Search

Epithelial sodium channel (ENaC) are integral to maintaining salt and water homeostasis in various biological tissues, including the kidney, lung, and colon. They enable the selective reabsorption of sodium ions, which is a process critical for controlling blood pressure, electrolyte balance, and overall fluid volume. ENaC activity is finely controlled through proteolytic activation, a process wherein specific enzymes, or proteases, cleave ENaC subunits, resulting in channel activation and increased sodium reabsorption. This regulatory mechanism plays a pivotal role in adapting sodium transport to different physiological conditions. In this review article, we provide an in-depth exploration of the role of proteolytic activation in regulating ENaC activity. We elucidate the involvement of various proteases, including furin-like convertases, cysteine, and serine proteases, and detail the precise cleavage sites and regulatory mechanisms underlying ENaC activation by these proteases. We also discuss the physiological implications of proteolytic ENaC activation, focusing on its involvement in blood pressure regulation, pulmonary function, and intestinal sodium absorption. Understanding the mechanisms and consequences of ENaC proteolytic activation provides valuable insights into the pathophysiology of various diseases, including hypertension, pulmonary disorders, and various gastrointestinal conditions. Moreover, we discuss the potential therapeutic avenues that emerge from understanding these mechanisms, offering new possibilities for managing diseases associated with ENaC dysfunction. In summary, this review provides a comprehensive discussion of the intricate interplay between proteases and ENaC, emphasizing the significance of proteolytic activation in maintaining sodium and fluid balance in both health and disease. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostasin and matriptase are serine proteases co-expressed on the cell membrane in almost all epithelial cells. They reciprocally activate each other to maintain epithelial integrity. In cancers, matriptase is an oncoprotein with key roles in tumor initiation and progression, whereas prostasin acts in the opposite way. A subgroup of Burkitt lymphoma ectopically over-express matriptase without co-expressing prostasin. Reducing the matriptase expression level via small interfering RNAs in the lymphoma cells reduced tumor growth in vitro and in vivo. We hypothesized that an endowment of prostasin in the lymphoma cells can regulate the expression and function of matriptase. We show that prostasin can be introduced to the cancer cells via exosomes to initiate the prostasin–matriptase protease activation cascade and remove matriptase. The method of assembling this protease cascade in B cells via exosomes could be further exploited in animal models for developing alternative treatments for lymphoma. Prostasin and matriptase are extracellular membrane serine proteases with opposing effects in solid epithelial tumors. Matriptase is an oncoprotein that promotes tumor initiation and progression, and prostasin is a tumor suppressor that reduces tumor invasion and metastasis. Previous studies have shown that a subgroup of Burkitt lymphoma have high levels of ectopic matriptase expression but no prostasin. Reducing the matriptase level via small interfering RNAs in B lymphoma cells impeded tumor xenograft growth in mice. Here, we report a novel approach to matriptase regulation in B cancer cells by prostasin via exosomes to initiate a prostasin–matriptase protease activation cascade. The activation and shedding of matriptase were monitored by measuring its quantity and trypsin-like serine protease activity in conditioned media. Sustained activation of the protease cascade in the cells was achieved by the stable expression of prostasin. The B cancer cells with prostasin expression presented phenotypes consistent with its tumor suppressor role, such as reduced growth and increased apoptosis. Prostasin exosomes could be developed as an agent to initiate the prostasin–matriptase cascade for treating B lymphoma with further studies in animal models. [ABSTRACT FROM AUTHOR]

Hepatocyte growth factor activator inhibitor (HAI)-2 is an integral membrane Kunitz-type serine protease inhibitor that regulates the proteolysis of matriptase and prostasin in a cell-type selective manner. The cell-type selective nature of HAI-2 function depends largely on whether the inhibitor and potential target enzymes are targeted to locations in close proximity. The N -glycan moiety of HAI-2 can function as a subcellular targeting signal. HAI-2 is synthesized with 1 of 2 different N -glycan modifications: one of oligomannose-type, which largely remains in the endoplasmic reticulum/GA, and another of complex-type, which is targeted toward the apical surface in vesicle-like structures, and could function as an inhibitor of matriptase and prostasin. HAI-2 contains 2 putative N-glycosylation sites, Asn-57 and Asn-94, point mutations of which were generated and characterized in this study. The protein expression profile of the HAI-2 mutants indicates that Asn-57, and not Asn-94, is responsible for the N-glycosylation of both HAI-2 species, suggesting that the form with oligomannose-type N -glycan is the precursor of the form with complex-type N -glycan. Unexpectedly, the vast majority of non-glycosylated HAI-2 is synthesized into multiple disulfide-linked oligomers, which lack protease inhibitory function, likely due to distorted conformations caused by the disarrayed disulfide linkages. Although forced expression of HAI-2 in HAI-2 knockout cells artificially enhances HAI-2 oligomerization, disulfide-linked HAI-2 oligomers can also be observed in unmodified cells. These results suggest that N-glycosylation on Asn-57 is required for folding into a functional HAI-2 with full protease suppressive activity and correct subcellular targeting signal. [ABSTRACT FROM AUTHOR]

