Proprotein convertase 5/6a is associated with bone morphogenetic protein-2-induced squamous cell differentiation.

Squamous metaplasia in airway epithelium is a pathological process arising from abnormal remodeling/repair responses to injury. Proteolytic maturation of many growth and differentiation factors involved in tissue remodeling is controlled by proprotein convertases (PCs). However, the role of these convertases in airway remodeling remains poorly understood. Using a retinoic acid deficiency-induced squamous metaplasia model of cultured human nasal epithelial cells (HNECs), we observed a significant increase in the expression of PC5/6A, a PC member, and bone morphogenetic protein-2 (BMP-2), a candidate substrate for PC5/6A. Specific lentiviral short hairpin RNA-mediated PC5/6A knockdown decreased BMP-2 expression and maturation, decreased expression of squamous cell markers, and increased expression of ciliated cell markers. Decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK), a PC inhibitor, and LDN-193189, a BMP receptor inhibitor, suppressed squamous differentiation, promoted mucociliary differentiation, and down-regulated the BMP-2/Smad1/5/8/p38 signaling pathways. Dec-RVKR-CMK also decreased expression of PC5/6A, but not furin, another PC member, suggesting the involvement of PC5/6A in squamous differentiation of HNECs. Overexpression of PC5/6A and BMP-2 in the human nasal epithelial cell line RPMI-2650 demonstrated that PC5/6A can activate BMP-2. Under retinoic acid-sufficient culture conditions for mucociliary differentiation of HNECs, short-term expression of PC5/6A by the adenovirus system and addition of exogenous BMP-2 induced squamous differentiation. Furthermore, PC5/6A and BMP-2 were highly expressed in metaplastic squamous epithelium of human nasal polyps. Taken together, PC5/6A is involved in squamous differentiation of HNECs, possibly through up-regulation of the BMP-2/pSmad1/5/8/p38 signaling pathway, pointing to a potential therapeutic target for the prevention of chronic airway diseases that exhibit squamous metaplasia.

In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate.

The proprotein convertase PC5/6 cleaves protein precursors after basic amino acids and is essential for implantation in CD1/129/Sv/C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdf11-related phenotypes such as altered anteroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdf11-independent phenotypes, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC5/6 and Gdf11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PC5/6 for Gdf11 essentially resides in the presence of a P1' Asn in the RSRR downward arrowN cleavage motif. This work identifies Gdf11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase.

The cell-specific pattern of cholecystokinin peptides in endocrine cells versus neurons is governed by the expression of prohormone convertases 1/3, 2, and 5/6.

Most peptide hormone genes are, in addition to endocrine cells, also expressed in neurons. The peptide hormone cholecystokinin (CCK) is expressed in different molecular forms in cerebral neurons and intestinal endocrine cells. To understand this difference, we examined the roles of the neuroendocrine prohormone convertases (PC) 1/3, PC2, and PC5/6 by measurement of proCCK, processing intermediates and bioactive, alpha-amidated, and O-sulfated CCK peptides in cerebral and jejunal extracts of null mice, controls, and in the PC5/6-expressing SK-N-MC cell-line. In PC1/3 null mice, the synthesis of bioactive CCK peptide in the gut was reduced to 3% of the translational product, all of which was in the form of alpha-amidated and tyrosine O-sulfated CCK-22, whereas the neuronal synthesis in the brain was largely unaffected. This is opposite to the PC2 null mice in which only the cerebral synthesis was affected. SK-N-MC cells, which express neither PC1/3 nor PC2, synthesized alone the processing intermediate, glycine-extended CCK-22. Immunocytochemistry confirmed that intestinal endocrine CCK cells in wild-type mice express PC1/3 but not PC2. In contrast, cerebral CCK neurons contain PC2 and only little, if any, PC1/3. Taken together, the data indicate that PC1/3 governs the endocrine and PC2 the neuronal processing of proCCK, whereas PC5/6 contributes only to a modest endocrine synthesis of CCK-22. The results suggest that the different peptide patterns in the brain and the gut are due to different expression of PCs.

The cysteine-rich domain of the secreted proprotein convertases PC5A and PACE4 functions as a cell surface anchor and interacts with tissue inhibitors of metalloproteinases.

The proprotein convertases PC5, PACE4 and furin contain a C-terminal cysteine-rich domain (CRD) of unknown function. We demonstrate that the CRD confers to PC5A and PACE4 properties to bind tissue inhibitors of metalloproteinases (TIMPs) and the cell surface. Confocal microscopy and biochemical analyses revealed that the CRD is essential for cell surface tethering of PC5A and PACE4 and that it colocalizes and coimmunoprecipitates with the full-length and C-terminal domain of TIMP-2. Surface-bound PC5A in TIMP-2 null fibroblasts was only observed upon coexpression with TIMP-2. In COS-1 cells, plasma membrane-associated PC5A can be displaced by heparin, suramin, or heparinases I and III and by competition with excess exogenous TIMP-2. Furthermore, PC5A and TIMP-2 are shown to be colocalized over the surface of enterocytes in the mouse duodenum and jejunum, as well as in liver sinusoids. In conclusion, the CRD of PC5A and PACE4 functions as a cell surface anchor favoring the processing of their cognate surface-anchored substrates, including endothelial lipase.

