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At least 19 recordsLinked to original sources
PubMed2005

Identification of hepatocyte growth factor activator inhibitor-1B as a potential physiological inhibitor of prostasin.

Prostasin is a trypsin-like serine protease that is glycosylphosphatidylinositol-anchored to the epithelial cell surface, from where it can be released in a soluble form. We undertook a co-expression search using the Genesis Enterprise System Database from Gene Logic to identify prostasin inhibitors, on the assumption that prostasin and its natural inhibitors may have a similar gene expression pattern. We found the expression profile of prostasin in normal human tissues to correlate highly with hepatocyte growth factor activator inhibitor-1B (HAI-1B) and its splice variant HAI-1. Soluble HAI-1B (sHAI-1B), comprising the entire extracellular domain, formed a 1:1 complex with purified prostasin in protein binding assays and inhibited prostasin enzymatic activity with an IC(50) of 66 +/- 15 nM. Two sHAI-1B mutants with inactivated N- and C-terminal Kunitz domains (KD1 and KD2) were used to show that the interaction of sHAI-1B with prostasin is mediated by KD1. In agreement, KD1 (Thr(246)-Val(303)) alone potently inhibited prostasin activity (IC(50) = 4.7 +/- 0.5 nM). Furthermore, prostasin was isolated with two major HAI-1/1B fragments (40 and 58 kDa) from OVCAR3 cell medium, demonstrating that prostasin.HAI-1/1B complexes are formed naturally. Moreover, when prostasin and HAI-1B were co-expressed in Chinese hamster ovary cells, complexes of prostasin with HAI-1B were detected on the cell membrane as well as in the culture medium, suggesting that preformed complexes were shed from the cell surface. The identification of HAI-1B as a potential physiological regulator of prostasin function, as described herein, may further the investigation of the role of prostasin in normal physiology and cancer.

Alternative Splicing↗
PubMed2006

Prostasin regulates epithelial monolayer function: cell-specific Gpld1-mediated secretion and functional role for GPI anchor.

Prostasin, a trypsinlike serine peptidase, is highly expressed in prostate, kidney, and lung epithelia, where it is bound to the cell surface, secreted, or both. Prostasin activates the epithelial sodium channel (ENaC) and suppresses invasion of prostate and breast cancer cells. The studies reported here establish mechanisms of membrane anchoring and secretion in kidney and lung epithelial cells and demonstrate a critical role for prostasin in regulating epithelial monolayer function. We report that endogenous mouse prostasin is glycosylphosphatidylinositol (GPI) anchored to the cell surface and is constitutively secreted from the apical surface of kidney cortical collecting duct cells. Using site-directed mutagenesis, detergent phase separation, and RNA interference approaches, we show that prostasin secretion depends on GPI anchor cleavage by endogenous GPI-specific phospholipase D1 (Gpld1). Secretion of prostasin by kidney and lung epithelial cells, in contrast to prostate epithelium, does not depend on COOH-terminal processing at conserved Arg(322). Using stably transfected M-1 cells expressing wild-type, catalytically inactive, or chimeric transmembrane (not GPI)-anchored prostasins we establish that prostasin regulates transepithelial resistance, current, and paracellular permeability by GPI anchor- and protease activity-dependent mechanisms. These studies demonstrate a novel role for prostasin in regulating epithelial monolayer resistance and permeability in kidney epithelial cells and, furthermore, show specifically that prostasin is a critical regulator of transepithelial ion transport in M-1 cells. These functions depend on the GPI anchor as well as the peptidase activity of prostasin. These studies suggest that cell-specific Gpld1- or peptidase-dependent pathways for prostasin secretion may control prostasin functions in a tissue-specific manner.

Animals↗
PubMed2006

Androgen regulation of prostasin gene expression is mediated by sterol-regulatory element-binding proteins and SLUG.

