Tolloid cleavage activates latent GDF8 by priming the pro‐complex for dissociation

Growth differentiation factor 8 (GDF8)/myostatin is a latent TGF‐β family member that potently inhibits skeletal muscle growth. Here, we compared the conformation and dynamics of precursor, latent, and Tolloid‐cleaved GDF8 pro‐complexes to understand structural mechanisms underlying latency and activation of GDF8. Negative stain electron microscopy (EM) of precursor and latent pro‐complexes reveals a V‐shaped conformation that is unaltered by furin cleavage and sharply contrasts with the ring‐like, cross‐armed conformation of latent TGF‐β1. Surprisingly, Tolloid‐cleaved GDF8 does not immediately dissociate, but in EM exhibits structural heterogeneity consistent with partial dissociation. Hydrogen–deuterium exchange was not affected by furin cleavage. In contrast, Tolloid cleavage, in the absence of prodomain–growth factor dissociation, increased exchange in regions that correspond in pro‐TGF‐β1 to the α1‐helix, latency lasso, and β1‐strand in the prodomain and to the β6′‐ and β7′‐strands in the growth factor. Thus, these regions are important in maintaining GDF8 latency. Our results show that Tolloid cleavage activates latent GDF8 by destabilizing specific prodomain–growth factor interfaces and primes the growth factor for release from the prodomain.     

Prodomains of transforming growth factor beta (TGFbeta) superfamily members specify different functions: extracellular matrix interactions and growth factor bioavailability

The specific functions of the prodomains of TGFβ superfamily members are largely unknown. Interactions are known between prodomains of TGFβ-1-3 and latent TGFβ-binding proteins and between prodomains of BMP-2, -4, -7, and -10 and GDF-5 and fibrillins, raising the possibility that latent TGFβ-binding proteins and fibrillins may mediate interactions with all other prodomains of this superfamily. This possibility is tested in this study. Results show that the prodomain of BMP-5 interacts with the N-terminal regions of fibrillin-1 and -2 in a site similar to the binding sites for other bone morphogenetic proteins. However, in contrast, the prodomain of GDF-8 (myostatin) interacts with the glycosaminoglycan side chains of perlecan. The binding site for the GDF-8 prodomain is likely the heparan sulfate chain present on perlecan domain V. These results support and extend the emerging concept that TGFβ superfamily prodomains target their growth factor dimers to extracellular matrix macromolecules. In addition, biochemical studies of prodomain·growth factor complexes were performed to identify inactive complexes. For some members of the superfamily, the prodomain is noncovalently associated with its growth factor dimer in an inactive complex; for others, the prodomain·growth factor complex is active, even though the prodomain is noncovalently associated with its growth factor dimer. Results show that the BMP-10 prodomain, in contrast to BMP-4, -5, and -7 prodomains, can inhibit the bioactivity of the BMP-10 growth factor and suggest that the BMP-10 complex is like TGFβ and GDF-8 complexes, which can be activated by cleavage of the associated prodomain.     

Targeting of bone morphogenetic protein growth factor complexes to fibrillin

Both latent transforming growth factor-beta (TGF-beta)-binding proteins fibrillins are components of microfibril networks, and both interact with members of the TGF-beta family of growth factors. Interactions between latent TGF-beta-binding protein-1 and TGF-beta and between fibrillin-1 and bone morphogenetic protein-7 (BMP-7) are mediated by the prodomain of growth factor complexes. To extend this information, investigations were performed to test whether stable complexes are formed by additional selected TGF-beta family members. Using velocity sedimentation in sucrose gradients as an assay, complex formation was demonstrated for BMP-7 and growth and differentiation factor-8 (GDF-8), which are known to exist in prodomain/growth factor complexes. Comparison of these results with complex formation by BMP-2, BMP-4 (full-length and shortened propeptides), BMP-10, and GDF-5 allowed us to conclude that all, except for BMP-2 and the short BMP-4 propeptides, formed complexes with their growth factors. Using surface plasmon resonance, binding affinities between fibrillin and all propeptides were determined. Binding studies revealed that the N-terminal end of fibrillin-1 serves as a universal high affinity docking site for the propeptides of BMP-2, -4, -7, and -10 and GDF-5, but not GDF-8, and located the BMP/GDF binding site within the N-terminal domain in fibrillin-1. Rotary shadowing electron microscopy of molecules of BMP-7 complex bound to fibrillin-1 confirmed these findings and also showed that prodomain binding targets the growth factor to fibrillin. Immunolocalization of BMP-4 demonstrated fibrillar staining limited to certain tissues, indicating tissue-specific targeting of BMP-4. These data implicate the fibrillin microfibril network in the extracellular control of BMP signaling and demonstrate differences in how prodomains target their growth factors to the extracellular space.     

Developmental roles of the BMP1/TLD metalloproteinases

The astacin family (M12A) of the metzincin subclan MA(M) of metalloproteinases has been detected in developing and mature individuals of species that range from hydra to humans. Functions of this family of metalloproteinase vary from digestive degradation of polypeptides, to biosynthetic processing of extracellular proteins, to activation of growth factors. This review will focus on a small subgroup of the astacin family; the bone morphogenetic protein 1 (BMP1)/Tolloid (TLD)-like metalloproteinases. In vertebrates, the BMP1/TLD-like metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating mineralization of the ECM of hard tissues. Roles in ECM formation include: processing of the C-propeptides of procollagens types I-III, to yield the major fibrous components of vertebrate ECM; proteolytic activation of the enzyme lysyl oxidase, necessary to formation of covalent cross-links in collagen and elastic fibers; processing of NH2-terminal globular domains and C-propeptides of types V and XI procollagen chains to yield monomers that are incorporated into and control the diameters of collagen type I and II fibrils, respectively; processing of precursors for laminin 5 and collagen type VII, both of which are involved in securing epidermis to underlying dermis; and maturation of small leucine-rich proteoglycans. The BMP1/TLD-related metalloproteinases are also capable of activating the vertebrate transforming growth factor-beta (TGF-beta)-like "chalones" growth differentiation factor 8 (GDF8, also known as myostatin), and GDF11 (also known as BMP11), involved in negative feedback inhibition of muscle and neural tissue growth, respectively; by freeing them from noncovalent latent complexes with their cleaved prodomains. BMP1/TLD-like proteinases also liberate the vertebrate TGF-beta-like morphogens BMP2 and 4 and their invertebrate ortholog decapentaplegic, from latent complexes with the vertebrate extracellular antagonist chordin and its invertebrate ortholog short gastrulation (SOG), respectively. The result is formation of the BMP signaling gradients that form the dorsal-ventral axis in embryogenesis. Thus, BMP1/TLD-like proteinases appear to be key to regulating and orchestrating formation of the ECM and signaling by various TGF-beta-like proteins in morphogenetic and homeostatic events.     

The role and regulation of GDF11 in Smad2 activation during tailbud formation in the Xenopus embryo

A key role for phosphorylation of Smad2 by TGFβ superfamily ligands in the axial patterning of early embryos is well established. The regulation and role of Smad2 signaling in post-neurula embryonic patterning, however, is less well understood. While a variety of TGFβ superfamily ligands are implicated in various stages of anterior–posterior patterning, the ligand GDF11 has been shown to have a particular role in post-gastrula patterning in the mouse. Mouse GDF11 is specifically localized to the developing tail and is essential for normal posterior axial patterning. Mature GDF11 ligand is inhibited by its own prodomain, and extracellular proteolysis of this prodomain is thought to be necessary for GDF11 activity. The contribution of this proteolytic regulatory mechanism to Smad activation during embryogenesis in vivo, and to the development of posterior pattern, has not been characterized. We investigate here the role of Xenopus GDF11 in the activation of Smad2 during the development of tailbud-stage embryos, and the role of this activation in larval development. We also demonstrate that the activity of BMP-1/Tolloid-like proteases is necessary for the normal GDF11-dependent activation of Smad2 phosphorylation during post-gastrula development. These data demonstrate that GDF11 has a central role in the activation of Smad2 phosphorylation in tailbud stage Xenopus embryos, and provide the first evidence that BMP-1/Tolloid-mediated prodomain cleavage is important for activation of GDF11 in vivo.     

