Overexpression of HSP-70 Attenuates Increases in [Ca2+]iand Protects Human Epidermoid A-431 Cells after Chemical Hypoxia☆

Abstract This laboratory previously reported that thermotolerance diminishes the NaCN-induced increase in intracellular free calcium concentrations ([Ca 2+ ] i ) in human epidermoid A-431 cells and that blocking this increase protects the cells from NaCN toxicity. In this study, we report that cell viability after exposure to NaCN (10 mM, 1 h) is enhanced by the overexpression of HSP-70 resulting from heat shock (45°C, 10 min), treatment with a protein kinase C activator phorbol 12 myristate 13-acetate (PMA; 1 μM, 4 h), or HSP-70 cDNA transfection. Because the toxicity of NaCN is mediated by increases in [Ca 2+ ] i , we sought to determine whether the overexpression of HSP-70 might protect the cells by altering the [Ca 2+ ] i response induced by NaCN. Basal [Ca 2+ ] i in vector-, HSF1 cDNA-, and HSP-70 cDNA-transfected cells was 114 ± 11 ( n = 11), 95 ± 5 ( n = 6), and 151 ± 11 ( n = 15) nM, respectively, suggesting that HSP-70 metabolism is associated with maintenance of resting [Ca 2+ ] i . Removal of external Ca 2+ reduced the resting [Ca 2+ ] i in all of these cells. With external Ca 2+ present, NaCN increased [Ca 2+ ] i by 97 ± 21% in vector-transfected cells and 111 ± 5% in HSF1 vector-transfected cells but by only 27 ± 8% in HSP-70 cDNA-transfected cells. Heat shock or PMA treatment of vector- or HSF1 cDNA-transfected cells to induce HSP-70 also attenuated the NaCN-induced increase in [Ca 2+ ] i , perhaps because of a decrease in V max for the uptake of external Ca 2+ . Removal of external Ca 2+ or treatment with inhibitors of Na + /Ca 2+ exchangers eliminated the NaCN-induced increase in [Ca 2+ ] i in HSP-70 cDNA-transfected cells, but ryanodine treatment did not. HSP-70 cDNA transfection also reduced Ca 2+ mobilization stimulated by various Ca 2+ -mobilizing agents. The results suggest that HSP-70 overexpression protects cells from NaCN cytotoxicity, perhaps by attenuating the [Ca 2+ ] i response.