast to the lack of ILs in the MFH cells, moderate IL-6 and very weak IL-1beta expression was detected in the osteosarcoma cells. These results suggest that variation in expression pattern of these cytokines in osteosarcomas and MFHs might be involved in differences in histological appearance and biological behavior, including metastatic ability, between these two mesenchyme-derived tumor types.

Topics: 4-Hydroxyaminoquinoline-1-oxide; Animals; Base Sequence; DNA Primers; Gene Expression Regulation; Histiocytoma, Benign Fibrous; Interleukins; Osteosarcoma; Rats; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Cultured rodent osteoblastic cells reiterate the phenotypic differentiation and maturation of osteoblasts seen in vivo. As previously shown, the human osteosarcoma cell line HOS 58 represents a differentiated stage of osteoblast development. The potential of HOS 58 for still further in vitro differentiation suggests the line can serve as a model of osteoblast maturation. Using this cell line, we have investigated the influence of 1,25-(OH)2-D3 (D3), TGF-beta and Dexamethasone (Dex) on proliferation and on the protein and mRNA levels of alkaline phosphatase (AP), procollagen 1 (Col 1), and osteocalcin (Oc), as well as mineralization during 28 days in culture. AP mRNA and protein were highly expressed throughout the culture period with further increase of protein AP activity at constant gene expression levels. A differentiation inhibiting effect of either TGF-beta or Dex was seen. Col 1 was investigated without the use of ascorbic acid and showed only minor changes during culture time or stimulation. The gene expression for Oc increased continually whereas protein synthesis peaked at confluence and decreased thereafter. TGF-beta and Dex treatments decreased Oc mRNA and protein levels. Stimulation by D3 was maximal at day 7 with a decrease thereafter. HOS 58 cells showed no mineralization capacity when stimulated with different agents, as measured by energy-dispersive X-ray microanalysis. This was not due to absence of Cbfa1 expression. In conclusion, the HOS 58 osteosarcoma cell line represents a differentiated cell line with highly expressed and physiologically regulated AP expression during further differentiation in culture. We observed a dissociation between osteocalcin gene expression and protein secretion which may contribute to the lack of mineralization in this cell line.

Topics: Adult; Alkaline Phosphatase; Animals; Blotting, Northern; Calcification, Physiologic; Calcitriol; Collagen Type I; Core Binding Factor alpha Subunits; Dexamethasone; DNA Primers; DNA-Binding Proteins; Gene Expression; Gene Expression Regulation, Enzymologic; Humans; Male; Mice; Microscopy, Electron, Scanning; Osteocalcin; Osteosarcoma; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Thrombospondin-1 (TSP-1), a multifunctional matrix protein, affects tumor growth through modulation of angiogenesis and other stromal biological functions. In several of nine human cancer cell lines derived from liver, brain, pancreas, and bone, expression of TSP-1 was up-regulated in response to the two most representative growth factors, epidermal growth factor (EGF) and transforming growth factor beta1 (TGFbeta1). Expression of TSP-1 was markedly enhanced in hepatic HuH-7 cells by EGF but not by TGFbeta1. In contrast, expression of TSP-1 was markedly enhanced by TGFbeta1, but not by EGF, in osteosarcoma MG63 cells. EGF induced activation of TSP-1 promoter-driven luciferase activity in HuH-7 cells, and the elements between -267 and -71 on the 5' region of TSP-1 gene containing two GC boxes to which Sp1 bound, were found to be responsible for the promoter activation by EGF. However, EGF did not alter TSP-1 mRNA stability in hepatic cells. On the other hand, no such enhancement of the TSP-1 promoter activity by TGFbeta1 appeared in MG63 cells. Enhanced expression of TSP-1 by TGFbeta1 in MG63 cells was partially blocked by exogenous addition of SB203580, an inhibitor of p38 mitogen-activated protein kinase. TGFbeta was found to induce marked elongation of TSP-1 mRNA longevity in osteosarcoma cells when mRNA degradation was assayed in the presence of alpha-amanitin. The up-regulation of TSP-1 by EGF and TGFbeta might play a critical role in modulation of angiogenesis and formation of matrices in tumor stroma.

Topics: Bone Neoplasms; Carcinoma, Hepatocellular; Epidermal Growth Factor; Gene Expression Regulation; Humans; Liver Neoplasms; Osteosarcoma; RNA Stability; Thrombospondin 1; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

Runx factors control lineage commitment and are transcriptional effectors of Smad signaling. Genetic defects in these pathways interfere with normal development. The in situ localization of Runx and Smad proteins must impact the mechanisms by which these proteins function together in gene regulation. We show that the integration of Runx and Smad signals is mediated by in situ interactions at specific foci within the nucleus. Activated Smads are directed to these subnuclear foci only in the presence of Runx proteins. Smad-Runx complexes are associated in situ with the nuclear matrix, and this association requires the intranuclear targeting signal of Runx factors. The convergence of Smad and Runx proteins at these sites supports transcription as reflected by BrUTP labeling and functional cooperativity between the proteins. Thus, Runx-mediated intranuclear targeting of Smads is critical for the integration of two distinct pathways essential for fetal development.

Topics: Amino Acid Substitution; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Neoplasms; Cell Nucleus; Core Binding Factor alpha Subunits; DNA-Binding Proteins; HeLa Cells; Humans; Mutagenesis, Site-Directed; Neoplasm Proteins; Osteosarcoma; Phosphoproteins; Rats; Recombinant Proteins; Signal Transduction; Smad5 Protein; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The biocompatibility of an orthopedic implant depends on the effect of the implant on bone-forming cells, osteoblasts. Changes in osteoblastic proliferation, maturation and differentiation are important events in ossification that enable monitoring the effect of the implant. Transforming growth factor-beta (TGF-beta) is known to suppress osteoblast proliferation and, on the other hand, to induce the maturation and differentiation of osteoblasts. Moreover, osteoblasts produce TGF-beta, which is embedded in the bone matrix and activated by bone-resorbing osteoclasts. TGF-beta inhibits osteoclastic activity. Here, we show for the first time the effect of nickel titanium shape memory metal (NiTi) on osteoblastic cytokine expression. In this study, we measured the levels of TGF-beta with enzyme-linked immunosorbent assay (ELISA) from a ROS-17/2.8 osteosarcoma cell line cultured on different metal alloy discs. ELISA results were proportioned to total DNA content of the samples. We compared NiTi, to stainless steel (Stst), pure titanium (Ti) and pure nickel (Ni). The TGF-beta1/DNA value in the NiTi group (0.0007 +/- 0.0003) was comparable with those seen in the Stst (0.0008 +/- 0.0001) and Ti (0.0007 +/- 0.0001) groups. The concentration in the Ni group was lower (0.0006 +/- 0.0003), though not statistically significantly so. In addition, the effect of surface roughness on TGF-beta1 production was studied. We compared three different grades of roughness in three differently hot-rolled alloys: NiTi. hot-rolled at 950 degrees C. Ti alloy hot-rolled at 850 degrees C (TiI) and the same Ti alloy hot-rolled at 1,050 degrees C (TiII). We found that increasing roughness of the NiTi surface increased the TGF-beta1 concentration. On the other hand, all roughness groups of TiII showed low levels of TGF-beta1. while a rough TiI surface induced similar TGF-beta1, expression as rough NiTi. Further, these same measurements made with interleukine 6 (IL-6) were found to be under the detection limit in these cultures. We conclude that a rough NiTi surface promotes TGF-beta1 expression in ROS-17/2.8 cells.

Topics: Animals; Bone Neoplasms; Cell Division; Cell Line; Dental Alloys; DNA, Neoplasm; Nickel; Osteosarcoma; Rats; Titanium; Transforming Growth Factor beta; Tumor Cells, Cultured

The mechanism of metastasis of osteosarcoma cells to other bones has not yet fully been clarified. The purpose of the present study was to examine whether various factors involve the formation of osteosarcoma metastatic foci in other bones. Immunohistochemically, CD31 expression in osteosarcoma with no bone metastasis and osteosarcoma with bone metastasis was noted in 10 and 75% of cases, respectively. Met/hepatocyte growth factor (HGF) receptor expression in osteosarcoma with no bone metastasis and osteosarcoma with bone metastasis was noted in 90 and 25% of cases, respectively. Bone morphogenetic protein (BMP) expression in osteosarcoma with no bone metastasis and osteosarcoma with bone metastasis was noted in 20 and 75% of cases, respectively. Metastasis of osteosarcoma cells to other bones was significantly correlated with expression of BMP and CD31 and with no expression of Met/HGF receptor protein in osteosarcoma cells. In contrast, expression of insulin-like growth factor receptor in osteosarcoma cells did not correlate significantly with bone metastasis. These results suggest that formation of metastatic foci of osteosarcoma cells in other bones is regulated by CD31, which is associated with migration between endothelial cells, by BMP, which can induce and activate various mesenchymal cells affecting bone formation, and by escape of effect by HGF, which promotes differentiation of osteosarcoma cells.

Topics: Adolescent; Adult; Aged; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Bone Neoplasms; Child; Hepatocyte Growth Factor; Humans; Immunoenzyme Techniques; Lung Neoplasms; Middle Aged; Neoplasm Metastasis; Osteosarcoma; Platelet Endothelial Cell Adhesion Molecule-1; Receptor, IGF Type 1; Transforming Growth Factor beta

Certain cells of the periodontium are necessary for the regeneration of tissues that are destroyed as a result of periodontal disease. There has been debate regarding which cells are the primary participants in periodontal regeneration. It is a well-known fact that osteoblasts are essential in new bone formation, but controversy surrounds the role that gingival fibroblasts may play in the regeneration of the hard tissues of the periodontium. If gingival fibroblasts could contribute to the regeneration of these tissues, they might provide an additional source of progenitor cells. The bone morphogenetic proteins are potent stimulators of cell differentiation and have been shown to induce new bone formation in many experimental models. This project investigated the ability of recombinant human bone morphogenetic protein-2 (rhBMP-2) to (1) enhance the production of markers of osteoblastlike cells (osteocalcin and mineralization in culture) in human osteosarcoma cells (MG63) and to (2) induce the expression of an osteoblast phenotype in cultured human gingival fibroblasts (HGFs). MG63 cells and pooled HGFs were exposed to varying concentrations of rhBMP-2 for 24, 48, and 72 hours after 9 days in culture, and osteocalcin levels were measured by enzyme immunosorbent assay in the cell supernatants. In addition, the cells were exposed to rhBMP-2 for 72 hours after 18 days in culture, and mineralization was determined by the Von Kossa stain. The rhBMP-2 had an inhibitory effect on both osteocalcin production and mineralization (p < 0.05) in MG63 cells compared with untreated controls. In addition, increasing doses of rhBMP-2 inhibited both osteocalcin and mineralization in HGF cells. These results suggest that HGFs can express an osteoblastic phenotype when exposed to rhBMP-2; however, rhBMP-2 has inhibitory effects at higher rhBMP-2 doses in both cell types and may, in fact, be inhibitory to MG63 cells.

Topics: Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Calcification, Physiologic; Fibroblasts; Gingiva; Humans; Osteoblasts; Osteocalcin; Osteosarcoma; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

A chemical exchange of the silica gel layer forming on the surface of bioactive glasses is thought to be the principal reaction for bone-bioactive glass bonding. The contribution of biological molecules on cell-bioactive glass interaction is largely unknown. To further analyze the mechanisms involved in efficient bone bonding to bioactive glass, Saos-2 osteoblastic cells with proven osteogenic phenotype were cultured for 4, 7 and 14 days on two bioactive glasses with different Si contents. Culture plates and dishes made of bioactive (BAG, 53 % SiO2), biocompatible (BCG, 58% SiO2) and control (GO) glasses were extensively conditioned with phosphate buffer and DMEM medium before seeding the cells. Northern hybridization was used for analysis of mRNA levels of collagen type I (Col-I), alkaline phosphatase (ALP) and bone morphogenetic protein-2 (BMP-2). A significant increase was observed in Col-I mRNA levels in cells grown on the two bioactive glasses when compared with those grown on controls at 4 and 7 days (p < 0.04). The mRNA level for ALP in the cultures of bioactive glasses-made plates and dishes was also increased over control at 7 days (p < 0.02) and remained this way between BAG and G0 at 14 days. Striking differences in BMP-2 mRNA levels existed between BAG and G0 plates and dishes at 7 days (p < 0.05). BMP-2 mRNA level in BAG group was higher than in BCG group at 4, 7 and 14 days, but without statistical significance. Saos-2 osteoblastic cells with strong ALP staining were mostly seen on BAG plates under a light microscope. In confocal microscopy, a bright FITC-stained F-actin ring was present in the cytoplasm of cells grown on BAG dish, demonstrating an active functional status. Stimulation of the expression of BMP-2 and other bone mRNAs by bioactive glasses in osteoblastic cells suggests biological involvement of bone related growth factors, peptides and cytokines in bone-bioactive glass bonding.

Topics: Alkaline Phosphatase; Biocompatible Materials; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Collagen; Gene Expression Regulation; Glass; Humans; Kinetics; Osteoblasts; Osteosarcoma; RNA, Messenger; Silicon Dioxide; Time Factors; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits cell growth. Previous reports have suggested the existence of plasma membrane IGFBP-3 receptors that could mediate direct, IGF-independent effects. Thus far, however, the only well-defined putative IGFBP-3 receptor is the type V transforming growth factor-beta (TGF-beta) receptor, a membrane glycoprotein that mediates TGF-beta-induced growth inhibition in selected cells. The aim of the study was to test whether IGFBP-3 and TGF-beta exert short-term effects in an osteosarcoma cell line that produces no IGF but contains type 1 IGF receptors.. DNA synthesis and apoptosis in Saos-2/B-10 cells were measured in response to IGF-I, IGF-II, IGFBP-3 and TGF-beta2, and to type 1 IGF receptor ligands with poor affinity for IGFBP-3 ([QAYL]-IGF-I and insulin).. IGF-I and IGF-II stimulated thymidine incorporation into DNA and suppressed apoptosis in a dose-dependent manner with maximal effects at 1 and 3 nM respectively. TGF-beta2 slightly increased thymidine incorporation into DNA but had no effect on apoptosis. IGFBP-3 had no effect by itself. Whereas it blocked the above effects of 1 nmol/l IGF-I, it did not block those of 1 nmol/l [QAYL]-IGF-I or 100 nmol/l insulin.. IGFBP-3 does not affect DNA synthesis or apoptosis in an IGF-independent manner in IGF-responsive osteosarcoma cells. It therefore appears to act essentially by sequestration of IGF.

Topics: Apoptosis; Blotting, Western; Cell Division; DNA Fragmentation; DNA, Neoplasm; Enzyme-Linked Immunosorbent Assay; Growth Inhibitors; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Osteosarcoma; Receptor, IGF Type 1; Receptor, Insulin; Signal Transduction; Somatomedins; Transforming Growth Factor beta; Tumor Cells, Cultured

The incidence of osteosarcoma is increased 500-fold in patients who inherit mutations in the RB gene. To understand why the retinoblastoma protein (pRb) is specifically targeted in osteosarcoma, we studied its function in osteogenesis. Loss of pRb but not p107 or p130 blocks late osteoblast differentiation. pRb physically interacts with the osteoblast transcription factor, CBFA1, and associates with osteoblast-specific promoters in vivo in a CBFA1-dependent fashion. Association of pRb with CBFA1 and promoter sequences results in synergistic transactivation of an osteoblast-specific reporter. This transactivation function is lost in tumor-derived pRb mutants, underscoring a potential role in tumor suppression. Thus, pRb functions as a direct transcriptional coactivator promoting osteoblast differentiation, which may contribute to the targeting of pRb in osteosarcoma.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Genes, Reporter; Genes, Retinoblastoma; Humans; Mice; Mice, Knockout; Mutagenesis, Site-Directed; Neoplasm Proteins; Neoplasms; Oncogene Proteins, Viral; Osteoblasts; Osteocalcin; Osteogenesis; Osteosarcoma; Papillomavirus E7 Proteins; Promoter Regions, Genetic; Protein Binding; Recombinant Fusion Proteins; Recombinant Proteins; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transforming Growth Factor beta

Small latent TGF-beta consists of latency associated peptide (LAP) bound to the 25 kDa TGF-beta by noncovalent interactions. Small latent TGF-beta is secreted from cells and deposited into the extracellular matrix as covalent complexes with its binding proteins, LTBPs. Four LTBPs have been molecularly cloned and their structures contain repetitive sequences. The 3rd 8-Cys repeats of LTBP-1, -3 and -4 are able to associate with small latent TGF-beta. We analyzed by RT-PCR the expression of LTBPs 1-4 in a panel of cultured human cell lines including fibroblasts of different origin, endothelial cells and immortalized keratinocytes. LTBPs were expressed in an overlapping manner, but differences in their expression levels were detected. SV-40 transformed human embryonic lung fibroblasts contained less of the mRNAs for the LTBPs, suggesting that malignant transformation leads to decrease in LTBP expression. A novel alternatively spliced form of LTBP-4 lacking the 3rd 8-Cys repeat (LTBP-4delta8-Cys3rd) was identified. LTBP-4delta8-Cys3rd does not bind TGF-beta and it was found to be expressed in the same tissues as the full length LTBP-4. The exon-intron structure of LTBP-4 around the 3rd 8-Cys repeat was similar to those of LTBP-2 and -3. LTBP-4delta8-Cys3rd was produced by alternative splicing over two exons. In addition, HL-60 promyelocytic leukemia cells expressed a splice variant lacking only one exon of this region. The expression of the non-TGF-beta-binding variant of LTBP-4 may be important for the regulation of TGF-beta deposition in tissues. Since LTBPs are a part of the extracellular matrix microfibrils, the LTBP-4delta8-Cys3rd protein may also be involved in various structural functions not related to TGF-beta signaling.

Topics: Adaptor Proteins, Signal Transducing; Alternative Splicing; Carrier Proteins; Cell Line, Transformed; Culture Media, Conditioned; Cysteine; DNA, Complementary; Fibroblasts; Gene Expression; Humans; Immunoblotting; Kidney; Latent TGF-beta Binding Proteins; Lung; Osteosarcoma; Phenotype; Repetitive Sequences, Amino Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Skin; Transforming Growth Factor beta; Tumor Cells, Cultured; Umbilical Veins

Bone morphogenetic protein-7 (BMP-7) affects differentiation of preosteoblasts enabling the resultant cells to respond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-mobilizing inositol 1,4,5-trisphosphate (InsP3) receptor are integral to stimulus-secretion coupling in osteoblasts, the hypothesis that BMP-7 affects InsP3 receptor expression was examined in the G-292 human osteosarcoma cell line and in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein, alkaline phosphatase, alpha1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured long term in the presence of ascorbic acid and beta-glycerophosphate, G-292 cells underwent further osteoblastic differentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with an increase in InsP3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292 cells increased InsP3 receptor density 2.4-fold with no apparent change in affinity. Immunoblot analysis with antibodies specific for type I, type II, and type III InsP3 receptors revealed that BMP-7 (50 ng/ml) treatment resulted in a specific increase (206+/-8%) in the type I receptor. Reverse transcription-polymerase chain reaction and Northern blot analyses of G-292 and primary human osteoblasts confirmed an increase in type I InsP3 receptor mRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase InsP3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca2+-mobilizing secretory hormones during osteoblast differentiation.

