BMP Binding Research Articles

TMF suppresses chondrocyte hypertrophy in osteoarthritic cartilage by mediating the FOXO3a/BMPER pathway.

Osteoarthritis (OA) is a disease of the joints, characterized by chronic inflammation, cartilage destruction and extracellular matrix (ECM) remodeling. Aberrant chondrocyte hypertrophy promotes cartilage destruction and OA development. Collagen X, the biomarker of chondrocyte hypertrophy, is upregulated by runt-related transcription factor 2 (Runx2), which is mediated by the bone morphogenetic protein 4 (BMP4)/Smad1 signaling pathway. BMP binding endothelial regulator (BMPER), a secreted glycoprotein, acts as an agonist of BMP4. 5,7,3',4'-tetramethoxyflavone (TMF) is a natural flavonoid derived from Murraya exotica L. Results of our previous study demonstrated that TMF exhibits chondroprotective effects against OA development through the activation of Forkhead box protein O3a (FOXO3a) expression. However, whether TMF suppresses chondrocyte hypertrophy through activation of FOXO3a expression and inhibition of BMPER/BMP4/Smad1 signaling remains unknown. Results of the present study revealed that TMF inhibited collagen X and Runx2 expression, inhibited BMPER/BMP4/Smad1 signaling, and activated FOXO3a expression; thus, protecting against chondrocyte hypertrophy and OA development. However, BMPER overexpression and FOXO3a knockdown impacted the protective effects of TMF. Thus, TMF inhibited chondrocyte hypertrophy in OA cartilage through mediating the FOXO3a/BMPER signaling pathway.

Modelling the structure of Short Gastrulation and generation of a toolkit for studying its function in Drosophila.

ABSTRACTA BMP gradient is essential for patterning the dorsal-ventral axis of invertebrate and vertebrate embryos. The extracellular BMP binding protein Short Gastrulation (Sog) in Drosophila plays a key role in BMP gradient formation. In this study, we combine genome editing, structural and developmental approaches to study Sog function in Drosophila. We generate a sog knockout fly stock, which allows simple reintegration of altered versions of the sog coding sequence. As proof-of-principle, we test the requirement for two cysteine residues that were previously identified as targets for palmitoylation, which has been proposed to enhance Sog secretion. However, we show that the sogC27,28S mutant is viable with only very mild phenotypes, indicating that these residues and their potential modification are not critical for Sog secretion in vivo. Additionally, we use experimental negative stain EM imaging and hydrodynamic data to validate the AlphaFold structure prediction for Sog. The model suggests a more compact shape than the vertebrate ortholog Chordin and conformational flexibility between the C-terminal von Willebrand C domains. We discuss how this altered compactness may contribute to mechanistic differences in Sog and Chordin function during BMP gradient formation.This article has an associated First Person interview with the first author of the paper.

Cytogenomic aberrations in isolated multicystic dysplastic kidney in children.

Background:Multicystic dysplastic kidney (MCDK) is a common form of congenital kidney anomaly. The cause of MCDK is unknown. We investigated whether MCDK in children is linked to cytogenomic aberrations.Methods:We conducted Array Comparative Genomic Hybridization (aCGH) in 10 unrelated children with MCDK. The pattern of inheritance was determined by real-time PCR in patients and their biological parents.Results:Pathogenic aberrations were detected in three patients: a deletion at 7p14.3 with a size of 2.07 Mb housing 12 genes, including BBS9 and BMPER; a duplication at 16p13.11p12.3 with a size of 3.28 Mb that included more than 20 genes; and monosomy X for a female patient. The deletion at 7p14.3 was inherited from patient’s father, while the duplication at 16p13.11p12.3 was derived from patient’s mother.Conclusions:Up to 30% of patients with MCDK possess cytogenomic aberrations. BBS9 and BMPER variants have been reported to result in cystic kidney dysplasia, suggesting possible pathogenic function for the deletion at 7p14.3 in children with MCDK. The duplication at 16p13.11p12.3 was not reported previously to associate with MCDK. Both variations were inherited from parents, indicating hereditary contributions in MCDK. Thus, aCGH is an informative tool to unravel pathogenic mechanisms of MCDK.

Crossveinless is a direct transcriptional target of Trachealess and Tango in Drosophila tracheal precursor cells.

