Heat shock-induced loss of the glucocorticoid receptor protein in cultured cells.
Effects of heat shock on the glucocorticoid receptor (GR) hormone binding activity and its degradation have been studied in mouse AtT-20 pituitary tumor cells in culture. Heat shock of cultured cells rapidly (30 min at 42 degrees C) enhanced the incorporation of [35S]methionine into heat shock protein 70 (hsp70), hsp90, and other unidentified hsp's. Maximum induction of newly synthesized hsp's occurred after 2-4 h at 42 degrees C. The initiation of the heat shock response rapidly and progressively decreased the total cellular GR hormone binding activity, and 4 h of heat shock resulted in the loss of about 80% of the initial hormone binding. This loss of GR hormone binding occurred at times when the general protein synthesis pattern or cellular viability was not significantly affected. In addition, cycloheximide prevented subsequent heat shock-induced loss of cellular hormone binding, suggesting that protein synthesis is required for this effect. By labeling of cellular GR with [3H]dexamethasone 21-mesylate at 37 degrees C and then transferring the cells to 42 degrees C, it was shown that heat shock caused an actual degradation (loss) of the 94 kDa GR protein. In addition, heat shock also caused loss of immunoreactive 94 kDa GR as detected by anti-GR (BuGR2) in a Western blot assay. A comparison of the loss of hormone binding and the disappearance of 94 kDa GR band during heat shock of AtT-20 cells followed similar kinetics (t1/2 = 1 h). Finally, the residual GR remaining in the cells after heat shock did not associate normally with the cell nucleus, perhaps indicating that a substantial alteration in GR structure had occurred. These studies suggest that the initiation of the shock response, and possibly the elevated levels of hsp's themselves, may be involved in the specific degradation of cellular GR. Therefore, heat shocked cells are likely to be refractile to the glucocorticoids.
Ali
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Gene Expression Laboratory, Pennington Biomedical Research Center, Baton Rouge, LA 70808.
Vedeckis
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WV
D000345:Affinity Labels; D000818:Animals; D015153:Blotting, Western; D003907:Dexamethasone / Q000031:analogs & derivatives; D006360:Heat-Shock Proteins / Q000096:biosynthesis*; D006358:Hot Temperature; D007700:Kinetics; D011869:Radioligand Assay; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D006360:Heat-Shock Proteins; D011965:Receptors, Glucocorticoid; D013462:Sulfur Radioisotopes; D003907:Dexamethasone; C031710:dexamethasone 21-methanesulfonate