Prostasin

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Thomas H Bugge - One of the best experts on this subject based on the ideXlab platform.

  • loss of hai 2 in mice with decreased Prostasin activity leads to an early onset intestinal failure resembling congenital tufting enteropathy
    PLOS ONE, 2018
    Co-Authors: Roman Szabo, Thomas H Bugge
    Abstract:

    : 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.

  • Loss of HAI-2 in mice with decreased Prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy.
    PloS one, 2018
    Co-Authors: Roman Szabo, Thomas H Bugge
    Abstract:

    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.

  • HAI-2 is dispensable for pre-natal development in Prss8R44Q/R44Q mice.
    2018
    Co-Authors: Roman Szabo, Thomas H Bugge
    Abstract:

    (A-C). Western blot detection of SDS-stable complexes between HAI-1 (H1), HAI-2 (H2), and protein nexin-1 (PN-1) and wildtype (A), catalytically-inactive S238A (B), and zymogen-locked R44Q (C) variants of Prostasin after pre-incubation with (A-C, lanes 2, 4, 6, and 8) or without (A-C, lanes 1, 3, 5, and 7) recombinant human matriptase. HAI-1 and HAI-2 efficiently formed SDS-stable complexes with wildtype and catalytically-inactive Prostasin after zymogen conversion (A and B, lanes 4 and 6), whereas PN-1 only formed complex with a wildtype Prostasin (A and B, lane 8). No complexes were detected between the R44Q variant of Prostasin and any of the three inhibitors (C, lanes 4, 6, and 8). Incubation with matriptase leads to a reduction in apparent molecular weight of Prostasin both before (A, lanes 9 and 10) and after (A, lanes 11 and 12) de-glycosylation, indicating proteolytic processing of Prostasin zymogen. Positions of Prostasin zymogen (black arrowhead) and activated double-chain Prostasin (grey arrowhead) are indicated on the right. Location of Prostasin/HAI-1 (blue asterisk), Prostasin/HAI-2 (green asterisk) and Prostasin/PN-1 (red asterisk) are shown directly on the blot. Positions of protein molecular weight markers is shown on the left. (D). Western blot detection of Prostasin and HAI-2 after co-immunoprecipitation from E11.5 mouse placental tissues. Placental extracts from control (Spint2+/+;Prss8+/+, C, lanes 1 and 4), and HAI-2-expressing (Spint2+/+;Prss8R44Q/R44Q (Zy, lanes 2 and 5) or HAI-2-deficient (Spint2-/-; Prss8R44Q/R44Q, 0, lanes 3 and 6) Prostasin zymogen-locked embryos were incubated with anti-HAI-2 (lanes 1–3) or anti-Prostasin (lanes 4–6) antibody, then analyzed by Western blot using anti-Prostasin (black arrowhead, top panel) or anti-HAI-2 (red arrowheads, bottom panel) antibodies. The two proteins co-immunoprecipitated in mice expressing wildtype, but not R44Q Prostasin. (E). Distribution of HAI-2 genotypes among newborn mice from Spint2+/−; Prss8R44Q/+ breeding pairs. Loss of HAI-2 (Spint2-/-) leads to a complete embryonic lethality in mice expressing at least one wildtype allele (Prss8+/+ or Prss8R44Q/+, collectively labeled as Prss8+) of Prostasin (Spint2-/-;Prss8+, P

  • Delineation of proteolytic and non-proteolytic functions of the membrane-anchored serine protease Prostasin.
    Development (Cambridge England), 2016
    Co-Authors: Roman Szabo, Diane E Peters, Taliya Lantsman, Thomas H Bugge
    Abstract:

    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 (Prss8(Ki/Ki)) to show that the physiological and pathological functions of Prostasin vary in their dependence on its catalytic activity. Whereas Prostasin null mice exhibited partial embryonic and complete perinatal lethality, Prss8(Ki/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.

  • distinct developmental functions of Prostasin cap1 prss8 zymogen and activated Prostasin
    Journal of Biological Chemistry, 2016
    Co-Authors: Daniel H. Madsen, Stine Friis, Thomas H Bugge
    Abstract:

    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.

Karl X. Chai - One of the best experts on this subject based on the ideXlab platform.

