Bone Morphogenetic Protein 2B Research Articles

Zebrafish as a model for the study of wound healing in hyperglycemia

Background: For preclinical studies of diabetic wound therapy, zebrafish have many orthologous signalling pathways that play essential roles in wound healing and regeneration. Objectives: This study compares the expression of four essential wound-healing genes and caudal fin regeneration in hyperglycemic zebrafish (Danio rerio) to normal zebrafish. Methods: Hyperglycemia was induced by using the intraperitoneal injection method with 350 mg/BW streptozotocin on days one, three, and five. The regeneration of the zebrafish's caudal fin was observed on day 5 after amputation using a stereomicroscope, followed by sampling of the blastema to analyse gene expression. Caudal fin regeneration was analysed using Zeiss Zen 3.3 blue documentation; blood glucose levels were measured using a glucometer; and relative gene expression analysis of sonic hedgehog (shh), insulin-like growth factor 2a (igf2a), bone morphogenetic protein 2b (bmp2b), collagen 1a2 (col1a2) was performed by qRT-PCR method. Results: The glucose level in the hyperglycemia group was 203.2 mg/dL, and that in the normal group was 59.5 mg/dL. The authors found that igf2a, shh, bmp2b, and col1a2 expression were all downregulated in the hyperglycemia group and decreased in caudal fin regeneration. Conclusion: This novel adult zebrafish model of hyperglycemia significantly impairs gene expression and caudal fin regeneration.

Toxic effects of the insecticide tolfenpyrad on zebrafish embryos: Cardiac toxicity and mitochondrial damage.

Tolfenpyrad, a highly effective and broad-spectrum insecticide and acaricide extensively utilized in agriculture, presents a potential hazard to nontarget organisms. This study was designed to explore the toxic mechanisms of tolfenpyrad on zebrafish embryos. Between 24 and 96 h after exposure of the fertilized embryos to tolfenpyrad at concentrations ranging from 0.001 to 0.016 mg/L (96 h-LC50 = 0.017 mg/L), lethal effects were apparent, accompanied with notable anomalies including pericardial edema, increased pericardial area, diminished heart rate, and an elongated distance between the venous sinus and the arterial bulb. Tolfenpyrad elicited noteworthy alterations in the expression of genes pertinent to cardiac development and apoptosis, with the most pronounced changes observed in the cardiac development-related genes of bone morphogenetic protein 2b (bmp2b) and p53 upregulated modulator of apoptosis (puma). The findings underscore that tolfenpyrad induces severe cardiac toxicity and mitochondrial damage in zebrafish embryos. This data is imperative for a comprehensive assessment of tolfenpyrad risks to aquatic ecosystems, particularly considering the limited knowledge regarding its detrimental impact on aquatic vertebrates.

Characterization and potential function of 7-dehydrocholesterol reductase (dhcr7) and lathosterol 5-desaturase (sc5d) in Cynoglossus semilaevis sexual size dimorphism

The typical sexual size dimorphism (SSD) phenomenon of Chinese tongue sole (Cynoglossus semilaevis) seriously restricts the sustainable development of the fishing industry. Previous transcriptome analysis has found a close relationship between the steroid biosynthesis and C. semilaevis SSD. The 7-dehydrocholesterol reductase (dhcr7) and lathosterol 5-desaturase (sc5d) are two genes in the steroid biosynthesis pathway, playing important roles in lipid synthesis, cellular metabolism, and growth. The present study assessed their roles in the mechanism of C. semilaevis SSD. The quantitative polymerase chain reaction (qPCR) results showed that C. semilaevis dhcr7 was mainly expressed in female livers, and C. semilaevis sc5d was highly expressed in female livers and gonads. Dual-luciferase experiment showed that dhcr7 and sc5d promoters had strong transcriptional activity. The transcription factors E2F transcription factor 1 (E2F1), and CCAAT enhancer binding protein alpha (C/EBPα) significantly regulated the transcriptional activity of dhcr7 and sc5d promoters, respectively. Furthermore, small interfering RNA (siRNA) knockdown results showed that expression levels of several genes [SREBF chaperone (scap), membrane-bound transcription factor peptidase, site 1 (mbtps1), fatty acid synthase (fasn), sonic hedgehog (shh), bone morphogenetic protein 2b (bmp2b) and AKT serine/threonine kinase 1 (akt1)] were suppressed. Protein subcellular localization results indicated that Dhcr7 and Sc5d were both specifically distributed in the cytoplasm, with co-localization been observed. The present study provides evidence that dhcr7 and sc5d might regulate C. semilaevis sexual size dimorphism by involving in energy homeostasis and cell cycle, or by affecting PI3K-Akt and Shh signaling pathways. The detailed roles of these steroid biosynthesis genes regulating C. semilaevis SSD needed more information.