Furin-like proprotein convertases are central regulators of the membrane type matrix metalloproteinase-pro-matrix metalloproteinase-2 proteolytic cascade in atherosclerosis.

BACKGROUND: Accumulation of macrophages and their in situ expression of matrix metalloproteinases (MMPs) are important determinants of plaque stability. Activation of membrane-bound MT1-MMP, the major activator of pro-MMP-2, requires intracellular endoproteolytic cleavage of its precursor protein. This type of activation typically requires suitable furin-like proprotein convertases (PCs), specifically furin and PC5. The present study was done to investigate the function of MT1-MMP as well as furin-like PCs in mononuclear inflammatory cells. METHODS AND RESULTS: Macrophage differentiation of human monocytic THP-1 cells was accompanied by increased expression of furin, PC5, and MT1-MMP. Some pro-MMP-2 activation was found in macrophages, but pro-MMP-2 level or activation was not enhanced after stimulation with the proinflammatory mediators tumor necrosis factor-alpha or lipopolysaccharide. However, culturing of macrophages in conditioned medium from serum-starved vascular smooth muscle cells, which constitutively secrete pro-MMP-2, resulted in a strong pro-MMP-2 activation. Inhibition of furin-like PCs with the specific pharmacological inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) inhibited MT1-MMP activation in macrophages. Dec-CMK or furin-specific small interfering RNA significantly inhibited macrophage MT1-MMP-dependent activation of vascular smooth muscle cell-derived pro-MMP-2. Flow cytometry demonstrated that human circulating monocytes express furin and PC5, and MT1-MMP and immunohistochemistry revealed their colocalization in macrophages in advanced human atherosclerotic lesions. CONCLUSIONS: Furin-like PCs (furin and PC5) play a central role in a MT-MMP-MMP-2 proteolytic cascade, involving provision of macrophage MT1-MMP for the activation of pro-MMP-2 synthesized by other cells. Furin and PC5 are expressed in human peripheral blood mononuclear cells and colocalize with MT1-MMP in macrophages in the atherosclerotic plaque, supporting the hypothesis that they are potential targets in atherosclerosis.

Proprotein convertases furin and PC5: targeting atherosclerosis and restenosis at multiple levels.

Several growth factors, chemokines, adhesion molecules, and proteolytic enzymes important for cell-cell/cell-matrix interactions in atherosclerosis and restenosis are initially synthesized as inactive precursor proteins. Activation of proproteins to biologically active molecules is regulated by limited endoproteolytic cleavage at dibasic amino acid residues. This type of activation typically requires the presence of suitable proprotein convertases (PCs). The PC-isozymes furin and PC5 are expressed in human atherosclerotic lesions and have been found to be up-regulated, following vascular injury in animal models in vivo. In vitro, these PCs can regulate vascular smooth muscle cell and macrophage functions and signaling events, through activation of pro-alpha-integrins and/or pro-membrane-type matrix metalloproteinases. Integrins link the cytoskeleton with the extracellular matrix and mediate bidirectional signaling and mechanotransduction, whereas matrix metalloproteinases are the major matrix-degrading enzymes. Both activities are required for cell recruitment to the intima. Furthermore, cleavage of extracellular matrix molecules by matrix metalloproteinases potentially contributes to weakening of the fibrous cap, promoting plaque rupture. Based on these recent in vitro and in vivo data, furin and PC5 are potential contributors to the initiation, progression, and complications of atherosclerosis and restenosis. Targeting these PCs may provide future anti-atherosclerotic therapies.

Endoproteolytic activation of alpha(v) integrin by proprotein convertase PC5 is required for vascular smooth muscle cell adhesion to vitronectin and integrin-dependent signaling.