Topics: Alkaline Phosphatase; Ascorbic Acid; Blotting, Northern; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Proteins; Calcification, Physiologic; Calcium Channels; Cell Differentiation; Cells, Cultured; Collagen; ErbB Receptors; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Glycerophosphates; Humans; Immunoblotting; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Integrin-Binding Sialoprotein; Osteoblasts; Osteocalcin; Osteosarcoma; Phosphatidylinositols; Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Receptors, Growth Factor; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Implant surface roughness and chemical composition, as well as other factors, affect the ability of osteogenic cells to form bone adjacent to an implant. The same principles may also apply to the tooth root and some reports have shown that surface modification of the root may lead to improved restoration of the periodontal apparatus. The most common of these surface modification techniques involves demineralization with citric acid or treatment with tetracycline to expose collagen fibrils. In addition, during normal bone remodeling, osteoclasts demineralize the extracellular matrix, leaving resorption pits and exposed collagen fibrils. In this study, the effect of different dentin surface-preparation techniques on osteoblasts were compared.. Slices of sperm whale dentin were mechanically polished and surfaces were treated with tetracycline-HCl (TCN) or were cultured with mouse bone marrow cells to create a surface with osteoclast (OC) resorption pits or left untreated. Profilometry, x-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM) were used to evaluate the 3 different dentin surfaces. MG63 osteoblast-like cells were cultured on the 3 different surfaces and the effect of dentin surface preparation technique on MG63 cell proliferation (cell number), differentiaton (alkaline phosphatase specific activity of isolated cells and cell layer lysates; osteocalcin production), and local factor production (transforming growth factor (TGF)-beta1 and prostaglandin E2 (PGE2) compared.. Profilometry showed the polished and TCN surfaces were smooth with comparable Ra values, whereas the OC surfaces were slightly rougher due to resorption pits which covered 3.7% of the surface. XPS measurements showed that TCN treatment reduced the Ca and P content of the surface, indicating that it had dissolved the mineral. Osteoclast-resorption also reduced the Ca and P content, but to a lesser extent. MG63 cell proliferation on polished dentin and tissue culture polystyrene was equivalent. In contrast, cells grown on the TCN- and OC-treated surfaces exhibited increased proliferation. No effect of surface treatment on cell alkaline phosphatase activity was observed, but activity in the cell layer lysates was increased on the TCN- and OC-treated surfaces. Osteocalcin production was reduced on all dentin surfaces, but the greatest reduction was found on the TCN-treated surface. Production of both TGF-beta1 and PGE2 was increased on the treated surfaces. All effects were greatest in cultures grown on the TCN-treated dentin.. These data indicate that demineralization of the dentin surface promotes proliferation of osteoblasts and early differentiation events like production of alkaline phosphatase and autocrine mediators such as PGE2 and TGF-beta1. However, later differentiation events like osteocalcin production are decreased. Osteoclast-mediated bone resorption elicits similar responses; less than 4% of the dentin surface resulted in approximately 75% of the response caused by TCN treatment. These observations suggest that greater attention should be paid to the effects of osteoclastic resorption in designing methods for enhancing bone and cementum formation adjacent to root surfaces.

Topics: Alkaline Phosphatase; Analysis of Variance; Animals; Calcium; Cell Count; Cell Differentiation; Cell Division; Collagen; Dentin; Dinoprostone; Electron Probe Microanalysis; Extracellular Matrix; Humans; Mice; Microscopy, Electron, Scanning; Osteoblasts; Osteocalcin; Osteoclasts; Osteosarcoma; Phosphorus; Protein Synthesis Inhibitors; Tetracycline; Transforming Growth Factor beta; Tumor Cells, Cultured; Whales

Bone morphogenetic protein (BMP)-2, a member of the BMP family, plays an important role in osteoblast differentiation and bone formation. To discover small molecules that induce BMP-2, a luciferase reporter vector containing the 5'-flanking promoter region of the human BMP-2 gene was constructed and transfected into human osteosarcoma (HOS) cells. By the screening of an in-house natural product library with stably transfected HOS cells, a fungal metabolite, compactin, known as an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was isolated. The stimulation of the promoter activity by compactin seemed to be specific for BMP-2 gene in HOS cells, since it had little effect on BMP-4 or SV40 promoter activity and the stimulation was not observed in Chinese hamster ovary (CHO) cells. RT-PCR analysis and alkaline phosphatase assay revealed that compactin induced an increase in the expression of BMP-2 mRNA and protein. Like compactin, simvastatin also activated the BMP-2 promoter, whereas pravastatin did not. The statin-mediated activation of BMP-2 promoter was completely inhibited by the downstream metabolite of HMG-CoA reductase, mevalonate, indicating that the activation was a result of the inhibition of the enzyme. These results suggest that statins, if they are selectively targeted to bone, have beneficial effects in the treatment of osteoporosis or bone fracture.

Topics: Anti-Bacterial Agents; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Bone Neoplasms; Cloning, Molecular; Disulfides; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Indole Alkaloids; Lovastatin; Mevalonic Acid; Osteoporosis; Osteosarcoma; Pravastatin; Promoter Regions, Genetic; RNA, Messenger; Simvastatin; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The current studies were intended to determine whether the anabolic effects of calcitonin (CT) on human osteoblast-line cells were (1) unique to osteosarcoma cells or also evident in osteoblast-line cells derived from normal human bone; and/or (2) associated with effects on several insulin-like growth factor (IGF) system components. Preliminary studies identified several osteoblastic cell lines, derived from normal human bone, which showed calcitonindependent increases in cell proliferation, alkaline phosphatase activity, and/or (45)Ca uptake (P < 0.05-P < 0.001). Two of these cell lines-(human vertebrae) HBV-155 and HBV-163-were included with the human osteosarcoma cell line, SaOS-2, in most of our subsequent studies of calcitonin effects on selected IGF system components: IGF-II, IGF-I, and IGF binding proteins -3, -4, and -5. The results of those studies revealed that a 48 hour exposure to salmon CT caused a dose-dependent (0.03-3 mU/ml) increase in the net extracellular level of IGF-II (r = 0.96, P < 0.01) in serum-free cultures of SaOS-2 cells, with a maximal 60% increase at the highest tested dose (P < 0.02). Similar effects were seen with HBV-163 cells (r = 0.90, P < 0.01) and HBV-155 cells (r = 0.55, P < 0.02). The effect of calcitonin on the extracellular level of IGF-II was biphasic with respect to time: it decreased at 6 hours (P < 0.005 and P < 0.001, for SaOS-2 cells and HBV-163 cells, respectively) and increased at 24 hours (P < 0.02 and P < 0.05). These calcitonin-dependent increases in the extracellular level of IGF-II were associated with parallel increases in IGF-I (P < 0.005 for SaOS-2 cells and P < 0.03 for HBV-163 cells), but calcitonin did not affect the extracellular level of transforming growth factor (TGF)-beta. The calcitonin-dependent changes in IGF-II were not associated with changes in the extracellular levels of IGF binding proteins -3, -4, or -5. Finally, our studies showed that two other members of the CT superfamily-CT gene-related peptide and amylin-did not mimic the effect of CT to increase the extracellular level of IGF-II. Together, these data demonstrate that human osteoblast-line cells derived from normal human bone can respond to CT, and that those responses can include CT dose- and time-dependent increases in the extracellular levels of IGF-I and IGF-II.

Topics: Alkaline Phosphatase; Calcitonin; Calcium; Culture Media, Serum-Free; DNA; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Humans; Insulin-Like Growth Factor Binding Proteins; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Osteoblasts; Osteosarcoma; Thymidine; Transforming Growth Factor beta; Tumor Cells, Cultured

Embryonic enamel matrix proteins are hypothesized to be involved in the formation of acellular cementum during tooth development, suggesting that these proteins can be used to regenerate periodontal tissues. Enamel matrix protein derived from embryonic porcine tooth germs is used clinically, but the mechanisms by which it promotes the formation of cementum, periodontal ligament, and bone are not well understood.. This study examined the response of osteoblasts at 3 stages of osteogenic maturation to porcine fetal enamel matrix derivative (EMD). Proliferation (cell number and [3H]-thymidine incorporation), differentiation (alkaline phosphatase and osteocalcin), matrix synthesis ([35S]-sulfate incorporation; percentage of collagen production), and local factor production (prostaglandin E2 [PGE2] and transforming growth factor-beta 1 [TGF-beta1]) were measured in cultures of 2T9 cells (pre-osteoblasts which exhibit osteogenesis in response to bone morphogenetic protein-2 [BMP-2]), MG63 human osteoblast-like osteosarcoma cells, and normal human osteoblasts (NHOst cells).. EMD regulated osteoblast proliferation and differentiation, but the effects were cell-specific. In 2T9 cell cultures, EMD increased proliferation but had no effect on alkaline phosphatase-specific activity. EMD decreased proliferation of MG63 cells and increased cellular alkaline phosphatase and osteocalcin production. There was no effect on collagen synthesis, proteoglycan sulfation, or PGE2 production; however, TGF-beta1 content of the conditioned media was increased. There was a 60-fold increase in cell number in third passage NHOst cells cultured for 35 days in the presence of EMD. EMD also caused a biphasic increase in alkaline phosphatase that was maximal at day 14.. EMD affects early states of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD enhances differentiation.

Topics: Alkaline Phosphatase; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Count; Cell Differentiation; Cell Division; Cell Line; Cell Lineage; Collagen; Culture Media, Conditioned; Dental Enamel Proteins; Dinoprostone; Humans; Osteoblasts; Osteocalcin; Osteogenesis; Osteosarcoma; Radiopharmaceuticals; Sulfur Radioisotopes; Swine; Thymidine; Transforming Growth Factor beta; Tritium; Tumor Cells, Cultured

We have investigated the effect of transforming growth factor-beta1 (TGF-beta1) on the in vitro adhesion activity of the rat osteosarcoma cell lines (ROS 17/2.8) to extracellular matrix substrata, including fibronectin, type I and IV collagen, as well as laminin. The interaction of Arg-Gly-Asp (RGD) and rhodostomin, an RGD containing snake venom, with TGF-beta1 on the cell adhesion was also evaluated. The results showed that incubation with various concentration of TGF-beta1 (1-15 ng/ml) significantly increased the adhesion activity (1.4 to 2.5 folds) of ROS 17/2.8 to fibronectin and type I collagen (p<0.01), whereas the adhesion activity to laminin and type IV collagen was slightly elevated (1.1 to 1.5 folds). The peak effect of TGF-beta1 on the cell adhesion occurred after pretreatment of ROS 17/2.8 with TGF-beta1 for 6 hours. Treatment with Arg-Gly-Asp-Ser (RGDS) and rhodostomin effectively suppressed the TGF-beta1-enhanced adhesion activity to fibronectin and type I collagen. This study demonstrated that the up-regulated cell adhesion activity of ROS 17/2.8 cells by the TGF-beta1 can be inhibited by the rhodostomin.

Topics: Animals; Cell Adhesion; Extracellular Matrix; Oligopeptides; Osteosarcoma; Peptides; Rats; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Pulsed electromagnetic field stimulation has been used to promote the healing of chronic nonunions and fractures with delayed healing, but relatively little is known about its effects on osteogenic cells or the mechanisms involved. The purpose of this study was to examine the response of osteoblast-like cells to a pulsed electromagnetic field signal used clinically and to determine if the signal modulates the production of autocrine factors associated with differentiation. Confluent cultures of MG63 human osteoblast-like cells were placed between Helmholtz coils and exposed to a pulsed electromagnetic signal consisting of a burst of 20 pulses repeating at 15 Hz for 8 hours per day for 1, 2, or 4 days. Controls were cultured under identical conditions, but no signal was applied. Treated and control cultures were alternated between two comparable incubators and, therefore, between active coils; measurement of the temperature of the incubators and the culture medium indicated that application of the signal did not generate heat above the level found in the control incubator or culture medium. The pulsed electromagnetic signal caused a reduction in cell proliferation on the basis of cell number and [3H]thymidine incorporation. Cellular alkaline phosphatase-specific activity increased in the cultures exposed to the signal, with maximum effects at day 1. In contrast, enzyme activity in the cell-layer lysates, which included alkaline phosphatase-enriched extracellular matrix vesicles, continued to increase with the time of exposure to the signal. After 1 and 2 days of exposure, collagen synthesis and osteocalcin production were greater than in the control cultures. Prostaglandin E2 in the treated cultures was significantly reduced at 1 and 2 days, whereas transforming growth factor-beta1 was increased; at 4 days of treatment, however, the levels of both local factors were similar to those in the controls. The results indicate enhanced differentiation as the net effect of pulsed electromagnetic fields on osteoblasts, as evidenced by decreased proliferation and increased alkaline phosphatase-specific activity, osteocalcin synthesis, and collagen production. Pulsed electromagnetic field stimulation appears to promote the production of matrix vesicles on the basis of higher levels of alkaline phosphatase at 4 days in the cell layers than in the isolated cells, commensurate with osteogenic differentiation in response to transforming growth factor-beta1. The results i

Topics: Alkaline Phosphatase; Biomarkers; Cell Differentiation; Cell Division; Cell Line; Collagen; Dinoprostone; Electric Stimulation; Electromagnetic Fields; Extracellular Matrix Proteins; Humans; Osteoblasts; Osteocalcin; Osteosarcoma; Transforming Growth Factor beta; Tumor Cells, Cultured

The effects of recombinant human bone morphogenetic protein-2 (rhBMP-2) on cell growth were studied in three human osteosarcoma cell lines, NOS-1, HuO9, and HuO-3N1; one human prostate cancer cell line, PC-3; and one human breast cancer cell line, OCUB-1M. The growth of these cell lines was not promoted by rhBMP-2 at concentrations of 50, 100, 250, and 500 ng/ml, as evaluated by colorimetric 3 (4,5-dimethyl-thiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. Furthermore, the protein induced osteogenic differentiation, characterized by increased alkaline phosphatase activity, and increased production of type I collagen and gamma-carboxylated osteocalcin in NOS-1 cells. The results of this study may suggest the feasibility of using rhBMP-2 for the reconstruction of bone defects caused by malignant tumors, although the data are still preliminary and require further investigation.

Topics: Alkaline Phosphatase; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cell Division; Humans; Osteoblasts; Osteocalcin; Osteogenesis; Osteosarcoma; Peptide Fragments; Procollagen; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Collagenase 3 (MMP-13) is a recently identified member of the matrix metalloproteinase (MMP) gene family that is expressed at high levels in diverse human carcinomas and in articular cartilage from arthritic patients. In addition to its expression in pathological conditions, collagenase 3 has been detected in osteoblasts and hypertrophic chondrocytes during fetal ossification. In this work, we have evaluated the possibility that Cbfa1 (core binding factor 1), a transcription factor playing a major role in the expression of osteoblastic specific genes, is involved in the expression of collagenase 3 during bone formation. We have functionally characterized a Cbfa motif present in the promoter region of collagenase 3 gene and demonstrated, by cotransfection experiments and gel mobility shift assays, that this element is involved in the inducibility of the collagenase 3 promoter by Cbfa1 in osteoblastic and chondrocytic cells. Furthermore, overexpression of Cbfa1 in osteoblastic cells unable to produce collagenase 3 leads to the expression of this gene after stimulation with transforming growth factor beta. Finally, we show that mutant mice deficient in Cbfa1, lacking mature osteoblasts but containing hypertrophic chondrocytes which are also a major source of collagenase 3, do not express this protease during fetal development. These results provide in vivo evidence that collagenase 3 is a target of the transcriptional activator Cbfa1 in these cells. On the basis of these transcriptional regulation studies, together with the potent proteolytic activity of collagenase 3 on diverse collagenous and noncollagenous bone and cartilage components, we proposed that this enzyme may play a key role in the process of bone formation and remodeling.

Topics: 3T3 Cells; Animals; Blotting, Northern; Bone Development; Collagenases; Core Binding Factor Alpha 1 Subunit; Core Binding Factors; DNA Primers; DNA-Binding Proteins; Drosophila Proteins; Electrophoresis, Polyacrylamide Gel; Embryo, Mammalian; HeLa Cells; Humans; Luciferases; Matrix Metalloproteinase 13; Metalloendopeptidases; Mice; Mice, Transgenic; Models, Genetic; Multigene Family; Neoplasm Proteins; Nuclear Proteins; Osteosarcoma; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Permanent human osteosarcoma cell lines are important tools for the study of bone cancer. As representative of an osteoblastic phenotype, they partly reflect their normal osteoblastic counterparts and, thus, may represent appropriate models to investigate the mechanisms involved in bone remodelling and in haematopoietic differentiation. In the present work, we describe a new human cell line, CAL 72, obtained from an osteosarcoma of the knee of a 10-year-old boy. These cells grow in continuous culture, and karyotypic analysis has revealed clonal abnormalities in number and structure, especially loss of chromosome Y. These cells exhibit morphological, immuno-histochemical and molecular characteristics of the osteoblastic lineage. Using RT-PCR, we have shown that the CAL 72 cell line expresses high levels of mRNA coding for several cytokines, such as G-CSF, GM-CSF, IL-1beta and IL-6. In view of this expression profile, the CAL 72 phenotype appears to be closer to normal primary osteoblasts than other reported osteosarcomas. Moreover, these cells express mRNA for both HGF and its receptor c-MET, suggesting that this autocrine loop might contribute to the invasiveness of the tumour from which CAL 72 originated.

Topics: Alkaline Phosphatase; Animals; Biomarkers; Bone Neoplasms; Child; Cytokines; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hepatocyte Growth Factor; Humans; Interleukin-1; Interleukin-6; Male; Mice; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Osteoblasts; Osteocalcin; Osteopontin; Osteosarcoma; Proto-Oncogene Proteins c-met; Receptors, Parathyroid Hormone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Biomarkers, Tumor; Bone Neoplasms; Humans; Osteosarcoma; Protein Isoforms; Transforming Growth Factor beta

For the first time a non natural BMP-variant (EHBMP-2) with osteoinductive properties was produced by expression in E. coli through specific mutation of the amino acid sequence. The substitution of 12 N-terminal amino acids by a nonsense sequence results in a neglectible affinity of EHBMP-2 to the extracellular matrix. In vitro EHBMP-2 induces dose-dependent cartilage formation in neonatal muscle tissue. Single intramuscular implantation in mice results in the formation of an ossicle with functional active bone marrow. The size of the ossicle depends on the amount of implanted EHBMP-2 and can significantly be increased by the combination with a collagen carrier. The largest bone formation is observed after injection of EHBMP-2 containing collagen suspensions. In rats a stronger osteoinductive activity can be achieved by coupling of EHBMP-2 to collagen discs than by coupling natural BMP-2 to the same collagen carrier. Critical size defects in rats' mandibular angels can be restored by the combination of granular collagenous bone matrix (ICBM) with EHBMP-2. Further investigations have to show whether the altered pharmacokinetics of EHBMP-2 has advantages regarding its therapeutical use and tissue-engineering.

