Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor β1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.     

Use of Bmp1/Tll1 doubly homozygous null mice and proteomics to identify and validate in vivo substrates of bone morphogenetic protein 1/tolloid-like metalloproteinases

Bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD), two proteinases encoded by Bmp1, provide procollagen C-proteinase (pCP) activity that converts procollagens I to III into the major fibrous components of mammalian extracellular matrix (ECM). Yet, although Bmp1(-/-) mice have aberrant collagen fibrils, they have residual pCP activity, indicative of genetic redundancy. Mammals possess two additional proteinases structurally similar to BMP-1 and mTLD: the genetically distinct mammalian Tolloid-like 1 (mTLL-1) and mTLL-2. Mice lacking the mTLL-1 gene Tll1 are embryonic lethal but have pCP activity levels similar to those of the wild type, suggesting that mTLL-1 might not be an in vivo pCP. In vitro studies have shown BMP-1 and mTLL-1 capable of cleaving Chordin, an extracellular antagonist of BMP signaling, suggesting that these proteases might also serve to modulate BMP signaling and to coordinate the latter with ECM formation. However, in vivo evidence of roles for BMP-1 and mTLL-1 in BMP signaling in mammals is lacking. To remove functional redundancy obscuring the in vivo functions of BMP-1-related proteases in mammals, we here characterize Bmp1 Tll1 doubly null mouse embryos. Although these appear morphologically indistinguishable from Tll1(-/-) embryos, biochemical analysis of cells derived from doubly null embryos shows functional redundancy removed to an extent enabling us to demonstrate that (i) products of Bmp1 and Tll1 are responsible for in vivo cleavage of Chordin in mammals and (ii) mTLL-1 is an in vivo pCP that provides residual activity observed in Bmp1(-/-) embryos. Removal of functional redundancy also enabled use of Bmp1(-/-) Tll1(-/-) cells in a proteomics approach for identifying novel substrates of Bmp1 and Tll1 products.     

Inactivation of bone morphogenetic protein 1 (<Emphasis Type="Italic">Bmp1</Emphasis>) and tolloid-like 1 (<Emphasis Type="Italic">Tll1</Emphasis>) in cells expressing type I collagen leads to dental and periodontal defects in mice

Bone morphogenetic protein 1 (BMP1) and tolloid-like 1 (TLL1) belong to the BMP1/tolloid-like proteinase family, which cleaves secretory proteins. The constitutive deletion of the Bmp1 or Tll1 genes causes perinatal or embryonic lethality in mice. In this study, we first studied the β-galactosidase activity in mice in which an IRES-lacZ-Neo cassette was inserted in the intron of either the Bmp1 or the Tll1 gene; the β-galactosidase activities were used to reflect the expression of endogenous Bmp1 and Tll1, respectively. Our X-gal staining results showed that the odontoblasts in the tooth and cells in the periodontal ligament express both Bmp1 and Tll1. We then created Bmp1 ^flox/flox and Tll1 ^flox/flox mice by removing the IRES-lacZ-Neo cassette. By breeding 2.3 kb Col1a1-Cre mice with the Bmp1 ^flox/flox and Tll1 ^flox/flox mice, we further generated Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice in which both Bmp1 and Tll1 were inactivated in the Type I collagen-expressing cells. We employed X-ray radiography, histology and immunohistochemistry approaches to characterize the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice. Our results showed that the molars of the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice had wider predentin, thinner dentin and larger pulp chambers than those of the normal controls. The dentinal tubules of the molars in the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice appeared disorganized. The level of dentin sialophosphoprotein in the molars of the 6-week-old Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice was lower than in the normal controls. The periodontal ligaments of the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice were disorganized and had less fibrillin-1. Our findings indicate that the proteinases encoded by Bmp1 and Tll1 genes play essential roles in the development and maintenance of mouse dentin and periodontal ligaments.     

Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.     

Genetic interaction between Bmp2 and Bmp4 reveals shared functions during multiple aspects of mouse organogenesis

Vertebrate Bmp2 and Bmp4 diverged from a common ancestral gene and encode closely related proteins. Mice homozygous for null mutations in either gene show early embryonic lethality, thereby precluding analysis of shared functions. In the current studies, we present phenotypic analysis of compound mutant mice heterozygous for a null allele of Bmp2 in combination with null or hypomorphic alleles of Bmp4. Whereas mice lacking a single copy of Bmp2 or Bmp4 are viable and have subtle developmental defects, compound mutants show embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, skeleton, eye and heart. Within the heart, BMP2 and BMP4 function coordinately to direct normal lengthening of the outflow tract, proper positioning of the outflow vessels, and septation of the atria, ventricle and atrioventricular canal. Our results identify numerous BMP4-dependent developmental processes that are also very sensitive to BMP2 dosage, thus revealing novel functions of Bmp2.     

Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis

Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis.     

bmp1 and mini fin are functionally redundant in regulating formation of the zebrafish dorsoventral axis

Drosophila metalloproteinase Tolloid (TLD) is responsible for cleaving the antagonist Short gastrulation (SOG), thereby regulating signaling by the bone morphogenetic protein (BMP) Decapentaplegic (DPP). In mice there are four TLD-related proteinases, two of which, BMP1 and mammalian Tolloid-like 1 (mTLL1), are responsible for cleaving the SOG orthologue Chordin, thereby regulating signaling by DPP orthologues BMP2 and 4. However, although TLD mutations markedly dorsalize Drosophila embryos, mice doubly homozygous null for BMP1 and mTLL1 genes are not dorsalized in early development. Only a single TLD-related proteinase has previously been reported for zebrafish, and mutation of the zebrafish TLD gene (mini fin) results only in mild dorsalization, manifested by loss of the most ventral cell types of the tail. Here we identify and map the zebrafish BMP1 gene bmp1. Knockdown of BMP1 expression results in a mild tail phenotype. However, simultaneous knockdown of mini fin and bmp1 results in severe dorsalization resembling the Swirl (swr) and Snailhouse (snh) phenotypes; caused by defects in major zebrafish ventralizing genes bmp2b and bmp7, respectively. We conclude that bmp1 and mfn gene products functionally overlap and are together responsible for a key portion of the Chordin processing activity necessary to formation of the zebrafish dorsoventral axis.     

Fibronectin Binds and Enhances the Activity of Bone Morphogenetic Protein 1

Bone morphogenetic protein-1-like proteinases play key roles in formation of the extracellular matrix (ECM) in vertebrates via biosynthetic processing of precursors into mature functional proteins involved in ECM assembly. Such processing includes proteolytic activation of the zymogen for lysyl oxidase. Fibronectin (FN) is an abundant protein component of the ECM that is capable of regulating manifold cellular functions through its interactions with various ECM and cell surface proteins. It was previously shown that proteolytic activation of lysyl oxidase is much reduced in cultures of FN-null mouse embryo fibroblasts (MEFs). Here we demonstrate that cellular fibronectin, the form produced by fibroblasts and various other tissue cell types, and plasma fibronectin bind BMP1 with dissociation constants (K_D) of ∼100 nm, consistent with a physiological role. Also consistent with such a role, cellular fibronectin FN is shown to positively regulate BMP1 processing activity against Chordin, probiglycan, and type I procollagen in vitro. Endogenous FN and BMP1 are demonstrated to co-localize in cell layers and to form complexes in culture medium. In addition, processing of endogenous BMP1 substrates Chordin, probiglycan, and procollagen is demonstrated to be strikingly reduced in cultures of FN^−/− MEFs compared with FN^+/− MEF cultures despite similar levels of endogenous BMP1. These data support the conclusion that FN binds BMP1-like proteinases in vivo and that FN is an important determinant of the in vivo activity levels of BMP1-like proteinases.Fibronectin (FN)³ is a noncollagenous extracellular matrix (ECM) glycoprotein of relatively high abundance that regulates a wide variety of cellular functions, including adhesion, migration, proliferation, differentiation, and apoptosis (1–4). FN is secreted as a disulfide-bonded dimer, and each subunit comprises 12 type I, 2 type II, and 15–17 type III FN modules as well as a “variable” (V) region that lacks homology to other protein domains (3). FN is found as two different major forms, plasma fibronectin (pFN), a soluble form synthesized by hepatocytes, and cellular fibronectin (cFN), which is locally expressed by many other cell types in various tissues (5). Both forms can be assembled into a fibrillar ECM by cultured fibroblasts (6). Differences between cFN and pFN arise from alternative RNA splicing in three regions; two type III repeats (designated EDA and EDB) and the V region. EDA and EDB are present in cFN but absent from pFN, whereas although only one subunit of the pFN dimer contains the V region, almost all cFN subunits contain this region (7). These differences in domain structure contribute to distinct functions for pFN and cFN; cFN plays roles in the dynamic tissue modeling of early embryogenesis and wound healing (8), whereas pFN subserves roles in hemostasis and thrombosis and immune responses (3, 9–11) and provides a reservoir for deposition in tissue (12).BMP1-like proteinases are evolutionary conserved extracellular metalloproteinases that play multiple roles in fostering ECM formation and activating TGFβ-like growth factors (13). These proteinases biosynthetically convert a variety of precursors into mature functional proteins with roles in ECM formation, including collagen types I-III, V, VII, and XI, laminin 332, and the small leucine-rich proteoglycans biglycan and osteoglycin. One important example is the zymogen for lysyl oxidase (LOX), an enzyme essential to formation of the covalent cross-links responsible for providing collagen and elastic fibers with much of their tensile strength (14). Recently, FN was reported to bind LOX in vitro (15). It was also suggested to positively regulate the proteolytic activation of LOX, as dramatically decreased processing of the zymogen for LOX was observed in FN^−/− mouse embryo fibroblast (MEF) cultures compared with FN^+/− MEF cultures even though equal amounts of BMP1 proteinase were produced by MEFs of the two different genotypes (15). These observations prompted the present study to determine whether FN might be involved in modulating the activities of BMP1-like proteinases. Herein, we provide evidence for direct interaction between FN and BMP1. BMP1 is shown to bind multiple FN sites via its non-protease domains, with affinities in the ∼100 nm range for cFN and pFN. This is a range congruent with K_D values (30–800 nm) previously estimated for binding of FN to its integrin receptors (16, 17) and is, thus, consistent with the likelihood of in vivo FN-BMP1 interactions. Moreover, cFN is shown to positively regulate BMP1 processing activity against a number of substrates in vitro. Consistent with the in vitro evidence of FN-BMP1 interactions, we demonstrate FN-BMP1 co-localization and the existence of FN-BMP1 complexes in cell cultures. Also demonstrated is a striking decrease in the processing of various endogenous BMP1 substrates in cultures of FN^−/− MEFs compared with FN^+/− MEF cultures. Implications of the data, which support the conclusion that FN positively regulates BMP1 activities in vivo, are discussed.     

