transforming-growth-factor-beta and Uterine-Neoplasms

Uterine fibroids (UFs) are benign tumors of the female genital tract made of the smooth muscle of the uterus. UF growth depends mostly on the influence of the steroid hormones and selected growth factors. Transforming growth factor β (TGF-βs) is a polypeptide that consists of three isoforms: TGF-β1, TGF-β2, and TGF-β3. At present, TGF-β is considered to be one of the key factors in the pathophysiology of UFs. It plays a major role in cellular migration within the tumor, stimulates tumor growth, and enhances tumor metabolism. As a consequence of various dependencies, the synthesis and release of TGF-β in a UF tumor is increased, which results in excessive extracellular matrix production and storage. High concentrations or overexpression of TGF-β mediators may be responsible for clinically symptomatic UFs. The aim of this review was to check the available evidence for the influence of the TGF-β family on UF biology. We conducted their search in PubMed of the National Library of Medicine with the use of the following selected keywords: "uterine fibroid", "leiomyoma", and "transforming growth factor β". After reviewing the titles and abstracts, more than 115 full articles were evaluated. We focused on the TGF-β-related molecular aspects and their influence on the most common symptoms that are associated with UFs. Also, we described how the available data might implicate the current medical management of UFs.

Topics: Aromatase Inhibitors; Estrogens; Female; Gonadotropin-Releasing Hormone; Humans; Leiomyoma; Progesterone; Protein Isoforms; Transforming Growth Factor beta; Uterine Neoplasms

Hyaluronan and versican are extracellular matrix (ECM) components that are enriched in the provisional matrices that form during the early stages of development and disease. These two molecules interact to create pericellular "coats" and "open space" that facilitate cell sorting, proliferation, migration, and survival. Such complexes also impact the recruitment of leukocytes during development and in the early stages of disease. Once thought to be inert components of the ECM that help hold cells together, it is now quite clear that they play important roles in controlling cell phenotype, shaping tissue response to injury and maintaining tissue homeostasis. Conversion of hyaluronan-/versican-enriched provisional matrix to collagen-rich matrix is a "hallmark" of tissue fibrosis. Targeting the hyaluronan and versican content of provisional matrices in a variety of diseases including, cardiovascular disease and cancer, is becoming an attractive strategy for intervention.

Topics: Animals; Cell Proliferation; Cells, Cultured; Collagen; Disease Models, Animal; Extracellular Matrix; Female; Fibrosis; Gene Expression Regulation; Humans; Hyaluronic Acid; Leiomyosarcoma; Mice; Muscle, Smooth, Vascular; Platelet-Derived Growth Factor; RNA, Small Interfering; Transforming Growth Factor beta; Uterine Neoplasms; Versicans

Leiomyomas are believed to derive from the transformation of myometrial smooth muscle cells/connective tissue fibroblasts. Although the identity of the molecule(s) that initiate such cellular transformation and orchestrate subsequent growth is still unknown, conventional evidence indicates that ovarian steroids are essential for leiomyoma growth. Ovarian steroid action in their target cell/tissue is mediated in part through local expression of various growth factors, cytokines, and chemokines. These autocrine/paracrine molecules with proinflammatory and profibrotic activities serve as major contributing factors in regulating cellular transformation, cell growth and apoptosis, angiogenesis, cellular hypertrophy, and excess tissue turnover, events central to leiomyoma growth. This review addresses the key regulatory functions of proinflammatory and profibrotic mediators and their molecular mechanisms, downstream signaling that regulates cellular events that result in transformation, and commitments of specific cells into forming a cellular environment with a possible role in development and subsequent growth of leiomyomas.

Topics: Female; Humans; Inflammation Mediators; Leiomyoma; MicroRNAs; Phenotype; Transforming Growth Factor beta; Uterine Neoplasms

Topics: Danazol; Diagnosis, Differential; Diagnostic Imaging; Female; Gonadotropin-Releasing Hormone; HMGA Proteins; Humans; Hysterectomy; Hysteroscopy; Leiomyoma; Prognosis; Transforming Growth Factor beta; Uterine Neoplasms

Virtually all known cytokines have been demonstrated to be expressed in the placenta and associated fetal and maternal membranes during normal gestation. In addition to playing their traditional roles as modulators of immunological function, cytokines derived from the placenta and extraplacental membranes, together with other locally-derived growth factors, appear to be implicated in various aspects of implantation and placental development. Imbalances in the intrauterine cytokine milieu around the time of implantation and invasion may play a causative role in disorders associated with early pregnancy failure, and are also associated with the abnormal trophoblast development seen in gestational trophoblastic disease. Cytokines thus appear to be an important component of a paracrine/autocrine communication network operating within the feto-maternal interface to ensure the successful establishment of pregnancy.

Topics: Abortion, Habitual; Chemokines; Cytokines; Embryo Implantation; Female; Growth Substances; Humans; Interferons; Maternal-Fetal Exchange; Placenta; Placentation; Pregnancy; Transforming Growth Factor beta; Trophoblastic Neoplasms; Uterine Neoplasms

Leiomyomas are a significant problem in women's health. An understanding of the biology of these tumors and how their growth is regulated is emerging from in vitro studies using tissue specimens and cultured cells. These studies have clarified how the ovarian steroid hormones regulate growth of uterine SMCs and how the ovarian steroid ligand-receptor system has been altered in leiomyomas. Such information will allow investigators to identify steroid hormone antagonists and steroid hormone receptor modulators that may be useful for treatment of leiomyomas. We are now also developing a much better understanding of the growth factors that are produced by SMCs of leiomyoma tumors. These growth factors not only regulate the proliferation, apoptosis, and extra-cellular matrix production of the SMCs but also regulate proliferation and migration of vascular endothelial cells. Targeting these growth factors and their receptors can reduce leiomyoma growth through two different mechanisms. One targets the SMCs and the other targets the vascular system that supports the growth of the tumor. Another important lesson that can be learned from reading the scientific literature is that there are striking similarities between the biology of uterine leiomyomas and other pathologic diseases that involve mesenchymally derived cells. These include benign keloids, other fibrotic diseases such as pulmonary fibrosis, and vascular diseases such as atherosclerosis. Compounds that are developed to treat these conditions may also be beneficial for treatment of uterine leiomyomas. The next few years will undoubtedly yield many new drug discoveries for these diseases.

Topics: Angiogenesis Inducing Agents; Female; Fibrosis; Humans; In Vitro Techniques; Leiomyoma; Somatomedins; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

To evaluate the clinical features and the expression of transforming growth factor-beta3 (TGF-beta3) and connective tissue growth factor (CTGF) in myometrium and uterine leiomyomas after preoperative treatment with gonadotropin-releasing hormone-analogs (GnRH-a) and tibolone.. Twenty-three patients received 3.75 mg leuprolide acetate depot for 4 months. Twenty-two patients received the same therapy plus 2.5 mg tibolone daily. Patients underwent uterine surgery after therapy. Twenty-two untreated patients underwent surgery directly. Hematologic tests, bone mineral density (BMD) measurement, and ultrasonographic evaluation of uterine volume were performed before and after treatment. Menorrhagia and pelvic pain were evaluated with a visual analog scale. Hot flushes were recorded in daily diaries. Immunohistochemical expression of TGF-beta3 and CTGF in myometrium and myoma samples was evaluated semiquantitatively.. After therapy, hemoglobin and iron levels similarly increased in both groups. BMD significantly decreased only in the GnRH-a group. Uterine volume similarly decreased in both groups. No patient had menorrhagia or pelvic pain at the end of therapy. The number of hot flushes increased after the first month in the GnRH-a group; in the GnRH-a plus tibolone group, it remained constant and was lower. In untreated cases, TGF-beta3 and CTGF smooth muscle cell immunoexpression was lower in myometrium than in leiomyomas. After medical treatment, growth factor immunoexpression remained unchanged in myometrial samples and was reduced in leiomyomas. Endothelial cells showed strong immunopositivity, both in untreated and in treated cases.. This study focuses on the effects of GnRH-a and tibolone on TGF-beta3 and CTGF expression in myometrium and myomas and supports the hypothesis of a pathogenetic role of these growth factors in uterine fibromatosis.

Topics: Adult; Antineoplastic Agents, Hormonal; Bone Density; Connective Tissue Growth Factor; Drug Therapy, Combination; Female; Hemoglobins; Hot Flashes; Humans; Immediate-Early Proteins; Immunohistochemistry; Injections, Subcutaneous; Intercellular Signaling Peptides and Proteins; Iron; Leiomyoma; Leuprolide; Myometrium; Neoadjuvant Therapy; Norpregnenes; Transforming Growth Factor beta; Transforming Growth Factor beta3; Uterine Neoplasms

Topics: Cell Culture Techniques, Three Dimensional; Cell Proliferation; Estrogens; Extracellular Matrix; Female; Fibrosis; Humans; Leiomyoma; Phosphorylation; Polybrominated Biphenyls; Signal Transduction; Transforming Growth Factor beta; Uterine Neoplasms

Topics: Female; Humans; Intercellular Signaling Peptides and Proteins; Leiomyoma; Mifepristone; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factors; Uterine Neoplasms; Vascular Endothelial Growth Factor A

Transforming growth factor β (TGF-β) and leucine-rich α-2-glycoprotein 1 (LRG1) play significant roles in the pathogenicity of uterine leiomyomas (ULMs). The current study aimed to assess the diagnostic values of serum TGF-β and LRG1 in terms of the presence and severity of ULMs.. Premenopausal women with ULMs (n=44) together with age-adjusted ULM-free individuals (n=41) were incorporated into the study. ULMs were detected and evaluated using transvaginal ultrasonography. Serum levels of TGF-β and LRG1 were quantified by enzyme-linked immunosorbent assay.. Mean concentrations of serum TGF-β and LRG1 were significantly higher in the group of patients with ULMs compared to the control group (p<0.05). The volume of the largest leiomyoma was positively correlated with the levels of TGF-β (r = 0.414, p= 0.005) and LRG1 (r = 0.341, p= 0.023). The receiver-operating characteristics analysis demonstrated moderate and robust values of area under the curve for TGF-β (0.755) and LRG1 (0.90), respectively.. Increases in serum levels of TGF-β and LRG1 is associated with the incidence and severity of ULMs. LRG1 in particular but also TGF-β may be able to serve as reliable biomarkers for the diagnosis and monitoring of ULMs.

