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.
Tong
Zhenyue
Z
Cardiovascular Research Institute, and Department of Medicine, University of California at San Francisco, San Francisco 94143, USA.
Illek
Beate
B
Bhagwandin
Vikash J
VJ
Verghese
George M
GM
Caughey
George H
GH
D007611:Aprotinin / Q000494:pharmacology; D002460:Cell Line; D003550:Cystic Fibrosis / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D053503:Epithelial Sodium Channels; D000939:Epitopes; D004926:Escherichia coli; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology*; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism; D012995:Solubility; D014357:Trypsin / Q000494:pharmacology; D000099271:Prostasin
D007611:Aprotinin; D053503:Epithelial Sodium Channels; D000939:Epitopes; D017261:Glycosylphosphatidylinositols; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D012964:Sodium; D015222:Sodium Channels; D014357:Trypsin; D000099271:Prostasin