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-respon sive genes gadd153(chop) and grp78, Herein, we used a renal epithelial cell line, LLC-PK1, to investigate the mechanism(s) whereby DTTox act ivates a molecular stress response, DTTox activated both grp78 and gad d153 transcriptionally, but gadd153 mRNA stability also increased sugg esting that both transcriptional and posttranscriptional mechanisms ar e involved. DTTox did not activate hsp70 transcription indicating that a heat shock response was not induced. Structure-activity studies sho wed that DTTox analogues lacking the intramolecular disulfide were ina ctive, Furthermore, the ring-open intermolecular di sulfide form of DT Tox, a-hydroxyethyl disulfide, was only a weak inducer of grp78 and ga dd153 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 c ells were not undergoing oxidative stress; however, the NADPH/NADP(+) ratio decreased slightly indicating that reducing equivalents were con sumed. 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 cel ls tolerant to the potent nephrotoxicant S-(1,1,2,2-tetrafluoroethyl)- L-cysteine. Bacitracin, an inhibitor of plasma membrane oxidoreductase s, blocked DTTox reduction and gene activation as well as DTTox induce d tolerance, Thus, activation of stress genes and induction of cellula r tolerance by DTTox is mediated by a novel mechanism involving cellul ar oxidoreductases.