Topics: Adult; Biomarkers; Cross-Sectional Studies; Female; Glycoproteins; Humans; Leiomyoma; Prospective Studies; ROC Curve; Transforming Growth Factor beta; Uterine Neoplasms

Topics: Cadmium; Cell Line, Tumor; Extracellular Matrix; Female; Humans; Leiomyoma; Signal Transduction; Transforming Growth Factor beta; Uterine Neoplasms

To study the transforming growth factor beta (TGF-β) signaling pathway in interactions with estrogen receptor alpha (ERα) signaling pathway mediating the growth of human uterine leiomyoma (UL) activated by phenolic environmental estrogens (EEs).. The subcultured UL cells were used to determine the validation of TGF-β3 for the viability of human UL cells using CCK-8 assay, mRNA expressions of ERα, and c-fos by quantitative reverse transcription polymerase chain reaction method, and expressions of p-Smad3, SnoN, and c-fos proteins by Western blot assay in each treatment group.. Compared with each of EEs or TGF-β3 treatment, slightly decrease in the proliferation rate of UL was detected in the coexistence of each EE with TGF-β3. Interestingly, mRNA expressions of ERα and c-fos reduced in the setting of coexistence of TGF-β3 and EEs. Somehow, the expression of p-Smad3 and c-fos proteins significantly decreased in each of E2, bisphenol A (BPA), nonylphenol (NP), and octylphenol (OP) group, as well as the expression of SnoN protein significantly reduced only in BPA and NP groups, followed by TGF-β3 treatment. With the overlaid action of ICI 182,780, the expression of p-Smad3 protein significantly increased in OP group, but slightly increased in E2, BPA, NP, and OP groups. However, compared with the control group, the expression of SnoN and c-fos proteins significantly decreased in the same setting.. Both ERα signaling pathway and TGF-β signaling pathway have different roles in governing UL cell proliferation. The phenolic EEs can be a promoter to the proliferation of UL cells, which is mediated by ERα signaling pathway and cross-talked with TGF-β signaling pathway.

Topics: Adult; Apoptosis; Benzhydryl Compounds; Cell Proliferation; Estrogen Receptor alpha; Estrogens, Non-Steroidal; Female; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Leiomyoma; Middle Aged; Phenols; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

Uterine fibroids, or leiomyoma, are the most common benign tumors in women of reproductive age. In this work, the effect of silencing the mediator complex subunit 12 (Med12) gene in human uterine fibroid cells was evaluated. The role of Med12 in the modulation of Wnt/β-catenin and cell proliferation-associated signaling was evaluated in human uterine fibroid cells. Med12 was silenced in the immortalized human uterine fibroid cell line (HuLM) using a lentivirus-based Med12 gene-specific RNA interference strategy. HuLM cells were infected with lentiviruses carrying Med12-specific short hairpin RNA (shRNA) sequences or a nonfunctional shRNA scrambled control with green fluorescence protein. Stable cells that expressed low levels of Med12 protein were characterized. Wnt/β-catenin signaling, sex steroid receptor signaling, cell cycle-associated, and fibrosis-associated proteins were measured. Med12 knockdown cells showed significantly (P < 0.05) reduced levels of Wnt4 and β-catenin proteins as well as cell proliferation, as compared with scrambled control cells. Med12 knockdown cells also showed reduced levels of cell cycle-associated cyclin D1, Cdk1, and Cdk2 proteins as well as reduced activation of p-extracellular signal-regulated kinase, p-protein kinase B, and transforming growth factor (TGF)-β signaling pathways as compared with scrambled control cells. Moreover, TGF-β-regulated fibrosis-related proteins such as fibronectin, collagen type 1, and plasminogen activator inhibitor-1 were significantly (P < 0.05) reduced in Med12 knockdown cells as compared with scrambled control cells. Together, these results suggest that Med12 plays a key role in the regulation of HuLM cell proliferation through the modulation of Wnt/β-catenin, cell cycle-associated, and fibrosis-associated protein expression.

Topics: beta Catenin; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Collagen Type I; Cyclic AMP-Dependent Protein Kinases; Estrogen Receptor alpha; Female; Fibronectins; Gene Silencing; Humans; Leiomyoma; Mediator Complex; Proto-Oncogene Proteins c-akt; Smad Proteins; Transforming Growth Factor beta; Uterine Neoplasms; Wnt Signaling Pathway

The aim of this paper is to study the participation of transforming growth factor-β (TGF-β) signaling pathway in mediating the growth of human uterine leiomyoma (UL) activated by phenolic environmental estrogens (EEs), via the interaction between TGF-β and ER signaling pathways. The UL cells were prepared by primary culture and subculture methods. To validate the role of TGF-β3 (5 ng/ml) for the viability of human uterine leiomyoma cells, CCK-8 assay was performed in each of five treatment groups including E2 group (E2 10(9) mol/l), BPA group (bisphenol A 10 μmol/l), NP group (nonylphenol 32 μmol/l), OP group (octylphenol 8 μmol/l), or control group (DMSO only). Subsequently, qRT-PCR was applied to detect mRNA expressions of ERα and c-fos, while western blot assay was used to test the expressions of p-Smad3, SnoN, and c-fos proteins in all settings mentioned above; the expressions were compared among different groups, and also in settings with and without synchronous treatment of ICI 182,780. Primarily cultured UL cells were successfully established. Compared with the control group, there were statistically significant increases in the proliferation rate of the UL cells in all EE groups or treated with TGF-β3 only (p < 0.05). Nevertheless, a slight decrease in proliferation rate of UL was detected in coexistence with TGF-β3 in all EE groups (p > 0.05). Interestingly, mRNA expressions of ERα and c-fos reduced in the setting of coexistence of TGF-β3 and EEs compared to isolated EE treatment (p < 0.05). Compared with the control group, the expression of p-Smad3 and c-fos proteins significantly decreased (p < 0.05) in each of E2, BPA, NP, and OP group, and the expression of SnoN protein also significantly reduced only in BPA and NP groups (p < 0.05), followed by TGF-β3 treatment. When adding ICI 182,780, the expression of p-Smad3 protein significantly increased in OP group (p < 0.05), but slightly increased in E2, BPA, NP, and OP groups (p > 0.05). However, compared with the control group, the expressions of SnoN and c-fos proteins significantly decreased (p < 0.05) after adding ICI182,780. Moreover, there was a significant statistical difference in the expression of p-Smad3, SnoN, and c-fos proteins between pre- and post-treatment of ICI 182,780 in all groups (p < 0.05). The ERα signaling pathway and TGF-β signaling pathway have different roles in the control of UL cell proliferation. The phenolic EEs can be a promoter of UL cell proliferation, which is medi

Topics: Adult; Benzhydryl Compounds; Cell Proliferation; Cell Survival; Estrogen Receptor alpha; Estrogens; Female; Humans; Leiomyoma; Middle Aged; Phenol; Phenols; Proto-Oncogene Proteins c-fos; RNA, Messenger; Signal Transduction; Smad Proteins; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta3; Uterine Neoplasms

Topics: Biomarkers; Humans; Leiomyoma; Transforming Growth Factor beta; Uterine Neoplasms; Vitamin D Deficiency

To determine whether transforming growth factor (TGF)-β3 is a paracrine signal secreted by leiomyoma that inhibits bone morphogenetic protein (BMP)-mediated endometrial receptivity and decidualization.. Experimental.. Laboratory.. Women with symptomatic leiomyomas.. Endometrial stromal cells (ESCs) and leiomyoma cells were isolated from surgical specimens. Leiomyoma-conditioned media (LCM) was applied to cultured ESC. The TGF-β was blocked by two approaches: TGF-β pan-specific antibody or transfection with a mutant TGF-β receptor type II. Cells were then treated with recombinant human BMP-2 to assess BMP responsiveness.. Expression of BMP receptor types 1A, 1B, 2, as well as endometrial receptivity mediators HOXA10 and leukemia inhibitory factor (LIF).. Enzyme-linked immunosorbent assay showed elevated TGF-β levels in LCM. LCM treatment of ESC reduced expression of BMP receptor types 1B and 2 to approximately 60% of pretreatment levels. Preincubation of LCM with TGF-β neutralizing antibody or mutant TGF receptor, but not respective controls, prevented repression of BMP receptors. HOXA10 and LIF expression was repressed in recombinant human BMP-2 treated, LCM exposed ESC. Pretreatment of LCM with TGF-β antibody or transfection with mutant TGF receptor prevented HOXA10 and LIF repression.. Leiomyoma-derived TGF-β was necessary and sufficient to alter endometrial BMP-2 responsiveness. Blockade of TGF-β prevents repression of BMP-2 receptors and restores BMP-2-stimulated expression of HOXA10 and LIF. Blockade of TGF signaling is a potential strategy to improve infertility and pregnancy loss associated with uterine leiomyoma.

Topics: Adult; Bone Morphogenetic Protein 2; Case-Control Studies; Cells, Cultured; Decidua; Embryo Implantation; Endometrium; Female; Humans; Leiomyoma; Middle Aged; Pregnancy; Recombinant Proteins; Transforming Growth Factor beta; Uterine Neoplasms

Uterine carcinosarcomas (UCS) are rare (3-4%) but highly aggressive, accounting for a disproportionately high (16.4%) mortality among uterine malignancies. Transforming growth factor beta (TGFβ) is a multifunctional cytokine that regulates important cellular processes including epithelial-mesenchymal transition (EMT). Existence of biphasic elements and a report demonstrating amplification of TGFβ at 19q13.1 prompted us to investigate the role of TGFβ signaling in UCS.Here we demonstrated the components of TGFβ pathway are expressed and functional in UCS. TGFβ-I induced significant Smad2/3 phosphorylation, migration and EMT responses in UCS cell lines which could be attenuated by the TGFβ receptor I (TGFβR-I) or TGFβ receptor I/II (TGFβR-I/II) inhibitor developed by Eli Lilly and company. Importantly, TGFβ-I induced proliferation was c-Myc dependent, likely through activation of cell cycle. c-Myc was induced by nuclear translocation of nuclear factor of activated T cells (NFAT-1) in response to TGFβ-I. Inhibition of NFAT-1 or TGFβR-I blocked c-Myc induction, cell cycle progression and proliferation in UCS. In corroboration, mRNA levels of c-Myc were elevated in recurrent versus the non-recurrent UCS patient samples. Interestingly, in the absence of exogenous TGFβ the TGFβR-I/II inhibitor enhanced proliferation likely through non-Smad pathways. Thus, inhibition of TGFβR-I could be efficacious in treatment of UCS.

Topics: Carcinosarcoma; Cell Movement; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Humans; Signal Transduction; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

Uterine fibroids (UF) are the most common benign tumor of the female reproductive tract. The aim of this study was to explore the role of lipopolysaccharide (LPS)-induced activation of TLR4/NF-κB signaling pathway on stromal fibroblasts in the pathogenesis of UF. Here, TLR4/NF-κB signaling pathway was more activated in UF, and UF cells (UFC) and UF derived fibroblasts (TAF) than in smooth muscle tissues, smooth muscle cell (SMC) and myometrial fibroblasts (fib) respectively. After lipopolysaccharide (LPS) stimulation, the activity of fib was enhanced, characterized by the increased expression of fibroblast activation protein (FAP), and increased secretion of collagen I and transforming growth factor-β (TGF-β). Moreover, TLR4 inhibitor (VIPER) and siTLR4 can represses LPS-activated fibroblasts and TLR4/NF-κB signaling transduction pathways in fib and UFC cells. Co-cultured with LPS-activated fibroblast enhanced fibroblast activation and TLR4/NF-κB signaling. In conclusion, LPS treatment activated TLR4/NF-κB signaling pathway on fibroblasts, which may involve in the development of UF. Our study indicated reproductive tract infection may be associated with fibroid pathogenesis through TLR4/NF-κB signaling. Targeting NF-κB with inhibitors may hold promises of treating uterine fibroid.

