THE MOLECULAR RESPONSE TO REDUCTIVE STRESS IN LLC-PK1 RENAL EPITHELIAL-CELLS - COORDINATE TRANSCRIPTIONAL REGULATION OF GADD153 AND GRP78 GENES BY THIOLS

Organic thiols are toxic to eukaryotic cells. Treatment of cells with thiols activates expression of grp78, but it is not known if, like oth er forms of stress, there is a battery of stress response genes that a re induced by thiols. In LLC-PK1 renal epithelial cells, mRNAs for bot h grp78 and gadd153 were induced by thiols with similar time, concentr ation and structure-activity dependence. Dithiothreitol (DTT) was the most potent reductant and inducer of gene expression among the thiols tested. Nuclear run-on assays demonstrated that DTT activated both grp 78 and gadd153 genes transcriptionally. A hamster gadd153 promoter con struct which contains enhancer elements necessary for gadd153 activati on was stably integrated into the LLC-PK1 cell genome and was activate d by DTT. Although auto-oxidation,of thiols can generate active oxygen species, transcriptional activation of the gadd153 promoter was not d ue to formation of hydrogen peroxide or superoxide since neither catal ase nor superoxide dismutase prevented activation of the gadd153 promo ter by DTT. The concentration dependence for activation of the gadd153 promoter correlated with inhibition of dome formation and protein syn thesis, two toxic effects of DTT in LLC-PK1 cells. Thus, both grp78 an d gadd153 are members of a gene battery which is responsive to reducti ve stress. There appears to be considerable, but not complete, overlap between the upstream signaling pathways for activation of both genes.