Stress- and Growth-Related Gene Expression Are Independent of

Sampath Ramachandiran, Qihong Huang, Jing Dong, Serrine S. Lau, and Terrence J. Monks. Chemical Research in Toxicology 2002 15 (12), 1635-1642...
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Chem. Res. Toxicol. 2000, 13, 111-117

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Stress- and Growth-Related Gene Expression Are Independent of Chemical-Induced Prostaglandin E2 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. PGA2, a metabolite of PGE2, 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 PGE2 synthesis and gene expression in a renal proximal tubular epithelial cell line (LLC-PK1). TGHQ (200 µM, 2 h) increases PGE2 synthesis (2-3-fold) in LLC-PK1 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 PGE2 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-PK1 cells from TGHQ-mediated cytotoxicity. Only catalase, however, completely blocks TGHQ-mediated PGE2 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 PGE2 synthesis, it does not affect TGHQ-mediated induction of gadd153 or hsp70 expression. In contrast, a stable PGE2 analogue, 11-deoxy-16,16-dimethyl-PGE2 (DDM-PGE2), which protects LLC-PK1 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 PGE2 synthesis and stress gene expression, acute TGHQ-mediated increases in gadd153 and hsp70 mRNA levels are independent of PGE2 synthesis.

Introduction Prostaglandins (PGs)1 modulate a wide variety of cellular functions, including gene expression, growth, and differentiation. In particular, the cyclopentenone prostaglandins of the A and J series exhibit growth inhibitory * 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: [email protected]. † 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, deferoxamine 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)hydroquinone.

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 PGA2, a dehydration product of PGE2, in human diploid fibroblasts (6). Similarly, PGA2 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-PGE2, a stable synthetic PGE2 analogue, protects rats against mild carbon tetrachlorideinduced liver and kidney damage (12). Renal proximal tubular anoxia is also associated with increases in phospholipase A2 activity, preceded by arachidonic acid release, and the breakdown of membrane phospholipids (13). Misoprostol, a stable PGE1 analogue, is cytoprotective 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-

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ment of a tissue’s regenerative capacity by PGs may contribute to PG-induced cytoprotection (10, 15). Additionally, PGs protect isolated hepatic and renal cells against carbon tetrachloride- and hypoxic-induced cytotoxicity, respectively, suggesting that cytoprotection occurs at the cellular level (14, 16). Consistent with these latter observations, prolonged exposure (8-24 h) to both PGE2 and 11-deoxy-16,16-dimethyl-PGE2 (DDM-PGE2), a stable PGE2 analogue, protects renal proximal tubule epithelial cells (LLC-PK1) against 2,3,5-tris(glutathionS-yl)hydroquinone (TGHQ)-induced cytotoxicity (17). Cytoprotection appears to be mediated via a receptor which is pharmacologically distinct from known PGE receptor (EP) subtypes, and is associated with a protein kinase C (PKC)-related signal transduction pathway (17). In addition to an acute stimulation of PG synthesis, oxidative stress can lead to the induction of early stress response genes, such as gadd153 and hsp70 (18, 19). Gadd153 is related to the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors, and although capable of dimerizing with C/EBP transcription factors, it does not possess intrinsic DNA binding activities (20). Gadd153 is therefore considered a negative regulator of C/EBP transcription factors and is associated with growth arrest (20). Heat shock proteins (HSPs) constitute a large family of stress response genes. HSPs are constitutively expressed, in addition to being induced by heat and other stressors (21). The abundance of HSPs is coupled to their multiple functions, including protein chaperoning across membranes, participation in protein refolding associated with degradation, and protection against oxidative injury (21-23). Of particular interest is the protective role of HSPs against reactive oxygen species (ROS) generated during inflammation (24). TGHQ (see structure) is a redox active metabolite (25) of the nephrocarcinogen, hydroquinone (26, 27).

Administration of TGHQ to male rats causes renal proximal tubular necrosis, and doses of TGHQ as low as 7.5 µmol/kg (iv) cause significant increases in blood urea nitrogen levels, enzymuria, and glucosuria (25). Furthermore, TGHQ is nephrocarcinogenic (28), and it catalyzes 8-oxodeoxyguanosine formation in vitro (29). Incubation of the supF gene with TGHQ, followed by transfection and replication in human AD293 embryonic kidney cells, causes a significant increase in the mutation frequency (30). Both TGHQ (17) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC; see structure) (31) cause cytotoxicity in LLC-PK1 cells, a proximal tubular epithelial cell line. Here, we have investigated the ability of TGHQ and DCVC to stimulate cellular PGE2 synthesis, and compared the effects of this acute in situ production of PGE2, with a brief exposure to exogenous DDM-PGE2 and PGE2, on stress gene expression in LLC-PK1 cells.

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Experimental Procedures Caution: TGHQ is nephrotoxic and nephrocarcinogenic in rats and must be handled with care. This compound should therefore be handled with protective clothing and in a wellventilated fume hood. Chemicals and Reagents. TGHQ was synthesized and purified as previously described (32). 11-Deoxy-16,16-dimethylPGE2 (DDM-PGE2), PGE2, and 5,8,11,14-eicosatetraenoic acid [arachidonic acid (AA)] were purchased from Caymen Chemical (Ann Arbor, MI). Plasmid pBluescript containing a 600 bp fragment of cDNA of gadd153 was kindly provided by N. J. Holbrook (National Institute on Aging, Gerontology Research Center, Bethesda, MD). Plasmid pAT153 containing a 2.3 kb fragment of hsp70 was obtained from American Type Culture Collection. S. Fisher (M. D. Anderson Cancer Center, Houston, TX) provided 7S ribosomal RNA cDNA. Procedures for cDNA probe preparation have been described elsewhere (19). S-(1,2Dichlorovinyl)-L-cysteine (DCVC) was a generous gift from J. L. Stevens (University of Vermont, Burlington, VT). Cell Culture and Treatment Conditions. The New Hampshire minipig-derived renal proximal tubular epithelial cell line (LLC-PK1) was purchased from the American Type Culture Collection (CL101, passage 185). Cells were maintained in Dulbecco’s modified Eagle’s medium with 4.5 g/L D-glucose (DMEM; GIBCO BRL, Grand Island, NY), supplemented with 10% fetal bovine serum (FBS; JRH Bioscience, Lenexa, KS) at 37 °C in a humidified atmosphere containing 5% CO2. Culture dishes were seeded at starting cell densities of 2 × 105 cells/ well for 24-well plates, 9 × 105 cells/well for 6-well plates, and 2 × 106 cells/100 mm dishes, and experiments were conducted on cells grown for 7-10 days (approximately 4-7 days postconfluent) with a medium change on day 5. Experiments with TGHQ were conducted in DMEM with 25 mM HEPES (pH 7.4). DCVC treatments were conducted in Earle’s balanced salt solution [EBSS; 5.4 mM KCl, 1.7 mM MgSO4, 106.4 mM NaCl, 26.2 mM NaHCO3, 1 mM NaH2PO4, 5.6 mM D-(+)-glucose, 25 mM HEPES, and 1.8 mM CaCl2 (pH 7.4)]. DMEM with 25 mM HEPES supplemented with 10% FBS was used in gene expression studies with DDM-PGE2, according to the PG-mediated cytoprotection protocol previously described (17). In some cases, cells were pretreated with 20 µM arachidonic acid for 15 min prior to and during both TGHQ and DCVC treatment, to increase the sensitivity of the medium PGE2 measurements. A 0.1 mM aspirin pretreatment (30 min) prior to TGHQ exposure (200 µM, 2 h) was used to inhibit cyclooxygenase activity and had no effect on cell viability with this dosing regimen. Viability was assessed using the lysosomal neutral red accumulation assay (33). Quantitation of Prostaglandin E2. Following exposure of cells to TGHQ, media were immediately removed, frozen on dry ice, and stored at -70 °C until further analysis. A PGE2 125I radioimmunoassay kit (NEN Du Pont, Boston, MA) was used to quantitate medium PGE2 levels. LLC-PK1 cells produce low constitutive levels of PGE2 (34, 35). Commercially available PGE2 125I radioimmunoassay kits, which allow the direct measurement of PGE2 as low as 1 pg, offer increased sensitivity compared to the hepatic radioreceptor assay of Lifschitz et al., which requires an extraction step with a 75% recovery efficiency, and has a limit of sensitivity to only 62 pg (34-36). Lifschitz (34, 35) suggested that LLC-PK1 cells produce little or no constitutive PGE2, possibly due to a defect in cyclooxygenase activity, and that levels of PGE2 did not increase following treatment of LLC-PK1 cells with either calcium ionophore (A23187), butyrate, or arachidonic acid. However, Roszinski et al. (37) later reported the ability to detect elevations in the levels of PGE2 release utilizing the radioimmunoassay following calcium ionophore (A23187) treatment and arachidonic acid supplementation in LLC-PK1 cells. Evaluation of Gene Expression by Northern Blot Analysis. Cells were treated as described above in a 10 mL culture volume and were subsequently scraped and homogenized in 3-6 mL of RNAzol or UltraSpec RNA reagent (Biotecx Laboratories,

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Figure 1. TGHQ-induced cytotoxicity (9, A) and PGE2 synthesis (O, A and B) and the effects of arachidonic acid (AA) supplementation on TGHQ-induced PGE2 synthesis (B) in LLCPK1 cells. Cells [except for the without AA curve (b) in panel B] were supplemented with 20 µM AA (15 min) prior to 0, 200, 500, and 1000 µM TGHQ exposure for 120 min in 6-well plates (1 mL volumes). Medium was collected, frozen immediately, and stored at -70 °C until PGE2 analysis by radioimmunoassay. Viability was assessed using the neutral red lysosomal accumulation assay. Mean PGE2 control levels were 7 ( 2 (without AA) and 30 ( 5 pg/mL (with AA). PGE2 and cytotoxicity data are expressed as means ( SE (n ) 3) and are representative of three experiments producing similar results. Asterisks and secants denote values that are significantly different from their respective control values at P < 0.05. Inc., Houston, TX) per 100 mm Petri dish. Isolation of total RNA, electrophoresis, and hybridization conditions were as previously described (19). Quantitation of Autoradiograms. Data from Northern blot analysis were quantified by densitometric analysis (Imaging Research Inc. or NIH Image public domain software) or with a Packard InstantImager Electronic Autoradiography System (Packard Instrument Co., Meriden, CT). All densitometric and imager count values (CPM) were normalized for loading with 7S rRNA. Statistics. Statistical significance (P < 0.05) was determined with one-way ANOVA followed by Student-Newman-Keuls post hoc analysis where appropriate.

Results Early Responses to Cellular Injury Induced by TGHQ. TGHQ is a potent renal proximal tubular toxicant in vivo (25) and in vitro (Figure 1A). Lysosomal uptake and accumulation of neutral red dye is a sensitive and early indicator of chemical insult and generally occurs prior to membrane and mitochondrial damage (33). TGHQ (200 µM, 2 h) also induced a 2-3-fold increase in PGE2 synthesis and release in LLC-PK1 cells (Figure 1B, -AA curve). Supplementing cells with arachidonic acid (20 µM, AA curve) for 15 min prior to TGHQ exposure enhanced the sensitivity of PGE2 measurements (Figure 1B) without affecting cell viability (data not shown). Therefore, cells were supplemented with AA in all subsequent experiments. Increases in PGE2 synthesis occurred as early as 30 min following TGHQ exposure (data not shown). Interestingly, a 2 h exposure to TGHQ (200 µM), while only causing a 5% reduction in cell viability, produced a 2-3-fold increase in PGE2 levels (Figure 1A). In contrast to TGHQ, another proximal tubular toxicant with a differing toxicological mechanism of action, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), only elevated PGE2 synthesis in LLC-PK1 cells after marked reductions in cell viability (Figure 2). A 10% reduction in cell viability by DCVC (500 µM, 3 h) did not alter PGE2 synthesis (Figure 2). Indeed, a 3-4-fold increase in PGE2 levels occurred only after a 9 h exposure of LLC-PK1 cells to 500 µM DCVC, which was accompanied by a 68% reduction in cell viability (Figure 2).