Comparative transcriptome analyses and identification of candidate genes involved in vertebral abnormality of bighead carp Hypophthalmichthys nobilis

Body deformity occurs both in wild and farmed fishes, which is one of the most challenging problems for aquaculture industry. In most cases, such body deformities are linked to skeletal deformities. Currently, very limited information is available on skeletal deformities of farmed fish species which may be caused by genetic factor. In this study, we performed muscle and vertebra transcriptome analyses in body deformity and normality of bighead carp Hypophthalmichthys nobilis (from one meiotic gynogenesis family) using RNA-Seq. A total of 43,923 and 44,416 unigenes were predicted in muscles and vertebrae, respectively. Based on these data, we further explored the gene expression profiles in gynogenetic normal and abnormal bighead carp. No differentially expressed gene (DEG) was found in transcriptome data of muscles. Totally, 20 key DEGs were identified in transcriptome data of vertebrae, such as low density lipoprotein-related protein 2 (lrp2), bone morphogenetic protein 2B (bmp2b) and collagen alpha-1(IV) (col4a1). 12 potential pathways were also identified in vertebra transcriptome data, which were mainly involved in development, growth, cytoskeleton and energy metabolism, such as MAPK signaling pathway, regulation of actin cytoskeleton and TGF-beta signaling pathway. Results of this study will be informative for the understanding of genetic mechanisms for body shape formation and also provide potential candidate genes for selection program involved in body shape and skeletal development in H. nobilis.

Four and a Half LIM Domains 1b (Fhl1b) Is Essential for Regulating the Liver versus Pancreas Fate Decision and for β-Cell Regeneration.

The liver and pancreas originate from overlapping embryonic regions, and single-cell lineage tracing in zebrafish has shown that Bone morphogenetic protein 2b (Bmp2b) signaling is essential for determining the fate of bipotential hepatopancreatic progenitors towards the liver or pancreas. Despite its pivotal role, the gene regulatory networks functioning downstream of Bmp2b signaling in this process are poorly understood. We have identified four and a half LIM domains 1b (fhl1b), which is primarily expressed in the prospective liver anlage, as a novel target of Bmp2b signaling. fhl1b depletion compromised liver specification and enhanced induction of pancreatic cells from endodermal progenitors. Conversely, overexpression of fhl1b favored liver specification and inhibited induction of pancreatic cells. By single-cell lineage tracing, we showed that fhl1b depletion led lateral endodermal cells, destined to become liver cells, to become pancreatic cells. Reversely, when fhl1b was overexpressed, medially located endodermal cells, fated to differentiate into pancreatic and intestinal cells, contributed to the liver by directly or indirectly modulating the discrete levels of pdx1 expression in endodermal progenitors. Moreover, loss of fhl1b increased the regenerative capacity of β-cells by increasing pdx1 and neurod expression in the hepatopancreatic ductal system. Altogether, these data reveal novel and critical functions of Fhl1b in the hepatic versus pancreatic fate decision and in β-cell regeneration.

Regulation of the Activin-Inhibin-Follistatin System by Bone Morphogenetic Proteins in the Zebrafish Ovary1

In the zebrafish, the dynamic expression of the activin-inhibin-follistatin system during folliculogenesis and its exclusive localization (except follistatin) in follicle cells suggests that the system plays important roles in follicle development and that its expression is subject to tight controls, probably by external factors including those derived from the oocyte. We have previously identified zebrafish bone morphogenetic proteins (BMPs) as oocyte factors that may act on follicle cells; however, the targets of BMPs in the follicle cells remain unknown. Considering their spatiotemporal expression in the follicle, we hypothesized that members of the activin-inhibin-follistatin system in follicle cells could be potential target genes of BMPs. In the present study, we developed a novel coculture system to co-incubate zebrafish bone morphogenetic protein 2b or 4 (zfBMP2b/4)-producing Chinese hamster ovary (CHO) cells with zebrafish follicle cells. During incubation, the zfBMPs secreted from the CHO cells would act directly on the follicle cells in a paracrine manner. Our results showed that all activin beta subunits (inhbaa, inhbab, and inhbb) were down-regulated by both zfBMP2b and zfBMP4, while follistatin (fst, an activin-binding protein) and inhibin alpha (inha, an activin antagonist) were significantly up-regulated. The specificity of bone morphogenetic protein (BMP) actions was confirmed by short interfering RNA knockdown of zfBMP4 expression in the CHO cells. The robust response of inha to zfBMPs, together with our previous observation that inha expression surged at the full-grown stage prior to oocyte maturation, led us to hypothesize that the full-grown oocyte may signal upper levels of the hypothalamic-pituitary-gonadal axis its readiness to mature by releasing BMPs, which in turn stimulate inhibin production. As an ovarian hormone and activin antagonist, inhibin may suppress the action of activin in the pituitary to reduce follicle-stimulating hormone but increase luteinizing hormone (LH) biosynthesis. Meanwhile, by increasing the local follistatin level and reducing the activin production, BMPs could help prevent precocious maturation before preovulatory LH surge.

