Modulation of bone morphogenetic protein antagonists to stimulate clinical osteogenesis

Bone morphogenetic proteins (BMPs) are important for the development and functioning of a wide variety of tissues and organ systems. Their ability to induce bone formation has been harnessed for clinical application. Specifically, local application of BMPs into fractures and fusions has shown some efficacy in inducing bone formation. However, clinical success has not been as robust as might be expected from the results obtained using animal models. This difference may be due to a number of mechanisms regulating BMP activity in vivo. One class of major regulators is the extracellular antagonist (e.g. Noggin, Gremlin, DAN), the dysfunction of which has been shown to result in ectopic bone formation in animal models and human disease. We hypothesize that local application of BMPs at high concentrations induces increased production of BMP antagonists, thereby limiting BMP activity and clinical efficacy. Therapies blocking the function of BMP antagonists should therefore result in enhanced BMP activity and increased bone formation. Furthermore, titrated systemic regulation of BMP antagonist may potentially reverse osteoporosis. Our collective experience with the clinical use of BMP illustrates the importance of understanding mechanisms of endogenous antagonism and regulation in the exogenous application of a protein as a therapeutic.     

Bone morphogenetic protein signaling through ACVR1 and BMPR1A negatively regulates bone mass along with alterations in bone composition

Bone quantity and bone quality are important factors in determining the properties and the mechanical functions of bone. This study examined the effects of disrupting bone morphogenetic protein (BMP) signaling through BMP receptors on bone quantity and bone quality. More specifically, we disrupted two BMP receptors, Acvr1 and Bmpr1a, respectively, in Osterix-expressing osteogenic progenitor cells in mice. We examined the structural changes to the femora from 3-month old male and female conditional knockout (cKO) mice using micro-computed tomography (micro-CT) and histology, as well as compositional changes to both cortical and trabecular compartments of bone using Raman spectroscopy. We found that the deletion of Acvr1 and Bmpr1a, respectively, in an osteoblast-specific manner resulted in higher bone mass in the trabecular compartment. Disruption of Bmpr1a resulted in a more significantly increased bone mass in the trabecular compartment. We also found that these cKO mice showed lower mineral-to-matrix ratio, while tissue mineral density was lower in the cortical compartment. Collagen crosslink ratio was higher in both cortical and trabecular compartments of male cKO mice. Our study suggested that BMP signaling in osteoblast mediated by BMP receptors, namely ACVR1 and BMPR1A, is critical in regulating bone quantity and bone quality.     

The role of bone morphogenetic proteins in myeloma cell survival

Multiple myeloma is characterized by slowly growing clones of malignant plasma cells in the bone marrow. The malignant state is frequently accompanied by osteolytic bone disease due to a disturbed balance between osteoblasts and osteoclasts. Bone morphogenetic proteins (BMPs) are present in the bone marrow and are important for several aspects of myeloma pathogenesis including growth and survival of tumor cells, bone homeostasis, and anemia. Among cancer cells, myeloma cells are particularly sensitive to growth inhibition and apoptosis induced by BMPs and therefore represent good models to study BMP receptor usage and signaling. Our review highlights and discusses the current knowledge on BMP signaling in myeloma.     

骨形态发生蛋白9基因敲除小鼠的构建

目的运用CRISR/Cas9技术敲除小鼠基因组中Bmp9基因片段,构建Bmp9基因敲除小鼠。方法根据Bmp9基因的外显子序列,设计一段sgRNA并合成。sgRNA体外转录后和Cas9mRNA混合后显微注射受精卵细胞,注射后的受精卵细胞移植至受体动物获得子代小鼠。提取子代小鼠基因组DNA测序鉴定其基因型。基因型鉴定正确的小鼠与野生型交配后筛选纯合子小鼠。同时取纯合子小鼠心脏、肝、脾、肺、肾,匀浆后提取总RNA和总蛋白,通过qPCR、WB和免疫组化检测BMP9在各组织中的表达。结果设计并合成20bp的sgRNA并进行体外转录,显微注射并回植后得到基因突变小鼠,连续交配后得F2代纯合子。测序结果显示,突变小鼠存在两种基因型,一种为5bp缺失突变,另一种为13bp缺失并伴有1bp插入突变。与野生型C57BL/6相比,qPCR、WB和免疫组化结果均表明基因敲除小鼠肝中BMP9表达显著降低。结论利用CRISPR/Cas9技术成功构建出了BMP9基因敲除小鼠。     

Formation of stable homomeric and transient heteromeric bone morphogenetic protein (BMP) receptor complexes regulates Smad protein signaling

The type I and type II bone morphogenetic protein receptors (BMPRI and BMPRII) are present at the plasma membrane as monomers and homomeric and heteromeric complexes, which are modulated by ligand binding. The complexes of their extracellular domains with ligand were shown to form heterotetramers. However, the dynamics of the oligomeric interactions among the full-length receptors in live cell membranes were not explored, and the roles of BMP receptor homodimerization were unknown. Here, we investigated these issues by combining patching/immobilization of an epitope-tagged BMP receptor at the cell surface with measurements of the lateral diffusion of a co-expressed, differently tagged BMP receptor by fluorescence recovery after photobleaching (FRAP). These studies led to several novel conclusions. (a) All homomeric complexes (without or with BMP-2) were stable on the patch/FRAP time scale (minutes), whereas the heterocomplexes were transient, a difference that may affect signaling. (b) Patch/FRAP between HA- and myc-tagged BMPRII combined with competition by untagged BMPRIb showed that the heterocomplexes form at the expense of homodimers. (c) Stabilization of BMPRII·BMPRIb heterocomplexes (but not homomeric complexes) by IgG binding to same-tag receptors elevated phospho-Smad formation both without and with BMP-2. These findings suggest two mechanisms that may suppress the tendency of preformed BMP receptor hetero-oligomers to signal without ligand: (a) competition between homo- and heterocomplex formation, which reduces the steady-state level of the latter, and (b) the transient nature of the heterocomplexes, which limits the time during which BMPRI can be phosphorylated by BMPRII in the heterocomplex.     

Bone morphogenetic protein 2 promotes osteogenesis of bone marrow stromal cells in type 2 diabetic rats via the Wnt signaling pathway

Type 2 diabetes mellitus impairs osteogenesis in bone marrow stromal cells (BMSCs). Bone morphogenetic protein 2 (BMP2) has been extensively applied for bone defect restoration and has been shown to activate the Wnt signaling pathway. The objective of this study was to investigate the effects of BMP2 on the cell proliferation and osteogenesis of type 2 diabetic BMSCs in rats and explore whether BMP2 induced osteogenesis via the stimulation of Wnt signaling pathway. The cell experiments were divided into DM (diabetic BMSCs), BMP25 (induced with 25 ng/ml BMP2), BMP100 (induced with 100 ng/ml BMP2) and BMP25  + XAV groups. All cells with or without the different concentrations of BMP2 were cultured under the same experimental conditions. The in vitro results indicated that BMP2 enhanced cell proliferation by 130%–157% and osteogenic differentiation by approximately two-fold in type 2 diabetic BMSCs. The expression levels of β-catenin, cyclin D1, Runx2 and c-myc related to the Wnt signaling pathway were also upregulated from 180% to 212% in BMP2-induced type 2 diabetic rat BMSCs, while the level of GSK3β decreased to 43%. In BMP2-induced type 2 diabetic BMSCs with calcium phosphate cement (CPC) scaffolds for osteoblast study in vivo, the appearance of newly formed bone dramatically increased to 175% compared with type 2 diabetic BMSCs. These data demonstrated that BMP2 enhanced bone regeneration in diabetic BMSCs by stimulating the Wnt signaling pathway with the accumulation of β-catenin and the depressed expression of GSK3β. Diabetic BMSCs associated with BMP2 might be a potential tissue-engineered construct for bone defects in type 2 diabetes mellitus.     

Changes in bone morphogenetic protein receptor-IB localisation regulate osteogenic responses of human bone cells to bone morphogenetic protein-2

Cell responses to bone morphogenetic proteins (BMP) depend on the expression and surface localisation of transmembrane receptors BMPR-IA, -IB and -II. The present study shows that all three antigens are readily detected in human bone cells. However, only BMPR-II was found primarily at the plasma membrane, whereas BMPR-IA was expressed equally in the cytoplasm and at the cell surface. Notably, BMPR-IB was mainly intracellular, where it was associated with a number of cytoplasmic structures and possibly the nucleus. Treatment with transforming growth factor β1 (TGF-β1) caused rapid translocation of BMPR-IB to the cell surface, mediated via the p38 mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) pathways. The TGF-β1-induced increase in surface BMPR-IB resulted in significantly elevated BMP-2 binding and Smad1/5/8 phosphorylation, although the receptor was subsequently internalised and the functional response to BMP-2 consequently down-regulated. The results show, for the first time, that BMPR-IB is localised primarily in intracellular compartments in bone cells and that TGF-β1 induces rapid surface translocation from the cytoplasm to the cell surface, resulting in increased sensitivity of the cells to BMP-2.     