Understanding the transcriptional pathways controlling tissue-specific gene expression is critical to unraveling the complex regulatory networks that underlie developmental mechanisms. Here, we assessed how the Drosophila crossveinless (cv) gene, that encodes a BMP-binding factor, is transcriptionally regulated in the developing embryonic tracheal system. We identify an upstream regulatory region of cv that promotes reporter gene expression in the tracheal precursors. We further demonstrate that this promoter region is directly responsive to the basic, helix-loop-helix-PAS domain factors Trachealess (Trh) and Tango (Tgo), that function to specify tracheal fate. Moreover, cv expression in embryos is lost in trh mutants, and the integrity of the Trh/Tgo binding sites are required for promoter-lacZ expression. These findings for the first time elucidate the transcriptional regulation of one member of a family of BMP binding proteins, that have diverse functions in animal development.

BBP-Functionalized Biomimetic Nanofibrous Scaffold Can Capture BMP2 and Promote Osteogenic Differentiation.

Bone morphogenetic proteins (BMPs, e.g., BMP2 and 7) are potent mediators for bone repair, however, their clinical use has been limited by their safety and cost-effectiveness. Therefore, innovative strategies that can improve the efficacy of BMPs, and thereby, use a lower dose of exogenous BMPs are highly desired. Inspired by the natural interaction between extracellular matrix (ECM) and growth factors, we hypothesize that bone matrix-mimicking nanofibrous scaffold functionalized with BMP binding moieties can selectively capture and stabilize BMPs, and thereby, promote BMP-induced osteogenic differentiation. To test our hypothesis, a gelatin nanofibrous scaffold was fabricated using thermally induced phase separation together with a porogen leaching technique (TIPS&P) and functionalized by a BMP-binding peptide (BBP) through cross-linking. Our data indicated that BBP decoration largely improved the BMP2 binding and retention capacity of the nanofibrous scaffolds without compromising their macro/microstructure and mechanical properties. Importantly, the BBP-functionalized gelatin scaffolds were able to significantly promote BMP2-induced osteogenic differentiation. Moreover, BBP alone was able to significantly stimulate endogenous BMP2 expression and improve osteogenic differentiation. Compared to other affinity-based drug delivery strategies, e.g., heparin and antibody-mediated growth factor delivering techniques, we expect BBP-functionalized scaffolds will be a safer, more feasible and selective strategy for endogenous BMP stimulating and binding. Therefore, our data suggests a promising application of using the BBP-decorated gelatin nanofibrous scaffold to stimulate/capture BMPs and promote endogenous bone formation in situ in contrast to relying on the administration of high doses of exogenous BMPs and transplantation of cells.

Spatio-Temporal Gene Expression Profiling during In Vivo Early Ovarian Folliculogenesis: Integrated Transcriptomic Study and Molecular Signature of Early Follicular Growth.

BackgroundThe successful achievement of early ovarian folliculogenesis is important for fertility and reproductive life span. This complex biological process requires the appropriate expression of numerous genes at each developmental stage, in each follicular compartment. Relatively little is known at present about the molecular mechanisms that drive this process, and most gene expression studies have been performed in rodents and without considering the different follicular compartments.ResultsWe used RNA-seq technology to explore the sheep transcriptome during early ovarian follicular development in the two main compartments: oocytes and granulosa cells. We documented the differential expression of 3,015 genes during this phase and described the gene expression dynamic specific to these compartments. We showed that important steps occurred during primary/secondary transition in sheep. We also described the in vivo molecular course of a number of pathways. In oocytes, these pathways documented the chronology of the acquisition of meiotic competence, migration and cellular organization, while in granulosa cells they concerned adhesion, the formation of cytoplasmic projections and steroid synthesis. This study proposes the involvement in this process of several members of the integrin and BMP families. The expression of genes such as Kruppel-like factor 9 (KLF9) and BMP binding endothelial regulator (BMPER) was highlighted for the first time during early follicular development, and their proteins were also predicted to be involved in gene regulation. Finally, we selected a data set of 24 biomarkers that enabled the discrimination of early follicular stages and thus offer a molecular signature of early follicular growth. This set of biomarkers includes known genes such as SPO11 meiotic protein covalently bound to DSB (SPO11), bone morphogenetic protein 15 (BMP15) and WEE1 homolog 2 (S. pombe)(WEE2) which play critical roles in follicular development but other biomarkers are also likely to play significant roles in this process.ConclusionsTo our knowledge, this is the first in vivo spatio-temporal exploration of transcriptomes derived from early follicles in sheep.