Background: A wide range of disorders can be detected in the urine. Tumor-modifying proteins in the urine may serve as a diagnostic tool for cancer patients and the alterations in their profiles may indicate efficacies of chemotherapy, radiotherapy, and surgery. Methods: We focused on urinary proteomes of patients with prostate cancer and identified tumor-modifying proteins in the samples before and after prostatectomy. Protein array analysis was conducted to evaluate a differential profile of tumor-promoting cytokines, while mass spectrometry-based global proteomics was conducted to identify tumor-suppressing proteins. Results: The result revealed striking differences by prostatectomy. Notably, the urine from the post-prostatectomy significantly decreased the tumorigenic behaviors of prostate tumor cells as well as breast cancer cells. We observed that angiogenin, a stimulator of blood vessel formation, was reduced in the post-prostatectomy urine. By contrast, the levels of three cell-membrane proteins such as prostasin (PRSS8), nectin 2 (PVRL2), and nidogen 1 (NID1) were elevated and they acted as extracellular tumor-suppressing proteins. These three proteins, given extracellularly, downregulated tumorigenic genes such as Runx2, Snail, and transforming growth factor beta and induced apoptosis of tumor cells. However, the role of NID1 differed depending on the location, and intracellular NID1 was tumorigenic and reduced the percent survival. Conclusions: This study demonstrated that prostatectomy remarkably altered the profile of urinary proteomes, and the post-prostatectomy urine provided tumor-suppressive proteomes. The result sheds novel light on the dynamic nature of the urinary proteomes and a unique strategy for predicting tumor suppressors. [ABSTRACT FROM AUTHOR]

Aims/hypothesis: Diabetes is associated with an increased risk of cancer. Prostasin is an epithelial sodium channel stimulator that has been associated with suppression of tumours, glucose metabolism and hyperglycaemia-associated tumour pathology. However, the association between prostasin, diabetes and cancer mortality has not been well investigated in humans. We aim to investigate the associations between plasma prostasin and diabetes, and to explore whether prostasin has an effect on cancer mortality risk in individuals with hyperglycaemia. Methods: Plasma prostasin was measured using samples from the Malmö Diet and Cancer Study Cardiovascular Cohort, and statistical analysis was performed from both sex-specific quartiles and per 1 SD. The cross-sectional association between plasma prostasin and diabetes was first studied in 4658 participants (age 57.5 ± 5.9 years, 39.9% men). After excluding 361 with prevalent diabetes, the associations of prostasin with incident diabetes and cancer mortality risk were assessed using Cox regression analysis. The interactions between prostasin and blood glucose levels as well as other covariates were tested. Results: The adjusted OR for prevalent diabetes in the 4th vs 1st quartile of prostasin concentrations was 1.95 (95% CI 1.39, 2.76) (p for trend <0.0001). During mean follow-up periods of 21.9 ± 7.0 and 23.5 ± 6.1 years, respectively, 702 participants developed diabetes and 651 died from cancer. Prostasin was significantly associated with the incidence of diabetes. The adjusted HR for diabetes in the 4th vs 1st quartile of prostasin concentrations was 1.76 (95% CI 1.41, 2.19) (p for trend <0.0001). Prostasin was also associated with cancer mortality There was a significant interaction between prostasin and fasting blood glucose for cancer mortality risk (p for interaction =0.022), with a stronger association observed in individuals with impaired fasting blood glucose levels at baseline (HR per 1 SD change 1.52; 95% CI 1.07, 2.16; p=0.019). Conclusions/interpretation: Plasma prostasin levels are positively associated with diabetes risk and with cancer mortality risk, especially in individuals with high blood glucose levels, which may shed new light on the relationship between diabetes and cancer. [ABSTRACT FROM AUTHOR]

Objective: The study was performed to investigate the association of hypertension in pregnancy with prostasin gene polymorphism in Pakistani females. Methods: This case-control study was performed at University of Karachi, Pakistan from April 2018 to May 2019. A total of 160 females, including 90 hypertensives and 70 healthy pregnant females, were recruited by purposive sampling after obtaining informed written consent. Genotyping was performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). Results: The frequencies of the TC and CC genotypes were higher in hypertensive pregnant females compared to healthy controls. A significant difference was evident for CC (P=0.012) genotype; however, no significant difference was observed for TC (P=0.49) and TT genotypes (P=0.06) between control and hypertensive groups. The adjusted odds ratio for CC genotype was 6.2 (P=0.025) and 1.48 (P=0.44) for TC genotype compared to the TT genotype. There was a significantly higher prevalence of the C allele of the prostasin gene at rs12597511 in the hypertensive group, suggesting that this allele is a risk factor for hypertension and cardiovascular diseases. Conclusion: C allele at rs12597511 of prostasin gene demonstrate as a risk factor for having hypertension in pregnancy. [ABSTRACT FROM AUTHOR]

Copyright of Saudi Medical Journal is the property of Saudi Medical Journal and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Extracellular matrix (ECM) assembly and remodelling is critical during development and organ morphogenesis. Dysregulation of ECM is implicated in many pathogenic conditions, including cancer. The type II transmembrane serine protease matriptase and the serine protease prostasin are key factors in a proteolytic cascade that regulates epithelial ECM differentiation during development in vertebrates. Here, we show by rescue experiments that the Drosophila proteases Notopleural (Np) and Tracheal-prostasin (Tpr) are functional homologues of matriptase and prostasin, respectively. Np mediates morphogenesis and remodelling of apical ECM during tracheal system development and is essential for maintenance of the transepithelial barrier function. Both Np and Tpr degrade the zona pellucida-domain (ZP-domain) protein Dumpy, a component of the transient tracheal apical ECM. Furthermore, we demonstrate that Tpr zymogen and the ZP domain of the ECM protein Piopio are cleaved by Np and matriptase in vitro. Our data indicate that the evolutionarily conserved ZP domain, present in many ECM proteins of vertebrates and invertebrates, is a novel target of the conserved matriptase-prostasin proteolytic cascade. [ABSTRACT FROM AUTHOR]

