Regulation Of Bone Morphogenetic Protein Signaling Research Articles

Chordin-like 1 and Twisted Gastrulation 1 Regulate BMP Signaling following Kidney Injury

Stimulation of the bone morphogenetic protein (BMP) pathway protects the kidney from acute and chronic injury. Numerous regulators in the kidney control BMP signaling, offering many targets for therapeutic manipulation. Here, we screened for modulators of BMP signaling in the ischemia-sensitive S3 segment and found that Chordin-like 1 is expressed in this segment of both the mouse and human nephron. Chordin-like 1 specifically antagonizes BMP7, which is expressed in the neighboring distal nephron, and this depends on the presence of the protein Twisted gastrulation. Upon ischemia-induced degeneration of the S3 segment, we observed a reduction in Chordin-like 1 expression coincident with intense BMP signaling in tubules of the recovering kidney. Restored expression accompanied proximal tubule epithelia redifferentiation, again coincident with decreased BMP signaling. We propose that Chordin-like 1 reduces BMP7 signaling in healthy proximal tubules, and the loss of this activity upon sloughing of injured epithelia promotes BMP7 signaling in repopulating, dedifferentiated epithelia. As regenerating epithelia differentiate, Chordin-like 1 is again expressed, antagonizing BMP7. These data suggest a mechanism for dynamic regulation of renoprotective BMP7 signaling in the S3 segment of the proximal tubule.

NDST1-dependent heparan sulfate regulates BMP signaling and internalization in lung development

Heparan sulfate proteoglycans (HSPGs) are required for various signaling pathways, one of which is the bone morphogenetic protein (BMP) signaling pathway. N-deacetylase/N-sulfotransferase-1 (NDST1) participates in synthesizing heparan sulfate (HS) chains of HSPGs, and is involved in bone and lung development. Here, we report that in spite of the redundant expression of Ndst2, Ndst3 and Ndst4 genes, Ndst1(-/-) mice display defective differentiation of lung cells and increased cell proliferation. Loss of Ndst1 in the lung enhances downstream BMP signaling in vivo. Noggin, which is an antagonist of BMP, can rescue the Ndst1(-/-) lung morphogenetic defects in explant cultures. Further studies in vitro indicated that loss of Ndst1 significantly impairs BMP internalization by decreasing BMP binding to endogenous HS. Exogenous heparin can rescue both the BMP signaling and BMP internalization abnormalities in Ndst1(-/-) lung. Thus, we propose that HS regulates BMP signaling by controlling the balance between BMP binding to HS, and that BMP receptors and NDST1-dependent modification are essential for this process. The results suggest that NDST1-dependent HS is essential for proper functioning of BMP in embryonic lung development.

Kekkon5 is an extracellular regulator of BMP signaling

Precise spatial and temporal control of Drosophila Bone Morphogenetic Protein (BMP) signaling is achieved by a host of extracellular factors that modulate ligand distribution and activity. Here we describe Kekkon5 (Kek5), a transmembrane protein containing leucine-rich repeats (LRRs), as a novel regulator of BMP signaling in Drosophila. We find that loss or gain of kek5 disrupts crossvein development and alters the early profile of phosphorylated Mad and dSRF in presumptive crossvein cells. kek5 phenotypic effects closely mimic those observed with Short gastrulation (Sog), but do not completely recapitulate the effects of dominant negative BMP receptors. We further demonstrate that Kek5 is able to antagonize the BMP ligand Glass bottom boat (Gbb) and that the Kek5 LRRs are required for BMP inhibitory activity, while the Ig domain is dispensable in this context. Our identification of Kek5 as a modulator of BMP signaling supports the emerging notion that LIG proteins function as diverse regulators of cellular communication.

Gremlin-mediated Decrease in Bone Morphogenetic Protein Signaling Promotes Pulmonary Fibrosis