  • Ibuprofen regulates the expression and function of membrane-associated serine proteases Prostasin and matriptase.
    BMC cancer, 2015
    Co-Authors: Andreas C. Chai, Karl X. Chai, Andrew L. Robinson, Li-mei Chen
    Abstract:

    Background The glycosylphosphatidylinositol-anchored extracellular membrane serine protease Prostasin is expressed in normal bladder urothelial cells. Bladder inflammation reduces Prostasin expression and a loss of Prostasin expression is associated with epithelial-mesenchymal transition (EMT) in human bladder transitional cell carcinomas. Non-steroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of various cancers including bladder cancer, but the molecular mechanisms underlying the anticancer effect of NSAIDs are not fully understood.

  • regulation of the matriptase Prostasin cell surface proteolytic cascade by hepatocyte growth factor activator inhibitor 1 during epidermal differentiation
    Journal of Biological Chemistry, 2010
    Co-Authors: Ya-wen Chen, Li-mei Chen, Karl X. Chai, Jehng-kang Wang, Michael D. Johnson, Feng-pai Chou, Chiuyuan Chen, Ellen A Rorke, Richard L Eckert, Chenyong Lin
    Abstract:

    Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of Prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates Prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and Prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, Prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-Prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) Prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active Prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active Prostasin to act on their substrates.

  • Prostasin regulates human placental trophoblast cell proliferation via the epidermal growth factor receptor signaling pathway
    Human reproduction (Oxford England), 2010
    Co-Authors: Wen-long Gao, Mengqian Chen, Karl X. Chai, Yan-ling Wang, Li-mei Chen
    Abstract:

    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. METHODS: An immortalized normal human CT cell line (B6Tert-1) was used as an in vitro cell model. Prostasin expression in B6Tert- 1 cells was knocked down by transfection of a short interfering RNA. Lentivirus-mediated expression of recombinant human Prostasin under tetracycline regulation was performed to obtain stable B6Tert-1 cell sublines that over-expressed Prostasin. Changes in cell proliferation and EGFR signaling were evaluated by immunocytochemistry for Ki67 and western blot analysis, respectively, in B6Tert-1 cells with knocked-down or increased Prostasin expression. RESULTS: Prostasin knock-down in B6Tert-1 cells resulted in inhibition of cell proliferation, in association with down-regulated EGFR protein expression (both P < 0.05 versus control) as well as reduced phosphorylation of c-raf, mitogen-activated protein kinase (MAPK) kinases (MEK1/2) and extracellular signal-regulated kinases (Erk1/2) (all P < 0.05 versus control). Over-expression of Prostasin led to up-regulation of the EGFR protein, but had no effect on cell proliferation or phosphorylation of MAPK signaling molecules in the B6Tert-I cells. CONCLUSIONS: Prostasin may regulate trophoblast cell proliferation via modulating the EGFR-MAPK signaling pathway.

  • Prostasin regulates inos and cyclin d1 expression by modulating protease activated receptor 2 signaling in prostate epithelial cells
    The Prostate, 2009
    Co-Authors: Li-mei Chen, Meghan L. Hatfield, Karl X. Chai
    Abstract:

    BACKGROUND Prostasin is down-regulated during inflammation and in invasive cancers, and plays a role in regulation of inflammatory gene expression and invasion. METHODS We used the human benign prostatic hyperplasia cell line BPH-1 to investigate gene expression changes associated with siRNA-mediated loss of Prostasin expression. Quantitative PCR and/or western blotting were used to evaluate the expression changes of iNOS, ICAM-1, cyclin D1, IL-6, and IL-8. Gene expression changes were also evaluated in the presence of a PAR-2 antagonist. The PC-3 human prostate cancer cell line was used for evaluation of gene expression in response to Prostasin re-expression. RESULTS Prostasin silencing in BPH-1 was associated with up-regulation of iNOS, ICAM-1, IL-6, and IL-8, and down-regulation of cyclin D1; as well as reduced proliferation and invasion. The iNOS up-regulation and cyclin D1 down-regulation associated with Prostasin silencing were inhibited by a PAR-2 antagonist. Re-expression of Prostasin, a serine active-site mutant, and a GPI-anchor-free mutant, in the PC-3 cells resulted in PAR-2 and cyclin D1 transcription up-regulation. Transcription up-regulation of IL-6 and IL-8 was associated with re-expression of the serine active-site mutant Prostasin in the PC-3 cells. Transcription up-regulation of IL-8, but to a lesser extent, was also observed in PC-3 cells expressing the wild-type Prostasin. Expression of a serine protease active Prostasin, GPI-anchored or anchor-free, prevented the IL-6 induction in response to PAR-2. The GPI-anchor-free Prostasin also prevented the IL-8 induction. CONCLUSIONS Prostasin plays a negative regulatory role on PAR-2-mediated signaling in prostate epithelial cells. Prostate 69: 1790–1801, 2009. © 2009 Wiley-Liss, Inc.

