Increases in HSF1 Translocation and Synthesis in Human Epidermoid A-431 Cells: Role of Protein Kinase C and [Ca 2+ ] i

Abstract

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⁺β]_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 HSP-70 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 [³βP]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 µmol/L PMA were completely blocked by preincubating cells with either 2 µmol/L staurosporine in the presence of extracellular Ca²⁺ or 100 µmol/L BAPTA-am in absence of extracellular Ca²⁺. Like PMA, the increases induced by ionomycin were also inhibited by 100 µmol/L BAPTA-am in absence of extracellular Ca²⁺. Furthermore, Western blots show that 1 µmol/L PMA or ionomycin induced maximal increase in HSP-70 after 7 hours of continuous incubation with either agent. When cells were simultaneously treated with 1µmol/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.