Members of the transforming growth factor (TGF)-beta superfamily, including TGF-betas and bone morphogenetic proteins (BMPs), are essential for the maintenance of tissue homeostasis and regeneration after injury. We have observed that the BMP antagonist, gremlin, is highly up-regulated in idiopathic pulmonary fibrosis (IPF). To investigate the role of gremlin in the regulation of BMP signaling in pulmonary fibrosis. Progressive asbestos-induced fibrosis in the mouse was used as a model of human IPF. TGF-beta and BMP expression and signaling activities were measured from murine and human fibrotic lungs. The mechanism of gremlin induction was analyzed in cultured lung epithelial cells. In addition, the possible therapeutic role of gremlin inhibition was tested by administration of BMP-7 to mice after asbestos exposure. Gremlin mRNA levels were up-regulated in the asbestos-exposed mouse lungs, which is in agreement with the human IPF biopsy data. Down-regulation of BMP signaling was demonstrated by reduced levels of Smad1/5/8 and enhanced Smad2 phosphorylation in asbestos-treated lungs. Accordingly, analyses of cultured human bronchial epithelial cells indicated that asbestos-induced gremlin expression could be prevented by inhibitors of the TGF-beta receptor and also by inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase pathways. BMP-7 treatment significantly reduced hydroxyproline contents in the asbestos-treated mice. The TGF-beta and BMP signaling balance is important for lung regenerative events and is significantly perturbed in pulmonary fibrosis. Rescue of BMP signaling activity may represent a potential beneficial strategy for treating human pulmonary fibrosis.

Von Willebrand Factor Type C Domain-containing Proteins Regulate Bone Morphogenetic Protein Signaling through Different Recognition Mechanisms

Bone morphogenetic protein (BMP) function is regulated in the extracellular space by many modulator proteins, including those containing a von Willebrand factor type C (VWC) domain. The function of the VWC domain-containing proteins in development and diseases has been extensively studied. The structural basis, however, for the mechanism by which BMP is regulated by these proteins is still poorly understood. By analyzing chordin, CHL2 (chordin-like 2), and CV2 (crossveinless 2) as well as their individual VWC domains, we show that the VWC domain is a versatile binding module that in its multiple forms and environments can expose a variety of binding specificities. Three of four, two of three, and one of five VWCs from chordin, CHL2, and CV2, respectively, can bind BMPs. Using an array of BMP-2 mutant proteins, it can be demonstrated that the binding-competent VWC domains all use a specific subset of BMP-2 binding determinants that overlap with the binding site for the type II receptors (knuckle epitope) or for the type I receptors (wrist epitope). This explains the competition between modulator proteins and receptors for BMP binding and therefore the inhibition of BMP signaling. A subset of VWC domains from CHL2 binds to the Tsg (twisted gastrulation) protein similar to chordin. A stable ternary complex consisting of BMP-2, CHL2, and Tsg can be formed, thus making CHL2 a more efficient BMP-2 inhibitor. The VWCs of CV2, however, do not interact with Tsg. The present results show that chordin, CHL2, and CV2 regulate BMP-2 signaling by different recognition mechanisms.

Selective Inhibitory Effects of Smad6 on Bone Morphogenetic Protein Type I Receptors

The inhibitory Smads, Smad6 and Smad7, play pivotal roles in negative regulation of transforming growth factor-beta (TGF-beta) family signaling as feedback molecules as well as mediators of cross-talk with other signaling pathways. Whereas Smad7 acts as a ubiquitous inhibitor of Smad signaling, Smad6 has been shown to effectively inhibit bone morphogenetic protein (BMP) signaling but only weakly TGF-beta/activin signaling. In the present study, we have found that Smad6 inhibits signaling from the activin receptor-like kinase (ALK)-3/6 subgroup in preference to that from the ALK-1/2 subgroup of BMP type I receptors. The difference is attributable to the interaction of Smad6 with these BMP type I receptors. The amino acid residues responsible for Smad6 sensitivity of ALK-3 were identified as Arg-238, Phe-264, Thr-265, and Ala-269, which map to the N-terminal lobe of the ALK-3 kinase domain. Although Smad6 regulates BMP signaling through multiple mechanisms, our findings suggest that interaction with type I receptors is a critical step in the function of Smad6.

Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP signaling

Basic helix-loop-helix (bHLH) transcription factors are known as key regulators for mesenchymal differentiation. The present study showed that overexpression of Twist-1, a bHLH transcription factor, suppresses bone morphogenetic protein (BMP)-induced osteoblast differentiation, and downregulation of endogenous Twist-1 enhances BMP signaling. Maximal inhibition of BMP signaling was observed when Twist-1 was bound to E47, which markedly enhanced the stability of Twist-1. Co-immunoprecipitation assays revealed that Twist-1 formed a complex with Smad4 and histone deacetylase (HDAC) 1 in MC3T3-E1 cells stably expressing Twist-1. With trichostatin, an HDAC inhibitor, osteogenic factors such as alkaline phosphatase, Runx2 and osteopontin increased. Those results suggested that Twist-1 inhibited BMP signaling by recruiting HDAC1 to Smad4. Furthermore, the inhibitory effects of Twist-1 on BMP signaling were overcome by Id1 through induction of Twist-1 degradation. These findings suggest that Twist-1 can act as an inhibitor of BMP signaling, and Id1 can regulate BMP signaling through a positive feedback loop repressing Twist-1 function. These two molecules may therefore regulate differentiation of mesenchymal cells into progeny such as osteoblasts by controlling BMP signaling.

