Expression, purification and osteogenic bioactivity of recombinant human BMP-4, -9, -10, -11 and -14

Bone morphogenetic proteins (BMPs) are cytokines from the TGF-β superfamily, with important roles during embryonic development and in the induction of bone and cartilage tissue differentiation in the adult body. In this contribution, we report the expression of recombinant human BMP-4, BMP-9, BMP-10, BMP-11 (or growth differentiation factor-11, GDF-11) and BMP-14 (GDF-5), using Escherichia coli pET-25b vector. BMPs were overexpressed, purified by affinity his-tag chromatography and shown to induce the expression of early markers of bone differentiation (e.g. smad-1, smad-5, runx2/cbfa1, dlx5, osterix, osteopontin, bone sialoprotein and alkaline phosphatase) in C2C12 cells and in human adipose stem cells. The described approach is a promising method for producing large amounts of different recombinant BMPs that show potential for novel biomedical applications.     

Bone Morphogenetic Protein-2 Is a Regulator of Cell Adhesion

Bone morphogenetic proteins (BMPs) are a group of peptide growth factors closely related to transforming growth factors-β. The BMPs are suggested to play an essential role in bone development and they are strong candidate molecules to be used clinically to improve fracture healing. BMPs are also involved in the differentiation of several other tissues during embryogenesis. Here, we show that human recombinant BMP-2 regulates cell–matrix interactions by modifying the expression of integrin-type receptors. The synthesis of α3 integrin was down-regulated by BMP-2 in two osteogenic sarcoma-derived cell lines, Saos-2 and HOS, and also in human fetal chondrocytes. BMP-2 had no effect on the expression of α1, α2, α5, α6, and αV integrins. BMP-2 reduced the expression of α3 integrin subunit at mRNA level. Laminin-5 was shown to be the ligand for α3β1 integrin on Saos cells and BMP-2 decreased the ability of Saos cells to attach to it. These results suggest that BMP-2 has a specific effect on the α3β1 integrin-mediated cell adhesion to laminin-5. Given the fact that BMP-2 is expressed in osteosarcomas, in addition to in bone, this mechanism is putatively important especially in bone development and tumors. We also studied the effect of BMP-2 on a human keratinocyte cell line, HaCaT. In HaCaT cells, the expression of α2 integrin was strongly down-regulated by BMP-2, whereas its effect on the expression of α3 integrin was smaller. We suggest that the effects of BMP-2 may be partially mediated by specifically altered cell adhesion.     

Expression of bone morphogenic proteins and receptors at the injured growth plate cartilage in young rats.

The injured growth plate cartilage is often repaired by bony tissue, resulting in impaired bone growth in children. Bone morphogenic proteins (BMPs) are important for bone fracture repair, and as a step to characterize potential involvement of BMPs in bony repair of injured growth plate, expression of BMPs and receptors (BMP-R) was examined by quantitative RT-PCR and immunohistochemistry in rat injured tibial growth plate. During the inflammatory response on day 1, slightly increased expression of BMP-3, BMP-4, BMP-R1a, and BMP-R2 was observed, with immunostaining seen among inflammatory cells at the injury site. During mesenchymal infiltration and osteogenic responses on days 3-14, moderately increased expression of BMP-2, -3, -4, -7, and BMP-R1a was found, with immunostaining observed among infiltrated mesenchymal cells and differentiated osteoblasts lining bony trabeculae. During maturation phase on days 14-25, only BMP-7 was seen upregulated slightly and was localized in osteoblasts and marrow cells at the injury site. The temporospatial expression of BMPs and receptors at the injured growth plate suggests potential involvement of BMP-3 and -4 in regulating the inflammatory response or as its mediators in modulating downstream events, and BMP-2, -3, -4, and -7 in the fibrogenic and osteogenic responses, and BMP-7 in bone remodeling at the injured growth plate.     

