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.

Spatiotemporal expression of proprotein convertase subtilisin/kexin type 5 in the development of anorectal malformations in fetal rats.

This study examined the expression patterns of proprotein convertase subtilisin/kexin type 5 (Pcsk5) during anorectal development in normal and anorectal malformations (ARM) rat embryos, determine the possible role of Pcsk5 in the pathogenesis of ARM. An ARM rat model was developed by the administration of ethylenethiourea gestational day 10 (GD10). Embryos were harvested by surgical excision from GD13 to GD16, and the spatiotemporal expression of Pcsk5 was evaluated, using immunohistochemistry staining, Western blotting and real time RT-PCR. Immunohistochemistry staining in normal embryos revealed that Pcsk5 was abundantly expressed on the epithelium of the cloaca (CL) on GD13. On GD14 and GD15, positive cells were noted on the urorectal septum and the thin anal membrane. However, the epithelium of the CL of ARM embryos only faintly expressed Pcsk5 from GD13 to GD15. Western blotting and real time RT-PCR showed time-dependent increase of Pcsk5 expression in the developing hindgut. Pcsk5 expression levels were lower in the ARM group from GD14 to GD16 (p ≤ 0.05). These results indicate that downregulation of Pcsk5 during cloaca development into the rectum and urethra might be related to the formation of ARMs.

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.

Pcsk5 is required in the early cranio-cardiac mesoderm for heart development.

BACKGROUND: Loss of proprotein convertase subtilisin/kexin type 5 (Pcsk5) results in multiple developmental anomalies including cardiac malformations, caudal regression, pre-sacral mass, renal agenesis, anteroposterior patterning defects, and tracheo-oesophageal and anorectal malformations, and is a model for VACTERL/caudal regression/Currarino syndromes (VACTERL association - Vertebral anomalies, Anal atresia, Cardiac defects, Tracheoesophageal fistula and/or Esophageal atresia, Renal & Radial anomalies and Limb defects). RESULTS: Using magnetic resonance imaging (MRI), we examined heart development in mouse embryos with zygotic and cardiac specific deletion of Pcsk5. We show that conditional deletion of Pcsk5 in all epiblastic lineages recapitulates all developmental malformations except for tracheo-esophageal malformations. Using a conditional deletion strategy, we find that there is an essential and specific requirement for Pcsk5 in the cranio-cardiac mesoderm for cardiogenesis, but not for conotruncal septation or any other aspect of embryonic development. Surprisingly, deletion of Pcsk5 in cardiogenic or pharyngeal mesodermal progenitors that form later from the cranio-cardiac mesoderm does not affect heart development. Neither is Pcsk5 essential in the neural crest, which drives conotruncal septation. CONCLUSIONS: Our results suggest that Pcsk5 may have an essential and early role in the cranio-cardiac mesoderm for heart development. Alternatively, it is possible that Pcsk5 may still play a critical role in Nkx2.5-expressing cardiac progenitors, with persistence of mRNA or protein accounting for the lack of effect of deletion on heart development.

PCSK5 mutation in a patient with the VACTERL association.

BACKGROUND: The VACTERL association is a typically sporadic, non-random collection of congenital anomalies that includes vertebral defects, anal atresia, cardiac defects, tracheoesophageal fistula with esophageal atresia, renal anomalies, and limb abnormalities. Although several chromosomal aberrations and gene mutations have been reported as disease-causative, these findings have been sparsely replicated to date. CASE PRESENTATION: In the present study, whole exome sequencing of a case with the VACTERL association uncovered a novel frameshift mutation in the PCSK5 gene, which has been reported as one of the causative genes for the VACTERL association. Although this mutation appears potentially pathogenic in its functional aspects, it was also carried by the healthy father. Furthermore, a database survey revealed several other deleterious variants in the PCSK5 gene in the general population. CONCLUSIONS: Further studies are necessary to clarify the etiological role of the PCSK5 mutation in the VACTERL association.

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.

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.

Furin is the primary in vivo convertase of angiopoietin-like 3 and endothelial lipase in hepatocytes.