Glypican LON-2 Is a Conserved Negative Regulator of BMP-like Signaling in Caenorhabditis elegans

Bone morphogenetic protein (BMP) pathways are required for a wide variety of developmental and homeostatic decisions, and mutations in signaling components are associated with several diseases. An important aspect of BMP control is the extracellular regulation of these pathways. We show that LON-2 negatively regulates a BMP-like signaling pathway that controls body length in C. elegans. lon-2 acts genetically upstream of the BMP-like gene dbl-1, and loss of lon-2 function results in animals that are longer than normal. LON-2 is a conserved member of the glypican family of heparan sulfate proteoglycans, a family with several members known to regulate growth-factor signaling in many organisms. LON-2 is functionally conserved because the Drosophila glypican gene dally rescues the lon-2(lf) body-size defect. We show that the LON-2 protein binds BMP2 in vitro, and a mutant variation of LON-2 found in lon-2(e2140) animals diminishes this interaction. We propose that LON-2 binding to DBL-1 negatively regulates this pathway in C. elegans by attenuating ligand-receptor interactions. This is the first report of a glypican directly interacting with a growth-factor pathway in C. elegans and provides a mechanistic model for glypican regulation of growth-factor pathways.

Hypoxia Regulates Bone Morphogenetic Protein Signaling Through C-Terminal–Binding Protein 1

Bone morphogenetic protein receptor 2 (BMPR2) mutations have been linked to familial pulmonary arterial hypertension (PAH), but the molecular pathways leading to this severe pathology remain poorly characterized. We report that hypoxia, a paramount stimulus for the development of pulmonary hypertension, suppresses the expression of inhibitor of differentiation 1 (Id1), a downstream target of the BMPR2 pathway, in human pulmonary artery smooth muscle cells (HPASMC). This attenuation of BMP signaling by hypoxia is conveyed through a repression of the transcriptional activity of the BMP responsive element (BRE) through mechanisms involving the transcriptional corepressor C-terminal-binding protein 1 (CtBP-1) and histone deacetylases (HDACs). Concordantly, overexpression of CtBP-1 suppressed BMP signaling, whereas small interfering RNA against CtBP-1 efficiently enhanced BMP stimulation of Id1 gene expression. Scavengers of reactive oxygen species had no effect on the hypoxic regulation of Id1, but, significantly, enhancement of the intracellular NADH/NAD(+) ratio mimicked the effects of hypoxia. These results indicate that attenuation of BMP signaling can occur through modulation of CtBP-1 activity by hypoxia-induced changes in the NADH/NAD(+) ratio. Our findings, taken in context with the observed prevalence of pulmonary arterial hypertension associated with BMPR2 mutations, define converging molecular pathways that lead to the development of pulmonary hypertension, through either genetic or epigenetic loss of function of components of the BMP signaling pathway.

Regulation of BMP signaling

Bone morphogenetic proteins (BMPs), members of transforming growth factor-beta (TGF-beta) superfamily, bind to two different serine/threonine kinase receptors, and mediate their signals through Smad-dependent and -independent pathways. Intracellular signaling of BMPs is regulated by various modifiers and post-transcriptional modifications including phosphorylation and ubiquitination.

