BMPs and BMPRs in chicken ovary and effects of BMP-4 and -7 on granulosa cell proliferation and progesterone production in vitro

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are now known to have important roles in mammalian ovarian folliculogenesis. This study determined the expression of the mRNA encoding for BMPs and their receptors in the chicken ovary and explored possible roles for them. The expression of the mRNA for BMP-2, -4, -6, -7, and BMPR-IA, -IB, and -II was determined and quantified by a semiquantitative RT-PCR. The mRNAs for all the BMPs and receptors determined were present in both the granulosa (G) and theca (T) cells of the F1, F2, and F3 follicles. All BMP mRNAs increased in G cells with follicular development, whereas only BMP-7 mRNA had this trend in the T cells. BMP-2, -4, and -6 mRNAs in T were similar between follicles. BMPR-IA mRNA was similar in F2G and F3G but lower in F1G. BMPR-IB mRNA was similar in G of all follicles, and BMPR-II mRNA increased with development. In the T, each receptor subtype showed equal distribution between follicles. mRNA levels for BMPR-IB and -II were higher in G than in T, suggesting that the G is a major target for BMPs. BMP-4 and -7 stimulated basal, IGF-I-, and gonadotropin-stimulated progesterone production by cultured G cells, with differential responses between cells from the F1 and F3/4. This suggests involvement in follicular differentiation. BMP-4 and -7 reversed the inhibitory effects of transforming growth factor (TGF)-alpha on basal and gonadotropin-stimulated G cell progesterone production, with greater effect in the F1 than in the F3/4. This effect suggests an important role for BMPs interacting with TGF-alpha in modulating the effects of gonadotropins and IGF-I on follicular differentiation. Finally, BMP-7 stimulated G cell proliferation, but BMP-4 inhibited TGF-alpha+ IGF-I- and/or FSH-stimulated G cell proliferation, suggesting a role in the control of follicular growth during development. These effects of BMP-4 and -7 on the G cell function showed relationships with the expression levels of the BMPs and the BMPR-II.     

Osteogenic protein-1 differentially regulates the mRNA expression of bone morphogenetic proteins and their receptors in primary cultures of osteoblasts

The mRNA expression patterns of several bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in long-term primary cultures of fetal rat calvaria (FRC) cells were examined by Northern analysis. Their temporal orders of expression were correlated with those of several biochemical markers characteristic of osteoblastic cell differentiation. Distinct temporal patterns of expression of BMPs and BMPRs during osteoblastic cell differentiation were observed. BMP-2 and BMP-7 mRNA levels did not change significantly. BMP-4 mRNA expression increased and reached a peak prior to matrix formation. BMP-5 mRNA expression increased during the mineralization phase and BMP-6 mRNA expression increased throughout all phases of cell differentiation. Effects of BMP-7 (Osteogenic Protein-1; OP-1) on the expression patterns of several other members of the BMP family and the receptors were also studied. OP-1 downregulated the BMP-4, -5, and -6 mRNA levels by a maximal of 2-fold, 1.5-fold, and 6-fold, respectively. OP-1 did not change significantly the OP-1 and BMP-2 mRNA expression. Of the three type I BMPR examined, OP-1 upregulated ActR-I and BMPR-IA mRNA expression slightly but with statistical significance. OP-1 downregulated BMPR-IB mRNA expression slightly. OP-1 upregulated BMPR-II mRNA expression by a maximum of 2-fold. Our findings demonstrate that OP-1 differentially regulates the mRNA expression of several related members of the BMP family and their receptors in osteoblasts. The observations suggest that OP-1 action on osteoblastic cells involves a complex regulation of gene expression of related members of the BMP family and their receptors in a cell differentiation stage dependent manner.     

Role of bone morphogenetic proteins in human prostate cancer pathogenesis and development of bone metastases: immunohistochemical study

Bone morphogenetic proteins (BMP) have the ability to induce ectopic bone formation. The findings of their expression in prostate cancers have been linked with specifically tumor progression to bone and development of osteosclerotic metastases. We investigated the expression pattern of BMP-2/4, -6 and -7 and the receptors BMPR-IA,-IB and -II in normal human prostate, organ-localized and metastatic prostate cancers. The expression we also examined in skeletal metastases caused by prostate cancer. In localized prostate cancers we found increased expression of BMP-6 and decreased expression of BMP-2/4 and -7. In metastatic prostate cancers the expression of examined BMPs decreased. The expression of BMPRs showed the tendency to be lower with progression of prostate cancer but the expression of BMPR-II was completely absent in metastatic prostate cancers. In bone metastases caused by prostate cancer we found high expression of BMP-2/4, -6 and -7. Decreased expression of BMPs and lose of BMPR-II expression, could suggest that the influence of BMPs on prostate cancer cells is inhibited and plays an important role in prostate cancer pathogenesis. High expression of osteogenic BMPs in prostate cancer bone metastases could explain their osteosclerotic properties.     