Matriptase and prostasin, acting as a tightly coupled proteolytic cascade, were reported to be required for epidermal barrier formation in mouse skin. Here we show that, in human skin, matriptase and prostasin are expressed with an inverse pattern over the course of differentiation. Matriptase was detected primarily in epidermal basal keratinocytes and the basaloid cells in the outer root sheath of hair follicles and the sebaceous gland, where prostasin was not detected. In contrast, prostasin was detected primarily in differentiated cells in the epidermal granular layer, the inner root sheath of hair follicles, and the sebaceous gland, where matriptase expression is negligible. While co-expressed in the middle stage of differentiation, prostasin was detected as polarized patches, and matriptase at intercellular junctions. Targeting to different subcellular localizations is also observed in HaCaT human keratinocytes, in which matriptase was detected primarily at intercellular junctions, and prostasin primarily on membrane protrusion. Furthermore, upon induction of zymogen activation, free active prostasin remains cell-associated and free active matriptase is rapidly shed into the extracellular milieu. Our data suggest that matriptase and prostasin likely function as independent entities in human skin rather than as a tightly coupled proteolytic cascade as observed in mouse skin.

Prostasin (CAP1/PRSS8) is a glycosylphosphatidylinositol (GPI)-anchored serine protease that is essential for epithelial development and overall survival in mice. Prostasin is regulated primarily by the transmembrane serine protease inhibitor, hepatocyte growth factor activator inhibitor (HAI)-2, and loss of HAI-2 function leads to early embryonic lethality in mice due to an unregulated prostasin activity. We have recently reported that critical in vivo functions of prostasin can be performed by proteolytically-inactive or zymogen-locked variants of the protease. Here we show that the zymogen form of prostasin does not bind to HAI-2 and, as a result, loss of HAI-2 does not affect prenatal development and survival of mice expressing only zymogen-locked variant of prostasin (Prss8 R44Q). Indeed, HAI-2-deficient mice homozygous for R44Q mutation (Spint2-/-;Prss8R44Q/R44Q) are born in the expected numbers and do not exhibit any obvious developmental abnormality at birth. However, postnatal growth in these mice is severely impaired and they all die within 4 to 7 days after birth due to a critical failure in the development of small and large intestines, characterized by a widespread villous atrophy, tufted villi, near-complete loss of mucin-producing goblet cells, loss of colonic crypt structure, and bleeding into the intestinal lumen. Intestines of Spint2-/-;Prss8R44Q/R44Q mice showed altered expression of epithelial junctional proteins, including reduced levels of EpCAM, E-cadherin, occludin, claudin-1 and -7, as well as an increased level of claudin-4, indicating that the loss of HAI-2 compromises intestinal epithelial barrier function. Our data indicate that the loss of HAI-2 in Prss8R44Q/R44Q mice leads to development of progressive intestinal failure that at both histological and molecular level bears a striking resemblance to human congenital tufting enteropathy, and may provide important clues for understanding and treating this debilitating human disease. [ABSTRACT FROM AUTHOR]

The membrane-bound serine proteases prostasin and matriptase and the Kunitz-type protease inhibitors HAI-1 and HAI-2 are all expressed in human skin and may form a tightly regulated proteolysis network, contributing to skin pathophysiology. Evidence from other systems, however, suggests that the relationship between matriptase and prostasin and between the proteases and the inhibitors can be context-dependent. In this study the in vivo zymogen activation and protease inhibition status of matriptase and prostasin were investigated in the human skin. Immunohistochemistry detected high levels of activated prostasin in the granular layer, but only low levels of activated matriptase restricted to the basal layer. Immunoblot analysis of foreskin lysates confirmed this in vivo zymogen activation status and further revealed that HAI-1 but not HAI-2 is the prominent inhibitor for prostasin and matriptase in skin. The zymogen activation status and location of the proteases does not support a close functional relation between matriptase and prostasin in the human skin. The limited role for HAI-2 in the inhibition of matriptase and prostasin is the result of its primarily intracellular localization in basal and spinous layer keratinocytes, which probably prevents the Kunitz inhibitor from interacting with active prostasin or matriptase. In contrast, the cell surface expression of HAI-1 in all viable epidermal layers renders it an effective regulator for matriptase and prostasin. Collectively, our study suggests the importance of tissue distribution and subcellular localization in the functional relationship between proteases and protease inhibitors. [ABSTRACT FROM AUTHOR]

Background: Clostridium difficile is the causative agent of C. difficile infection (CDI) that could be manifested by diarrhea, pseudomembranous colitis or life-threatening toxic megacolon. The spread of certain strains represents a significant economic burden for health-care. The epidemic successful strains are also associated with severe clinical features of CDI. Therefore, a proteomic study has been conducted that comprises proteomes released from in vitro cultured panel of eight different PCR ribotypes (RTs) and employs the combination of shotgun proteomics and label-free quantification (LFQ) approach. Results: The comparative semi-quantitative analyses enabled investigation of a total of 662 proteins. Both hierarchical clustering and principal component analysis (PCA) created eight distinctive groups. From these quantifiable proteins, 27 were significantly increased in functional annotations. Among them, several known factors connected with virulence were identified, such as toxin A, B, binary toxin, flagellar proteins, and proteins associated with Pro-Pro endopeptidase (PPEP-1) functional complex. Comparative analysis of protein expression showed a higher expression or unique expression of proteins linked to pathogenicity or iron metabolism in RTs 027 and 176 supporting their genetic relatedness and clinical importance at the proteomic level. Moreover, the absence of putative nitroreductase and the abundance of the Abc-type fe3+ transport system protein were observed as biomarkers for the RTs possessing binary toxin genes (027, 176 and 078). Higher expression of selected flagellar proteins clearly distinguished RTs 027, 176, 005 and 012, confirming the pathogenic role of the assembly in CDI. Finally, the histidine synthesis pathway regulating protein complex HisG/HisZ was observed only in isolates possessing the genes for toxin A and B. Conclusions: This study showed the applicability of the LFQ approach and provided the first semi-quantitative insight into the proteomes released from in vitro cultured panel of eight RTs. The observed differences pointed to a new direction for studies focused on the elucidation of the mechanisms underlining the CDI nature. [ABSTRACT FROM AUTHOR]