Topics: Collagen Type I; Endopeptidases; Female; Fibroblasts; Gelatinases; Humans; Leiomyoma; Lipopolysaccharides; Membrane Proteins; Muscle, Smooth; NF-kappa B; Serine Endopeptidases; Signal Transduction; Toll-Like Receptor 4; Transforming Growth Factor beta; Uterine Neoplasms

Data assessing the role of various genetic alterations in uterine carcinosarcoma (CS), particularly the transforming growth factors-β (TGFβ) that play a crucial role in many cellular processes, including proliferation, differentiation, adhesion and migration, are scarce. TGFβ exert their effects through specific receptors and associated auxiliary receptors. In the current study, we investigated the expression of TGFβ isoforms and their receptors, as well as selected genes in a case of CS. We applied the real-time fluorescence detection PCR method with FAM dye-labeled TaqMan specific probes. In a comparison to the normal counterpart, TGFB1, TGFB2, TGFBRII, TGFBR3, ENG and CD109 were all down-regulated in uterine CS samples at different extents. BIRC5 and hTERT, markers of tumor survival, were up-regulated in CS as compared with normal counterparts. A concomitant increase of the hypoxia marker HIF1A expression pattern was noted, whereas the expression of GPR120, responsible for free fatty acids sensing, was not different in both counterparts evaluated. In conclusion, deregulation of various cellular mechanisms in uterine CS is associated with alterations at many levels - cell growth and proliferation, apoptosis, and impaired response to stimuli from extracellular environment.

Topics: Aged; Apoptosis; Carcinosarcoma; Cell Proliferation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Humans; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Uterine Neoplasms

Endoglin (CD105), an accessory receptor of transforming growth factor-β, is expressed in vascular endothelial cells. Recently, it was reported that endoglin expression was significantly associated with poorer survival in several cancers. In this study, we evaluated the role of endoglin in uterine leiomyosarcoma. We examined the expression of endoglin in 22 uterine leiomyosarcomas and the association between their expression and the outcome. Additionally, to evaluate the function of endoglin, we used SKN cells, a human uterine leiomyosarcoma cell line. We generated SKN cells stably transfected with plasmids encompassing shRNA targeting endoglin (shEng cells), and compared the ability of proliferation, migration, and invasion to control shRNA-transfected cells (shCon cells). We compared the level of VEGF and matrix metalloproteinases (MMP) in culture supernatants of shEndoglin and shControl cells. Nine patients were endoglin-positive and 13 patients were -negative. The endoglin-positive group had a significantly poorer overall survival and progression-free survival than the endoglin-negative group. In an in vitro study, there was no difference in cell proliferation between shEng and shCon cells. On the other hand, shEng cells showed a lower ability for migration and invasion than shControl cells. The activity of MMP-9 and VEGF level in the supernatant from shEng cells were lower than in shCon cells. In uterine leiomyosarcoma, endoglin expression was associated with a poor prognosis. It was suggested that endoglin up-regulated invasion and VEGF secretion. The investigation of endoglin may lead to a new strategy in uterine leiomyosarcoma therapy.

Topics: Adult; Aged; Antigens, CD; Cell Line, Tumor; Endoglin; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Immunohistochemistry; Leiomyosarcoma; Middle Aged; Neoplasm Invasiveness; Receptors, Cell Surface; Signal Transduction; Transforming Growth Factor beta; Uterine Neoplasms; Vascular Endothelial Growth Factor A

Previously, we found that high doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. In this study, using microarray analysis and Ingenuity Pathways Analysis™, we identified genes (up- or down-regulated, ≥ 1.5 fold, P ≤ 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 μg/ml) in UtLM cells. Downregulation of TGF-β signaling pathway genes, activin A, activin B, Smad3, TGF-β2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real- time RT-PCR studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that downregulation of activin A and Smad3, both members of the TGF-β pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas.

Topics: Activins; Anticarcinogenic Agents; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p15; Down-Regulation; Female; Genistein; Humans; Leiomyoma; Oligonucleotide Array Sequence Analysis; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation; Uterine Neoplasms

To investigate the effects of the antifibrotic drug halofuginone on extracellular matrix production, cell proliferation, and apoptosis of cultured myometrial and leiomyoma smooth muscle cells.. Comparative and controlled experimental research study.. University research laboratory.. Leiomyoma and myometrial tissues were obtained from eight different patients at the time of elective hysterectomy.. The effects of halofuginone on cell proliferation were assessed by tritiated thymidine uptake assays and cell count assays. Effects on TGFbeta1, collagen type I, and collagen type III mRNA levels were assessed by quantitative real-time polymerase chain reaction. Effects on apoptosis were assayed using a chemiluminescent assay to measure changes in caspase 3 and 7.. Halofuginone inhibited cell proliferation of both leiomyoma and autologous myometrial cells in a dose-dependent manner by inhibiting DNA synthesis within 24 hours and later inducing apoptosis (as measured by increased caspase 3/7) by 48-72 hours. Halofuginone also significantly reduced collagen type I (alpha1) and collagen type III (alpha1) mRNA levels, as well as the profibrotic factor TGFbeta1 mRNA levels in both cell types.. These results provide evidence to support the use of the antifibrotic drug halofuginone as a novel drug treatment for uterine leiomyomas.

Topics: Apoptosis; Cell Proliferation; Collagen Type I; Collagen Type III; Female; Fibrosis; Growth Inhibitors; Humans; Leiomyoma; Myocytes, Smooth Muscle; Myometrium; Piperidines; Quinazolinones; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

Aberrant expression of microRNAs (miRNAs), including miR-21, and alteration of their target genes stability have been associated with cellular transformation and tumorigenesis. We investigated the expression, regulation and function of miR-21 in leiomyomas which develop from myometrial cellular transformation. The results indicated that miR-21 is over-expressed in leiomyomas with specific elevation during the secretory phase of the menstrual cycle and in women who received Depo-Provera and oral contraceptives, but reduced due to GnRHa therapy (P < 0.05). Bioinformatic analysis of microarray gene expression profiles previously obtained from the above cohorts, and myometrial smooth muscle cells (MSMC) and leiomyoma smooth muscle cells (LSMC) treated with GnRHa, transforming growth factor (TGF)-beta and TGF-beta receptor type II (TGF-betaRII) antisense oligomer, indicated that a number of miR-21-predicted target genes were co-expressed and differentially regulated in these cohorts. Gain- and loss-of-function of miR-21 in MSMC, LSMC, transformed LSMC and leiomyosarcoma cell line (SKLM-S1) resulted in differential expression of many genes, including some of the miR-21-predicted/validated target genes, PTEN, PDCD4 and E2F1, and TGF-betaRII, in a cell-specific manner. Gain-of miR-21 function in MSMC and LSMC reduced TGF-beta-induced expression of fibromodulin and TGF-beta-induced factor (P < 0.05), and moderately altered the rate of cell growth and caspase-3/7 activity in these cells. We concluded that miR-21 is aberrantly expressed and hormonally regulated in leiomyomas where, through functional interaction with ovarian steroids and the TGF-beta signaling pathway, either directly or indirectly regulates a number of genes whose products are critical in leiomyoma growth and regression as well as their potential cellular transformation.

Topics: Adult; Blotting, Western; Cell Proliferation; Cells, Cultured; Computational Biology; E2F1 Transcription Factor; Female; Gonadotropin-Releasing Hormone; Humans; Leiomyoma; Leiomyosarcoma; MicroRNAs; Middle Aged; Myocytes, Smooth Muscle; Oligonucleotide Array Sequence Analysis; PTEN Phosphohydrolase; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

Uterine leiomyomas are highly prevalent and symptomatic tumors of women in their reproductive years. The morbidity caused by these tumors is directly related to increasing size. Leiomyoma cells do not rapidly proliferate; instead, the tumors grow primarily due to excessive production of disorganized extracellular matrix (ECM). The aberrant ECM results from excessive production of collagen subtypes and proteoglycans, increased profibrotic cytokines including transforming growth factors beta1 and beta3, and decreased or disrupted matrix metalloproteinases. These alterations result in the development of an ECM that is exceptionally stable. As a result, therapeutic interventions must redirect leiomyoma cells toward extracellular matrix dissolution, rather than solely inhibiting cell proliferation. Gonadotropin-releasing hormone analogues and selective progesterone receptor modulators with demonstrated clinical efficacy provide such a change in abnormal extracellular matrix formation by leiomyoma cells, inhibiting and reversing the fibrotic process. Novel therapies using pathways distinct from gonadal hormones, including antifibrotics, retinoic acid, peroxisome-proliferator-activated receptor gamma ligands, and curcumin, provide promise for a future with improved therapeutic options for women suffering from uterine leiomyomas.

Topics: Collagen; Extracellular Matrix; Female; Hormones; Humans; Leiomyoma; Matrix Metalloproteinases; Prevalence; Proteoglycans; Research; Terminology as Topic; Transforming Growth Factor beta; Uterine Neoplasms

This study aimed to determine the immunohistochemical characteristics of intramural leiomyomata that enlarged during controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF).. For this retrospective case-control clinical study and immunohistochemical analysis, 5 patients with enlarged intramural leiomyomata during COH for IVF, who had undergone myomectomy immediately after a failed IVF cycle, were recruited retrospectively. Fifteen consecutive patients who had had myomectomy for intramural leiomyomata <5 cm, but had never undergone any infertility treatment, served as the control group. Histological examinations and immunohistochemical staining with proliferating cell nuclear antigen (PCNA), transforming growth factor-beta (TGF-beta) and fibronectin were performed on all specimens taken from the study and control groups. The main outcome measures were defined as histological and immunohistochemical scores.. Hematoxylin and eosin as well as PCNA staining showed increased mitotic index, cellularity and proliferation index in these growing leiomyomata (study group). There were neither increased mitoses nor cellularity in the leiomyomata of the control group, while PCNA, TGF-beta and fibronectin scores in the study group were significantly higher than those in the control group (p < 0.001).. This study is the first to report increased TGF-beta, PCNA, cellularity and mitosis in leiomyomata enlarging during COH for IVF. Further studies with larger sample size are needed to determine the role of TGF-beta, PCNA and fibronectin in the growth of leiomyomata during COH.