BACKGROUND: Prostasin is downregulated in hormone-refractory prostate cancers (HRPC). The mechanisms by which androgens regulate prostasin expression are unclear. METHODS: LNCaP cells were treated with dihydrotestosterone (DHT), and mRNA expression of prostasin, SREBPs, SNAIL, and SLUG was examined by real-time PCR following reverse transcription. A human prostasin promoter was evaluated in HEK-293 cells co-transfected with transcription factor cDNAs. Regulation of endogenous prostasin expression by transfected SREBP-2 or SLUG was evaluated. Expression of SNAIL and SLUG mRNA in DU-145 cells treated with epidermal growth factor (EGF) was examined. RESULTS: Prostasin mRNA expression in LNCaP cells was not responsive to DHT treatment. DHT marginally upregulated mRNA expression of SREBP-1c, SREBP-2, and SNAIL, but not SREBP-1a, while dramatically increased SLUG mRNA expression, in a dose-dependent manner. Co-transfection of prostasin promoter and SREBP cDNA in HEK-293 cells resulted in stimulation of promoter activity at approximately twofold by SREBP-1c, and up to sixfold by SREBP-2; while co-transfection with SNAIL or SLUG cDNA resulted in repression of promoter activity to 43% or 59%, respectively. Co-transfection of the SLUG cDNA negated SREBP-2's stimulation of prostasin promoter in a dose-dependent manner. Transfection of an SREBP-2 cDNA in HEK-293 and DU-145 resulted in upregulation of prostasin while transfection of a SLUG cDNA in LNCaP repressed prostasin expression. EGF upregulated SNAIL and SLUG mRNA in DU-145. CONCLUSIONS: DHT regulates prostasin expression in prostate cells via SREBP stimulation and SLUG repression of prostasin promoter. SLUG is upregulated by DHT and EGF, providing a molecular mechanism by which epithelial cell-specific genes are silenced during prostate cancer development and progression.

Androgens↗
PubMed2001

Prostasin is a glycosylphosphatidylinositol-anchored active serine protease.

A recombinant human prostasin serine protease was expressed in several human cell lines. Subcellular fractionation showed that this serine protease is synthesized as a membrane-bound protein while a free-form prostasin is secreted into the culture medium. Prostasin was identified in nuclear and membrane fractions. Membrane-bound prostasin can be released by phosphatidylinositol-specific phospholipase C treatment, or labeled by [(3)H]ethanolamine, indicating a glycosylphosphatidylinositol anchorage. A prostasin-binding protein was identified in mouse and human seminal vesicle fluid. Both the secreted and the membrane-bound prostasin were able to form a covalently linked 82-kDa complex when incubated with seminal vesicle fluid. The complex formation between prostasin and the prostasin-binding protein was inhibited by a prostasin antibody, heparin, and serine protease inhibitors. Prostasin's serine protease activity was inhibited when bound to the prostasin-binding protein in mouse seminal vesicle fluid. This study indicates that prostasin is an active serine protease in its membrane-bound form.

Animals↗
PubMed2004

Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line.

Prostasin is a tryptic peptidase expressed in prostate, kidney, lung, and airway. Mammalian prostasins are related to Xenopus channel-activating protease, which stimulates epithelial Na+ channel (ENaC) activity in frogs. In human epithelia, prostasin is one of several membrane peptidases proposed to regulate ENaC. This study tests the hypothesis that prostasin can regulate ENaC in cystic fibrosis epithelia in which excessive Na+ uptake contributes to salt and water imbalance. We show that prostasin mRNA and protein are strongly expressed by human airway epithelial cell lines, including immortalized JME/CF15 nasal epithelial cells homozygous for the DeltaF508 cystic fibrosis mutation. Epithelial cells transfected with vectors encoding recombinant soluble prostasin secrete active, tryptic peptidase that is highly sensitive to inactivation by aprotinin. When studied as monolayers in Ussing chambers, JME/CF15 cells exhibit amiloride-sensitive, transepithelial Na+ currents that are markedly diminished by aprotinin, suggesting regulation by serine-class peptidases. Overproduction of membrane-anchored prostasin in transfected JME/CF15 cells does not augment Na+ currents, and trypsin-induced increases are small, suggesting that baseline serine peptidase-dependent ENaC activation is maximal in these cells. To probe prostasin's involvement in basal ENaC activity, we silenced expression of prostasin using short interfering RNA targeting of prostasin mRNA's 3'-untranslated region. This drops ENaC currents to 26 +/- 9% of baseline. These data predict that prostasin is a major regulator of ENaC-mediated Na+ current in DeltaF508 cystic fibrosis epithelia and suggest that airway prostasin is a target for therapeutic inhibition to normalize ion current in cystic fibrosis airway.

Aprotinin↗
PubMed2001

Activation of epithelial sodium channels by prostasin in Xenopus oocytes.