The prodomain of BMP-7 targets the BMP-7 complex to the extracellular matrix

Biochemical and biophysical methods are used to show that BMP-7 is secreted as a stable complex consisting of the processed growth factor dimer noncovalently associated with its two prodomain propeptide chains and that the BMP-7 complex is structurally similar to the small transforming growth factor beta (TGFbeta) complex. Because the prodomain of TGFbeta interacts with latent TGFbeta-binding proteins, a family of molecules homologous to the fibrillins, the prodomain of BMP-7 was tested for binding to fibrillin-1 or to LTBP-1. The BMP-7 prodomain and BMP-7 complex, but not the separated growth factor dimer, interact with N-terminal regions of fibrillin-1. This interaction may target the BMP-7 complex to fibrillin microfibrils in the extracellular matrix. Immunolocalization of BMP-7 in tissues like the kidney capsule and skin reveals co-localization with fibrillin. However, BMP-7 immunolocalization in other tissues known to be active sites for BMP-7 signaling is not apparent, suggesting that immunolocalization of BMP-7 in certain tissues represents specific extracellular storage sites. These studies suggest that the prodomains of TGFbeta-like growth factors are important for positioning and concentrating growth factors in the extracellular matrix. In addition, they raise the possibility that prodomains of other TGFbeta-like growth factors interact with fibrillins and/or LTBPs and are also targeted to the extracellular matrix.     

Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin

Growth differentiation factor 9 (GDF9) is an oocyte-expressed member of the transforming growth factor beta (TGF-beta) superfamily and is required for normal ovarian follicle development and female fertility. GDF9 acts as a paracrine factor and affects granulosa cell physiology. Only a few genes regulated by GDF9 are known. Our microarray analysis has identified gremlin as one of the genes up-regulated by GDF9 in cultures of granulosa cells. Gremlin is a known member of the DAN family of bone morphogenetic protein (BMP) antagonists, but its expression and function in the ovary are unknown. We have investigated the regulation of gremlin in mouse granulosa cells by GDF9 as well as other members of the TGF-beta superfamily. GDF9 and BMP4 induce gremlin, but TGF-beta does not. In addition, in cultures of granulosa cells, gremlin negatively regulates BMP4 signaling but not GDF9 activity. The expression of gremlin in the ovary was also examined by in situ hybridization. A distinct change in gremlin mRNA compartmentalization occurs during follicle development and ovulation, indicating a highly regulated expression pattern during folliculogenesis. We propose that gremlin modulates the cross-talk between GDF9 and BMP signaling that is necessary during follicle development because both ligands use components of the same signaling pathway.     

BMP1 controls TGFbeta1 activation via cleavage of latent TGFbeta-binding protein

Transforming growth factor beta1 (TGFbeta1), an important regulator of cell behavior, is secreted as a large latent complex (LLC) in which it is bound to its cleaved prodomain (latency-associated peptide [LAP]) and, via LAP, to latent TGFbeta-binding proteins (LTBPs). The latter target LLCs to the extracellular matrix (ECM). Bone morphogenetic protein 1 (BMP1)-like metalloproteinases play key roles in ECM formation, by converting precursors into mature functional proteins, and in morphogenetic patterning, by cleaving the antagonist Chordin to activate BMP2/4. We provide in vitro and in vivo evidence that BMP1 cleaves LTBP1 at two specific sites, thus liberating LLC from ECM and resulting in consequent activation of TGFbeta1 via cleavage of LAP by non-BMP1-like proteinases. In mouse embryo fibroblasts, LAP cleavage is shown to be predominantly matrix metalloproteinase 2 dependent. TGFbeta1 is a potent inducer of ECM formation and of BMP1 expression. Thus, a role for BMP1-like proteinases in TGFbeta1 activation completes a novel fast-forward loop in vertebrate tissue remodeling.     

Cumulin,an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family,Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation, and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modeling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC, activating both SMAD2/3 and SMAD1/5/8 signaling pathways and promoting proliferation and expression of a set of genes associated with oocyte-regulated GC differentiation. Cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at notably improving oocyte quality as assessed by subsequent day 7 embryo development. Our results support a model of activation for human GDF9 dependent on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals.     