The proprotein convertases (PCs) furin, PC5/6, and PACE4 exhibit unique and/or complementary functions. Their knock-out (KO) in mice resulted in strong and specific phenotypes demonstrating that, in vivo, these PCs are unique and essential during development. However, they also exhibit redundant functions. Liver angiopoietin-like 3 (ANGPTL3) inhibits lipolysis by binding to lipoprotein lipases. It is found in the plasma as full length and truncated forms. The latter is more active and generated by cleavage at a furin-like site. Endothelial lipase (EL) binds heparin sulfate proteoglycans on cell surfaces and catalyzes the hydrolysis of HDL phospholipids. EL activity is regulated by two endogenous inhibitors, ANGPTL3 and ANGPTL4, and by PCs that inactivate EL through cleavage releasing the N-terminal catalytic and C-terminal lipid-binding domains. Herein, because furin and PC5/6 complete KOs are lethal, we used mice lacking furin or PC5/6 specifically in hepatocytes (hKO) or mice completely lacking PACE4. In primary hepatocytes, ANGPTL3 was processed into a shorter form of ANGPTL3 intracellularly by furin only, and extracellularly mainly by PACE4. In vivo, the absence of furin in hepatocytes reduced by ∼50% the circulating levels of cleaved ANGPTL3, while the lack of PACE4 had only a minor effect. Analysis of the EL processing in primary hepatocytes and in vivo revealed that it is mostly cleaved by furin. However, the lack of furin or PC5/6 in hepatocytes and complete PACE4 KO did not appreciably modify plasma HDL levels or EL activity. Thus, inhibition of furin in liver would not be expected to modify the plasma lipid profiles.

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.

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.

HMC-1 human mast cells synthesize neurotensin (NT) precursor, secrete bioactive NT-like peptide(s) and express NT receptor NTS1.

OBJECTIVE AND DESIGN: To determine if mast cells synthesize the inflammatory peptide, neurotensin (NT), secrete immunoreactive and bioactive NT, and express the NT receptor NTS1. MATERIALS: HMC-1 cells, pleural mast cells from Sprague-Dawley rats, LAD2 mast cells, and human cord blood mast cells were used. TREATMENT: HMC-1 cells were stimulated with NT, C48/80, mastoparan, or PGE(2). For changes in cutaneous vascular permeability, anesthetized rats were injected intravenously with Evans Blue dye and intradermally with saline, NT, histamine, diphenhydramine, and C48/80. METHODS: RT-PCR was used to identify RNA transcripts. Histamine was measured by fluorometric assay. In vivo cutaneous vascular permeability assays, radio-immunoassays for NT, Western blotting for the NT precursor protein and NTS1 protein from HMC-1 cells and tissues from rats were used. Immunohistochemistry was used to identify NT precursor-like proteins in HMC-1 mast cells. RESULTS: HMC-1 cells express mRNAs for NT precursor, PC5A processing enzyme and NTS1 receptor. Human cord blood mast cells and LAD2 mast cells express mRNA transcripts for NT precursor and NTS1. Western blotting showed NT precursor and NTS1 receptor in HMC1. Rat tissues with high numbers of mast cells contained NT precursor proteins. NT-like peptides from HMC-1 displayed NT-like bioactivity. CONCLUSIONS: HMC-1 mast cells synthesize and secrete immunoreactive and bioactive NT-like peptide(s) and express the NT receptor, suggesting that NT from mast cells might serve autocrine and paracrine roles.

Use of zebrafish and knockdown technology to define proprotein convertase activity.

The Zebrafish (Danio rerio) is a powerful and well-established tool used extensively for the study of early vertebrate development and as a model of human diseases. Zebrafish genes orthologous to their mammalian counterparts generally share conserved biological function. Protein knockdown or overexpression can be effectively achieved by microinjection of morpholino antisense oligonucleotides (MOs) or mRNA, respectively, into developing embryos at the one- to two-cell stage. Correlating gene expression patterns with the characterizing of phenotypes resulting from over- or underexpression can reveal the function of a particular protein. The microinjection technique is simple and results are reproducible. We defined the expression pattern of the proprotein convertase PCSK5 within the lateral line neuromasts and various organs including the liver, gut and otic vesicle by whole-mount in situ hybridization (ISH) and immunofluorescence (IF). MO-mediated knockdown of zebrafish PCSK5 expression generated embryos that display abnormal neuromast deposition within the lateral line system resulting in uncoordinated patterns of swimming.

Inactivation of endothelial proprotein convertase 5/6 decreases collagen deposition in the cardiovascular system: role of fibroblast autophagy.

Proprotein convertase (PC) 5/6 belongs to a family of secretory proteases involved in proprotein proteolysis. Several studies suggest a role for PC5/6 in cardiovascular disease. Because lethality at birth of mice lacking PC5/6 precluded elucidation of its function in the adult, we generated mice in which the gene of PC5/6 (pcsk5) is specifically inactivated in endothelial cells (ecKO), which are viable and do not exhibit overt abnormalities. In order to uncover the function of PC5/6 in the cardiovascular system, the effect of ecKO was studied in aging mice. In 16 to 18-month-old ecKO mice, the left ventricle (LV) mass, media cross-sectional area of aorta and coronary arteries, and media-to-lumen ratio of mesenteric arteries were decreased. The LV presented decreased diastolic function, and mesenteric arteries showed decreased stiffness. Collagen was decreased in the LV myocardial interstitium and perivascularly in coronary arteries and aorta. Cardiovascular hypotrophy likely develops with aging, since no significant changes were observed in 2-month-old ecKO mice. Fibroblasts, as a source of collagen in myocardium and vasculature, may play a role in the decrease in collagen deposition. Fibroblasts co-cultured with ecKO endothelial cells showed decreased collagen production, decreased insulin-like growth factor (IGF)-1/Akt/mTOR signaling, and enhanced autophagic activation. PC5/6 inactivation in endothelial cells results in cardiovascular hypotrophy associated with decreased collagen deposition, decreased LV diastolic function, and vascular stiffness, suggesting a trophic role of endothelial PC5/6 in the cardiovascular system, likely mediated by IGF-1/Akt/mTOR signaling and control of autophagy.