  • Hepsin activates Prostasin and cleaves the extracellular domain of the epidermal growth factor receptor
    Molecular and Cellular Biochemistry, 2009
    Co-Authors: Mengqian Chen, Li-mei Chen, Chenyong Lin, Karl X. Chai
    Abstract:

    The epithelial extracellular serine protease activation cascade involves matriptase (PRSS14) and Prostasin (PRSS8), capable of modulating epidermal growth factor receptor (EGFR) signaling. Matriptase activates Prostasin by cleaving in the amino-terminal pro-peptide region of Prostasin, presumably at the Arg residue of position 44 (R44) of the full-length human Prostasin. Using an Arg-to-Ala mutant (R44A) human Prostasin, we showed in this report that the cleavage of Prostasin by matriptase is at Arg44. This Prostasin proteolytic activation site is also cleaved by hepsin (TMPRSS1) to produce active Prostasin capable of forming a covalent complex with protease nexin 1 (PN-1). An amino-terminal truncation of EGFR in the extracellular domain (ECD) was observed when the receptor was co-expressed with hepsin. Hepsin and matriptase appear to cleave the EGFR ECD at different sites, while the hepsin cleavage is not affected by active Prostasin, which enhances the matriptase cleavage of EGFR. Using hepsin as the Prostasin-activating protease in cells co-transfected with EGFR, we showed that active Prostasin does not cleave the EGFR ECD directly in the cellular context. Purified active Prostasin also does not cleave purified EGFR. Hepsin cleavage of EGFR is not dependent on receptor tyrosine phosphorylation, while the hepsin-cleaved EGFR is phosphorylated at Tyr1068 and no longer responsive to EGF stimulation. The cleavage of EGFR by hepsin does not result in increased phosphorylation of the downstream extracellular signal-regulated kinases (Erk1/2), an event inducible by the matriptase-Prostasin cleavage of EGFR. The role of hepsin serine protease should be considered in future studies of epithelial biology concerning matriptase, Prostasin, and EGFR.

Kenichiro Kitamura - One of the best experts on this subject based on the ideXlab platform.

  • Sodium absorption stimulator Prostasin (PRSS8) has an anti-inflammatory effect via downregulation of TLR4 signaling in inflammatory bowel disease
    Journal of gastroenterology, 2020
    Co-Authors: Yoshihiko Sugitani, Kenichiro Kitamura, Atsushi Nishida, Osamu Inatomi, Masashi Ohno, Takayuki Imai, Masahiro Kawahara, Akira Andoh
    Abstract:

    Prostasin (PRSS8) is a stimulator of epithelial sodium transport. In this study, we evaluated alteration of Prostasin expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD) and investigated the role of Prostasin in the gut inflammation. Prostasin expression was evaluated by immunohistochemical staining. Dextran sodium sulfate (DSS)-colitis was induced in mice lacking Prostasin specifically in intestinal epithelial cells (PRSS8ΔIEC mice). In colonic mucosa of healthy individuals, Prostasin was strongly expressed at the apical surfaces of epithelial cells, and this was markedly decreased in active mucosa of both ulcerative colitis and Crohn’s disease. DSS-colitis was exacerbated in PRSS8ΔIEC mice compared to control PRSS8lox/lox mice. Toll-like receptor4 (TLR4) expression in colonic epithelial cells was stronger in DSS-treated PRSS8ΔIEC mice than in DSS-treated PRSS8 lox/lox mice. NF-κB activation in colonic epithelial cells was more pronounced in DSS-treated PRSS8ΔIEC mice than in DSS-treated PRSS8lox/lox mice, and the mRNA expression of inflammatory cytokines was significantly higher in DSS-treated PRSS8ΔIEC mice. Broad-spectrum antibiotic treatment completely suppressed the exacerbation of DSS-colitis in PRSS8ΔIEC mice. The mRNA expression of tight junction proteins and mucosal permeability assessed using FITC-dextran were comparable between DSS-treated PRSS8lox/lox and DSS-treated PRSS8ΔIEC mice. Prostasin has an anti-inflammatory effect via downregulation of TLR4 expression in colonic epithelial cells. Reduced Prostasin expression in IBD mucosa is linked to the deterioration of local anti-inflammatory activity and may contribute to the persistence of mucosal inflammation.