Differential effects of osteogenic protein-1 (BMP-7) on gene expression of BMP and GDF family members during differentiation of the mouse MC615 chondrocyte cells

The mRNA expression patterns of several bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs) in long-term cultures of the clonal mouse chondrocyte cell line MC615 were examined. Distinct spatial and temporal patterns of expression of BMPs and GDFs were observed. The temporal orders of expression were correlated with those of several biochemical markers characteristic of chondrocytic cell differentiation. BMP-1, -2, -5, and -6 mRNA expression increased throughout the chondrogenic process and BMP-4 mRNA expression was not changed. GDF-1 and -3 mRNA expression increased throughout the chondrogenic process, and GDF-5, -6, -8, and -9 mRNA expressions were not changed. Effects of osteogenic protein-1 (OP-1, BMP-7) on the expression patterns of several other members of the BMP family and of the GDF family were also examined. OP-1 downregulated the BMP-1, -4, -5, and -6 mRNA expression by a maximal 3-, 5-, 2.5-, and 3-fold, respectively. The BMP-2 mRNA expression was not changed significantly by a low concentration of OP-1, but was increased at 200 ng/ml at day 7 of treatment. In contrast to the BMPs, OP-1 upregulated significantly the six GDF members examined (GDF-1, -3, -5, -6, -8, and -9) by three- to four-fold. Our findings demonstrate that OP-1 differentially regulates the mRNA expression of several related members of the BMP family and upregulates the mRNA expression of several members of the GDF family. The observations suggest that OP-1 action on cartilage differentiation involves a complex regulation of gene expression of several members of the BMP and the GDF family.     

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.     

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.     

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.     

Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.     

Cytokine-specific induction of the TGF-beta inducible early gene (TIEG): regulation by specific members of the TGF-beta family

Select members of the TGF-beta family of cytokines play key regulatory roles in skeletal development, structure, and turnover. This laboratory has previously reported that TGF-beta treatment of immortalized normal human fetal osteoblast (hFOB) cells results in the rapid induction of the mRNA levels of a TGF-beta inducible early gene (TIEG) followed by changes in cell proliferation and bone matrix protein production. Previous studies have also shown that nonmembers of the TGF-beta superfamily showed little or no induction of TIEG mRNA. This article further addresses the cytokine specificity of this TIEG induction by examining whether activin and select bone morphogenetic proteins, (BMP-2, BMP-4, and BMP-6), which are representative of different subfamilies of this superfamily, also induce the expression of TIEG in hFOB cells. However, TGF-beta remained the most potent of these cytokines, inducing TIEG mRNA steady-state levels at 0.1 ng/ml, with a maximum induction of 24-fold at 2.0 ng/ml. The BMP-2 (16-fold), BMP-4 (4-fold), and activin (1-3-fold) also induced TIEG mRNA levels, but at reduced degrees compared to TGF-beta (24-fold), and only at much higher cytokine concentrations, e.g., 50-100 ng/ml, compared to 2 ng/ml for TGF-beta. BMP-6 showed no effect on TIEG mRNA levels. The TIEG protein levels generally correlated with the mRNA steady-state levels. As with TGF-beta, BMP-2 treatment of hFOB cells was shown by confocal microscopy to induce a rapid translocation of the TIEG protein to the nucleus. In summary, the relative potencies of these TGF-beta family members to induce TIEG expression generally follows the general osteoinductive capacity of these cytokines, with TGF-beta > BMP-2 > BMP-4 > activin > BMP-6.     

Diverse Bone Morphogenetic Protein Expression Profiles and Smad Pathway Activation in Different Phenotypes of Experimental Canine Mammary Tumors

Background

BMPs are currently receiving attention for their role in tumorigenesis and tumor progression. Currently, most BMP expression studies are performed on carcinomas, and not much is known about the situation in sarcomas.

Methodology/Principal Findings

We have investigated the BMP expression profiles and Smad activation in clones from different spontaneous canine mammary tumors. Spindle cell tumor and osteosarcoma clones expressed high levels of BMPs, in particular BMP-2, -4 and -6. Clones from a scirrhous carcinoma expressed much lower BMP levels. The various clones formed different tumor types in nude mice but only clones that expressed high levels of BMP-6 gave bone formation. Phosphorylated Smad-1/5, located in the nucleus, was detected in tumors derived from clones expressing high levels of BMPs, indicating an active BMP signaling pathway and BMP-2 stimulation of mammary tumor cell clones in vitro resulted in activation of the Smad-1/5 pathway. In contrast BMP-2 stimulation did not induce phosphorylation of the non-Smad pathway p38 MAPK. Interestingly, an increased level of the BMP-antagonist chordin-like 1 was detected after BMP stimulation of non-bone forming clones.

Conclusions/Significance

We conclude that the specific BMP expression repertoire differs substantially between different types of mammary tumors and that BMP-6 expression most probably has a biological role in bone formation of canine mammary tumors.     

