Urine peptidome in combination with transcriptomics analysis highlights MMP7, MMP14 and PCSK5 for further investigation in chronic kidney disease.

Chronic kidney disease (CKD) is characterized by the loss of kidney function. The molecular mechanisms underlying the development and progression of CKD are still not fully understood. Among others, the urinary peptidome has been extensively studied, with several urinary peptides effectively detecting disease progression. However, their link to proteolytic events has not been made yet. This study aimed to predict the proteases involved in the generation of CKD-associated urinary excreted peptides in a well-matched (for age, sex, lack of heart disease) case-control study. The urinary peptide profiles from CKD (n = 241) and controls (n = 240) were compared and statistically analyzed. The in-silico analysis of the involved proteases was performed using Proteasix and proteases activity was predicted based on the abundance changes of the associated peptides. Predictions were cross-correlated to transcriptomics datasets by using the Nephroseq database. Information on the respective protease inhibitors was also retrieved from the MEROPS database. Totally, 303 urinary peptides were significantly associated with CKD. Among the most frequently observed were fragments of collagen types I, II and III, uromodulin, albumin and beta-2-microglobulin. Proteasix predicted 16 proteases involved in their generation. Through investigating CKD-associated transcriptomics datasets, several proteases are highlighted including members of matrix metalloproteinases (MMP7, MMP14) and serine proteases (PCSK5); laying the foundation for further studies towards elucidating their role in CKD pathophysiology.

In Vivo Analysis of the Contribution of Proprotein Convertases to the Processing of FGF23.

Fibroblast growth factor 23 (FGF23) is a hormone secreted from fully differentiated osteoblasts and osteocytes that inhibits phosphate reabsorption by kidney proximal tubules. The full-length (i.e., intact) protein mediates FGF23 endocrine functions, while endoproteolytic cleavage at a consensus cleavage sequence for the proprotein convertases (PCs) inactivates FGF23. Two PCs, furin and PC5, were shown to cleave FGF23 in vitro at RHTR179↓, but whether they are fulfilling this function in vivo is currently unknown. To address this question, we used here mice lacking either or both furin and PC5 in cell-specific manners and mice lacking the paired basic amino acid-cleaving enzyme 4 (PACE4) in all cells. Our analysis shows that furin inactivation in osteoblasts and osteocytes results in a 25% increase in circulating intact FGF23, without any significant impact on serum phosphate levels, whether mice are maintained on a normal or a low phosphate diet. Under conditions of iron deficiency, FGF23 is normally processed in control mice, but its processing is impaired in mice lacking furin in osteoblasts and osteocytes. In contrast, FGF23 is normally cleaved following erythropoietin or IL-1ß injections in mice lacking furin or both furin and PC5, and in PACE4-deficient mice. Altogether, these studies suggest that furin is only partially responsible for FGF23 cleavage under certain conditions in vivo. The processing of FGF23 may therefore involve the redundant action of multiple PCs or of other peptidases in osteoblasts, osteocytes and hematopoietic cells.

MicroRNA­338­3p regulates age­associated osteoporosis via targeting PCSK5.

Bone loss is a disease that is highly associated with aging. This deleterious health condition has become a public concern worldwide, and there is an urgent need to discover more novel therapeutic strategies for the development of age­associated osteoporosis. The present study aimed to explore the association between proprotein convertase subtilisin/kexin type 5 (PCSK5) and microRNA(miR)­338­3p in bone­formation and bone­loss processes. Western blotting assay and reverse transcription­quantitative PCR were employed to analyze PCSK5 and miR­338­3p expression levels in bone mesenchymal stem cells (BMSCs). Dual­luciferase reporter and RNA pull­down assays were used to determine the target. For osteoblastic differentiation verification, alkaline phosphatase activity, osteocalcin secretion detection, bone formation­related indicators (osterix, runt­related gene 2, osteopontin and bone sialoprotein), hematoxylin and eosin staining and Alizarin Red S staining were performed. The findings of the present study indicated that the expression level of PCSK5 was higher in BMSCs from young rat samples, whereas the expression level of miR­338­3p was higher in BMSCs from samples of old rats. Experimental results also revealed that unlike miR­338­3p, downregulation of PCSK5 inhibited osteoblastic differentiation and osteogenesis by inhibiting alkaline phosphatase, osteocalcin, osterix, runt­related transcription factor 2, osteopontin, bone sialoprotein and mineralized nodule formation. Overall, the results suggested that miR­338­3p could suppress age­associated osteoporosis by regulating PCSK5.