Secreted phosphoprotein 24 kD inhibits nerve root inflammation induced by bone morphogenetic protein-2

Background contextBone morphogenetic protein-2 (BMP-2) has been used to successfully promote spine fusion, but side-effects including nerve inflammation have been observed. PurposeTo investigate the direct neurotoxic effects of BMP-2 and test the hypotheses that the use of BMP binding proteins, such as secreted phosphoprotein 24 kD (Spp24), can reduce or eliminate these effects. Study designIn vitro experiments and in vivo analysis in a rodent model. MethodsIn vitro, dorsal root ganglion cells were cultured in the presence of BMP-2 with and without Spp24 and calcitonin gene-related peptide and Substance P, markers of neuroinflammation, were measured by immunohistochemistry. In vivo, rats underwent a left-sided laminotomy at L5 to expose the S1 nerve root and were randomized into four different groups according to the intervention at the laminotomy site: collagen sponge only (no BMP-2 or Spp24), BMP-2 in a collagen sponge only, BMP-2 in a collagen sponge+an empty collagen sponge to act as a barrier, and BMP-2 in a collagen sponge+Spp24 in a collagen sponge to act as a barrier. Functional evaluation was done using the Basso, Beattie, and Bresnahan scale and immunohistochemical analyses were performed using calcitonin gene-related peptide and Substance P staining. ResultsThe neuroinflammatory effects of BMP-2 in vitro were ameliorated by the addition of Spp24. Similarly, in vivo, Spp24 reduced the expression of markers on neuroinflammation in animals treated with BMP-2 and also improved the function after BMP-2 administration. ConclusionsThese results confirm that BMP binding proteins have great potential as adjuvant therapies to limit BMP-2 related side-effects in spine surgery.

A complex signaling network involving protein kinase CK2 is required for hepatitis C virus core protein-mediated modulation of the iron-regulatory hepcidin gene expression.

Hepatitis C virus (HCV) infection is associated with hepatic iron overload and elevated serum iron that correlate to poor antiviral responses. Hepcidin (HAMP), a 25-aa cysteine-rich liver-specific peptide, controls iron homeostasis. Its expression is up-regulated in inflammation and iron excess. HCV-mediated hepcidin regulation remains controversial. Chronic HCV patients possess relatively low hepcidin levels; however, elevated HAMP mRNA has been reported in HCV core transgenic mice and HCV replicon-expressing cells. We investigated the effect of HCV core protein on HAMP gene expression and delineated the complex interplay of molecular mechanisms involved. HCV core protein up-regulated HAMP promoter activity, mRNA, and secreted protein levels. Enhanced promoter activity was abolished by co-transfections of core with HAMP promoter constructs containing mutated/deleted BMP and STAT binding sites. Dominant negative constructs, pharmacological inhibitors, and silencing experiments against STAT3 and SMAD4 confirmed the participation of both pathways in HAMP gene regulation by core protein. STAT3 and SMAD4 expression levels were found increased in the presence of HCV core, which orchestrated SMAD4 translocation into the nucleus and STAT3 phosphorylation. To further understand the mechanisms governing the core effect, the role of the JAK/STAT-activating kinase CK2 was investigated. A CK2-dominant negative construct, a CK2-specific inhibitor, and RNAi interference abrogated the core-induced increase on HAMP promoter activity, mRNA, and protein levels, while CK2 acted in synergy with core to significantly enhance HAMP gene expression. Therefore, HCV core up-regulates HAMP gene transcription via a complex signaling network that requires both SMAD/BMP and STAT3 pathways and CK2 involvement.

Inhibition of MicroRNA-302 (miR-302) by Bone Morphogenetic Protein 4 (BMP4) Facilitates the BMP Signaling Pathway

The signaling pathway mediated by BMPs plays an essential role during development as well as the maintenance of homeostasis in adult. Aberrant activation or inactivation of BMP signaling can lead to developmental defects and various human disorders. To fine-tune its activity, BMP signaling is regulated both positively and negatively by extrinsic and intrinsic regulatory factors that modulate binding of ligand to the receptors, and the activity of receptors and their dedicated signal transducers, the Smad proteins. Upon BMP binding to the receptor complex, Smad proteins translocate to the nucleus and modulate gene expression transcriptionally by directly associating with the promoter region of target genes, or post-transcriptionally through modulation of microRNA (miRNA) synthesis. In this study, we demonstrate that BMP signaling down-regulates transcription of the miRNA-302∼367 gene cluster. We show that the type II BMP receptor (BMPRII) is a novel target of miR-302. Upon overexpression, miR-302 targets a partially complementary sequence localized in the 3'-untranslated region (UTR) of BMPRII transcripts and leads to destabilization of the transcripts and down-regulation of BMP signaling. We propose that the negative regulatory loop of BMP4-miR-302-BMPRII is a potential mechanism for the maintenance and fine-tuning of the BMP signaling pathway in various systems.