Topics: Adult; Case-Control Studies; Female; Fertilization in Vitro; Fibronectins; Follicle Stimulating Hormone; Gene Expression; Hormones; Humans; Immunohistochemistry; Infertility, Female; Leiomyoma; Mitosis; Ovary; Ovulation Induction; Proliferating Cell Nuclear Antigen; Retrospective Studies; Transforming Growth Factor beta; Uterine Neoplasms

Some authors suggest that growth factors are intermediate regulatory elements through which the ovarian hormones exert their growth-stimulatory effects on uterine leiomyomas.. It was decided to compare the amounts of transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) in myometrium and in uterine leiomyomas of various weights (small: less than 10 g and large: more than 100 g). The tissues were homogenised and extracted with 1M acetic acid or with 0.05 M Tris-HCl, pH 7.6. The extracts were assayed for TGF-beta and PDGF with the use of the ELISA technique.. The Tris-HCl was more efficient at extracting solvent than 1M of acetic acid. Both myometrium and leiomyomas contained nanogram amounts of extractable TGF-beta and picogram amounts of PDGF. Western immunoblotting demonstrated that both factors exist as stable complexes, probably with extracellular matrix components. The PDGF/TGF-beta ratio in Tris-HCl extracts was higher in leiomyomas than in myometrium and it increased during tumour growth.. It is known that low concentrations of TGF-beta induce proliferation of cells by stimulating autocrine PDGF secretion. Higher concentrations of TGF-beta1 evoke a reverse effect by the down-regulation of the PDGF receptor and by direct growth inhibition. The increase in the PDGF/TGF-beta ratio during tumour growth seems be important in tumour biology. The low amount of TGF-beta eliminates the inhibitory effect of this factor on cell proliferation and stimulates both autocrine PDGF secretion and promotes the synthesis of PDGF receptors. It is thus possible to bind more PDGF by myometrial cells resulting in a hyperplasia of myometrium and enhancement of extracellular matrix synthesis.

Topics: Adult; Extracellular Matrix; Female; Humans; Leiomyoma; Middle Aged; Multiprotein Complexes; Myometrium; Platelet-Derived Growth Factor; Transforming Growth Factor beta; Uterine Neoplasms

Smooth muscle tumors of uterus have been reported to contain considerable number of mast cells, especially cellular leiomyoma. However, to our knowledge the mechanism by which mast cells increased in them is not known. The purpose of this study was to reveal the different mast cell subsets in smooth muscle tumors of uterus and to investigate the mechanism of local increase of mast cells.. Tissue sections from 85 uterine smooth muscle tumors were studied using immunohistochemical double labeling techniques, including 40 cases of ordinary leiomyomas, 30 cases of cellular leiomyomas and 15 cases of leiomyosarcomas. The sections were double immunostained for mast cell tryptase and chymase, mast cell tryptase and ki-67, mast cell tryptase and chemokines (i.e., CCL2, CCL5, CCL11, TGFbeta), as well as tryptase and CCR3.. MC(TC)-type of mast cells was the predominant type in ordinary leiomyoma and cellular leiomyoma, whereas MC(T)-type was seldom found in them. There was no MC(C) in smooth muscle tumors. The total intratumoral number of mast cells in cellular leiomyoma group was significantly higher than that in both leiomyosarcoma and ordinary leiomyoma (P<0.01). Mast cells proliferation was rarely detected in smooth muscle tumors, as revealed by constant negative labeling of the proliferation marker Ki-67 in mast cells. Almost all mast cells (tryptase positive) in smooth muscle tumors were also CCL2, CCL5, CCL11 and TGFbeta positive. Expressions of CCL5 and CCL11 in tumor cells in cellular leiomyoma were all significantly higher than that in both ordinary leiomyoma and leiomyosarcoma (P<0.01). While the expression of TGFbeta in tumor cells in cellular leiomyoma was not significantly different from that in ordinary leiomyoma, expression of CCL2 was not observed in smooth muscle tumor cells. There were positive correlations between CCL5 and the number of mast cells (r(s)=0.801, P<0.01) and between CCL11 and the number of mast cells (r(s)=0.744, P<0.01) in smooth muscle tumors as well. The vast majority of the mast cells in cellular leiomyoma were CCR3 positive.. Using the monoclonal anti-mast cell tryptase antibody could detect all mast cells in smooth muscle tumor. The increased intratumoral mast cell counts in cellular leiomyoma might be the result of mast cells recruitment from the peripheral blood rather than local mast cells proliferation. CCL5 and CCL11, which are expressed by smooth muscle tumor cells, are possibly responsible for the recruitment of mast cells in uterine cellular leiomyoma. Whether they combine to CCR3 expressed by mast cells need further study.

Topics: Chemokine CCL11; Chemokine CCL2; Chemokine CCL5; Chemokines, CC; Female; Humans; Ki-67 Antigen; Leiomyoma; Leiomyosarcoma; Mast Cells; Transforming Growth Factor beta; Uterine Neoplasms

Transforming growth factor beta (TGF-beta), which generally stimulates the growth of mesenchymally derived cells but inhibits the growth of epithelial cells, has been proposed as a possible target for cancer therapy. However, concerns have been raised that whereas inhibition of TGF-beta signaling could be efficacious for lesions in which TGF-beta promotes tumor development and/or progression, systemic pharmacologic blockade of this signaling pathway could also promote the growth of epithelial lesions.. We examined the effect of a TGF-beta inhibitor on mesenchymal (leiomyoma) and epithelial (renal cell carcinoma) tumors in Eker rats, which are genetically predisposed to develop these tumors with a high frequency.. Blockade of TGF-beta signaling with the ALK5/type I TGF-beta R kinase inhibitor, SB-525334, was efficacious for uterine leiomyoma; significantly decreasing tumor incidence and multiplicity, and reducing the size of these mesenchymal tumors. However, SB-525334 was also mitogenic and antiapoptotic for epithelial cells in the kidney and exacerbated the growth of epithelial lesions present in the kidneys of these animals.. Although pharmacologic inhibition of TGF-beta signaling with SB-525334 may be efficacious for mesenchymal tumors, inhibition of this signaling pathway seems to promote the development of epithelial tumors.

Topics: Activin Receptors, Type I; Animals; Apoptosis; Carcinoma, Renal Cell; Female; Imidazoles; Kidney Neoplasms; Leiomyoma; Mitosis; Protein Serine-Threonine Kinases; Quinoxalines; Rats; Rats, Inbred Strains; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Uterine Neoplasms

Abelson (Abl) interactor 2 (Abi-2) has been considered as a key regulator of cell/tissue structural organization and is differentially expressed in leiomyomas. The objective of this study was to evaluate the expression of Abi-2 in leiomyoma/myometrium during the menstrual cycle and following GnRH analogue (GnRHa) therapy, as well as regulation by transforming growth factor (TGF)-beta1 in leiomyoma and myometrial smooth muscle cells (LSMC and MSMC).. We used real-time PCR, Western blotting and immunohistochemistry to determine the expression of Abi-2 in paired leiomyoma and myometrium (n = 27) from proliferative (n = 8) and secretory (n = 12) phases of the menstrual cycle and from patients who received GnRHa therapy (n = 7). Time-dependent action of TGF-beta1 (2.5 ng/ml) and GnRHa (0.1 microM) on Abi-2 expression was determined in LSMC and MSMC.. Leiomyomas express elevated levels of Abi-2 as compared with myometrium from the proliferative but not the secretory phase of the menstrual cycle, with a significant reduction following GnRHa therapy (P < 0.05). Western blotting showed a similar trend in Abi-2 protein expression in leiomyoma/myometrial tissue extracts, which was immunolocalized in LSMC and MSMC, connective tissue fibroblasts and arterial walls. The expression of Abi-2 in LSMC and MSMC was increased by TGF-beta1 (2.5 ng/ml) and was inhibited by GnRHa (0.1 microM) in a time- and cell-dependent manner, and pretreatment with Smad3 SiRNA and U0126, an MEK-1/2 inhibitor, respectively, reversed their actions.. Based on the menstrual cycle-dependent expression, the influence of GnRHa therapy, and regulation by TGF-beta in LSMC/MSMC, we conclude that Abi-2 may have a key regulatory function in leiomyomas cellular/tissue structural organization during growth and regression.

Topics: Adaptor Proteins, Signal Transducing; Adult; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Gonadotropin-Releasing Hormone; Humans; Leiomyoma; MAP Kinase Signaling System; Menstrual Cycle; Myocytes, Smooth Muscle; Myometrium; Transforming Growth Factor beta; Uterine Neoplasms

Connective tissue growth factor (CTGF; CCN2) is considered to serve as downstream midiator of TGF-beta action in tissue fibrosis. We tested this hypothesis in paired leiomyoma and myometrium by evaluating the expression of TGF-beta1/TGF-beta3 and CCN2, the other members of the CCN family, CCN3 and CCN4, as well as fibulin-1C and S100A4, calcium-binding proteins that interact with CCNs. The regulatory function of TGF-beta1 on the expression of these genes was further evaluated using leiomyoma (L) and myometrial (M) smooth muscle cells (SMC). Real-time PCR, Western blotting and immunohistochemistry revealed that leiomyomas and myometrium express CCNs, fibulin-1C and S100A4, whose levels of expression with the exception of fibulin-1C were lower in leiomyomas and inversely correlated with the expression of TGF-beta1 and TGF-beta3 (P<0.05). The expression of these genes was menstrual cycle-independent and GnRHa therapy increased the expression of CCN2 in leiomyomas, while inhibiting CCN3, CCN4 and S100A4 in myometrium (P<0.05). TGF-beta (2.5 ng/ml) in a time- and cell-dependent manner, and through MAPK and Smad pathways, differentially regulated the expression of these genes in LSMC and MSMC. We concluded that CCNs, fibulin-1C and S100A4 are expressed in leiomyomas/myometrium with relative expression levels inversely correlating with TGF-betas and influenced by GnRHa and TGF-beta regulatory actions. The results suggest that unlike other fibrotic disorders, CCN2 (CTGF), at least at tissue level, may not serve as a downstream mediator of TGF-beta action in leiomyomas.

Topics: Blotting, Western; Calcium-Binding Proteins; Cells, Cultured; Connective Tissue Growth Factor; Female; Gene Expression; Gonadotropin-Releasing Hormone; Humans; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Leiomyoma; Myocytes, Smooth Muscle; Myometrium; Nephroblastoma Overexpressed Protein; RNA, Messenger; RNA, Small Interfering; S100 Calcium-Binding Protein A4; S100 Proteins; Smad3 Protein; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta3; Tumor Cells, Cultured; Uterine Neoplasms

This study was conducted to evaluate the effects of a novel selective progesterone receptor modulator (SPRM) asoprisnil on the expression of growth factors and their receptors and on growth factor-induced proliferation of cultured uterine leiomyoma and matching myometrial cells.. The expression of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor (TGFbeta3) was assessed by immunocytochemistry and semi-quantitative RT-PCR. The expression of phosphorylated EGF receptor (p-EGFR), IGF-I receptor alpha subunit (IGF-IRalpha) and phosphorylated TGFbeta receptor type II (p-TGFbeta RII) was assessed by Western blot analysis. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay.. Treatment with 10(-7) M asoprisnil decreased EGF, IGF-I and TGFbeta3 mRNA and protein expression as well as p-EGFR, IGF-IRalpha and p-TGFbeta RII protein expression in leiomyoma cells cultured for 72 h. EGF (100 ng/ml), IGF-I (100 ng/ml) and TGFbeta3 (10 ng/ml) increased the number of viable leiomyoma cells cultured for 72 h, whereas the concomitant treatment with 10(-7) M asoprisnil antagonized the growth factor-induced increase in leiomyoma cell proliferation. In cultured myometrial cells, however, asoprisnil affected neither the growth factor and their receptor expression nor the cell proliferation.. Asoprisnil inhibits the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured leiomyoma cells without affecting their expressions in myometrial cells.