SNP-adjacent super enhancer network mediates enhanced osteogenic differentiation of MSCs in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a rheumatic disease with pathological osteogenesis that causes bony ankylosis and even deformity over time. Mesenchymal stem cells (MSCs) are multipotent stem cells that are the main source of osteoblasts. We previously demonstrated that enhanced osteogenic differentiation of MSCs from AS patients (ASMSCs) is related to pathological osteogenesis in AS. However, the more concrete mechanism needs further exploration. Super enhancers (SEs) are dense clusters of stitched enhancers that control cell identity determination and disease development. Single-nucleotide polymorphisms (SNPs) regulate the formation and interaction of SEs and denote genes accounting for AS susceptibility. Via integrative analysis of multiomic data, including histone 3 lysine 27 acetylation (H3K27ac), chromatin immunoprecipitation sequencing (ChIP-seq), SNPs and RNA sequencing (RNA-seq) data, we discovered a transcription network mediated by AS SNP-adjacent SEs (SASEs) in ASMSCs and identified key genes, such as Toll-like receptor 4 (TLR4), interleukin 18 receptor 1 (IL18R1), insulin-like growth factor binding protein 4 (IGFBP4), transportin 1 (TNPO1) and proprotein convertase subtilisin/kexin type 5 (PCSK5), which are pivotal in osteogenesis and AS pathogenesis. The SASE-regulated network modulates the enhanced osteogenic differentiation of ASMSCs by synergistically activating the PI3K-Akt, NF-kappaB and Hippo signaling pathways. Our results emphasize the crucial role of the SASE-regulated network in pathological osteogenesis in AS, and the preferential inhibition of ASMSC osteogenic differentiation by JQ1 indicates that SEs may be attractive targets in future treatment for new bone formation in AS.

Smad3 signalling affects high glucose-induced podocyte injury via regulation of the cytoskeletal protein transgelin.

AIM: The aim of the present study was to characterize the role of Smad3 signalling on high glucose-induced podocyte injury. METHODS: Synchronized conditionally immortalized mouse podocyte cell line (MPC5) cells were treated with either D-glucose alone or D-glucose plus the Smad3 inhibitor SIS3. The distribution of F-actin and transgelin in a high glucose-induced model of podocyte injury were examined by immunofluorescence. Levels of transgelin and Smad3 signalling proteins in MPC5 cells were determined by Western blot. RESULTS: A disordered distribution of F-actin, as well as co-localization of F-actin and transgelin, was observed in podocytes exposed to high glucose. Increased levels of transgelin were first observed 10 minutes after treatment with glucose, suggesting that this protein is sensitive to hyperglycaemic injury. Levels of phosphorylated Smad3 and cleaved caspase 3 increased significantly with glucose stimulation. Moreover, expression of the downstream protein c-Myc, but not JAK1/STAT3, was induced in conditions of high glucose. The Smad3-specific inhibitor SIS3 prevented the effects of high glucose on Smad3 phosphorylation, expression of transgelin and c-Myc, caspase 3 cleavage and cytoskeletal organization. Expression of the tumour suppressor protein p15INK4B increased after podocyte injury but was unaffected by Smad3 inhibition, suggesting that Smad3 regulation of high glucose-induced podocyte injury occurs through a p15INK4B -independent mechanism. CONCLUSION: Smad3 signalling plays a critical role in the modulation of hyperglycaemic injury. Targeted inhibition of the Smad3 pathway may offer a novel route for treatment of podocyte damage, especially in cases of diabetic nephropathy.

Jagged1 promotes mineralization in human bone-derived cells.

OBJECTIVES: The present study aimed to investigate the expression of Notch signaling components during osteogenic differentiation in vitro and bone healing in vivo. In addition, the influence of Notch signaling on osteogenic differentiation of human bone-derived cells was examined. METHODS: Gene expression profiling of osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro (GSE80614) and bone healing period of murine tibial fracture in vivo (GSE99388) was downloaded from Gene Expression Omnibus database. The expression of Notch signaling components was obtained from bioinformatic tools. Human bone-derived cells were isolated from alveolar and iliac bone. Cells were seeded on Jagged1 immobilized surface. Osteogenic marker gene expression and mineralization were examined using real-time polymerase chain reaction and alizarin red s staining, respectively. RESULTS: From bioinformatic analysis of gene expression profiling, various Notch signaling components were differentially expressed during osteogenic differentiation of human bone marrow-derived mesenchymal stromal cells in vitro and bone healing period of murine tibial fracture in vivo. The common genes differentially regulated of these two datasets were Hes1, Aph1a, Nsctn, Furin, Adam17, Hey1, Pcsk5, Nedd4, Jag1, Heyl, Notch3, Dlk1, and Hey2. For an in vitro analysis, the mineral deposition markedly increased after seeding human bone-derived cells on Jagged1 immobilized surface, correspondingly with the increase of ALP mRNA expression. Jagged1 treatment downregulated TWIST2 mRNA expression in both human alveolar and iliac bone-derived cells. CONCLUSION: Notch signaling is regulated during osteogenic differentiation and bone healing. In addition, the activation of Notch signaling promotes osteogenic differentiation in human alveolar and iliac bone-derived cells. Therefore, Notch signaling manipulation could be a useful approach for enhancing bone regeneration.