  • Urinary Prostasin in normotensive individuals : correlation with the aldosterone to renin ratio and urinary sodium
    Hypertension research : official journal of the Japanese Society of Hypertension, 2013
    Co-Authors: Oliviero Olivieri, Francesca Pizzolo, Patrizia Guarini, Laura Chiecchi, Annalisa Castagna, Ricciarda Raffaelli, Muthukumar Gunasekaran, Letizia Consoli, Gian Luca Salvagno, Kenichiro Kitamura
    Abstract:

    Prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease, activates the epithelial sodium (Na) channel (ENaC), and Prostasin is released in extracellular fluids, including urine. Previous data have suggested a direct association between urinary Prostasin and the activation of an aldosterone-driven pathway, but a quantitative association has never been demonstrated in normotensive subjects. Similarly, physiological relationships with natriuresis or possible gender- or female hormone-related changes in urinary Prostasin concentrations have never been investigated. We measured urinary Prostasin by enzyme-linked immunosorbent assay in 43 healthy normotensive subjects of similar age presenting different urinary Na levels and in 15 women during the menstrual cycle and after oral estro-progestinic contraceptive (OC) therapy. Exosomal urinary Prostasin was also estimated by western blotting of samples from six healthy subjects twice during the morning. Urinary Prostasin presented a wide range of values (from 0.5 to 18.9 nM) without gender differences. It was positively correlated with the aldosterone to renin ratio (ARR) but not with circulating aldosterone or renin individually. Urinary Prostasin was directly correlated with U-Na levels (up to 200 nmol Na), whereas it decreased for higher Na concentrations. In women, no significant changes of Prostasin concentration were observed during menstrual phases. After OC therapy, Prostasin increased (from 2.37±1.27 to 4.85±5.28 nM), although the increase was not statistically different (P=0.07). Prostasin was detectable in urinary exosomes and displayed a pattern similar to urinary Prostasin in relation to urinary Na. In conclusion, urinary Prostasin correlates with the ARR, and it is physiologically modulated by natriuresis in normotensive individuals.

  • Proteolytic activation of the epithelial sodium channel and therapeutic application of a serine protease inhibitor for the treatment of salt-sensitive hypertension
    Clinical and Experimental Nephrology, 2012
    Co-Authors: Kenichiro Kitamura, Kimio Tomita
    Abstract:

    Proteases are involved in numerous essential biological processes including blood clotting, controlled cell death, and tissue differentiation. Prostasin, a glycosylphosphatidylinositol-anchored serine protease, has been identified as a potential regulator of the epithelial sodium channel (ENaC) function in the kidney, lung, and airways. ENaC is composed of three homologous subunits α, β, and, γ. The dual cleavage of α subunit by furin and γ subunit by Prostasin and furin releases inhibitory segments from ENaC, leading to the channel activation. Protease nexin-1, an endogenous Prostasin inhibitor, inhibits ENaC activity through the suppression of Prostasin activity, strongly suggesting the possibility that a coordinated regulation of serine proteases and serine protease inhibitors plays a key role in the sodium handling in the kidney. Camostat mesilate (CM), a synthetic serine protease inhibitor, reduced Prostasin activity and subsequently decreased ENaC current. Oral administration of CM to Dahl salt-sensitive rats resulted in a significant decrease in blood pressure with an elevation of the urinary sodium/potassium ratio. These findings suggest that synthetic serine protease inhibitors such as CM might represent a new class of antihypertensive drugs in patients with salt-sensitive hypertension.