BACKGROUND: Integrins play an important role for vascular smooth muscle cell (VSMC) migration during the development of atherosclerosis and restenosis. Integrin alpha(v)-subunit consists of disulphide-bound 125-kDa heavy and 25-kDa light chains, which are generated by endoproteolytic cleavage. This type of activation requires the presence of suitable proprotein convertases (PCs). Based on ex vivo and in vitro data, the PC5 isozyme has been suggested to be the major integrin convertase. We have recently demonstrated that PC5 is upregulated during vascular remodeling in rodents, colocalizing with alpha(v) in VSMCs. The aim of this study was to investigate the activation of alpha(v) by PCs in VSMCs and its consequences for alpha(v)-dependent cell functions. METHODS AND RESULTS: Immunoblotting demonstrated that inhibition of PC activity by the specific pharmacological inhibitor dec-CMK inhibits alpha(v) cleavage in VSMCs. These results were confirmed using PC5-specific antisense oligonucleotides. PC5-antisense oligonucleotides and dec-CMK inhibited VSMC adhesion to the alpha(v)beta3/beta5 ligand vitronectin (both P<0.05). Furthermore, PC5-asODNs inhibited VSMC migration on vitronectin-coated wells (P<0.05). Inhibition of PC activity and consequently alpha(v) cleavage inhibited the adhesion-dependent focal adhesion kinase(Y397)-autophosphorylation and subsequent Akt activation, whereas phosphorylation of extracellular signal-regulated kinase 1/2 was not affected. In human endarterectomy lesions, PC5 colocalized with alpha(v) integrin in VSMCs in the atherosclerotic plaques. CONCLUSIONS: The present study demonstrates that alpha(v) endoproteolytic activation is necessary for integrin-mediated adhesion and migration as well as signaling and requires PC5 in VSMCs. The colocalization of PC5 and alpha(v) in human carotid plaques indicates that PC5 might play a key role for alpha(v) activation in vivo.

Proprotein convertase 5/6 is critical for embryo implantation in women: regulating receptivity by cleaving EBP50, modulating ezrin binding, and membrane-cytoskeletal interactions.

Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

Posttranslational activation of bone morphogenetic protein 2 is mediated by proprotein convertase 6 during decidualization for pregnancy establishment.

Bone morphogenetic proteins (BMPs) require major posttranslational modifications to become biologically active. One such key modification is endoproteolytic cleavage of the initially synthesized nonactive precursor protein to release the mature ligand. Here we show in a physiological context of uterine stromal decidualization that BMP2 cleavage is mediated by proprotein convertase 5/6 (PC6). Decidualization is a uterine remodeling event critical for embryo implantation. Deletion or knockdown of either BMP2 or PC6 inhibits decidualization causing implantation failure and female infertility. In this study we provide biochemical and physiological evidence that PC6 proteolytically activates BMP2. We used freshly isolated primary human endometrial stromal cells and demonstrated that PC6 was the sole member of the PC family significantly up-regulated during decidualization. The precursor form of BMP2 was reduced, whereas its active form was increased during decidualization. Inhibition of PC6 activity inhibited decidualization, and this was accompanied by a total blockade of BMP2 activation. Addition of recombinant active BMP2 partially rescued the decidualization arrest caused by PC6 inhibition. PC6 processed BMP2 at the KREKR(282) downward arrow cleavage site, and mutating this site prevented the cleavage. This study thus demonstrates for the first time that the proteolytic activation and thus bioavailability of BMP2 is controlled by PC6.

Membrane type-1 matrix metalloprotease-independent activation of pro-matrix metalloprotease-2 by proprotein convertases.

Matrix metalloprotease-2 is implicated in many biological processes and degrades extracellular and non-extracellular matrix molecules. Matrix metalloprotease-2 maintains a latent state through a cysteine-zinc ion pairing which, when disrupted, results in full enzyme activation. This pairing can be disrupted by a conformational change or cleavage within the propeptide. The best known activation mechanism for pro-matrix metalloprotease-2 occurs via cleavage of the propeptide by membrane type-1 matrix metalloprotease. However, significant residual activation of pro-matrix metalloprotease-2 is seen in membrane type-1 matrix metalloprotease knockout mice and in fibroblasts treated with metalloprotease inhibitors. These findings indicate the presence of a membrane type-1 matrix metalloprotease-independent activation mechanism for pro-matrix metalloprotease-2 in vivo, which prompted us to explore an alternative activation mechanism for pro-matrix metalloprotese-2. In this study, we demonstrate membrane type-1 matrix metalloprotease-independent propeptide processing of matrix metalloprotease-2 in HEK293F and various tumor cell lines, and show that proprotein convertases can mediate the processing intracellularly as well as extracellularly. Furthermore, processed matrix metalloprotease-2 exhibits enzymatic activity that is enhanced by intermolecular autolytic cleavage. Thus, our experimental data, taken together with the broad expression of proprotein convertases, suggest that the proprotein convertase-mediated processing may be a general activation mechanism for pro-matrix metalloprotease-2 in vivo.

Vg1 has specific processing requirements that restrict its action to body axis patterning centers.

Unlike most transforming growth factor-beta (TGF-beta) superfamily members, Vg1 has been shown not to produce gross phenotypic alterations in Xenopus embryos when overexpressed by mRNA injection. Experiments with artificial chimeric constructs and a recently identified second allele of Vg1 suggest that this may be due to unusually stringent requirements for proteolytic processing. We provide biological and biochemical evidence that cleavage by two distinct proteolytic enzymes is required for effective activation of Vg1. We demonstrate a tightly restricted overlap in expression patterns of Vg1 with the proteases required to release the mature peptide. The data presented may account for the long-standing observation that the vast majority of Vg1 protein, in vivo, is present in its unprocessed form. Taken together, these observations provide a plausible mechanism for local action of Vg1 consistent with requirements imposed by current models of pattern formation in the developing body axis.