BMP-1及BMP家族在背-腹轴图式形成中的作用

骨形态发生蛋白（bone morphogenetic proteins, BMPs）是一类在发育过程中起重要作用的分子。除BMP-1外,其他BMP分子均属于转化生长因子-β（transforming growth factor-β, TGF-β）/BMP超家族的发育信号分子。在胚胎发育过程中,这些信号分子通过形成浓度梯度对背—腹轴各向异性分化进行调控。它们借助细胞表面受体的识别进行信号传导,参与调控细胞分化、增殖等活动。而BMP-1则属于细胞外基质金属蛋白酶超家族中的Tolloid蛋白酶家族。BMP-1通过水解其他BMP的抑制物（如脊索发生素,Chordin）,达到促进其他BMP信号传导的目的。BMP-1、BMP和Chordin三者通过相互制约与相互促进等一系列作用,在背—腹沿线建立起稳定的BMP信号梯度。本文就BMP浓度梯度的形成及其稳态维持的机制进行回顾与总结。并在此基础上,对各个物种间BMP浓度梯度形成机制的异同,以及可能存在的协同进化进行比较、分析和讨论。     

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.     

Differential gene expression of bone morphogenetic protein 15 and growth differentiation factor 9 during in vitro maturation of porcine oocytes and early embryos

Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) belong to the TGF-beta superfamily and are involved in the regulation of folliculogenesis. Though there are many reports concerning the expression and regulation of GDF9 in the process of oocyte maturation, expression of BMP15 during oocyte maturation is still not clearly understood. It has been reported that BMP15 and GDF9 expression is important in folliculogeneiss and that the regulation of these two proteins is complex and species-specific. In this report, we investigated the expression of BMP15 and GDF9 genes during in vitro maturation (IVM) at 0, 6, 12, 18, 24, 30, 36, 42 and 48 h for porcine oocytes. Porcine GDF9 gene was found to be highly expressed in immature oocytes and declined slowly during the oocyte maturation process. BMP15mRNA and its encoded protein were expressed at low levels in immature oocytes and increased to the highest level at 18 h of IVM, which coincides with the time of cumulus cell expansion. Thus, these two genes were differentially expressed during the oocyte maturation process and BMP15 is specifically expressed during cumulus cell expansion in porcine oocytes.     

Crystal structure analysis reveals a spring‐loaded latch as molecular mechanism for GDF‐5–type I receptor specificity

Dysregulation of growth and differentiation factor 5 (GDF‐5) signalling, a member of the TGF‐β superfamily, is strongly linked to skeletal malformation. GDF‐5‐mediated signal transduction involves both BMP type I receptors, BMPR‐IA and BMPR‐IB. However, mutations in either GDF‐5 or BMPR‐IB lead to similar phenotypes, indicating that in chondrogenesis GDF‐5 signalling seems to be exclusively mediated through BMPR‐IB. Here, we present structural insights into the GDF‐5:BMPR‐IB complex revealing how binding specificity for BMPR‐IB is generated on a molecular level. In BMPR‐IB, a loop within the ligand‐binding epitope functions similar to a latch allowing high‐affinity binding of GDF‐5. In BMPR‐IA, this latch is in a closed conformation leading to steric repulsion. The new structural data now provide also a molecular basis of how phenotypically relevant missense mutations in GDF‐5 might impair receptor binding and activation.     

WFIKKN1 and WFIKKN2 bind growth factors TGFβ1, BMP2 and BMP4 but do not inhibit their signalling activity

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP domain, a follistatin domain, an immunoglobulin domain, two Kunitz-type protease inhibitor domains and an NTR domain. Recent experiments have shown that both proteins have high affinity for growth and differentiation factor (GDF)8 and GDF11. Here we study the interaction of WFIKKN proteins with several additional representatives of the transforming growth factor (TGF)β family using SPR measurements. Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN proteins bind TGFβ1, bone morphogenetic protein (BMP)2 and BMP4 with relatively high affinity (K(d) ～ 10(-6) m). To assess the biological significance of these interactions we studied the effect of WFIKKN proteins on the activity of GDF8, GDF11, TGFβ1, BMP2 and BMP4 using reporter assays. These studies revealed that WFIKKN1 and WFIKKN2 inhibited the biological activity of GDF8 and GDF11 in the nanomolar range, whereas they did not inhibit the activities of TGFβ1, BMP2 and BMP4 even in the micromolar range. Our data indicate that WFIKKN proteins are antagonists of GDF8 and GDF11, but in the case of TGFβ1, BMP2 and BMP4 they function as growth factor binding proteins. It is suggested that the physical association of WFIKKN proteins with these growth factors may localize their action and thus help to establish growth factor gradients in the extracellular space.     

