Regulation of calcineurin activity in insulin-secreting cells: stimulation by Hsp90 during glucocorticoid-induced apoptosis.
Previously, we described that apoptotic cell death induced by the synthetic glucocorticoid dexamethasone (dex) is inhibited by calcineurin inhibitors, FK506 and deltamethrin, in insulin-secreting cells. The aim of the present study was to examine the mechanism of dex-dependent activation of calcineurin. In INS-1 cells cultured up to 4d with dex (100 nmol/l), the percentage of apoptosis, quantified by condensed nuclei and TUNEL positive cells, increased from 1% to 10.9%. FK506 inhibited dex-mediated cell death. Apoptosis was significantly higher at glucose concentrations that induce [Ca(2+)](i) oscillations than at low, non-stimulatory glucose. Dex had no acute effect on [Ca(2+)](i). Calcineurin activity, measured in control and dex-treated cell homogenates, revealed that maximal activity and the sensitivity to the substrate RII peptide was unaltered. However, dex treatment significantly increased enzyme activity at submaximal, physiological Ca(2+) concentrations. Dex did not stimulate the Ca(2+)-dependent protease calpain, known to activate calcineurin by cleavage, as no cleaved calcineurin was detectable. Furthermore, the calpain inhibitor ALLN did not counteract dex-dependent cell death. Western blotting revealed that in dex-treated cells heat shock protein 90 (Hsp90), a component of the glucocorticoid receptor (GR) known to stimulate calcineurin, was increased while calcineurin protein levels were unchanged. In immunoprecipitates with calcineurin antibodies, Hsp90 was only detected in dex-treated cell homogenates. These data suggest that dex-induced apoptosis involves release of Hsp90 from the stimulated GR complex, subsequent binding to and activation of calcineurin, that may contribute to dex-mediated cell death in the presence of high glucose.
Ranta
Felicia
F
Institute of Physiology, University of Tübingen, Gmelinstrasse 5, D-72076 Tübingen, Germany.
Düfer
Martina
M
Stork
Björn
B
Wesselborg
Sebastian
S
Drews
Gisela
G
Häring
Hans-Ulrich
HU
Lang
Florian
F
Ullrich
Susanne
S
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D019703:Calcineurin / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D016257:Fura-2; D005947:Glucose / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D013379:Substrate Specificity / Q000187:drug effects; D016559:Tacrolimus / Q000494:pharmacology
D065095:Calcineurin Inhibitors; D018841:HSP90 Heat-Shock Proteins; D003907:Dexamethasone; D019703:Calcineurin; D002154:Calpain; D005947:Glucose; D002118:Calcium; D016257:Fura-2; D016559:Tacrolimus