  • Regulation of renal sodium handling through the interaction between serine proteases and serine protease inhibitors
    Clinical and Experimental Nephrology, 2010
    Co-Authors: Kenichiro Kitamura, Kimio Tomita
    Abstract:

    Sodium balance, extracellular fluid volume, and ultimately blood pressure are maintained by precise regulation of the activity of epithelial sodium channels (ENaC). Multiple mechanisms such as hormones, intracellular factors, and other regulatory factors contribute to regulation of ENaC activity. Prostasin, a glycosylphosphatidylinositol-anchored serine protease, has been identified as an activator of ENaC that increases its open probability. Furin cleaves αENaC at two sites and γENaC at one site at the Golgi. Prostasin cleaves γENaC at one site that is distinct from the furin site at the plasma membrane. Dual cleavage of α- and γ-subunit releases inhibitory segments from ENaC, leading to channel activation. Protease nexin-1 (PN-1), an endogenous Prostasin inhibitor, inhibits ENaC activity through suppression of Prostasin activity. Aldosterone and transforming growth factor-β1 reciprocally regulate expression of Prostasin, PN-1, and ENaC in renal epithelial cell, resulting in sodium retention or natriuresis, respectively. These findings strongly suggest the possibility that coordinated regulation of serine protease, serpin, and ENaC expression plays a key role in sodium handling in the kidney.

  • urinary Prostasin in humans relationships among Prostasin aldosterone and epithelial sodium channel activity
    Hypertension Research, 2009
    Co-Authors: Aya Koda, Kimio Tomita, Naoki Wakida, Kazuhiro Toriyama, Kazutoshi Yamamoto, Hiromi Iijima, Kenichiro Kitamura
    Abstract:

    Prostasin, a membrane-bound serine protease, is known to increase the activity of the epithelial sodium channel (ENaC). Gene expression of Prostasin was shown to be regulated by aldosterone, which increases the rate of sodium reabsorption through ENaC. To clarify the physiological relationships among Prostasin, aldosterone and ENaC, we developed a specific radioimmunoassay (RIA) for human Prostasin. Prostasin levels in urine were determined in 26 normotensive and 121 hypertensive subjects. Aldosterone content in urine and plasma, urinary Na/K ratio and other clinical parameters were also measured. We observed a highly significant correlation between Prostasin and aldosterone concentration in urine (correlation coefficient: 0.673, P<0.0001). A significant correlation was also found between urinary Prostasin and plasma aldosterone concentration (correlation coefficient: 0.229, P<0.05). In addition, urinary Prostasin excretion was inversely correlated with urinary Na/K ratio (correlation coefficient: -0.425, P<0.0001). In conclusion, we developed a Prostasin-specific RIA and applied it to the clinical study. Our findings suggest that urinary Prostasin level is strongly correlated with urinary or plasma aldosterone level and may serve as a surrogate marker for ENaC activation in hypertensive patients. However, it is not clear, at the present time, whether endogenous aldosterone regulates Prostasin expression or vice versa.

Kimio Tomita - One of the best experts on this subject based on the ideXlab platform.

  • Proteolytic activation of the epithelial sodium channel and therapeutic application of a serine protease inhibitor for the treatment of salt-sensitive hypertension
    Clinical and Experimental Nephrology, 2012
    Co-Authors: Kenichiro Kitamura, Kimio Tomita
    Abstract:

    Proteases are involved in numerous essential biological processes including blood clotting, controlled cell death, and tissue differentiation. Prostasin, a glycosylphosphatidylinositol-anchored serine protease, has been identified as a potential regulator of the epithelial sodium channel (ENaC) function in the kidney, lung, and airways. ENaC is composed of three homologous subunits α, β, and, γ. The dual cleavage of α subunit by furin and γ subunit by Prostasin and furin releases inhibitory segments from ENaC, leading to the channel activation. Protease nexin-1, an endogenous Prostasin inhibitor, inhibits ENaC activity through the suppression of Prostasin activity, strongly suggesting the possibility that a coordinated regulation of serine proteases and serine protease inhibitors plays a key role in the sodium handling in the kidney. Camostat mesilate (CM), a synthetic serine protease inhibitor, reduced Prostasin activity and subsequently decreased ENaC current. Oral administration of CM to Dahl salt-sensitive rats resulted in a significant decrease in blood pressure with an elevation of the urinary sodium/potassium ratio. These findings suggest that synthetic serine protease inhibitors such as CM might represent a new class of antihypertensive drugs in patients with salt-sensitive hypertension.