Temporal and spatial action of Tolloid (Mini fin) and Chordin to pattern tail tissues

In vertebrates, a bone morphogenetic protein (BMP) signaling pathway patterns all ventral cell fates along the embryonic axis. BMP activity is positively regulated by Tolloid, a metalloprotease, that can eliminate the activity of the BMP antagonist Chordin. A tolloid mutant in zebrafish, mini fin (mfn), exhibits a specific loss of ventral tail tissues. Here, we investigate the spatial and temporal requirements for Tolloid (Mfn) in dorsoventral patterning of the tail. Through chimeric analyses, we found that Tolloid (Mfn) functions cell non-autonomously in the ventral-most vegetal cells of the gastrula or their derivatives. We generated a tolloid transgene under the control of the inducible hsp70 promoter and demonstrate that tolloid (mfn) is first required at the completion of gastrulation. Although tolloid is expressed during gastrulation and dorsally and ventrally within the tail bud, our results indicate that Tolloid (Mfn) acts specifically in the ventral tail bud during a ∼4 h period extending from the completion of gastrulation to early somitogenesis stages to regulate BMP signaling. Examination of the temporal requirements of Chordin activity by overexpression of the hsp70-tolloid transgene indicates that Chordin is required both during and after gastrulation for proper patterning of the tail, contrasting Tld's requirement only during post-gastrula stages. We hypothesize that the gastrula role of Chordin in tail patterning is to generate the proper size domains of cells to enter the ventral and dorsal tail bud, whereas post-gastrula Chordin activity patterns the derivatives of the tail bud. Thus, fine modulation of BMP signaling levels through the negative and positive actions of Chordin and Tolloid, respectively, patterns tail tissues.

OAZ Regulates Bone Morphogenetic Protein Signaling through Smad6 Activation

The intensity and duration of activation of a signal transduction system are important determinants of the specificity of the cellular response to the stimulus. It is unclear how different cells can generate a signal of varying intensity and duration in response to the same cytokine. We investigated the role of the transcriptional activator and Smad1/4 cofactor OAZ in regulating bone morphogenetic protein (BMP) signaling. We demonstrate that upon BMP4 stimulation, an OAZ-Smad1/4 complex binds to and activates the gene encoding Smad6, a specific inhibitor of the BMP pathway. Removal of endogenous OAZ from pluripotent embryonal carcinoma cells prevents the induction of Smad6 by BMP4 and extends the period of detection of phosphorylated Smad1 after BMP stimulation. Conversely, in cells that do not normally express OAZ, such as myoblasts and smooth muscle cells, forced OAZ expression leads to faster and higher Smad6 induction in response to BMP4, decrease of Smad1 phosphorylation, and attenuation of BMP-mediated responses. Our results demonstrate that OAZ can alter the intensity and duration of the BMP stimulus through Smad6 and indicate that the tissue-specific expression of OAZ is a critical determinant of the cellular response to the BMP signal.

Bone Morphogenetic Protein 4 Promotes Pulmonary Vascular Remodeling in Hypoxic Pulmonary Hypertension

We show that 1 of the type II bone morphogenetic protein (BMP) receptor ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated in hypoxia-induced pulmonary hypertension (PH). Furthermore, heterozygous null Bmp4(lacZ/+) mice are protected from the development of hypoxia-induced PH, vascular smooth muscle cell proliferation, and vascular remodeling. This is associated with a reduction in hypoxia-induced Smad1/5/8 phosphorylation and Id1 expression in the pulmonary vasculature. In addition, pulmonary microvascular endothelial cells secrete BMP4 in response to hypoxia and promote proliferation and migration of vascular smooth muscle cells in a BMP4-dependent fashion. These findings indicate that BMP4 plays a dominant role in regulating BMP signaling in the hypoxic pulmonary vasculature and suggest that endothelium-derived BMP4 plays a direct, paracrine role in promoting smooth muscle proliferation and remodeling in hypoxic PH.

Dorsal-ventral patterning and neural induction in Xenopus embryos.

We review the current status of research in dorsal-ventral (D-V) patterning in vertebrates. Emphasis is placed on recent work on Xenopus, which provides a paradigm for vertebrate development based on a rich heritage of experimental embryology. D-V patterning starts much earlier than previously thought, under the influence of a dorsal nuclear -Catenin signal. At mid-blastula two signaling centers are present on the dorsal side: The prospective neuroectoderm expresses bone morphogenetic protein (BMP) antagonists, and the future dorsal endoderm secretes Nodal-related mesoderm-inducing factors. When dorsal mesoderm is formed at gastrula, a cocktail of growth factor antagonists is secreted by the Spemann organizer and further patterns the embryo. A ventral gastrula signaling center opposes the actions of the dorsal organizer, and another set of secreted antagonists is produced ventrally under the control of BMP4. The early dorsal -Catenin signal inhibits BMP expression at the transcriptional level and promotes expression of secreted BMP antagonists in the prospective central nervous system (CNS). In the absence of mesoderm, expression of Chordin and Noggin in ectoderm is required for anterior CNS formation. FGF (fibroblast growth factor) and IGF (insulin-like growth factor) signals are also potent neural inducers. Neural induction by anti-BMPs such as Chordin requires mitogen-activated protein kinase (MAPK) activation mediated by FGF and IGF. These multiple signals can be integrated at the level of Smad1. Phosphorylation by BMP receptor stimulates Smad1 transcriptional activity, whereas phosphorylation by MAPK has the opposite effect. Neural tissue is formed only at very low levels of activity of BMP-transducing Smads, which require the combination of both low BMP levels and high MAPK signals. Many of the molecular players that regulate D-V patterning via regulation of BMP signaling have been conserved between Drosophila and the vertebrates.

Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling.

Heparin-binding epidermal growth factor (HB-EGF) and betacellulin (BTC) are activating ligands for EGF receptor (EGFR/ErbB1) and ErbB4. To identify their physiological functions, we disrupted mouse HB-EGF and BTC alleles by homologous recombination. Most HB-EGF(-/-) mice died before weaning, and survivors had enlarged, dysfunctional hearts and reduced lifespans. Although BTC(-/-) mice were viable and fertile and displayed no overt defects, the lifespan of double null HB-EGF(-/-)/BTC(-/-) mice was further reduced, apparently due to accelerated heart failure. HB-EGF(-/-) newborns had enlarged and malformed semilunar and atrioventricular heart valves, and hypoplastic, poorly differentiated lungs. Defective cardiac valvulogenesis was the result of abnormal mesenchymal cell proliferation during remodeling, and was associated with dramatic increases in activated Smad1/5/8. Consistent with the phenotype, HB-EGF transcripts were localized to endocardial cells lining the margins of wild-type valves. Similarly defective valvulogenesis was observed in newborn mice lacking EGFR and tumor necrosis factor-alpha converting enzyme (TACE). These results suggest that cardiac valvulogenesis is dependent on EGFR activation by TACE-derived soluble HB-EGF, and that EGFR signaling is required to regulate bone morphogenetic protein signaling in this context.

BMP Receptors Keep SANE During Development

Raju et al. used a yeast two-hybrid screen for proteins binding to Xenopus Smad1 and identified SANE (Smad1 antagonistic effector), a novel protein with sequence similarity to nuclear envelope proteins. SANE bound to type I bone morphogenetic protein (BMP) receptors, as well as to Smad1, and inhibited BMP signaling. BMPs, members of the transforming growth factor-β (TGF-β) family, help regulate bone formation, neural cell fate determination, and embryonic patterning during development. BMPs bind to a receptor complex containing type I and type II receptors, which leads to phosphorylation of Smads1 and 5 and Smad translocation to the nucleus. The authors used Western analysis of Smad1 immunoprecipitates to confirm that SANE interacted with Smad1 in Xenopus embryos and to show that Smad1 did not bind to a SANE mutant lacking the COOH-terminus. SANE also bound to Smad5. SANE mRNA injected into Xenopus embryos specifically blocked BMP signaling, whereas the SANE mutant that did not bind Smad1 did not. SANE also blocked BMP-stimulated osteoblastic differentiation in cultured mouse C2C12 cells. SANE reduced Smad1 phosphorylation in embryos derived from fertilized eggs injected with BMP-4 mRNA and inhibited BMP-4-dependent nuclear translocation of Smad1 in NIH 3T3 cells. Western analysis of SANE immunoprecipitates indicated that SANE also bound to BMP type I receptors; unlike SANE binding to Smad1, this interaction did not require the SANE COOH-terminus. These data suggest that SANE is an inhibitor of BMP signaling with a novel mechanism that involves interaction with both the BMP receptor and pathway-specific Smads. G. P. Raju, N. Dimova, P. S. Klein, H.-C. Huang, SANE, a novel LEM domain protein, regulates bone morphogenetic protein signaling through interaction with Smad1. J. Biol. Chem. 278 , 428-437 (2003). [Abstract] [Full Text]

SANE, a Novel LEM Domain Protein, Regulates Bone Morphogenetic Protein Signaling through Interaction with Smad1