Prostasin, a novel serine protease, was purified from seminal fluid, and its cDNA sequence was determined. Expression of prostasin was detected in human tissues, including prostate, kidney, and lung, as well as bodily fluids, including seminal fluid and urine. However, its physiologic role in the human body is not known. Recently, a novel regulatory mechanism by which serine proteases activate epithelial sodium channel in the Xenopus oocyte was identified. Therefore, it was hypothesized that prostasin could activate sodium currents, and a rat prostasin cDNA clone was isolated to investigate its physiologic function. Rat prostasin mRNA is expressed predominantly in kidney, and lower levels of expression were detected in prostate, lung, colon, stomach, and skin. These all are epithelial tissues in which the epithelial sodium channel (ENaC) is expressed. Coexpression of rat prostasin and rat ENaC in Xenopus oocytes increased the amiloride-sensitive sodium current by twofold. Preincubation of oocytes that expressed prostasin with aprotinin did not result in an increase in sodium current, compared with the control. The removal of aprotinin from the bath solution resulted in a twofold increase of the current only in oocytes that expressed prostasin, which indicates that protease activity of prostasin is required for the ENaC activation. Expression of rat prostasin had no effect on the potassium current when expressed with rat renal outer medulla K channel, which shows specificity of prostasin action for ENAC: These results indicate that prostasin acts as an extracellular regulator of ENAC:

Amiloride↗
PubMed2001

Down-regulation of prostasin serine protease: a potential invasion suppressor in prostate cancer.

BACKGROUND: Prostasin is a serine protease predominantly expressed in normal prostate epithelial cells. The biological function of prostasin has not been determined. METHODS: Western blot and RT-PCR analyses were used to examine the expression of prostasin in prostate cancer cell lines. Immunohistochemistry was used to evaluate prostasin protein expression in human prostate cancer. An in vitro Matrigel invasion assay was used to test the invasiveness of prostate cancer cell lines forced to express recombinant prostasin. RESULTS: Both prostasin protein and mRNA were found to be expressed in normal human prostate epithelial cells and a non-invasive human prostate cancer cell line, the LNCaP, but neither was found in invasive human prostate cancer cell lines DU-145 and PC-3. Prostasin mRNA expression was absent in invasive prostate cancer cell lines of a transgenic mouse model. Immunohistochemistry analysis showed that prostasin protein expression is down-regulated in high-grade prostate cancer. Transfection of DU-145 and PC-3 cells with a full-length human prostasin cDNA restored prostasin expression and reduced the in vitro invasiveness by 68 and 42%, respectively. CONCLUSIONS: Our data indicate that prostasin may be implicated in normal prostate biology and is able to suppress prostate cancer invasion in vitro.

Blotting, Western↗
PubMed2006

Evidence for a matriptase-prostasin proteolytic cascade regulating terminal epidermal differentiation.

Recent gene ablation studies in mice have shown that matriptase, a type II transmembrane serine protease, and prostasin, a glycosylphosphatidylinositol-anchored membrane serine protease, are both required for processing of the epidermis-specific polyprotein, profilaggrin, stratum corneum formation, and acquisition of epidermal barrier function. Here we present evidence that matriptase acts upstream of prostasin in a zymogen activation cascade that regulates terminal epidermal differentiation and is required for prostasin zymogen activation. Enzymatic gene trapping of matriptase combined with prostasin immunohistochemistry revealed that matriptase was co-localized with prostasin in transitional layer cells of the epidermis and that the developmental onset of expression of the two membrane proteases was coordinated and correlated with acquisition of epidermal barrier function. Purified soluble matriptase efficiently converted soluble prostasin zymogen to an active two-chain form that formed SDS-stable complexes with the serpin protease nexin-1. Whereas two forms of prostasin with molecular weights corresponding to the prostasin zymogen and active prostasin were present in wild type epidermis, prostasin was exclusively found in the zymogen form in matriptase-deficient epidermis. These data suggest that matriptase, an autoactivating protease, acts upstream from prostasin to initiate a zymogen cascade that is essential for epidermal differentiation.

Animals↗
PubMed2002

Adenovirus-mediated human prostasin gene delivery is linked to increased aldosterone production and hypertension in rats.