Regulation of the proprotein convertases expression and activity during regenerative angiogenesis: Role of hypoxia-inducible factor (HIF).

Proprotein convertases (PCs) are involved in various physiological and pathological processes ranging from embryogenesis to carcinogenesis. Here, using the zebrafish fin regeneration model, we report induced expression of furin and PC5 but not PACE4 and PC7 during fin regeneration that is associated with increased PC activity. Stabilization of HIF by cobalt chloride (CoCl2) further increases these processes. The use of the general PC-inhibitor decanoyl-RVKR-cholromethyl ketone (CMK) inhibited control and CoCl2-induced PC activity. CoCl2 inhibits embryonic zebrafish ZF4 cell proliferation and caudal fin regeneration that is associated with the expression of the anti-proliferative genes P21, P16, PC3 and P53 in ZF4 cells and in non-regenerating stump tissues. In contrast, during fin regeneration, HIF stabilization failed to promote the expression of these anti-proliferative genes and maintained high expression of cyclin D. Further analysis revealed that CoCl2 maintained the formation of immature regenerating vasculature that was associated with amplified expression of OCT4 and various angiogenic factors reported to be PC substrates and/or downstream effectors. These findings revealed that while furin and PC5 expression/activity and their substrates/effectors are regulated during fin regeneration, HIF stabilization by CoCl2 has the potential to modulate these processes and impact on the regenerative process and vessels organization.

Thrombin activation of protein C requires prior processing by a liver proprotein convertase.

Protein C, a secretory vitamin K-dependent anticoagulant serine protease, inactivates factors Va/VIIIa. It is exclusively synthesized in liver hepatocytes as an inactive zymogen (proprotein C). In humans, thrombin cleavage of the propeptide at PR221↓ results in activated protein C (APC; residues 222-461). However, the propeptide is also cleaved by a furin-like proprotein convertase(s) (PCs) at KKRSHLKR199↓ (underlined basic residues critical for the recognition by PCs), but the order of cleavage is unknown. Herein, we present evidence that at the surface of COS-1 cells, mouse proprotein C is first cleaved by the convertases furin, PC5/6A, and PACE4. In mice, this cleavage occurs at the equivalent site, KKRKILKR198↓, and requires the presence of Arg198 at P1 and a combination of two other basic residues at either P2 (Lys197), P6 (Arg193), or P8 (Lys191) positions. Notably, the thrombin-resistant R221A mutant is still cleaved by these PCs, revealing that convertase cleavage can precede thrombin activation. This conclusion was supported by the fact that the APC-specific activity in the medium of COS-1 cells is exclusively dependent on prior cleavage by the convertases, because both R198A and R221A lack protein C activity. Primary cultures of hepatocytes derived from wild-type or hepatocyte-specific furin, PC5/6, or complete PACE4 knock-out mice suggested that the cleavage of overexpressed proprotein C is predominantly performed by furin intracellularly and by all three proprotein convertases at the cell surface. Indeed, plasma analyses of single-proprotein convertase-knock-out mice showed that loss of the convertase furin or PC5/6 in hepatocytes results in a ∼30% decrease in APC levels, with no significant contribution from PACE4. We conclude that prior convertase cleavage of protein C in hepatocytes is critical for its thrombin activation.

Posttranslational removal of α-dystroglycan N terminus by PC5/6 cleavage is important for uterine preparation for embryo implantation in women.