Topics: Adult; Blotting, Western; Cell Proliferation; Cells, Cultured; Down-Regulation; Epidermal Growth Factor; ErbB Receptors; Estrenes; Female; Gene Expression; Humans; Insulin-Like Growth Factor I; Leiomyoma; Middle Aged; Myometrium; Oximes; Proteoglycans; Receptor, IGF Type 1; Receptors, Growth Factor; Receptors, Progesterone; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

Microarray gene expression profiling revealed fibromodulin (FMOD) is among differentially expressed genes in leiomyoma (L) and myometrium. Using realtime PCR, western blotting and immunohistochemistry, we validated the expression of FMOD in paired leiomyoma and myometrium (N = 20) during the menstrual cycle, from women who received gonadotropin-releasing hormone analogue (GnRHa) therapy (N = 7) and in leiomyoma and myometrial (M) smooth muscle cells (SMC) due to transforming growth factor (TGF)-beta and GnRHa treatment. The results indicated that FMOD is expressed at significantly higher levels in leiomyoma as compared to myometrium from proliferative phase (two- to three-folds; P < 0.05), but not the secretory phase of the menstrual cycle, whereas GnRHa therapy reduced FMOD expression to levels detected in myometrium from proliferative phase (P = 0.05). By using western blotting and immunohistochemistry immunoreactive FMOD was detected in leiomyoma and myometrial tissue-extract and in LSMC and MSMC, connective tissue fibroblasts and arterial walls. In a time- and cell-dependent manner, TGF-beta1 (2.5 ng/ml) increased the expression of FMOD in MSMC, whereas GnRHa (0.1 microM) inhibited that in MSMC and LSMC (P < 0.05). The effect of TGF-beta and GnRHa on FMOD expression was reversed following pretreatment of LSMC and MSMC with Smad3 SiRNA and U0126 (MEK1/2 inhibitor), respectively. In summary, menstrual cycle-dependent expression of FMOD and suppression following GnRHa therapy in leiomyoma and myometrium, as well as differential regulation by TGF-beta and GnRHa in vitro suggests that FMOD, a key regulator of tissue organization, plays a critical role in leiomyoma fibrotic characteristics.

Topics: Extracellular Matrix Proteins; Female; Fibromodulin; Gonadotropin-Releasing Hormone; Humans; Leiomyoma; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Myometrium; Proteoglycans; Smad3 Protein; Transforming Growth Factor beta; Uterine Neoplasms

GnRH analog (GnRHa) and TGF-beta act directly on leiomyoma/myometrial smooth muscle cells (LSMCs and MSMCs) regulating diverse activities resulting in leiomyoma growth and regression. Because GnRH and TGF-beta receptor signaling is in part mediated through the MAPK pathway, we determined whether the contribution of MAPK/ERK and transcriptional activation of c-fos and c-jun, result in differential regulation of type I collagen, fibronectin, and plasminogen activator inhibitor 1 (PAI-1) gene expression, whose products are known to influence extracellular matrix turnover, which is critical in leiomyoma growth and GnRHa-induced regression. We found that GnRHa and TGF-beta in a dose- and time-dependent manner increased the level of phosphorylated ERK1/2 (pERK1/2) in LSMCs and MSMCs. GnRHa and TGF-beta increased ERK1/2 nuclear accumulation resulting in differential regulation of c-fos and c-jun mRNA expression via downstream signaling from MAPK kinase (MEK)1/2, because pretreatment with U0126, a synthetic inhibitor of MEK1/2, abolished basal and GnRHa- and TGF-beta-induced pERK1/2 and the expression of c-fos and c-jun. LSMCs and MSMCs also express fibronectin, type I collagen, and PAI-1 mRNA, and GnRHa and TGF-beta altered their expression in a cell-specific manner through MEK1/2. We concluded that GnRHa and TGF-beta acting through a MAPK/ERK pathway and transcriptional activation of c-fos/c-jun results in differential regulation of specific genes whose products may in part influence the outcome of leiomyoma growth and regression.

Topics: Collagen Type I; Enzyme Activation; Female; Fibronectins; Gene Expression Regulation; Genes, fos; Genes, jun; Gonadotropin-Releasing Hormone; Humans; Leiomyoma; Mitogen-Activated Protein Kinases; Myocytes, Smooth Muscle; Myometrium; Plasminogen Activator Inhibitor 1; Transforming Growth Factor beta; Uterine Neoplasms

Extravillous trophoblast (EVT) cells of the human placenta proliferate, migrate, and invade the pregnant uterus and its vasculature in order to nourish the fetus. However, the normal EVT cell proliferation, migration, and invasiveness are exquisitely controlled in situ by decidua-produced transforming growth factor-beta (TGF-beta), whereas EVT cancer (choriocarcinoma) cells are TGF-beta-resistant. We found that these cells lack in expression of Smad3, a key transcription factor involved in TGF-beta signaling pathway. To test whether Smad3 restitution restores TGF-beta response in choriocarcinoma cells, we produced a Smad3-expressing cell line (JAR-smad3/c). Since anti-invasive effect of TGF-beta in the normal EVT cells was partly mediated by an upregulation of tissue inhibitor of metalloprotease (TIMP)-1, we examined whether Smad3-restituted JAR cells have restored TGF-beta response of TIMP-1 upregulation. The expression of TIMP-1 mRNA was found to be low in JAR and JAR-smad3/c cells. Moreover, the basal level of secreted TIMP-1 protein was very low in these cells as compared to the normal EVT cells. TGF-beta1 upregulated TIMP-1 mRNA and secreted protein in Smad3-restituted JAR cells as well as in the normal EVT cells, whereas no effect was detected in Smad3-deficient (wild-type) JAR cells. We had earlier shown that Smad3-restituted JAR cells had also restored TGF-beta response of plasminogen activator inhibitor-1 upregulation. However, in vitro functional analysis revealed that, in contrast to the normal EVT cells, anti-invasive action of TGF-beta was not restored in Smad3-restituted JAR cells. Thus, additional factors (possibly low expression of Smad4 and/or other unknown factors) may contribute to refractoriness to anti-invasive action of TGF-beta in JAR cells.

Topics: Cell Line; Choriocarcinoma; DNA-Binding Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Pregnancy; RNA, Messenger; Smad3 Protein; Tissue Inhibitor of Metalloproteinase-1; Trans-Activators; Transfection; Transforming Growth Factor beta; Trophoblasts; Tumor Cells, Cultured; Up-Regulation; Uterine Neoplasms

To investigate whether phorbol 12-myristate 13-acetate (PMA) modifies the invasive ability of trophoblast cells by regulating their cytokine productions.. Reverse transcriptase-polymerase chain reaction was used to examine the effect of PMA on the expression of cytokines which regulated the invasive ability of trophoblast cells.. Prior to PMA treatment, expressions of the cytokins including hepatocyte growth factor (HGF), interleukin (IL)-1beta, insulin-like growth factor (IGF)-II, transforming growth factor (TGF)- beta and vascular endothelial growth factor (VEGF) were all detected in JAR cells, only with the exception of IGF-I. After incubation with 100 nmol/L PMA for 24 h, the cells showed strong expression of IL-1beta, HGF and IGF-II, with reduced expression of TGF-beta2 and TGF-beta3.. By regulating the autocrine of these cytokines, PMA exercises its effect to enhance the invasive ability of trophoblast or choriocarcinoma cells.

Topics: Cell Line; Choriocarcinoma; Cytokines; Endothelial Growth Factors; Female; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Neoplasm Invasiveness; Pregnancy; RNA, Messenger; Somatomedins; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Trophoblasts; Uterine Neoplasms; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Transforming growth factor-beta1 is the prototype of a bimodal regulator of cell growth, which can either inhibit or stimulate the proliferation of smooth muscle cells. Part of transforming growth factor-beta1-mediated stimulation of growth is associated with the increased production of platelet-derived growth factor. The conversion of latent-to-active transforming growth factor-beta provides a pivotal mechanism for the regulation of the biologic activity of transforming growth factor-beta. We investigated the differential expression and production of the active form of transforming growth factor-beta1 in the myometrium and leiomyoma throughout the menstrual cycle. We also studied the mitogenic effects of transforming growth factor-beta1 and platelet derived growth factor on myometrial and leiomyoma cells in culture.. Myometrium and leiomyoma tissue pairs were obtained from 28 women who underwent hysterectomy. Total RNA from each tissue was extracted, and Northern blot analysis was performed for the detection of TGF-beta1 messenger RNA. Active and total transforming growth factor-beta1 protein was quantified with enzyme-linked immunosorbent assay. Cell proliferation of cultured human myometrial and leiomyoma cells that are treated with TGF-beta1 (0.01-1 ng/mL), anti-transforming growth factor-beta antibody (0.01-10 ng/mL), or platelet-derived growth factor (10 ng/mL) was assessed by the [(3)H]thymidine incorporation method.. Overall, the transforming growth factor-beta1 messenger RNA level in myometrial samples was 1.2-fold higher than in the leiomyoma samples (P <.05). Active transforming growth factor-beta1 protein levels in follicular and luteal phase myometrial and leiomyoma samples were significantly greater than the levels in samples from women with atrophic endometrium (P < 0.05). Transforming growth factor-beta1, at low concentrations (0.01 ng/mL), induced an increase in cell proliferation (2- to 3-fold; P <.05). When cells were treated with anti-transforming growth factor-beta antibody, there was a larger magnitude of increase observed (7- to 20-fold; P <.05). Platelet-derived growth factor (10 ng/mL) consistently increased the rate of cell proliferation both in myometrium and leiomyoma cells (5- to 6-fold; P <.05).. Levels of active transforming growth factor-beta1 that were produced in follicular and luteal phases indicate a stimulatory role for ovarian hormones. The finding that transforming growth factor-beta1, only at low concentrations, stimulates cell proliferation mainly in leiomyoma cells is in agreement with the bimodal and dose-dependent effects of transforming growth factor-beta1 that is observed in smooth muscle cells of other tissues. The persistent and high rate of cell proliferation with platelet-derived growth factor suggests that the growth stimulatory effect of transforming growth factor-beta1 may be mediated through its up-regulatory effect on platelet-derived growth factor.