Latent transforming growth factor beta-binding proteins-2 and -3 inhibit the proprotein convertase 5/6A.

The basic amino acid-specific proprotein convertase 5/6 (PC5/6) is an essential secretory protease, as knock-out mice die at birth and exhibit multiple homeotic transformation defects, including impaired bone morphogenesis and lung structure. Some of the observed defects were attributed to impaired processing of the TGFß-like growth differentiating factor 11 precursor (proGdf11). In this work we present evidence that the latent TGFß-binding proteins 2 and 3 (LTBP-2 and -3) inhibit the extracellular processing of proGdf11 by PC5/6A. This is partly due to the binding of LTBPs in the endoplasmic reticulum to the zymogen proPC5/6A, thus allowing the complex to exit the endoplasmic reticulum and be sequestered as an inactive zymogen in the extracellular matrix but not at the cell surface. This results in lower levels of PC5/6A in the media, without affecting those of PACE4, Furin, or a soluble form of PC7. The secreted soluble protease-specific activity of PC5/6A or a variant lacking the C-terminal Cys-rich domain (PC5/6-ΔCRD) is significantly decreased when co-expressed with LTBPs in cells. A similar enzymatic inhibition seems to apply to PACE4 and Furin. In situ hybridization analyses revealed extensive co-localization of PC5/6 and LTBP-3 mRNAs in mice at embryonic day 15.5 and post partum day 1. In conclusion, this is the first time that a zymogen of the proprotein convertases was shown to exit the endoplasmic reticulum in the presence of LTBPs, representing a potential novel mechanism for the regulation of PC5/6A activity, e.g. in tissues such as bone and lung where LTBP-3 and PC5/6 co-localize.

Embryo implantation is closely associated with dynamic expression of proprotein convertase 5/6 in the rabbit uterus.

BACKGROUND: Proprotein convertase 5/6 (PC5/6) is critical for embryo implantation in women, regulating both uterine epithelial receptivity and stromal cell decidualization. PC5/6 is likewise essential for implantation in mice, but involved only in decidualization. An alternative animal model is required to address the function of PC5/6 in the uterine epithelium. This study aimed to establish whether PC5/6 is associated with embryo implantation in rabbits. METHODS: Virgin New-Zealand white rabbits aged 3-4 moths were mated with males of the same strain, or pseudo-pregnancy induced. After mating, uterine tissues were collected over a 10 day (d) period (n = 3 per time point) for RNA, protein and histological analyses to determine the temporal and spatial uterine expression pattern of PC5/6 during the initial stages of pregnancy or induced pseudo-pregnancy. RESULTS: PC5/6 mRNA was up-regulated just prior to embryo attachment on d6, and the elevated expression was maintained throughout implantation on d6.5-10. Western analysis revealed a preferential up-regulation of PC5/6 in the implantation sites. Immunohistochemical analysis identified that both the amount and cellular localization of PC5/6 changed with increasing pregnancy stages. Before embryo attachment, PC5/6 was low and localised in the luminal and glandular epithelium. It increased on d6.5 in the basal glands and mucosal folds, and then strongly intensified on d7-10 in the multinucleated luminal symplasma and decidual cells at the site of embryo implantation. In contrast, the pseudo-pregnant uterus displayed relatively low and static PC6 mRNA expression throughout the 10 days, with no obvious changes in either PC5/6 level or cellular localization. CONCLUSIONS: These findings demonstrate that embryo implantation in the rabbit is closely associated with dynamic expression of uterine PC5/6, and that the rabbit may be an appropriate model to investigate the function of PC5/6 in the uterine epithelium during embryo attachment.

Role of PCSK5 expression in mouse ovarian follicle development: identification of the inhibin α- and ß-subunits as candidate substrates.

Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFß) superfamily. Inhibin is a heterodimer of α- and ß-subunits, whereas activin is a homodimer of ß-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase) that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and ß-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK), a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and ß-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and ß(B)-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability.