Stress- and Growth-Related Gene Expression Are Independent of Chemical-Induced Prostaglandin E 2 Synthesis in Renal Epithelial Cells Kelly M. Towndrow, Jozef J. W. M. Mertens, † Jeongmi K. Jeong, ‡ Thomas J. Weber, § Terrence J. Monks, and Serrine S. Lau* Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712 Received September 9, 1999 Cellular stress can initiate prostaglandin (PG) biosynthesis which, through changes in gene expression, can modulate cellular functions, including cell growth. PGA 2 , a metabolite of PGE 2 , induces the expression of stress response genes, including gadd153 and hsp70, in HeLa cells and human diploid fibroblasts. PGs, gadd153, and hsp70 expression are also influenced by the cellular redox status. Polyphenolic glutathione conjugates retain the ability to redox cycle, with the concomitant generation of reactive oxygen species. One such conjugate, 2,3,5-tris- (glutathion-S-yl)hydroquinone (TGHQ), is a potent nephrotoxic and nephrocarcinogenic metabolite of the nephrocarcinogen, hydroquinone. We therefore investigated the effects of TGHQ on PGE 2 synthesis and gene expression in a renal proximal tubular epithelial cell line (LLC-PK 1 ). TGHQ (200 µM, 2 h) increases PGE 2 synthesis (2-3-fold) in LLC-PK 1 cells with only minor (5%) reductions in cell viability. This response is toxicant-specific, since another proximal tubular toxicant, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), stimulates PGE 2 synthesis only after massive (68%) reductions in cell viability. Consistent with the ability of TGHQ to generate an oxidative stress, both deferoxamine mesylate and catalase protect LLC-PK 1 cells from TGHQ-mediated cytotoxicity. Only catalase, however, completely blocks TGHQ-mediated PGE 2 synthesis, implying a major role for hydrogen peroxide in this response. TGHQ induces the early (60 min) expression of gadd153 and hsp70. However, while inhibition of cyclooxygenase with aspirin prevents TGHQ-induced PGE 2 synthesis, it does not affect TGHQ-mediated induction of gadd153 or hsp70 expression. In contrast, a stable PGE 2 analogue, 11-deoxy-16,- 16-dimethyl-PGE 2 (DDM-PGE 2 ), which protects LLC-PK 1 cells against TGHQ-mediated cytotoxicity, modestly elevates the levels of gadd153 and hsp70 expression. In addition, catalase and, to a lesser extent, deferoxamine mesylate block TGHQ-induced gene expression. Therefore, although TGHQ-induced generation of reactive oxygen species is required for PGE 2 synthesis and stress gene expression, acute TGHQ-mediated increases in gadd153 and hsp70 mRNA levels are independent of PGE 2 synthesis. Introduction Prostaglandins (PGs) 1 modulate a wide variety of cellular functions, including gene expression, growth, and differentiation. In particular, the cyclopentenone pros- taglandins of the A and J series exhibit growth inhibitory and antitumor activities (1-5). Induction of early stress response genes, such as the growth arrest and DNA damage inducible gene 153 (gadd153), heat shock protein 70 (hsp70), c-fos, and Egr-1, is modulated by PGA 2 ,a dehydration product of PGE 2 , in human diploid fibro- blasts (6). Similarly, PGA 2 elevates the levels of gadd153 and heat shock protein mRNA expression in HeLa cells (3, 7, 8). Prevention or reduction of chemically induced liver damage by PGs is also well documented (9-11). For example, 16,16-dimethyl-PGE 2 , a stable synthetic PGE 2 analogue, protects rats against mild carbon tetrachloride- induced liver and kidney damage (12). Renal proximal tubular anoxia is also associated with increases in phospholipase A 2 activity, preceded by arachidonic acid release, and the breakdown of membrane phospholipids (13). Misoprostol, a stable PGE 1 analogue, is cytoprotec- tive against renal ischemic and mercuric chloride-induced injury in male Sprague-Dawley rats (14). However, the mechanism(s) of PG-mediated cytoprotection remains unclear. Alterations in blood flow, increases in membrane stability, changes in toxicant metabolism, and enhance- * To whom all correspondence should be addressed: Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, TX 78712. Telephone: (512) 471-5190. Fax: (512) 471-5002. E-mail: slau@mail.utexas.edu. † Present address: WIL Research Laboratories, Inc., 1407 George Rd., Ashland, OH 44805. ‡ Present address: Laboratory of Cardiovascular Disease Research, National Institute of Health, Rebublic of Korea, #5 Nokbun-dong, Eunpyung-gu, Seoul 122-701, Korea. § Present address: Pacific Northwest National Laboratory, 902 Batelle Blvd., P7-56, Richland, WA 99352. 1 Abbreviations: AA, arachidonic acid; CAT, catalase; DFX, defer- oxamine mesylate; DDM-PGE2, 11-deoxy-16,16-dimethyl-prostaglandin E2; DCVC, S-(1,2-dichlorovinyl)-L-cysteine; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GSH, glutathione; gadd, growth arrest and DNA damage inducible gene; hsp, heat shock protein gene; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; PG, prostaglandin; PKC, protein kinase C; ROS, reactive oxygen species; TX, thromboxane; TGHQ, 2,3,5-tris-(glutathion-S-yl)hydro- quinone. 111 Chem. Res. Toxicol. 2000, 13, 111-117 10.1021/tx990160s CCC: $19.00 © 2000 American Chemical Society Published on Web 02/02/2000