transforming-growth-factor-beta and Bone-Diseases--Metabolic

There have been a number of reports that chronic antiepileptic drug (AEDs) therapy is associated with abnormal bone and calcium metabolism, osteoporosis/osteomalacia, and increased risk of fractures. Bony adverse effects of long term antiepileptic drug therapy have been reported for more than four decades but the exact molecular mechanism is still lacking. Several mechanisms have been proposed regarding AEDs induced bone loss; Hypovitaminosis D, hyperparathyroidism, estrogen deficiency, calcitonin deficiency. Transforming growth factor-β (TGF- β) is abundant in bone matrix and has been shown to regulate the activity of osteoblasts and osteoclasts in vitro. All isoforms of TGF- β are expressed in bone and intricately play role in bone homeostasis by modulating estrogen level. Ovariectomised animal have shown down regulation of TGF- β in bone that could also be a probable target of AEDs therapy associated bone loss. One of the widely accepted hypotheses regarding the conventional drugs induced bone loss is hypovitaminosis D which is by virtue of their microsomal enzyme inducing effect. However, despite of the lack of enzyme inducing effect of certain newer antiepileptic drugs, reduced bone mineral density with these drugs have also been reported. Thus an understanding of bone biology, pathophysiology of AEDs induced bone loss at molecular level can aid in the better management of bone loss in patients on chronic AEDs therapy. This review focuses mainly on certain new molecular targets of AEDs induced bone loss.

Topics: Anticonvulsants; Bone Density; Bone Diseases, Metabolic; Osteoporosis; Transforming Growth Factor beta

Almost all patients with chronic liver diseases (CLD) show altered bone metabolism. Depending on the etiology, this manifests in a severe osteoporosis in up to 75% of the affected patients. Due to high prevalence, the generic term hepatic osteodystrophy (HOD) evolved, describing altered bone metabolism, decreased bone mineral density, and deterioration of bone structure in patients with CLD. Once developed, HOD is difficult to treat and increases the risk of fragility fractures. Existing fractures affect the quality of life and, more importantly, long-term prognosis of these patients, which presents with increased mortality. Thus, special care is required to support the healing process. However, for early diagnosis (reduce fracture risk) and development of adequate treatment strategies (support healing of existing fractures), it is essential to understand the underlying mechanisms that link disturbed liver function with this bone phenotype. In the present review, we summarize proposed molecular mechanisms favoring the development of HOD and compromising the healing of associated fractures, including alterations in vitamin D metabolism and action, disbalances in transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) signaling with histone deacetylases (HDACs) as secondary regulators, as well as alterations in the receptor activator of nuclear factor kappa B ligand (RANKL)-osteoprotegerin (OPG) system mediated by sclerostin. Based on these mechanisms, we give an overview on the limitations of early diagnosis of HOD with established serum markers.

Topics: Animals; Bone Diseases, Metabolic; Bone Morphogenetic Proteins; Calcium; Humans; Liver Diseases; Transforming Growth Factor beta; Vitamin D

Topics: Bone and Bones; Bone Diseases, Metabolic; Bone Remodeling; Bone Resorption; Cytokines; Glycoproteins; Humans; Insulin-Like Growth Factor I; Insulin-Like Growth Factor II; Interleukins; Osteoblasts; Osteoclasts; Osteoprotegerin; Receptors, Cytokine; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Human bone marrow stroma contains pluripotent mesenchymal progenitor cells that can give rise to many mesenchymal lineages, including chondroblasts, adipocytes or osteoblasts. The differentiation of these cells towards a specific lineage is dependent on hormonal and local factors activating specific transcription factors. Attempts have been recently made to identify osteoprogenitor cells in the human bone marrow and to identify the molecular mechanisms responsible for lineage-specific differentiation of human bone marrow stromal cells. Using a clonal pluripotent human bone marrow stromal cell line with tri-potential characteristics, we have provided evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of human bone marrow stromal cells. We have also shown that administration of TGFss that regulates the expression of specific osteoblast and adipocyte transcription factors can promote osteoblast differentiation and inhibit adipocyte conversion of rat marrow stromal cells in vivo. This indicates that the reciprocal relationship between osteoblastogenesis and adipogenesis can be manipulated in vivo in order to improve bone formation. Future studies will have to identify key signals for lineage-specific differentiation of human marrow stromal cells. This may result in the development of therapeutic strategies to promote the differentiation of these cells towards the osteoblast lineage and to inhibit excessive bone marrow adipogenesis associated with aging.

Topics: Adipocytes; Aging; Animals; Bone Diseases, Metabolic; Bone Marrow Cells; Cell Differentiation; Cell Line; Cell Lineage; Chondrocytes; Gene Expression Regulation; Humans; Models, Biological; Osteoblasts; Stromal Cells; Transcription Factors; Transforming Growth Factor beta

Topics: Animals; Biglycan; Bone Diseases, Metabolic; Cell Differentiation; Collagen; Decorin; Extracellular Matrix Proteins; Humans; Leucine; Osteoblasts; Proteoglycans; Repetitive Sequences, Amino Acid; Transforming Growth Factor beta

Bone loss is one of the most common complications of immobilization after spinal cord injury (SCI). Whether transforming growth factor (TGF)-β signaling plays a role in SCI-induced disuse bone loss has not been determined. Thus, 16-week-old male mice underwent sham or spinal cord contusion injury to cause complete hindlimb paralysis. Five days later, 10 mg/kg/day control (IgG) or anti-TGF-β1,2,3 neutralizing antibody (1D11) was administered twice weekly for 4 weeks. Femurs were examined by micro-computed tomography (micro-CT) scanning and histology. Bone marrow (BM) supernatants were analyzed by enzyme-linked immunosorbent assay for levels of procollagen type 1 intact N-terminal propeptide (P1NP), tartrate-resistant acid phosphatase (TRAcP-5b), receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG), and prostaglandin E2 (PGE2). Distal femoral micro-CT analysis showed that SCI-1D11 mice had significantly (P < .05) attenuated loss of trabecular fractional bone volume (123% SCI-1D11 vs 69% SCI-IgG), thickness (98% vs 81%), and connectivity (112% vs 69%) and improved the structure model index (2.1 vs 2.7). Histomorphometry analysis revealed that osteoclast numbers were lower in the SCI-IgG mice than in sham-IgG control. Biochemically, SCI-IgG mice had higher levels of P1NP and PGE2 but similar TRAcP-5b and RANKL/OPG ratio to the sham-IgG group. The SCI-1D11 group exhibited higher levels of P1NP but similar TRAcP-5b, RANKL/OPG ratio, and PGE2 to the sham-1D11 group. Furthermore, 1D11 treatment prevented SCI-induced hyperphosphorylation of tau protein in osteocytes, an event that destabilizes the cytoskeleton. Together, inhibition of TGF-β signaling after SCI protects trabecular bone integrity, likely by balancing bone remodeling, inhibiting PGE2 elevation, and preserving the osteocyte cytoskeleton.

Topics: Animals; Antibodies, Neutralizing; Bone and Bones; Bone Diseases, Metabolic; Bone Marrow; Bone Remodeling; Bone Resorption; Cancellous Bone; Cytoskeleton; Dinoprostone; Disease Models, Animal; Homeostasis; Male; Mice; Mice, Inbred C57BL; Osteocytes; Osteoporosis; Osteoprotegerin; Peptides; Phosphorylation; RANK Ligand; Signal Transduction; Smad2 Protein; Spinal Cord Injuries; Transforming Growth Factor beta; X-Ray Microtomography

It is well established that smoking has detrimental effects on bone integrity and is a preventable risk factor for metabolic bone disorders. Following orthopedic surgeries, smokers frequently show delayed fracture healing associated with many complications, which results in prolonged hospital stays. One crucial factor responsible for fracture repair is the recruitment and differentiation of mesenchymal stem cells (MSCs) at early stages, a mechanism mediated by transforming growth factor β (TGF-β). Although it is known that smokers frequently have decreased TGF-β levels, little is known about the actual signaling occurring in these patients. We investigated the effect of cigarette smoke on TGF-β signaling in MSCs to evaluate which step in the pathway is affected by cigarette smoke extract (CSE). Single-cell-derived human mesenchymal stem cell line (SCP-1 cells) were treated with CSE concentrations associated with smoking up to 20 cigarettes a day. TGF-β signaling was analyzed using an adenovirus-based reporter assay system. Primary cilia structure and downstream TGF-β signaling modulators (Smad2, Smad3, and Smad4) were analyzed by Western blot and immunofluorescence staining. CSE exposure significantly reduced TGF-β signaling. Intriguingly, we observed that protein levels of phospho-Smad2/3 (active forms) as well as nuclear translocation of the phospho-Smad3/4 complex decreased after CSE exposure, phenomena that affected signal propagation. CSE exposure reduced the activation of TGF-β modulators under constitutive activation of TGF-β receptor type I (ALK5), evidencing that CSE affects signaling downstream of the ALK5 receptor but not the binding of the cytokine to the receptor itself. CSE-mediated TGF-β signaling impaired MSC migration, proliferation, and differentiation and ultimately affected endochondral ossification. Thus, we conclude that CSE-mediated disruption of TGF-β signaling in MSCs is partially responsible for delayed fracture healing in smokers.

Topics: Bone Diseases, Metabolic; Cell Differentiation; Cell Line; Cell Movement; Cell Proliferation; Cilia; Humans; Mesenchymal Stem Cells; Receptor, Transforming Growth Factor-beta Type I; Signal Transduction; Smad Proteins; Tobacco Smoke Pollution; Transforming Growth Factor beta

Surface-modified metal implants incorporating different ions have been employed in the biomedical field as bioactive dental implants with good osseointegration properties. However, the molecular mechanism through which surface coatings exert the biological activity is not fully understood, and the effects have been difficult to achieve, especially in the osteopenic bone. In this study, We examined the effect of zinc-modified calcium silicate coatings with two different Zn contents to induce osteogenic differentiation of rat bone marrow-derived pericytes (BM-PCs) and osteogenetic efficiency in ovariectomised rabbits. Ti-6Al-4V with zinc-modified calcium silicate coatings not only enhanced proliferation but also promoted osteogenic differentiation and mineralized matrix deposition of rat BM-PCs as the zinc content and culture time increased in vitro. The associated molecular mechanisms were investigated by Q-PCR and Western blotting, revealing that TGF-β/Smad signaling pathway plays a direct and significant role in regulating BM-PCs osteoblastic differentiation on Zn-modified coatings. Furthermore, in vivo results that revealed Zn-modified calcium silicate coatings significantly promoted new bone formation around the implant surface in osteopenic rabbits as the Zn content and exposure time increased. Therefore, Zn-modified calcium silicate coatings can improve implant osseointegration in the condition of osteopenia, which may be beneficial for patients suffering from osteoporosis-related fractures.

Topics: Animals; Bone Diseases, Metabolic; Calcium Compounds; Cell Differentiation; Cells, Cultured; Coated Materials, Biocompatible; Female; Osseointegration; Osteogenesis; Pericytes; Rabbits; Signal Transduction; Silicates; Smad Proteins; Transforming Growth Factor beta; Zinc

Deletion of TIEG1/KLF10 in mice results in an osteopenic skeletal phenotype with significant decreases in both bone mineral density and content throughout the skeleton. Calvarial osteoblasts isolated from TIEG1 knockout (KO) mice display numerous changes in gene expression and exhibit significant delays in their mineralization rates relative to wild-type (WT) controls. Here, we demonstrate that loss of TIEG1 expression in osteoblasts results in decreased levels of Osterix mRNA. Suppression of TIEG1 expression in WT osteoblasts leads to decreased Osterix expression while restoration of TIEG1 expression in TIEG1 KO osteoblasts results in increased levels of Osterix. Transient transfection and chromatin immunoprecipitation assays reveal that TIEG1 directly binds to and activates the Osterix promoter and demonstrate that the zinc finger-containing DNA binding domain of TIEG1 is necessary for this regulation. Furthermore, we reveal that TIEG1 expression is essential for the induction of Osterix expression by important bone-related cytokines such as TGFβ and BMP2 in osteoblast cells. Taken together, these data implicate an important role for TIEG1 in regulating the expression of Osterix, a master regulator of osteoblast differentiation and bone formation, and suggest that decreased expression of Osterix, as well as impaired TGFβ and BMP2 signaling, contribute to the observed osteopenic bone phenotype of TIEG1 KO mice.

Topics: Animals; Bone Diseases, Metabolic; Bone Morphogenetic Protein 2; Cells, Cultured; DNA-Binding Proteins; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Osteoblasts; Signal Transduction; Sp7 Transcription Factor; Transcription Factors; Transforming Growth Factor beta

Fibrillin-1 (FBN1) deficiency-induced systemic sclerosis is attributed to elevation of interleukin-4 (IL4) and TGF-β, but the mechanism underlying FBN1 deficiency-associated osteopenia is not fully understood. We show that bone marrow mesenchymal stem cells (BMMSCs) from FBN1-deficient (Fbn1(+/-)) mice exhibit decreased osteogenic differentiation and increased adipogenic differentiation. Mechanistically, this lineage alteration is regulated by IL4/IL4Rα-mediated activation of mTOR signaling to down-regulate RUNX2 and up-regulate PPARγ2, respectively, via P70 ribosomal S6 protein kinase (P70S6K). Additionally, we reveal that activation of TGF-β/SMAD3/SP1 signaling results in enhancement of SP1 binding to the IL4Rα promoter to synergistically activate mTOR pathway in Fbn1(+/-) BMMSCs. Blockage of mTOR signaling by osteoblastic-specific knockout or rapamycin treatment rescues osteopenia phenotype in Fbn1(+/-) mice by improving osteogenic differentiation of BMMSCs. Collectively, this study identifies a previously unrecognized role of the FBN1/TGF-β/IL4Rα/mTOR cascade in BMMSC lineage selection and provides experimental evidence that rapamycin treatment may provide an anabolic therapy for osteopenia in Fbn1(+/-) mice.

Topics: Adipogenesis; Animals; Bone Diseases, Metabolic; Cell Differentiation; Cells, Cultured; Female; Fibrillin-1; Fibrillins; Flow Cytometry; Immunoblotting; Immunosuppressive Agents; Male; Mesenchymal Stem Cells; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microfilament Proteins; Microscopy, Fluorescence; Osteogenesis; Receptors, Cell Surface; RNA Interference; Scleroderma, Systemic; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Type 2 diabetes mellitus (T2DM) is one of the most frequent metabolic disorders in industrialized countries. Among other complications, T2DM patients have an increased fracture risk and delayed fracture healing. We have demonstrated that supraphysiological glucose and insulin levels inhibit primary human osteoblasts׳ maturation. We aimed at developing a more physiologically relevant in vitro model to analyze T2DM-mediated osteoblast changes. Therefore, SCP-1-immortalized pre-osteoblasts were differentiated with T2DM or control (non-obese and obese) sera. Between both control groups, no significant changes were observed. Proliferation was significantly increased (1.69-fold), while AP activity and matrix mineralization was significantly reduced in the T2DM group. Expression levels of osteogenic marker genes and transcription factors were altered, e.g. down-regulation of RUNX2 and SP-7 or up-regulation of STAT1, in the T2DM group. Active TGF-β levels were significantly increased (1.46-fold) in T2DM patients׳ sera. SCP-1 cells treated with these sera showed significantly increased TGF-β signaling (2.47-fold). Signaling inhibition effectively restored osteoblast maturation in the T2DM group. Summarizing our data, SCP-1 cells differentiated in the presence of T2DM patients׳ serum exhibit reduced osteoblast function. Thus, this model has a high physiological impact, as it can identify circulating factors in T2DM patients׳ blood that may affect bone function, e.g. TGF-β.

Topics: Adult; Aged; Alkaline Phosphatase; Bone Diseases, Metabolic; Case-Control Studies; Cell Differentiation; Cell Line; Diabetes Mellitus, Type 2; Female; Gene Expression; Humans; Male; Middle Aged; Osteoblasts; Transforming Growth Factor beta

There are contradictory results about the effect of angiotensin-converting enzyme inhibitors (ACEIs) on bone. This study was performed to address the skeletal renin-angiotensin system (RAS) activity and the effects of the ACEI, captopril, on the bone of streptozotocin-induced type 1 diabetic mice. Histochemical assessment on bone paraffin sections was conducted by Safranin O staining and tartrate-resistant acid phosphatase staining. Micro-computed tomography was performed to analyze bone biological parameters. Gene and protein expression were determined by real-time polymerase chain reaction and immunoblotting, respectively. Type 1 diabetic mice displayed osteopenia phenotype and captopril treatment showed no osteoprotective effects in diabetic mice as shown by the reduction of bone mineral density, trabecular thickness and bone volume/total volume. The mRNA expression of ACE and renin receptor, and the protein expression of renin and angiotensin II were markedly up-regulated in the bone of vehicle-treated diabetic mice compared to those of non-diabetic mice, and these molecular changes of skeletal RAS components were effectively inhibited by treatment with captopril. However, treatment with captopril significantly elevated serum tartrate-resistant acid phosphatase 5b levels, reduced the ratio of osteoprotegerin/receptor activator of nuclear factor-κB ligand expression, increased carbonic anhydrase II mRNA expression and the number of matured osteoclasts and decreased transforming growth factor-β and osteocalcin mRNA expression in the tibia compared to those of diabetic mice. The present study demonstrated that the use of the ACEI, captopril, has no beneficial effect on the skeletal biological properties of diabetic mice. However, this could be attributed, at least partially, to its suppression of osteogenesis and stimulation of osteoclastogenesis, even though it could effectively inhibit high activity of local RAS in the bone of diabetic mice.

Topics: Acid Phosphatase; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Bone and Bones; Bone Density; Bone Diseases, Metabolic; Captopril; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Isoenzymes; Male; Mice; Mice, Inbred DBA; NF-kappa B; Osteocalcin; Osteoclasts; Random Allocation; Renin; Renin-Angiotensin System; RNA, Messenger; Streptozocin; Tartrate-Resistant Acid Phosphatase; Tibia; Transforming Growth Factor beta; Up-Regulation

TGF-β is abundantly produced in the skeletal system and plays a crucial role in skeletal homeostasis. E-selectin ligand-1 (ESL-1), a Golgi apparatus-localized protein, acts as a negative regulator of TGF-β bioavailability by attenuating maturation of pro-TGF-β during cartilage homeostasis. However, whether regulation of intracellular TGF-β maturation by ESL-1 is also crucial during bone homeostasis has not been well defined. Here, we show that Esl-1(-/-) mice exhibit a severe osteopenia with elevated bone resorption and decreased bone mineralization. In primary culture, Esl-1(-/-) osteoclast progenitors show no difference in osteoclastogenesis. However, Esl-1(-/-) osteoblasts show delayed differentiation and mineralization and stimulate osteoclastogenesis more potently in the osteoblast-osteoclast coculture, suggesting that ESL-1 primarily acts in osteoblasts to regulate bone homeostasis. In addition, Esl-1(-/-) calvaria exhibit an elevated mature TGF-β/pro-TGF-β ratio, with increased expression of TGF-β downstream targets (plasminogen activator inhibitor-1, parathyroid hormone-related peptide, connective tissue growth factor, and matrix metallopeptidase 13, etc.) and a key regulator of osteoclastogenesis (receptor activator of nuclear factor κB ligand). Moreover, in vivo treatment with 1D11, a pan-TGF-β antibody, significantly improved the low bone mass of Esl-1(-/-) mice, suggesting that elevated TGF-β signaling is the major cause of osteopenia in Esl-1(-/-) mice. In summary, our study identifies ESL-1 as an important regulator of bone remodeling and demonstrates that the modulation of TGF-β maturation is pivotal in the maintenance of a homeostatic bone microenvironment and for proper osteoblast-osteoclast coupling.

Topics: Animals; Antibodies; Bone Diseases, Metabolic; Bone Remodeling; Bone Resorption; Calcification, Physiologic; Cell Differentiation; Cell Lineage; Cells, Cultured; Femur; Gene Expression Profiling; Gene Expression Regulation; Homeostasis; Mice; Organ Size; Osteoblasts; Osteoclasts; Osteogenesis; Phenotype; Radiography; Receptors, Fibroblast Growth Factor; Sialoglycoproteins; Signal Transduction; Transforming Growth Factor beta

Microfibril-associated glycoprotein-1 (MAGP1), together with the fibrillins, are constitutive components of vertebrate microfibrils. Mice deficient in MAGP1 (murine MAGP1 knockout animals (Mfap2(-/-)); MAGP1Δ) is appropriate develop progressive osteopenia and reduced whole bone strength, and have elevated numbers of osteoclasts lining the bone surface. Our previous studies suggested that the increased osteoclast population was associated with elevated levels of receptor activator of NF-κB ligand (RANKL), a positive regulator of osteoclast differentiation. To explore the relationship between RANKL expression and osteoclast differentiation in MAGP1 deficiency, oophorectomy (OVX) was used to stimulate RANKL expression in both WT and MAGP1Δ animals. Bone loss following OVX was monitored using whole body DEXA and in vivo µCT. While WT mice exhibited significant bone loss following OVX, percent bone loss was reduced in MAGP1Δ mice. Further, serum RANKL levels rose significantly in OVX WT mice, whereas, there was only a modest increase in RANKL following OVX in the mutant mice due to already high baseline levels. Elevated RANKL expression was normalized when cultured MAGP1Δ osteoblasts were treated with a neutralizing antibody targeting free TGFβ. These studies provide support for increased RANKL expression associated with MAGP1 deficiency and provide a link to altered TGF-β signaling as a possible causative signaling pathway regulating RANKL expression in MAGP1Δ osteoblasts.

Topics: Animals; Bone Diseases, Metabolic; Bone Resorption; Cell Differentiation; Cells, Cultured; Contractile Proteins; Extracellular Matrix Proteins; Female; Mice; Mice, Knockout; Microfibrils; Osteoblasts; Osteoclasts; Osteogenesis; Ovariectomy; RANK Ligand; RNA Splicing Factors; Signal Transduction; Transforming Growth Factor beta

Reduced bone mineral density (osteopenia) is a poorly characterized manifestation of pediatric and adult patients afflicted with Marfan syndrome (MFS), a multisystem disorder caused by structural or quantitative defects in fibrillin-1 that perturb tissue integrity and TGFβ bioavailability. Here we report that mice with progressively severe MFS (Fbn1(mgR/mgR) mice) develop osteopenia associated with normal osteoblast differentiation and bone formation. In vivo and ex vivo experiments, respectively, revealed that adult Fbn1(mgR/mgR) mice respond more strongly to locally induced osteolysis and that Fbn1(mgR/mgR) osteoblasts stimulate pre-osteoclast differentiation more than wild-type cells. Greater osteoclastogenic potential of mutant osteoblasts was largely attributed to Rankl up-regulation secondary to improper TGFβ activation and signaling. Losartan treatment, which lowers TGFβ signaling and restores aortic wall integrity in mice with mild MFS, did not mitigate bone loss in Fbn1(mgR/mgR) mice even though it ameliorated vascular disease. Conversely, alendronate treatment, which restricts osteoclast activity, improved bone quality but not aneurysm progression in Fbn1(mgR/mgR) mice. Taken together, our findings shed new light on the pathogenesis of osteopenia in MFS, in addition to arguing for a multifaceted treatment strategy in this congenital disorder of the connective tissue.

Topics: Alendronate; Animals; Aorta; Aortic Aneurysm; Bone Diseases, Metabolic; Bone Morphogenetic Proteins; Bone Resorption; Disease Models, Animal; Fibrillin-1; Fibrillins; Losartan; Marfan Syndrome; Mice; Mice, Inbred C57BL; Microfilament Proteins; Mutation; Osteoblasts; Osteoclasts; Osteogenesis; Spine; Tomography, X-Ray Computed; Transforming Growth Factor beta

Recombinant bone morphogenetic proteins (BMPs) show promise in treating the orthopedic complications associated with neurofibromatosis type 1 (NF1), such as congenital pseudarthrosis of the tibia. Minimal scientific information regarding the effects of BMP in the context of NF1 is available. As abnormalities in both bone formation and resorption have been documented in Nf1-deficient mice, we hypothesized that inadequate BMP-induced bone formation could be augmented by cotreatment with the bisphosphonate zoledronic acid (ZA). First, primary osteoblasts isolated from wild type (Nf1(+/+)) and Nf1-deficient (Nf1(+/-)) mice were cultured in the presence and absence of BMP-2. While Nf1(+/-) cells exhibited less osteogenic potential than Nf1(+/+) cells, alkaline phosphatase expression and matrix mineralization for both genotypes were enhanced by BMP-2 treatment. To model this response in vivo, 20 microg BMP-2 was implanted intramuscularly into the quadriceps of mice to induce heterotopic bone. Radiographs revealed significantly less net bone formation in Nf1(+/-) mice compared to Nf1(+/+) controls. To test the effect of an antiresorptive agent, mice were cotreated twice weekly from postoperative day 3 with 0.02 mg/kg ZA or with saline. ZA treatment led to a synergistic increase in the amount of heterotopic bone in both Nf1(+/+) and Nf1(+/-) mice compared with saline controls, as measured by DEXA and histomorphometry. Thus, the anabolic deficiency noted in Nf1(+/-) mice is amenable to stimulation by BMP-2, but mineralized tissue formation remains below that of Nf1(+/+) controls. Bisphosphonate combination therapy is superior to BMP therapy alone in terms of net bone production in vivo in both wild-type and Nf1-deficient mice.

Topics: Animals; Bone Density Conservation Agents; Bone Diseases, Metabolic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Survival; Cells, Cultured; Diphosphonates; Disease Models, Animal; Drug Therapy, Combination; Energy Metabolism; Female; Femur; Haplotypes; Imidazoles; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurofibromatosis 1; Neurofibromin 1; Osteoblasts; Skull; Tibia; Transforming Growth Factor beta; Zoledronic Acid

Expression of the Wnt signaling inhibitor, DKK1 by multiple myeloma cells is correlated with lytic bone disease in multiple myeloma. However, the mechanism(s) by which DKK1 contributes to this process is not clear. Herein, we analyzed the functional role of canonical Wnt signaling and Dkk1 inhibition of this pathway in bone morphogenic protein (BMP)-2-induced osteoblast differentiation. Osteoblast differentiation was measured by alkaline phosphatase (ALP) activity in murine (C2C12) and human pre-osteoblast (hFOB1.19) and osteoblast-like (Saos-2 and MG63) cell lines. Cytoplasmic beta-catenin protein was separated by E-cadherin-GST pull-down assay and analyzed by Western blotting. A dominant negative form of beta-catenin, Dkk1 and TCF reporter constructs were transfected into C2C12 cells. C2C12 cells were also transfected with siRNA specific to LRP5/6 to knockdown receptor expression. Canonical Wnt signaling was activated in these cell lines in response to Wnt3a as assessed by increased cytoplasmic, non-phosphorylated beta-catenin and TCF/LEF transcription activity. Recombinant Dkk1 and plasma from MM patients containing high levels of Dkk1 blocked Wnt3a-induced beta-catenin accumulation. Importantly, Dkk1 abrogated BMP-2 mediated osteoblast differentiation. The requirement for Wnt signaling in osteoblast differentiation was confirmed by the following observations: 1) overexpression of Dkk1 decreased endogenous beta-catenin and ALP activity; 2) silencing of Wnt receptor mRNAs blocked ALP activity; and 3) a dominant negative form of beta-catenin eliminated BMP-2-induced ALP activity. Furthermore, Wnt3a did not increase ALP activity nor did BMP-2 treatment result in beta-catenin stabilization indicating that cooperation between these two pathways is required, but they are not co-regulated by either ligand. These studies have revealed that autocrine Wnt signaling in osteoblasts is necessary to promote BMP-2-mediated differentiation of pre-osteoblast cells, while Wnt signaling alone is not capable of inducing such differentiation. Dkk1 inhibits this process and may be a key factor regulating pre-osteoblast differentiation and myeloma bone disease.

Topics: Alkaline Phosphatase; Animals; beta Catenin; Bone Diseases, Metabolic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cell Line; Core Binding Factor Alpha 1 Subunit; Humans; Intercellular Signaling Peptides and Proteins; LDL-Receptor Related Proteins; Low Density Lipoprotein Receptor-Related Protein-5; Low Density Lipoprotein Receptor-Related Protein-6; Mice; Multiple Myeloma; Osteoblasts; RNA, Small Interfering; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Wnt Proteins

Nephroblastoma overexpressed (Nov), a member of the Cyr 61, connective tissue growth factor, Nov (CCN) family of proteins, is expressed by osteoblasts, but its function in cells of the osteoblastic lineage is not known. We investigated the effects of Nov overexpression by transducing murine ST-2 stromal and MC3T3 osteoblastic cells with a retroviral vector where Nov is under the control of the cytomegalovirus promoter. We also examined the skeletal phenotype of transgenic mice expressing Nov under the control of the human osteocalcin promoter. Overexpression of Nov in ST-2 cells inhibited the appearance of mineralized nodules and decreased alkaline phosphatase activity and osteocalcin mRNA levels. Nov overexpression inhibited the effect of bone morphogenetic protein (BMP)-2 on the phosphorylation of Smad 1/5/8; on the transactivation of 12xSBE-Oc-pGL3, a BMP/Smad signaling reporter construct, and of Wnt 3 on cytoplasmic beta-catenin levels; and on the transactivation of the Wnt/beta-catenin signaling reporter construct 16xTCF-Luc. Nov overexpression did not activate Notch or transforming growth factor beta signaling. Glutathione S-transferase pulldown assays demonstrated direct Nov-BMP interactions. Nov transgenic mice exhibited osteopenia. In conclusion, Nov binds BMP-2 and antagonizes BMP-2 and Wnt activity, and its overexpression inhibits osteoblastogenesis and causes osteopenia.

Topics: Alkaline Phosphatase; Animals; Bone Diseases, Metabolic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Line; Connective Tissue Growth Factor; Female; Gene Expression; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Nephroblastoma Overexpressed Protein; Osteocalcin; Osteogenesis; Promoter Regions, Genetic; Protein Binding; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Wnt Proteins

Biochemical experiments have shown that Smad6 and Smad ubiquitin regulatory factor 1 (Smurf1) block the signal transduction of bone morphogenetic proteins (BMPs). However, their in vivo functions are largely unknown. Here, we generated transgenic mice overexpressing Smad6 in chondrocytes. Smad6 transgenic mice showed postnatal dwarfism with osteopenia and inhibition of Smad1/5/8 phosphorylation in chondrocytes. Endochondral ossification during development in these mice was associated with almost normal chondrocyte proliferation, significantly delayed chondrocyte hypertrophy, and thin trabecular bone. The reduced population of hypertrophic chondrocytes after birth seemed to be related to impaired bone growth and formation. Organ culture of cartilage rudiments showed that chondrocyte hypertrophy induced by BMP2 was inhibited in cartilage prepared from Smad6 transgenic mice. We then generated transgenic mice overexpressing Smurf1 in chondrocytes. Abnormalities were undetectable in Smurf1 transgenic mice. Mating Smad6 and Smurf1 transgenic mice produced double-transgenic pups with more delayed endochondral ossification than Smad6 transgenic mice. These results provided evidence that Smurf1 supports Smad6 function in vivo.

Topics: Animals; Animals, Newborn; Bone and Bones; Bone Diseases, Metabolic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage; Cell Differentiation; Cell Size; Chondrocytes; Disease Models, Animal; DNA-Binding Proteins; Down-Regulation; Dwarfism; Fetus; Gene Expression Regulation, Developmental; Mice; Mice, Transgenic; Osteogenesis; Phosphorylation; Smad Proteins; Smad1 Protein; Smad6 Protein; Trans-Activators; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Human transforming growth factor-beta1 (TGFB1) is a family of polypeptides that regulate cell growth, cell differentiation, and cell function as a multifunctional regulator of cellular activity. TGFB1 is produced by osteoblasts and stored in substantial amounts in the bone matrix, which is an important regulator of both skeletal development and homeostasis of bone metabolism. In the present study, we identified four new polymorphisms in TGFB1 and examined whether these polymorphisms are risk factors for osteoporosis. We have sequenced all exons including in the promoter region up to -1,800bp to identify additional genetic polymorphisms in TGFB1. Four novel polymorphisms were newly identified: one in 5' region (g.14129555_14129557dupAGG), one in promoter region (g.14128838C>T), and two in intron (g.14106505G>A and g.14106215G>A). Two known SNPs (g.14128554C>T and g.14127139T>C) were also confirmed. The frequencies of each SNP were 0.479 (g.14129555_14129557dupAGG), 0.007 (g.14128838C>T), 0.478 (g.14128554C>T), 0.476 (g.14127139T>C), 0.016 (g.14106505G>A), and 0.004 (g.14106215G>A) in the Korean population (n=1,885), respectively. Haplotypes and their frequencies were estimated by EM algorithm, and linkage disequilibrium coefficients (mid R:/D'/: and r2) between polymorphism pairs were calculated. We analyzed genetic associations of TGFB1 polymorphisms and haplotypes with spinal bone mineral density (BMD) value of 433 postmenopausal Korean women. By statistical analysis, we could not find any associations with spinal BMD. The information from this study of the critical TGFB1 would be useful for genetic studies of other diseases.

Topics: Asian People; Bone Density; Bone Diseases, Metabolic; Female; Genetic Variation; Humans; Osteoporosis; Osteoporosis, Postmenopausal; Postmenopause; Spine; Transforming Growth Factor beta; Transforming Growth Factor beta1

Osteoporosis is a disease that is strongly genetically determined and polymorphisms present in a range of candidate genes may be involved. A number of previous studies have shown an association between the T869C functional polymorphism of the gene for transforming growth factor beta (TGF beta) and bone mineral density (BMD) and fracture, but these studies have been limited to relatively small studies of selected subjects. In a population-based study of 1337 white women over age 70 we examined the TGF beta T869 polymorphism in relation to BMD, calcaneal quantitative ultrasound (QUS), and prevalent and incident fracture. The TGF beta C allele was observed in 50% of the subjects and was associated with reduced hip BMD at all sites (2.8% total hip, 2.4% femoral neck, 2.6% intertrochanter, and 3.4% trochanter) compared to the TGF beta TT genotype. The TGF beta C allele was also associated with a reduction in the QUS parameters BUA, SOS, and stiffness of 0.87%, 0.26%, and 2.4%, respectively, compared to the TGF beta TT genotype. After adjustment for body mass index in an analysis of variance model, the effect of the TGF beta C allele remained significant at the total hip, the femoral neck, and the trochanter, and for the QUS SOS and stiffness parameters. The TGF beta C allele was associated with an increase in osteoporosis [T score < or =-2.5 SD; odds ratio (OR) 2.07; 95% confidence interval (CI) 1.19-3.60] and prevalent fracture (1.37; 95% CI 1.06-1.75). After adjustment for BMD and QUS stiffness, the association of the TGF beta C allele with prevalent fracture was still present (OR 1.40; 95% CI 1.04-1.89), suggesting that the effect of the C allele on fracture was independent of a reduction in BMD and QUS stiffness. Subjects with normal BMD and a TGF beta C allele had an increased risk of incident fracture over 3 years compared to subjects with normal BMD and a TGF beta TT genotype (relative risk 3.95; 95% CI 1.52-10.29). This association was not found in osteopenic or in osteoporotic subjects, indicating a BMD-TGF beta C allele interaction in relation to the association of the TGF beta C allele with fracture risk. These findings are of potential clinical usefulness, as the TGF beta T869C genotype could be used, in conjunction with other genetic and clinical information, to determine an individual's risk of osteoporosis.

Topics: Aged; Bone Density; Bone Diseases, Metabolic; Calcaneus; Female; Fractures, Bone; Gene Frequency; Genetic Predisposition to Disease; Humans; Incidence; Osteoporosis; Polymorphism, Genetic; Prevalence; Risk Factors; Transforming Growth Factor beta; Ultrasonography

Osteoporosis is a disease manifested in drastic bone loss resulting in osteopenia and high risk for fractures. This disease is generally divided into two subtypes. The first, post-menopausal (type I) osteoporosis, is primarily related to estrogen deficiency. The second, senile (type II) osteoporosis, is mostly related to aging. Decreased bone formation, as well as increased bone resorption and turnover, are thought to play roles in the pathophysiology of both types of osteoporosis. In this study, we demonstrate in murine models for both type I (estrogen deficiency) and type II (senile) osteopenia/osteoporosis that reduced bone formation is related to a decrease in adult mesenchymal stem cell (AMSC) number, osteogenic activity, and proliferation. Decreased proliferation is coupled with increased apoptosis in AMSC cultures obtained from osteopenic mice. Recombinant human bone morphogenetic protein (rhBMP-2) is a highly osteoinductive protein, promoting osteogenic differentiation of AMSCs. Systemic intra-peritoneal (i.p.) injections of rhBMP-2 into osteopenic mice were able to reverse this phenotype in the bones of these animals. Moreover, this change in bone mass was coupled to an increase in AMSCs numbers, osteogenic activity, and proliferation as well as a decrease in apoptosis. Bone formation activity was increased as well. However, the magnitude of this response to rhBMP-2 varied among different stains of mice. In old osteopenic BALB/c male mice (type II osteoporosis model), rhBMP-2 systemic treatment also restored both articular and epiphyseal cartilage width to the levels seen in young mice. In summary, our study shows that AMSCs are a good target for systemically active anabolic compounds like rhBMP-2.

Topics: Aging; Alkaline Phosphatase; Animals; Apoptosis; Bone and Bones; Bone Diseases, Metabolic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cartilage; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Female; Humans; Male; Mesoderm; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Osteogenesis; Osteoporosis; Ovariectomy; Recombinant Proteins; Stem Cells; Transforming Growth Factor beta

Cyclosporine A (CsA), a potent immunosuppressant used in transplantation, induces increased formation with excess resorption in the rat with resultant osteopenia. These findings are confirmed in the human model. Transforming growth factor-beta (TGF-beta) is reported to be involved in the coupling of bone formation with resorption and in vivo and in vitro stimulates osteoblasts, and in vitro inhibits osteoclasts. CsA stimulates secretion of TGF-beta1 in humans, which, while improving immunosuppression, may also contribute to renal toxicity. This study was performed determine whether exogenously administered TGF-beta would modify the bone effects of CsA. Male Sprague-Dawley rats, 6 months of age, were randomized to receive: TGF-beta and CsA vehicle (group A); TGF-beta 5 microg/kg three times per week and CsA vehicle (group B); TGF-beta vehicle and CsA 10 mg/kg (group C); or TGF-beta 5 microg/kg three times per week and CsA 10 mg/kg (group D). These were compared with control over 28 days. CsA, but not TGF-beta, increased serum 1,25(OH)(2)D levels throughout the study. CsA increased osteocalcin (BGP), but TGF-beta negated this effect. Histomorphometry confirmed the known effects of CsA, whereas TGF-beta alone had no effect. However, in combination, TGF-beta blocked CsA's effect and increased osteoblast recruitment and activity, as reflected by increased percent mineralizing surface, percent osteoid perimeter, bone formation rate (bone volume referent), and activation frequency. Thus, it appears as if TGF-beta administration may have potential in modulating the deleterious bone effects of CsA.

Topics: Animals; Bone Diseases, Metabolic; Bone Resorption; Cyclosporine; Ergocalciferols; Male; Osteocalcin; Parathyroid Hormone; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Smad3 is a well-characterized intracellular effector of the transforming growth factor beta (TGF-beta) signaling pathway and was implicated recently in the potentiation of vitamin D receptor (VDR)-mediated signaling. Given that both TGF-beta and vitamin D are important regulators of bone remodeling, it is expected that Smad3 plays an integral role in normal maintenance of bone. However, the exact mechanisms by which Smad3 functions in bone remodeling are unknown. Here, we show that mice with targeted deletion of Smad3 are osteopenic with less cortical and cancellous bone compared with wild-type littermates. Decreases in bone mineral density (BMD) in Smad3 null mice reflect the inability of osteoblasts to balance osteoclast activity, although osteoclast numbers are normal and vitamin D mediated serum calcium homeostasis is maintained. The osteopenia of Smad3 null mice is attributed to a decreased rate of bone formation associated with increased osteocyte number and apoptosis. These findings are supported by studies with isolated primary osteoblasts that show TGF-beta can no longer inhibit the differentiation of osteoblasts in the absence of Smad3; yet, TGF-beta-stimulated proliferation remains intact. Together these data support a model that a loss of Smad3 increases the osteocyte fate of the osteoblast and decreases the duration of osteoblast function by shortening lifespan, ultimately resulting in osteopenia.

Topics: Animals; Apoptosis; Bone and Bones; Bone Diseases, Metabolic; Calcium; Cell Count; Cell Differentiation; DNA-Binding Proteins; Female; Homeostasis; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

Topics: Animals; Anticholesteremic Agents; Bone Diseases, Metabolic; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Bone Resorption; Female; Humans; Hypercholesterolemia; Male; Models, Biological; Osteoporosis; Transforming Growth Factor beta

We have previously hypothesized that the osteopenic changes seen in the skeletons of old male BALB/c mice are due to reductions in the availability and/or synthesis of bone TGF-beta which results in fewer, less osteogenic marrow osteoprogenitor cells (CFU-f; OPCs) and lower levels of bone formation. Among other things, this hypothesis would predict that introducing exogenous TGF-beta into old mice (growth factor replacement) should stimulate marrow CFU-f and increase bone formation. In the present study, we have tested this prediction and, indirectly the hypothesis, by injecting human recombinant TGF-beta1, i.p., into both young adult (4 month) and old mice (24 month). The effects of the growth factor on the skeleton were then assessed by measurements of trabecular bone volume, bone formation, fracture healing, and the number, proliferative, apoptotic, and alkaline phosphatase activity of marrow CFU-f/OPCs. Our data show that the introduction of 0.5 or 5.0 ug/day of TGF-beta1 into old mice for 20 days 1) increases trabecular bone volume, bone formation and the mineral apposition rate, 2) augments fracture healing, 3) increases the number and size of CFU-f colonies, and 4) increases proliferation and diminishes apoptosis of CFU-f in primary bone marrow cultures. Importantly, these stimulatory effects of injected growth factor are apparently age-specific, i.e., they are either not seen in young animals or, if seen, are found at much lower levels. While these observations do not exclude other possible mechanisms for the osteopenia of old mice, they provide further support for the hypothesis that, with age, diminished TGF-beta synthesis or availability results in a reduction in the marrow osteoprogenitor pool and bone formation. The findings also demonstrate that the latter changes can be reversed, at least transiently, by introducing exogenous TGF-beta1.

Topics: Age Factors; Alkaline Phosphatase; Animals; Bone Diseases, Metabolic; Bone Marrow Cells; Bone Matrix; Cell Division; Cells, Cultured; Femur; Fracture Healing; Male; Mice; Mice, Inbred BALB C; Recombinant Proteins; Ribs; Stem Cells; Tibia; Transforming Growth Factor beta

The effects of tail suspension hypokinesia on the gene expression for TGF-beta2 at different sites within bone were evaluated. TGF-beta2 mRNA signal levels were determined quantitatively by an image analysis system. The osteopenia induced by tail suspension was verified by histomorphometry. In the periosteum of nonsuspended control rats, TGF-beta2 mRNA was highly expressed in the preosteoblasts and osteoblast-rich cambial layers; very little signal was present within the middle and outer fibroblastic layers. Gene expression was significantly reduced in suspended rats, and this was evident both in terms of the number of silver grains in unit area or length of tissue and in each osteoblast and preosteoblast. Hypokinesia also reduced the expression of TGF-beta2 mRNA level in cortical and trabecular bone osteocytes, but did not adversely affect the mRNA level in chondrocytes in growth plate. The results affirm the site-specific response of TGF-beta2 gene expression in rats, and suggest that the cortical and trabecular bone osteopenia associated with hypokinesia in rats may be associated with a deficit in osteoblastic and osteocytic TGF-beta2 level.

Topics: Animals; Body Weight; Bone Diseases, Metabolic; Femur; Hindlimb Suspension; Humerus; Image Processing, Computer-Assisted; In Situ Hybridization; Osteoblasts; Periosteum; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta

One of the universal characteristics of the long bones and spines of middle-age and older mammals is a loss in bone mass (osteopenia). In humans, if this bone loss is severe enough, it results in osteoporosis, a skeletal disorder characterized by a markedly increased incidence of fractures with sequelae that may include pain, loss of mobility, and in the event of hip fracture, even death within a relatively few months of injury. An important contributing factor to the development of osteoporosis appears to be a diminution in the number and activity of osteoblasts responsible for synthesizing new bone matrix. The findings in the present and other similar studies suggest that this reduction in osteoblast number and activity is due to an age-related diminution in the size and osteogenic potential of the bone marrow osteoblast progenitor cell (OPC or CFU-f) compartment. We previously postulated that these regressive changes in the OPC/CFU-f compartment occurred in old animals because of a reduction in the amount and/or activity of TGF-beta1, an autocrine growth factor important in the promotion of OPC/CFU-f proliferation and differentiation. In support of this hypothesis, we now report that (1) the osteogenic capacity of the bone marrow of 24-month-old BALB/c mice, as assessed in vivo, is markedly reduced relative to that of 3-4-month-old animals, (2) that the matrix of the long bones of old mice contains significantly less TGF-beta than that of young mice, (3) that OPC's/CFU-f's isolated from old mice produce less TGF-beta in vitro than those recovered from young mice, and (4) that OPC's/CFU-f's from old mice express significantly more TGF-beta receptor (Types I, II, and III) than those of young animals and that such cells are more responsive in vitro to exogenous recombinant TGF-beta1. We also find that colony number and proliferative activity of OPC's/CFU-f's of young mice and old mice, respectively, are significantly reduced when incubated in the presence of neutralizing TGF-beta1 antibody. Collectively, these data are consistent with the hypothesis that in old male mice the reduction in the synthesis and, perhaps, availability from the bone matrix of TGF-beta1 contributes to a diminution in the size and development potential of the bone marrow osteoprogenitor pool.

Topics: Aging; Animals; Bone Development; Bone Diseases, Metabolic; Bone Marrow Cells; Male; Mice; Mice, Inbred BALB C; Recombinant Proteins; Transforming Growth Factor beta

A previous study showed that skeletal unloading induced by hindlimb suspension for 14 days in rats reduces osteoblastic cell proliferation, inhibits skeletal growth and bone formation and induces metaphyseal bone loss. This study investigated the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) in this model. In vitro analysis showed that rhBMP-2 (25-100 ng/ml, 48-96 h) increased alkaline phosphatase activity, an early marker of osteoblast differentiation, in rat neonatal calvaria cells and adult marrow stromal cells, showing that rhBMP-2 induced the differentiation of osteoblast precursor cells in vitro. In contrast, rhBMP-2 did not increase rat calvaria or marrow stromal cell proliferation. Biochemical and histomorphometric analysis showed that systemic infusion with rhBMP-2 (2 microg/kg/day) in unloaded rats had no significant effect on serum osteocalcin levels and on histomorphometric indices of bone formation. Accordingly, rhBMP-2 infusion did not prevent the decreased skeletal growth, trabecular bone bone volume and bone mineral content induced by unloading. The present data indicate that, although rhBMP-2 stimulates osteoblastic cell differentiation, rhBMP-2 infusion is not effective in increasing bone formation and in preventing trabecular bone loss induced by unloading in rats.

Topics: Alkaline Phosphatase; Animals; Animals, Newborn; Body Weight; Bone Density; Bone Development; Bone Diseases, Metabolic; Bone Marrow Cells; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cell Division; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Activation; Femur; Humans; Osteoblasts; Osteocalcin; Rats; Recombinant Proteins; Skull; Stem Cells; Stromal Cells; Tibia; Transforming Growth Factor beta

We investigated the effect of recombinant human transforming growth factor beta 2 (rhTGF-beta 2) administration on trabecular bone loss induced by unloading in rats. Hind limb suspension for 14 d inhibited bone formation and induced osteopenia as shown by decreased bone volume, calcium and protein contents in long bone metaphysis. Systemic infusion of rhTFG-beta 2 (2 micrograms/kg per day) maintained normal bone formation rate, and prevented the decrease in bone volume, bone mineral content, trabecular thickness and number induced by unloading. In vitro analysis of tibial marrow stromal cells showed that rhTGF-beta 2 infusion in unloaded rats increased the proliferation of osteoblast precursor cells, but did not affect alkaline phosphatase activity or osteocalcin production. Northern blot analysis of RNA extracted from the femoral metaphysis showed that rhTGF-beta 2 infusion in unloaded rats increased steady-state levels of type I collagen mRNA but not alkaline phosphatase mRNA levels. rhTGF-beta 2 infusion at the dose used had no effect on metaphyseal bone volume and formation, osteoblast proliferation or collagen expression in control rats. The results show that systemic administration of rhTGF-beta 2 enhances osteoblast precursor cell proliferation and type I collagen expression by osteoblasts, and prevents the impaired bone formation and osteopenia induced by unloading.

Topics: Animals; Biomechanical Phenomena; Bone and Bones; Bone Density; Bone Development; Bone Diseases, Metabolic; Bone Marrow; Bone Marrow Cells; Cell Division; Cells, Cultured; Collagen; Humans; Male; Rats; Rats, Wistar; Recombinant Proteins; Reference Values; RNA, Messenger; Transforming Growth Factor beta

We previously reported that bone marrow stromal cells produce insulin-like growth factors (IGF-I and -II), and that medium conditioned by marrow stromal cells stimulates osteoblast proliferation in vitro. The present study employed the rat tail-suspension model to unload the hindlimbs. It was designed to test the hypothesis that the development of osteopenia or osteoporosis could be due to a deficit in the osteogenic function of marrow stromal cells. Although tail suspension suppressed body weight during the first 3 days of an 11-day pair-fed study, the overall weight gain recorded by these animals was normal. Nevertheless, bone growth was inhibited by suspension. Similarly, the total adherent marrow stromal cell population harvested from the femurs and tibias was decreased by tail suspension, and only half the normal number of fibroblastic stromal cell colonies grew when they were cultured. The proliferation of alkaline-phosphatase-positive cells in the stroma was also inhibited. Northern hybridization revealed that the messenger RNA level for transforming growth factor-beta 2 and IGF-II in stromal cell was reduced by tail suspension. The production of IGF-II by marrow stromal cells was also decreased. The steady-state level of five different transcript sizes of IGF-I mRNA was altered differentially by tail suspension. Osteopontin mRNA was also reduced in marrow stromal cells from tail-suspended rats compared with the normal rats. These data suggest that skeletal unloading not only alters the mRNA level for growth factors and peptide production, but also affects the proliferation and osteogenic differentiation of marrow stromal cells. These changes may be responsible for the reduced bone formation in osteopenia and osteoporosis.

Topics: Animals; Bone Diseases, Metabolic; Bone Marrow Cells; Cell Adhesion; Cell Differentiation; Cell Division; Cells, Cultured; Collagen; Femur; Gene Expression Regulation, Developmental; Male; Osteoblasts; Osteogenesis; Osteopontin; Osteoporosis; Random Allocation; Rats; Rats, Wistar; RNA, Messenger; Sialoglycoproteins; Somatomedins; Stromal Cells; Tail; Tibia; Transforming Growth Factor beta; Weight-Bearing