Low-level pyrene exposure causes cardiac toxicity in zebrafish (Danio rerio) embryos

It is widely accepted that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils is cardiotoxic. Although PAHs toxic endpoints show strong correlation with the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, and is thought to be a potent inducer of cytochrome P4501A, the action mechanism of PAHs on vertebrate cardiovascular development and disease is unclear. Herein, we address the cardiac developmental effects of exposure to the weak AhR agonist pyrene on the early life-stages of zebrafish. Embryos were exposed to 0, 0.05, 0.5, 5, and 50nmol/L pyrene up to 72h post-fertilization (hpf). Pyrene-treated embryos showed dose-dependent heart abnormalities, such as pericardial edema and cardiac looping defects. Changes in AhR1a, AhR1b, AhR2, and Cyp1A expression were assessed by real-time RT-PCR. The results showed that low-level pyrene failed to alter these genes expression. However, the homeodomain transcription factor Nkx2.5, which plays an essential role in the development of the cardiovascular system, was down-regulated in a dose-dependent manner by pyrene exposure. The bone morphogenetic protein 2b (Bmp2b), which has been identified as the upstream gene of Nkx2.5, also was inhibited in a dose-dependent manner after treatment with pyrene. Taken together, these data indicated that embryonic exposure of zebrafish to low-level environmental pyrene disrupt normal cardiac development and alter expression of defective cardiac differentiation related genes.

Expression Levels of Bone Morphogenetic Protein 2b in Fins of Adult Japanese Medaka (Oryzias latipes) Exposed to Sex Steroid Hormones

Expression Levels of Bone Morphogenetic Protein 2b in Fins of Adult Japanese Medaka (Oryzias latipes) Exposed to Sex Steroid Hormones

Zebrafish Fins as a Model System for Skeletal Human Studies

Recent studies on the morphogenesis of the fins of Danio rerio (zebrafish) during development and regeneration suggest that a number of inductive signals involved in the process are similar to some of those that affect bone and cartilage differentiation in mammals and humans. Akimenko et al. (2002) has shown that bone morphogenetic protein-2b (BMP2b) is involved in the induction of dermal bone differentiation during fin regeneration. Many other groups have also shown that molecules from the transforming growth factor-beta superfamily (TGFβ), including BMP2, are effective in promoting chondrogenesis and osteogenesis in vivo in higher vertebrates, including humans. In the present study, we review the state of the art of this topic by a comparative analysis of skeletal tissue development, regeneration and renewal processes in tetrapods, and fin regeneration in fishes. A general conclusion of this study states that lepidotrichia is a special skeletal tissue different to cartilage, bone, enamel, or dentine in fishes, according to its extracellular matrix (ECM) composition. However, the empirical analysis of inducing signals of skeletal tissues in fishes and tetrapods suggests that lepidotrichia is different to any responding features with main skeletal tissues. A number of new inductive molecules are arising from fin development and regeneration studies that might establish an empirical basis for further molecular approaches to mammal skeletal tissues differentiation. Despite the tissue dissimilarity, this empirical evidence might finally lead to clinical applications to skeletal disorders in humans.

Inhibition of BMP signaling during zebrafish fin regeneration disrupts fin growth and scleroblast differentiation and function

The zebrafish caudal fin provides a simple model to study molecular mechanisms of dermal bone regeneration. We previously showed that misexpression of Bone morphogenetic protein 2b (Bmp2b) induces ectopic bone formation within the regenerate. Here we show that in addition to bmp2b and bmp4 another family member, bmp6, is involved in fin regeneration. We further investigated the function of BMP signaling by ectopically expressing the BMP signaling inhibitor Chordin which caused: (1) inhibition of regenerate outgrowth due to a decrease of blastema cell proliferation and downregulation of msxb and msxC expression and (2) reduced bone matrix deposition resulting from a defect in the maturation and function of bone-secreting cells. We then identified targets of BMP signaling involved in regeneration of the bone of the fin rays. runx2a/b and their target col10a1 were downregulated following BMP signaling inhibition. Unexpectedly, the sox9a/b transcription factors responsible for chondrocyte differentiation were detected in the non-cartilaginous fin rays, sox9a and sox9b were not only differentially expressed but also differentially regulated since sox9a, but not sox9b, was downregulated in the absence of BMP signaling. Finally, this analysis revealed the surprising finding of the expression, in the fin regenerate, of several factors which are normally the signatures of chondrogenic elements during endochondral bone formation although fin rays form through dermal ossification, without a cartilage intermediate.

Growth factor responsiveness of human articular chondrocytes: Distinct profiles in primary chondrocytes, subcultured chondrocytes, and fibroblasts

The objectives of this study were to establish a growth factor response profile for adult human articular chondrocytes, to determine whether this is unique for chondrocytes or influenced by the differentiation status of the cells, and to characterize growth factor interactions. It is shown that transforming growth factor-beta (TGF-beta) is the most potent mitogen among a variety of factors tested. All three isoforms of TGF-beta caused similar dose-dependent increases in chondrocyte proliferation. Other members of the TGF-beta family, including bone morphogenetic protein 2B (BMP2B), activin, and inhibin, did not detectably increase chondrocyte proliferation. Platelet-derived growth factor-AA (PDGF-AA), basic fibroblast growth factor (bFGF), and insulin-like growth factor 1 (IGF-1) also stimulated proliferation but were less effective than TGF-beta. In contrast to findings with other cell types, the effects of TGF-beta on chondrocyte proliferation were not dependent on the endogenous production of PDGF. The cytokines Interleukin 1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) gave no stimulation, but IL-1 inhibited chondrocyte proliferation induced by TGF-beta or serum. This response profile was characteristic for primary chondrocytes from human adults and distinct from subcultured (dedifferentiated) chondrocytes or skin fibroblasts. The latter preferentially responded to PDGF, and IL-1 caused greater increases in proliferation than TGF-beta. In summary, these results describe growth factor responses that are characteristic for chondrocytes and provide a basis for the analysis of changes in chondrocyte growth proliferation that occur in aging and tissue injury.

Purification and Characterization of a Bone-Inducing Protein From a Murine Osteosarcoma (Dunn Type)

A protein fraction capable of eliciting cartilage or bone formation in vivo was purified more than 100,000-fold from a murine osteosarcoma (Dunn type). Intramuscular implantation of as little as 20 ng of the purified protein with 2 mg of pure skin collagen consistently induced ectopic new bone formation. The apparent molecular size of the purified protein was 32 kd on sodium dodecylsulfate-polyacrylamide gel electrophoresis. On reduction, the 32-kd protein split into subunits with the same partial amino acid sequences, and these partial sequences were identical to those of human bone morphogenetic protein-2B (BMP-4) which is assumed to be a member of the transforming growth factor-beta superfamily.

516850 Mammalian expression of the bone morphogenetic protein-2B using BMP2a/BMP2B fusion: R Glenn Hammonds, Anthony J Mason assigned to Genentech Inc

516850 Mammalian expression of the bone morphogenetic protein-2B using BMP2a/BMP2B fusion: R Glenn Hammonds, Anthony J Mason assigned to Genentech Inc

Recombinant human bone morphogenetic protein 2B stimulates PC12 cell differentiation: potentiation and binding to type IV collagen.

Bone morphogenetic protein 2B (BMP 2B, also known as BMP 4) induces cartilage and bone morphogenesis in ectopic extraskeletal sites. BMP 2B is one of several bone morphogenetic proteins which along with activins and inhibins are members of the transforming growth factor-beta (TGF-beta) family. Both BMP 2B and activin A, but not TGF-beta 1, induce rat pheochromocytoma PC12 neuronal cell differentiation and expression of VGF, a nervous system-specific mRNA. PC12 cells exhibited approximately 2,500 receptors per cell for BMP 2B with an apparent dissociation constant of 19 pM. Extracellular matrix components, including fibronectin, laminin, and collagen type IV potentiated the activity of BMP and activin A, with the latter being the most active. Direct experiments demonstrated that radioiodinated BMP 2B bound to collagen type IV better than to either laminin or fibronectin. These data demonstrate a common neurotrophic activity of both BMP 2B and activin A, and suggest that these regulatory molecules alone and in conjunction with extracellular matrix components may play a role in both the development and repair of nervous tissue.