Topics: Amino Acid Substitution; Animals; Bone Matrix; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Chick Embryo; Collagen; Humans; Injections, Intramuscular; Mandible; Mice; Mice, Inbred ICR; Mouth Rehabilitation; Osteogenesis; Osteosarcoma; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), recombinant human transforming growth factor (rhTGF)-beta 1 and recombinant human bone morphogenetic protein (rhBMP)-2 on differentiation in four different canine osteosarcoma cell lines (POS53B, 53C, 53D and 14A) were examined using markers specifically expressed by phenotypic osteoblasts. 1,25(OH)2D3 increased alkaline phosphatase (ALP) activity in one cell line, osteocalcin production in two lines and type I collagen production in three lines. RhTGF-beta 1 increased ALP activity in one clonal cell, osteocalcin production in one clonal cell and type I collagen production in two clonal cells. RhBMP-2 increased ALP activity in all clonal cells, osteocalcin production in two clonal cells and type I collagen production in three clonal cells. Thus, these agents induced differentiation in osteosarcoma cells at different efficacies. Electron microscopic study revealed that these agents increased cellular activity in all cell lines with no evidence of degeneration of cell organelle by drug cytotoxicity. In some cultures treated with either 1,25(OH)2D3 or rhBMP-2, apoptotic cells were observed. Based on the change in markers, rhBMP-2 and 1,25(OH)2D3 seemed to be more effective than rhTGF-beta 1. These agents are potential inducers of apoptosis.

Topics: Alkaline Phosphatase; Animals; Apoptosis; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Neoplasms; Calcitriol; Calcium Channel Agonists; Cell Differentiation; Collagen; Dogs; Humans; Osteocalcin; Osteosarcoma; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Bone morphogenetic proteins, which are capable of inducing mesenchymal tissue to form bone in mammals, have been implicated as important in normal skeletal development. The expression of bone morphogenetic proteins and their receptors were studied in 36 osteosarcoma specimens, six Ewing's sarcomas, 20 synovial sarcomas, and 20 chondrosarcomas by reverse transcriptase-polymerase chain reaction, and the findings were correlated with clinical data. Bone morphogenetic protein-2, and -4 messages were detected in most sarcoma samples. Bone morphogenetic protein-6 expression was detected in 22 of 32 osteosarcomas and seven of eight chondrosarcomas. Bone morphogenetic protein-7 and receptor IB were not detected in sarcoma samples but were detected in three osteosarcoma cell lines and one malignant fibrous histiocytoma cell line. Expression of bone morphogenetic protein receptor II was found in 25 of 36 osteosarcomas, eight of 20 chondrosarcomas, four of six Ewing's sarcomas, and 15 of 20 synovial sarcoma samples. Expression of bone morphogenetic protein type II receptor was found to correlate with metastasis in osteosarcomas, which suggests that the bone morphogenetic protein pathway may participate in tumor aggressiveness or progression. The expression of bone morphogenetic protein receptor II in metastatic synovial sarcoma and dedifferentiated chondrosarcoma lesions also supports this hypothesis. The current study showed that the ligands for bone morphogenetic protein receptors, bone morphogenetic proteins-2, -4, and -6 also are expressed in osteosarcoma and other sarcoma tissues, indicating a potential for autocrine or paracrine growth stimulation in these tumors.

Topics: Adult; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein 7; Bone Morphogenetic Protein Receptors; Bone Morphogenetic Protein Receptors, Type I; Bone Morphogenetic Protein Receptors, Type II; Bone Morphogenetic Proteins; Cell Differentiation; Chondrosarcoma; Female; Gene Expression Regulation, Neoplastic; Histiocytoma, Benign Fibrous; Humans; Male; Mesoderm; Osteogenesis; Osteosarcoma; Protein Serine-Threonine Kinases; Receptors, Cell Surface; Receptors, Growth Factor; Sarcoma; Sarcoma, Ewing; Sarcoma, Synovial; Transforming Growth Factor beta; Tumor Cells, Cultured

This study aimed at clarifying the role of Aminopeptidase N (APN), a Zn2+-dependent ectopeptidase localized on the cell surface of human osteosarcoma cell lines treated with proinflammatory cytokines. We investigated the proinflammatory cytokines interleukin-1 beta (IL-1beta), IL-6 and tumor necrosis factor alpha (TNF-alpha) as well as the anti-inflammatory cytokine transforming growth factor beta (TGF-beta) for their influence on APN regulation. Soluble IL-6 receptor (sIL-6R) was always used together with IL-6 to achieve a stable effect. In addition, the invasive potential of the osteosarcoma cell lines MG63 and HOS was examined. Competitive RT-PCR and Ala-pNA activity assays revealed that IL-6 and sIL-6R significantly increased the mRNA expression and activity of APN in both osteosarcoma cell lines. Although IL-1beta significantly stimulated APN mRNA expression in both cell lines, it influenced the enzyme activity only in MG63. TNF-alpha and TGF-beta, however, had an effect neither on mRNA expression nor on the enzyme activity of APN in both cell lines. In the Matrigel invasion assay, IL-6 and sIL-6R significantly up-regulated the transmigration of these cell lines, whereas other cytokines did not. The up-regulated invasion was inhibited by bestatin, a specific inhibitor of APN. Cellular migration correlated highly with APN activity (r = 0.79, P < 0.002). These findings suggest that APN contributes to the invasive potential of human osteosarcomas enhanced by IL-6 and SIL-6R.

Topics: Bone Neoplasms; CD13 Antigens; Humans; Interleukin-1; Interleukin-6; Neoplasm Invasiveness; Osteosarcoma; Receptors, Interleukin; Receptors, Interleukin-6; Recombinant Fusion Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We cloned a genomic fragment of the 5'-flanking region of the gene encoding bone morphogenetic protein-6 (BMP-6) and assessed its promoter activity. Primer extension revealed the presence of one major transcription start site 178 bp upstream of the translation start site. The promoter region lacked a canonical TATA box but did contain a GC-rich region. A putative tramtrack responsive element, a Drosophila transcriptional factor regulating the segment polarity, was found in the promoter region. Known steroid hormonal responsive elements, however, were not found. Although BMP-6 is classified as a member of the vgr-1 family, the structure of the promoter was similar to that of BMP-2 and 4.

Topics: Base Sequence; Bone Morphogenetic Protein 6; Bone Morphogenetic Proteins; Cloning, Molecular; Fibrosarcoma; Genes, Reporter; Humans; Luciferases; Molecular Sequence Data; Osteosarcoma; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Biosynthesis; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Direct cell-cell interactions are fundamental for tissue development and differentiation. We have studied the expression and function of cadherins in human osteoblasts during in vitro differentiation. Using reverse transcription-polymerase chain reaction and mRNA hybridization, we found that human trabecular bone osteoblasts (HOBs), osteoprogenitor marrow stromal cells (BMCs), and the osteogenic sarcoma lines, SaOS-2 and MG-63, expressed mRNA for cadherin-11 (C11) and N-cadherin (N-cad). HOBs and BMCs also expressed low levels of cadherin-4 (C4) mRNA. C11 was the most abundant cadherin protein present in human osteoblasts, and its expression was unaffected by bone morphogenetic protein-2 (BMP-2) treatment of either BMCs or HOBs. Likewise, N-cad mRNA did not change during BMP-2 incubation. Conversely, C4 protein, undetectable in transformed cell lines, was down-regulated by BMP-2 treatment of normal cells. Both C11 and C4 were localized to sites of cell-cell contact in both HOBs and BMCs, colocalized with beta-catenin, and bands corresponding to cadherins were coimmunoprecipitated by a beta-catenin antibody, findings indicative of functional cadherins. A decapeptide containing the HAV motif of human N-cad partially inhibited Ca2+-dependent cell-cell adhesion and completely prevented BMP-2-induced stimulation of alkaline phosphatase activity by BMCs. Thus, human osteoblasts and their progenitor cells express a repertoire of multiple cadherins. Cadherin-mediated cell-to-cell adhesion is critical for normal human osteoblast differentiation.

Topics: beta Catenin; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cadherins; Cell Adhesion; Cell Communication; Cell Differentiation; Cell Line, Transformed; Cells, Cultured; Cytoskeletal Proteins; Humans; Osteoblasts; Osteosarcoma; Polymerase Chain Reaction; RNA, Messenger; Stem Cells; Trans-Activators; Transforming Growth Factor beta

Little is known about bone and cartilage tumors at the molecular level; thus, the identification of genes associated with these tumors may be useful as markers and therapeutic targets. To address this issue and to test the hypothesis that abnormal expression of one or more growth factors in the transforming growth factor-beta superfamily is associated with musculoskeletal neoplasia, degenerate primers based on the conserved sequences in these genes were made for screening tumor samples by reverse transcription-polymerase chain reaction. First, these primers were used to obtain a comparative profile between a low-grade chondrosarcoma and its dedifferentiated high-grade counterpart in the same patient. This experiment identified an amplified DNA product in the high-grade sample that was identical to osteogenic protein-1/bone morphogenetic protein-7. Osteogenic protein-1 mRNA expression was 17-fold greater in this high-grade sample than in the low-grade one. Osteogenic protein-1 was highly expressed (three of three) in human osteosarcoma cell lines but was not expressed (zero of four) in normal osteoblast samples. Screening for gene expression of osteogenic protein-1 in 57 osteosarcomas and chondrosarcomas indicated that 44% (range: 38-52%) of them were positive for osteogenic protein-1 mRNA. Screening of breast and prostate tumors revealed a similar association with osteogenic protein-1 mRNA expression.

Topics: Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Neoplasms; Breast Neoplasms; Chondrosarcoma; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Osteosarcoma; Prostatic Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

Even though adjuvant chemotherapy has improved the 5-year survival rate of osteosarcoma patients, a significant percentage of patients eventually die from lung metastasis. Since transforming growth faCtor-beta (TGF-beta) has been demonstrated to be related to the tumor progression, we investigated the clinical implications of the presence of TGF-beta isoforms in 16 human osteosarcoma tissue. There were 10 males and 6 females with a mean age of 20.8 years of age (range, 8 to 57 years). Biopsied specimen before chemotherapy was fixed in 10% formalin, demineralized and followed by paraffin embedding. The locations of tumor included femur (10), tibia (3), humerus (1), fibula (1), and ilium (1). Histologic subtypes included osteoblastic (11), chondroblastic (2), and fibroblastic (3). All patients were followed for a minimum of 1 year (range 12 to 44 months) or to the development of lung metastasis. Five patients (31.3%) developed subsequent lung metastasis during the follow up. We used immunohistochemistry technique to investigate the presence of the TGF-beta isoforms in osteosarcoma tissue and its relationship to the subsequent pulmonary metastasis. The results showed the presence of one or more TGF-beta isoforms in tumor cells in osteosarcoma tissues (13 of 16, 81.3%) in all of the subtypes. However, minimal presence of TGF-beta isoforms was shown in the tumor bone matrix. The expression of TGF-beta1 or TGF-beta3 isoforms was associated with a higher rate of subsequent lung metastasis (p < 0.05, chi-square test). Further research is warranted to determine the utility of routine TGF-beta analysis in the clinical practice.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Bone Neoplasms; Child; Female; Humans; Immunohistochemistry; Isomerism; Lung Neoplasms; Male; Middle Aged; Neoplasm Metastasis; Osteosarcoma; Time Factors; Tissue Distribution; Transforming Growth Factor beta

Osteoblasts produce a 100 kDa soluble form of latent transforming growth factor beta (TGF-beta) as well as a 290 kDa form containing latent TGF-beta binding protein-1 (LTBP1), which targets the latent complex to the matrix for storage. The nature of the soluble and stored forms of latent TGF-beta in chondrocytes, however, is not known. In the present study, resting zone and growth zone chondrocytes from rat costochondral cartilage were cultured to fourth passage and then examined for the presence of mRNA coding for LTBP1 protein. In addition, the matrix and media were examined for LTBP1 protein and latent TGF-beta. Northern blots, RT-PCR, and in situ hybridization showed that growth zone cells expressed higher levels of LTBP1 mRNA in vitro than resting zone cells. Immunohistochemical staining for LTBP1 revealed fine fibrillar structures around the cells and in the cell matrix. When the extracellular matrix of these cultures was digested with plasmin, LTBP1 was released, as determined by immunoprecipitation. Both active and latent TGF-beta1 were found in these digests by TGF-beta1 ELISA and Western blotting. Immunoprecipitation demonstrated that the cells also secrete LTBP1 which is not associated with latent TGF-beta, in addition to LTBP1 that is associated with the 100 kDa latent TGF-beta complex. These studies show for the first time that latent TGF-beta is present in the matrix of costochondral chondrocytes and that LTBP1 is responsible for storage of this complex in the matrix. The data suggest that chondrocytes are able to regulate both the temporal and spatial activation of latent TGF-beta, even at sites distant from the cell, in a relatively avascular environment.

Topics: Animals; Carrier Proteins; Cells, Cultured; Chondrocytes; Extracellular Matrix; Gene Expression; Growth Plate; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Osteoblasts; Osteosarcoma; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

Studies on osteosarcoma cell lines point to the potential importance of transforming growth factor beta (TGF beta) as an autocrine factor which controls the growth of human osteosarcomas. To define further the role of TGF beta isoforms in these neoplasms, a series of 27 osteosarcomas was studied using immunohistochemical, mRNA in situ hybridization, and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. All 14 central high-grade osteosarcomas, two telangiectatic osteosarcomas, and one high-grade surface osteosarcoma showed cytoplasmic immunoreactivity for TGF beta 1, -2, and -3. The expression of TGF beta 1 was moderate or diffuse in 14 cases (82.3 per cent), while low expression was detected in only three cases (17.7 per cent). For TGF beta 2 and -3, only moderate or diffuse staining was observed. Low-grade parosteal and periosteal osteosarcomas showed low or undetectable levels of TGF beta 1, while TGF beta 2 and -3 were moderately or diffusely expressed. Finally, three dedifferentiated parosteal osteosarcomas were diffusely positive for TGF beta 1, -2, and -3 in the high-grade component, while in the low-grade component, available for analysis in two of these cases, TGF beta 1 was demonstrated in a few neoplastic cells, and TGF beta 2 and -3 maintained a diffuse distribution. Statistical analysis of these data showed that high-grade osteosarcomas had a significantly higher expression of TGF beta 1 than low-grade osteosarcomas, while levels of TGF beta 2 and -3 were comparable in the two groups (p < 0.001; p = 0.3; p = 0.3, respectively; Fisher's exact test). Similarly, mRNA levels of TGF beta 1 detected by in situ hybridization were significantly higher (p = 0.04, Fisher's exact test) in high-grade osteosarcoma variants, while no differences were found for TGF beta 2 and -3 mRNA (p = 1.0; p = 0.2, respectively; Fisher's exact test). In addition, mRNA analysis performed by RT-PCR in seven cases (five high-grade and two low-grade osteosarcomas) confirmed the presence of high levels of TGF beta 1 in high-grade osteosarcomas, while low-grade tumours had low or absent mRNA expression. In conclusion, this positive association suggests that TGF beta 1 may be involved in determining the aggressive clinical behaviour of high-grade osteosarcomas.

Topics: Bone Neoplasms; Humans; Immunohistochemistry; In Situ Hybridization; Isomerism; Osteosarcoma; Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily and are crucial factors in the process of bone formation. Despite knowledge on their wide distribution and expression, however, there is very little information on the biological factors that affect gene transcription of these osteoinductive agents. To investigate this aspect of BMP gene regulation we have studied the effect of a number of factors known to affect osteogenic cells. Northern analysis showed modulation of the expression of BMP-2 and BMP-4 mRNAs in two human osteosarcoma cell lines, MG63 and Saos-2, by prostaglandin E2 (PGE2), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interferon-alpha (IFN-alpha), retinoic acid and 1,25(OH)2 vitamin D3. mRNA expressions of the normally used "housekeeping genes", glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin, were found to be susceptible to influence by some of the factors used. Hence, an oligo(dT)15-18 probe was used to reliably estimate the relative quantities of mRNA present for normalization of data. In general, all factors down-regulated mRNA expressions of BMP-2 and BMP-4 in MG63 cells. IL-6 completely abolished detectable expression of BMP-2 mRNA, which was also greatly reduced by IL-1beta, retinoic acid and 1,25(OH)2 vitamin D3. PGE2 had similar influences on BMP-2 and BMP-4 expressions, showing reductions to approximately 60% of normal. In Saos-2 cells only 1,25(OH)2 vitamin D3 had any great effect on BMP-2 expression, which was down-regulated to approximately 60% of control values. BMP-4 was down-regulated by IFN-alpha (approximately 60%) and IL-1beta (approximately 20%). We conclude that BMPs are subject to regulation by a variety of factors and that this is dependent on the stage of the cell in the osteogenic lineage. Furthermore, the use of GAPDH and beta-actin genes as "housekeeping genes" in expression-modulation studies must be treated with care.

Topics: Actins; Blotting, Northern; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cholecalciferol; Dinoprostone; Gene Expression Regulation; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Interferon-alpha; Interleukin-1; Interleukin-6; Oligonucleotide Probes; Osteosarcoma; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

Fetal rat calvarial-derived osteoblasts in vitro (ROB) reinitiate a developmental program from growth to differentiation concomitant with production of a bone tissue-like organized extracellular matrix. To identify novel genes which may mediate this sequence, we isolated total RNA from three stages of the cellular differentiation process (proliferation, extracellular matrix maturation, and mineralization), for screening gene expression by the differential mRNA display technique. Of 15 differentially displayed bands that were analyzed by Northern blot analysis, one prominent 310 nucleotide band was confirmed to be proliferation-stage specific. Northern blot analysis showed a 600-650 nt transcript which was highly expressed in proliferating cells and decreased to trace levels after confluency and throughout the differentiation process. We have designated this transcript PROM-1 (for proliferating cell marker). A full length PROM-1 cDNA of 607 bp was obtained by 5' RACE. A short open reading frame encoded a putative 37 amino acid peptide with no significant similarity to known sequences. Expression of PROM-1 in the ROS 17/2.8 osteosarcoma cell line was several fold greater than in normal diploid cells and was not downregulated when ROS 17/2.8 cells reached confluency. The relationship of PROM-1 expression to cell growth was also observed in diploid fetal rat lung fibroblasts. Hydroxyurea treatment of proliferating osteoblasts blocked PROM-1 expression; however, its expression was not cell cycle regulated. Upregulation of PROM-1 in response to TGF-beta paralleled the stimulatory effects on growth as quantitated by histone gene expression. In conclusion, PROM-1 represents a small cytoplasmic polyA containing RNA whose expression is restricted to the exponential growth period of normal diploid cells; the gene appears to be deregulated in tumor derived cell lines.

