K(+)-induced HSP-72 expression is mediated via rapid Ca(2+) influx in renal epithelial cells.
Pathophysiological stimuli, including hypoxia, lead to K(+) efflux from the intracellular lumen to the extracellular space, thereby increasing local tissue K(+) concentrations and depolarizing resident cells. In this study, we investigated the effects of increased extracellular K(+) concentrations ([K(+)](e)) on heat shock protein (HSP) expression in the porcine proximal tubule epithelial cell line LLC-PK(1). We analyzed HSP-25, HSP-72, HSC-73, and HSP-90 protein expression by Western blot analyses and HSP-72 promoter activity by luciferase reporter gene assays using the proximal 1,440 bp of the HSP-72 promoter. Elevating [K(+)](e) from 20 to 50 mM increased HSP-72 protein expression and promoter activity but did not affect HSP-25, HSC-73, or HSP-90 levels. Addition of identical concentrations of sodium chloride did not increase HSP-72 expression to a similar amount. The Ca(2+) channel blocker diltiazem and the Ca(2+)-specific chelator EGTA-AM abolished high [K(+)](e)-induced HSP-72 expression by 69.7 and 75.2%, respectively, indicating that the transcriptional induction of HSP-72 involves Ca(2+) influx. As measured by confocal microscopy using the Ca(2+) dye fluo 3-AM, we also observed a rapid increase of intracellular Ca(2+) concentration as early as 30 s after placing LLC-PK(1) cells in high [K(+)](e). We further analyzed whether Ca(2+) influx was necessary for induction of HSP-72 expression by high [K(+)](e) using Ca(2+)-free medium. Here, induction of HSP-72 in response to high [K(+)](e) was completely abolished. Our data thus demonstrate activation of a protective cellular response to ionic stress, e.g., elevated K(+) concentrations, by specifically increasing protein levels of HSP-72.
Eickelberg
O
O
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520-8023, USA. oliver.eickelberg@yale.edu
Geibel
J
J
Seebach
F
F
Giebisch
G
G
Kashgarian
M
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D004110:Diltiazem / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D017930:Genes, Reporter; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal; D011188:Potassium / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013552:Swine; D019284:Thapsigargin / Q000494:pharmacology; D019459:Urothelium / Q000166:cytology / Q000378:metabolism
D002121:Calcium Channel Blockers; D004791:Enzyme Inhibitors; D050884:HSP72 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011993:Recombinant Fusion Proteins; D004533:Egtazic Acid; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D019284:Thapsigargin; C046770:EGTA acetoxymethyl ester; D012964:Sodium; D004110:Diltiazem; D011188:Potassium; D002118:Calcium