Embryo implantation requires a healthy embryo and a receptive endometrium (inner lining of the uterus); endometrial receptivity acquisition involves considerable epithelial surface remodeling. Dystroglycan (DG), a large cell surface glycoprotein, consists of α- and ß-subunits; ß-DG anchors within the plasma membrane whereas α-DG attaches extracellularly to ß-DG. The glycosylated central α-DG mediates adhesion, but it is obstructed by its large N terminus (α-DG-N); α-DG-N removal enables DG's adhesive function. We demonstrate here that full-length α-DG in the human endometrial epithelium is a barrier for embryo attachment and that removal of α-DG-N by proprotein convertase 5/6 (PC6; a protease critical for implantation) regulates receptivity. This was evidenced by: 1) α-DG contains a PC6-cleavage site near α-DG-N, and PC6 cleaves a peptide harboring such a site; 2) PC6 knockdown reduces α-DG-N removal from endometrial epithelial cell surface and blastocyst adhesion; 3) mutating the PC6-cleavage site prevents α-DG-N removal, causing cell surface retention of full-length α-DG and loss of adhesiveness; 4) α-DG-N is removed from endometrial tissue in vivo for receptivity and uterine fluid α-DG-N reflects tissue removal and receptivity. We thus identified α-DG-N removal as an important posttranslational control of endometrial receptivity and uterine fluid α-DG-N as a potential biomarker for receptivity in women.

Development of a high-throughput assay for human proprotein convertase 5/6 for detecting uterine receptivity.

Embryo implantation requires a healthy embryo and a receptive uterus. In women, the inner lining of the uterus, the endometrium, remains in a hostile state and becomes receptive for embryo implantation for only a short period during each menstrual cycle. Determining endometrial receptivity is vital in in vitro fertilization (IVF) treatment because the timing of embryo transfer needs to be synchronized with endometrial receptivity. We have previously demonstrated that proprotein convertase 5/6A (PC6) is highly expressed in the receptive endometrium and that PC6 is critical for receptivity establishment in women. Furthermore, endometrial PC6 is secreted into the uterine fluid, and levels correlate with receptivity status. Detection of PC6 in uterine fluids, therefore, would provide a nonsurgical assessment of endometrial receptivity. However, to date no assays are available for human PC6. In this study, we produced three PC6 monoclonal antibodies (mAbs) and developed a sandwich enzyme-linked immunosorbent assay (ELISA) for PC6 detection in human uterine fluids. The PC6 mAbs were confirmed to be highly specific to PC6, and the ELISA detected PC6 in human uterine fluids with a significantly higher level during the receptive phase. This newly established PC6 ELISA provides an important tool in the development of noninvasive strategies to detect endometrial receptivity in women.

Proprotein convertase 5/6 cleaves platelet-derived growth factor A in the human endometrium in preparation for embryo implantation.

Establishment of endometrial receptivity is vital for successful embryo implantation. Proprotein convertase 5/6 (referred to as PC6) is up-regulated in the human endometrium specifically at the time of epithelial receptivity. PC6, a serine protease of the proprotein convertase family, plays an important role in converting precursor proteins into their active forms through specific proteolysis. The proform of platelet-derived growth factor A (pro-PDGFA) requires PC cleavage to convert to the active-PDGFA. We investigated the PC6-mediated activation of PDGFA in the human endometrium during the establishment of receptivity. Proteomic analysis identified that the pro-PDGFA was increased in the conditioned medium of HEC1A cells in which PC6 was stably knocked down by small interfering RNA (PC6-siRNA). Western blot analysis demonstrated an accumulation of the pro-PDGFA but a reduction in the active-PDGFA in PC6-siRNA cell lysates and medium compared with control. PC6 cleavage of pro-PDGFA was further confirmed in vitro by incubation of recombinant pro-PDGFA with PC6. Immunohistochemistry revealed cycle-stage-specific localization of the active-PDGFA in the human endometrium. During the non-receptive phase, the active-PDGFA was barely detectable. In contrast, it was localized specifically to the apical surface of the luminal and glandular epithelium in the receptive phase. Furthermore, the active-PDGFA was detected in uterine lavage with levels being significantly higher in the receptive than the non-receptive phase. We thus identified that the secreted PDGFA may serve as a biomarker for endometrial receptivity. This is also the first study demonstrating that the active-PDGFA localizes to the apical surface of the endometrium during receptivity.

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.

Small molecule proprotein convertase inhibitors for inhibition of embryo implantation.