Topics: Amino Acid Sequence; Animals; Base Sequence; Biomarkers; Blotting, Northern; Calcitriol; Cell Differentiation; Cell Division; Cells, Cultured; Diploidy; DNA Primers; DNA, Complementary; Gene Expression Regulation, Developmental; Gene Expression Regulation, Neoplastic; Genetic Techniques; Hydroxyurea; Lung; Molecular Sequence Data; Osteoblasts; Osteosarcoma; Polymerase Chain Reaction; Proteins; Rats; RNA, Messenger; Sequence Analysis, DNA; Skull; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Bone morphogenetic proteins (BMPs) are novel growth and differentiation factors that act on mesenchymal stem cells to initiate new bone formation in vivo and promote the growth and differentiation of cells in the osteoblastic lineage. In the present study, we examined the effects of recombinant human osteogenic protein-1 (also known as BMP-7) on the expression of related members of the BMP family using SaOS-2 and U2-OS, two human osteosarcoma cell strains. Evaluation of BMP-2, -4, and -6 mRNA expression indicates that OP-1 stimulated the mRNA levels of BMP-6 in both SaOS-2 cells (threefold) and U2-OS cells (fivefold) after 24 hours of treatment, while decreasing the mRNA levels of BMP-4 in SaOS-2 cells (80%) and BMP-2 and BMP-4 in U2-OS cells by 50% and 72%, respectively. BMP-2 mRNA expression, as examined by Northern blot analysis, was below detectable limits in SaOS-2 cultures. These results demonstrate that OP-1 modulates the mRNA expression of related members of the BMP family, suggesting a possible mode of action of OP-1 on the growth and differentiation of cells in the osteoblastic lineage in vitro.

Topics: Blotting, Northern; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Neoplasms; Humans; Osteosarcoma; Recombinant Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Indian hedgehog (Ihh) was recently reported to be expressed in chondrocytes and to regulate chondrocyte differentiation. This report examined the expression of Ihh in osteoblastic cells and its regulation by calcitropic cytokines. We found that Ihh messenger RNA (mRNA) was expressed as a single 2.5-kilobase band at a modest level in rat osteoblastic osteosarcoma ROS17/2.8 cells. In sharp contrast to the previous observation of dpp regulation of hedgehog expression in Drosophila embryos, bone morphogenetic protein-2 did not affect Ihh expression in these cells. On the other hand, treatment with 2 ng/ml transforming growth factor-beta1 (TGFbeta1) increased the steady state level of Ihh mRNA 2- to 4-fold. Western blot analysis of the cell lysates using antisera also showed enhancement of the Ihh protein level by TGFbeta1 treatment. The effect of TGFbeta1 on Ihh mRNA abundance started within 3 h, peaked at 24 h and lasted at least 48 h after the initiation of the treatment. The effect of TGFbeta1 on the increase in Ihh mRNA was dose dependent, starting at 0.2 ng/ml and saturating at 2 ng/ml. Neither actinomycin D nor cycloheximide blocked this effect. Experiments using 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole showed an enhancement of Ihh mRNA stability by TGFbeta1, indicating the presence of posttranscriptional regulation. We then examined the effects of TGFbeta1 on Ihh mRNA in osteoblast-enriched cells isolated from neonatal rat calvariae. TGFbeta1 also enhanced Ihh mRNA expression in these cells. Our data indicate for the first time that Ihh is one of the members of the cytokines produced by osteoblastic cells and that the expression of Ihh is regulated posttranscriptionally by TGFbeta.

Topics: Animals; Blotting, Northern; Blotting, Western; Drosophila Proteins; Gene Expression Regulation; Half-Life; Hedgehog Proteins; Insect Proteins; Osteoblasts; Osteosarcoma; Rats; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Homeobox gene expression in osteoblast-like cells was investigated using the polymerase chain reaction (PCR). A total of 13 homeobox genes was detected in U-2 OS (human osteosarcoma) and MC3T3-E1 (mouse osteoblast) cells by sequencing cloned PCR products. Using specific primers, a different pattern of Hox gene expression was shown for the neuroblastoma cell line SK-N-SH relative to U-2 OS and MC3T3-E1. Additionally, we showed that expression of HOXC6 in U-2 OS and SK-N-SH was differentially regulated by rhBMP-2, TGF-beta and activin-A. This suggests that specific Hox genes may be target genes for TGF-beta superfamily members, and allows us to further understand the complex functions of these growth factors and how they relate to growth and development.

Topics: Animals; Base Sequence; Bone Neoplasms; Gene Expression; Genes, Homeobox; Humans; Mice; Molecular Sequence Data; Neuroblastoma; Osteosarcoma; Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Cells, Cultured

We analysed the glucocorticoid receptor (GR) function and its ability to modulate cell-cell interactions between the PA-III rat prostate cancer and UMR 106 osteoblast-like rat osteosarcoma cells as an in vitro model for studying GR function in PA-III cell-induced tumor and blastic reaction in rat bone. Intact GR was detected by ligand binding assays, DNA band-shift, and GR trans-activation analysis of PA-III and UMR 106 cells transiently transfected with the mouse mammary tumor virus thymidine kinase-chloramphenicol acetyltransferase reporter gene. Dexamethasone and transforming growth factor beta 1 (TGFbeta1) inhibited the growth of PA-III and UMR 106 cells. Dexamethasone's inhibition of PA-III and UMR 106 cells was reversed by anti-TGFbeta1 antibody and exogenous insulin-like growth factor I (IGF-I). Exogenous IGF-I, urokinase-type plasminogen activator (uPA), UMR 106 conditioned media (CM) and PA-III CM stimulated the proliferation of PA-III and UMR 106 cells. The mitogenic activity exerted by uPA and PA-III CM in UMR 106 cells was completely neutralized by anti-IGF-I specific antibody. In addition, dexamethasone up-regulated TGFbeta1 mRNA and down-regulated uPA mRNA expression in PA-III cells without affecting TGFbeta1 and uPA mRNA expression in UMR 106 cells. These data suggested that TGFbeta1, uPA, and IGF-I mediate at least in part cell-cell interactions and GR function in PA-III prostate cancer and UMR 106 osteosarcoma cells.

Topics: Animals; Bone Neoplasms; Cell Communication; Cell Division; Dexamethasone; DNA; Humans; Insulin-Like Growth Factor I; Male; Osteosarcoma; Prostatic Neoplasms; Rats; Receptors, Glucocorticoid; RNA, Messenger; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Transforming growth factor-beta1 (TGF-beta1) induces increased extracellular matrix deposition. Bone morphogenetic protein-1 (BMP-1) also plays key roles in regulating vertebrate matrix deposition; it is the procollagen C-proteinase (PCP) that processes procollagen types I-III, and it may also mediate biosynthetic processing of lysyl oxidase and laminin 5. Here we show that BMP-1 is itself up-regulated by TGF-beta1 and that secreted BMP-1, induced by TGF-beta1, is either processed to an active form or remains as unprocessed proenzyme, in a cell type-dependent manner. In MG-63 osteosacrcoma cells, TGF-beta1 elevated levels of BMP-1 mRNA approximately 7-fold and elevated levels of mRNA for mammalian tolloid (mTld), an alternatively spliced product of the BMP1 gene, to a lesser extent. Induction of RNA was dose- and time-dependent and cycloheximide-inhibitable. Secreted BMP-1 and mTld, induced by TGF-beta1 in MG-63 and other fibrogenic cell cultures, were predominantly in forms in which proregions had been removed to yield activated enzyme. TGF-beta1 treatment also induced procollagen N-proteinase activity in fibrogenic cultures, while expression of the procollagen C-proteinase enhancer (PCPE), a glycoprotein that stimulates PCP activity, was unaffected. In contrast to fibrogenic cells, keratinocytes lacked detectable PCPE under any culture conditions and were induced by TGF-beta1 to secrete BMP-1 and mTld predominantly as unprocessed proenzymes.

Topics: Amino Acid Sequence; Ascorbic Acid; Bone Morphogenetic Protein 1; Bone Morphogenetic Proteins; Enhancer Elements, Genetic; Humans; Keratinocytes; Metalloendopeptidases; Metalloproteases; Molecular Sequence Data; Osteosarcoma; Procollagen N-Endopeptidase; Proteins; RNA, Messenger; Tolloid-Like Metalloproteinases; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

We previously found that TGF-beta1 inhibits PDGF mitogenicity in MG-63 cells and the inhibition is correlated with a suppression of the PDGF-induced receptor autophosphorylation. In this study, we analyze if all the PDGF receptor signaling pathways are similarly affected by the TGF-beta1 pretreatment. We show that TGF-beta1 suppresses PDGF-stimulated tyrosine phosphorylation of PLC-gamma1, and the phosphorylation of Erk. In contrast, the tyrosine phosphorylation of PI3-kinase is not affected. Thus, TGF-beta1 selectively suppresses two out of three PDGF receptor signaling pathways despite of its prominent inhibition of the PDGF-induced receptor autophosphorylation. The results also indicate that activation of the PI3-kinase pathway alone by PDGF is not sufficient in supporting the optimum growth of MG-63 cells under the culture conditions employed.

Topics: Humans; Osteosarcoma; Phosphorylation; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Recently we found that primary human osteoblast-like cells (HOBs) support hematopoietic progenitor cells (assayed by colony formation in methylcellulose) and long-term culture initiating (LTC-IC) activity in vitro. In the present investigation, we evaluate whether human osteosarcoma cells share in these activities. We observed that relative to controls, significantly fewer hematopoietic colonies were formed in the presence of HOS TE85, MG-63, SaOS-2, or U2-OS human osteosarcomas. In addition, neither MG-63 or SaOS-2 cells supported hematopoietic progenitor cell activity or LTC-IC activity in vitro. We established that the suppressive activity produced by the osteosarcomas is soluble, correlated with osteosarcoma cell number and is partially neutralized with antibody to TGF-beta 1,2,3. While it is clear that the osteosarcomas express several phenotypic characteristics of primary human osteoblasts, these data suggest that they may be functionally disregulated with regard to their ability to support normal hematopoiesis. For these reasons, caution should be exercised when evaluating osteoblastic and hematopoietic cell interactions based purely on the use of osteosarcoma cell lines.

Topics: Antibodies; Antigens, CD34; Bone Neoplasms; Cell Count; Colony-Forming Units Assay; Hematopoietic Stem Cells; Humans; Hydrogen-Ion Concentration; Leukotriene C4; Osteosarcoma; Transforming Growth Factor beta; Tumor Cells, Cultured

To begin delineating molecular mechanisms by which osteogenic protein-1 (OP-1) modulates its effect on the insulin-like growth factor (IGF) system in human skeletal cells, we evaluated time-course effects of OP-1 on the expression of IGFBP-3 messenger RNA (mRNA) in human SaOS-2 osteosarcoma cells and found that 100 ng/ml of OP-1 increased (maximum 10.7-fold at 24 h; P < 0.01) the level of IGFBP-3 mRNA in a time-dependent manner (from 3-36 h; treatment x time interaction, P < 0.001). The stimulatory effect of OP-1 on IGFBP-3 mRNA was not promoted by transcript stabilization; actually, OP-1 treatment selectively increased the decay of mRNA for IGFBP-3 (T1/2 = 5 h vs. 24 h for OP-1 and controls), but not for IGFBP-4 or beta-actin. Conversely, OP-1 acutely increased IGFBP-3 nuclear transcript abundance in total RNA samples ranging between 1-24 h of treatment. After 6 h of treatment, OP-1 produced an average 4-fold increase (P < 0.02; n = 4 experiments) in the level of IGFBP-3 nuclear transcripts vs. a 3-fold increase (P < 0.01; n = 2 experiments) in mRNA abundance. The OP-1 stimulated induction of IGFBP-3 nuclear transcript and mRNA expression was dependent on de novo protein synthesis. Transient transfection experiments were undertaken to isolate putative OP-1 stimulatory cis-elements within 1.8-kb of the IGFBP-3 5'-flanking region in SaOS-2 and TE-85 osteosarcoma cells. In these experiments, OP-1 did not stimulate IGFBP-3 proximal promoter activity in either cell line, thus suggesting that OP-1 reactive domains may be located either beyond the currently established 5'-flanking region, or within internal exon/intron regions of the IGFBP-3 gene. In conclusion, OP-1 treatment stimulates IGFBP-3 expression in human osteoblastic cells by a mechanism that largely promotes the production of IGFBP-3 nuclear transcripts, a process that requires de novo protein synthesis, and overrides an OP-1-induced targeted degradation of IGFBP-3 steady-state mRNA.

Topics: Actins; Base Sequence; Blotting, Northern; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Neoplasms; DNA Primers; DNA, Neoplasm; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor Binding Protein 4; Introns; Osteosarcoma; Polymerase Chain Reaction; Promoter Regions, Genetic; RNA, Messenger; Time Factors; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) is a multipotent growth factor affecting development, homeostasis, and tissue repair. In addition, increased expression of TGF-beta has been reported in different malignancies, suggesting a role for this growth factor in tumorigenesis.. Using immunohistochemistry, the expression, prevalence, and distribution of TGF-beta isoforms were evaluated in 25 high grade human osteosarcomas. The Cox proportional hazards models and Kaplan-Meier curves were calculated correlating disease free survival with TGF-beta expression.. Expression of one or more TGF-beta isoforms was found in all the osteosarcomas. Immunoreactivity for TGF-beta1 and TGF-beta3 generally was stronger than for TGF-beta2. The cytoplasm of the tumor cells showed stronger staining than their surrounding extracellular stroma. Most notably, osteoclasts showed strong to intense staining for all three isoforms. In 11 of 25 specimens angiogenic activity was noted with staining of multiple small vessels in the tumor stroma. Expression of TGF-beta3, but not of TGF-beta2 or TGF-beta1, related to disease progression, such that there was a statistically significant decrease in the disease free interval as the immunoreactivity for TGF-beta3 increased.. All osteosarcomas expressed TGF-beta in the cytoplasm of the tumor cells as well as in their extracellular stroma. The presence of TGF-beta in the endothelial and perivascular layers of small vessels in the tumor stroma suggests angiogenic activity of this growth factor. The expression of TGF-beta3 was correlated strongly with disease progression (P = 0.027). These data suggest that increased expression of TGF-beta isoforms, especially TGF-beta3, may play a role in osteosarcoma progression.

Topics: Adolescent; Adult; Aged; Biomarkers, Tumor; Bone Neoplasms; Child; Disease Progression; Disease-Free Survival; Female; Humans; Immunoenzyme Techniques; Male; Middle Aged; Osteosarcoma; Proportional Hazards Models; Survival Analysis; Transforming Growth Factor beta

Prostatic carcinoma cells have a propensity to metastasize to bone, and we propose that this phenomenon may be promoted by the adhesion of metastatic cells to bone matrix. Bone matrix is produced by osteoblasts, and we have developed an in vitro model of bone matrix by isolating the substratum deposited by human osteoblast-like U2OS cells. The collagenous nature of this matrix was demonstrated by the incorporation of [3H]proline and its subsequent release by purified collagenase. Both U2OS matrix and purified type I collagen stimulated the adhesion of human PC-3 prostatic carcinoma cells. Human laminin supported adhesion to a much lesser extent, and PC-3 cells did not adhere to fibronectin. Adhesion of PC-3 cells to U2OS matrix closely resembled adhesion to purified type I collagen with respect to (a) inhibition by a collagen-derived peptide and by antibodies raised against alpha 2 or beta 1 integrin collagen receptor subunits; (b) lack of inhibition by RGD (Arg-Gly-Asp) peptides; (c) stimulation by Mn2+ and Mg2+ ions but not by Ca2+ ion; and (d) stimulation by the phorbol ester PMA (phorbol 12-myristate 13-acetate). This adhesion was also stimulated (2.3-fold) by transforming growth factor beta (TGF-beta), which is a major bone-derived growth factor. We conclude that human osteoblast-like matrix is an adhesive substrate for PC-3 prostate carcinoma cells. This adhesion appears to be mediated by the interaction of alpha 2 beta 1 integrin on PC-3 cells with matrix-derived collagen. The stimulation of this adhesion by TGF-beta suggests that the co-expression of TGF-beta and type I collagen in bone may synergistically facilitate the adhesion of metastatic cells to bone matrix proteins and thereby increase their localization in the skeleton.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Bone and Bones; Carcinoma; Cell Adhesion; Cells, Cultured; Collagen; Extracellular Matrix; Humans; Integrin beta1; Male; Molecular Sequence Data; Osteoblasts; Osteosarcoma; Peptides; Phorbol Esters; Proline; Prostatic Neoplasms; Transforming Growth Factor beta; Tumor Cells, Cultured

Osteocalcin (OC), a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix, is down-regulated upon treatment with transforming growth factor (TGF)-beta 1. To address the potential role of OC gene expression in relation to TGF-beta 1 regulation of bone formation and resorption, we examined the transcriptional activity of the rat OC promoter after TGF-beta 1 treatment. 5' deletion analysis of rat OC promoter-chloramphenicol acetyltransferase constructs demonstrated that TGF-beta 1 treatment repressed chloramphenicol acetyltransferase activity by 2.4-fold in transient transfections of ROS 17/2.8 cells. A 29-bp region between -162 and -134 identified as the TGF-beta 1 response domain, conferred TGF-beta 1 responsiveness to the -108 to +24 rat OC basal promoter in an orientation dependent manner. Mutation of an activator protein-1/cAMP-response element-like motif (- 146 to -139) abolished TGF-beta 1 responsiveness of the construct. In vitro gel-mobility shift and competition assays using wild-type and mutated oligonucleotides and antibodies indicate that Fra-2, a Fos related transcription factor, binds to this motif. We show that Fra-2 is an activator of the OC promoter, and TGF-beta 1 inhibits this activation. Our results demonstrate that Fra-2 is hyperphosphorylated upon TGF-beta 1 treatment of ROS 17/2.8 cells. Additionally, treatment of cells with a staurosporine protein kinase C inhibitor abrogates TGF-beta 1 mediated down-regulation of the OC promoter activity. Together, these results demonstrate that TGF-beta 1 responsiveness of the rat osteocalcin gene in ROS 17/2.8 cells is mediated through an activator protein-1 like cis-acting element that interacts with Fra-2. Furthermore, our results are consistent with a critical role for TGF-beta 1 induced phosphorylation of Fra-2 in the repression of OC gene transcription.

