Regulation of apoAI processing by procollagen C-proteinase enhancer-2 and bone morphogenetic protein-1

Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations controlling the levels of risk factors associated with the development of the disease continues. Multiple genetic association studies suggest the involvement of procollagen C-proteinase enhancer-2 (PCPE2) in modulating HDL-C levels. Therefore biochemical and mechanistic studies were undertaken to determine whether there might be a basis for a role of PCPE2 in HDL biogenesis. Our studies indicate that PCPE2 accelerates the proteolytic processing of pro-apolipoprotein (apo) AI by enhancing the cleavage of the hexapeptide extension present at the N terminus of apoAI. Surface Plasmon Resonance and immunoprecipitation studies indicate that PCPE2 interacts with BMP-1 and pro-apoAI to form a ternary pro-apoAI/BMP-1/PCPE2 complex. The most favorable interaction among these proteins begins with the association of BMP-1 to pro-apoAI followed by the binding of PCPE2 which further stabilizes the complex. PCPE2 resides, along with apoAI, on the HDL fraction of lipoproteins in human plasma supporting a relationship between HDL and PCPE2. Taken together, the findings from our studies identify a new player in the regulation of apoAI post-translational processing and open a new avenue to the study of mechanisms involved in the regulation of apoAI synthesis, HDL levels, and potentially, cardiovascular disease.     

Identification of the minimal domain structure of bone morphogenetic protein-1 (BMP-1) for chordinase activity: chordinase activity is not enhanced by procollagen C-proteinase enhancer-1 (PCPE-1)

Bone morphogenetic protein 1 (BMP-1), which is a tolloid member of the astacin-like family of zinc metalloproteinases, is a highly effective procollagen C-proteinase (PCP) and chordinase. On the other hand, mammalian tolloid like-2 (mTLL-2) does not cleave chordin or procollagen; procollagen is cleaved by mTLL-2 in the presence of high levels of procollagen C-proteinase enhancer-1 (PCPE-1), for reasons that are unknown. We used these differences in activity between BMP-1 and mTLL-2 to narrow in on the domains in BMP-1 that specify PCP and chordinase activity. Using a domain swap approach, we showed that: 1) the metalloproteinase and CUB2 domains of BMP-1 are absolutely required for PCP activity; swaps with either of the corresponding domains in BMP-1 and mTLL-2 did not result in procollagen cleavage and 2) the proteinase domain of mTLL-2 can cleave chordin if coupled to the CUB1 domain of BMP-1. Therefore, the minimal structure for chordinase activity comprises a metalloproteinase domain (either from BMP-1 or from mTLL-2) and the CUB1 domain of BMP-1 (the CUB1 domain of mTLL-2 cannot substitute for the CUB1 domain of BMP-1). We showed that the minimal procollagen C-proteinase (BMP-1 lacking the EGF and CUB3 domain) was enhanced by PCPE-1 but not as well as BMP-1 retaining the CUB3 domain. Further studies showed that PCPE-1 had no effect on the ability of BMP-1 to cleave chordin. The data support a previously suggested mechanism of PCPE-1 whereby PCPE-1 interacts with procollagen, but in addition, the CUB3 domain of BMP-1 appears to augment the interaction.     

The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains

Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding.     

Procollagen C-proteinase enhancer stimulates procollagen processing by binding to the C-propeptide region only

Bone morphogenetic protein-1 (BMP-1) and the tolloid-like metalloproteinases control several aspects of embryonic development and tissue repair. Unlike other proteinases whose activities are regulated mainly by endogenous inhibitors, regulation of BMP-1/tolloid-like proteinases relies mostly on proteins that stimulate activity. Among these, procollagen C-proteinase enhancers (PCPEs) markedly increase BMP-1/tolloid-like proteinase activity on fibrillar procollagens, in a substrate-specific manner. Here, we performed a detailed quantitative study of the binding of PCPE-1 and of its minimal active fragment (CUB1-CUB2) to three regions of the procollagen III molecule: the triple helix, the C-telopeptide, and the C-propeptide. Contrary to results described elsewhere, we found the PCPE-1-binding sites to be located exclusively in the C-propeptide region. In addition, binding and enhancing activities were found to be independent of the glycosylation state of the C-propeptide. These data exclude previously proposed mechanisms for the action of PCPEs and also suggest new mechanisms to explain how these proteins can stimulate BMP-1/tolloid-like proteinases by up to 20-fold.     