Bone morphogenetic proteins act synergistically with haematopoietic cytokines in the differentiation of haematopoietic progenitors

We examined the effects of bone morphogenetic protein-2 (BMP-2), -3, -4, -5, -6, and -7 on the proliferation and differentiation of bone marrow CD34+ haematopoietic progenitors in semi-solid medium. The BMPs had no effect on haematopoietic colony development when added to medium containing erythropoietin (Epo) or Interleukin-3 plus Epo. Synergistic effects with the haematopoietic cytokines stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed. In conjunction with GM-CSF and Epo, BMP-4 increased the number of both erythroid and granulocyte/monocyte colonies formed in semi-solid medium (P<0.01). No other BMP stimulated erythroid colony development under these conditions, while BMP-3, BMP-7 (P<0.01), BMP-5, and BMP-6 (P<0.05) stimulated granulocyte/monocyte colony formation. BMP-7 acted synergistically with stem cell factor to increase granulocyte/monocyte colony formation but not erythroid colony formation. The other BMPs did not affect either erythroid or granulocyte/monocyte colony development under these conditions. These results suggest that individual BMPs form part of the complement of cytokines regulating the development of haematopoietic progenitors, and in particular, point to a role for BMP-4 in the control of definitive, as well as embryonic erythropoiesis.     

Blood cell induction in Xenopus animal cap explants: Effects of fibroblast growth factor, bone morphogenetic proteins, and activin

 Cultures of Xenopus blastula animal caps were used to explore the haematopoietic effects of three candidate inducers of mesoderm: basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMPs) and activin A. In response to either bFGF or activin A, explants expanded into egg-shaped structures, and beneath an outer layer of epidermis, a ventral mesodermal lining surrounded a fluid-filled cavity containing ”blood-like cells”. Immunocytochemistry identified some of these cells as early leukocytes, but erythrocytes were rare. BMP-2 or BMP-4 induced primitive erythrocytes as well as leukocytes, and a high concentration was required for these cells to differentiate in only a small proportion of explants. BMP-2 but not BMP-4 induced ventral mesoderm concomitantly. High concentrations of activin A dorsalized explants, which contained infrequent leukocytes, and an optimal combination of activin A and bFGF caused differentiation of muscle with few blood cells. By contrast, BMP-2 or BMP-4 plus activin A synergistically increased the numbers of both leukocytes and erythrocytes. Explants treated with BMPs plus activin contained a well organized cell mass in which yolk-rich cells mixed with blood cells and pigmented cells did not. BMP-2 plus bFGF also induced numerous leukocytes and fewer erythrocytes, but BMP-4 antagonized the leukopoietic effect of bFGF. The data suggest that the signalling pathways these three factors use to induce leukopoiesis overlap and that erythropoiesis may be activated when inducers are present in combination. Received: 3 August 1998 / Accepted: 7 October 1998     

Bone morphogenetic proteins (BMPs) expression in the femoral heads of patients with avascular necrosis

Avascular necrosis (AVN) is a disorder of the bone repair process which usually results in femoral head (FH) destruction. Bone morphogenetic proteins (BMPs) are the key proteins regulating bone remodelling and healing. BMPs gene expression levels were analyzed in the normal and necrotic sites of osteonecrotic FHs. Quantitative RT-PCR for BMP-2, -4, -6, -7 genes was performed in bone tissue samples from 47 osteonecrotic FHs. Protein levels of BMP-2, -4, -6 were estimated by Western Blot. Statistical analysis was performed using the Wilcoxon signed rank test. BMP-2 and BMP-6 mRNA levels were higher in the normal than the necrotic site (normal/necrotic: 16.8/6.8 and 1.75/1.64, respectively). On the contrary, BMP-4 mRNA levels were higher in the necrotic (0.75) than the normal (0.62), while BMP-7 mRNA levels were extremely low. At the protein level, BMP-2 continued to have a higher expression in the normal region (normal/necrotic: 0.67/0.64). BMP-4 and -6 were detected at higher levels in the necrotic site (normal/necrotic: 0.51/0.61 for BMP-4, 0.51/0.56 for BMP-6), while BMP-7 was not detectable. Different BMP levels between the normal and necrotic site, as well as discrepancies between the gene and protein expression pattern suggest a different regulation mechanism for BMPs between the two regions of FHs. The understanding of the expression pattern and the correlation of BMPs could lead to a more successful use in the prevention and treatment of AVN.     