Topics: Animals; Base Sequence; Binding Sites; DNA-Binding Proteins; Fos-Related Antigen-2; Gene Expression Regulation; Methylation; Molecular Sequence Data; Mutagenesis; Osteocalcin; Osteosarcoma; Phosphorylation; Promoter Regions, Genetic; Rats; Transcription Factor AP-1; Transcription Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The transforming growth factor beta (TGF-beta) family of growth factors control proliferation, extracellular matrix synthesis and/ or differentiation in a wide variety of cells. However, the molecular mechanisms governing ligand binding, receptor oligomerization and signal transduction remain incompletely understood. In this study, we utilized a set of antibodies selective for the extracellular and intracellular domains of the TGF-beta type-II receptor as probes to investigate the intrinsic kinase activity of this receptor and its physical association in multimeric complexes with type-I and type-III receptors. The type-II receptor immuno-precipitated from human osteosarcoma cells exhibited autophosphorylation and casein kinase activity that was markedly stimulated by polylysine yet was insensitive to heparin. Affinity cross-linking of 125I-TGF-beta 1 ligand to cellular receptors followed by specific immunoprecipitation demonstrated that type-II receptors form stable complexes with both type-I and type-III receptors expressed on the surfaces of both human osteosarcoma cells and rabbit chondrocytes. Pretreatment of the cultured cells with an antibody directed against a distinct extracellular segment of the type-II receptor (anti-TGF-beta-IIR-NT) effectively blocked the 125I-TGF-beta labelling of type-I receptors without preventing the affinity labelling of type-II or type-III receptors, indicating a selective disruption of the type-I/type-II hetero-oligomers. The anti-TGF-beta-IIR-NT antibodies also blocked the TGF-beta-dependent induction of the plasminogen activator inhibitor (PAI-1) promoter observed in mink lung epithelial cells. However, the same anti-TGF-beta-IIR-NT antibodies did not prevent the characteristic inhibition of cellular proliferation by TGF-beta 1, as determined by [3H]thymidine incorporation into DNA. The selective perturbation of PAI-1 promoter induction versus cell-cycle-negative regulation suggests that strategic disruption of TGF-beta type-I and -II receptor interactions can effectively alter specific cellular responses to TGF-beta signalling.

Topics: Amino Acid Sequence; Animals; Antibodies; Antibody Specificity; Cartilage, Articular; Casein Kinases; Cell Differentiation; Cell Line; Cells, Cultured; Cross-Linking Reagents; DNA; DNA Replication; Humans; Luciferases; Lung; Mink; Molecular Sequence Data; Osteosarcoma; Peptide Fragments; Phosphorylation; Promoter Regions, Genetic; Protein Kinases; Protein Serine-Threonine Kinases; Rabbits; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Signal Transduction; Succinimides; Thymidine; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

In this study, a somatic cell genetic approach was used to study the regulation of liver/bone/kidney alkaline phosphatase (ALPL) gene expression in osteoblasts. ALPL plays an important role in skeletal mineralization and serves as a good index of bone formation. A series of intertypic hybrids constructed by fusion of the human osteosarcoma TE-85 with the mouse fibrosarcoma La-t- demonstrated a 10-fold reduction of ALPL steady-state mRNA and enzyme activity, a phenomenon termed extinction. Hybrid subclones which reexpressed ALPL contained reduced numbers of fibroblast chromosomes compared to earlier passages. This suggests that a trans-acting negative regulatory factor expressed from the fibroblast genome regulates ALPL expression. Two factors known to influence ALPL expression are 1,25-dihydroxyvitamin D3 (1,25D3) and transforming growth factor-beta1 (TGFbeta1). 1,25D3 is involved in mobilizing bone calcium stores and TGFbeta1 plays a critical role in bone remodeling. The extinguished hybrids were exposed to 1,25D3, TGFbeta1, and a combination of these factors. For two hybrids, the combination induced reexpression of ALPL activity to levels comparable to the TE-85 parent, indicating a competition between the factors and the extinguisher(s). Neither factor alone could induce ALPL reexpression to the levels observed with the combination. In only one hybrid, the combination of factors synergistically increased ALPL expression. These data help define the cis sequence element(s) in the ALPL promoter which are involved in the negative regulation of this gene.

Topics: Alkaline Phosphatase; Animals; Base Sequence; Blotting, Northern; Bone and Bones; Calcitriol; Drug Synergism; Fibrosarcoma; Gene Expression Regulation, Enzymologic; Humans; Hybrid Cells; Kidney; Liver; Mice; Molecular Sequence Data; Osteosarcoma; Phenotype; Promoter Regions, Genetic; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Implants of defatted, freeze-dried Saos-2 human osteosarcoma cells grown to confluency induce de novo bone formation in athymic mice. These cells are also richly endowed with bone morphogenetic proteins and express mRNA for bone morphogenetic proteins 1, 2, 3, 4 and 6, as well as for transforming growth factor-beta 1. Our aim was to study whether the ability to induce bone formation is related to the level of expression of bone morphogenetic protein. We studied the osteoinductive abilities and levels of expression of bone morphogenetic protein of Saos-2 cells both during the growth phase and after confluency was reached. Subconfluent cells were at least 70% less effective in their osteoinductive ability than confluent cells. Comparison of bone morphogenetic protein mRNA expression in confluent and subconfluent cells revealed that the latter had lower expression of all the mRNAs studied. The expression of bone morphogenetic protein-1, bone morphogenetic protein-2, and bone morphogenetic protein-6 mRNAs was 2, 3, and 6 to 10-fold lower, respectively, in subconfluent cells. These results suggest that the ability of Saos-2 cells to induce de novo bone formation may be correlated with the relative expression of these proteins; the expression of bone morphogenetic proteins in Saos-2 cells also may be dependent on the cell cycle.

Topics: Animals; Biological Assay; Blotting, Northern; Bone and Bones; Bone Morphogenetic Proteins; Cell Count; Cell Cycle; Gene Expression; Humans; Mice; Mice, Nude; Osteosarcoma; Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Freeze-dried Saos-2, human osteosarcoma cells, and extracts of Saos-2 cells contain all components necessary to induce ectopic new bone and marrow when implanted into athymic Nu/Nu nuce. On the other hand, human osteosarcoma cells of the U-2 OS strain failed to induce bone formation under the same experimental conditions. Our aim was to compare the relative expressions of known osteoinductive factors including bone morphogenetic proteins (BMPs) and transforming growth factor-beta (TGF-beta) in these two cell lines in an attempt to explain the unique bone-inducing ability of the Saos-2 cells. Saos-2 cells expressed mRNA for BMP-1, -2, -3, -4,-6, and TGF-beta 1. The non-osteoinductive U-2 OS cells expressed BMP-2, -4, -5, -6, and -7 as well as TGF-beta 1 mRNA, while levels of BMP-1 and BMP-3 mRNA were either not detectable or detectable at a very low level in U-2 OS cells. The presence of BMP-1 and -4 protein was confirmed in Saos-2 cells by immunofluorescence, and TGF beta protein was demonstrated by bioassay in both cell types. These findings suggest that Saos-2 cells are endowed qualitatively and quantitatively with sufficient amounts of many bone morphogenetic proteins-especially BMP-1, -3, and -4-to confer osteoinductivity upon these cells. However, the absence of osteoinductivity in U-2 OS cells, despite significant mRNA expression levels of several bone morphogenetic proteins, suggests that, even though expression of one or more bone morphogenetic proteins may be present, it may not necessarily be sufficient to confer osteoinductivity upon U-2 OS cells. U-2 OS cells may be non-osteoinductive because (1) they contain inhibitors to the BMPs or secrete inhibitory binding proteins, (2) they do not process BMPs correctly, or (3) the BMPs are inappropriately localized and sequestered within the U-2 OS cells. Saos 2 cells may be osteoinductive because (1) they uniquely express BMP-1, (2) they express an appropriate combination of interactive BMPs at appropriate levels, and/or (3) the Saos-2 cells elaborate as-yet-unidentified osteoinductive factor(s).

Topics: Animals; Biological Assay; Blotting, Northern; Blotting, Western; Bone Morphogenetic Proteins; Bone Neoplasms; Fluorescent Antibody Technique; Gene Expression Regulation; Humans; Mice; Mice, Nude; Ossification, Heterotopic; Osteogenesis; Osteosarcoma; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Two human osteosarcoma cell lines, Hu09 and OST, were suspended in Matrigel (Becton Dickinson Labware, Bedford, Massachusetts) and implanted subcutaneously in the backs of nude mice. To study phenotypic changes of tumor cells and host cells, expression of mRNA for osteopontin (OPN), osteocalcin (OC), and osteonectin (ON) was analyzed by in situ hybridization. Bone tissue was formed in the tumors derived from Hu09 cells. OPN mRNA was transcribed predominantly in osteocyte-like cells within the bone, whereas OC mRNA was transcribed in osteoblast-like cells that surrounded the bone. ON mRNA was detected in both types of cells. The similarity of the expression pattern of OPN, OC, and ON during osteogenesis of Hu09 cells to that of normal skeletal development suggests that the bone formed in Hu09-implanted mice is the same as normal bone tissue. By DNA-DNA in situ hybridization using a human-specific Alu probe and a mouse-specific m-L1 probe, osteoblast-like cells in Hu09 tumorous bone were, however, of human origin, whereas osteocyte-like cells were of mouse origin. In the tumors derived from OST cells, no osteogenesis was observed during the experimental period, and the expression of OPN, OC, and ON was not detected in tumor cells. An endochondral bone formation was not evident when these cells were simply implanted into muscle tissue. An endochondral bone was, however, reactively induced in the host mUscle tissue either when 1 alpha-hydroxyvitamin D3 and all-transretinoic acid were administered to OST-implanted mice or when Hu09 cells were pretreated with dexamethasone before implantation. Hu09 implantation seems to be a useful tool not only for the study of the differentiation of osteosarcoma cells but also for the investigation of the mechanism of bone formation. This system, using Hu09 and OST, may provide us with a new tool for the isolation of the unidentified factors that induce or inhibit osteogenesis in vivo.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cell Adhesion; Cell Transplantation; Cells, Cultured; Cytokines; Dexamethasone; Humans; Hydroxycholecalciferols; In Situ Hybridization; Mice; Mice, Nude; Neoplasm Transplantation; Osteoblasts; Osteocalcin; Osteocytes; Osteogenesis; Osteonectin; Osteopontin; Osteosarcoma; Phosphoproteins; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Transplantation, Heterologous; Tretinoin

Fifteen archival human osteosarcoma specimens were examined by in situ hybridization for the expression of human and mouse transforming growth factor-beta (isoforms 1, 2, and 3), c-fos, and metalloproteinase (stromelysin-3 and matrilysin). Osteosarcoma subtypes were confirmed by review of patients' radiographs, histopathology, and age at diagnosis. The outcome and method of treatment were documented. The subtypes of osteosarcoma consisted of nine conventional osteosarcomas and two each of fibroblastic, telangiectatic, and post-radiation osteosarcomas. Each specimen was histologically examined under light microscopy, and then adjacent paraffin sections were assayed with sense and anti-sense RNA probes by in situ hybridization. The probes localized to the neoplastic cells, confirming the methodology of the technique. Human transforming growth factor-beta 1 had the most uniform binding affinity to the osteosarcomas examined and was more specific in binding than mouse transforming growth factor-beta 1. Specific mRNA encoding for the transforming growth factor-beta s, c-fos, and metalloproteinases are detectable in patterns within osteosarcoma cells, and collectively, their expression parallels the different histopathologic subtypes. The less differentiated subtypes (telangiectatic and post-radiation osteosarcomas) expressed the fewest molecular markers. Osteosarcoma is a heterogeneous tumor. Differential expression of matrilysin in osteosarcoma is the first reported detection of metalloproteinase activity in human skeletal sarcoma.

Topics: Adult; Animals; Bone Neoplasms; Child; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization; Matrix Metalloproteinase 3; Matrix Metalloproteinase 7; Metalloendopeptidases; Mice; Neoplasm Proteins; Neoplasms, Radiation-Induced; Neoplasms, Second Primary; Osteosarcoma; Proto-Oncogene Proteins c-fos; RNA Probes; RNA, Neoplasm; Transforming Growth Factor beta

The contraction of floating collagen gels is suggested to mimic the reorganization of collagenous matrix during development and tissue healing. Here, we have studied two osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed subclone of HOS cells, HOS-MNNG. Transforming growth factor-beta (TGF-beta), a putative regulator of bone fracture healing, increased collagen gel contraction by MG-63 and HOS-MNNG, but not by HOS cells. Our data show that TGF-beta-induced fibronectin synthesis is not sufficient for the process. Instead, anti-beta 1 integrin antibodies could prevent the contraction. There are three different integrin heterodimers that are known to mediate the cell-collagen interaction, namely alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1. In MG-63 cells TGF-beta increased the expression of alpha 2 beta 1 integrin and decreased the expression of alpha 3 beta 1 integrin, whereas alpha 1 beta 1 integrin is not expressed. HOS cells had no alpha 2 beta 1 integrin, neither did TGF-beta induce its expression. However, HOS-MNNG cells expressed more alpha 2 beta 1 integrin when treated with TGF-beta. Thus, we suggest that the mechanism of the enhanced collagen gel contraction by TGF-beta is the increased expression of alpha 2 beta 1 integrin heterodimer. To further test this hypothesis, we expressed a full-length alpha 2 integrin cDNA in HOS cells and in MG-63 cells. We obtained HOS cell clones that expressed alpha 2 beta 1 heterodimer, and the ability of these cells to contract collagen gels was greatly enhanced. Furthermore, the contraction by MG-63 cells transfected with alpha 2 integrin cDNA was enhanced, and the contraction by cells transfected with antisense oriented alpha 2 integrin cDNA was decreased. Thus, both in MG-63 and HOS cells the increased alpha 2 integrin expression alone was sufficient for the enhanced contraction of collagen gels. Furthermore, the amount of alpha 2 integrin is critical for the process, and its decrease leads to diminished ability to contract gels.

Topics: Antigens, CD; Collagen; DNA, Antisense; Fibronectins; Humans; Integrin beta1; Integrins; Methylnitronitrosoguanidine; Osteogenesis; Osteosarcoma; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Insulin-like growth factors (IGFs) are found in human circulation predominantly as part of a growth hormone (GH)-dependent complex of 125-150 kD, which is composed of three subunits: IGF-I or IGF-II, an acid stable IGF binding protein (IGFBP)-3, and an acid labile subunit (ALS). Although recent studies demonstrate that a number of cell types in culture secrete IGFs and IGFBP-3, very little is known with regard to the origin of circulating ALS. To test the hypothesis that human bone cells (HBCs), which produce abundant amounts of IGF-II and IGFBP-3, also produce ALS, we measured the IGF-I, IGF-II, IGFBP-3, and ALS levels using specific radioimmunoassays (RIAs) in the conditioned medium (CM) of untransformed normal HBCs and SaOS-2 osteosarcoma cells treated with various effectors (IGF-II, osteogenic protein-1 [OP-1, bone morphogenetic protein-7] and human GH) for 48 h. No detectable levels (< 3 ng/ml) of ALS were found in the CM of various HBC types under basal conditions. In contrast, CM collected from liver explants in culture contained significant amount of ALS prepared and assayed under identical conditions. The IGF-I level was also undetectable in the CM of various HBC types. In the IGF-II (3, 30 ng/ml)-treated HBC CM, the IGFBP-3 level was increased in a dose-dependent manner but neither IGF-I nor ALS could be detected. In the SaOS-2 cell culture, OP-1 (1, 100 ng/ml) increased both IGF-II and IGFBP-3 secretion but neither ALS nor IGF-I secretion. Treatment of HBCs with GH (1, 10, 100 ng/ml) had no significant effect on the secretion of either IGF-I, IGF-II, IGFBP-3, or ALS.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Bone and Bones; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Neoplasms; Carrier Proteins; Cells, Cultured; Culture Media, Conditioned; Culture Media, Serum-Free; Glycoproteins; Growth Hormone; Growth Inhibitors; Humans; Infant; Insulin-Like Growth Factor Binding Protein 3; Insulin-Like Growth Factor II; Liver; Molecular Weight; Osteoblasts; Osteosarcoma; Proteins; Radioimmunoassay; Recombinant Proteins; Ribs; Skull; Somatomedins; Transforming Growth Factor beta; Tumor Cells, Cultured

The representation of cyclins and cyclin-dependent kinases (cdks) was analyzed during progressive development of the bone cell phenotype in cultures of normal diploid rat calvarial osteoblasts. Three developmental stages were examined: (a) proliferation; (b) monolayer confluency; and (c) mineralization of the bone extracellular matrix. We demonstrate that the presence of cyclins and cdks is not restricted to the proliferation period. Consistent with their role in cell cycle progression, cdc2 and cdk2 decrease postproliferatively. However, cdk4 and cyclins A, B, and D1 persist in confluent cells. Cyclin E is significantly up-regulated during the extracellular matrix mineralization developmental period. Examination of the cytoplasmic levels of these cell cycle regulatory proteins indicates a marked increase in cyclin B in the late differentiation stage. The elevation of nuclear cyclin E and cytoplasmic cyclin B is not observed in osteoblasts maintained under culture conditions that do not support differentiation. Furthermore, treatment with transforming growth factor beta for 48 h during the proliferation period renders the cells incompetent for differentiation and abrogates the postproliferative up-regulation of cyclins B and E. Density-induced growth inhibition of ROS 17/2.8 osteosarcoma cells is not accompanied by up-regulation of nuclear cyclin E and cytoplasmic cyclin B when compared to the proliferation period. This observation is consistent with abrogation of both growth control and differentiation regulatory mechanisms in tumor cells. These results suggest that cell cycle regulatory proteins function not only during proliferation but may also play a role in normal diploid osteoblast differentiation.

Topics: Animals; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cell Nucleus; Cells, Cultured; Cyclins; Cytoplasm; Osteoblasts; Osteosarcoma; Rats; Subcellular Fractions; Transforming Growth Factor beta; Up-Regulation

This report describes skeletal site-related differences in human osteoblastic cell metabolism in studies of four patients. Northern analyses of the constitutive growth factor messenger ribonucleic acid (mRNA) expression pattern in mandibular and iliac crest-derived human osteoblastic cells (based on within-patient comparisons) revealed higher mRNA levels for strong mitogenic growth factors such as basic fibroblast growth factor (bFGF) and insulin-like growth factor II (IGF-II) in the rapidly proliferating and less alkaline phosphatase (ALP)-expressing mandibular osteoblastic cells compared to those in the lower bFGF and IGF-II mRNA levels in slowly proliferating iliac human osteoblastic cells exhibiting a higher ALP expression level. In contrast, transforming growth factor-beta (TGF beta) mRNA was more abundant in iliac human osteoblastic cells than in mandibular osteoblastic cells. Furthermore, we found that there was a proportionality, based on data from both sites, between the level of constitutive TGF beta mRNA and the response to exogenously administered bFGF or IGF-II. A comparable pattern of growth characteristics and mRNA expression was also observed in transformed human osteoblastic cells that had been subcloned in sublines expressing high and low levels of the human osteoblastic differentiation marker ALP. These findings are consistent with 1) skeletal site-related differences in human bone cell phenotypes, and 2) decreased IGF-II and bFGF expression and increased TGF beta expression and responsiveness to bFGF and IGF-II in human bone cells exhibiting a high ALP expression.