Uterine proprotein convertase (PC) 6 plays a critical role in embryo implantation and is pivotal for pregnancy establishment. Inhibition of PC6 may provide a novel approach for the development of non-hormonal and female-controlled contraceptives. We investigated a class of five synthetic non-peptidic small molecule compounds that were previously reported as potent inhibitors of furin, another PC member. We examined (i) the potency of these compounds in inhibiting PC6 activity in vitro; (ii) their binding modes in the PC6 active site in silico; (iii) their efficacy in inhibiting PC6-dependent cellular processes essential for embryo implantation using human cell-based models. All five compounds showed potent inhibition of PC6 activity in vitro, and in silico docking demonstrated that these inhibitors could adopt a similar binding mode in the PC6 active site. However, when these compounds were tested for their inhibition of decidualization of primary human endometrial stromal cells, a PC6-dependent cellular process critical for embryo implantation, only one (compound 1o) showed potent inhibition. The lack of activity in the cell-based assay may reflect the inability of the compounds to penetrate the cell membrane. Because compound's lipophilicity is linked to cell penetration, a measurement of lipophilicity (logP) was calculated for each compound. Compound 1o is unique as it appears the most lipophilic among the five compounds. Compound 1o also inhibited another crucial PC6-dependent process, the attachment of human trophoblast spheroids to endometrial epithelial cells (a model for human embryo attachment). We thus identified compound 1o as a potent small molecule PC6 inhibitor with pharmaceutical potential to inhibit embryo implantation. Our findings also highlight that human cell-based functional models are vital to complement the biochemical and in silico analyses in the selection of promising drug candidates. Further investigations for compound 1o are warranted in animal models to test its utility as an implantation-inhibiting contraceptive drug.

Proprotein convertases play an important role in regulating PKGI endoproteolytic cleavage and nuclear transport.

Nitric oxide and cGMP modulate vascular smooth muscle cell (SMC) phenotype by regulating cell differentiation and proliferation. Recent studies suggest that cGMP-dependent protein kinase I (PKGI) cleavage and the nuclear translocation of a constitutively active kinase fragment, PKGIγ, are required for nuclear cGMP signaling in SMC. However, the mechanisms that control PKGI proteolysis are unknown. Inspection of the amino acid sequence of a PKGI cleavage site that yields PKGIγ and a protease database revealed a putative minimum consensus sequence for proprotein convertases (PCs). Therefore we investigated the role of PCs in regulating PKGI proteolysis. We observed that overexpression of PCs, furin and PC5, but not PC7, which are all expressed in SMC, increase PKGI cleavage in a dose-dependent manner in human embryonic kidney (HEK) 293 cells. Moreover, furin-induced proteolysis of mutant PKGI, in which alanines were substituted into the putative PC consensus sequence, was decreased in these cells. In addition, overexpression of furin increased PKGI proteolysis in LoVo cells, which is an adenocarcinoma cell line expressing defective furin without PC activity. Also, expression of α1-PDX, an engineered serpin-like PC inhibitor, reduced PC activity and decreased PKGI proteolysis in HEK293 cells. Last, treatment of low-passage rat aortic SMC with membrane-permeable PC inhibitor peptides decreased cGMP-stimulated nuclear PKGIγ translocation. These data indicate for the first time that PCs have a role in regulating PKGI proteolysis and the nuclear localization of its active cleavage product, which are important for cGMP-mediated SMC phenotype.

Engineering of α1-antitrypsin variants with improved specificity for the proprotein convertase furin using site-directed random mutagenesis.

Furin, PACE4, PC5/6 and PC7 are members of the subtilisin-like proprotein convertase (SPC) family. Although these enzymes are known to play critical roles in various physiological and pathological events including cell differentiation, tumor growth, virus replication and the activation of bacterial toxins, their distinct functions are yet to be fully delineated. α1-PDX is an engineered α1-antitrypsin variant carrying the RXXR consensus motif for furin within its reactive site loop. However, α1-PDX inhibits other SPCs in addition to furin. In this work, we prepared various rat α1-antitrypsin variants containing Arg at the P1 site within the reactive site loop, and examined their respective selectivity. The novel α1-antitrypsin variant AVNR (AVPM(352)/AVNR) was identified as a highly selective inhibitor of furin. This variant formed a sodium dodecyl sulfate- and heat-stable furin/α1-antitrypsin complex and inhibited furin activity ex vivo and in vitro. Other SPC members including PACE4, PC5/6 and PC7 were not inhibited by the AVNR variant. Furin-mediated maturation of bone morphogenetic protein-4 was completely inhibited by ectopic expression of the AVNR variant. The AVNR variant should prove to be a useful inhibitor in identifying the specific role of furin.

Opposite roles of furin and PC5A in N-cadherin processing.