Expression of bone morphogenetic proteins during mandibular distraction osteogenesis

Distraction osteogenesis is a form of in vivo tissue engineering in which the gradual separation of cut bone edges results in the generation of new bone. In this study, the temporal and spatial expression of bone morphogenetic proteins (BMPs) 2, 4, and 7 was examined in a rabbit model of mandibular distraction osteogenesis. Fourteen skeletally mature male rabbits were studied. After osteotomy, a distractor was applied to one side of the mandible. After 1 week of latency, distraction was initiated at 0.25 mm every 12 hours for 3 weeks (distraction period), followed by a 3-week consolidation period. Two animals were killed each week after surgery. The generate bone was analyzed for the expression of BMP-2, -4, and -7 by using standard bone histological and immunohistochemical techniques. BMP-2 and -4 were highly expressed in osteoblastic cells during the distraction period and in chondrocytes during the consolidation period. BMP-7 demonstrated relatively minor expression in osteoblastic cells during the distraction period. All BMPs were strongly expressed in vascularized connective tissue during the distraction period. These data indicate that BMPs participate in the translation of mechanical stimuli into a biological response during mandibular distraction osteogenesis.     

The bone morphogenetic protein family and osteogenesis.

The BMPs (bone morphogenetic proteins) are a group of related proteins originally identified by their presence in bone-inductive extracts of demineralized bone. By molecular cloning, at least six related members of this family have been identified and are called BMP-2 through BMP-7. These molecules are part of the TGF-beta superfamily, based on primary amino acid sequence homology, including the absolute conservation of seven cysteine residues between the TGF-betas and the BMPs. The BMPs can be divided into subgroups with BMP-2 and BMP-4 being 92% identical, and BMP-5, BMP-6, and BMP-7 being an average of about 90% identical. To examine the individual activities of these molecules, we are producing each BMP in a mammalian expression system. In this system, each BMP is synthesized as a precursor peptide, which is glycosylated, processed to the mature peptide, and secreted as a homodimer. These reagents have been used to demonstrate that single molecules, such as BMP-2, are capable of inducing the formation of new cartilage and bone when implanted ectopically in a rodent assay system. Whether each of the BMPs possesses the same inductive activities in an animal is the subject of ongoing research. Based on the chondrogenic and osteogenic abilities of the BMPs in the adult animal, the expression of the mRNAs for the BMPs has been examined in the development of the embryonic skeleton by in situ hybridization. These studies demonstrate that the BMP mRNAs are spatially and temporally expressed appropriately for the proteins involved in the induction and development of cartilage and bone in the embryonic limb bud.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1

The activities of three bone morphogenetic proteins (BMPs), BMP-1, BMP-2 and BMP-3, on alkaline phosphatase activity, collagen synthesis and DNA synthesis were studied in cultured osteoblastic cells, MC3T3-E1. Treatment of cells with BMP-2 for 48 h induces an increase in cellular alkaline phosphatase activity. This stimulatory effect is evident at a concentration as low as 20 ng/ml of BMP-2 and becomes greater with increasing doses of BMP-2. The BMP-2-induced increase in alkaline phosphatase activity is enhanced by the presence of beta-estradiol, dexamethasone or 1 alpha, 25(OH)2D3. BMP-2 and BMP-3 slightly but significantly stimulate collagen synthesis. None of the BMPs stimulates DNA synthesis in MC3T3-E1 cells at doses tested. These results indicate that BMPs act directly on osteoblastic cells and stimulate the expression of the osteoblastic phenotypes.     