Topics: Antibodies; Blotting, Northern; Cell Division; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Humans; Hysterectomy; Leiomyoma; Menstrual Cycle; Myometrium; Platelet-Derived Growth Factor; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Uterine Neoplasms

To determine whether leiomyoma, adenomyosis and endometrial polyps are associated with changes in uterine cavity matrix metalloproteinases (MMP-2 and MMP-9) and cytokines.. Uterine cavity irrigation was performed in women with leiomyoma, adenomyosis and endometrial polyps, and in women with a normal uterus. MMP-2 and MMP-9 were assayed in the uterine washings by gelatin zymography. For individual subjects, the total MMP level was obtained by adding the semi-quantitative scores of band densities related to gelatinases in the zymograms. Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta1 (TGF-beta1) were measured using enzyme-linked immunosorbant assay (ELISA) kits.. The uterine cavity of patients with leiomyoma, adenomyosis and endometrial polyps had significantly higher MMP scores than controls. Although the mean IL-1beta levels were elevated in uteri harboring a pathology compared with the normal uteri, the cytokine was significantly elevated only in the adenomyotic group. Significantly elevated levels of IFN-gamma were found in uteri with leiomyoma and endometrial polyps. Uterine washings from leiomyoma and adenomyosis contained significantly elevated mean levels of TGF-beta1 compared with controls, while TNF-alpha was significantly higher only in leiomyoma. When uterine cytokine levels were compared in relation to individual MMP levels a significant relationship was found between TGF-beta1 and elevated levels of MMP-9 and total MMPs in leiomyoma. A significant relationship was also found between IL-1beta and elevated levels of MMP-2, MMP-9 and total MMPs in the endometrial polyp group.. The uterine cavity in leiomyoma, adenomyosis and endometrial polyps contains elevated levels of MMPs and cytokines compared with the normal uterus. In some pathologies elevated cytokines are associated with elevated MMPs.

Topics: Cytokines; Endometrial Neoplasms; Endometriosis; Female; Humans; Interferon-gamma; Interleukin-1; Leiomyoma; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinases; Polyps; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Uterine Diseases; Uterine Neoplasms; Uterus

Extravillous trophoblast (EVT) cells of the human placenta progressively lose their proliferative activity in situ as EVT cell columns migrate into and invade the decidua. It remains unclear whether this is due to a terminal differentiation of EVT cells along the invasive pathway with concomitant loss of proliferative ability, or a negative regulation by decidua-derived factors, or both mechanisms. Our earlier studies provided evidence for a negative regulation by a decidua-derived factor, transforming growth factor (TGF)-beta, which inhibited proliferation, migration, and invasiveness of first-trimester EVT cells in vitro. We further discovered that decidua also produces decorin, a proteoglycan that binds TGF-beta (and in some cases, inactivates TGF-beta), which is colocalized with TGF-beta in the decidual extracellular matrix. The present study used in vitro-propagated EVT cell lines to examine whether EVT cells retain their capacity for proliferation after the process of invasion; and whether decorin exerts any effect on EVT cell proliferation, migration, or invasiveness in a TGF-beta-dependent or TGF-beta-independent manner. We also examined whether trophoblastic cancer (choriocarcinoma) JAR and JEG-3 cells responded to decorin in a similar manner. Proliferation was measured using a colorimetric (MTT) cellularity assay and immunolabeling for the Ki-67 proliferation marker. Migration and invasiveness were measured in transwells by the ability of cells to cross 8-microm pores of polycarbonate membranes in the absence or presence of an additional matrigel barrier. These experiments revealed three points. First, EVT cells retained limited but significant proliferative ability in vitro after invading matrigel. Second, that decorin alone blocked EVT cell proliferation in a dose-dependent manner. This effect remained unaffected in an additional presence of TGF-beta, which exerted antiproliferative effects on its own. The antiproliferative effect of decorin was explained by an up-regulation of the p21 protein. Third, that decorin alone or TGF-beta alone exerted antimigratory and anti-invasive effects on EVT cells, but the addition of TGF-beta to decorin did not alter decorin action. And fourth, that choriocarcinoma cells were resistant to antiproliferative, antimigratory, and anti-invasive effects of decorin. These results suggest 1) that the invasive function of EVT cells is not associated with a terminal differentiation into a noncycling state; 2) that prol

Topics: Blotting, Western; Cell Division; Cell Movement; Cell Survival; Choriocarcinoma; Chorionic Villi; Cyclin-Dependent Kinases; Decidua; Decorin; Extracellular Matrix Proteins; Female; Humans; Neoplasm Invasiveness; Oncogene Protein p21(ras); Proteoglycans; Transforming Growth Factor beta; Trophoblasts; Tumor Cells, Cultured; Up-Regulation; Uterine Neoplasms

We used a PCR-based subtraction method, representational difference analysis of cDNA (cDNA--RDA), to identify genes with a higher expression in leiomyomas in comparison with the corresponding myometrium during the proliferative phase of the menstrual cycle. Increased expression of the genes for pregnancy-associated plasma protein A (PAPPA), tomoregulin, cellular retinoid acid binding protein 1 (CRABP1), zinc finger protein 185 (ZFP 185) and latent transforming growth factor beta binding protein 2 (LTBP2) was demonstrated in individual leiomyoma samples compared with corresponding myometrium. Additionally, a specific positive immunostaining of LTBP2 was found in the smooth muscle cells of both leiomyomas and myometrium. These genes may be part of previously unidentified molecular mechanisms responsible for the selective growth advantage of leiomyomas compared with myometrium. This work expands our knowledge about the molecular nature of leiomyomas and provides novel candidate genes to further explore in relation to their function during leiomyoma growth.

Topics: Adaptor Proteins, Signal Transducing; Carrier Proteins; Cytoskeletal Proteins; DNA-Binding Proteins; Endopeptidases; Female; Gene Expression; Gene Expression Profiling; Humans; Immunohistochemistry; Latent TGF-beta Binding Proteins; Leiomyoma; LIM Domain Proteins; Membrane Proteins; Myometrium; Neoplasm Proteins; Pregnancy Proteins; Pregnancy-Associated Plasma Protein-A; Retinol-Binding Proteins; Retinol-Binding Proteins, Cellular; Retinol-Binding Proteins, Plasma; Transforming Growth Factor beta; Uterine Neoplasms; Uterus; Zinc Fingers

Proliferation, migration, and invasiveness of the normal placental extravillous trophoblast (EVT) cells are negatively regulated by transforming growth factor-beta (TGF-beta), whereas malignant EVT (JAR and JEG-3 choriocarcinoma) cells are resistant to TGF-beta. These malignant cells were found to have lost the expression of Smad3. Present study examined whether Smad3 restitution in JAR cells could restore TGF-beta response. We produced a stable Smad3 cDNA-transfected clone (JAR-smad3/c) which exhibited further upregulation of Smad3 in the presence of TGF-beta1. Since anti-invasive effects of TGF-beta in the normal EVT cells were shown to be mediated in part by plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA), we compared the expression of PAI-1 and uPA in the normal EVT, JAR, and JAR-smad3/c cells in the presence or absence of TGF-beta1. The basal levels of PAI-1 mRNA and secreted PAI-1 and uPA proteins were found to be very low in JAR and JAR-smad3/c cells, as compared to the normal EVT cells. However, TGF-beta1 upregulated PAI-1 and downregulated uPA in JAR-smad3/c cells, but not in JAR cells. Thus, resistance of choriocarcinoma cells to anti-invasive effects of TGF-beta may, at least in part, be due to loss of Smad3 expression.

Topics: Cell Line; Choriocarcinoma; DNA-Binding Proteins; Female; Humans; Plasminogen Activator Inhibitor 1; Pregnancy; RNA, Messenger; RNA, Neoplasm; Smad3 Protein; Trans-Activators; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Trophoblasts; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Uterine Neoplasms

Transforming growth factor-betas (TGF betas) are multifunctional peptides that regulate growth and differentiation in a variety of cells. The goals of this study were to compare expression of the TGF beta isoforms in normal myometrium and benign leiomyoma tumors of the uterus and to examine the effects of TGF betas on cell proliferation and collagen production by these cells in vitro. Myometrium and leiomyoma tissues were obtained from patients undergoing elective hysterectomies. Tissues were processed for ribonucleic acid (RNA) and were also established as primary cell cultures. Northern blot analysis showed that the levels of TGF beta 1 messenger RNAs (mRNAs) were similar between leiomyoma and myometrium, whereas leiomyoma showed 5-fold higher levels of expression of TGF beta 3 mRNA than autologous myometrium. Expression of TGF beta 3 protein detected by immunohistochemistry was much more intense in leiomyoma tissues than in corresponding myometrium. Levels of both TGF beta 1 and TGF beta 3 increased with increasing cell density for leiomyoma and myometrium smooth muscle cells cultured in vitro. Effects of TGF beta 1 and TGF beta 3 on cell proliferation were assessed by measuring changes in DNA synthesis with the tritiated thymidine incorporation assay. The doses of TGF betas tested were 0, 0.1, 1.0, and 10.0 ng/mL. All three doses of TGF beta 1 and TGF beta 3 inhibited DNA synthesis in myometrium smooth muscle cells by 31--54%. Concomitant treatment with an immunoneutralizing antibody to TGF beta 1--3 reversed this inhibitory effect. In contrast, TGF beta 1 had no effect on leiomyoma smooth muscle cells, whereas TGF beta 3 increased DNA synthesis by leiomyoma cells. Combined treatment with the immunoneutralizing antibody prevented this increase. Treatment of leiomyoma and myometrial cells with the TGF beta immunoneutralizing antibody for 24 h caused a 45--60% reduction in collagen type I and type III mRNA levels, suggesting that endogenous TGF betas are important for collagen production. These results support the hypothesis that alterations in the TGF beta system produce loss of sensitivity to the antiproliferative effects of TGF beta, and increased expression of TGF beta 3 may contribute to the growth of these tumors.

Topics: Cell Division; Cells, Cultured; Female; Humans; Leiomyoma; Muscle, Smooth; Myometrium; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta3; Tumor Cells, Cultured; Uterine Neoplasms

We had earlier shown that TGF-beta controls proliferation, migration, and invasiveness of normal human trophoblast cells, whereas premalignant and malignant trophoblast cells are resistant to TGF-beta. To identify signaling defects responsible for TGF-beta resistance in premalignant and malignant trophoblasts, we have compared the expression of TGF-beta signaling molecules in a normal trophoblast cell line (HTR-8), its premalignant derivative (RSVT2/C), and two choriocarcinoma cell lines (JAR and JEG-3). RT-PCR analysis revealed that all these cell lines expressed the mRNA of TGF-beta1, -beta2, and -beta3, TGF-beta receptors type I, II, and III, and post-receptor signaling genes smad2, smad3, smad4, smad6, and smad7 with the exception that TGF-beta2 and smad3 were undetectable in JAR and JEG-3 cells. Immunoblot analysis confirmed the absence of smad3 protein in choriocarcinoma cells. Treatment with TGF-beta1 induced smad3 phosphorylation and smad3 translocation to the nucleus in the normal and premalignant trophoblast cells. These results suggest that loss of smad3 may account for a functional disruption in the TGF-beta signaling pathway in choriocarcinomas, but not in the premalignant trophoblast.