BMP and BMP receptor expression during murine organogenesis

Cell–cell communication is critical for regulating embryonic organ growth and differentiation. The Bone Morphogenetic Protein (BMP) family of transforming growth factor β (TGFβ) molecules represents one class of such cell–cell signaling molecules that regulate the morphogenesis of several organs. Due to high redundancy between the myriad BMP ligands and receptors in certain tissues, it has been challenging to address the role of BMP signaling using targeting of single Bmp genes in mouse models. Here, we present a detailed study of the developmental expression profiles of three BMP ligands (Bmp2, Bmp4, Bmp7) and three BMP receptors (Bmpr1a, Bmpr1b, and BmprII), as well as their molecular antagonist (noggin), in the early embryo during the initial steps of murine organogenesis. In particular, we focus on the expression of Bmp family members in the first organs and tissues that take shape during embryogenesis, such as the heart, vascular system, lungs, liver, stomach, nervous system, somites and limbs. Using in situ hybridization, we identify domains where ligand(s) and receptor(s) are either singly or co-expressed in specific tissues. In addition, we identify a previously unnoticed asymmetric expression of Bmp4 in the gut mesogastrium, which initiates just prior to gut turning and the establishment of organ asymmetry in the gastrointestinal tract. Our studies will aid in the future design and/or interpretation of targeted deletion of individual Bmp or Bmpr genes, since this study identifies organs and tissues where redundant BMP signaling pathways are likely to occur.     

The bone morphogenetic protein 1/Tolloid-like metalloproteinases.

A decade ago, bone morphogenetic protein 1 (BMP1) was shown to provide the activity necessary for proteolytic removal of the C-propeptides of procollagens I-III: precursors of the major fibrillar collagens. Subsequent studies have shown BMP1 to be the prototype of a small group of extracellular metalloproteinases that play manifold roles in regulating formation of the extracellular matrix (ECM). Soon after initial cloning of BMP1, genetic studies showed the related Drosophila proteinase Tolloid (TLD) to be necessary for the formation of the dorsal-ventral axis in early embryogenesis. It is now clear that the BMP1/TLD-like proteinases, conserved in species ranging from Drosophila to humans, act in dorsal-ventral patterning via activation of transforming growth factor beta (TGFbeta)-like proteins BMP2, BMP4 (vertebrates) and decapentaplegic (arthropods). More recently, it has become apparent that the BMP1/TLD-like proteinases are activators of a broader subset of the TGFbeta superfamily of proteins, with implications that these proteinases may be key in orchestrating the formation of ECM with growth factor activation and BMP signaling in morphogenetic processes.     