The membrane-associated serine proteases matriptase and prostasin are believed to function in close partnership. Their zymogen activation has been reported to be tightly coupled, either as a matriptase-initiated proteolytic cascade or through a mutually dependent mechanism involving the formation of a reciprocal zymogen activation complex. Here we show that this putative relationship may not apply in the context of human matriptase and prostasin. First, the tightly coupled proteolytic cascade between matriptase and prostasin might not occur when modest matriptase activation is induced by sphingosine 1-phospahte in human mammary epithelial cells. Second, prostasin is not required and/or involved in matriptase autoactivation because matriptase can undergo zymogen activation in cells that do not endogenously express prostasin. Third, matriptase is not required for and/or involved in prostasin activation, since activated prostasin can be detected in cells expressing no endogenous matriptase. Finally, matriptase and prostasin both undergo zymogen activation through an apparently un-coupled mechanism in cells endogenously expressing both proteases, such as in Caco-2 cells. In these human enterocytes, matriptase is detected primarily in the zymogen form and prostasin predominantly as the activated form, either in complexes with protease inhibitors or as the free active form. The negligible levels of prostasin zymogen with high levels of matriptase zymogen suggests that the reciprocal zymogen activation complex is likely not the mechanism for matriptase zymogen activation. Furthermore, high level prostasin activation still occurs in Caco-2 variants with reduced or absent matriptase expression, indicating that matriptase is not required and/or involved in prostasin zymogen activation. Collectively, these data suggest that any functional relationship between natural endogenous human matriptase and prostasin does not occur at the level of zymogen activation. [ABSTRACT FROM AUTHOR]

Rationale: In cystic fibrosis (CF) a reduction in airway surface liquid (ASL) height compromises mucociliary clearance, favoring mucus plugging and chronic bacterial infection. Inhibitors of the epithelial sodium channel (ENaC) have therapeutic potential in CF airways to reduce hyperstimulated sodium and fluid absorption to levels that can restore airway hydration.Objectives: To determine whether a novel compound (QUB-TL1) designed to inhibit protease/ENaC signaling in CF airways restores ASL volume and mucociliary function.Methods: Protease activity was measured using fluorogenic activity assays. Differentiated primary airway epithelial cell cultures (F508del homozygotes) were used to determined ENaC activity (Ussing chamber recordings), ASL height (confocal microscopy), and mucociliary function (by tracking the surface flow of apically applied microbeads). Cell toxicity was measured using a lactate dehydrogenase assay.Measurements and Main Results: QUB-TL1 inhibits extracellularly located channel activating proteases (CAPs), including prostasin, matriptase, and furin, the activities of which are observed at excessive levels at the apical surface of CF airway epithelial cells. QUB-TL1-mediated CAP inhibition results in diminished ENaC-mediated Na(+) absorption in CF airway epithelial cells caused by internalization of a prominent pool of cleaved (active) ENaCγ from the cell surface. Importantly, diminished ENaC activity correlates with improved airway hydration status and mucociliary clearance. We further demonstrate QUB-TL1-mediated furin inhibition, which is in contrast to other serine protease inhibitors (camostat mesylate and aprotinin), affords protection against neutrophil elastase-mediated ENaC activation and Pseudomonas aeruginosa exotoxin A-induced cell death.Conclusions: QUB-TL1 corrects aberrant CAP activities, providing a mechanism to delay or prevent the development of CF lung disease in a manner independent of CF transmembrane conductance regulator mutation. [ABSTRACT FROM AUTHOR]

The membrane-anchored serine proteases prostasin (PRSS8) and matriptase (ST14) initiate a cell surface proteolytic pathway essential for epithelial function. Mice expressing only catalytically inactive prostasin are viable, unlike prostasin null mice, indicating that at least some prostasin functions are non-proteolytic. Here we used knock-in mice expressing catalytically inactive prostasin (Prss8Ki/Ki) to show that the physiological and pathological functions of prostasin vary in their dependence on its catalytic activity. Whereas prostasin nullmice exhibited partial embryonic and complete perinatal lethality, Prss8Ki/Ki mice displayed normal prenatal and postnatal survival. Unexpectedly, catalytically inactive prostasin caused embryonic lethality in mice lacking its cognate inhibitors HAI-1 (SPINT1) or HAI-2 (SPINT2). Proteolytically inactive prostasin, unlike the wild-type protease, was unable to activate matriptase during placentation. Surprisingly, all essential functions of prostasin in embryonic and postnatal development were compensated for by loss of HAI-1, indicating that prostasin is only required for mouse development and overall viability in the presence of this inhibitor. This study expands our knowledge of non-proteolytic functions of membrane-anchored serine proteases and provides unexpected new data on the mechanistic interactions between matriptase and prostasin in the context of epithelial development. [ABSTRACT FROM AUTHOR]

Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer (OVC) is the deadliest of all gynecologic cancers, primarily as a consequence of asymptomatic progression. The complex nature of OVC creates challenges for early detection, and there is a lack of specific and sensitive biomarkers suitable for screening and detecting early stage OVC. Methods: Potential OVC biomarkers were identified by bioinformatic analysis. Candidates were further screened for differential expression in a library of OVC cell lines. OVC-specific overexpression of a candidate gene, PRSS8, which encodes prostasin, was confirmed against 18 major human cancer types from 390 cancer samples by qRT-PCR. PRSS8 expression profiles stratified by OVC tumor stage-, grade- and subtype were generated using cDNA samples from 159 OVC samples. Cell-specific expression and localization of prostasin was determined by immunohistological tissue array analysis of more than 500 normal, benign, and cancerous ovarian tissues. The presence of prostasin in normal, benign, and OVC serum samples was also determined. Results: Gene expression analysis indicated that PRSS8 was expressed in OVC at levels more than 100 fold greater than found in normal or benign ovarian lesions. This overexpression signature was found in early stages of OVC and was maintained in higher stages and grades of OVC. The PRSS8 overexpression signature was specific for OVC and urinary bladder cancer among 18 human cancer types. The majority of ovarian cell lines overexpressed PRSS8. In situ hybridization and histopathology studies of OVC tissues indicated that overexpression of prostasin was largely localized to tumor epithelium and was absent in neighboring stroma. Significantly higher levels of prostasin were found in early stage OVC serum samples compared to benign ovarian and normal donor samples. Conclusions: The abundant amounts of secreted prostasin found in sera of early stage OVC can potentially be used as a minimally invasive screening biomarker for early stage OVC. Overexpression of PRSS8 mRNA and high levels of prostasin in multiple subtypes of early stage ovarian tumors may provide clinical biomarkers for early detection of OVC, which can potentially be used with CA125 and HE4. [ABSTRACT FROM AUTHOR]

The membrane-anchored serine prostasin (CAP1/PRSS8) is essential for barrier acquisition of the interfollicular epidermis and for normal hair follicle development. Consequently, prostasin null mice die shortly after birth. Prostasin is found in two forms in the epidermis: a one-chain zymogen and a two-chain proteolytically active form, generated by matriptase-dependent activation site cleavage. Here we used gene editing to generate mice expressing only activation site cleavage-resistant (zymogen-locked) endogenous prostasin. Interestingly, these mutant mice displayed normal interfollicular epidermal development and postnatal survival, but had defects in whisker and pelage hair formation. These findings identify two distinct in vivo functions of epidermal prostasin: a function in the interfollicular epidermis, not requiring activation site cleavage, that can be mediated by the zymogen-locked version of prostasin and a proteolysis-dependent function of activated prostasin in hair follicles, dependent on zymogen conversion by matriptase. [ABSTRACT FROM AUTHOR]

Background. Diabetic nephropathy (DN) is associated with hypertension, expanded extracellular volume and impaired renal Na+ excretion. It was hypothesized that aberrant glomerular filtration of serine proteases in DN causes proteolytic activation of the epithelial sodium channel (ENaC) in the kidney by excision of an inhibitory peptide tract from the y subunit. Methods. In a cross-sectional design, urine, plasma and clinical data were collected from type 1 diabetic patients with DN (n =19) and matched normoalbuminuric type 1 diabetics (controls, n = 20). Urine was examined for proteases by western immunoblotting, patch clamp and ELISA. Urine exosomes were isolated to elucidate potential cleavage of γENaC by a monoclonal antibody directed against the 'inhibitory' peptide tract. Results. Compared with control, DN patients displayed significantly higher blood pressure and urinary excretion of plasmin(ogen), prostasin and urokinase that correlated directly with urine albumin. Western blotting confirmed plasmin, prostasin and urokinase in urine from the DN group predominantly. Urine from DN evoked a significantly larger amiloride- sensitive inward current in single collecting duct cells compared with controls. Immunoblotting of urine exosomes showed aquaporin 2 in all patient samples. Exosomes displayed a virtual absence of intact yENaC while moieties compatible with cleavage by furin only, were shown in both groups. Proteolytic cleavage by the extracellular serine proteases plasmin or prostasin was observed in DN samples predominantly. Conclusion. DN is associated with increased urinary excretion of plasmin, prostasin and urokinase and proteolytic activation of ENaC that might contribute to impaired renal Na+ excretion and hypertension. [ABSTRACT FROM AUTHOR]

During maturation, the α- and γ-subunits of the epithelial Na+ channel (ENaC) undergo proteolytic processing by furin. Cleavage of the γ-subunit by furin at the consensus site γRKRR143 and subsequent cleavage by a second protease at a distal site strongly activate the channel. For example, coexpression of prostasin with ENaC increases both channel function and cleavage at the γRKRK186 site. We generated a polyclonal antibody that recognizes the region 144-186 in the γ-subunit (anti-γ43) to determine whether prostasin promotes the release of the intervening tract between the putative furin and γRKRK186 cleavage sites. Anti-γ43 precipitated both full-length (93 kDa) and furin-processed (83 kDa) γ-subunits from extracts obtained from oocytes expressing αβHA-γ-V5 channels, but only the fulllength (93 kDa) γ-subunit from oocytes expressing αβHA-γ-V5 channels and either wild-type or a catalytically inactive prostasin. Although both wild-type and catalytically inactive prostasin activated ENaCs in an aprotinin-sensitive manner, only wild-type prostasin bound to aprotinin beads, suggesting that catalytically inactive prostasin facilitates the cleavage of the γ-subunit by an endogenous protease in Xenopus oocytes. As dietary salt restriction increases cleavage of the renal γ-subunit, we assessed release of the 43-mer inhibitory tract on rats fed a low-Na+ diet. We found that a low-Na+ diet increased γ-subunit cleavage detected with the anti-γ antibody and dramatically reduced the fraction precipitated with the anti-γ43 antibody. Our results suggest that the inhibitory tract dissociates from the γ-subunit in kidneys from rats on a low-Na+ diet. [ABSTRACT FROM AUTHOR]