Identification of amino acid residues in bone morphogenetic protein-1 important for procollagen C-proteinase activity

Bone morphogenetic protein (BMP)-1, which belongs to the tolloid subgroup of astacin-like zinc metalloproteinases, cleaves the C-propeptides of procollagen at the physiologic site and is, therefore, a procollagen C-proteinase (PCP). Cleavage occurs between a specific alanine or glycine residue (depending on the procollagen chain) and an invariant aspartic acid residue in each of the three chains of procollagen. To learn more about how BMP-1 exhibits PCP activity we mapped the primary structure of BMP-1 onto the x-ray crystal structure of astacin and identified residues in the metalloproteinase domain of BMP-1 for subsequent site-directed mutagenesis studies. Recombinant wild-type and mutant BMP-1 were expressed in COS-7 cells and assayed for PCP activity using type I procollagen as the substrate. We showed that substitution of alanine for Glu(94), which occurs in the HEXXH zinc-binding motif of BMP-1, abolishes PCP activity. Furthermore, mutation of residues Lys(87) and Lys(176), which are located in the S1' pocket of the enzyme and are therefore adjacent to the P1' residue in the substrate, reduced the proteolytic activity of BMP-1 by approximately 50%. A surprising observation was that mutation of Cys(66) reduced the activity to 20%, suggesting that this residue is crucial for activity. Further experiments showed that Cys(66) and Cys(63), which are located in the tolloid-specific sequence Cys(63)-Gly(64)-Cys(65)-Cys(66) in the active site, most likely form a disulfide bridge.     

Insights into how CUB domains can exert specific functions while sharing a common fold: conserved and specific features of the CUB1 domain contribute to the molecular basis of procollagen C-proteinase enhancer-1 activity

Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known three-dimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies.     

Procollagen VII self-assembly depends on site-specific interactions and is promoted by cleavage of the NC2 domain with procollagen C-proteinase

Procollagen VII is a homotrimer of 350-kDa proalpha1(VII) chains. Each chain has a central collagenous domain flanked by a noncollagenous amino-terminal NC1 domain and a carboxy-terminal NC2 domain. After secretion from cells, procollagen VII molecules form antiparallel dimers with a 60 nm overlap. These dimers are stabilized by disulfide bonds formed between cysteines present in the NC2 domain and cysteines present in the triple-helical domain. Electron microscopy has provided direct evidence for the existence of collagen VII dimers, but the dynamic process of dimer formation is not well understood. In the present study, we tested the hypothesis that, during dimer formation, the NC2 domain of one procollagen VII molecule specifically recognizes and binds to the triple-helical region adjacent to Cys-2625 of another procollagen VII molecule. We also investigated the role of processing of the NC2 domain by the procollagen C-proteinase/BMP-1 in dimer assembly. We engineered mini mouse procollagen VII variants consisting of intact NC1 and NC2 domains and a shortened triple helix in which the C-terminal region encompassing Cys-2625 was either preserved or substituted with the region encompassing Cys-1448 derived from the N-terminal part of the triple-helical domain. The results indicate that procollagen VII self-assembly depends on site-specific interactions between the NC2 domain and the triple-helical region adjacent to Cys-2625 and that this process is promoted by the cleavage of the NC2 by procollagen C-proteinase/BMP1.     

Bone morphogenetic protein-1 (BMP-1). Identification of the minimal domain structure for procollagen C-proteinase activity

Bone morphogenetic protein-1 (BMP-1) is a shorter spliced variant of mammalian tolloid (mTld), both of which cleave the C-propeptides of type I procollagen during the synthesis of extracellular matrix collagen fibrils. The fact that BMP-1 and mTld both exhibit procollagen C-proteinase (PCP) activity and that BMP-1 is the smaller variant might indicate that BMP-1 comprises the minimal required sequences for PCP activity. BMP-1 comprises a metalloproteinase domain, three CUB domains, and an epidermal growth factor (EGF)-like domain, which is located between the second and third CUB (complement components C1r/C1s, the sea urchin protein Uegf, and BMP-1) domains. In this study we showed the following. 1) The CUB1 domain is required for secretion of the molecule. Domain swapping experiments, in which CUB1 and other CUB domains were interchanged, resulted in retention of the proteins by cells. Therefore, CUB1 and its location immediately adjacent to the metalloproteinase domain are essential for secretion of the protein. 2) Mutants lacking the EGF-like and CUB3 domains exhibited full C-proteinase activity. In contrast, mutants lacking the CUB2 domain were poor C-proteinases. 3) Further studies showed that Glu-483 on the beta4-beta5 loop of CUB2 is essential for C-proteinase activity of BMP-1. In conclusion, the study showed that the minimal domain structure for PCP activity is considerably shorter than expected and comprises the metalloproteinase domain and the CUB1 and CUB2 domains of BMP-1.     

A role for cardiotrophin-1 in myocardial remodeling induced by aldosterone

Hyperaldosteronim is associated with left ventricular (LV) hypertrophy (LVH) and fibrosis. Cardiotrophin (CT)-1 is a cytokine that induces myocardial remodeling. We investigated whether CT-1 mediates aldosterone (Aldo)-induced myocardial remodeling in two experimental models. Wistar rats were treated with Aldo-salt (1 mg·kg(-1)·day(-1)) with or without spironolactone (200 mg·kg(-1)·day(-1)) for 3 wk. Wild-type (WT) and CT-1-null mice were infused with Aldo (1 mg·kg(-1)·day(-1)) for 3 wk. Hemodynamic parameters were analyzed. LVH, fibrosis, inflammation, and CT-1 expression were evaluated in both experimental models by histopathological analysis, RT-PCR, Western blot analysis, and ELISA. Hypertensive Aldo-treated rats exhibited increased LV end-diastolic pressure and -dP/dt compared with controls. The cardiac index, LV cross-sectional area and wall thickness, cardiomyocyte size, collagen deposition, and inflammation were increased in Aldo-salt-treated rats. Myocardial expression of molecular markers assessing LVH and fibrosis as well as CT-l levels were also augmented by Aldo-salt. Spironolactone treatment reversed all the above effects. CT-1 correlated positively with hemodynamic, histological, and molecular parameters showing myocardial remodeling. In WT and CT-1-null mice, Aldo infusion did not modify blood pressure. Whereas Aldo treatment induced LVH, fibrosis, and inflammation in WT mice, the mineralocorticoid did not provoke cardiac remodeling in CT-1-null mice. In conclusion, in experimental hyperaldosteronism, the increase in CT-1 expression was associated with parameters showing LVH and fibrosis. CT-1-null mice were resistant to Aldo-induced LVH and fibrosis. These data suggest a key role for CT-1 in cardiac remodeling induced by Aldo independent of changes in blood pressure levels.     

Interaction properties of the procollagen C-proteinase enhancer protein shed light on the mechanism of stimulation of BMP-1

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.     

Expression of calcium-binding protein regucalcin mRNA in fetal rat liver is stimulated by calcium administration

The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, -actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic -actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.     

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.     

Effects of the absence of procollagen C-endopeptidase enhancer-2 on myocardial collagen accumulation in chronic pressure overload

Cardiac interstitial fibrillar collagen accumulation, such as that associated with chronic pressure overload (PO), has been shown to impair left ventricular diastolic function. Therefore, insight into cellular mechanisms that mediate excessive collagen deposition in the myocardium is pivotal to this important area of research. Collagen is secreted as a soluble procollagen molecule with NH(2)- and COOH (C)-terminal propeptides. Cleavage of these propeptides is required for collagen incorporation to insoluble collagen fibrils. The C-procollagen proteinase, bone morphogenic protein 1, cleaves the C-propeptide of procollagen. Procollagen C-endopeptidase enhancer (PCOLCE) 2, an enhancer of bone morphogenic protein-1 activity in vitro, is expressed at high levels in the myocardium. However, whether the absence of PCOLCE2 affects collagen content at baseline or after PO induced by transverse aortic constriction (TAC) has never been examined. Accordingly, in vivo procollagen processing and deposition were examined in wild-type (WT) and PCOLCE2-null mice. No significant differences in collagen content or myocardial stiffness were detected in non-TAC (control) PCOLCE2-null versus WT mice. After TAC-induced PO, PCOLCE2-null hearts demonstrated a lesser collagen content (PCOLCE2-null TAC collagen volume fraction, 0.41% ± 0.07 vs. WT TAC, 1.2% ± 0.3) and lower muscle stiffness compared with WT PO hearts [PCOLCE2-null myocardial stiffness (β), 0.041 ± 0.002 vs. WT myocardial stiffness, 0.065 ± 0.001]. In addition, in vitro, PCOLCE2-null cardiac fibroblasts exhibited reductions in efficiency of C-propeptide cleavage, as demonstrated by increases in procollagen α1(I) and decreased levels of processed collagen α1(I) versus WT cardiac fibroblasts. Hence, PCOLCE2 is required for efficient procollagen processing and deposition of fibrillar collagen in the PO myocardium. These results support a critical role for procollagen processing in the regulation of collagen deposition in the heart.     

Apoptosis in rat placenta is zone-dependent and stimulated by glucocorticoids

Apoptosis, or physiological cell death, is elevated in the placenta of human pregnancies complicated by fetal growth retardation, suggesting that placental apoptosis may be a key factor in the overall control of feto-placental growth. The present study used DNA internucleosomal fragmentation analysis to characterize apoptosis in the two morphologically and functionally distinct regions of the rat placenta, the basal and labyrinth zones, during the last week of pregnancy (Days 16, 22, and 23). In addition, because glucocorticoids are potent inhibitors of feto-placental growth and can stimulate apoptosis in other tissues, we examined whether dexamethasone treatment in vivo induces placental apoptosis. DNA fragmentation was clearly evident in both placental zones at each stage of pregnancy, with higher levels evident in the basal zone compared with the labyrinth zone on Days 22 and 23. TUNEL analysis, which identifies dying cells in situ, demonstrated positive staining of cells in the basal zone, particularly giant trophoblast cells. Dexamethasone treatment increased DNA fragmentation in the basal zone but not the labyrinth zone. Similarly, maternal treatment with carbenoxolone, which can enhance local concentrations of endogenous glucocorticoid by inhibition of 11 beta-hydroxysteroid dehydrogenase, also increased DNA fragmentation in the basal zone but not in the labyrinth zone. These effects of dexamethasone and carbenoxolone on placental apoptosis were associated with reduced placental and fetal weights. In conclusion, this study shows that apoptosis occurs in both zones of the rat placenta, particularly in the basal zone near term, and is elevated after increased glucocorticoid exposure in vivo. These data support the hypothesis that placental apoptosis is an important player in the regulation of feto-placental growth, and establish the rat as a useful model to study the endocrine control of placental apoptosis.     

L-NAME-induced protein remodeling and fibrosis in the rat heart

The aim of the present study was to determine whether NO deficiency itself or rather the elevation of systolic blood pressure is responsible for the protein and structural remodeling of the heart during hypertension induced by long-term treatment by nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). Three groups of rats were investigated. The first group served as control. In the second group L-NAME was given in the dose of 20 mg/kg/day in the drinking water and in the third group L-NAME was given in the dose of 40 mg/kg/day during 4 weeks. While L-NAME treatment in both doses caused essentially the same increase in systolic blood pressure (SBP), NO synthase activity and cGMP concentration in the left ventricle decreased by 17% and 13%, respectively in the 20 mg/kg/day L-NAME group and by 69% and 27%, respectively in the 40 mg/kg/day L-NAME group. The protein profile of the left ventricle in both L-NAME groups was characterized by an increased concentration of metabolic proteins. Nevertheless, a significant increase in the concentration of pepsin-soluble collagenous proteins and the concentration of hydroxyproline in pepsin-insoluble collagenous proteins was found only in the group receiving 40 mg/kg/day L-NAME. The morphometric evaluation revealed a significant increase in myocardial fibrosis in both L-NAME groups. However, this was more pronounced in the 40 mg/kg/day L-NAME group. It is concluded that NO deficiency resulted in significant enhancement of fibrotic tissue growth in proportion to the administered L-NAME dose, while SBP was increased similarly in both L-NAME groups. Thus, NO deficiency rather than hemodynamic changes appears to be crucially involved in collagenous protein and fibrotic tissue changes of the left ventricle in hypertension induced by L-NAME.     

Expression of calcium-binding protein regucalcin mRNA in rat liver is stimulated by calcitonin: The hormonal effect is mediated through calcium

The involvement of a hypocalcemic hormone calcitonin (CT) in the expression of hepatic Ca²⁺-binding protein regucalcin mRNA was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). A single oral administration of calcium chloride (100 mg Ca/100 g body weight) to rats induced a remarkable increase in the serum calcium concentration and a corresponding elevation of the liver calcium content during 120 min after the administration. Thyroparathyroidectomy (TPTX) did not cause a significant increase in the liver calcium content after calcium administration. Hepatic regucalcin mRNA level was markedly elevated by calcium administration; the level was about 180% of controls at 60 min after the administration. This increase was completely abolished by TPTX. A single subcutaneous administration of CT (synthetic eel CT; 25–100 MRC mU/100 g) to TPTX rats received oral administration of calcium (100 mg/100 g) produced a remarkable increase in hepatic regucalcin mRNA levels; the level was about 280% of controls with the dose of 25 MRC mU CT/100 g. The present finding suggests that the expression of hepatic mRNA is stimulated by CT, and that the hormonal effect is mediated through Ca²⁺ in rat liver.