Heat Shock Protein 90β1 Is Essential for Polyunsaturated Fatty Acid-induced Mitochondrial Ca2+ Efflux

摘要

Nonesterified fatty acids may influence mitochondrial function by alterations in gene expression, metabolism, and/or mitochondrial Ca2+ ([Ca2+]m) homeostasis. We have previously reported that polyunsaturated fatty acids induce Ca2+ efflux from mitochondria, an action that may deplete [Ca2+]m and thus contribute to nonesterified fatty acid-responsive mitochondrial dysfunction. Here we show that the chaperone protein heat shock protein 90 β1 (hsp90β1) is required for polyunsaturated fatty acid-induced mitochondrial Ca2+ efflux (PIMCE). Retinoic acid induced differentiation of human teratocarcinoma NT2 cells in association with attenuation of PIMCE. Proteomic analysis of mitochondrial proteins revealed that hsp90β1, among other proteins, was reduced in retinoic acid-differentiated cells. Blockade of PIMCE in NT2 cells by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, a known inhibitor of the chaperone activity of hsp90, and hsp90β1 RNA interference demonstrated that hsp90β1 is essential for PIMCE. We also show localization of hsp90β1 in mitochondria by Western blot and immunofluorescence. Distinctive effects of inhibitors binding to the N or C terminus of hsp90 on PIMCE in isolated mitochondria suggested that the C terminus of hsp90β1 plays a critical role in PIMCE. Nonesterified fatty acids may influence mitochondrial function by alterations in gene expression, metabolism, and/or mitochondrial Ca2+ ([Ca2+]m) homeostasis. We have previously reported that polyunsaturated fatty acids induce Ca2+ efflux from mitochondria, an action that may deplete [Ca2+]m and thus contribute to nonesterified fatty acid-responsive mitochondrial dysfunction. Here we show that the chaperone protein heat shock protein 90 β1 (hsp90β1) is required for polyunsaturated fatty acid-induced mitochondrial Ca2+ efflux (PIMCE). Retinoic acid induced differentiation of human teratocarcinoma NT2 cells in association with attenuation of PIMCE. Proteomic analysis of mitochondrial proteins revealed that hsp90β1, among other proteins, was reduced in retinoic acid-differentiated cells. Blockade of PIMCE in NT2 cells by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin, a known inhibitor of the chaperone activity of hsp90, and hsp90β1 RNA interference demonstrated that hsp90β1 is essential for PIMCE. We also show localization of hsp90β1 in mitochondria by Western blot and immunofluorescence. Distinctive effects of inhibitors binding to the N or C terminus of hsp90 on PIMCE in isolated mitochondria suggested that the C terminus of hsp90β1 plays a critical role in PIMCE. Defective mitochondrial function has been observed in type 2 diabetes and is proposed to be a major contributing factor in the pathogenesis and progression of the disease (1Lowell B.B. Shulman G.I. Science. 2005; 307: 384-387Crossref PubMed Scopus (1603) Google Scholar, 2Kelley D.E. He J. Menshikova E.V. Ritov V.B. Diabetes. 2002; 51: 2944-2950Crossref PubMed Scopus (1766) Google Scholar). Although the mechanism leading to mitochondrial dysfunction in diabetes remains under intensive investigation, a critical role for nonesterified fatty acids (NEFA) 2The abbreviations used are: NEFAnonesterified fatty acid(s)PDHpyruvate dehydrogenaseFAfatty acid(s)PUFApolyunsaturated fatty acid(s)PIMCEpolyunsaturated fatty acid-induced mitochondrial Ca2+ effluxhspheat shock proteinRAretinoic acidRNAiRNA interferencePBSphosphate-buffered saline17-DMAG17-(dimethylaminoethylamino)-17-demethoxygeldanamycinFBSfetal bovine serumCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidHPLChigh pressure liquid chromatographyLAlinoleic acid. and/or fatty acid metabolites is emphasized by an increasing body of evidence (3Reaven G.M. Hollenbeck C. Jeng C-Y. Wu M.S. Chen Y.-D.I. Diabetes. 1988; 37: 1020-1024Crossref PubMed Scopus (0) Google Scholar, 4Paolisso G. Tataranni P.A. Foley J.E. Bogardus C. Howard B.V. Ravussin E. Diabetologia. 1995; 38: 1213-1217Crossref PubMed Scopus (309) Google Scholar, 5Joseph J.W.