  • Regulation of renal sodium handling through the interaction between serine proteases and serine protease inhibitors
    Clinical and Experimental Nephrology, 2010
    Co-Authors: Kenichiro Kitamura, Kimio Tomita
    Abstract:

    Sodium balance, extracellular fluid volume, and ultimately blood pressure are maintained by precise regulation of the activity of epithelial sodium channels (ENaC). Multiple mechanisms such as hormones, intracellular factors, and other regulatory factors contribute to regulation of ENaC activity. Prostasin, a glycosylphosphatidylinositol-anchored serine protease, has been identified as an activator of ENaC that increases its open probability. Furin cleaves αENaC at two sites and γENaC at one site at the Golgi. Prostasin cleaves γENaC at one site that is distinct from the furin site at the plasma membrane. Dual cleavage of α- and γ-subunit releases inhibitory segments from ENaC, leading to channel activation. Protease nexin-1 (PN-1), an endogenous Prostasin inhibitor, inhibits ENaC activity through suppression of Prostasin activity. Aldosterone and transforming growth factor-β1 reciprocally regulate expression of Prostasin, PN-1, and ENaC in renal epithelial cell, resulting in sodium retention or natriuresis, respectively. These findings strongly suggest the possibility that coordinated regulation of serine protease, serpin, and ENaC expression plays a key role in sodium handling in the kidney.

  • urinary Prostasin in humans relationships among Prostasin aldosterone and epithelial sodium channel activity
    Hypertension Research, 2009
    Co-Authors: Aya Koda, Kimio Tomita, Naoki Wakida, Kazuhiro Toriyama, Kazutoshi Yamamoto, Hiromi Iijima, Kenichiro Kitamura
    Abstract:

    Prostasin, a membrane-bound serine protease, is known to increase the activity of the epithelial sodium channel (ENaC). Gene expression of Prostasin was shown to be regulated by aldosterone, which increases the rate of sodium reabsorption through ENaC. To clarify the physiological relationships among Prostasin, aldosterone and ENaC, we developed a specific radioimmunoassay (RIA) for human Prostasin. Prostasin levels in urine were determined in 26 normotensive and 121 hypertensive subjects. Aldosterone content in urine and plasma, urinary Na/K ratio and other clinical parameters were also measured. We observed a highly significant correlation between Prostasin and aldosterone concentration in urine (correlation coefficient: 0.673, P<0.0001). A significant correlation was also found between urinary Prostasin and plasma aldosterone concentration (correlation coefficient: 0.229, P<0.05). In addition, urinary Prostasin excretion was inversely correlated with urinary Na/K ratio (correlation coefficient: -0.425, P<0.0001). In conclusion, we developed a Prostasin-specific RIA and applied it to the clinical study. Our findings suggest that urinary Prostasin level is strongly correlated with urinary or plasma aldosterone level and may serve as a surrogate marker for ENaC activation in hypertensive patients. However, it is not clear, at the present time, whether endogenous aldosterone regulates Prostasin expression or vice versa.

  • Urinary Prostasin in humans: relationships among Prostasin, aldosterone and epithelial sodium channel activity
    Hypertension Research, 2009
    Co-Authors: Aya Koda, Kimio Tomita, Naoki Wakida, Kazuhiro Toriyama, Kazutoshi Yamamoto, Hiromi Iijima, Kenichiro Kitamura
    Abstract:

    Prostasin, a membrane-bound serine protease, is known to increase the activity of the epithelial sodium channel (ENaC). Gene expression of Prostasin was shown to be regulated by aldosterone, which increases the rate of sodium reabsorption through ENaC. To clarify the physiological relationships among Prostasin, aldosterone and ENaC, we developed a specific radioimmunoassay (RIA) for human Prostasin. Prostasin levels in urine were determined in 26 normotensive and 121 hypertensive subjects. Aldosterone content in urine and plasma, urinary Na/K ratio and other clinical parameters were also measured. We observed a highly significant correlation between Prostasin and aldosterone concentration in urine (correlation coefficient: 0.673, P

  • Camostat mesilate inhibits Prostasin activity and reduces blood pressure and renal injury in salt-sensitive hypertension.
    Journal of hypertension, 2009
    Co-Authors: Ai Maekawa, Kimio Tomita, Yutaka Kakizoe, Naoki Wakida, Naoki Shiraishi, Taku Miyoshi, Masataka Adachi, Kenichiro Kitamura
    Abstract:

