Reduction of trans-4,5-Dihydroxy-1,2-dithiane by Cellular Oxidoreductases Activates gadd153/chop andgrp78 Transcription and Induces Cellular Tolerance in Kidney Epithelial Cells

Abstract

trans-4,5-Dihydroxy-1,2-dithiane, the intramolecular disulfide form of dithiothreitol (DTTox) transcriptionally 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 disulfide form of DTTox, 2-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 DTToxinduced tolerance. Thus, activation of stress genes and induction of cellular tolerance by DTTox is mediated by a novel mechanism involving cellular oxidoreductases.

Endoplasmic Reticulum Stress Due to Altered Cellular Redox Status Positively Regulates Murine Hepatic CYP2A5 Expression

“…1) DTT ox induces CYP2A5 expression in primary mouse hepatocytes. In their studies of ER stress in renal epithelial cells (LLC-PK1), Stevens et al showed that the molecular response to DTT ox resembles reductive stress induced by DTT (Halleck et al, 1997). For example, reduction of DTT ox in LLC-PK1 cells correlated with induction of GRP78 expression.…”

“…Disulfide reduction of DTT ox to DTT by GSH and/or by NADPH-dependent oxidoreductase systems drives the redox potential of the ER toward a more reducing environment. This alteration in the ER redox status results in disruption of protein disulfide bond formation and protein folding, which in turn induces ER stress (Halleck et al, 1997). DTT ox evidently alters redox status specifically in the ER because only GRP78 is induced and not its cytosolic counterpart heat shock protein 70 (Halleck et al, 1997).…”

Endoplasmic Reticulum Chaperones GRP78 and Calreticulin Prevent Oxidative Stress, Ca2+ Disturbances, and Cell Death in Renal Epithelial Cells

“…2). Treating cells with DTTox, tunicamycin, or thapsigargin, agents that cause ER stress (5,65), increased mRNA for grp78 and synthesis of both GRP78 and GRP94 proteins (Fig. 3, A and B) in LLC-PK1 cells.…”

Endoplasmic Reticulum Stress Proteins Block Oxidant-induced Ca2+ Increases and Cell Death

“…Overexpression of calreticulin and Grp78, both of which are ER Ca 2ϩ -binding proteins and chaperones, increases the capacity of intracellular Ca 2ϩ stores (20,21) and prevents Ca 2ϩ toxicity (30,31 sensitizes cells to injury (30,31,39,40). Recently, we demonstrated that increasing the expression of ER stress protein genes protects renal epithelial cells against a subsequent challenge with the alkylating and acylating agents, iodoacetamide, or nephrotoxic cysteine conjugates, respectively (31,41). With iodoacetamide, protection required grp78 induction and was linked both to inhibition of lipid peroxidation and maintenance of low intracellular free Ca 2ϩ (31).…”