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Figure 2. Time-dependent effects of DCVC on viability (9) and PGE2 synthesis (O) in LLC-PK1 cells. Cells were supplemented with 20 µM AA (15 min) prior to 0, 3, 5, 9, and 12 h exposure to 500 µM DCVC (1 mL volumes) in 6-well plates. Medium was collected, frozen immediately, and stored at -70 °C until PGE2 analysis by radioimmunoassay. Viability was assessed using the neutral red lysosomal accumulation assay. Mean PGE2 control levels at 0 h were 127 ( 9 pg/mL. PGE2 and cytotoxicity data are expressed as means ( SE (n ) 3) and are representative of two experiments producing similiar results. Asterisks and secants denote values that are significantly different from their respective control values at P < 0.05.

Figure 3. Effects of catalase (CAT; A) and deferoxamine mesylate (DFX; B) on cell viability after exposure of LLC-PK1 cells to TGHQ. LLC-PK1 cells were either pre- (1 h) and cotreated with 10 mM DFX or cotreated with 10 units/mL catalase and 400 µM TGHQ for 2 h in 24-well plates (0.5 mL volumes). Viability was assessed using the neutral red lysosomal accumulation assay. Data are expressed as the means ( SE (n ) 4 for CAT; n ) 3 for DFX) and are representative of three experiments producing similar results. Asterisks denote values that are significantly different from control values at P < 0.05. Secants denote values that are significantly different from TGHQ values at P < 0.05.

Role of ROS in TGHQ-Mediated Cytotoxicity and PGE2 Synthesis. Polyphenolic glutathione (GSH) conjugates retain the ability to redox cycle, and the generation of an oxidative stress is implicated in quinol thioethermediated cytotoxicity (38). ROS can stimulate arachidonic acid metabolism, leading to the production of PGs (3941). Cotreatment of LLC-PK1 cells with catalase (10 units/mL) completely protected against TGHQ-induced cytotoxicity (Figure 3A) and blocked PGE2 synthesis from TGHQ exposure (Figure 4A), suggesting a role for hydrogen peroxide in both responses. However, although chelation of iron with deferoxamine mesylate (DFX, 10 mM), which presumably prevents hydroxyl radical formation, also diminished TGHQ-induced cytotoxicity (Figure 3B), it did not significantly prevent TGHQ-mediated PGE2 synthesis (Figure 4B). Effects of ROS and Prostaglandin E2 on gadd153 and hsp70 Gene Expression. Cellular stress, including DNA damage and oxidative stress, induces the expression of certain stress response genes, such as gadd153 and

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Figure 4. Effects of catalase (CAT; A) and deferoxamine mesylate (DFX; B) on PGE2 synthesis after exposure of LLCPK1 cells to TGHQ. LLC-PK1 cells were either pre- (1 h) and cotreated with 10 mM DFX or cotreated with 10 units/mL catalase and 400 µM TGHQ for 2 h. Cells were supplemented with 20 µM AA (15 min) prior to DFX, CAT, and TGHQ exposure. After treatment, medium was collected, frozen immediately, and stored at -70 °C until PGE2 analysis by radioimmunoassay. Mean PGE2 control levels were 56 ( 7 (CAT) and 61 ( 4 pg/mL (DFX). Data are expressed as the means ( SE (n ) 6) and are representative of at least three experiments producing similar results. Asterisks denote values that are significantly different from control values at P < 0.05. The secant denotes a value that is significantly different from TGHQ values at P < 0.05.

Figure 5. Concentration-dependent effects of TGHQ on gadd153 and hsp70 mRNA expression in LLC-PK1 cells. Cells were exposed to varying concentrations of TGHQ for 120 min. Total RNA was isolated and quantified by Northern blot analysis. Data were normalized for loading with 7S rRNA and are representative of at least two experiments producing similar results.

hsp70 (42-45). TGHQ dramatically induced the expression of both gadd153 and hsp70 mRNA, in a dose- and time-dependent manner (Figures 5 and 6). Induction of both genes occurred as early as 60 min after treatment with TGHQ, and was maximal after 120 min (Figure 6). A 120 min exposure to 200 µM TGHQ produced an ∼20fold increase in gadd153 mRNA and an 8-9-fold increase in hsp70 mRNA levels (Figure 5). Catalase cotreatment (10 units/mL) completely prevented TGHQ-induced elevations in gadd153 and hsp70 mRNA (Figure 7). While DFX partially prevented gadd153 and hsp70 expression following TGHQ exposure, inhibition of hsp70 gene expression may be minimized by the stimulatory properties of DFX alone (Figure 7). To probe the role of PGE2 in the acute expression of gadd153 and hsp70, we investigated the effects of DDMPGE2 and PGE2 on gadd153 and hsp70 stress gene expression in LLC-PK1 cells. Inhibition of cyclooxygenase by aspirin, while completely blocking TGHQ-mediated elevations in PGE2 levels in LLC-PK1 cells (Figure 8),

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Figure 6. Time-dependent effects of TGHQ on gadd153 and hsp70 mRNA expression in LLC-PK1 cells. Cells were exposed to 200 µM TGHQ for 0, 30, 60, 120, and 300 min. Total RNA was isolated and quantified by Northern blot analysis. Data were normalized for loading with 7S rRNA and are representative of at least two experiments producing similar results.

Figure 7. Effects of catalase and deferoxamine mesylate on gadd153 (A) and hsp70 (B) mRNA expression in LLC-PK1 cells following TGHQ exposure. LLC-PK1 cells were either pre- (1 h) and cotreated with 10 mM DFX or cotreated with 10 units/mL catalase and 200 µM TGHQ for 2 h. Total RNA was isolated and quantified by Northern blot analysis. Data were normalized for loading with 7S rRNA and are representative of at least two experiments producing similar results.

Figure 8. Effects of cyclooxygenase inhibition by aspirin on TGHQ-mediated PGE2 synthesis in LLC-PK1 cells. After cells were pretreated with 0.1 mM aspirin (30 min) prior to 200 µM TGHQ (2 h) exposure, medium was collected, frozen immediately, and stored at -70 °C until PGE2 analysis by radioimmunoassay. Cells were supplemented with 20 µM AA (15 min) prior to aspirin and TGHQ exposure. Mean PGE2 control levels were 97 ( 15 pg/mL. Data are expressed as means ( SE of three experiments. Asterisks denote values that are significantly different from control values at P < 0.05. The secant denotes a value that is significantly different from TGHQ values at P < 0.05.

had no effect on TGHQ-induced gadd153 and hsp70 gene expression (Figure 9). Furthermore, AA supplementation also had no effect on gene expression (data not shown).

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Discussion

Figure 9. Effects of cyclooxygenase inhibition by aspirin on TGHQ-mediated gadd153 and hsp70 mRNA expression in LLCPK1 cells. After cells were pretreated with 0.1 mM aspirin (30 min) and 20 µM arachidonic acid (AA, 15 min) prior to 200 µM TGHQ (2 h) exposure, total RNA was isolated and quantified by Northern blot analysis. Data were normalized for loading with 7S rRNA and are representative of at least two experiments producing similar results.

Figure 10. Effects of DDM-PGE2 and PGE2 exposure on gadd153 and hsp70 mRNA expression in LLC-PK1 cells. Cells were exposed to either 20 µM DDM-PGE2 or 20 µM PGE2 for 120 min. Total RNA was isolated and quantified by Northern blot analysis. Data were normalized for loading with 7S rRNA and are representative of at least two experiments producing similar results.

