HSP-72 SYNTHESIS IS PROMOTED BY INCREASE IN [CA2+](I) OR ACTIVATION OF G-PROTEINS BUT NOT PH(I) 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 (pH(i)), 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 pH(i), cellular cAMP, and [Ca2+](i) on HSP-72 induction. The reduction of pH(i) 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 Ca2+-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 pH(i) and cAMP, enhances HSP-72 induction.