The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation. [ABSTRACT FROM AUTHOR]

The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival. [ABSTRACT FROM AUTHOR]

The membrane-anchored serine protease prostasin (CAP1/ PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8-/-)mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast,mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8Cat-/Cat- mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8-/- and Prss8Cat-/Cat- mice born within the same litter. Furthermore, Prss8Cat-/Cat- mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this uniquemembrane-anchored serine protease. [ABSTRACT FROM AUTHOR]

Matriptase and prostasin are part of a cell surface proteolytic pathway critical for epithelial development and homeostasis. Here we have used a reconstituted cell-based system and transgenic mice to investigate the mechanistic interrelationship between the two proteases. We show that matriptase and prostasin form a reciprocal zymogen activation complex with unique features. Prostasin serves as a critical co-factor for matriptase activation. Unexpectedly, however, prostasin-induced matriptase activation requires neither prostasin zymogen conversion nor prostasin catalytic activity. Prostasin zymogen conversion to active prostasin is dependent on matriptase but does not require matriptase zymogen conversion. Consistent with these findings, wild type prostasin, activation cleavage site-mutated prostasin, and catalytically inactive prostasin all were biologically active in vivo when overexpressed in the epidermis of transgenic mice, giving rise to a severe skin phenotype. Our finding of non-enzymatic stimulation of matriptase activation by prostasin and activation of prostasin by the matriptase zymogen provides a tentative mechanistic explanation for several hitherto unaccounted for genetic and biochemical observations regarding these two membrane-anchored serine proteases and their downstream targets [ABSTRACT FROM AUTHOR]

Human and mouse marapsins (Prss27) are serine proteases preferentially expressed by stratified squamous epithelia. However, mouse marapsin contains a transmembrane anchor absent from the human enzyme. To gain insights into physical forms, activities, inhibition, and roles in epithelial differentiation, we traced tail loss in human marapsin to a nonsense mutation in an ancestral ape, compared substrate preferences of mouse and human marapsins with those of the epithelial peptidase prostasin, designed a selective substrate and inhibitor, and generated Prss27-null mice. Phylogenetic analysis predicts that most marapsins are transmembrane proteins. However, nonsense mutations caused membrane anchor loss in three clades: human/bonobo/chimpanzee, guinea pig/degu/tuco-tuco/mole rat, and cattle/yak. Most marapsin-related proteases, including prostasins, are type I transmembrane proteins, but the closest relatives (prosemins) are not. Soluble mouse and human marapsins are tryptic with subsite preferences distinct from those of prostasin, lack general proteinase activity, and unlike prostasins resist antiproteases, including leupeptin, aprotinin, serpins, and α2-macroglobulin, suggesting the presence of noncanonical active sites. Prss27-null mice develop normally in barrier conditions and are fertile without overt epithelial defects, indicating that marapsin does not play critical, non-redundant roles in development, reproduction, or epithelial differentiation. In conclusion, marapsins are conserved, inhibitor-resistant, tryptic peptidases. Although marapsins are type I transmembrane proteins in their typical form, they mutated independently into anchorless forms in several mammalian clades, including one involving humans. Similar pathways appear to have been traversed by prosemins and tryptases, suggesting that mutational tail loss is an important means of evolving new functions of tryptic serine proteases from transmembrane ancestors. [ABSTRACT FROM AUTHOR]

The type II transmembrane serine protease matriptase is a key regulator of epithelial barriers in skin and intestine. In skin, matriptase acts upstream of the glycosylphosphatidylinositol-anchored serine protease, prostasin, to activate the prostasin zymogen and initiate a proteolytic cascade that is required for stratum corneum barrier functionality. Here, we have investigated the relationship between prostasin and matriptase in intestinal epithelial barrier function. We find that similar to skin, matriptase and prostasin are components of a common intestinal epithelial barrier-forming pathway. Depletion of prostasin by siRNA silencing in Caco-2 intestinal epithelium inhibits barrier development similar to loss of matriptase, and the addition of recombinant prostasin to the basal side of polarized Caco-2 epithelium stimulates barrier forming changes similar to the addition of recombinant matriptase. However, in contrast to the proteolytic cascade in skin, prostasin functions upstream of matriptase to activate the endogenous matriptase zymogen. Prostasin is unable to proteolytically activate the matriptase zymogen directly but induces matriptase activation indirectly. Prostasin requires expression of endogenous matriptase to stimulate barrier formation since matriptase depletion by siRNA silencing abrogates prostasin barrier-forming activity. Active recombinant matriptase, however, does not require the expression of endogenous prostasin for barrier-forming activity. Together, these data show that matriptase and not prostasin is the primary effector protease of tight junction assembly in simple columnar epithelia and further highlight a spatial and tissue-specific aspect of cell surface proteolytic cascades. [ABSTRACT FROM AUTHOR]