Identification of receptors and signaling pathways for orphan bone morphogenetic protein/growth differentiation factor ligands based on genomic analyses

There are more than 30 human transforming growth factor beta/bone morphogenetic protein/growth differentiation factor (TGFbeta/BMP/GDF)-related ligands known to be important during embryonic development, organogenesis, bone formation, reproduction, and other physiological processes. Although select TGFbeta/BMP/GDF proteins were found to interact with type II and type I serine/threonine receptors to activate downstream Smad and other proteins, the receptors and signaling pathways for one-third of these TGFbeta/BMP/GDF paralogs are still unclear. Based on a genomic analysis of the entire repertoire of TGFbeta/BMP/GDF ligands and serine/threonine kinase receptors, we tested the ability of three orphan BMP/GDF ligands to activate a limited number of phylogenetically related receptors. We characterized the dimeric nature of recombinant GDF6 (also known as BMP13), GDF7 (also known as BMP12), and BMP10. We demonstrated their bioactivities based on the activation of Smad1/5/8-, but not Smad2/3-, responsive promoter constructs in the MC3T3 cell line. Furthermore, we showed their ability to induce the phosphorylation of Smad1, but not Smad2, in these cells. In COS7 cells transfected with the seven known type I receptors, overexpression of ALK3 or ALK6 conferred ligand signaling by GDF6, GDF7, and BMP10. In contrast, transfection of MC3T3 cells with ALK3 small hairpin RNA suppressed Smad signaling induced by all three ligands. Based on the coevolution of ligands and receptors, we also tested the role of BMPRII and ActRIIA as the type II receptor candidates for the three orphan ligands. We found that transfection of small hairpin RNA for BMPRII and ActRIIA in MC3T3 cells suppressed the signaling of GDF6, GDF7, and BMP10. Thus, the present approach provides a genomic paradigm for matching paralogous polypeptide ligands with a limited number of evolutionarily related receptors capable of activating specific downstream Smad proteins.     

Expression, purification, renaturation and activation of fish myostatin expressed in Escherichia coli: facilitation of refolding and activity inhibition by myostatin prodomain

Myostatin (growth and differentiation factor-8) is a member of the transforming growth factor-beta superfamily, is expressed mainly in skeletal muscle and acts as a negative growth regulator. Mature myostatin (C-terminal) is a homodimer that is cleaved post-translationally from the precursor myostatin, also yielding the N-terminal prodomain. We expressed in Escherichia coli three forms of fish myostatin: precursor, prodomain and mature. The three forms were over-expressed as inclusion bodies. Highly purified inclusion bodies were solubilized in a solution containing guanidine hydrochloride and the reducing agent DTT. Refolding (indicated by a dimer formation) of precursor myostatin, mature myostatin or a mixture of prodomain and mature myostatin was compared under identical refolding conditions, performed in a solution containing sodium chloride, arginine, a low concentration of guanidine hydrochloride and reduced and oxidized glutathione at 4 degrees C for 14 days. While precursor myostatin formed a reversible disulfide bond with no apparent precipitation, mature myostatin precipitated in the same refolding solution, unless CHAPS was included, and only a small proportion formed a disulfide bond. The trans presence of the prodomain in the refolding solution prevented precipitation of mature myostatin but did not promote formation of a dimer. Proteolytic cleavage of purified, refolded precursor myostatin with furin yielded a monomeric prodomain and a disulfide-linked, homodimeric mature myostatin, which remained as a latent complex. Activation of the latent complex was achieved by acidic or thermal treatments. These results demonstrate that the cis presence of the prodomain is essential for the proper refolding of fish myostatin and that the cleaved mature dimer exists as a latent form.