Reduction of trans-4,5-dihydroxy-1,2-dithiane by cellular oxidoreductases activates gadd153/chop and grp78 transcription and induces cellular tolerance in kidney epithelial cells

Trans-4,5-Dihydroxy-1,2-dithiane, the intramolecular disulfide form of dithiothreitol (DTTox) transcription ally activates the stress-responsive genes gadd153(chop) and grp78, Herein, we used a renal epithelial cell line, LLC-PK1, to investigate the mechanism(s) whereby DTTox activates a molecular stress response, DTTox activated both grp78 and gadd153 transcriptionally, but gadd153 mRNA stability also increased suggesting that both transcriptional and posttranscriptional mechanisms are involved. DTTox did not activate hsp70 transcription indicating that a heat shock response was not induced. Structure-activity studies showed that DTTox analogues lacking the intramolecular disulfide were inactive, Furthermore, the ring-open intermolecular di sulfide form of DTTox, a-hydroxyethyl disulfide, was only a weak inducer of grp78 and gadd153 but was a strong inducer of hsp70 mRNA and a potent oxidant that lowered the NADPH/NADP(+) ratio and depleted reduced glutathione (GSH). DTTox had little effect on the overall GSH and NADPH levels; thus cells were not undergoing oxidative stress; however, the NADPH/NADP(+) ratio decreased slightly indicating that reducing equivalents were consumed. LLC-PK1 cells reduced DTTox to DTT, and the kinetics as well as the concentration dependence for reduction correlated with induction of both grp78 and gadd153 mRNA Prior treatment with DTTox rendered cells tolerant to the potent nephrotoxicant S-(1,1,2,2-tetrafluoroethyl)-L-cysteine. Bacitracin, an inhibitor of plasma membrane oxidoreductases, blocked DTTox reduction and gene activation as well as DTTox induced tolerance, Thus, activation of stress genes and induction of cellular tolerance by DTTox is mediated by a novel mechanism involving cellular oxidoreductases.