We have previously shown that PGE2 and DDM-PGE2 protect LLC-PK1 cells against TGHQ-induced cellular damage (17). In contrast to the elevated in situ synthesis of cellular PGE2 following TGHQ exposure (Figure 1) which appeared to be insufficient to stimulate gadd153 and hsp70 mRNA (Figure 9), exposure of LLC-PK1 cells to 20 µM DDM-PGE2 (2 h) induced a 1.6- and 2.5-fold increase in hsp70 and gadd153 mRNA, respectively (Figure 10). PGE2 (20 µM, 2 h) was much less effective at stimulating gadd153 (1.7-fold) and hsp70 (1.2-fold) gene expression (Figure 10). Interestingly, EP receptor agonists, such as sulprostone (EP1/EP3 agonist) and 17phenyltrinor PGE2 (EP1 agonist), had no effect on gadd153 expression, but slightly stimulated hsp70 gene expression, to a lesser extent than that of DDM-PGE2 (data not shown).

Little is known about the role of endogenous PGE2 in the acute cellular response to injury. We report here that LLC-PK1 cells respond to chemical challenge by increasing PGE2 synthesis and release. Increases in PGE2 levels following exposure of LLC-PK1 cells to TGHQ occur prior to the onset of overt cytotoxicity (Figure 1). The biological reactivity of quinol thioethers lies in their ability to redox cycle and to subsequently generate an oxidative stress (38). Deferoxamine mesylate, an iron chelator, and catalase, a scavenger of hydrogen peroxide, both prevent TGHQ-mediated cytotoxicity in LLC-PK1 cells, supporting a role for the formation of ROS in this response (Figure 3). ROS can alter arachidonic acid metabolism (39, 40, 46). Cotreatment of LLC-PK1 cells with catalase completely blocks TGHQ-mediated induction of PGE2 synthesis (Figure 4A), gadd153 and hsp70 gene expression (Figure 7), and cytotoxicity (Figure 3A). However, whereas DFX protects LLC-PK1 cells against TGHQ exposure (Figure 3B), it does not significantly prevent increases in PGE2 synthesis (Figure 4B) and only partially prevents TGHQ-induced gadd153 and hsp70 gene expression (Figure 7). These data suggest that although hydrogen peroxide is essential for stimulating PGE2 synthesis and stress gene expression, both hydrogen peroxide and the subsequent formation of hydroxyl radicals are required for TGHQ-induced cytotoxicity. Consistent with our findings, treatment of cultured human endothelial cells with hydrogen peroxide increases PGI2 and thromboxane A2 (TXA2) synthesis (39). Similarly, in rat peritoneal exudate cells, E3330 [(2E)3-[5-(2,3-dimethoxy-6-methyl-1,4-benzoquinoyl)]-2-nonyl2-proponoic acid], a quinone derivative, inhibits the synthesis of TXB2 and LTB4, but induces PGE2 production (47). Chemical toxicants, and other stressors, stimulate the expression of early stress response genes, including gadd153 and hsp70 (42-44). Although exogenously supplied PGs stimulate early stress response gene expression, including gadd153 and hsp70 (3, 6-8), the role of constitutive PGE2 synthesis in gene expression is less clear. Inductions of gadd153, hsp70, and PGE2 are early responses to TGHQ exposure in LLC-PK1 cells, and occur prior to overt cytotoxicity (Figures 1 and 6). Similarly, LLC-PK1 cells undergo growth arrest following exposure to the quinol thioether 2-bromo-bis-(glutathion-S-yl)hydroquinone, as indicated by formation of DNA singlestrand breaks, a decreased level of DNA synthesis, and elevations in gadd153 expression (44). The ability of both catalase and deferoxamine to prevent quinol thioethermediated gadd153 induction is consistent with the modus operandi of quinol thioethers (44). However, TGHQmediated cellular PGE2 synthesis and gadd153 and hsp70 gene expression do not appear to be linked, because inhibition of cyclooxygenase with aspirin, while blocking PG synthesis (Figure 8), does not attentuate TGHQinduced gene expression (Figure 9). Stimulation of in situ production of PGE2 in LLC-PK1 cells appears to be toxicant-specific. Although TGHQ stimulates PGE2 synthesis before the onset of overt cytotoxicity (Figure 1A), increases in the levels of PGE2 synthesis following DCVC exposure only occur after cell viability is compromised and may be the result of overt membrane damage (Figure 2). The different responses to TGHQ and DCVC probably relate to differences in the