Topics: Choriocarcinoma; DNA-Binding Proteins; Female; Gene Expression; Humans; Phosphorylation; Pregnancy; Signal Transduction; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta; Trophoblastic Neoplasms; Trophoblasts; Tumor Cells, Cultured; Uterine Neoplasms

To investigate the expression of TGF-beta3 in leiomyoma and myometrium as well as the effect of TGF-beta3 on the expression of fibronectin and on the proliferation of leiomyoma and myometrial cells.. Observational and in vitro experimental study.. University medical center.. Women with (n = 18) leiomyoma.. First TGF-beta3 mRNA and protein levels in myometrium and leiomyoma were measured, and then myometrial and leiomyoma cells in culture were treated with TGF-beta3.. TGF-beta3 and fibronectin mRNA were evaluated by Northern analysis. Myometrial and leiomyoma cell proliferation was assessed with use of [(3)H]thymidine incorporation.. The TGF-beta3 mRNA level in the leiomyoma samples was 3.5-fold higher than in the myometrial samples. The highest TGF-beta3 mRNA level was observed in leiomyoma samples from midsecretory phase and was 5-fold higher than in proliferative phase samples. Fibronectin mRNA expression also was higher in the leiomyoma than in the myometrium. TGF-beta3 induced fibronectin expression in leiomyoma cells and directly stimulated myometrial and leiomyoma cell proliferation in cultures.. These findings suggest that TGF-beta3 may be mediating the growth-promoting effects of sex steroids on leiomyomas by playing a role in the fibrogenic process and cell proliferation that characterize these tumors.

Topics: Cell Division; Cells, Cultured; Female; Fibronectins; Gonadal Steroid Hormones; Humans; Leiomyoma; Myometrium; RNA, Messenger; Transforming Growth Factor beta; Uterine Neoplasms

To clarify the biological and pathological features of choriocarcinoma, we evaluated the in vitro production of cytokines by a choriocarcinoma cell line, BeWo. We measured the concentration of interleukin (IL)-6 and IL-8 in the culture media of BeWo cells after stimulation with various modulatory agents of cytokine expression by enzyme-linked immunosorbent assays. Northern blot analysis was used to examine the expression of IL-6 and IL-8 mRNA in these cells. A weak expression of IL-6 and IL-8 was detected in unstimulated BeWo cells by both methods. IL-6 transcription and secretion were dose-dependently enhanced by stimulation with IL-1beta, tumour necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1 and 12-O-tetradecanoyl phorbol-13-acetate (TPA). Forskolin, lipopolysaccharide and interferon-gamma had no effect on these cytokines production. The TNF-alpha-induced secretion of IL-6 was inhibited by dexamethasone. The TPA-induced production of IL-6 was inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and sphyngosine, suggesting the involvement of a protein kinase C-dependent pathway. Levels of IL-8 mRNA and protein were also dose-dependently increased by stimulation with IL-1beta, TNF-alpha and TPA. In contrast to IL-6, the expression of IL-8 was not affected by TGF-beta1. It is suggested that, in addition to the production of steroidal and non-steroidal hormones, these cytokines may serve as part of a cytokine network that modulates the proliferation and angiogenesis of choriocarcinomas.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adult; Blotting, Northern; Choriocarcinoma; Dexamethasone; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Humans; Interleukin-1; Interleukin-6; Interleukin-8; Pregnancy; RNA, Messenger; Sphingosine; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Uterine Neoplasms

In this work, we report the isolation of a factor from the culture supernatant of confluent fibroblasts from human cervix with the diagnosis of uterine myomatosis. This factor possesses the capacity to inhibit the proliferation of normal fibroblasts. The proliferation inhibitor factor (PIF) was purified from the culture supernatant by precipitation with 80% ammonium sulfate, and by molecular sieve chromatography. Our results indicate that PIF is a protein of 23 kDa, which is highly sensitive to trypsin treatment, and is thermolabile, since temperatures equal to, or above, 60 degrees C eliminate the protein activity in 15 to 20 min. Western blot analyses identified no cross reactions of the purified PIF with TGF-alpha, TNFalpha, IFNgamma, or IL-1beta, suggesting that PIF is a new protein belonging to the group of factors secreted by fibroblasts able to inhibit cellular proliferation.

Topics: Blotting, Western; Cell Count; Chromatography, Gel; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Fibroblasts; Gentian Violet; Growth Inhibitors; Humans; Interferon-gamma; Interleukin-1; Leiomyoma; Neoplasm Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Uterine Neoplasms

Fas-Fas ligand (FasL) interactions play a significant role in the immune privilege status of certain cell populations, and several cytokines and growth factors can modulate their expression. When a FasL-expressing cell binds a Fas-bearing immune cell, it triggers its death by apoptosis. In this study, we demonstrate that normal human endometrial epithelial but not stromal cells express FasL. Moreover, we showed that macrophage-conditioned media induced FasL expression by endometrial stromal cells in a dose-dependent manner. To elucidate which macrophage product was responsible for the up-regulation of FasL, endometrial stromal cell cultures were treated with the macrophage products platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta1, and basic fibroblast growth factor (bFGF). The first two (which are known to be elevated in the peritoneal fluid of women with endometriosis) induced a dose-dependent up-regulation of FasL expression, which was specifically inhibited by the antibody. Interestingly, bFGF (which is not elevated in peritoneal fluid of women with endometriosis) did not induce any response. These results suggest that the pro-inflammatory nature of the peritoneal fluid of women with endometriosis induces the FasL expression by regurgitated endometrial cells, and signals Fas-mediated cell death of activated immune cells. This could be a mechanism for endometrial cells to escape immune surveillance, implant and grow.

Topics: Cell Differentiation; Cells, Cultured; Choriocarcinoma; Culture Media, Conditioned; Endometrium; Fas Ligand Protein; Female; Fibroblast Growth Factor 2; Gene Expression Regulation; Humans; Leukemia, Myeloid; Ligands; Macrophages; Membrane Glycoproteins; Platelet-Derived Growth Factor; Pregnancy; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

Human myometrium and leiomyomas express granulocyte macrophage-colony-stimulating factor (GM-CSF), transforming growth factor-beta (TGFbeta), and their receptors. Overexpression of TGFbeta and, to a limited extent, GM-CSF has been associated with tissue fibrosis throughout the body, including leiomyomas. The objective of the present study was to determine the action of GM-CSF on leiomyoma and myometrial smooth muscle cells (LSMC and MSMC) and examine whether the action of GM-CSF is mediated through the induction of TGFbeta1 expression. Using competitive quantitative RT-PCR and enzyme-linked immunosorbent assay, we found that LSMC express significantly higher GM-CSF messenger ribonucleic acid (mRNA; 0.6 +/- 0.1 x 10(3) copies of mRNA/microg total RNA) and protein (0.75 +/- 0.2 ng/mL) than MSMC (0.5 +/- 0.1 x 10(2) copies of mRNA and 0.45 +/- 0.07 ng/mL protein; P < 0.05). In addition, LSMC expressed significantly higher TGFbeta1 mRNA (1.6 +/- 0.3 x 10(4) copies of mRNA/microg total RNA) than MSMC (2.4 +/- 0.4 x 10(3) copies) and synthesized and secreted more TGFbeta1 protein (1.7 +/- 0.2 vs. 0.5 +/- 0.02 ng/mL); whereas MSMC contained more cell-associated TGFbeta1 (56.2 +/- 1.2 ng/mL) than LSMC (35.2 +/- 1.2 ng/mL; P < 0.05). We found that GM-CSF (0.01-100 ng/mL) has limited mitogenic activity for LSMC but not for MSMC determined by the rate of [3H]thymidine incorporation and cell proliferation assay. However, GM-CSF at 1 ng/mL increased its own production, the expression of TGFbeta1 mRNA, the cell-associated TGFbeta1 protein content in both cell types, and TGFbeta1 released into the culture-conditioned medium of LSMC (P < 0.05). TGFbeta1 also increased its own mRNA and protein expression, but had no effect on cell-associated TGFbeta1 in both cell types (P < 0.05). Cotreatment of LSMC and MSMC with GM-CSF and TGFbeta1 induced changes similar to those produced by GM-CSF in both cells. In conclusion, our data suggest that GM-CSF is not a mitogen for MSMC and LSMC, but it regulates its own expression and the expression of TGFbeta1 by these cells, a regulatory interaction that may account for the GM-CSF-induced tissue fibrosis that occurs in leiomyomas.

Topics: Culture Media, Conditioned; DNA; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Leiomyoma; Muscle, Smooth; Myometrium; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

Tumor cells from eight freshly isolated cervical cancers (i.e., four adenocarcinomas and four squamous carcinomas) were analyzed for their production of the immune-inhibitory cytokine transforming growth factor-beta (TGF-beta) in vitro. All fresh adenocarcinomas secreted significant levels of TGF-beta (mean 397, range between 207 and 782 pg/ml/10(5) cells/48 hr). In contrast, no detectable TGF-beta was present in the supernatants from the four fresh squamous carcinoma cultures (P < 0.001). These data suggest that major differences in the secretion of the immunoinhibitory cytokine TGF-beta exist between squamous cell carcinomas and adenocarcinomas of the uterine cervix. Furthermore, these findings suggest that at least some of the differences in the natural biologic behavior, as well as in the response to radiation treatment, between these two histologic types of cervical cancer could be related to differences in secretion of this immune-inhibitory cytokine.

Topics: Adenocarcinoma; Carcinoma, Squamous Cell; Female; Humans; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

Human endometrium is unique since it is the only tissue in the body that bleeds at regular intervals. In addition, abnormal endometrial bleeding is one of the most common manifestations of gynecological diseases, and is a prime indication for hysterectomy. Here, we report on a novel human gene, endometrial bleeding associated factor (ebaf), whose strong expression in endometrium was associated with abnormal endometrial bleeding. In normal human endometrium, this gene was transiently expressed before and during menstrual bleeding. In situ hybridization showed that the mRNA of ebaf was expressed in the stroma without any significant mRNA expression in the endometrial glands or endothelial cells. The predicted protein sequence of ebaf showed homology with and structural features of the members of TGF-beta superfamily. Fluorescence in situ hybridization showed that the ebaf gene is located on human chromosome 1 at band q42.1. Thus, ebaf is a novel member of the TGF-beta superfamily and an endometrial tissue factor whose expression is associated with normal menstrual and abnormal endometrial bleeding.

