HSP-72 synthesis is promoted by increase in [Ca2+]i or activation of G proteins but not pHi or cAMP

The family of 70-kDa heat-shock proteins (HSP-70) is evolutionarily highly conserved and has been shown to enhance cell survival from thermal injury. This study characterized HSP-72 induction in human epidermoid A-431 cells exposed to 45 degrees C for 10 min and determined the relationship between HSP-72, intracellular pH (pHi), adenosine 3',5'-cyclic monophosphate (cAMP), G proteins, and intracellular cytosolic free Ca2+ concentration ([Ca2+]i). Heat shock induced HSP-72 production, which was dependent on both temperature and the duration of heating. This HSP-72 induction was confirmed by Western blot analysis. HSP-72 levels in cells that had been heated then returned to 37 degrees C were elevated at 2 h (1.5 +/- 0.1 x control), reached a maximum at 8 h (2.7 +/- 0.1 x control), and remained above baseline for up to 4 days. Levels of HSP-72 mRNA, determined by dot-blot analysis, reached a maximum at 2 h and returned to baseline within 8 h. Both actinomycin D and cycloheximide blocked HSP-72 induction. Because heating causes intracellular acidification and increases in cAMP and [Ca2+]i, we studied the effect of pHi, cellular cAMP, and [Ca2+]i on HSP-72 induction. The reduction of pHi to 6.9 by acid loading did not affect the basal level of HSP-72 in unheated cells. Treatment with pertussis toxin, cholera toxin, or forskolin, but not 8-bromo-cAMP, 3-isobutyl-1-methylxanthine, or N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide potentiated heat-induced HSP-72 production. Inhibition of the heat-induced increase in [Ca2+]i attenuated, but failed to completely block, heat-induced HSP-72 production, mRNA synthesis, and the heat-shock transcriptional factor-heat-shock element binding complex formation, which suggests there are Ca(2+)-dependent and -independent processes involved in HSP-72 synthesis. Our results show that an increase in [Ca2+]i or activation of G proteins, but not pHi and cAMP, enhances HSP-72 induction.