Aldosterone plays an important role in the regulation of blood pressure by modulating the activity of the epithelial sodium channel (ENaC) that consists of α-, β-, and γ-subunits. Aldosterone induces a molecular weight shift of γENaC from 85 to 70 kDa that is necessary for the channel activation. In vitro experiments demonstrated that a dual cleavage mechanism is responsible for this shift. It has been postulated that furin executes the primary cleavage in the Golgi and that the second cleavage is provided by other serine proteases such as prostasin or plasmin at the plasma membrane. However, the in vivo contribution of serine proteases to this cleavage remains unclear. To address this issue, we administered the synthetic serine protease inhibitor camostat mesilate (CM) to aldosterone-infused rats. CM decreased the abundance of the 70-kDa form of ENaC and led to a new 75-kDa form with a concomitant increase in the urinary Na-to-K ratio. Because CM inhibits the protease activity of serine proteases such as prostasin and plasmin, but not furin, our findings strongly indicate that CM inhibited the second cleavage of γENaC and subsequently suppressed ENaC activity. The results of our current studies also suggest the possibility that the synthetic serine protease inhibitor CM might represent a new strategy for the treatment of salt-sensitive hypertension in humans. [ABSTRACT FROM AUTHOR]

Epithelial Na Channels (ENaC) are responsible for the apical entry of Na+ in a number of different epithelia including the renal connecting tubule and cortical collecting duct. Proteolytic cleavage of γ-ENaC by serine proteases, including trypsin, furin, elastase, and prostasin, has been shown to increase channel activity. Here, we investigate the ability of another serine protease, tissue kallikrein, to regulate ENaC. We show that excretion of tissue kallikrein, which is secreted into the lumen of the connecting tubule, is stimulated following 5 days of a high-K+ or low-Na+ diet in rats. Urinary proteins reconstituted in a low-Na buffer activated amiloride-sensitive currents (INa) in ENaC-expressing oocytes, suggesting an endogenous urinary protease can activate ENaC. We next tested whether tissue kallikrein can directly cleave and activate ENaC. When rat ENaC-expressing oocytes were exposed to purified tissue kallikrein from rat urine (RTK), ENaC currents increased threefold in both the presence and absence of a soybean trypsin inhibitor (SBTI). RTK and trypsin both decreased the apparent molecular mass of cleaved cell-surface γ-ENaC, while immunodepleted RTK produced no shift in apparent molecular mass, demonstrating the specificity of the tissue kallikrein. A decreased effect of RTK on Xenopus ENaC, which has variations in the putative prostasin cleavage sites in γ-ENaC, suggests these sites are important in RTK activation of ENaC. Mutating the prostasin site in mouse γ-ENaC (γRKRK186QQQQ) abolished ENaC activation and cleavage by RTK while wild-type mouse ENaC was activated and cleaved similar to that of the rat. We conclude that tissue kallikrein can be a physiologically relevant regulator of ENaC activity. [ABSTRACT FROM AUTHOR]

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1-deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2-deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent. [ABSTRACT FROM AUTHOR]

Proteases activate the epithelia sodium channel (ENaC) by cleaving the large extracellular domains of the α- and γ-subunits and releasing peptides with inhibitory properties. Furin and prostasin activate mouse ENaC by cleaving the γ-subunit at sites flanking a 43 residue inhibitory tract (γE144-K186) To determine whether there is a minimal inhibitory region within this 43 residue tract, we generated serial deletions in the inhibitory tract of the γ-subunit in channels resistant to cleavage by furin and prostasin. We round that partial or complete deletion of a short segment in the 3,-subunit, R158-N171, enhanced channel activity. Synthetic peptides overlapping this segment in the γ-subunit further identified a key 11-mer tract. R158-F168 (RFLNLIPLLVF), which inhibited wild- type ENaC expressed in Xenopus laevis oocytes, and endogenous channels in mpkCCD cells and human airway epithelia. Further studies with amino acid-substituted peptides defined residues that are required for inhibition in this key 11-mer tract. The presence of the native γ inhibitory tract in ENaC weakened the intrinsic binding constant of the 11-mer peptide inhibitor, suggesting that the 7 inhibitory tract and the 11-mer peptide interact at overlapping sites within the channel. [ABSTRACT FROM AUTHOR]

The aim of this study is to elucidate the effects of interleukin-6 (IL-6) on the expression and activity of the epithelial sodium channel (ENaC), which is one of the key mechanisms underlying tubular sodium reabsorption. M1 cortical collecting duct cells were treated with IL-6 (100 ng/ml) for 12 h. Real-time polymerase chain reaction and immunoblotting were employed to examine the mRNA and protein abundance. Transepithelial voltage (Vte) and resistance (Rte) were measured with an ohm! voltmeter (EVOM, WPI). The equivalent current was calculated as the ratio of Vte tO Rte Treatment with IL-6 (n = 5) increased the mRNA abundance of γ-ENaC by 11 ± 7% (P = not significant), γ-ENaC by 78 ± 14% (P = 0.01), γ-ENaC by 185 ± 38% (P = 0.02), and prostasin by 29 ± 5% (P = 0.01), all normalized by β-actin. Treatment with IL-6 increased the protein expression of a-ENaC by 19 ± 3% (P = 0.001), β-ENaC by 89 ± 21% (P = 0.01), γ-ENaC by 36 ± 12% (P = 0.02), and prostasin by 33 ± 6% (P = 0.02). The amiloride-sensitive sodium current increased by 37 ± 5%, from 6.0 ± 0.4 to 8.2 ± 0.3 μA/cm2 (P < 0.01), in the cells treated with IL-6 compared with controls (P = 0.01). Aprotinin (28 μg/ml), a prostasin inhibitor, reduced the amiloride-sensitive sodium current by 61 ± 5%, from 6.1 ± 0.3 to 3.7 ± 0.2 μA/cm2 (P = 0.01). The magnitude of the IL-6-induced amiloride-sensitive sodium current in the presence of aprotinin dropped by 57 ± 2%, from 8.6 ± 0.2 to 4.9 ± 0.2 μA/cm2 (P < 0.01). This study has identified a novel function of IL-6, namely, IL-6 may activate ENaC. Therefore, renal inflammation mediated by IL-6 likely contributes to impaired pressure natriuresis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Prostasin is a glycosylphosphatidylinositol-anchored extracellular serine protease with a role in epidermal growth factor receptor (EGFR) signal modulation. EGFR signaling has been shown to be important for regulating cytotrophoblast (CT) cell proliferation in human placenta. We investigated the impact of prostasin expression regulation on this cellular function as well as the molecular mechanisms involved in human cytotrophoblastic cells. [ABSTRACT FROM PUBLISHER]