We recently demonstrated that lack of Furin-processing of the N-cadherin precursor (proNCAD) in highly invasive melanoma and brain tumor cells results in the cell-surface expression of a nonadhesive protein favoring cell migration and invasion in vitro. Quantitative polymerase chain reaction analysis of malignant human brain tumor cells revealed that of all proprotein convertases (PCs) only the levels of Furin and PC5A are modulated, being inversely (Furin) or directly (PC5A) correlated with brain tumor invasive capacity. Intriguingly, the N-terminal sequence following the Furin-activated NCAD site (RQKR↓DW(161), mouse nomenclature) reveals a second putative PC-processing site (RIRSDR↓DK(189)) located in the first extracellular domain. Cleavage at this site would abolish the adhesive functions of NCAD because of the loss of the critical Trp(161). This was confirmed upon analysis of the fate of the endogenous prosegment of proNCAD in human malignant glioma cells expressing high levels of Furin and low levels of PC5A (U343) or high levels of PC5A and negligible Furin levels (U251). Cellular analyses revealed that Furin is the best activating convertase releasing an ~17-kDa prosegment, whereas PC5A is the major inactivating enzyme resulting in the secretion of an ~20-kDa product. Like expression of proNCAD at the cell surface, cleavage of the NCAD molecule at RIRSDR↓DK(189) renders the U251 cancer cells less adhesive to one another and more migratory. Our work modifies the present view on posttranslational processing and surface expression of classic cadherins and clarifies how NCAD possesses a range of adhesive potentials and plays a critical role in tumor progression.

Cleavage of endometrial α-integrins into their functional forms is mediated by proprotein convertase 5/6.

BACKGROUND: Proprotein convertases (PCs) post-translationally activate a large number of protein precursors through limited cleavage. PC5/6 (PC6) in the human endometrium is tightly regulated during receptivity for embryo implantation. Integrins are transmembrane glycoproteins, some of which play an important role in the adhesive interactions between the trophoblast (blastocyst) and uterine epithelium at implantation. Integrins require PC cleavage for post-translational modification. We hypothesize that pro-integrin-αs in the endometrial epithelium are post-translationally cleaved by PC6 into functional subunits for the binding of blastocyst and adhesion of extracellular matrix proteins. METHODS AND RESULTS: We first used the endometrial epithelial cell line, HEC1A, into which siRNA specific to human PC6 (PC6-siRNA) or scrambled sequence (control) was stably transfected. The specific knockdown was confirmed by real-time RT-PCR. PC6-siRNA cells reduced their capacity to attach to trophoblast spheroids and bind to fibronectin compared with control. Knockdown of PC6 decreased cell surface presentation of functional integrins-α1, α2, α5, αV and αVß5. Western blot analysis demonstrated that PC6 was responsible for the post-translational cleavage of pro-integrin-α5 and integrin-αV into their heavy and light chains in HEC1A cells. We then isolated primary human endometrial epithelial cells and validated that PC6 mediated the post-translational cleavage of integrin-αs in these cells. CONCLUSIONS: This study implicates PC6 as a key regulatory protein essential for the attachment of the blastocyst to the endometrial epithelium through the processing of pro-integrin-αs. Compromised PC6 action reduces the post-translational modification of integrin-αs, thus compromising implantation.

Inhibition of proprotein convertase 5/6 activity: potential for nonhormonal women-centered contraception.

BACKGROUND: Proprotein convertase 5/6 (PC6) is critical for endometrial epithelial receptivity and stromal cell decidualization for embryo implantation in women. We hypothesized that inhibiting PC6 could block implantation for contraception. The aim of this study was to prove this concept using human cell models and rabbits. STUDY DESIGN: A potential PC6 inhibitor, C1239-PEG-Poly R, was biochemically confirmed to be a potent PC6 inhibitor. The potential contraceptive action of the inhibitor was then tested in decidualization of primary human endometrial stromal cells in a human trophoblast spheroid attachment model and in vivo in rabbits. RESULTS: The PC6 inhibitor C1239-PEG-Poly R inhibited in a dose-dependent manner both decidualization and spheroid attachment. Vaginal delivery of 200 µL of the inhibitor at a final concentration of 5 mM to rabbits over a 3-day period starting 6 days after mating resulted in a 60% decrease in implantation and, hence, pregnancy. CONCLUSIONS: This study presents proof of concept that PC6 inhibition has the potential to block embryo implantation, providing nonhormonal contraception for women.

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.