Prostasin has been demonstrated to be an activator of epithelial sodium channels in cultured renal and bronchial epithelial cells. In this study, we evaluated the effects of adenovirus-mediated gene transfer of human prostasin on blood pressure regulation and sodium reabsorption in Wistar rats. Expression of human prostasin mRNA was identified in rat adrenal gland, liver, kidney, heart, lung, and aorta, and immunoreactive human prostasin was detected in the circulation and urine of rats receiving prostasin gene transfer. A single injection of adenovirus carrying the prostasin gene caused prolonged increases in blood pressure for 3-4 wk. Blood pressure increase was accompanied by elevated plasma aldosterone levels and reduced plasma renin activity. The increase in blood pressure and plasma aldosterone levels as well as the reduction of plasma renin activity correlated with the expression of human prostasin transgene. Elevated plasma aldosterone levels were detected at 3 days after gene transfer before the development of hypertension, indicating that stimulation of mineralocorticoid production is the primary target of prostasin. Prostasin gene transfer significantly reduced urinary K(+) excretion but increased urinary Na(+) and kallikrein excretion. Elevated renal kallikrein levels promote natriuresis, which may lead to sodium escape and prevent further increases of blood pressure after prostasin gene transfer. In summary, these results suggest that prostasin participates in blood pressure and electrolyte homeostasis by regulating the renin-angiotensin-aldosterone and kallikrein-kinin systems.

Adenoviridae↗
PubMed2002

Regulation of prostasin by aldosterone in the kidney.

Prostasin is a serine protease present in mammalian urine that increases the activity of the epithelial sodium channel (ENaC) when the two are coexpressed in Xenopus oocytes. To determine if aldosterone, one of the principal regulators of urinary Na reabsorption by the distal nephron, affects prostasin expression, we examined prostasin mRNA and protein in a cultured mouse cortical collecting duct cell line (M-1), whole rats, and patients with primary aldosteronism. Aldosterone treatment of M-1 cells substantially increased prostasin expression and stimulated (22)Na uptake. Urinary excretion of prostasin in rats that were infused with aldosterone likewise increased by approximately 4-fold when compared with the vehicle-infused rats. Finally, urinary excretion of prostasin in patients with primary aldosteronism was substantially increased when compared with normal patients. Adrenalectomy reduced urinary prostasin excretion to control levels, whereas urinary prostasin levels were not altered in patients undergoing surgery for other reasons. In patients with primary aldosteronism, reduction in the urinary excretion of prostasin correlated with the increase in the urinary Na/K ratio. These findings, together with our previous report that prostasin activates the amiloride-sensitive Na currents through ENaC, demonstrate that prostasin regulates Na balance in vivo by virtue of its heightened expression in the presence of aldosterone.

Adrenalectomy↗
PubMed2001

Prostasin, a potential serum marker for ovarian cancer: identification through microarray technology.

BACKGROUND: Screening biomarkers for ovarian cancer are needed because of its late stage at diagnosis and poor survival. We used microarray technology to identify overexpressed genes for secretory proteins as potential serum biomarkers and selected prostasin, a serine protease normally secreted by the prostate gland, for further study. METHODS: RNA was isolated and pooled from three ovarian cancer cell lines and from three normal human ovarian surface epithelial (HOSE) cell lines. Complementary DNA generated from these pools was hybridized to a microarray slide, and genes overexpressed in the cancer cells were identified. Real-time quantitative polymerase chain reaction was used to examine prostasin gene expression in ovarian cancer and HOSE cell lines. Anti-prostasin antibodies were used to examine prostasin expression and to measure serum prostasin by an enzyme-linked immunosorbent assay in 64 case patients with ovarian cancer and in 137 control subjects. Previously determined levels of CA 125, an ovarian cancer marker, were available from about 70% of all subjects. All statistical tests were two-sided. RESULTS: Prostasin was detected by immunostaining more strongly in cancerous ovarian epithelial cells and stroma than in normal ovarian tissue. The mean level of serum prostasin was 13.7 microg/mL (95% confidence interval [CI] = 10.5 to 16.9 microg/mL) in 64 case patients with ovarian cancer and 7.5 microg/mL (95% CI = 6.6 to 8.3 microg/mL) in 137 control subjects (P<.001, after adjustment for the subject's age, year of collection, and specimen quality). In 14 of 16 case patients with both preoperative and postoperative serum samples, postoperative prostasin levels were statistically significantly lower than preoperative levels (P =.004). In 37 case patients with nonmucinous ovarian cancer and in 100 control subjects for whom levels of CA 125 and prostasin were available, the combination of markers gave a sensitivity of 92% (95% CI = 78.1% to 98.3%) and a specificity of 94% (95% CI = 87.4% to 97.7%) for detecting ovarian cancer. CONCLUSIONS: Prostasin is overexpressed in epithelial ovarian cancer and should be investigated further as a screening or tumor marker, alone and in combination with CA 125.

Adult↗
PubMed2003

Inhibition of prostasin secretion by serine protease inhibitors in the kidney.

A serine protease, prostasin, has been shown to stimulate the activity of amiloride-sensitive sodium channels (ENaC). Prostasin is a glycosylphosphatidylinositol-anchored protein that is found free in physiologic fluids and tissue culture medium, but the mechanism by which prostasin is secreted from the cells has not been elucidated. The current studies found that serine protease inhibitor aprotinin blocked the secretion of prostasin in a mouse cortical collecting duct (CCD) cell line (M-1 cells). A synthetic serine protease inhibitor, nafamostat mesilate (NM), which is commonly used for the treatment of pancreatitis and disseminated intravascular coagulation in Japan, also inhibited the secretion of prostasin in M-1 cells. Continuous infusion of NM into rats resulted in a substantial decrease in urinary prostasin and urinary sodium excretion. p-guanidinobenzoic acid and 6-amidino-2-naphtol, catalytically inactive metabolites of NM, had no effect on prostasin secretion both in M-1 cells and in rats. These findings suggest that a serine protease-sensitive mechanism is involved in the secretion of prostasin in vitro as well as in vivo. Potassium secretion in the CCD is tightly linked to sodium reabsorption through EnaC; therefore, NM-induced decrease in prostasin secretion and subsequent inhibition of ENaC activity could account for the side effects of hyponatremia and/or hyperkalemia that are found sometimes in patients treated with NM. The results indicate an important role for prostasin in sodium reabsorption in the kidney under pathophysiologic conditions.

Animals↗
PubMed2006

Prostasin attenuates inducible nitric oxide synthase expression in lipopolysaccharide-induced urinary bladder inflammation.

Prostasin is a glycosylphosphatidylinositol-anchored serine protease, with epithelial sodium channel activation and tumor invasion suppression activities. We identified the bladder as an expression site of prostasin. In the mouse, prostasin mRNA expression was detected by reverse transcription and real-time polymerase chain reaction in the bladder, and the prostasin protein was localized by immunohistochemistry in the urothelial cells. In mice injected intraperitoneally with bacterial lipopolysaccharide (LPS), bladder prostasin mRNA expression was downregulated, whereas the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interferon-gamma (IFN-gamma), TNF-alpha, IL-1beta, and IL-6 was upregulated. Viral promoter-driven expression of the human prostasin homolog in the bladder of transgenic mice attenuated the LPS induction of iNOS but did not abolish the induction. LPS induction of COX-2, TNF-alpha, IL-1beta, and IL-6 expression, however, was not reduced by prostasin transgene expression. Liposome-mediated delivery of prostasin-expressing plasmid into mouse bladder produced similar attenuation effects on LPS-induced iNOS expression, while not affecting COX-2 or cytokine induction. Mice receiving plasmid expressing a catalytic mutant prostasin did not manifest the iNOS induction attenuation phenotype. We propose a proteolytic mechanism for prostasin to intercept cytokine signaling during LPS-induced bladder inflammation.

Animals↗
PubMed2002

Prostasin serine protease inhibits breast cancer invasiveness and is transcriptionally regulated by promoter DNA methylation.

We have shown that prostasin serine protease is downregulated in high-grade prostate tumors and inhibits invasiveness of prostate cancer cell lines upon enforced reexpression. In our study, prostasin mRNA and protein were shown to be expressed in normal human mammary epithelial cells (NHMEC), the poorly invasive breast carcinoma cell line MCF-7 and the nonmetastatic breast carcinoma cell line MDA-MB-453, but absent in highly invasive and metastatic breast carcinoma cell lines MDA-MB-231 and MDA-MB-435s. Enforced reexpression of prostasin in MDA-MB-231 and MDA-MB-435s reduced the in vitro invasiveness of either cell line by 50%. Examination of the prostasin gene promoter and first exon revealed a GC-enriched region that contains transcription regulatory elements. The promoter and exon 1 region of the prostasin gene was investigated for DNA methylation in NHMEC and the carcinoma cell lines. The results revealed a methylation pattern that correlates with prostasin expression in these cells. Demethylation coupled with histone deacetylase inhibition resulted in reactivated expression of the prostasin mRNA in MDA-MB-231 and MDA-MB-435s cells. These results suggest that prostasin expression in breast cancer cells may be regulated by DNA methylation and that an absence of prostasin expression may contribute to breast cancer invasiveness and metastatic potential.

Blotting, Southern↗
PubMed2004

Regulation of prostasin expression and function in the prostate.

