The epithelial sodium channel (ENaC) constitutes the rate-limiting step for sodium absorption across airway epithelia, which in turn regulates airway surface liquid (ASL) volume and the efficiency of mucociliary clearance. This role in ASL volume regulation suggests that ENaC activity is influenced by local factors rather than systemic signals indicative of total body volume homeostasis. Based on reports that ENaC may be regulated by extracellular serine protease activity in Xenopus and mouse renal epithelia, we sought to identify proteases that serve similar functions in human airway epithelia. Homology screening of a human airway epithelial cDNA library identified two trypsin-like serine proteases (prostasin and TMPRSS2) that, as revealed by in situ hybridization, are expressed in airway epithelia. Functional studies in the Xenopus oocyte expression system demonstrated that prostasin increased ENaC currents 60 -80%, whereas TMPRSS2 markedly decreased ENaC currents and protein levels. Studies of primary nasal epithelial cultures in Ussing chambers revealed that inhibition of endogenous serine protease activity with aprotinin markedly decreased ENaC-mediated currents and sensitized the epithelia to subsequent channel activation by exogenous trypsin. These data, therefore, suggest that protease-mediated regulation of sodium absorption is a function of human airway epithelia, and prostasin is a likely candidate for this activity.
To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-L-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-␣]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.
HK gene delivery enhances the native angiogenic response to ischemia. Angiogenesis gene therapy with HK might be applicable to peripheral occlusive vascular disease.
We have purified a novel human serine proteinase, designated as prostasin, from seminal fluid (Yu et al., 1994). In the present study, we have cloned and characterized the full-length cDNA encoding prostasin and identified its tissue-specific expression and cellular localization. A cDNA fragment was obtained by polymerase chain reaction using degenerate oligonucleotide primers derived from the NH2-terminal and internal amino acid sequences. A full-length cDNA sequence encoding prostasin was obtained by amplification of the 5'- and 3'-ends of the cDNA. It contains a 1,032-base coding region, a 572-base 3'-noncoding region and a 138-base 5'-noncoding sequence. Prostasin cDNA encodes a protein of 343 amino acids, which consists of a 32-amino acid signal peptide and a 311-amino acid proprostasin. Proprostasin is then cleaved between Arg12 and Ile13 to generate a 12-amino acid light chain and a 299-amino acid heavy chain, which are associated through a disulfide bond. The deduced amino acid sequence of the heavy chain has 34-42% identity to human acrosin, plasma kallikrein, and hepsin. A potential N-glycosylation site at Asn127 and the catalytic triad of His53, Asp102, and Ser206 have been identified. The deduced prostasin has a unique 19-amino acid hydrophobic portion at the COOH terminus, which makes it suitable to anchor in the cell membrane. Carboxyl-terminal sequencing of purified prostasin indicates that the hydrophobic portion is removed and that there is a cleavage between Arg290 and Pro291 during secretion. Southern blot analysis, following a reverse transcription polymerase chain reaction, indicates that prostasin mRNA is expressed in prostate, liver, salivary gland, kidney, lung, pancreas, colon, bronchus, renal proximal tubular cells, and prostate carcinoma LNCaP cells. Cellular localization of prostasin mRNA was identified within epithelial cells of the human prostate gland by in situ hybridization histochemistry.
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.
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.Prostasin is a serine protease discovered in ejaculated human semen in 1994 (1). The molecular mass of prostasin is 40 kDa when examined by SDS-polyacrylamide gel electrophoresis (PAGE) 1 under reducing conditions. Prostasin displays trypsin-like enzymatic activities by hydrolyzing peptidyl fluorogenic substrates such as D-Pro-Phe-Arg-AMC. This trypsinlike enzymatic activity can be inhibited by aprotinin, antipain, leupeptin, and benzamidine. Prostasin is present at high levels in normal human semen (8.61 Ϯ 0.42 g/ml) and in the prostate gland (143.7 Ϯ 15.9 ng/mg). Lower amounts of prostasin can also be detected in other tissues. In the prostate gland, the prostasin protein is present in the epithelial cells as well as in the secretion inside the lumen. The full-length human prostasin mRNA has been deduced (2). The predicted mature prostasin peptide sequence has a potential carboxyl-terminal hydrophobic membrane anchorage domain followed by a short cytoplasmic tail. The translated amino acid residue sequence of prostasin is similar to those of human prostase, testisin, plasma kallikrein, coagulation factor XI, hepsin, plasminogen, and acrosin (2-4). A membrane-bound Xenopus kidney epithelial cell sodium channel-activating protease (CAP1) was found highly homologous to human prostasin, sharing 53% sequence identity at the amino acid level (5). Recently, the mouse counterpart of CAP1, mCAP1, has been cloned from a cortical collecting duct cell line (6). mCAP1 shares 77% amino acid sequence identity with human prostasin.Serine proteases play important roles in a diverse range of the body's normal physiological processes, and they are implicated in various pathological processes such as cardiovascular disorders and cancers (7). The prostate produces a number of serine proteases such as prostate-specific antigen (8), human glandular kallikrein (9), and the most recently discovered prostase (3). Some of these serine proteases are ...
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