Increases in HSF1 translocation and synthesis in human epidermoid A-431 Cells: role of protein kinase C and [Ca²⁺]_i

Background: It is known that heat shock increases both heat shock protein 70 kd (HSP-70), mRNA synthesis, and intracellular cytosolic free calcium concentration ([Ca²⁺]_i). The latter enhances the heat inducible form of HSP-70 production by increasing the complex formation between heat shock transcriptional factor (HSF) and heat shock elements (HSE). In this study, we investigated the effect of agonists (PMA; ionomycin) and antagonists (BAPTA; staurosporine) of protein kinase C (PKC), and calcium channel on translocation and synthesis of HSF1, and activation of HSP-70 gene in human epidermoid A-431 cells. Methods: Cells were incubated with poly 12-myristate 13-acetate (PMA) or ionomycin at different concentrations for various periods of time. Messenger RNAs of HSF and HSP70 were measured with RT-PCR. The HSP-70 protein was determined with Western blots, and HSF protein was measured by gel mobility retardation assay. Results: Significant increases in HSF binding to [32P]labeled HSE were found at 30 minutes in nuclear extract and at 4 hours in both nuclear and cytosol extracts. The PMA- and ionomycin-induced increases in HSF were in a concentration-dependent manner with a maximal increase at 10^-6 mol/L of each drug. Meanwhile, the mRNAs encoded for HSF1 and HSP-70, but not HSF2, were significantly increased and reached the maximum at 1 hour after the treatment. The PMA increased [Ca²⁺]_i by 92% because of Ca²⁺ influx. The increases in mRNA of HSF1 and HSP-70 induced by treatment with 1 umol/L PMA were completely blocked by preincubating cells with either 2 umol/L staurosporine in the presence of extracellular Ca²⁺ or 100 umol/L BAPTA-am in absence of extracellular Ca²⁺. Like PMA, the increases induced by ionomycin were also inhibited by 100 pmol/L BAPTA-am in absence of extracellular Ca²⁺. Furthermore, Western blots show that 1 umol/L PMA or ionomycin induced maximal increase in HSP-70 after 7hours of continuous incubation with either agent. When cells were simultaneously treated with 1 umol/L PMA and ionomycin together for 1 hour, the increase in HSP-70 and HSF1 mRNAs reached a greater level than the level stimulated by either drug alone. Conclusions: These results indicate that both PMA and ionomycin stimulate HSF1, but not HSF2, translocation and synthesis leading to the HSP-70 expression and that their effects are Ca²⁺-dependent.