    Prostasin, a glycosylphosphatidylinositol-anchored serine protease, regulates epithelial sodium channel (ENaC) activity. Sodium reabsorption through ENaC in distal nephron segments is a rate-limiting step in transepithelial sodium transport. Recently, proteolytic cleavage of ENaC subunits by Prostasin has been shown to activate ENaC. Therefore, we hypothesized that serine protease inhibitors could inhibit ENaC activity in the kidney, leading to a decrease in blood pressure. We investigated the effects of camostat mesilate, a synthetic serine protease inhibitor, and FOY-251, an active metabolite of camostat mesilate, on sodium transport in the mouse cortical collecting duct cell line (M-1 cells) and on blood pressure in Dahl salt-sensitive rats. Treatment with camostat mesilate or FOY-251 decreased equivalent current (Ieq) in M-1 cells in a dose-dependent manner and inhibited the protease activity of Prostasin in vitro. Silencing of the Prostasin gene also reduced equivalent current in M-1 cells. The expression level of Prostasin protein was not changed by application of camostat mesilate or FOY-251 to M-1 cells. Oral administration of camostat mesilate to Dahl salt-sensitive rats fed a high-salt diet resulted in a significant decrease in blood pressure with elevation of the urinary Na/K ratio, decrease in serum creatinine, reduction in urinary protein excretion, and improvement of renal injury markers such as collagen 1, collagen 3, transforming growth factor-beta1, and nephrin. These findings suggest that camostat mesilate can decrease ENaC activity in M-1 cells probably through the inhibition of Prostasin activity, and that camostat mesilate can have beneficial effects on both hypertension and kidney injury in Dahl salt-sensitive rats. Camostat mesilate might represent a new class of antihypertensive drugs with renoprotective effects in patients with salt-sensitive hypertension.

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  • Ibuprofen regulates the expression and function of membrane-associated serine proteases Prostasin and matriptase.
    BMC cancer, 2015
    Co-Authors: Andreas C. Chai, Karl X. Chai, Andrew L. Robinson, Li-mei Chen
    Abstract:

    Background The glycosylphosphatidylinositol-anchored extracellular membrane serine protease Prostasin is expressed in normal bladder urothelial cells. Bladder inflammation reduces Prostasin expression and a loss of Prostasin expression is associated with epithelial-mesenchymal transition (EMT) in human bladder transitional cell carcinomas. Non-steroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of various cancers including bladder cancer, but the molecular mechanisms underlying the anticancer effect of NSAIDs are not fully understood.

  • regulation of the matriptase Prostasin cell surface proteolytic cascade by hepatocyte growth factor activator inhibitor 1 during epidermal differentiation
    Journal of Biological Chemistry, 2010
    Co-Authors: Ya-wen Chen, Li-mei Chen, Karl X. Chai, Jehng-kang Wang, Michael D. Johnson, Feng-pai Chou, Chiuyuan Chen, Ellen A Rorke, Richard L Eckert, Chenyong Lin
    Abstract:

    Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of Prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates Prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and Prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, Prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-Prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) Prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active Prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active Prostasin to act on their substrates.

  • Prostasin regulates human placental trophoblast cell proliferation via the epidermal growth factor receptor signaling pathway
    Human reproduction (Oxford England), 2010
    Co-Authors: Wen-long Gao, Mengqian Chen, Karl X. Chai, Yan-ling Wang, Li-mei Chen
    Abstract:

    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. METHODS: An immortalized normal human CT cell line (B6Tert-1) was used as an in vitro cell model. Prostasin expression in B6Tert- 1 cells was knocked down by transfection of a short interfering RNA. Lentivirus-mediated expression of recombinant human Prostasin under tetracycline regulation was performed to obtain stable B6Tert-1 cell sublines that over-expressed Prostasin. Changes in cell proliferation and EGFR signaling were evaluated by immunocytochemistry for Ki67 and western blot analysis, respectively, in B6Tert-1 cells with knocked-down or increased Prostasin expression. RESULTS: Prostasin knock-down in B6Tert-1 cells resulted in inhibition of cell proliferation, in association with down-regulated EGFR protein expression (both P < 0.05 versus control) as well as reduced phosphorylation of c-raf, mitogen-activated protein kinase (MAPK) kinases (MEK1/2) and extracellular signal-regulated kinases (Erk1/2) (all P < 0.05 versus control). Over-expression of Prostasin led to up-regulation of the EGFR protein, but had no effect on cell proliferation or phosphorylation of MAPK signaling molecules in the B6Tert-I cells. CONCLUSIONS: Prostasin may regulate trophoblast cell proliferation via modulating the EGFR-MAPK signaling pathway.