CYR61 Regulates BMP-2-dependent Osteoblast Differentiation through the αvβ3 Integrin/Integrin-linked Kinase/ERK Pathway

Osteoporosis is one of the most common bone pathologies. A number of novel molecules have been reported to increase bone formation including cysteine-rich protein 61 (CYR61), a ligand of integrin receptor, but mechanisms remain unclear. It is known that bone morphogenetic proteins (BMPs), especially BMP-2, are crucial regulators of osteogenesis. However, the interaction between CYR61 and BMP-2 is unclear. We found that CYR61 significantly increases proliferation and osteoblastic differentiation in MC3T3-E1 osteoblasts and primary cultured osteoblasts. CYR61 enhances mRNA and protein expression of BMP-2 in a time- and dose-dependent manner. Moreover, CYR61-mediated proliferation and osteoblastic differentiation are significantly decreased by knockdown of BMP-2 expression or inhibition of BMP-2 activity. In this study we found integrin α_vβ₃ is critical for CYR61-mediated BMP-2 expression and osteoblastic differentiation. We also found that integrin-linked kinase, which is downstream of the α_vβ₃ receptor, is involved in CYR61-induced BMP-2 expression and subsequent osteoblastic differentiation through an ERK-dependent pathway. Taken together, our results show that CYR61 up-regulates BMP-2 mRNA and protein expression, resulting in enhanced cell proliferation and osteoblastic differentiation through activation of the α_vβ₃ integrin/integrin-linked kinase/ERK signaling pathway.     

Enhanced Osteogenesis of Adipose Derived Stem Cells with Noggin Suppression and Delivery of BMP-2

Bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs) by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with 10-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D in vitro environments using scaffolds consisting of chitosan (CH), chondroitin sulfate (CS), and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in bone regeneration.     

Canonical Wnts and BMPs cooperatively induce osteoblastic differentiation through a GSK3β-dependent and β-catenin-independent mechanism

Both BMPs and Wnts play important roles in the regulation of bone formation. We examined the molecular mechanism regulating cross-talk between BMPs and Wnts in the osteoblastic differentiation of C2C12 cells. Canonical Wnts (Wnt1 and Wnt3a) but not non-canonical Wnts (Wnt5a and Wnt11) synergistically stimulated ALP activity in the presence of BMP-4. Wnt3a and BMP-4 synergistically stimulated the expression of type I collagen and osteonectin. However, Wnt3a did not stimulate ALP activity that was induced by a constitutively active BMP receptor or Smad1. Noggin and Dkk-1 suppressed the synergistic effect of BMP-4 and Wnt3a, but Smad7 did not. Overexpression of β-catenin did not affect BMP-4-induced ALP activity. By contrast, inhibition or stimulation of GSK3β activity resulted in either stimulation or suppression of ALP activity, respectively, in the presence of BMP-4. Taken together, these findings suggest that BMPs and canonical Wnts may regulate osteoblastic differentiation, especially at the early stages, through a GSK3β-dependent but β-catenin-independent mechanism.     

BMPs promote proliferation and migration of endothelial cells via stimulation of VEGF-A/VEGFR2 and angiopoietin-1/Tie2 signalling

The differentiation, growth, and survival of endothelial cells (ECs) are regulated by multiple signalling pathways, such as vascular endothelial growth factors (VEGFs) and angiopoietins through their receptor tyrosine kinases, VEGF receptor (VEGFR) 2 and Tie2, respectively. Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-beta family, have been implicated in the development and maintenance of vascular systems. However, their effects on EC proliferation remain to be elucidated. In the present study, we show that BMPs induce the proliferation and migration of mouse embryonic stem cell (ESC)-derived endothelial cells (MESECs) and human microvascular endothelial cells (HMECs). Addition of BMP-4 to culture induced significant proliferation and migration of both types of ECs. BMP-4 also increased the expression and phosphorylation of VEGFR2 and Tie2. These findings suggest that BMP signalling activates endothelium via activation of VEGF/VEGFR2 and Angiopoietin/Tie2 signalling.     