Topics: Adult; Amino Acid Sequence; Base Sequence; Chromosome Mapping; Chromosomes, Human, Pair 1; DNA Primers; Endometriosis; Endometrium; Female; Humans; Leiomyoma; Menorrhagia; Menstrual Cycle; Menstruation Disturbances; Molecular Sequence Data; Multigene Family; Polymerase Chain Reaction; Sequence Homology, Amino Acid; Transforming Growth Factor beta; Uterine Neoplasms

Topics: Cell Division; Cell Line; Choriocarcinoma; Cytokines; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interferon-gamma; Interleukins; Lymphotoxin-alpha; Macrophage Colony-Stimulating Factor; Pregnancy; Transforming Growth Factor beta; Trophoblasts; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Uterine Neoplasms

The objective of the present study was to determine whether GnRH and GnRH receptor are expressed in myometrium and leiomyomata, and if GnRH analogs alone or in the presence of ovarian steroids can modulate the rate of DNA synthesis, proliferation, and transforming growth factor-beta 1 (TGF beta 1) production in myometrial smooth muscle cells in vitro. Reverse transcription-PCR revealed that leiomyomata, unaffected myometrium, and isolated myometrial smooth muscle cells express GnRH and GnRH receptor messenger ribonucleic acid. Furthermore, in a dose-dependent manner, GnRH agonist (leuprolide acetate) inhibited, but GnRH antagonist [D-pGlu1,D-Phe2,D-Trp3.6] (GnRH-Ant1) stimulated, the rate of [3H]thymidine incorporation into myometrial smooth muscle cells (P < 0.05), whereas GnRH-Ant2 (Ac-D-P-Cl-Phe1.2,D-Trp3,D-Arg6,D-Ala10) had no effect. 17 beta-Estradiol (E2) medroxyprogesterone acetate (MPA), and E2 plus MPA (1 micromol/L) stimulated the rate of DNA synthesis by smooth muscle cells (P < 0.05), which was inhibited by GnRH analogs used at 5 micromol/L (P < 0.05). GnRH analogs had no significant effect on myometrial smooth muscle cell proliferation, with the exception of GnRH-Ant1; however, they inhibited the stimulatory action of E2, MPA, and E2 plus MPA in a time-dependent manner (P < 0.05). These cells also synthesized and released approximately 1.32 +/- 0.02 ng/mL total (active plus latent) TGF beta 1, of which 0.73 +/- 0.02 ng/mL was in an active form. E2, MPA, E2 plus MPA, and GnRH analog treatments resulted in an increase in total TGF beta 1 production, whereas GnRH agonist and GnRH-Ant2, but not GnRH-An1, inhibited active TGF beta 1 (P < 0.05). GnRH analogs also inhibited the action of E2 plus MPA on total and active TGF beta 1 production, whereas GnRH-Ant1 further stimulated E2, MPA, or E2 plus MPA action on active TGF beta 1 production (P < 0.05). The data demonstrate for the first time that GnRH and GnRH receptor messenger ribonucleic acid are expressed in myometrium, leiomyomata, and myometrial smooth muscle cells. The local expression of GnRH and receptor along with the direct action of GnRH analogs on the smooth muscle cell DNA synthesis and TGF beta 1 production suggest an autocrine/paracrine role for GnRH in these tissues, a mechanism that may be involved in leiomyomata regression in women receiving GnRH agonist therapy.

Topics: Adult; Amino Acid Sequence; Base Sequence; Cell Division; DNA; Estradiol; Female; Gene Expression; Gonadotropin-Releasing Hormone; Humans; Leiomyoma; Leuprolide; Medroxyprogesterone Acetate; Molecular Sequence Data; Muscle, Smooth; Myometrium; Receptors, LHRH; Transforming Growth Factor beta; Uterine Neoplasms

The expression and cellular distribution of transforming growth factor-1 (TGF beta 1) through TGF beta 3 and TGF beta type I-III receptor messenger ribonucleic acid (mRNA) and protein were analyzed in leiomyomata from patients receiving GnRH agonist (GnRHa; leuprolide acetate) compared to those in untreated controls. Standard reverse transcription-PCR revealed that the unaffected myometrium and leiomyomata from leuprolide-treated and untreated patients express TGF beta 1-3 and TGF beta type I-III receptor mRNA. The myometrial and leiomyomata smooth muscle cells were the primary site of TGF beta 1-3 and TGF beta type I and II receptor mRNA and protein expression, as determined by in situ hybridization and immunohistochemical localization. These observations indicate that leiomyomata express a higher of level of TGF beta and TGF beta receptor mRNA and protein than unaffected myometrium during the secretory phase of the menstrual cycle, and women who received leuprolide acetate therapy had a substantially lower level of expression than untreated controls. Furthermore, competition-based quantitative reverse transcription-PCR using synthetic internal standards revealed that leiomyomata express a significantly higher number (copies per cell) of TGF beta type II receptor mRNA, followed by TGF beta 1, TGF beta type I receptor, TGF beta 2, and TGF beta 3 (P < 0.05). However, there was a significant decrease in the levels (copies per cell) of TGF beta 1, TGF beta 3, and TGF beta type I and type II receptor mRNA expression in leiomyomata from leuprolide-treated compared to untreated patients (P < 0.05). The data provide further evidence that leiomyomata express mRNA and protein for all components of the TGF beta system, and GnRHa therapy results in down-regulation of their expression. More specifically, these data suggest that TGF beta 1 and TGF beta 3 may play a more important role in leiomyomata growth than TGF beta 2, which leads us to propose that lowering TGF beta and receptor expression may have a direct effect on leiomyomata regression.

Topics: Adult; Female; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Leiomyoma; Leuprolide; Middle Aged; Polymerase Chain Reaction; Receptors, Transforming Growth Factor beta; RNA-Directed DNA Polymerase; RNA, Messenger; Transforming Growth Factor beta; Uterine Neoplasms

Fibroids (leiomyomata) are the commonest tumours in women, but their aetiology is unknown. Epidermal growth factor (EGF) and transforming growth factor-beta (TGF beta) may be important factors involved in fibroid growth. We examined the mRNA expression of these two growth factors in fibroids and corresponding myometrium from 20 women who underwent hysterectomy because of fibroids. We also examined these factors in samples of fibroids from 9 women who underwent myomectomy after pretreatment with luteinizing hormone-releasing hormone agonists. We found that both factors were expressed in the three types of tissue examined. We also found that there was no difference in the relative abundance of either of the two growth factors between the tissues studied. Despite the lack of difference, we postulate that EGF and TGF beta may be important in fibroid growth because of a possible interaction between the two factors in this tissue.

Topics: Autoradiography; Blotting, Northern; Epidermal Growth Factor; Estrogens; Female; Humans; Leiomyoma; Myometrium; RNA, Messenger; Transforming Growth Factor beta; Uterine Neoplasms

Human placental trophoblast invasion of the uterus is a highly controlled event. We had shown that transforming growth factor-beta (TGF-beta) produced in the pregnant uterus controls invasiveness and reduces proliferation of first trimester placental trophoblasts in vitro. The anti-invasive effect of TGF-beta was due, at least in part, to induction of tissue inhibitor of metalloproteinases (TIMP)-1. In the present study we compared the effects of TGF-beta on proliferation ([3H]-TdR incorporation) and invasiveness (3-day Matrigel invasion assay) of JAR and JEG-3 choriocarcinoma cells vs normal first trimester human trophoblast cells. Transcripts of type IV collagenases (72- and 92-kDa enzymes, i.e., gelatinases A and B) and their inhibitors (TIMP-1 and TIMP-2) in these cells were measured by Northern analysis, and secretion of gelatinases and plasminogen activators (PAs) was evaluated by gel zymography. The results revealed that: (a) TGF-beta inhibited invasiveness and proliferation of normal trophoblast but not JAR and JEG-3 choriocarcinoma cells; (b) gelatinase A mRNA, expressed by the normal trophoblast and JAR cells, was upregulated in the presence of TGF-beta; (c) gelatinase B mRNA was not detected in the total RNA preparations of treated or untreated normal trophoblast or choriocarcinoma cells; (d) TGF-beta significantly upregulated the levels of TIMP-1 mRNA in the normal trophoblasts, but this transcript was very low in treated as well as untreated choriocarcinoma cells; TGF-beta also upregulated the 3.5-kb TIMP-2 message in the normal trophoblast; (e) gelatin zymography revealed a distinct band of approximately 68-kDa (gelatinase A) in the conditioned media of normal trophoblast and JAR cells; however, TGF-beta did not change the level of secretion of this gelatinase; and (f) the normal trophoblast also exhibited significant PA secretion (casein zymography) which was reduced in the presence of TGF-beta. PA secretion by the malignant trophoblast cells was low and unaffected by TGF-beta. These findings suggest that choriocarcinoma cells may become refractory to the mechanisms which control normal trophoblast proliferation and invasiveness. Concurrent resistance to antiproliferative and anti-invasive molecules such as TGF-beta may be highly relevant to tumor progression.

Topics: Cell Division; Cells, Cultured; Choriocarcinoma; Collagenases; Dose-Response Relationship, Drug; Drug Resistance; Female; Gelatinases; Glycoproteins; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Metalloendopeptidases; Neoplasm Invasiveness; Plasminogen Activators; Pregnancy; Pregnancy Trimester, First; Proteins; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-2; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta; Trophoblasts; Tumor Cells, Cultured; Uterine Neoplasms

In an attempt to understand the antiproliferative effects of progestins in endometrial cancer, we have examined the effects of the potent progestin, medroxyprogesterone acetate (MPA), on the cell proliferation and the expression of transforming growth factor (TGF) alpha and beta genes in human endometrial adenocarcinoma cell lines. The two cell lines used were Ishikawa, var 1, and HEC-50. In addition, the effects of exogenous TGF-alpha and anti-epidermal growth factor (EGF) receptor monoclonal antibody on cell proliferation were determined. Incubation of both cell lines with MPA resulted in a time- and dose-dependent inhibition of cell proliferation. Half-maximal growth inhibition was observed at 0.6 nM. In Ishikawa cells, the relative abundance of TGF-alpha was significantly reduced by MPA. A significant decrease in TGF-alpha mRNA was apparent 6 h after exposure to MPA and a further decrease was seen 12-24 h after addition of the progestin. The concentration of TGF-alpha immunoreactivity in conditioned medium of MPA-treated cells was also significantly reduced compared to control cultures. MPA had no effect on TGF-alpha expression by HEC-50 cells. EGF mRNA was not detected by Northern blot analysis in either cell type. MPA had no significant effect on EGF receptor mRNA abundance but resulted in a small increase in EGF receptor number in Ishikawa cells. Anti-EGF receptor monoclonal antibody (0.6-6 nM) inhibited Ishikawa cell growth but had no effect on HEC-50 cell proliferation. Exogenous TGF-alpha stimulated proliferation of both cell lines, but Ishikawa cells were significantly more sensitive to exogenous TGF-alpha than HEC-50 cells. Furthermore, TGF-alpha could reverse the growth inhibitory effects of MPA on Ishikawa cells. A decrease in TGF-beta mRNA abundance was also observed in MPA-treated Ishikawa and HEC-50 cells. This effect was of small magnitude, variable, and only observed after prolonged exposure to MPA. These observations are consistent with the hypothesis that the antiproliferative effects of progestins on Ishikawa cells are mediated by decreased expression and autocrine action of TGF-alpha. Since similar growth inhibition is also seen in the HEC-50 cells in which progestins have no effect on TGF-alpha expression, additional mechanisms are likely to be involved in the antiproliferative effects of progestins in human endometrial cancer.

Topics: Antineoplastic Agents; Female; Gene Expression Regulation, Neoplastic; Humans; Medroxyprogesterone; Medroxyprogesterone Acetate; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor alpha; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms