Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling

Bone morphogenetic proteins (BMPs) function during various aspects of embryonic development including skeletogenesis. However, their biological functions after birth are less understood. To investigate the role of BMPs during bone remodeling, we generated a postnatal osteoblast-specific disruption of Bmpr1a that encodes the type IA receptor for BMPs in mice. Mutant mice were smaller than controls up to 6 months after birth. Irregular calcification and low bone mass were observed, but there were normal numbers of osteoblasts. The ability of the mutant osteoblasts to form mineralized nodules in culture was severely reduced. Interestingly, bone mass was increased in aged mutant mice due to reduced bone resorption evidenced by reduced bone turnover. The mutant mice lost more bone after ovariectomy likely resulting from decreased osteoblast function which could not overcome ovariectomy-induced bone resorption. In organ culture of bones from aged mice, ablation of the Bmpr1a gene by adenoviral Cre recombinase abolished the stimulatory effects of BMP4 on the expression of lysosomal enzymes essential for osteoclastic bone resorption. These results demonstrate essential and age-dependent roles for BMP signaling mediated by BMPRIA (a type IA receptor for BMP) in osteoblasts for bone remodeling.     

Syndecan-2 regulates transforming growth factor-beta signaling

Transforming growth factor-beta (TGF-beta) has multiple functions including increasing extracellular matrix deposition in fibrosis. It functions through a complex family of cell surface receptors that mediate downstream signaling. We report here that a transmembrane heparan sulfate proteoglycan, syndecan-2 (S2), can regulate TGF-beta signaling. S2 protein increased in the renal interstitium in diabetes and regulated TGF-beta-mediated increased matrix deposition in vitro. Transfection of renal papillary fibroblasts with S2 or a S2 construct that has a truncated cytoplasmic domain (S2DeltaS) promoted TGF-beta binding and S2 core protein ectodomain directly bound TGF-beta. Transfection with S2 increased the amounts of type I and type II TGF-beta receptors (TbetaRI and TbetaRII), whereas S2DeltaS was much less effective. In contrast, S2DeltaS dramatically increased the level of type III TGF-beta receptor (TbetaRIII), betaglycan, whereas S2 resulted in a decrease. Syndecan-2 specifically co-immunoprecipitated with betaglycan but not with TbetaRI or TbetaRII. This is a novel mechanism of control of TGF-beta action that may be important in fibrosis.     

Role of activin, transforming growth factor-beta and bone morphogenetic protein 15 in regulating zebrafish oocyte maturation

The transforming growth factor-beta (TGF-beta) superfamily is a large group of peptide growth and differentiation factors that have important functions in many physiological processes, including reproduction. We previously reported that several members of the TGF-beta superfamily, including activin-A, bone morphogenetic protein-15 (BMP-15) and TGF-beta1, regulate oocyte maturation in zebrafish. The aim of this study was to further examine the functions and mechanisms of these growth factors in regulating zebrafish oocyte maturation. First, the interaction among three regulators was examined. Overexpression of BMP-15 reduced the effect of activin-A on oocyte maturation. Inhibition of BMP-15 function or expression increased oocyte maturation but had no additive effect with activin-A. TGF-beta1 suppressed activin-A-, as well as BMP-15 antiserum-induced oocyte maturation. Second, the role of Smad 2, an intracellular mediator of activin and TGF-beta, in oocyte maturation was investigated. Western blot analysis revealed that both activin-A and TGF-beta1 activate Smad2 in zebrafish follicles. Injection of morpholino antisense olignucleotides against Smad2 into oocytes reduced Smad2 expression and completely blocked activin-A-induced oocyte maturation. Knockdown of Smad 2 also significantly decreased basal and hCG-induced oocyte maturation. These findings suggest that activin-A, TGF-beta1, and BMP-15 may target common gene(s) to regulate oocyte maturation and demonstrate that Smad2 plays an important role in oocyte maturation.     

Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells

Transforming growth factor beta (TGF-beta) activates Ras/MAPK signaling in many cell types. Because TGF-beta and BMP-2 exert similar effects, we examined if this signaling is stimulated by both factors and analyzed the relationship between this signaling and the Smads in osteoblasts. BMP-2 and TGF-beta stimulated Ras, MAPK, and AP-1 activities. The DNA binding activities of c-Fos, FosB/Delta FosB, Fra-1, Fra-2, and JunB were up-regulated whereas JunD activity was decreased. c-Fos, FosB/Delta FosB, and JunB were associated with Smad4. The stimulation of AP-1 by BMP-2 and TGF-beta was dependent on Smad signaling, and anti-Smad4 antibody interfered with AP-1 activity. Thus, BMP-2 and TGF-beta activate both Ras/MAPK/AP-1 and Smad signaling in osteoblasts with Smads modulating AP-1 activity. To determine the roles of MAPK in BMP-2 and TGF-beta function, we analyzed the effect of ERK and p38 inhibitors on the regulation of bone matrix protein expression and JunB and JunD levels by these two factors. ERK and p38 mediated TGF-beta suppression of osteocalcin and JunD as well as stimulation of JunB. p38 was essential in BMP-2 up-regulation of type I collagen, fibronectin, osteopontin, osteocalcin, and alkaline phosphatase activity whereas ERK mediated BMP-2 stimulation of fibronectin and osteopontin. Thus, ERK and p38 differentially mediate TGF-beta and BMP-2 function in osteoblasts.     

Interaction of transforming growth factor-beta 1 with alpha 2-macroglobulin. Role in transforming growth factor-beta 1 clearance.

It has been widely assumed that the interaction of transforming growth factor-beta 1 (TGF-beta 1) with its serum-binding protein, alpha 2-macroglobulin (alpha 2M), mediates the rapid clearance of TGF-beta 1 from the circulation. To test this, we have analyzed the effect of TGF-beta 1 binding on the conformational state of alpha 2M. Our results demonstrate that the binding of TGF-beta 1 to alpha 2M does not lead to the conformational change in the alpha 2M molecule that is required for the clearance of the alpha 2M.TGF-beta 1 complex via the alpha 2M receptor. Furthermore, endogenous TGF-beta 1 is associated with the conformationally unaltered slow clearance form of alpha 2M. Clearance studies in mice show that the half-life of 125I-TGF-beta 1 in the circulation (1.6 +/- 0.71 min) is not affected by blocking the alpha 2M receptor with excess conformationally altered alpha 2M. These results suggest that TGF-beta 1 is rapidly cleared from the circulation after injection by a pathway not involving alpha 2M.     

Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin

Growth differentiation factor 9 (GDF9) is an oocyte-expressed member of the transforming growth factor beta (TGF-beta) superfamily and is required for normal ovarian follicle development and female fertility. GDF9 acts as a paracrine factor and affects granulosa cell physiology. Only a few genes regulated by GDF9 are known. Our microarray analysis has identified gremlin as one of the genes up-regulated by GDF9 in cultures of granulosa cells. Gremlin is a known member of the DAN family of bone morphogenetic protein (BMP) antagonists, but its expression and function in the ovary are unknown. We have investigated the regulation of gremlin in mouse granulosa cells by GDF9 as well as other members of the TGF-beta superfamily. GDF9 and BMP4 induce gremlin, but TGF-beta does not. In addition, in cultures of granulosa cells, gremlin negatively regulates BMP4 signaling but not GDF9 activity. The expression of gremlin in the ovary was also examined by in situ hybridization. A distinct change in gremlin mRNA compartmentalization occurs during follicle development and ovulation, indicating a highly regulated expression pattern during folliculogenesis. We propose that gremlin modulates the cross-talk between GDF9 and BMP signaling that is necessary during follicle development because both ligands use components of the same signaling pathway.     

Microarray gene expression profiling of osteoarthritic bone suggests altered bone remodelling, WNT and transforming growth factor-beta/bone morphogenic protein signalling

Osteoarthritis (OA) is characterized by alterations to subchondral bone as well as articular cartilage. Changes to bone in OA have also been identified at sites distal to the affected joint, which include increased bone volume fraction and reduced bone mineralization. Altered bone remodelling has been proposed to underlie these bone changes in OA. To investigate the molecular basis for these changes, we performed microarray gene expression profiling of bone obtained at autopsy from individuals with no evidence of joint disease (control) and from individuals undergoing joint replacement surgery for either degenerative hip OA, or fractured neck of femur (osteoporosis [OP]). The OP sample set was included because an inverse association, with respect to bone density, has been observed between OA and the low bone density disease OP. Compugen human 19K-oligo microarray slides were used to compare the gene expression profiles of OA, control and OP bone samples. Four sets of samples were analyzed, comprising 10 OA-control female, 10 OA-control male, 10 OA-OP female and 9 OP-control female sample pairs. Print tip Lowess normalization and Bayesian statistical analyses were carried out using linear models for microarray analysis, which identified 150 differentially expressed genes in OA bone with t scores above 4. Twenty-five of these genes were then confirmed to be differentially expressed (P < 0.01) by real-time PCR analysis. A substantial number of the top-ranking differentially expressed genes identified in OA bone are known to play roles in osteoblasts, osteocytes and osteoclasts. Many of these genes are targets of either the WNT (wingless MMTV integration) signalling pathway (TWIST1, IBSP, S100A4, MMP25, RUNX2 and CD14) or the transforming growth factor (TGF)-beta/bone morphogenic protein (BMP) signalling pathway (ADAMTS4, ADM, MEPE, GADD45B, COL4A1 and FST). Other differentially expressed genes included WNT (WNT5B, NHERF1, CTNNB1 and PTEN) and TGF-beta/BMP (TGFB1, SMAD3, BMP5 and INHBA) signalling pathway component or modulating genes. In addition a subset of genes involved in osteoclast function (GSN, PTK9, VCAM1, ITGB2, ANXA2, GRN, PDE4A and FOXP1) was identified as being differentially expressed in OA bone between females and males. Altered expression of these sets of genes suggests altered bone remodelling and may in part explain the sex disparity observed in OA.     

Transforming growth factor-beta1, transforming growth factor-beta2, and transforming growth factor-beta3 enhance ovarian cancer metastatic potential by inducing a Smad3-dependent epithelial-to-mesenchymal transition

Transforming growth factor-beta (TGF-beta) is thought to play a role in the pathobiological progression of ovarian cancer because this peptide hormone is overexpressed in cancer tissue, plasma, and peritoneal fluid. In the current study, we investigated the role of the TGF-beta/Smad3 pathway in ovarian cancer metastasis by regulation of an epithelial-to-mesenchymal transition. When cancer cells were cultured on plastic, TGF-beta1, TGF-beta2, and TGF-beta3 induced pro-matrix metalloproteinase (MMP) secretion, loss of cell-cell junctions, down-regulation of E-cadherin, up-regulation of N-cadherin, and acquisition of a fibroblastoid phenotype, consistent with an epithelial-to-mesenchymal transition. Furthermore, Smad3 small interfering RNA transfection inhibited TGF-beta-mediated changes to a fibroblastic morphology, but not MMP secretion. When cancer cells were cultured on a three-dimensional collagen matrix, TGF-beta1, TGF-beta2, and TGF-beta3 stimulated both pro-MMP and active MMP secretion and invasion. Smad3 small interfering RNA transfection of cells cultured on a collagen matrix abrogated TGF-beta-stimulated invasion and MMP secretion. Analysis of Smad3 nuclear expression in microarrays of serous benign tumors, borderline tumors, and cystadenocarcinoma revealed that Smad3 expression could be used to distinguish benign and borderline tumors from carcinoma (P = 0.006). Higher Smad3 expression also correlated with poor survival (P = 0.031). Furthermore, a direct relationship exists between Smad3 nuclear expression and expression of the mesenchymal marker N-cadherin in cancer patients (P = 0.0057). Collectively, these results implicate an important role for the TGF-beta/Smad3 pathway in mediating ovarian oncogenesis by enhancing metastatic potential.     

Transforming growth factor-beta activity is potentiated by heparin via dissociation of the transforming growth factor-beta/alpha 2- macroglobulin inactive complex

The control of smooth muscle cell (SMC) proliferation is determined by the combined actions of mitogens, such as platelet-derived growth factor, and the opposing action of growth inhibitory agents, such as heparin and transforming growth factor-beta (TGF-beta). The present studies identify an interaction between heparin and TGF-beta in which heparin potentiates the biological action of TGF-beta. Using a neutralizing antibody to TGF-beta, we observed that the short term antiproliferative effect of heparin depended upon the presence of biologically active TGF-beta. This effect was observed in rat and bovine aortic SMC and in CCL64 cells, but not in human saphenous vein SMC. Binding studies demonstrated that the addition of heparin (100 micrograms/ml) to medium containing 10% plasma-derived serum resulted in a 45% increase in the specific binding of 125I-TGF-beta to cells. Likewise, heparin induced a twofold increase in the growth inhibitory action of TGF-beta at concentrations of TGF-beta near its apparent dissociation constant. Using 125I-labeled TGF-beta, we demonstrated that TGF-beta complexes with the plasma component alpha 2-macroglobulin, but not with fibronectin. Heparin increases the electrophoretic mobility of TGF-beta apparently by freeing TGF-beta from its complex with alpha 2-macroglobulin. Dextran sulfate, another highly charged antiproliferative molecule, but not chondroitin sulfate or dermatan sulfate, similarly modified TGF-beta's mobility. Relatively high, antiproliferative concentrations of heparin (1-100 micrograms/ml) were required to dissociate the TGF-beta/alpha 2-macroglobulin complex. Thus, it appears that the antiproliferative effect of heparin may be partially attributed to its ability to potentiate the biological activity of TGF-beta by dissociating it from alpha 2-macroglobulin, which normally renders it inactive. We suggest that heparin-like agents may be important regulators of TGF-beta's biological activity.     

Overexpression of bone morphogenetic protein 10 in myocardium disrupts cardiac postnatal hypertrophic growth

Postnatal cardiac hypertrophies have traditionally been classified into physiological or pathological hypertrophies. Both of them are induced by hemodynamic load. Cardiac postnatal hypertrophic growth is regarded as a part of the cardiac maturation process that is independent of the cardiac working load. However, the functional significance of this biological event has not been determined, mainly because of the difficulty in creating an experimental condition for testing the growth potential of functioning heart in the absence of hemodynamic load. Recently, we generated a novel transgenic mouse model (alphaMHC-BMP10) in which the cardiac-specific growth factor bone morphogenetic protein 10 (BMP10) is overexpressed in postnatal myocardium. These alphaMHC-BMP10 mice appear to have normal cardiogenesis throughout embryogenesis, but develop to smaller hearts within 6 weeks after birth. alphaMHC-BMP10 hearts are about half the normal size with 100% penetrance. Detailed morphometric analysis of cardiomyocytes clearly indicated that the compromised cardiac growth in alphaMHC-BMP10 mice was solely because of defect in cardiomyocyte postnatal hypertrophic growth. Physiological analysis further demonstrated that the responses of these hearts to both physiological (e.g. exercise-induced hypertrophy) and pathological hypertrophic stimuli remain normal. In addition, the alphaMHC-BMP10 mice develop subaortic narrowing and concentric myocardial thickening without obstruction by four weeks of age. Systematic analysis of potential intracellular pathways further suggested a novel genetic pathway regulating this previously undefined cardiac postnatal hypertrophic growth event. This is the first demonstration that cardiac postnatal hypertrophic growth can be specifically modified genetically and dissected out from physiological and pathological hypertrophies.     

CHIP regulates skeletal development and postnatal bone growth

C terminus of Hsc70-interacting protein (CHIP) is a chaperone-dependent and U-box containing E3 ubiquitin ligase. In previous studies, we found that CHIP regulates the stability of multiple tumor necrosis factor receptor-associated factor proteins in bone cells. In Chip global knockout (KO) mice, nuclear factor-κB signaling is activated, osteoclast formation is increased, osteoblast differentiation is inhibited, and bone mass is decreased in postnatal Chip KO mice. To determine the role of Chip in different cell types at different developmental stages, we created Chip^flox/flox mice. We then generated Chip conditional KO mice Chip^CMV and Chip^OsxER and demonstrated defects in skeletal development and postnatal bone growth in Chip conditional KO mice. Our findings indicate that Chip conditional KO mice could serve as a critical reagent for further investigations of functions of CHIP in bone cells and in other cell types.     

Binding of transforming growth factor-beta 1 to immobilized human alpha 2-macroglobulin.

Native alpha 2-macroglobulin (alpha 2M) and alpha 2M-methylamine were immobilized in 96-well microtiter plates. 125I-labeled transforming growth factor-beta 1 (TGF-beta 1) bound to both alpha 2M variants; however, greater binding was observed with alpha 2M-methylamine. Binding of 125I-TGF-beta 1 (0.2 nM) to immobilized alpha 2M-methylamine was inhibited by nonradiolabeled TGF-beta 1 (up to 74% with 0.4 microM TGF-beta 1). Approximately 10% of the TGF-beta 1-alpha 2M-methylamine complex was covalent. Treatment of alpha 2M-methylamine with iodoacetamide prior to immobilization completely eliminated covalent TGF-beta 1 binding; the total amount of 125I-TGF-beta 1-alpha 2M-methylamine complex detected was unchanged. The binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was not significantly inhibited by increasing the ionic strength to 1.0 M. Binding and complex dissociation were also unaffected by changes in pH within the range 6.9-8.9. Acidic pH dramatically decreased binding and promoted complex dissociation; no binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was detected at pH 3.5. The interaction of TGF-beta 1 with immobilized alpha 2M-methylamine was not significantly changed by 1.0 mM EDTA or 1.0 mM CaCl2. ZnCl2 (1.0 mM) completely eliminated binding. This result was not due to TGF-beta 1 precipitation or aggregation. Inhibition of 125I-TGF-beta 1 binding to alpha 2M-methylamine was 50% complete (IC50) with 30 microM ZnCl2. Native alpha 2M, thrombospondin, and alpha 2M-methylamine (in solution) decreased binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine. The IC50 values for these three proteins were 520, 160, and 79 nM, respectively. The TGF-beta 1-binding activity of native alpha 2M may have reflected, at least in part, trace-contamination with alpha 2M-proteinase complex.     

Cross-talk between bone morphogenetic protein and transforming growth factor-beta signaling is essential for exendin-4-induced insulin-positive differentiation of AR42J cells

A key goal of cellular engineering is to manipulate progenitor cells to become beta-cells, allowing cell replacement therapy to cure diabetes mellitus. As a paradigm for cell engineering, we have studied the molecular mechanisms by which AR42J cells become beta-cells. Bone morphogenetic proteins (BMPs), implicated in a myriad of developmental pathways, have not been well studied in insulin-positive differentiation. We found that the canonical intracellular mediators of BMP signaling, Smad-1 and Smad-8, were significantly elevated in AR42J cells undergoing insulin-positive differentiation in response to exendin-4 treatment, suggesting a role for BMP signaling in beta-cell formation. Similarly, endogenous BMP-2 ligand and ALK-1 receptor (activin receptor-like kinase-1; known to activate Smads 1 and 8) mRNAs were specifically up-regulated in exendin-4-treated AR42J cells. Surprisingly, Smad-1 and Smad-8 levels were suppressed by the addition of BMP-soluble receptor inhibition of BMP ligand binding to its receptor. Here, insulin-positive differentiation was also ablated. BMP-2 ligand antisense also strongly inhibited Smad-1 and Smad-8 expression, again with the abolition of insulin-positive differentiation. These results demonstrate a previously unrecognized key role for BMP signaling in mediating insulin-positive differentiation through the intracellular Smad signaling pathway. In short, BMP signaling may represent a novel downstream target of exendin-4 (glucagon-like peptide 1) signaling and potentially serve as an upstream regulator of transforming growth factor-beta isoform signaling to differentiate the acinar-like AR42J cells into insulin-secreting cells.     

Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling by bone morphogenetic proteins 2 and 4

Highly purified fractions of bone extracts capable of inducing ectopic bone formation have been reported to contain peptides corresponding to the mature active regions of the TGF-beta-like bone morphogenetic proteins (BMPs) 2-7, and to the prodomain region of the metalloproteinase BMP1. Co-purification of BMPs 2-7 with BMP1 prodomain sequences through the multiple biochemical steps used in these previous reports has suggested the possibility of interactions between the BMP1 prodomain and BMPs 2-7. Here we demonstrate that the BMP1 prodomain binds BMPs 2 and 4 with high specificity and with a KD of approximately 11 nM, in the physiological range. It is further demonstrated that the BMP1 prodomain is capable of modulating signaling by BMPs 2 and 4 in vitro and in vivo, and that endogenous BMP1 prodomain-BMP4 complexes exist in cell culture media and in tissues.