Reduced BMP Signaling Results in Hindlimb Fusion with Lethal Pelvic/Urogenital Organ Aplasia: A New Mouse Model of Sirenomelia

Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4^flox/flox) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4^flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.     

The mammalian Tolloid-like 1 gene, Tll1, is necessary for normal septation and positioning of the heart.

Mammalian Tolloid-like 1 (mTLL-1) is an astacin-like metalloprotease, highly similar in domain structure to the morphogenetically important proteases bone morphogenetic protein-1 (BMP-1) and Drosophila Tolloid. To investigate possible roles for mTLL-1 in mammalian development, we have used gene targeting in ES cells to produce mice with a disrupted allele for the corresponding gene, Tll1. Homozygous mutants were embryonic lethal, with death at mid-gestation from cardiac failure and a unique constellation of developmental defects that were apparently confined solely to the heart. Constant features were incomplete formation of the muscular interventricular septum and an abnormal and novel positioning of the heart and aorta. Consistent with roles in cardiac development, Tll1 expression was specific to precardiac tissue and endocardium in 7.5 and 8.5 days p.c. embryos, respectively. Tll1 expression was also high in the developing interventricular septum, where expression of the BMP-1 gene, Bmp1, was not observed. Cardiac structures that were not affected in Tll1-/- embryos either showed no Tll1 expression (atrio-ventricular cushions) or showed overlapping expression of Tll1 and Bmp1 (aortico-pulmonary septum), suggesting that products of the Bmp1 gene may be capable of functionally substituting for mTLL-1 at sites in which they are co-expressed. Together, the various data show that mTLL-1 plays multiple roles in formation of the mammalian heart and is essential for formation of the interventricular septum.     

Mammalian tolloid-like 1 binds procollagen C-proteinase enhancer protein 1 and differs from bone morphogenetic protein 1 in the functional roles of homologous protein domains

Bone morphogenetic protein 1 (BMP1) is the prototype of a subgroup of metalloproteinases with manifold roles in morphogenesis. Four mammalian subgroup members exist, including BMP1 and mammalian Tolloid-like 1 (mTLL1). Subgroup members have a conserved protein domain structure: an NH2-terminal astacin-like protease domain, followed by a fixed order of CUB and epidermal growth factor-like protein-protein interaction motifs. Previous structure/function studies have documented those BMP1 protein domains necessary for secretion, and activity against various substrates. Here we demonstrate that, in contradiction to previous reports, the most NH2-terminal CUB domain (CUB1) is not required for BMP1 secretion nor is the next CUB domain (CUB2) required for enzymatic activity. The same is true for mTLL1. In fact, secreted protease domains of BMP1 and mTLL1, devoid of CUB or epidermal growth factor-like domains, have procollagen C-proteinase (pCP) activity and activity for biosynthetic processing of biglycan, the latter with kinetics superior to those of the full-length proteins. Structure-function analyses herein also suggest differences in the functional roles played by some of the homologous domains in BMP1 and mTLL1. Surprisingly, although BMP1 has long been known to be Ca2+-dependent, a property previously assumed to apply to all members of the subgroup, mTLL1 is demonstrated to be independent of Ca2 levels in its ability to cleave some, but not all, substrates. We also show that pCP activities of only versions of BMP1 and mTLL1 with intact COOH termini are enhanced by the procollagen C-proteinase enhancer 1 (PCOLCE1) and that mTLL1 binds PCOLCE1, thus suggesting reappraisal of the accepted paradigm for how PCOLCE1 enhances pCP activities.     

Bmp2 and Bmp4 genetically interact to support multiple aspects of mouse development including functional heart development

Bone morphogenetic proteins (BMPs) have multiple roles during embryogenesis. Current data indicate that the dosage of BMPs is tightly regulated for normal development in mice. Since Bmp2 or Bmp4 homozygous mutant mice show early embryonic lethality, we generated compound heterozygous mice for Bmp2 and Bmp4 to explore the impact of lowered dosage of these BMP ligands. Genotyping pups bred between Bmp2 and Bmp4 heterozygous mice revealed that the ratio of adult compound heterozygous mice for Bmp2 and Bmp4 is much lower than expected. During embryogenesis, the compound heterozygous embryos showed several abnormalities, including defects in eye formation, body wall closure defects, and ventricular septal defects (VSD) in the heart. However, the ratio of the compound heterozygous embryos was the same as expected. Caesarean sections at E18.5 revealed that half of the compound heterozygotes died soon after birth, and the majority of the dead individuals exhibited VSD. Survivors were able to grow to adults, but their body weight was significantly lower than control littermates. They demonstrated progressive abnormalities in the heart, eventually showing a branched leaflet in atrioventricular valves. These results suggest that the dosage of both BMP2 and 4 is critical for functional heart formation during embryogenesis and after birth. genesis 47:374–384, 2009. © 2009 Wiley‐Liss, Inc.     