Novel contact-dependent bone morphogenetic protein (BMP) signaling mediated by heparan sulfate proteoglycans

We previously proposed a model that DALLY, a Drosophila glypican, acts as a trans co-receptor to regulate BMP signaling in the germ line stem cell niche. To investigate the molecular mechanisms of contact-dependent BMP signaling, we developed novel in vitro assay systems to monitor trans signaling using Drosophila S2 cells. Using immunoblot-based as well as single-cell assay systems, we present evidence that Drosophila glypicans indeed enhance BMP signaling in trans in a contact-dependent manner in vitro. Our analysis showed that heparan sulfate modification is required for the trans co-receptor activity of DALLY. Two BMP-like molecules, Decapentaplegic (DPP) and Glass bottom boat, can mediate trans signaling through a heparan sulfate proteoglycan co-receptor in S2 cells. The in vitro systems reflect the molecular characteristics of heparan sulfate proteoglycan functions observed previously in vivo, such as ligand specificity and biphasic activity dependent on the ligand dosage. In addition, experiments using a DALLY-coated surface suggested that DALLY regulates DPP signaling in trans by its effect on the stability of DPP protein on the surface of the contacting cells. Our findings provide the molecular foundation for novel contact-dependent signaling, which defines the physical space of the stem cell niche in vivo.     

Pleiotrophin regulates bone morphogenetic protein (BMP)-induced ectopic osteogenesis

We previously isolated pleiotrophin (PTN) from bovine bone as a protein and showed that it stimulated osteoblastic growth and differentiation. Further details of its function, however, have not been fully clarified. The aim of this paper was to elucidate the effects of PTN on bone morphogenetic protein (BMP)-induced ectopic osteogenesis. Recombinant human BMP (rhBMP)-2 (1.2 microg) was combined with a fibrous glass membrane, which had been established as an effective carrier. Various amounts of the purified bovine PTN (5, 10, 50, and 100 microg) or rhPTN (5 and 10 microg) were added to the rhBMP-2/carrier composites and implanted into rats subcutaneously as reported. It was found that the amount of bone induced in the system increased with the addition of 10 microg of either purified PTN or rhPTN. However, the amount of bone decreased with the addition of 50 or 100 microg of purified PTN dose-dependently, as judged by both alkaline phosphatase activity and calcium content in the retrieved implants. It was concluded that purified PTN or rhPTN, at ratios of concentration of 10-100 microg of PTN to 1.2 microg of rhBMP-2 in the carrier, regulated the ectopic bone-inducing activity of rhBMP-2.     

Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (～60 kDa) is processed into active BMP10 (～14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.     

Bone morphogenetic proteins: a critical review

Bone Morphogenetic Proteins (BMPs) are potent growth factors belonging to the Transforming Growth Factor Beta superfamily. To date over 20 members have been identified in humans with varying functions during processes such as embryogenesis, skeletal formation, hematopoesis and neurogenesis. Though their functions have been identified, less is known regarding levels of regulation at the extracellular matrix, membrane surface, and receptor activation. Further, current models of activation lack the integration of these regulatory mechanisms. This review focuses on the different levels of regulation, ranging from the release of BMPs into the extracellular components to receptor activation for different BMPs. It also highlights areas in research that is lacking or contradictory.     

Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.     

Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals

Functional interactions between cancer cells and the bone microenvironment contribute to the development of bone metastasis. Although the bone metastasis of prostate cancer is characterized by increased ossification, the molecular mechanisms involved in this process are not fully understood. Here, the roles of bone morphogenetic proteins (BMPs) in the interactions between prostate cancer cells and bone stromal cells were investigated. In human prostate cancer LNCaP cells, BMP-4 induced the production of Sonic hedgehog (SHH) through a Smad-dependent pathway. In mouse stromal MC3T3-E1 cells, SHH up-regulated the expression of activin receptor IIB (ActR-IIB) and Smad1, which in turn enhanced BMP-responsive reporter activities in these cells. The combined stimulation with BMP-4 and SHH of MC3T3-E1 cells cooperatively induced the expression of osteoblastic markers, including alkaline phosphatase, bone sialoprotein, collagen type II α1, and osteocalcin. When MC3T3-E1 cells and LNCaP cells were co-cultured, the osteoblastic differentiation of MC3T3-E1 cells, which was induced by BMP-4, was accelerated by SHH from LNCaP cells. Furthermore, LNCaP cells and BMP-4 cooperatively induced the production of growth factors, including fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) in MC3T3-E1 cells, and these may promote the proliferation of LNCaP cells. Taken together, our findings suggest that BMPs provide favorable circumstances for the survival of prostate cancer cells and the differentiation of bone stromal cells in the bone microenvironment, possibly leading to the osteoblastic metastasis of prostate cancer.