Osteogenin and recombinant bone morphogenetic protein 2B are chemotactic for human monocytes and stimulate transforming growth factor beta 1 mRNA expression.

Subcutaneous implantation of demineralized bone matrix initiates a sequence of developmental events, which culminate in endochondral bone formation. During early stages of development of matrix-induced implants, ED1, Ia-positive monocytes-macrophages were observed, suggesting that in the initial phases of the endochondral bone formation cascade, the bone-inductive protein osteogenin and related bone morphogenetic proteins (BMPs) might serve as potent chemoattractants to recruit circulating monocytes. In this investigation, we demonstrate that at concentrations of 10-100 fg/ml (0.3-3 fM), native bovine osteogenin and recombinant human BMP-2B (rhBMP-2B) induce the directed migration of human blood monocytes in vitro. This chemotactic response was associated with expression of BMP binding sites (receptors) on monocytes. About 750 receptors per cell were detected with an apparent dissociation constant of 200 pM. Both osteogenin and rhBMP-2B at higher concentrations (0.1-30 ng/ml) stimulated mRNA expression for an additional regulatory molecule, type beta 1 transforming growth factor (TGF-beta 1) in human monocytes. TGF-beta 1, in turn, is known to induce a cascade of events leading to matrix generation. Monocytes stimulated by TGF-beta are known to secrete a number of chemotactic and mitogenic cytokines that recruit endothelial and mesenchymal cells and promote their synthesis of collagen and associated matrix constituents. TGF-beta 1 in concert with these other cytokines and matrix components regulates chemotaxis, mesenchymal proliferation, differentiation, angiogenesis, and controlled synthesis of extracellular matrix. Our results demonstrate that osteogenin and related BMPs through their profound effects on monocyte recruitment and cytokine synthesis may promote additional successive steps in the endochondral bone formation cascade.

Chick limb bud mesodermal cell chondrogenesis: Inhibition by isoforms of platelet-derived growth factor and reversal by recombinant bone morphogenetic protein

Platelet-derived growth factor (PDGF) influences the proliferation and differentiation of a variety of cells. In this study, we have investigated the effect of PDGF isoforms on chondrogenesis by stage 24 chick limb bud mesoderm cells in culture. Synthesis of sulfated proteoglycans, an index of chondrogenesis, was inhibited by all three PDGF isoforms (PDGF-AA, PDGF-AB, and PDGF-BB). Application of PDGF isoforms during the first 2 days of culture, before the cells were overtly differentiating, resulted in decreased synthesis of sulfated proteoglycans. This was similar to when PDGF isoforms were present throughout the culture period. However, application of PDGF isoform during only the last 2 days of culture, did not inhibit cartilage matrix production. When chondrogenic and nonchondrogenic cells were separated from the cultures and replated, PDGF-AB and PDGF-BB inhibited the incorporation of sulfate by the chondrogenic cells. Recombinant bone morphogenetic protein 2B reversed the inhibitory effects of PDGF on sulfated proteoglycan synthesis and DNA synthesis. PDGF receptor binding analysis indicated that beta-receptors were predominant receptors present on the chondrogenic and nonchondrogenic cells of the stage 24 mesoderm. PDGF isoforms increased thymidine incorporation by 48 h in both high and low density cultures. However, at later periods, cell proliferation was inhibited by PDGF-AA and PDGF-AB but not by PDGF-BB. PDGF acted as a bifunctional modulator of mesodermal cell proliferation and thus may regulate chondrogenesis during limb differentiation and morphogenesis.

Natural bovine osteogenin and recombinant human bone morphogenetic protein-2B are equipotent in the maintenance of proteoglycans in bovine articular cartilage explant cultures.