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mechanisms of action of these two chemicals. The metabolism of DCVC by cysteine conjugate β-lyase produces a 1,2-dichlorovinylthiol intermediate, which further rearranges to an electrophilic thioketene. Thioketenes are highly reactive, and interact with cellular macromolecules, thereby initiating a series of events, including mitochondrial dysfunction and lipid peroxidation, that eventually results in cell death (31, 48). Although TGHQ possesses the ability to arylate cellular constituents (49, 50), it also retains the ability to redox cycle and generate an oxidative stress (38), which is likely the trigger for PGE2 synthesis (Figure 4A) and stress gene expression (Figure 7). In contrast to TGHQ, DCVC induces early elevations in gadd153 (2 h) and hsp70 (0.5 h) mRNA levels (51, 52), but only induces PGE2 synthesis as a late cellular response (9 h) and only after significant reductions in cell viability (Figure 2). Studies aimed at investigating the effects of exogenously added PGs often use concentrations that are usually 3-6 orders of magnitude higher than levels achieved after stimulation of cellular PGE2 synthesis (14, 6-8). In fact, exogenously added concentrations of PGE2 two orders of magnitude greater than the levels of PGE2 synthesized in situ following exposure of LLC-PK1 cells to TGHQ (Figure 1) only minimally induced gadd153 and hsp70 gene expression (Figure 10). Moreover, the cytoprotective PGE2 analogue, DDM-PGE2, modestly elevates gadd153 and hsp70 expression when added to LLC-PK1 cultures (Figure 10). Taken together, these data demonstrate differences in the cellular response to exogenously supplied PGs and PGs synthesized constitutively. Although it has been shown that exogenously added PGA2, a metabolite of PGE2, elevates gadd153 and hsp70 mRNA levels in HeLa cells (3, 7), the inability of aspirin to prevent TGHQ-induced stimulation of gene expression (Figure 9) indicates that TGHQ-mediated PGE2 synthesis and stress gene expression are unrelated in LLC-PK1 cells. The differences in PG-mediated regulation of stress gene expression between HeLa and LLC-PK1 cells are likely due to cell type selectivity and/or differences in the growth state of the cells. PGs generally exert their cellular actions via G-proteincoupled receptors (53), and in some cases, multiple PG receptors with opposite actions can be coexpressed in a particular cell type (54). Coexpression of both stimulatory and inhibitory PG receptors has recently been proposed as a possible model for homeostatic control of paracrine and autocrine signaling that enables the cell to buffer against locally high agonist concentrations (54). For example, PGE2 both stimulates and inhibits adenyl cyclase activity in platelets via activation of different prostanoid receptor subtypes, resulting in a slight net increase in cAMP levels (55). Therefore, coexpression of stimulatory and inhibitory prostanoid receptor subtypes and possible differences in prostanoid receptor subtype affinities and/or structural stability between DDM-PGE2 and PGE2 may contribute to the differences in the cellular response to exogenous and endogenous PGs we observed. Interestingly, certain eicosanoids, such as PGA, PGD, and PGJ2, can activate peroxisome proliferator-activated receptors (PPARs), which are nuclear hormone receptors, suggesting that PGs may also exert cellular effects independent of cell surface receptor activation (56). Consequently, DDM-PGE2-induced gadd153 and hsp70 mRNA expression likely occurs via activation of cell surface receptors. In contrast, stimulation of cellular

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PGE2 synthesis may cause both cell surface and nuclear receptor-dependent cellular responses. In summary, LLC-PK1 cells respond to TGHQ-generated ROS by increasing synthesis of PGE2 and by upregulating gadd153 and hsp70 gene expression. The acute stimulation of PGE2 synthesis in LLC-PK1 cells is toxicant-specific, and despite the ability of exogenous PGs to mildly induce stress gene expression, acute toxicantinduced stimulation of PGE2 synthesis and stress gene expression are not related.

Acknowledgment. This work was supported by NIH Grants GM56321 (to S.S.L.), ES77084 (NIEHS Center Grant), and ES07247 (NIEHS Training Grant to K.M.T.).

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