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that play important roles in bone formation, embryonic patterning, and epidermal-neural cell fate decisions. BMPs signal through pathway specific mediators such as Smads1 and 5, but the upstream regulation of BMP-specific Smads has not been fully characterized. Here we report the identification of SANE (Smad1 Antagonistic Effector), a novel protein with significant sequence similarity to nuclear envelop proteins such as MAN1. SANE binds to Smad1/5 and to BMP type I receptors and regulates BMP signaling. SANE specifically blocks BMP-dependent signaling in Xenopus embryos and in a mammalian model of bone formation but does not inhibit the TGF-beta/Smad2 pathway. Inhibition of BMP signaling by SANE requires interaction between SANE and Smad1, because a SANE mutant that does not bind Smad1 does not inhibit BMP signaling. Furthermore, inhibition appears to be mediated by inhibition of BMP-induced Smad1 phosphorylation, blocking ligand-dependent nuclear translocation of Smad1. These studies define a new mode of regulation for intracellular BMP/Smad1 signaling.

Gene profiling during neural induction in Xenopus laevis: regulation of BMP signaling by post-transcriptional mechanisms and TAB3, a novel TAK1-binding protein.

The earliest decision in vertebrate neural development is the acquisition of a neural identity by embryonic ectodermal cells. The default model for neural induction postulates that neural fate specification in the vertebrate embryo occurs by inhibition of epidermal inducing signals in the gastrula ectoderm. Bone morphogenetic proteins (BMPs) act as epidermal inducers, and all identified direct neural inducers block BMP signaling either intra- or extracellularly. Although the mechanism of action of the secreted neural inducers has been elucidated, the relevance of intracellular BMP inhibitors in neural induction is not clear. In order to address this issue and to identify downstream targets after BMP inhibition, we have monitored the transcriptional changes in ectodermal explants neuralized by Smad7 using a Xenopus laevis 5000-clone gastrula-stage cDNA microarray. We report the identification and initial characterization of 142 genes whose transcriptional profiles change in the neuralized explants. In order to address the potential involvement during neural induction of genes identified in the array, we performed gain-of-function studies in ectodermal explants. This approach lead to the identification of four genes that can function as neural inducers in Xenopus and three others that can synergize with known neural inducers in promoting neural fates. Based on these studies, we propose a role for post-transcriptional control of gene expression during neural induction in vertebrates and present a model whereby sustained BMP inhibition is promoted partly through the regulation of TGFbeta activated kinase (TAK1) activity by a novel TAK1-binding protein (TAB3).

Chordin-like CR domains and the regulation of evolutionarily conserved extracellular signaling systems

In fruit flies as well as in humans the Short gastrulation (Sog)/Chordin protein functions as an antagonist of the signaling of decapentaplegic (Dpp)/bone morphogenetic protein (BMP) in the extracellular space. Such antagonism inhibits Dpp/BMP signaling by blocking its binding to the receptor. Modulation of Dpp/BMP signaling is phylogenetically conserved and is a key step for the establishment of the dorso-ventral axis in vertebrates and invertebrates. Molecular studies have shown that the inhibitory activity of Chordin on BMP resides in specific cysteine-rich (CR) domains. Interestingly, Chordin-like CR domains are present in a growing number of extracellular proteins, several of which appear to be involved in BMP signaling regulation. We review here the conservation of the Chordin and Sog proteins, and in particular their functional domain, the CR domain. We discuss how the study of CR domains may provide a general mechanism for the regulation of growth factor signaling in the extracellular space.

Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases.

Bone morphogenetic proteins (BMPs) are derived from inactive precursor proteins by endoproteolytic cleavage. Here we show that processing of Nodal and Myc-tagged BMP4 is significantly enhanced by SPC1/Furin or SPC4/PACE4, providing direct evidence that regulation of BMP signaling is likely to be controlled by subtilisin-like proprotein convertase (SPC) activities. Nodal processing is dramatically enhanced if two residues adjacent to the precursor cleavage site are substituted with amino acids found at the equivalent positions of Activin, demonstrating that structural constraints at the precursor cleavage site limit the processing efficiency. However, in transfection assays, mature Nodal is undetectable either in culture supernatants or in cell lysates, despite efficient cleavage of the precursor protein, suggesting that mature Nodal is highly unstable. Domain swap experiments support this conclusion since mature BMP4 or Dorsalin are also destabilized when expressed in conjunction with the Nodal pro domain. By contrast, mature Nodal is stabilized by the Dorsalin pro domain, which mediates the formation of stable complexes. Collectively, these data show that the half-life of mature BMPs is greatly influenced by the identity of their pro regions.