Regulation of the cellular stress response by reactive electrophiles: The role of covalent binding and cellular thiols in transcriptional activation of the 70-kilodalton heat shock protein gene by nephrotoxic cysteine conjugates

The cytotoxicity of nephrotoxic cysteine conjugates (NCC) in the renal epithelial cell line, LLC-PK1, is due to the covalent binding of a reactive electrophilic metabolite produced from NCC metabolism by cysteine conjugate β-lyase. Covalent binding of NCC-derived reactive metabolites leads to a cascade of events including depletion of cellular non-protein sulfhydryls, increased cytosolic free calcium, and lipid peroxidation, which is ultimately responsible for cell death. We have used this model to investigate the signalling mechanism(s) through which reactive electrophiles increase synthesis of the 70-kD heat shock protein (HSP70). NCC treatment resulted in increased HSP70 synthesis as well as time- and dose-dependent increases in hsp70 mRNA in LLC-PK1 cells. The induction of hsp70 mRNA was blocked by actinomycin D, and nuclear run-on experiments showed that the hsp70 gene was transcriptionally activated. Inhibition of protein synthesis did not block the increase in hsp70 mRNA or transcriptional activation of the Asp70 gene suggesting that induction occurs due to activation of existing transcription factors. Inhibiting the covalent binding with a β-lyase inhibitor, aminooxyacetic acid, blocked the increase in hsp70 mRNA. Agents which do not alter binding but do prevent toxicity by blocking the rise in cytosolic free calcium and lipid peroxidation were not effective inhibitors of hsp70 mRNA accumulation. However, the thiol reducing agent, dithiothreitol, inhibited induction of hsp70 mRNA by NCC. The data suggest that covalent binding and alterations in cellular non-protein thiols serve as signals for activation of pre-existing transcription factors which increase hsp70 gene expression. It is proposed that reactive electrophiles may have a primary effect on protein conformation resulting in activation of the hsp70 gene.