Topics: Alkaline Phosphatase; Blotting, Northern; Bone and Bones; Cell Division; Cell Line; Cells, Cultured; DNA; Fibroblast Growth Factor 2; Fibroblasts; Gene Expression; Growth Substances; Humans; Ilium; Insulin-Like Growth Factor II; Kinetics; Mandible; Osteoblasts; Osteocalcin; Osteosarcoma; RNA, Messenger; Skin; Thymidine; Transforming Growth Factor beta; Tumor Cells, Cultured

Although 17 beta-estradiol (E2) replacement therapy has been shown to be effective in treating postmenopausal osteoporosis, the underlying mechanism remains unclear. The presence of low levels of functional endogenous estrogen receptor (ER) in some osteoblastic cells has been demonstrated, and the suggestion that the abundance of ER may be rate-limiting in the action of E2 on these cells has been made. To study the mechanism of ER in regard to E2-mediated effects, we stably transfected a human osteosarcoma cell line, SaOS-2, with an expression vector, pMV-7-ER, containing the human ER gene. We characterized six of the stably transfected clones. One of the stable clones, SaOS-2-ER, expressed extra copies of ER genes integrated into the genome as detected by Southern blot analysis, showed a significantly increased level of ER mRNA by RT-PCR, and contained an increased level of ER cytosolic protein as detected by an ER-specific EIA. The overexpressed ER was functional and sensitive to E2 in a dose-dependent fashion after transient transfection with a vector containing an estrogen response element (ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene. Scatchard analysis revealed a single high-affinity binding site with a Kd similar to values obtained for the ER in MCF-7 breast cancer cells. These SaOS-2-ER cells had altered osteoblast phenotypic features including growth inhibition, decreased basal alkaline phosphatase activity, and decreased IL-6 expression and secretion. In response to E2, a greater than 2-fold increase in TGF-beta 1 mRNA was quantitatively measured in these ER-overexpressing osteoblasts. These cells may provide a sensitive and unique model for understanding the mechanism of E2 and ER in overall bone metabolism.

Topics: Alkaline Phosphatase; Base Sequence; Binding Sites; Blotting, Northern; Blotting, Southern; Bone Neoplasms; Breast Neoplasms; Cell Division; Chloramphenicol O-Acetyltransferase; Cloning, Molecular; DNA, Complementary; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Interleukin-6; Molecular Sequence Data; Osteoblasts; Osteosarcoma; Polymerase Chain Reaction; Receptors, Estrogen; RNA, Messenger; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Saos-2 cultured human osteosarcoma cells contain an extractable bone inducing agent that can induce heterotopic bone in the muscle of Nu/Nu mice. A semipurified GuHCl extract of Saos-2 cells also can promote healing and complete bony union in otherwise non-healing surgically induced defects of rat femur. Northern blot analyses indicate expression of mRNAs for bone morphogenetic proteins (BMP)-1, 2, 3, 4, 6 and transforming growth factor beta (TGF beta) in Saos-2 cells, and BMP-2, 3, 4, 5, 7 and TGF beta in nonosteoinductive U20S human osteosarcoma cells. Saos-2 cells exceeded U20S cells in expression levels of BMP-1, 3, 4 and TGF beta, whereas U20S cells expressed higher levels of BMP-2, 6 and also expressed trace amounts of BMP-5 and 7 not seen in Saos-2 cells. The authors hypothesize that Saos-2 cells contain an optimal admixture of known bone growth factors plus possible other unknown components that, acting alone or in combination with bone morphogenetic protein and/or TGF beta, can induce bone. Although bone inducing agent-induced heterotopic bones have half lives of only a few weeks, the reparative bone induced by bone inducing agent in femoral defects gives every indication of being permanent and self-sustaining. This suggests a fundamental difference between heterotopic and orthotopic osteoprogenitor cells with those involved in orthotopic bone repair more closely resembling the committed or determined osteoprogenitor cells of marrow as described by Friedenstein.

Topics: Animals; Blotting, Northern; Bone Development; Bone Morphogenetic Proteins; Bone Neoplasms; Bone Regeneration; Gene Expression; Growth Substances; Humans; Mice; Mice, Nude; Osteosarcoma; Protein Biosynthesis; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) is present in high levels in bone and plays an important role in osteoblast growth and differentiation. In order to dissect the molecular mechanisms of action of TGF-beta on osteoblasts, the effects of TGF-beta on the steady state mRNA levels of c-fos, c-jun, and jun-B proto-oncogenes on normal human osteoblast-like cells (hOB) and a transformed human osteoblast cell line (MG-63) were measured. Treatment of hOBs with 2 ng/ml of TGF-beta 1 resulted in a rapid increase in c-fos mRNA levels as early as 15 min post-treatment. A maximum (10-fold) increase was observed at 30 min after TGF-beta treatment followed by a decrease to control values. Similar responses were measured whether the cells were rapidly proliferating or quiescent. TGF-beta 1 induced jun-B mRNA levels more gradually with steady increase initially observed at 30 min and a maximum induction measured at 2 h post-TGF-beta treatment. In contrast, TGF-beta treatment caused a time dependent decrease in the c-jun mRNA levels, an opposite pattern to that of jun-B mRNA. Treatment of hOBs with TGF-beta 1 in the presence of actinomycin-D abolished TGF-beta 1 induction of c-fos mRNA, suggesting that TGF-beta action is mediated via transcription. In the presence of cycloheximide, TGF-beta causes super-induction of c-fos mRNA at 30 min, indicating that the c-fos expression by TGF-beta is independent of new protein synthesis. Further, transfection of 3 kb upstream region of jun-B promoter linked to a CAT reporter gene into ROS 17/2.8 cells was sufficient to be regulated by TGF-beta 1. Interestingly, TGF-beta treatment also increased the mRNA levels of TGF-beta 1 itself at 4 h post TGF-beta treatment, with a maximum increase observed at 14 h of treatment. TGF-beta 1 treatment for 30 min were sufficient to cause a delayed increase in TGF-beta protein secretion within 24 h. These data support that TGF-beta has major effects on hOB cell proto-oncogene expression and that the nuclear proto-oncogenes respond as rapid, early genes in a cascade model of hormone action.

Topics: Animals; Gene Expression Regulation, Neoplastic; Humans; Osteoblasts; Osteosarcoma; Proto-Oncogene Mas; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Proto-Oncogenes; Rats; Recombinant Proteins; RNA, Messenger; Transforming Growth Factor beta

We analyzed glucocorticoid receptor function using ligand binding assays, DNA band-shift analysis and trans-activation of the murine mammary tumor virus-thymidine kinase-chloramphenicol acetyltransferase reporter gene in transiently transfected MG-63 human osteosarcoma cells. Dexamethasone increased the distribution of MG-63 cells in the G1/G0 phase of the cell cycle, thus decreasing the rate of DNA synthesis and cell growth. Its effect on MG-63 cell growth was neutralized by RU486 and anti-transforming growth factor beta 1 (TGF beta 1) antibody. In addition, (i) dexamethasone increased the levels of active TGF beta 1 in MG-63-conditioned media without significantly altering the expression of TGF beta 1 mRNA in MG-63 cells and (ii) TGF beta 1 inhibited proliferation of MG-63 cells. Therefore, we conclude that glucocorticoid receptor function is mediated by the activation of latent-TGF beta 1 in MG-63 osteosarcoma cells.

Topics: Base Sequence; Blotting, Northern; Cell Cycle; Cell Division; Dexamethasone; Genes, Reporter; Humans; Molecular Sequence Data; Osteosarcoma; Receptors, Glucocorticoid; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

We investigated the contribution of c-fos protooncogene in the mitogenic effect of transforming growth factor-beta (TGF beta) in serum-deprived, confluent rat calvaria osteoblastic cells. The TGF beta-induced growth in these cells was associated with an immediate and transient c-fos mRNA accumulation, similar to the inductive effect of fetal calf serum. To assess the role of c-fos in the response to TGF beta, we used a c-fos antisense (AS) oligonucleotide displaying duplex formation with rat c-fos mRNA. Studies of AS and sense (S) uptake by osteoblastic cells demonstrated that incorporation of labeled oligomers was maximal at 2 h, and the incorporated AS oligonucleotide remained intact for 24 h. Immunofluorescence analysis of c-Fos-labeled cells demonstrated that AS, but not S, oligonucleotide reduced c-Fos protein expression, suggesting specific efficient inhibition of c-fos translation by the AS oligomer. Proliferation assays showed that cell growth induced by fetal calf serum was inhibited by the AS, but not by the S oligonucleotide, in both normal rat osteoblasts and ROS 17/2.8 osteosarcoma cells, demonstrating efficient and specific blockage of cell growth by the AS oligomer. The mitogenic effect of TGF-beta was abolished in cells cultured in the presence of AS, whereas S had no effect, showing that c-fos is required for TGF beta-induced osteoblast cell growth. The results show that the induction of c-fos is implicated in the mitogenic effect of TGF beta in osteoblastic cells and provide a cellular mechanism involved in the response of these cells to TGF beta.

Topics: Animals; Base Sequence; Blotting, Northern; Cell Division; Fluorescent Antibody Technique; Gene Expression Regulation; Genes, fos; Growth Substances; Molecular Sequence Data; Osteoblasts; Osteosarcoma; Proto-Oncogene Proteins c-fos; Rats; RNA, Antisense; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

Previous studies demonstrated that insulin-like growth factors (IGFs) are important autocrine and paracrine mitogens for human bone cells in vitro and that IGF binding proteins (IGFBPs) are important regulators of the biologic actions of IGFs. Thus, the actions of IGFs may be determined not only by their concentrations but also by the type and amount of IGFBPs produced by human bone cells at a local site in bone. In this study, we sought to determine the effects of dexamethasone, 1,25-(OH)2D3, and parathyroid hormone (PTH) on the secretion of IGFBP-3 in human osteosarcoma cell lines. Serum-free cultures of low- and high-alkaline phosphatase (ALP) SaOS-2, MG-63, and TE89 human osteosarcoma cells were treated for 24 or 48 h with the effectors and the conditioned media used for determination of IGFBP-3 using a radioimmunoassay. We report that (1) the basal rate of IGFBP-3 secretion (ng/mg cellular protein) was dependent upon cell type, with TE89 > low-ALP Saos-2 > MG-63 > high-ALP SaOS-2 cells, and did not correlate with either basal cell proliferation or basal cellular ALP activity; (2) dexamethasone (10(-12)-10(-7) M) inhibited IGFBP-3 secretion in a dose-dependent manner in low-ALP SaOS-2, MG-63, and TE89 cells but not in high-ALP SaOS-2 cells; (3) 1,25-(OH)2D3 (10(-11)-10(-8) M) stimulated IGFBP-3 secretion in a dose-dependent manner in MG-63, low-ALP SaOS-2, and high-ALP SaOS-2 cells, and the coaddition of TGF-beta and 1,25-(OH)2D3 increased synergistically IGFBP-3 secretion and cellular ALP activity in MG-63 cells; and (4) human PTH-(1-34) (0.1-100 ng/ml) had no significant effect on IGFBP-3 secretion in MG-63, low-ALP SaOS-2, or high-ALP SaOS-2 cells. We conclude that such agents as dexamethasone, 1,25-(OH)2D3, and PTH differentially regulate IGFBP-3 secretion in human osteosarcoma cells in vitro.

Topics: Alkaline Phosphatase; Analysis of Variance; Bone and Bones; Calcitriol; Carrier Proteins; Cell Division; Dexamethasone; Dose-Response Relationship, Drug; Humans; Insulin-Like Growth Factor Binding Proteins; Osteosarcoma; Parathyroid Hormone; Radioimmunoassay; Somatomedins; Transforming Growth Factor beta; Tumor Cells, Cultured

We recently showed that osteogenic protein-1(OP-1), a bone morphogenetic protein member of TGF-beta superfamily, induces endochondral bone formation in vivo, and stimulates growth and differentiation of osteoblasts in rat calvarial-derived cell cultures. In the present study, we examined the effect of OP-1 on cell growth and expression of markers that are characteristic of osteoblast phenotype using the clonal rat osteosarcoma cells (ROS 17/2.8). A comparison of OP-1 and TGF-beta 1 effects on cell growth showed that, both OP-1 and TGF-beta 1 inhibited DNA synthesis up to 90 percent and 60 percent of the controls at concentrations of 10 ng/ml and 1 ng/ml, respectively, in serum-free medium. In the presence of 5% serum, TGF-beta 1 did not have any significant inhibitory effects while 40 ng OP-1/ml inhibited the DNA synthesis up to 80% of the controls. Examination of collagen synthesis showed that 40 ng OP-1/ml increased the expression of type I collagen mRNA, and thus increased collagen synthesis (4-fold), as examined by collagenase-digestible protein. Evaluation of markers that are characteristic of the osteoblast phenotype demonstrated that OP-1 stimulated cAMP production in response to PTH (10-fold at 200 ng/ml), alkaline phosphatase specific activity (ALPase) (4-fold at 80 ng/ml), and osteocalcin (OC) synthesis (4.5-fold at 40 ng/ml). Northern blot analysis revealed that OP-1 increased mRNA expression for both ALPase and OC in a dose-dependent manner. These data collectively demonstrate that OP-1 suppresses cell proliferation and stimulates the expression of markers characteristic of osteoblast phenotype in rat clonal osteoblastic osteosarcoma cells (ROS 17/2.8).

Topics: Alkaline Phosphatase; Animals; Biomarkers; Blotting, Northern; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cell Differentiation; Cell Division; Collagen; Cyclic AMP; DNA; Gene Expression; Osteoblasts; Osteocalcin; Osteosarcoma; Phenotype; Proteins; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

Treatment of human MG-63 osteosarcoma cells with human recombinant transforming growth factor beta 1 (TGF-beta 1) was found to inhibit cell proliferation. In addition, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced osteocalcin synthesis was greatly influenced by TGF-beta 1. Dose- and time-dependent inhibition was seen both in medium osteocalcin and the corresponding mRNA concentrations. Furthermore, TGF-beta 1 decreased osteocalcin synthesis modulated negatively by dexamethasone or positively by retinoic acid. The stability of osteocalcin mRNA was not decreased by the TGF-beta 1 treatment, but in vitro transcription assays demonstrated diminished osteocalcin gene transcription caused by the TGF-beta 1 treatment. Binding of vitamin D receptor (VDR) to an oligonucleotide probe containing the osteocalcin vitamin D response element (VDRE) was not influenced by TGF-beta 1, however. Incubation of the cells with the serine/threonine kinase inhibitor H-7 did not block the ability of TGF-beta 1 to decrease osteocalcin synthesis but caused a further inhibition. Also, the 1,25(OH)2D3-induced osteocalcin synthesis was decreased by H-7 treatment, suggesting that phosphorylation as such is involved in the transcriptional activation mechanism of VDR. These results demonstrate that TGF-beta 1 is a strong inhibitor of the synthesis of osteocalcin, a calcium binding protein participating in bone mineralization, by counteracting the stimulatory effects of other hormones on its synthesis. We further suggest that TGF-beta 1 affects the synthesis of osteocalcin at the level of transcription through mechanism(s) different from the serine/threonine kinase pathway.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Base Sequence; Bone Neoplasms; Calcitriol; Cell Division; Dexamethasone; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation, Neoplastic; Humans; Isoquinolines; Molecular Sequence Data; Oligonucleotide Probes; Osteocalcin; Osteosarcoma; Phosphorylation; Piperazines; Protein Kinase C; Receptors, Calcitriol; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Vitamin D-Binding Protein

Fibronectin (FN) is an important constituent of the extracellular matrix in bone. Its hormonal regulation in this tissue has not been extensively studied. We, therefore, examined the effects of PTH, estrogen, and transforming growth factor beta on the production of FN by human and rat osteoblast-like cells. Confluent cells were stabilized for 48 h under estrogen-replete (10(-9) M 17 beta-estradiol) conditions and then continued under these same conditions or withdrawn from estrogen for varying periods of time. PTH over the range 10(-11)-(10(-8) M caused a dose-dependent increase in FN production [P < 0.001 by analysis of variance (ANOVA)] such that at the highest dose, FN production was increased 11-fold. Estrogen withdrawal for 96 h caused a significant diminution in PTH-induced FN production (P < 0.005 by two-way ANOVA). Estrogen withdrawal over the of period 48-144 h caused a progressive diminution in PTH-induced FN production such that differences in mean values for estrogen-replete vs. deficient conditions were greater at 144 than 48 h (P < 0.05). The estrogen effect was titratable over the range 10(-11)-10(-9) M, and the inactive congener 17 alpha-estradiol failed to prevent the inhibitory effect of estrogen withdrawal on PTH-induced FN production. Interestingly, estrogen withdrawal had absolutely no effect on transforming growth factor-beta-induced FN production. Northern analysis demonstrated no effect of PTH on steady-state FN messenger RNA levels in Saos-2 cells under either estrogen-replete or estrogen-deficient conditions, suggesting that PTH effects an increase in FN production via a posttranscriptional mechanism in these cells. We conclude that PTH stimulates FN production in human and rat osteoblast-like cells, and under estrogen-deficient conditions this effect is significantly diminished. The modulatory effect of estrogen is not a universal phenomenon because transforming growth factor-beta-induced FN production is unaffected by estrogen withdrawal.

Topics: Aged; Analysis of Variance; Animals; Blotting, Northern; Bone and Bones; Bone Neoplasms; Cells, Cultured; Dose-Response Relationship, Drug; Estrogens; Female; Fibronectins; Humans; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

The effects of porcine bone morphogenetic protein (BMP) on DNA and collagen synthesis in cultured rat osteogenic sarcoma cells and mink lung epithelial cells were studied and compared with the effects induced by transforming growth factor-beta 1 (TGF-beta 1). In both cells, DNA synthesis was slightly but significantly increased by BMP whereas it was notably decreased by TGF-beta 1. The inhibitory action of TGF-beta 1 overrode the activation of DNA synthesis by BMP when the cells were incubated together with TGF-beta 1 and BMP. In osteogenic sarcoma cells, collagen synthesis was enhanced by both BMP and TGF-beta 1, but the stimulatory action of BMP was weaker than that of TGF-beta 1. In epithelial cells, TGF-beta 1 increased collagen synthesis but BMP induced no significant changes. No synergistic effects of TGF-beta 1 and BMP on collagen synthesis were observed in both cells. The present study demonstrates the possibility of direct actions of BMP and TGF-beta 1 on cultured rat osteogenic sarcoma cells and mink lung epithelial cells in vitro.

Topics: Animals; Bone and Bones; Bone Morphogenetic Proteins; Bone Neoplasms; Cell Division; Cell Line; Collagen; DNA; Dose-Response Relationship, Drug; Epithelial Cells; Epithelium; Lung; Mink; Osteosarcoma; Proteins; Rats; Swine; Transforming Growth Factor beta; Tumor Cells, Cultured

Previous studies had shown that binding of TGF-beta to the small proteoglycan decorin results in its inactivation. Indeed, in osteosarcoma cells the addition of decorin prevented the TGF-beta 1-mediated up-regulation of biglycan synthesis. However, the down-regulation of proteoglycan-100 remained unaltered. Even in the presence of a 100,000-fold molar excess of decorin, TGF-beta 1 was fully active in U937 monocytes with respect to the inhibition of cell proliferation. There was no inhibition of the TGF-beta-mediated stimulation of the retraction of fibroblast-populated collagen lattices. Thus, the formation of TGF-beta/decorin complexes leads to the neutralization of distinct effects only.