Fractone-heparan sulfates mediate BMP-7 inhibition of cell proliferation in the adult subventricular zone

Bone morphogenetic protein-7 (BMP-7) is a heparin-binding growth factor that inhibits cell proliferation in the subventricular zone (SVZ) of the lateral ventricle, the primary neurogenic niche in the adult brain. However, the physiological mechanisms regulating the activity of BMP-7 in the SVZ are unknown. Here, we report the inhibitory effect of BMP-7 on cell proliferation through the anterior SVZ after intracerebroventricular injection in the adult mouse. To determine whether the inhibition of cell proliferation induced by BMP-7 is dependant on heparin-binding, heparitinase-1 was intracerebroventricularly injected to N-desulfate heparan sulfate proteoglycans before BMP-7 was injected. Heparatinase-1 drastically reduced the inhibitory effect of BMP-7 on cell proliferation in the SVZ. To determine where BMP-7 binds within the niche, we visualized biotinylated-BMP-7 after intracerebroventricular injection, using streptavidin Texas red on frozen brain sections. BMP-7 binding was seen as puncta in the SVZ at the location of fractones, the particulate specialized extracellular matrix of the SVZ, which have been identified primarily by N-sulfated heparan sulfate immunoreactivity (NS-HS+). BMP binding was also seen in NS-HS+ blood vessels of the SVZ. Injection of heparitinase-1 prior to biotinylated BMP-7 resulted in the absence of signal for biotinylated-BMP-7 in the fractones and blood vessels, indicating that the binding is heparan sulfate dependant. These results indicate that BMP-7 requires heparan sulfates to bind and inhibit cell proliferation in the SVZ neurogenic niche. Heparan sulfates concentrated in fractones and SVZ blood vessels emerge as a functional stem cell niche component involved in growth factor activity.

Enhancement of recombinant human BMP-7 bone formation with bmp binding peptide in a rodent femoral defect model

Bone morphogenetic binding peptide (BBP) is an 18.5 kDa fragment of a bone matrix protein peptide. A rat femoral defect model was used to test the effect of BBP combined with recombinant human bone morphogenetic protein-7 (rhBMP-7) to induced bone healing. Two doses of BBP (500 and 1000 µg) were tested with two doses of rhBMP-7 (2 and 5 µg), and the results were compared with a positive control (10 µg rhBMP-7). Bone healing was evaluated by radiology, manual palpation, microcomputed tomography, and histology. The high dose of 10 µg of rhBMP-7 resulted in a consistent 100% bone union rate and a mature histological appearance on histology, and was used as a positive control. When 1000 µg of BBP was combined with lower doses of BMP-7 (2 µg rhBMP-7 or 5 µg rhBMP-7) significant differences were seen in radiographic scores, manual palpation, and bone volume, when compared to 2 µg rhBMP-7 or 5 µg rhBMP-7 alone. The combination of 1000 µg of BBP and 5 µg rhBMP-7 also achieved 100% fusion rate, induced a larger amount of bone formation, and yielded similar maturity of bone marrow when compared with the high dosage 10 µg rhBMP-7 group. This study demonstrated that when combined together, BBP can enhance the bone healing of rhBMP-7. Improved healing imparted by the addition of BBP may result in lesser amounts of rhBMP-7 needed to achieve union in the clinical setting.

P38 Regulates Expression of Osteoblast-specific Genes by Phosphorylation of Osterix

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

Binding between Crossveinless-2 and Chordin Von Willebrand Factor Type C Domains Promotes BMP Signaling by Blocking Chordin Activity

BackgroundCrossveinless-2 (CV2) is an extracellular BMP modulator protein of the Chordin family, which can either enhance or inhibit BMP activity. CV2 binds to BMP2 via subdomain 1 of the first of its five N-terminal von Willebrand factor type C domains (VWC1). Previous studies showed that this BMP binding is required for the anti-, but not for the pro-BMP effect of CV2. More recently, it was shown that CV2 can also bind to the BMP inhibitor Chordin. However, it remained unclear which domains mediate this binding, and whether it accounts for an anti- or pro-BMP effect.Principal FindingsHere we report that a composite interface of CV2 consisting of subdomain 2 of VWC1 and of VWC2-4, which are dispensable for BMP binding, binds to the VWC2 domain of Chordin. Functional data obtained in zebrafish embryos indicate that this binding of Chordin is required for CV2's pro-BMP effect, which actually is an anti-Chordin effect and, at least to a large extent, independent of Tolloid-mediated Chordin degradation. We further demonstrate that CV2 mutant versions that per se are incapable of BMP binding can attenuate the Chordin/BMP interaction.ConclusionsWe have physically dissected the anti- and pro-BMP effects of CV2. Its anti-BMP effect is obtained by binding to BMP via subdomain1 of the VWC1 domain, a binding that occurs in competition with Chordin. In contrast, its pro-BMP effect is achieved by direct binding to Chordin via subdomain 2 of VWC1 and VWC2-4. This binding seems to induce conformational changes within the Chordin protein that weaken Chordin's affinity to BMP. We propose that in ternary Chordin-CV2-BMP complexes, both BMP and Chordin are directly associated with CV2, whereas Chordin is pushed away from BMP, ensuring that BMPs can be more easily delivered to their receptors.

BMP Binding Peptide Reduces the Inflammatory Response to rhBMP-2 and rhBMP-7 in a Rodent Model of Soft Tissue Inflammation

BMP-2 and BMP-7 are used to enhance bone formation in spine surgery but the use of these materials is associated with side effects including inflammation, especially in the soft tissues of the neck. BBP binds BMP-2 and BMP-7 and imparts a “slow release” property to collagen carrier.

Extracellular regulation of BMP signaling

In the developing organism, cells differentiate, divide and die as part of groups of hundreds or thousands of cells called 'morphogenetic fields'. Fields have the remarkable property of self-regulation: for example, if the forelimb field is bisected, each half can give rise to a complete limb after transplantation, as discovered by Ross Harrison in 1918. Therefore, cells in the morphogenetic field are capable of long-range communication with each other in order to ascertain their position [1]. This positional information is relayed in the extracellular space in the form of concentration gradients of specific classes of extracellular molecules called 'morphogens' that trigger cellular responses by binding and activating cell surface receptors. Here, we focus on a family of morphogens called 'Bone Morphogenetic Proteins' (BMPs), which has provided a new paradigm for signaling regulation in the extracellular space.

Crossveinless 2 is an essential positive feedback regulator of Bmp signaling during zebrafish gastrulation

Signaling by bone morphogenetic proteins (Bmps) plays a pivotal role in developmental and pathological processes, and is regulated by a complex interplay with secreted Bmp binding factors, including Crossveinless 2 (Cvl2). Although structurally related to the Bmp antagonist Chordin, Crossveinless 2 has been described to be both a Bmp agonist and antagonist. Here, we present the first loss-of-function study of a vertebrate cvl2 homologue, showing that zebrafish cvl2 is required in a positive feedback loop to promote Bmp signaling during embryonic dorsoventral patterning. In vivo, Cvl2 protein undergoes proteolytic cleavage and this cleavage converts Cvl2 from an anti- to a pro-Bmp factor. Embryonic epistasis analyses and protein interaction assays indicate that the pro-Bmp function of Cvl2 is partly accomplished by competing with Chordin for binding to Bmps. Studies in cell culture and embryos further suggest that the anti-Bmp effect of uncleaved Cvl2 is due to its association with the extracellular matrix, which is not found for cleaved Cvl2. Our data identify Cvl2 as an essential pro-Bmp factor during zebrafish embryogenesis, emphasizing the functional diversity of Bmp binding CR-domain proteins. Differential proteolytic processing as a mode of regulation might account for anti-Bmp effects in other contexts.

BMP4 supports noradrenergic differentiation by a PKA-dependent mechanism

Differentiation of neural crest-derived noradrenergic neurons depends upon signaling mediated downstream of BMP binding to cognate receptors and involving cAMP. Compiled data from many groups suggest that neurogenesis and cell type-specific noradrenergic marker gene regulation is coordinated through the expression and function of the basic helix–loop–helix DNA binding protein HAND2 and the homeodomain DNA binding protein Phox2a. However, information detailing how BMP-mediated signaling and signaling through cAMP are coordinated has been lacking. We now provide compelling data suggesting that differentiation of noradrenergic sympathetic ganglion neurons depends upon both canonical and non-canonical pathways of BMP-mediated signaling. The non-canonical pathway involves the activation of protein kinase A (PKA) independent of cAMP. This is a novel mechanism in neural crest-derived cells and is necessary to support neurogenesis as well as aspects of DBH promoter regulation involving HAND2 phosphorylation and dimerization. The expression of transcripts encoding HAND2 and Phox2a is regulated via canonical BMP signaling and thus affects both neurogenesis and cell type-specific gene expression. Interestingly, cAMP- and MapK-mediated signaling modulate specific target sites in both the canonical and non-canonical BMP pathways. Activity of MapK is required for HAND2 transcription and thus affects neurogenesis. Signaling affected by cAMP is necessary for the transcription of Phox2a as well as regulation of DBH promoter transactivation by Phox2a and HAND2. We suggest a comprehensive model that shows how BMP- and cAMP-mediated intracellular signaling integrate neurogenesis and cell type-specific noradrenergic marker gene expression and function.

Facilitated Transport of a Dpp/Scw Heterodimer by Sog/Tsg Leads to Robust Patterning of the Drosophila Blastoderm Embryo

Patterning the dorsal surface of the Drosophila blastoderm embryo requires Decapentaplegic (Dpp) and Screw (Scw), two BMP family members. Signaling by these ligands is regulated at the extracellular level by the BMP binding proteins Sog and Tsg. We demonstrate that Tsg and Sog play essential roles in transporting Dpp to the dorsal-most cells. Furthermore, we provide biochemical and genetic evidence that a heterodimer of Dpp and Scw, but not the Dpp homodimer, is the primary transported ligand and that the heterodimer signals synergistically through the two type I BMP receptors Tkv and Sax. We propose that the use of broadly distributed Dpp homodimers and spatially restricted Dpp/Scw heterodimers produces the biphasic signal that is responsible for specifying the two dorsal tissue types. Finally, we demonstrate mathematically that heterodimer levels can be less sensitive to changes in gene dosage than homodimers, thereby providing further selective advantage for using heterodimers as morphogens.

BMP binding peptide: a BMP-2 enhancing factor deduced from the sequence of native bovine bone morphogenetic protein/non-collagenous protein

Forty years ago, Marshall Urist described a partially purified extract of demineralized bone matrix which induced the formation of ectopic bone. This substance, bone morphogenetic protein/non-collagenous protein (BMP/NCP), was never purified to homogeneity but other investigators used similar starting materials to clone a number of recombinant BMPs. Urist recognized that his material probably contained the BMPs which had been cloned by others but always contended that it contained another, more potent, bone inducing material which differed significantly in its physical and chemical properties from the known BMPs. We have used Urist's protocol to isolate a protein that has the chemical and physical properties of Urist's “BMP”. It is an 18.5 kD fragment of the bone matrix protein, SPP-24. This fragment contains the cystatin-like domain of SPP-24. We have located a 19 amino acid region which is similar to the TGF-β/BMP-binding region of fetuin, a member of the cystatin family of protease inhibitors. A cyclic peptide, which we call BMP binding peptide (BBP) was generated using this sequence. The peptide avidly bound rhBMP-2 with a K D of 3 × 10 −5 M. When implanted alone in mouse muscle, the peptide frequently induced dystrophic calcification. When implanted with rhBMP-2, the peptide enhanced the osteogenic activity of the recombinant molecule. We hypothesize that Urist's “BMP” was a fragment of SPP-24 which influenced bone induction by binding to bone morphogenetic proteins. BBP may be clinically useful because of its effects on other bone-inducing substances.

The pro-BMP activity of Twisted gastrulation is independent of BMP binding.

The determination of the vertebrate dorsoventral body axis is regulated in the extracellular space by a system of interacting secreted molecules consisting of BMP, Chordin, Tolloid and Twisted Gastrulation (Tsg). Tsg is a BMP-binding protein that forms ternary complexes with BMP and Chordin. We investigated the function of Tsg in embryonic patterning by generating point mutations in its two conserved cysteine-rich domains. Surprisingly, Tsg proteins with mutations in the N-terminal domain were unable to bind BMP, yet ventralized the embryo very effectively, indicating strong pro-BMP activity. This hyperventralizing Tsg activity required an intact C-terminal domain and could block the anti-BMP activity of isolated BMP-binding modules of Chordin (CRs) in embryonic assays. This activity was specific for CR-containing proteins as it did not affect the dorsalizing effects of Noggin or dominant-negative BMP receptor. The ventralizing effects of the xTsg mutants were stronger than the effect of Chordin loss-of-function in Xenopus or zebrafish. The results suggest that xTsg interacts with additional CR-containing proteins that regulate dorsoventral development in embryos.