BACKGROUND: The invasion suppressor prostasin is down-regulated in prostate cancer, but the mechanism is unknown. A prostasin-binding protein (PBP) was found in the seminal vesicles, but its identity remains unclear. METHODS: Genomic Southern blot analysis using methylation sensitive restriction endonucleases was employed to examine the prostasin gene promoter region in prostate cancer cell lines. RT-PCR was employed to examine prostasin expression under demethylation, histone deacetylase inhibition, and nerve growth factor (NGF) treatment. Liquid column chromatography was employed to purify the PBP from mouse seminal vesicles. The PBP was further characterized by amino acid sequence analysis, recombinant protein expression, protease inhibition and binding assays. Immunohistochemistry and Western blot analysis were used to evaluate PBP expression in the prostate and prostate cancer cells. RESULTS: Promoter DNA methylation partly causes the prostasin down-regulation in DU-145 and PC-3 cells, while prostasin expression can be induced by NGF. The PBP is identified to be protease nexin-1 (PN-1), a serpin. PN-1 inhibits prostasin's serine protease activity, is expressed by prostate epithelial cells (PrECs) and prostate cancer cells, and capable of binding to membrane-anchored prostasin. CONCLUSIONS: Prostasin's expression and function are regulated by factors in the prostate tissue environment.

Amyloid beta-Protein Precursor↗
PubMed2005

Inhibition of prostasin expression by TGF-beta1 in renal epithelial cells.

BACKGROUND: Prostasin has been shown to be involved in the regulation of sodium handling in the kidney. TGF-beta1 has been demonstrated to suppress alphaENaC expression and sodium uptake. Therefore, we hypothesized that TGF-beta1 may regulate prostasin expression to modulate sodium reabsorption in the kidney. METHODS: To determine if TGF-beta1 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-beta1 on the mRNA and protein abundance of prostasin, and amiloride-sensitive (22)Na uptake was determined. RESULTS: Treatment of M-1 cells with 20 ng/mL of TGF-beta1 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-beta1-mediated inhibition, and found that TGF-beta1 increases IkappaBalpha expression in M-1 cells. TGF-beta1 reduced endogenous prostasin mRNA and protein expression in M-1 cells by 50 +/- 12% and 44 +/- 12%, respectively, and the amiloride-sensitive (22)Na uptake by 35.9 +/- 4.8%. CONCLUSION: Our findings indicate the possibility that TGF-beta1 transcriptionally inhibits prostasin expression by the induction of IkappaBalpha and the subsequent inhibition of NF-kappaB/Rel activity in M-1 cells, and also suggest the possibility that TGF-beta1 might inhibit sodium reabsorption through a reduction in prostasin expression and subsequent inhibition of ENaC activity.

Amiloride↗
PubMed2004

In vivo induction of prostasin mRNA in colonic epithelial cells by dietary sodium depletion and aldosterone infusion in rats.

BACKGROUND: Parallel induction of prostasin, a novel serine protease, together with epithelial sodium channel (ENaC) in the colon, may be essential for physiological response to increased circulating aldosterone. The aim of the present study was to investigate whether aldosterone induces prostasin mRNA in parallel with enhanced expression of ENaC in colonic epithelial cells. METHODS: Sprague-Dawley rats were maintained on a sodium-depleted diet or subjected to continuous aldosterone infusion up to 4 weeks. Rats were necropsied at 1, 2, or 4 weeks after the beginning of each treatment. Blood was immediately collected and the large intestine was removed. Plasma aldosterone and arginine-vasopressin (AVP) levels were measured by radio-immunoassay. Epithelial cells were isolated from the right and left colon and RNA was extracted. Expression of prostasin and the alpha-, beta-, and gamma-subunits of ENaC was evaluated by quantitative RT-PCR or Northern blot analysis. In another series of experiments, T84 cells were stimulated with aldosterone, dexamethasone, and AVP alone or in combination, and prostasin mRNA was measured by quantitative RT-CPR. RESULTS: Treatment with sodium-depleted diet and aldosterone infusion resulted in an increase of plasma aldosterone and induction of prostasin mRNA in the left colon. Expression of three subunits of ENaC also increased in the left colon. Induction of prostasin mRNA was observed when T84 cells were stimulated with corticosteroids plus AVP in vitro. CONCLUSIONS: Aldosterone has a pivotal role for increasing expression of prostasin in epithelial cells of the left colon. AVP may have a synergistic effect on aldosterone-mediated prostasin induction.

Aldosterone↗
PubMed2004

Biochemical characterization of prostasin, a channel activating protease.

Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1', P2', and P3' displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4' had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC alpha- and beta-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1' explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate.

Animals↗