Topics: Biglycan; Cell Division; Collagen; Decorin; Down-Regulation; Extracellular Matrix Proteins; Humans; Monocytes; Osteosarcoma; Proteoglycans; Transforming Growth Factor beta; Tumor Cells, Cultured

We investigated the role of protein kinase C (PKC) in osteoblast function using a set of putative PKC modulating factors and an in situ peptide substrate-based kinase assay in different types of osteoblastic cells. Primary calvarial rat osteoblastic cells (ROB) and ROS 17/2.8 osteosarcoma cells showed an equally high PKC activity when a maximal dose of PKC-activating phorbol ester was applied. The osteosarcoma cell line UMR 106-01 showed only 5-10% of this maximal PKC activity. All 3 cell types responded to 10 U/ml thrombin with a 2-fold stimulation of PKC activity. However, no distinct direct effects of parathyroid hormone (bPTH (1-34)) or transforming growth factor-beta 2 (TGF-beta 2) were found in either of the cell types. The thrombin-induced stimulation of PKC was associated with an increase in the PTH-mediated cAMP response of ROB. Down-regulation of PKC-activity was found when ROB were treated for 24 h with phorbol ester and, interestingly, also after a 24 h treatment with bPTH (1-34) and TGF-beta 2. We conclude that differences in PKC activity exist among osteoblastic cell types, which may be related to their different proliferative activity. Direct PKC activation may lead to modulation of the cAMP signaling pathway. Down-regulation of PKC activity by bPTH (1-34) and TGF-beta 2 provides an interesting possible mechanism for the long-term regulation of signal transduction.

Topics: Amino Acid Sequence; Animals; Calcium; Cell Division; Cells, Cultured; Cyclic AMP; DNA; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Molecular Sequence Data; Osteoblasts; Osteosarcoma; Parathyroid Hormone; Peptide Fragments; Phorbol Esters; Protein Kinase C; Rats; Signal Transduction; Substrate Specificity; Teriparatide; Thrombin; Transforming Growth Factor beta; Tumor Cells, Cultured

Recent studies have shown that osteogenic protein (OP)-1 or bone morphogenetic protein (BMP)-7 increases proliferation and differentiation of human bone cells (HBCs) in culture and modulates production of IGF system components. In order to study the mechanism by which OP-1 causes these effects, we sought to test the hypothesis that the effects of OP-1 are mediated at least in part by specific receptors (for OP-1) in HBCs. Binding studies with serum-free cultures of normal HBCs and human osteosarcoma cells showed a maximum binding of 15-25% for [125I]OP-1; the binding was time- and temperature-dependent in different experiments. Scatchard analysis of [125]OP-1 binding to TE85 human osteosarcoma cells showed at least two binding sites, about 30,000 and 60,000 per cell with apparent Kd of 2.5 x 10(-10)M and 1 x 10(-9)M, respectively. [125]OP-1 binding to TE85 cells was displaced by unlabeled OP-1 (16-1000 ng/ml), with a 50% displacement at 250 ng/ml. BMP-2 effectively displaced [125]OP-1 binding to HBCs while TGF-beta 1 did not. Affinity cross-linking studies showed that [125]OP-1 interacted specifically with three binding sites with apparent M(r) of 34, 65 and > 205kDa. The findings of this study demonstrate that the effects of OP-1 on HBCs may be mediated in part via BMP-specific receptors.

Topics: Binding Sites; Bone and Bones; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cross-Linking Reagents; Humans; In Vitro Techniques; Kinetics; Osteosarcoma; Protein Binding; Proteins; Receptors, Cell Surface; Temperature; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

A bone and cartilage enzyme with both 5'-nucleotide phosphodiesterase I and nucleotide pyrophosphohydrolase (NTPPPH) activity modulates physiologic mineralization and pathologic chondrocalcinosis by generating inorganic pyrophosphate. We hypothesized that, as for alkaline phosphatase, expression of an NTPPPH gene can be shared by cells from bone, cartilage, and liver and by certain leukocytes. Recently, we demonstrated the hepatocyte and murine plasma cell membrane glycoprotein PC-1 to have both 5'-nucleotide phosphodiesterase I and NTPPPH activity. We detected polypeptides cross-reactive with PC-1 in human U20S osteosarcoma cells, articular chondrocytes, homogenized human knee cartilages, human knee synovial fluids, hepatoma cells, and murine plasmacytoma cells. Constitutive low abundance PC-1 mRNA expression was detected in U20S cells and chondrocytes by a nested RNA-PCR assay and by Northern blotting. TGF beta is known to substantially increase NTPPPH activity in primary osteoblast cultures. We demonstrated that TGF beta 1 increased NTPPPH activity and the level of PC-1 mRNA and immunoprecipitable [35S]-methionine-labeled PC-1 polypeptides in U20S cells. The identification of PC-1 as an NTPPPH expressed in cells derived from bone and cartilage may prove useful in furthering the understanding of the role of NTPPPH i n physiologic and pathologic mineralization.

Topics: Animals; Base Sequence; Bone and Bones; Cartilage; Gene Expression Regulation, Enzymologic; Humans; Liver; Membrane Glycoproteins; Mice; Molecular Sequence Data; Osteosarcoma; Phosphodiesterase I; Phosphoric Diester Hydrolases; Pyrophosphatases; RNA, Messenger; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is a polypeptide with multiple physiological functions. Isoforms of this growth factor have important roles in control of the cell cycle, in regulation of cell-cell interactions and in growth and development. Malignant transformation has been shown to be associated with increased expression of TGF-beta. Since bone is the largest storage site and producer of TGF-beta, we speculated on the existence of an autocrine mechanism in osteosarcoma, a malignant bone tumor. Expression of TGF-beta cell surface receptors, effects on growth of TGF-beta and TGF-beta antibodies and production of 2 TGF-beta isoforms were studied in a panel of 7 osteosarcoma cell lines. In contrast to most previous reports on the effects of TGF-beta on osteosarcoma cell growth, we found a mitogenic effect of TGF-beta 1 in 4 of 7 osteosarcoma cell lines. Receptor profiles for TGF-beta were aberrant in 5 of the 7 cell lines tested, and production of TGF-beta 1 and TGF-beta 2 varied among cell lines. Addition of anti-TGF-beta antagonized the effects of endogenous TGF-beta. Our results suggest a potential role of TGF-beta in autocrine growth control of osteosarcoma cells.

Topics: Antibodies; Bone Neoplasms; Cell Division; Culture Media; DNA, Neoplasm; Humans; Isomerism; Osteosarcoma; Receptors, Transforming Growth Factor beta; Stimulation, Chemical; Transforming Growth Factor beta; Tumor Cells, Cultured

Autocrine production of growth factors has been shown to be involved in the multistep process of tumorigenesis. The ability of suramin, a polyanionic anti-parasitic drug, to block growth factor-induced cell proliferation makes it a potential antineoplastic drug. We studied the effects of suramin on seven osteosarcoma cell lines. Using clinically achievable concentrations of suramin (50-400 micrograms/ml), we found a time- and dose-dependent inhibition of [3H]thymidine incorporation. We also showed that suramin is able, dose-dependently, to prevent binding of transforming growth factor (TGF)-beta 1 to its receptors. DNA synthesis inhibition by suramin was attenuated by TGF-beta 1 in some cell lines. Two cell lines that were inhibited by TGF-beta 1 were affected similarly by suramin as cell lines that were stimulated by TGF-beta 1. In conclusion, in five out of seven osteosarcoma cell lines, we showed a correlation between inhibition of growth factor-stimulated mitogenesis and binding of TGF-beta 1 to its receptor. Similar effects in TGF-beta 1-inhibited osteosarcoma cell lines suggest involvement of other mechanisms and/or growth factors. However, suramin proves to be a potent inhibitor of osteosarcoma cell proliferation in vitro.

Topics: Bone Neoplasms; Cell Division; DNA, Neoplasm; Dose-Response Relationship, Drug; Humans; Osteosarcoma; Receptors, Transforming Growth Factor beta; Suramin; Transforming Growth Factor beta; Tumor Cells, Cultured

We have previously shown that bone organ cultures produce large amounts of latent transforming growth factor beta (TGF beta), which lacks latent TGF beta-binding protein (LTBP). In this study we used the known osteoblast-like cell lines UMR-106, ROS 17/2.8, and MG63 as models to further examine latent TGF beta expression in bone. We found that the osteosarcoma cell line UMR-106 secreted latent TGF beta almost exclusively as a 100-kDa complex lacking LTBP. ROS 17/2.8 cells produced both the 100-kDa complex and also a 290-kDa complex containing the fibroblastic (190 kDa) form of LTBP. MG63 cells (like human foreskin fibroblasts) expressed almost exclusively the 290-kDa complex. To investigate the regulation of latent TGF beta complexes in bone cells we assessed the effects of TGF beta 1 treatment on expression of active and latent TGF beta. TGF beta 1 induced secretion of latent but not active TGF beta in all cell types examined. In human foreskin fibroblast cells, TGF beta 1 and LTBP mRNA were expressed concomitantly. In contrast, in osteosarcoma cell lines autoinduction of TGF beta 1 mRNA was associated with either a delayed increase or no change in LTBP mRNA. In UMR-106 cells LTBP message was virtually undetectable. We postulate that the expression of different latent TGF beta forms by osteoblast-like cells may reflect their maturation states and that different latent TGF beta complexes may have different functions, for example as secretory forms or as matrix storage forms.

Topics: Animals; Antibodies; Blotting, Northern; Bone and Bones; Carrier Proteins; Cell Line; Chromatography, High Pressure Liquid; Fibroblasts; Glyceraldehyde-3-Phosphate Dehydrogenases; Homeostasis; Humans; Immunoblotting; Male; Osteoblasts; Osteosarcoma; Rabbits; RNA, Messenger; Skin; Transforming Growth Factor beta; Tumor Cells, Cultured

The effect of transforming growth factor beta1 (TGF-beta1) on the expression of mRNA for the parathyroid hormone receptor and binding of iodinated parathyroid hormone-related protein in ROS 17/2.8 osteosarcoma cells was evaluated. TGF-beta1 stimulated a 2-7-fold increase in steady state mRNA levels for the parathyroid hormone receptor at a maximal dose of 5 ng/ml, with increased levels of expression at 6 h of TGF-beta1-incubation, and peak levels at 8-24 h. Receptor binding studies revealed a significant increase in PTHrP-specific binding with TGF-beta1 doses as low as 0.5 ng/ml and a 55% increase in numbers of receptors with no alteration in binding affinity with 5.0 ng/ml TGF-beta1. Time course studies indicated that receptor binding was increased at 24 h with peak levels reached at 48 h of treatment. PTH-stimulated cAMP levels were significantly increased in ROS 17/2.8 cells treated with TGF-beta1 (0.5 ng/ml) for 48 h. These data indicate that TGF-beta1 upregulates steady-state mRNA, ligand binding and PTH/PTHrP receptor signaling in rat osteosarcoma cells. The effects of TGF-beta1 on bone may be attributed in part to regulation of the PTH/PTHrP receptor at the molecular level.

Topics: Animals; Osteosarcoma; Parathyroid Hormone; Parathyroid Hormone-Related Protein; Proteins; Rats; Receptors, Parathyroid Hormone; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

During studies of mitogens in prostate, PSA quantities as low as 2.5 ng/mL caused cultured osteoblast cells to proliferate beyond controls (p = 0.05). Investigation of this novel mitogenicity suggested the use of several mechanisms by PSA, namely: 1) the activation of latent hTGF-beta in PC-3 conditioned medium, PSA treated conditioned medium stimulated DNA uptake in UMR-106 cells to 78% of acid treated conditioned medium, while DNA incorporation was less than controls with anti-hTGF-beta neutralizing IgG; and 2) the proteolytic modulation of cell surface receptors with temporary contact inhibition, PSA significantly stimulated cell detachment while hTGF-beta enhanced cell attachment of confluent Saos-2 cells above controls. Clinically, these results suggest that PSA may provide a mechanism for both tumor spread and the osteoblastic metastasis so common to prostate cancer.

Topics: Animals; Cell Adhesion; Cell Division; Hydrolysis; Osteoblasts; Osteosarcoma; Plasminogen Activators; Prostate-Specific Antigen; Rats; Transforming Growth Factor beta; Tumor Cells, Cultured

This study investigated the effect of bone resorbing factors on the pericellular fibrinolytic system of osteosarcoma NY cells. Parathyroid hormone (PTH), prostaglandin E2, (PGE2) or tumor necrosis factor alpha (TNF-alpha) enhanced the secretion of urokinase-type plasminogen activator (u-PA) antigen and suppressed the secretion of plasminogen activator inhibitor-1 (PAI-1) antigen to the conditioned medium. The former two factors also increased u-PA antigen in the cell surface. Transforming growth factor beta (TGF-beta) enhanced u-PA antigen, but its activity was suppressed due to the increased secretion of PAI-1. The binding assay of [125I]DFP-u-PA to NY cells revealed the presence of a single class of binding sites with a Kd of 5.51 nM and Bmax of 0.92 x 10(5) binding sites/cell. PTH or PGE2 increased Bmax 1.4-fold and enhanced the u-PA receptor (u-PAR) mRNA level 1.4-fold or 2.4-fold, respectively. However, TGF-beta did not alter either the Kd or u-PAR mRNA level. Thus, pericellular fibrinolytic activity by u-PA/u-PAR and PAI-1 is modulated by bone resorbing factors.

Topics: Bone Resorption; Dinoprostone; Humans; Osteosarcoma; Parathyroid Hormone; Plasminogen Activator Inhibitor 1; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator

Based on information from partial amino acid sequences of a protein with bone-inducing activity that was purified from a murine osteosarcoma (Dunn type), a cDNA library of the sarcoma was screened to clone a gene complementary to the protein. The cloned cDNA was amplified and transfected into Chinese hamster ovarian (CHO) cells for expression. When the protein produced by the transfected cell line was implanted in combination with pure carrier collagen into allogeneic mice, ectopic ossicles consistently developed at implanted sites within two weeks. The nucleotide sequence of the cDNA and its deduced amino acid sequence were homologous to those of human bone morphogenetic protein-4 (also BMP-2B). In addition, the cDNA and deduced amino acid sequences were identical to those proposed for murine BMP-4 derived from the normal murine fetus. It is postulated that the cloned cDNA encodes the protein responsible for bone formation induced by implantation of devitalized Dunn-type osteosarcoma tissue or cells. The protein product was identified as murine BMP-4, a member of the TGF-beta gene family.

Topics: Amino Acid Sequence; Animals; Base Sequence; Bone Morphogenetic Proteins; Cloning, Molecular; Cricetinae; Cricetulus; DNA, Circular; Gene Expression; Gene Library; Growth Substances; Mice; Mice, Inbred ICR; Molecular Sequence Data; Neoplasm Proteins; Osteosarcoma; Polymerase Chain Reaction; Proteins; Sarcoma, Experimental; Transfection; Transforming Growth Factor beta

Gallium nitrate, a group IIIa metal salt, has been found to be clinically effective for the treatment of accelerated bone resorption in cancer-related hypercalcemia and Paget's disease. Here we report the effects of gallium nitrate on osteocalcin mRNA and protein levels on the rat osteoblast-like cell line ROS 17/2.8. Gallium nitrate reduced both constitutive and vitamin D3-stimulated osteocalcin protein levels in culture medium by one-half and osteocalcin mRNA levels to one-third to one-tenth of control. Gallium nitrate also inhibited vitamin D3 stimulation of osteocalcin and osteopontin mRNA levels but did not affect constitutive osteopontin mRNA levels. Among several different metals examined, gallium was unique in its ability to reduce osteocalcin mRNA levels without decreasing levels of other mRNAs synthesized by ROS 17/2.8 cells. The effects of gallium nitrate on osteocalcin mRNA and protein synthesis mimic those seen when ROS 17/2.8 cells are exposed to transforming growth factor beta 1 (TGF beta 1); however, TGF-beta 1 was not detected in gallium nitrate-treated ROS 17/2.8 cell media. Use of the RNA polymerase II inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that gallium nitrate did not alter the stability of osteocalcin mRNA. Transient transfection assays using the rat osteocalcin promoter linked to the bacterial reporter gene chloramphenicol acetyltransferase indicated that gallium nitrate blocked reporter gene expression stimulated by the osteocalcin promoter. This is the first reported effect of gallium nitrate on isolated osteoblast cells.

Topics: Animals; Antineoplastic Agents; Blotting, Northern; Carrier Proteins; Cholecalciferol; Gallium; Osteoblasts; Osteocalcin; Osteopontin; Osteosarcoma; Rats; RNA, Messenger; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Controversy exists regarding the effect of fluoride on human osteoblast proliferation. To learn more of the cellular action of fluoride, we chose the clonal osteoblast cell line HOS TE85 as a model system. In these phenotypically osteoblast-like cells, sodium fluoride stimulated [3H]thymidine incorporation in a dose-dependent manner over the concentration range 1 x 10(-5)-2 x 10(-4) M. The fluoride-induced stimulation of [3H]thymidine uptake was dependent on cell density, being optimal at subconfluent cell numbers. Stimulation of [3H]thymidine uptake was inhibited by anti-transforming growth factor beta but not by antibody to insulin-like growth factor I or beta 2-microglobulin. Transforming growth factor beta was shown to be a biphasic stimulator of [3H]thymidine uptake in HOS TE85, with maximal stimulation occurring at 0.5 nM transforming growth factor beta. In the presence of fluoride the cells were more sensitive to stimulation by this growth factor, with maximum effect occurring at 0.1 nM. Fluoride did not increase mRNA for transforming growth factor beta following either 8 or 24 h of exposure. We conclude that fluoride activates osteoblast proliferation by modulating the cellular sensitivity to transforming growth factor beta, a known stimulator of bone growth.

Topics: Antibodies; Blotting, Northern; Cell Count; Cell Division; DNA; Dose-Response Relationship, Drug; Humans; Osteoblasts; Osteosarcoma; RNA, Messenger; Sodium Fluoride; Spectrophotometry, Ultraviolet; Thymidine; Transforming Growth Factor beta; Tumor Cells, Cultured

Injections of parathyroid hormone (PTH) result in increased bone formation in several species. Work in our laboratory and others has shown a stimulation of bone cell proliferation and growth factor production by PTH. Our purpose was to study the effects of PTH on a human bone cell line using TE-85 human osteosarcoma cells as a model. After 24 h treatment, PTH caused an increase in cell proliferation as measured by cell counts and [3H]-thymidine incorporation. Proliferation was not inhibited by an anti-transforming growth factor beta (TGF beta) antibody which could abolish stimulation by exogenous TGF beta. PTH did not stimulate cAMP production, alkaline phosphatase activity or production of insulin-like growth factors I or II (IGF-I or IGF-II) in TE-85 cells. Although basal TE-85 proliferation was slowed by incubation with the calcium channel blocking agent verapamil, PTH still caused an increase in growth rate. We conclude that PTH directly stimulates TE-85 proliferation via a mechanism not involving increased adenylate cyclase activity or increased secretion of IGF-I, IGF-II or TGF beta and may stimulate bone formation in vivo by activating some other mitogenic signal to increase bone cell proliferation.