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to β2-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by β2-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance. [ABSTRACT FROM AUTHOR]

Abstract: Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a membrane-associated Kunitz-type serine protease inhibitor that regulates cell surface and extracellular serine proteases involved in tissue remodeling and tumorigenesis, such as HGFA, matriptase, prostasin and hepsin. We generated HAI-1-deficient mice, which died in utero due to placental defects. The HAI-1 −/− placental labyrinth exhibited a complete failure of vascularization and a compact morphology of the trophoblast layer. Immunofluorescent staining of collagen IV and laminin and electron microscopy analysis revealed that this aberrant labyrinth architecture was associated with disrupted basement membranes located at the interface of chorionic trophoblasts and allantoic mesoderm. Unlike the placental labyrinth, basement membranes and vasculogenesis were normal in embryo and yolk sac. Therefore, basement membrane defects appear to be the underlying cause for the greatly impaired vascularization and trophoblast branching in HAI-1 −/− placentas. In wild-type placentas, the expression of matriptase and prostasin co-localized with their physiological inhibitor HAI-1 to the labyrinthine trophoblast cells in proximity to basement membranes. In HAI-1 −/− placentas, both the localization and expression of the two proteases remained unchanged, implying uncontrolled proteolytic activities of the two enzymes. Our study demonstrates the important role of HAI-1 in maintaining the integrity of basement membrane most likely by regulating extracellular proteolytic activities during placental development. [Copyright &y& Elsevier]

Prostasin has been shown to regulate sodium handling in the kidney. Recently, a serine protease inhibitor, protease nexin-1 (PN-1), was identified as an endogenous inhibitor for prostasin. Therefore, we hypothesized that PN-1 may regulate sodium reabsorption by reducing prostasin activity, and that expression of PN-1 was regulated by transforming growth factor-β1 (TGF-β1) or aldosterone, like prostasin. cRNAs for epithelial sodium channel (ENaC), prostasin, and PN-1 were expressed in Xenopus oocytes, and the amiloride-sensitive sodium currents (INa) were measured. The effect of TGF-β1 and aldosterone on the mRNA and protein abundance of PN-1 and ENaC was detected by real-time polymerase chain reaction and immunoblotting in M-1 cells. Expression of PN-1 substantially decreased prostasin-induced INa by approximately 68% in oocytes. Treatment of M-1 cells with 20 ng/ml TGF-β1 significantly increased protein expression of PN-1 by 3.8±0.5-fold, whereas administration of 10−6 M aldosterone markedly decreased protein expression of PN-1 to 53.7±6.7%. Basolateral, but not apical, application of TGF-β1 significantly reduced Ieq. To elucidate the involvement of PN-1 in basal ENaC activity, we silenced the expression of PN-1 by using short-interfering RNA. This increased Ieq by 1.6±0.1-fold. Our study indicates that PN-1 could have a natriuretic role by inhibiting prostasin activity and suggests the possibility that aldosterone and TGF-β reciprocally regulate the expression of PN-1 in renal epithelial cells contributing to salt retention or natriuresis, respectively by an additional mechanism. PN-1 could represent a new factor that contributes to regulation of ENaC activity in the kidney.Kidney International (2006) 70, 1432–1438. doi:10.1038/sj.ki.5001787; published online 30 August 2006 [ABSTRACT FROM AUTHOR]

Inhibition of prostasin expression by TGF-β1 in renal epithelial cells.Background.Prostasin has been shown to be involved in the regulation of sodium handling in the kidney. TGF-β1 has been demonstrated to suppressαENaC expression and sodium uptake. Therefore, we hypothesized that TGF-β1 may regulate prostasin expression to modulate sodium reabsorption in the kidney.Methods.To determine if TGF-β1 has an effect on prostasin expression, we isolated 2.9 kb of the rat prostasin promoter, and measured its transcriptional activity with a luciferase assay in mouse cortical collecting duct cell line (M-1). The effect of TGF-β1 on the mRNA and protein abundance of prostasin, and amiloride-sensitive22Na uptake was determined.Results.Treatment of M-1 cells with 20 ng/mL of TGF-β1 for 24 hours significantly decreased the promoter activity by 50± 1%, and the inhibitory effect was dose dependent over the range of 0.1 to 20 ng/mL. We identified a 50 bp region (−410 to−360) containing c-Rel–like sequence in prostasin promoter that is responsible for the TGF-β1–mediated inhibition, and found that TGF-β1 increases IκBα expression in M-1 cells. TGF-β1 reduced endogenous prostasin mRNA and protein expression in M-1 cells by 50± 12% and 44± 12%, respectively, and the amiloride-sensitive22Na uptake by 35.9± 4.8%.Conclusion.Our findings indicate the possibility that TGF-β1 transcriptionally inhibits prostasin expression by the induction of IκBα and the subsequent inhibition of NF-κB/Rel activity in M-1 cells, and also suggest the possibility that TGF-β1 might inhibit sodium reabsorption through a reduction in prostasin expression and subsequent inhibition of ENaC activity. [ABSTRACT FROM AUTHOR]