Osteocyte control of bone formation via sclerostin, a novel BMP antagonist.
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There is an unmet medical need for anabolic treatments to restore lost bone. Human genetic bone disorders provide insight into bone regulatory processes. Sclerosteosis is a disease typified by high bone mass due to the loss of SOST expression. Sclerostin, the SOST gene protein product, competed with the type I and type II bone morphogenetic protein (BMP) receptors for binding to BMPs, decreased BMP signaling and suppressed mineralization of osteoblastic cells. SOST expression was detected in cultured osteoblasts and in mineralizing areas of the skeleton, but not in osteoclasts. Strong expression in osteocytes suggested that sclerostin expressed by these central regulatory cells mediates bone homeostasis. Transgenic mice overexpressing SOST exhibited low bone mass and decreased bone strength as the result of a significant reduction in osteoblast activity and subsequently, bone formation. Modulation of this osteocyte-derived negative signal is therapeutically relevant for disorders associated with bone loss. (+info)
Reduction of osteophyte formation and synovial thickening by adenoviral overexpression of transforming growth factor beta/bone morphogenetic protein inhibitors during experimental osteoarthritis.
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OBJECTIVE: Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor beta (TGFbeta) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFbeta and BMP during papain-induced OA-like changes in mice. METHODS: We used adenoviral overexpression of TGFbeta and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan-specific TGFbeta antagonist called murine latency-associated peptide 1 (mLAP-1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFbeta/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss. RESULTS: Intraarticular injection of papain caused increased protein expression of several TGFbeta and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP-1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFbeta inhibitor mLAP-1 increased articular cartilage PG loss. CONCLUSION: Our results indicate a pivotal role of endogenous TGFbeta in the development of osteophytes and synovial thickening, implicating endogenous TGFbeta in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFbeta signifies the protective role of TGFbeta in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA. (+info)
Decreased expression of bone morphogenetic protein (BMP) receptor type II correlates with insensitivity to BMP-6 in human renal cell carcinoma cells.
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PURPOSE: Bone morphogenetic proteins (BMPs) are members of a family of pleiotropic growth factors that play a critical role during renal development as well as maintaining kidney homeostasis. In the present study, we investigated the potential role of BMP receptors (BMPRs) in renal cell carcinoma (RCC) cells. EXPERIMENTAL DESIGN: Immunohistochemistry was used to investigate the expression of BMPRs in human RCC tissues. As an in vitro model of RCC, three cell lines were used: 112, 117, and 181. Northern blot, immunoblot, and reverse transcription-PCR were used to study the expression of BMPRs in the cell lines. Finally, cells were transfected using LipofectAMINE. RESULTS: Normal human kidney tissues express the three BMPRs: types RIA, RIB, and RII. In contrast, human RCC cells frequently exhibit a loss of expression of BMP-RII. In tissue culture, BMP-6 inhibits in a dose-dependent manner the proliferation of 112 cells but not of 117 and 181 cells. Assays for BMPRs demonstrated that 117 and 181 cells express low levels of BMP-RII RNA. When these two BMP-6 resistant cell lines were infected with the adenovirus containing the constitutively active form of BMP-RIA or -RIB in combination with a BMP-6-responsive luciferase reporter construct, luciferase activity increased. Finally, when these cell lines were transfected with BMP-RII, BMP-6-sensitivity was restored. CONCLUSIONS: These results demonstrate that human RCC tissues frequently have decreased levels of expression of BMP-RII and that the human RCC cell lines 117 and 181 are resistant to the growth-inhibitory effect of BMP-6 because they have decreased levels of expression of BMP-RII. (+info)
Characterization of growth factor-binding structures in heparin/heparan sulfate using an octasaccharide library.
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Heparan sulfate (HS) chains interact with various growth and differentiation factors and morphogens, and the most interactions occur on the specific regions of the chains with certain monosaccharide sequences and sulfation patterns. Here we generated a library of octasaccharides by semienzymatic methods by using recombinant HS 2-O-sulfotransferase and HS 6-O-sulfotransferase, and we have made a systematic investigation of the specific binding structures for various heparin-binding growth factors. An octasaccharide (Octa-I, DeltaHexA-GlcNSO(3)-(HexA-GlcNSO(3))(3)) was prepared by partial heparitinase digestion from completely desulfated N-resulfated heparin. 2-O- and 6-O-sulfated Octa-I were prepared by enzymatically transferring one to three 2-O-sulfate groups and one to three 6-O-sulfate groups per molecule, respectively, to Octa-I. Another octasaccharide containing 3 units of HexA(2SO(4))-GlcNSO(3)(6SO(4)) was prepared also from heparin. This octasaccharide library was subjected to affinity chromatography for interactions with fibroblast growth factor (FGF)-2, -4, -7, -8, -10, and -18, hepatocyte growth factor, bone morphogenetic protein 6, and vascular endothelial growth factor, respectively. Based upon differences in the affinity to those octasaccharides, the growth factors could be classified roughly into five groups: group 1 needed 2-O-sulfate but not 6-O-sulfate (FGF-2); group 2 needed 6-O-sulfate but not 2-O-sulfate (FGF-10); group 3 had the affinity to both 2-O-sulfate and 6-O-sulfate but preferred 2-O-sulfate (FGF-18, hepatocyte growth factor); group 4 required both 2-O-sulfate and 6-O-sulfate (FGF-4, FGF-7); and group 5 hardly bound to any octasaccharides (FGF-8, bone morphogenetic protein 6, and vascular endothelial growth factor). The approach using the oligosaccharide library may be useful to define specific structures required for binding to various heparin-binding proteins. Octasaccharides with the high affinity to FGF-2 and FGF-10 had the activity to release them, respectively, from their complexes with HS. Thus, the library may provide new reagents to specifically regulate bindings of the growth factors to HS. (+info)
Sox5 and Sox6 are needed to develop and maintain source, columnar, and hypertrophic chondrocytes in the cartilage growth plate.
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Sox5 and Sox6 encode Sry-related transcription factors that redundantly promote early chondroblast differentiation. Using mouse embryos with three or four null alleles of Sox5 and Sox6, we show that they are also essential and redundant in major steps of growth plate chondrocyte differentiation. Sox5 and Sox6 promote the development of a highly proliferating pool of chondroblasts between the epiphyses and metaphyses of future long bones. This pool is the likely cellular source of growth plates. Sox5 and Sox6 permit formation of growth plate columnar zones by keeping chondroblasts proliferating and by delaying chondrocyte prehypertrophy. They allow induction of chondrocyte hypertrophy and permit formation of prehypertrophic and hypertrophic zones by delaying chondrocyte terminal differentiation induced by ossification fronts. They act, at least in part, by down-regulating Ihh signaling, Fgfr3, and Runx2 and by up-regulating Bmp6. In conclusion, Sox5 and Sox6 are needed for the establishment of multilayered growth plates, and thereby for proper and timely development of endochondral bones. (+info)
Expression of bone morphogenetic protein-6 and bone morphogenetic protein receptors in myoepithelial cells of canine mammary gland tumors.
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To study the ectopic chondrogenesis in canine mammary mixed tumors, the expression of bone morphogenetic protein-6 (BMP-6) and specific BMP receptors (BMPRs), BMPR-IA, BMPR-IB, and BMPR-II, was examined using immunohistochemical and immunoblot analysis in 39 canine mammary gland tumors. Immunohistochemically, BMP-6 and all three types of BMPRs were coexpressed in the myoepithelial cells and chondrocytes in six of eight benign mixed tumors. In complex adenomas, myoepithelial cells showed an expression pattern of BMP-6, BMPR-IA, and BMPR-II similar to those in benign mixed tumors, whereas immunoreactivity for BMPR-IB was very mild. The myoepithelial cells proliferating within the basement membrane showed more intense immunoreactivity for BMP-6 and all BMPRs as compared with those proliferating in the interstitial areas. Western blotting analysis revealed immunopositive bands at 40-45 kDa for BMP-6 in the samples from simple and complex adenomas and benign mixed tumors. The BMPR-IB-specific bands at 45 kDa were most detected in benign mixed tumors. Because among BMPRs, BMPR-IB is thought to be the major receptor for BMP-6 for primary chondrogenesis, these findings suggest that the expression of BMP and its receptors on the myoepithelial cells might play a role in the ectopic cartilage formation in canine mammary gland tumors, especially in benign mixed tumors. (+info)
Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin.
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Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed BMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K(d) 0.28 +/- 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary. (+info)
Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells.
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Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and play an important role in development and in many cellular processes. We have found that BMP-2, BMP-6, and BMP-9 induce the most potent osteogenic differentiation of mesenchymal stem cells. Expression profiling analysis has revealed that the Inhibitors of DNA binding/differentiation (Id)-1, Id-2, and Id-3 are among the most significantly up-regulated genes upon BMP-2, BMP-6, or BMP-9 stimulation. Here, we sought to determine the functional role of these Id proteins in BMP-induced osteoblast differentiation. We demonstrated that the expression of Id-1, Id-2, and Id-3 genes was significantly induced at the early stage of BMP-9 stimulation and returned to basal levels at 3 days after stimulation. RNA interference-mediated knockdown of Id expression significantly diminished the BMP-9-induced osteogenic differentiation of mesenchymal progenitor cells. Surprisingly, a constitutive overexpression of these Id genes also inhibited osteoblast differentiation initiated by BMP-9. Furthermore, we demonstrated that BMP-9-regulated Id expression is Smad4-dependent. Overexpression of the three Id genes was shown to promote cell proliferation that was coupled with an inhibition of osteogenic differentiation. Thus, our findings suggest that the Id helix-loop-helix proteins may play an important role in promoting the proliferation of early osteoblast progenitor cells and that Id expression must be down-regulated during the terminal differentiation of committed osteoblasts, suggesting that a balanced regulation of Id expression may be critical to BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells. (+info)