Topics: Adenylyl Cyclases; Alkaline Phosphatase; Cell Division; Cyclic AMP; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Osteosarcoma; Parathyroid Hormone; Transforming Growth Factor beta; Tumor Cells, Cultured; Verapamil

Transforming growth factor beta (TGF-beta) is now recognized as an important growth regulator and modulator in bone, where it apparently acts in an autocrine or paracrine fashion. In an effort to help elucidate how TGF-beta may interact with parathyroid hormone (PTH) to influence bone turnover, we examined the idea that TGF-beta might alter the number or affinity of PTH receptors in osteoblastic bone cells, PTH receptor binding was assessed in cultured ROS 17/2.8 cells using [125I]PTHrP-(1-34) as labeled ligand. Specific binding to intact cells was measured in the presence of up to 1 microM unlabeled rPTH-(1-34), and cAMP in cell extracts was determined by RIA. Incubation of ROS cells with 2 ng/ml of TGF-beta for the maximally effective time of 3 days increased the number of PTH binding sites (Bmax) by 47 +/- 13%, with no change in the KD (3 nM). TGF-beta also increased the intracellular cAMP response to 0.3 nM rPTH-(1-34) (ED50) by 53 +/- 22%. Both effects were dose dependent, with 1-4 ng/ml of TGF-beta producing maximal effects, and both effects were blocked by the protein synthesis inhibitor cycloheximide (2-5 microM). Since TGF-beta induced comparable increases in both PTH binding and cAMP formation, the findings suggest that TGF-beta can increase the number of functional PTH receptors in cultured ROS 17/2.8 cells. This effect may reflect an action of TGF-beta to slow replication and promote differentiated functions in these cells.

Topics: Animals; Cyclic AMP; DNA; Osteosarcoma; Parathyroid Hormone; Protein Biosynthesis; Rats; Receptors, Cell Surface; Receptors, Parathyroid Hormone; Transforming Growth Factor beta; Tumor Cells, Cultured

Using MG-63 cells as a model system capable of partial osteoblastic differentiation, we have examined the effect of growth on extracellular matrix. MG-63 cell matrix and purified type I collagen induced a morphological change characterized by long cytoplasmic processes reminiscent of those seen in osteocytes. Concurrent biochemical changes involving bone marker proteins included increased specific activity of cell-associated alkaline phosphatase and increased secretion of osteonectin (up to 2.5-fold for each protein); all changes occurred without alterations in the growth kinetics of the MG-63 cells. The increase in alkaline phosphatase activity was maximal on days 6-8 following seeding; increased osteonectin secretion was most prominent immediately following seeding; all changes decreased as cells reached confluence. Growing cells on type I collagen resulted in an increased induction of alkaline phosphatase activity by 1,25(OH)2D3 (with little change in the 1,25(OH)2D3 induction of osteonectin and osteocalcin secretion), and increased TGF-beta induction of alkaline phosphatase activity as well (both TGF-beta 1 and TGF-beta 2). Both the 1,25(OH)2D3 and TGF-beta effects appeared to be synergistic with growth on type I collagen. These studies support the hypothesis that bone extracellular matrix may play an important role in osteoblastic differentiation and phenotypic expression.

Topics: Alkaline Phosphatase; Animals; Bone and Bones; Calcitriol; Cell Division; Collagen; Humans; Osteoblasts; Osteonectin; Osteosarcoma; Phenotype; Rats; Species Specificity; Substrate Specificity; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth beta (TGF-beta) has been proposed to have a role in bone remodeling by affecting the differentiation and activity of osteoblasts and osteoclasts and by inhibiting the production of proteinases, such as plasminogen activators (PAs). Studies on PAs have largely been based on data from nonhuman and fetal cell lines, however. The purpose of this study was to investigate the effect of TGF-beta on the PA activity of normal human osteoblast-like cells and to compare this with its action on the human osteosarcoma cell line MG-63. The action of interleukin-1 beta (IL-1 beta) was also assessed because it has been shown to increase PA activity in other connective tissue cell types. Normal osteoblast-like cells had low to undetectable basal urokinase (uPA) and tissue plasminogen activator (tPA) activity, which was significantly stimulated by TGF-beta 1. This action was shown to be dependent on transcription and new protein synthesis. TGF-beta 2 had a similar action. IL-1 beta did not stimulate PA activity. In contrast, the MG-63 cell line had high basal tPA and uPA activities. TGF-beta 1 decreased basal PA activity, the effect being most marked for uPA activity. IL-1 beta stimulated uPA and tPA activity. TGF-beta 1 inhibited IL-1 beta-stimulated uPA activity, but the effect on tPA was more variable. This study has shown that TGF-beta has opposite effects on the PA activity of the two osteoblast-like cell types studied. Care must therefore be used before extrapolating data from one cell type to another.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Bone Remodeling; Cells, Cultured; Cycloheximide; Dactinomycin; Electrophoresis, Polyacrylamide Gel; Humans; Indomethacin; Interleukin-1; Osteoblasts; Osteosarcoma; Recombinant Proteins; Tissue Plasminogen Activator; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Transforming growth factor beta (TGF beta) and 1,25-dihydroxyvitamin D3 (1,25D3), when added simultaneously to a human osteosarcoma cell line, MG-63, induce alkaline phosphatase activity 40-70-fold over basal levels, 6-7-fold over 1,25D3 treatment alone, and 15-20-fold over TGF beta treatment alone. TGF beta and 1,25D3 synergistically increased alkaline phosphatase specific activity in both matrix vesicles and plasma membrane isolated from the cultures, but the specific activity was greater in and targeted to the matrix vesicle fraction. Inhibitor and cleavage studies proved that the enzymatic activity was liver/bone/kidney alkaline phosphatase. Preincubation of MG-63 cells with TGF beta for 30 min before addition of 1,25D3 was sufficient for maximal induction of enzyme activity. Messenger RNA for liver/bone/kidney alkaline phosphatase was increased 2.1-fold with TGF beta, 1.7-fold with 1,25D3, and 4.8-fold with the combination at 72 h. Human alkaline phosphatase protein as detected by radioimmunoassay was stimulated only 6.3-fold over control levels with the combination. This combination of factors was tested for their effect on production of three other osteoblast cell proteins: collagen type I, osteocalcin, and fibronectin. TGF beta inhibited 1,25D3-induced osteocalcin production, whereas both factors were additive for fibronectin and collagen type I production. TGF beta appears to modulate the differentiation effects of 1,25D3 on this human osteoblast-like cell and thereby retain the cell in a non-fully differentiated state.

Topics: Alkaline Phosphatase; Blotting, Northern; Bone and Bones; Calcitriol; Cell Differentiation; Cell Membrane; Collagen; Drug Synergism; Enzyme Induction; Fibronectins; Humans; Kidney; Liver; Osteocalcin; Osteosarcoma; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta (TGF beta) treatment of rat osteoblast-rich calvarial cells or of the clonal osteogenic sarcoma cells, UMR 106-01, resulted in dose-dependent inhibition of plasminogen activator (PA) activity, and increased production of 3.2 kb mRNA and protein for PA inhibitor -1 (PAI-1). Although tissue-type PA (tPA) protein was not measured, TGF beta did not influence production of mRNA for tPA. Production of 2.3 kb mRNA for urokinase-type PA (uPA) was also increased by TGF beta in a dose-dependent manner. The effects of TGF beta on synthesis of mRNA for PAI-1 and uPA were maintained when protein synthesis was inhibited, and were abolished by inhibition of RNA synthesis. Although uPA had not been detected previously as a product of rat osteoblasts, treatment of lysates of osteoblast-like cells with plasmin yielded a band of PA activity on reverse fibrin autography, corresponding to a low Mr form of uPA. Untreated conditioned media from normal osteoblasts or UMR 106-01 cells contained no significant TGF beta activity, but activity could be detected in acidified medium. Treatment of conditioned media with plasmin resulted in activation of approximately 50% of the TGF beta detectable in acidified media. The results identify several effects of TGF beta on the PA-PA inhibitor system in osteoblasts. Net regulation of tPA activity through the stimulatory actions of several calciotropic hormones and the promotion of PAI-1 formation by TGF beta could determine the amount of osteoblast-derived TGF beta activated locally in bone. Stimulation of osteoblast production of mRNA for uPA could reflect effects on the synthesis of sc-uPA, a precursor for the active form of the enzyme.

Topics: Animals; Cycloheximide; Dactinomycin; Fibrinolysin; Osteoblasts; Osteosarcoma; Plasminogen Activators; Plasminogen Inactivators; Protein Biosynthesis; Rats; RNA, Messenger; Tissue Plasminogen Activator; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator

Confluent cultures of rat bone cells synthesize several forms of secreted phosphoprotein 1 (SPP-1, osteopontin), the major phosphorylated forms of which migrate at 55 and 44 kDa on 15% cross-linked SDS-PAGE gels and correspond to the transformation-associated proteins pp 69 and pp 62. A clonal rat calvarial cell line (RCA 11), which expressed the highest level of SPP-1, produced only the 55 kDa form of the phosphorylated protein, whereas normal rat calvarial cells enriched in osteoblastic cells (RC IV cells) produced mostly the 55 kDa form, with small amounts of the 44 kDa form. In contrast, a 44 kDa form was the major [32PO4]-labelled SPP-1 synthesized by a rat osteocarcoma cell line (ROS 17/2.8 cells) with lesser amounts of the 55 kDa SPP-1. When [35S]methionine was used to measure protein synthesis, only the 55 kDa SPP-1 could be clearly detected in confluent cultures of each cell population, indicating that the 55 kDa SPP-1 is the prominent form of SPP-1 synthesized by each cell population. Following treatment of the normal rat bone cells for 24 h with osteotropic hormones (vit D3, PTH and RA), growth factors (PDGF, EGF, TGF-beta), a tumor promoter (TPA) and a plant lectin (Con A), the 55 kDa [35S]methionine labelled SPP-1 was increased 1.7-8.3-fold. Similar, but generally lower responses were observed in the clonal RCA 11 cell line, whereas the ROS 17/2.8 cells were more refractory, showing only a strong response to vit D3. In general, vit D3 produced the strongest stimulation in all populations with TGF-beta producing a good response in the non-transformed cells and RA in the RC IV cells. In contrast, PTH was inhibitory in both RCA 11 and ROS 17/2.8 cells. In most, but not all, cases the alteration in SPP-1 synthesis reflected similar changes in SPP-1 mRNA and in the intensity of the [32PO4]-labelled 55 kDa SPP-1. Collectively, these studies demonstrate that bone cells produce several forms of SPP-1 which are differentially regulated in normal and transformed cells through both transcriptional and posttranscriptional mechanisms.

Topics: Animals; Calcitriol; Cell Line; Cell Line, Transformed; Cells, Cultured; Concanavalin A; Epidermal Growth Factor; Growth Substances; Nucleic Acid Hybridization; Osteopontin; Osteosarcoma; Parathyroid Hormone; Platelet-Derived Growth Factor; Precipitin Tests; RNA, Messenger; Sialoglycoproteins; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta; Tretinoin

The metabolites of cycloxyegenase and lipoxygenase pathways are known to play an important role in the bone metabolism involving osteoclast and osteoblast interaction, bone resorption and morphogenesis. The recently discovered growth factor TGF-beta is abundant in bone and some of its intracellular and extracellular effects depend on de novo synthesis of eicosanoids. However, the effect of TGF-beta on the synthesis and the release of eicosanoid by bone cells is essentially unknown. In the present study we have identified the main eicosanoid metabolites produced by osteogenic osteosarcoma cell-line SAOS1 and investigated how production and release of these is affected by TGF-beta. We found that the leukotriene C4 is the main metabolite produced by these cells and that TGF-beta induces concentration-dependent, quantitative and qualitative alterations in eicosanoid production and release by human osteogenic osteosarcoma cells SAOS1.

Topics: Arachidonic Acid; Arachidonic Acids; Cell Line; Chromatography, High Pressure Liquid; Culture Media; Dose-Response Relationship, Drug; Eicosanoids; Humans; Kinetics; Lipoxygenase; Osteosarcoma; Transforming Growth Factor beta

Clinical observations have demonstrated a positive effect of estrogens and androgens on the maintenance of structural bone integrity. This study examines the direct effects of androgenic hormones on the osteoblast-like human osteosarcoma cell line, HOS TE85. Employing radiolabeled dihydrotestosterone (DHT), 2800 saturable, high-affinity (dissociation constant = 0.66 nM) androgen binding sites were detected per HOS TE85 cell. Androgen binding was specific in that DHT and testosterone (T) displayed significantly greater competition than the progestins, progesterone and medroxyprogesterone. The expression of androgen receptors in HOS TE85 cells was further substantiated by Northern analysis. A human androgen receptor complementary DNA probe revealed a 9.5 kilobase transcript which corresponds to the predominant human androgen receptor transcript detected in human male reproductive tissues. Androgens were also found to elicit biological responses in HOS TE85 cells. Physiological concentrations of DHT and T decreased HOS TE85 cell proliferation as assessed by cell count. This finding suggests that DHT may also play a role in osteoblast differentiation. In support of this hypothesis, treatment with T (24 h, 10 nM) enhanced the abundance of both alpha 1(I)-procollagen messenger RNA (mRNA) (5-fold) and transforming growth factor-beta mRNA (2.2 fold). The nonaromatizable androgen DHT (24 h, 10 nM) elicited an increase in the steady state concentration of alpha 1(I)-procollagen mRNA similar to the increase observed with T treatment. Thus, in addition to the recent discovery of estradiol receptors and estrogenic regulation of HOS TE85 cells, it is now evident that these osteoblast-like osteosarcoma cells also express high affinity androgen binding sites and can respond biologically to androgens.

Topics: Androgens; Binding, Competitive; Cell Division; Homeostasis; Humans; Osteoblasts; Osteosarcoma; Procollagen; Receptors, Androgen; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and heparin-binding growth factor-1 (HBGF-1) stimulated the proliferation of a variant of the human osteosarcoma cell line, MG-63-LS (LS = low serum). Transforming growth factor beta (TGF-beta) completely inhibited cell growth in basal medium supplemented with 2% fetal calf serum (FCS), blocked PDGF- and EGF-stimulated cell proliferation, and modulated that of HBGF-1. PDGF, but not EGF or HBGF-1, activated the inositol trisphosphate/diacylglycerol (IP3/DAG) second message system in a dose-dependent manner. EGF inhibited phosphoinositol lipid turnover and HBGF-1 and TGF-beta stimulated phosphatidylinositol hydrolysis to produce inositol phosphate (IP) but not IP3. Preincubation of quiescent cells with TGF-beta for 30-40 minutes prior to the addition of PDGF resulted in an inhibition of PDGF-induced production of IP3. This suggested that TGF-beta was an indirect inhibitor and blocked PDGF-stimulated cell growth in part by interfering with the generation of the second messenger, IP3.

Topics: Cell Division; Cell Line; Fibroblast Growth Factor 1; Humans; Hydrolysis; Inositol; Inositol 1,4,5-Trisphosphate; Kinetics; Osteosarcoma; Phosphatidylinositols; Platelet-Derived Growth Factor; Transforming Growth Factor beta

It has long been thought that the process of bone remodeling is regulated by the chain reactions of bone cells involving chemical mediators, growth factors and synthesis of extracellular matrix proteins etc. In this context, it has also been recognized that physical stimulation is an important factor in the regulation of bone remodeling. Thus, it is vitally important to understand whether the physical stimulation can induce the cellular events regarding autocrine regulation of protein synthesis. This study was conducted to examine the effects of hydrostatic intermittent compressive force (ICF) on the synthesis of the transforming growth factor beta (TGF-beta) and matrix phosphoproteins which may play an important role in the process of bone remodeling. The rat osteosarcoma cells (ROS 17/2.8) were cultured with DMEM containing 10% FCSP. ICF was applied to sub-confluent cells at 130 mb, 15/min cycle for 48h. ICF increased TGF-beta activity of the conditioned medium. This was assessed by its capacity to promote anchorage independent growth of NRK 49F cells and to inhibit the growth of human hepatoma cells (Hep-3B). Furthermore, ICF stimulated the synthesis of the phosphoproteins with Mr. 75 KDa by about 1.4 fold which was visualized by SDS-PAGE on 5-15% gradient gel. Immunoprecipitation of the phosphoproteins with rat osteopontin antibody revealed that the 75 KDa phosphoprotein was identical to osteopontin. The 75 KDa osteopontin synthesis was inhibited by the addition of TGF-beta antibody in a dose dependent manner. These results suggested that ICF stimulated the synthesis of TGF-beta and osteopontin in ROS 17/2.8 cells and that the osteopontin synthesis could be regulated by TGF-beta.

Topics: Animals; Bone Matrix; Bone Remodeling; Hydrostatic Pressure; Osteopontin; Osteosarcoma; Phosphoproteins; Physical Stimulation; Rats; Sialoglycoproteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta (TGF beta) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone-forming cells. TGF beta has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast-like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGF beta plays a role in regulating mineral deposition in the matrix, the effects of TGF beta on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGF beta (0.1-10 ng/ml) to examine the influence of cell maturation on response to TGF beta. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high-density cultures, there was further stimulation at 10 ng/ml. There was a dose-dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low-density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose-dependent increase in phospholipase A2-specific activity in the plasma membranes and matrix vesicles of both high- and low-density cultures. In agreement with previous studies, TGF beta inhibited cellular proliferation 50%. The results show that addition of TGF beta stimulates the activity of enzymes associated with calcification. The effect of TGF beta is dependent on the stage of maturation of the cell. This study indicates that TGF beta may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.

Topics: Alkaline Phosphatase; Animals; Cell Count; Cell Differentiation; Cell Membrane; Extracellular Matrix; Osteoblasts; Osteosarcoma; Phospholipases A; Phospholipases A2; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Characterization of the polypeptides present in bone-inductive protein extracts from bovine bone has led to the cloning of seven regulatory molecules, six of which are distantly related to transforming growth factor beta. The three human bone morphogenetic proteins (BMPs) we describe herein, BMP-5, BMP-6, and BMP-7, show extensive sequence similarity to BMP-2, a molecule that by itself is sufficient to induce de novo bone formation in vivo. The additive or synergistic contribution of these BMP-2-related molecules to the osteogenic activity associated with demineralized bone is strongly implicated by the presence of these growth factors in the most active fractions of highly purified bone extract.

Topics: Amino Acid Sequence; Base Sequence; Bone and Bones; Cell Line; Cloning, Molecular; DNA, Neoplasm; Gene Library; Humans; Molecular Sequence Data; Oligonucleotide Probes; Osteosarcoma; Proteins; RNA, Messenger; RNA, Neoplasm; Sequence Homology, Nucleic Acid; Transforming Growth Factor beta