BMP-Non-Responsive Sca1+CD73+CD44+ Mouse Bone Marrow Derived Osteoprogenitor Cells Respond to Combination of VEGF and BMP-6 to Display Enhanced Osteoblastic Differentiation and Ectopic Bone Formation

Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.     

Cell responses to bone morphogenetic proteins and peptides derived from them: Biomedical applications and limitations

The bone morphogenetic proteins (BMPs) are cytokines of the transforming growth factor beta family. Some BMPs such as BMP-2 and BMP-7 play a major role in the development of the skeleton and the maintenance of homeostasis during bone remodelling. To date, only BMP-2 and BMP-7 have been approved by the Food and Drug Administration for specific orthopaedic applications. However, due to BMP cost, peptides derived from their knuckle epitope with osteogenic properties have been developed. BMPs are involved in many other biological events, including embryogenesis, angiogenesis and cancer. BMPs therefore have great biomedical potential as osteogenic factors and as anti-cancer agents. This review focuses on the use of BMPs and their derived peptides in biomedical delivery systems and gene therapy.     

Bone morphogenetic protein signaling in prostate cancer cell lines

Prostate cancer is the most commonly diagnosed malignancy in men and is often associated with bone metastases. Prostate cancer bone lesions can be lytic or schlerotic, with the latter predominating. Bone morphogenetic proteins (BMPs) are a family of growth factors, which may play a role in the formation of prostate cancer osteoblastic bone metastases. This study evaluated the effects of BMPs on prostate cancer cell lines. We observed growth inhibitory effects of BMP-2 and -4 on LNCaP, while PC-3 was unaffected. Flow cytometric analysis determined that LNCaP cell growth was arrested in G(1) after bone morphogenetic protein-2 treatment. Treatment of LNCaP and PC-3 with BMP-2 and -4 activated downstream signaling pathways involving SMAD-1, up-regulation of p21(CIP1/WAF1) and changes in retinoblastoma (Rb) phosphorylation. Interestingly, bone morphogenetic protein-2 treatment stimulated a 2.7-fold increase in osteoprotegerin (OPG), a molecule, which inhibits osteoclastogenesis, production in PC-3.     

Systemically administered bone morphogenetic protein-6 restores bone in aged ovariectomized rats by increasing bone formation and suppressing bone resorption

Although recombinant human bone morphogenetic proteins (BMPs) are used locally for treating bone defects in humans, their systemic effect on bone augmentation has not been explored. We have previously demonstrated that demineralized bone (DB) from ovariectomized (OVX) rats cannot induce bone formation when implanted ectopically at the subcutaneous site. Here we showed in vitro that 17beta-estradiol (E2) specifically induced expression of Bmp6 mRNA in MC3T3-E1 preosteoblastic cells and that bone extracts from OVX rats lack BMPs. Next we demonstrated that 125I-BMP-6 administered systemically accumulated in the skeleton and also restored the osteoinductive capacity of ectopically implanted DB from OVX rats. BMP-6 applied systemically to aged OVX rats significantly increased bone volume and mechanical characteristics of both the trabecular and cortical bone, the osteoblast surface, serum osteocalcin and osteoprotegerin levels, and decreased the osteoclast surface, serum C-telopeptide, and interleukin-6. E2 was significantly less effective, and was not synergistic with BMP-6. Animals that discontinued BMP-6 therapy maintained bone mineral density gains for another 12 weeks. BMP-6 increased in vivo the bone expression of Acvr-1, Bmpr1b, Smad5, alkaline phosphatase, and collagen type I and decreased expression of Bmp3 and BMP antagonists, chordin and cerberus. These results show, for the first time, that systemically administered BMP-6 restores the bone inductive capacity, microarchitecture, and quality of the skeleton in osteoporotic rats.