Purification and properties of a fibrinogenase from the venom of Naja nigricollis

A fibrinogenolytic proteinase from the venom of Naja nigricollis was purified by chromatography on Bio-Rex 70 and Phenyl-Sepharose. The purified enzyme, designated proteinase F1, was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis, and consisted of a single chain with a molecular weight of 58 000. Purified proteinase F1 had approximately 15-fold more proteinase activity than the crude venom, based on its ability to inactive α₂-macroglobulin. The enzyme acted on only the Aα-chain of fibrinogen and left the Bβ- and γ-chains intact. The pH optimum for this fibrinogenolytic activity was in the range of pH 8 to 10. In addition to its activity on fibrinogen, proteinase F1 was active on α₂-macroglobulin and fibronectin, but did not degrade casein, hemoglobin or bovine serum albumin. The enzyme was not inhibited by inhibitors of serine proteinases, cysteine proteinases or acid proteinases, but only by the metalloproteinase inhibitor, EDTA. The inhibition by EDTA could be prevented by Zn²⁺, but not by Ca²⁺ or Mg²⁺.     

Procollagen C proteinase enhancer 1 genes are important determinants of the mechanical properties and geometry of bone and the ultrastructure of connective tissues

Procollagen C proteinases (pCPs) cleave type I to III procollagen C propeptides as a necessary step in assembling the major fibrous components of vertebrate extracellular matrix. The protein PCOLCE1 (procollagen C proteinase enhancer 1) is not a proteinase but can enhance the activity of pCPs ~10-fold in vitro and has reported roles in inhibiting other proteinases and in growth control. Here we have generated mice with null alleles of the PCOLCE1 gene, Pcolce, to ascertain in vivo roles. Although Pcolce^−/− mice are viable and fertile, Pcolce^−/− male, but not female, long bones are more massive and have altered geometries that increase resistance to loading, compared to wild type. Mechanical testing indicated inferior material properties of Pcolce^−/− male long bone, apparently compensated for by the adaptive changes in bone geometry. Male and female Pcolce^−/− vertebrae both appeared to compensate for inferior material properties with thickened and more numerous trabeculae and had a uniquely altered morphology in deposited mineral. Ultrastructurally, Pcolce^−/− mice had profoundly abnormal collagen fibrils in both mineralized and nonmineralized tissues. In Pcolce^−/− tendon, 100% of collagen fibrils had deranged morphologies, indicating marked functional effects in this tissue. Thus, PCOLCE1 is an important determinant of bone mechanical properties and geometry and of collagen fibril morphology in mammals, and the human PCOLCE1 gene is identified as a candidate for phenotypes with defects in such attributes in humans.     

Inhibition of bone morphogenetic protein 1 by native and altered forms of alpha2-macroglobulin

The four mammalian bone morphogenetic protein 1 (BMP1)-like proteinases act to proteolytically convert procollagens to the major fibrous components of the extracellular matrix. They also activate lysyl oxidase, an enzyme necessary to the covalent cross-linking that gives collagen fibrils much of their tensile strength. Thus, these four proteinases are attractive targets for interventions designed to limit the excess formation of fibrous collagenous matrix that characterizes fibrosis. Although it has previously been reported that the serum protein alpha(2)-macroglobulin is unable to inhibit the astacin-like proteinases meprin alpha and meprin beta, we herein demonstrate alpha(2)-macroglobulin to be a potent inhibitor of the similar BMP1-like proteinases. BMP1 is shown to cleave the alpha(2)-macroglobulin "bait" region, at a single specific site, which resembles the sites at which BMP1-like proteinases cleave the C-propeptides of procollagens I-III. alpha(2)-Macroglobulin is an irreversible inhibitor that is shown to bind bone morphogenetic protein 1 in a covalent complex. It is also demonstrated that genetically modified alpha(2)-macroglobulin, in which the native bait region is replaced by sequences flanking the probiglycan BMP1 cleavage site, is enhanced approximately 24-fold in its ability to inhibit BMP1, and is capable of inhibiting the biosynthetic processing of procollagen I by cells. These findings suggest possible therapeutic interventions involving ectopic expression of modified versions of alpha(2)-macroglobulin in the treatment of fibrotic conditions.     

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.