BMP-2/4 and BMP-6/7 differentially utilize cell surface receptors to induce osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily of growth factors and are used clinically to induce new bone formation. The purpose of this study was to evaluate receptor utilization by BMP-2, BMP-4, BMP-6, and BMP-7 in primary human mesenchymal stem cells (hMSC), a physiologically relevant cell type that probably mediates the in vivo effects of BMPs. RNA interference-mediated gene knockdown revealed that osteoinductive BMP activities in hMSC are elicited through the type I receptors ACVR1A and BMPR1A and the type II receptors ACVR2A and BMPR2. BMPR1B and ACVR2B were expressed at low levels and were not found to play a significant role in signaling by any of the BMPs evaluated in this study. Type II receptor utilization differed significantly between BMP-2/4 and BMP-6/7. A greater reliance on BMPR2 was observed for BMP-2/4 relative to BMP-6/7, whereas ACVR2A was more critical to signaling by BMP-6/7 than BMP-2/4. Significant differences were also observed for the type I receptors. Although BMP-2/4 used predominantly BMPR1A for signaling, ACVR1A was the preferred type I receptor for BMP-6/7. Signaling by both BMP-2/4 and BMP-6/7 was mediated by homodimers of ACVR1A or BMPR1A. A portion of BMP-2/4 signaling also required concurrent BMPR1A and ACVR1A expression, suggesting that BMP-2/4 signal in part through ACVR1A/BMPR1A heterodimers. The capacity of ACVR1A and BMPR1A to form homodimers and heterodimers was confirmed by bioluminescence resonance energy transfer analyses. These results suggest different mechanisms for BMP-2/4- and BMP-6/7-induced osteoblastic differentiation in primary hMSC.     

Osteogenic protein-1 and interleukin-6 with its soluble receptor synergistically stimulate rat osteoblastic cell differentiation

Osteogenic Protein-1 (OP-1, BMP-7), a member of the bone morphogenetic protein family, stimulates synthesis of biochemical markers characteristic of the osteoblastic and chondrocytic phenotypes and induces new bone formation. Interleukin-6 (IL-6), a cytokine produced by a wide variety of cells, appears to interact with other factors producing different biological effects. In the present study, we showed that OP-1 action in fetal rat calvaria (FRC) cells was enhanced by the combination of IL-6 and the soluble receptor IL-6sR. OP-1 alone induced alkaline phosphatase (AP) activity by 4- to 5-fold above the control. Exogenous IL-6 soluble receptor (IL-6sR) synergistically stimulated the OP-1-induced AP activity and mineralized bone nodule formation by an additional 3-fold. The stimulation was IL-6sR concentration-dependent. The combination of IL-6 and IL-6sR synergistically stimulated OP-1 action by an additional 6- to 7-fold. BMPR-II receptor mRNA expression in FRC cells treated with OP-1 and IL-6 plus IL-6sR was stimulated further, while BMPR-IA, -IB, and ActR-I expressions were not affected. The intracellular signaling molecules Smad2 and Smad5 mRNA expressions were not changed under these conditions. The expression of selected BMP family members (BMP-3, -4, and -6) was altered in FRC cells treated with OP-1 in combination with IL-6 and IL-6sR. The combination of IL-6 and IL-6sR reduced the OP-1-stimulated BMP-3 mRNA levels and enhanced the suppressive effect of OP-1 on BMP-4 and -6 mRNA expressions. In conclusion, the present results demonstrate that exogenous IL-6 and IL-6sR synergistically stimulate OP-1 action in primary cultures of rat osteoblastic cells. One possible mechanism of synergy involves differential regulation of the effects of OP-1 on the expression of the type II BMP receptor and several other BMPs.     

Effect of bone morphogenetic proteins-4, -5 and -6 on DNA synthesis and expression of bone-related proteins in cultured human periodontal ligament cells

Bone morphogenetic proteins (BMPs) have multiple functions in the development and growth of skeletal and extraskeletal tissues. Therefore, BMPs may regulate the regeneration of periodontal tissue. To investigate this issue, we examined the effects of BMP-4, -5 and -6 on DNA synthesis and the expression of bone-related proteins in cultures of human periodontal ligament (HPL) cells. The expression of bone-related proteins was determined by Real-time polymerase chain reaction and enzyme linked immunosorbent assay in cultures of HPL cells. DNA synthesis was estimated by measuring bromoderoxyuridine incorporation. It was found that BMP-4, -5 and -6 enhanced DNA synthesis dose-dependently. BMP-4 and -5 increased the levels of osteopontin, BMP-2, alkaline phosphatase and core binding factor alpha 1 mRNAs. BMP-6 stimulated the expression of osteopontin, BMP-2, ALPase and osteoprotegerin. These findings show that BMP-4, -5 and -6 have different actions on the expression of bone-related proteins and may play a role in the regeneration of periodontal tissue by promoting cell proliferation and protein expression.     

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.