Osteogenin and related bone morphogenetic proteins are members of the transforming growth factor-beta superfamily, and were isolated by their ability to induce cartilage and bone formation in vivo. The influence of osteogenin, purified from bovine bone, and of recombinant human bone morphogenetic protein-2B (BMP-2B) has been examined in bovine articular cartilage explants. Both differentiation factors stimulated in a dose-dependent manner the synthesis of proteoglycans and decreased their rate of degradation. At a dose of 30 ng/ml, proteoglycan synthesis was increased to levels observed with either 20 ng/ml insulin-like growth factor I, 10 ng/ml transforming growth factor-beta, or 20% fetal bovine serum. This increase of biosynthetic rates above basal medium levels was observed in young, adolescent, and adult tissues. Analysis of the size of the newly synthesized proteoglycans, the glycosaminoglycan chain size, and the glycosaminoglycan type of explants treated with osteogenin or BMP-2B were very comparable to each other, and to proteoglycans isolated from cartilage treated with either insulin-like growth factor I or fetal bovine serum. These results demonstrate that osteogenin and BMP-2B alone are capable of stimulating and maintaining the chondrocyte phenotype in vitro.

Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: Comparison with TGF-β1

The biologic effects of recombinant human bone morphogenetic protein-2b (BMP-2b = BMP-4) were studied and compared with transforming growth factor-beta 1 (TGF-beta 1) in fetal rat osteoblast-like (ROB) cells. Similar to the effects of TGF-beta 1, BMP-2b stimulated DNA and collagen synthesis as well as protein accumulation. Unlike TGF-beta 1, which inhibited alkaline phosphatase activity, BMP-2b enhanced enzyme activity eight-to ninefold over the control level. The present study demonstrates direct actions of BMP-2b on bone-associated cells to stimulate osteogenic phenotypes in vitro and provides a cellular mechanism for the induction of bone formation by BMP-2b in vivo.

Stimulation of chondrogenesis in limb bud mesoderm cells by recombinant human bone morphogenetic protein 2B (BMP-2B) and modulation by transforming growth factor β1 and β2

Bone morphogenetic protein 2B (BMP-2B) also called BMP-4 is one of a family of cartilage and bone-inductive proteins derived from bone matrix and belongs to the transforming growth factor β (TGF-β) superfamily. These bone-inductive proteins isolated from adult bone may be involved in bone repair. However, they may also play a role in cartilage and bone formation during embryonic development. To test whether BMP-2B influences cartilage formation by embryonic cells, recombinant human BMP-2B was applied to cultured limb bud mesoderm plated at three different densities. BMP-2B stimulated cartilage formation as assessed by Alcian blue staining and incorporation of radioactive sulfate into sulfated proteoglycans. Cells cultured at all three densities in the presence of 10 ng/ml BMP-2B formed a nearly continuous sheet of cartilage with abundant extracellular matrix and type II collagen. In addition, when cells were cultured in 0.5% serum in the presence of 10 ng/ml of BMP-2B for 5 days there was an increase in alkaline phosphatase as detected by histochemical and biochemical methods. Transforming growth factor β isoforms (TGF- β 1 and TGF- β 2) inhibited sulfate incorporation into proteoglycans in a dose-dependent manner. This inhibition by TGFβ was overcome by recombinant BMP-2B. This study demonstrates that recombinant BMP-2B stimulates cartilage formation by chick limb bud mesoderm in vitro and is further modulated by TGF-β isoforms.

Identification and characterization of cellular binding proteins (receptors) for recombinant human bone morphogenetic protein 2B, an initiator of bone differentiation cascade.

Bone morphogenetic protein 2B (BMP 2B), is a heparin-binding bone differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously. The molecular mechanism of action of this differentiation factor is not known, and as a first step we have examined BMP 2B-responsive cells for the presence of specific cellular binding proteins. Using 125I-labeled BMP 2B, specific high-affinity binding sites for recombinant human BMP 2B on MC3T3 E1 osteoblast-like cells as well as on NIH 3T3 fibroblasts were identified. Platelet-derived growth factor, insulin-like growth factor 1, basic fibroblast growth factor, epidermal growth factor, and transforming growth factor beta did not compete for the binding of radiolabeled BMP 2B. The binding of BMP 2B is a time- and temperature-dependent process. Chemical crosslinking of radiolabeled BMP showed two components (apparent size, 200 and 70 kDa in MC3T3 E1 cells and 200 and 90 kDa in NIH 3T3 cells). A minor component at 60 kDa was also detected in both cell lines. Scatchard analysis of the binding data showed a high-affinity receptor with an apparent dissociation constant of 128 +/- 40 pM in MC3T3 E1 cells. These data demonstrate specific, high-affinity cell-surface binding proteins for BMP 2B.