  • Prostasin regulates inos and cyclin d1 expression by modulating protease activated receptor 2 signaling in prostate epithelial cells
    The Prostate, 2009
    Co-Authors: Li-mei Chen, Meghan L. Hatfield, Karl X. Chai
    Abstract:

    BACKGROUND Prostasin is down-regulated during inflammation and in invasive cancers, and plays a role in regulation of inflammatory gene expression and invasion. METHODS We used the human benign prostatic hyperplasia cell line BPH-1 to investigate gene expression changes associated with siRNA-mediated loss of Prostasin expression. Quantitative PCR and/or western blotting were used to evaluate the expression changes of iNOS, ICAM-1, cyclin D1, IL-6, and IL-8. Gene expression changes were also evaluated in the presence of a PAR-2 antagonist. The PC-3 human prostate cancer cell line was used for evaluation of gene expression in response to Prostasin re-expression. RESULTS Prostasin silencing in BPH-1 was associated with up-regulation of iNOS, ICAM-1, IL-6, and IL-8, and down-regulation of cyclin D1; as well as reduced proliferation and invasion. The iNOS up-regulation and cyclin D1 down-regulation associated with Prostasin silencing were inhibited by a PAR-2 antagonist. Re-expression of Prostasin, a serine active-site mutant, and a GPI-anchor-free mutant, in the PC-3 cells resulted in PAR-2 and cyclin D1 transcription up-regulation. Transcription up-regulation of IL-6 and IL-8 was associated with re-expression of the serine active-site mutant Prostasin in the PC-3 cells. Transcription up-regulation of IL-8, but to a lesser extent, was also observed in PC-3 cells expressing the wild-type Prostasin. Expression of a serine protease active Prostasin, GPI-anchored or anchor-free, prevented the IL-6 induction in response to PAR-2. The GPI-anchor-free Prostasin also prevented the IL-8 induction. CONCLUSIONS Prostasin plays a negative regulatory role on PAR-2-mediated signaling in prostate epithelial cells. Prostate 69: 1790–1801, 2009. © 2009 Wiley-Liss, Inc.

  • Hepsin activates Prostasin and cleaves the extracellular domain of the epidermal growth factor receptor
    Molecular and Cellular Biochemistry, 2009
    Co-Authors: Mengqian Chen, Li-mei Chen, Chenyong Lin, Karl X. Chai
    Abstract:

    The epithelial extracellular serine protease activation cascade involves matriptase (PRSS14) and Prostasin (PRSS8), capable of modulating epidermal growth factor receptor (EGFR) signaling. Matriptase activates Prostasin by cleaving in the amino-terminal pro-peptide region of Prostasin, presumably at the Arg residue of position 44 (R44) of the full-length human Prostasin. Using an Arg-to-Ala mutant (R44A) human Prostasin, we showed in this report that the cleavage of Prostasin by matriptase is at Arg44. This Prostasin proteolytic activation site is also cleaved by hepsin (TMPRSS1) to produce active Prostasin capable of forming a covalent complex with protease nexin 1 (PN-1). An amino-terminal truncation of EGFR in the extracellular domain (ECD) was observed when the receptor was co-expressed with hepsin. Hepsin and matriptase appear to cleave the EGFR ECD at different sites, while the hepsin cleavage is not affected by active Prostasin, which enhances the matriptase cleavage of EGFR. Using hepsin as the Prostasin-activating protease in cells co-transfected with EGFR, we showed that active Prostasin does not cleave the EGFR ECD directly in the cellular context. Purified active Prostasin also does not cleave purified EGFR. Hepsin cleavage of EGFR is not dependent on receptor tyrosine phosphorylation, while the hepsin-cleaved EGFR is phosphorylated at Tyr1068 and no longer responsive to EGF stimulation. The cleavage of EGFR by hepsin does not result in increased phosphorylation of the downstream extracellular signal-regulated kinases (Erk1/2), an event inducible by the matriptase-Prostasin cleavage of EGFR. The role of hepsin serine protease should be considered in future studies of epithelial biology concerning matriptase, Prostasin, and EGFR.

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