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Mitogen-Activated Protein Kinases Contribute to Reactive Oxygen Species-Induced Cell Death in Renal Proximal Tubule Epithelial Cells Sampath Ramachandiran,† Qihong Huang,†,‡ Jing Dong, Serrine S. Lau, and Terrence J. Monks* Center for Molecular & Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712 Received July 9, 2002
Extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinase (MAPK) were all rapidly activated in a ROS-dependent manner during 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-mediated oxidative stress and oncotic cell death in renal proximal tubule epithelial cells (LLC-PK1). TGHQ-induced phosphorylation of ERK1/2 and JNK MAPKs required epidermal growth factor receptor (EGFR) activation, whereas p38 MAPK activation was EGFR independent. In contrast to their established roles in cell survival, TGHQ-activated ERK1/2 and p38 MAPK (but not JNK) appear to contribute to cell death, since inhibition of ERK1/2 or p38 MAPKs with PD098059 or SB202190 respectively, attenuated TGHQ-mediated cell death. TGHQ increased AP-1 and NFκB DNA-binding activity, but whereas pharmacological inhibition of ERK1/2 or p38 MAPKs attenuated AP-1 DNA binding activity, it potentiated TGHQ-mediated NFκB activation. Consistent with a role for NFκB activation in the cytoprotective response to ROS in renal epithelial cells, an anti-NFκB peptide SN50 suppressed the protective effects of ERK inhibition (PD098059 treatment). The data provide evidence that the activation of MAPKs by ROS in renal epithelial cells plays an important role in oncotic cell death, and NF-kB is involved in the cytoprotective effects of PD098059.
Introduction The mitogen-activated protein kinase (MAPK)1 signaling cascades are activated not only by growth factors but also by stresses such as heat shock, genotoxicants, UV irradiation, and inflammatory cytokines (1). The MAPK family comprises three major subfamilies: extracellularsignal regulated kinases (ERK), c-Jun N-terminal/stressactivated protein kinases (JNK/SAPK), and p38 MAPK. The activation of MAPKs is dependent on the dual phosphorylation of threonine and tyrosine residues by upstream MAPK kinase (MEK) and other MKKs. Activated MAPKs play a key role in activating transcription factors and down stream kinases, leading to the induction of immediate-early gene expression, and subsequent changes in other cellular processes (1, 2). ERKs are activated by phorbol esters and growth factors, including epidermal growth factor (EGF). Growth factor-mediated activation is initiated by binding to cell surface receptor tyrosine kinases which activate the small G-protein Ras, triggering a phosphorylation cascade and * To whom correspondence should be addressed. Telephone: (512) 471-5190. Fax: (512) 471-5002. E-mail:
[email protected]. † These authors contributed equally to this work ‡ Present address: Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT 06877. 1 Abbreviations: AP-1, activator protein-1; DMEM, Dulbecco’s modified Eagle’s medium; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERKs, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinases; MAPKs, mitogen-activated protein kinases; MEK, MAPK kinase; NFκB, nuclear factor kappa B.; ROS, reactive oxygen species; TGHQ, 2,3,5-tris-(glutathion-S-yl)hydroquinone.
the sequential activation of Raf, MEK, and ERK1/2 (1). Activated ERK1/2 phosphorylate and activate transcription factors, including Elk1 and c-Myc, and downstream kinases, including pp90 ribosomal S6 kinase, MAPKintegrating kinases, and mitogen- and stress-activated protein kinase (MSK) (1). A similar cascade is engaged in respone to genotoxic stress, including UVC irradiation (3), and hydrogen peroxide (4, 5), although the magnitude of ERK activation is generally less than that following mitogen treatment. Stress-activated ERK1/2 likely function to protect cells against apoptosis, and to enhance cell survival after UV irradiation (6), hydrogen peroxide (7), heat shock (8), and growth factor deprivation (9). Consequently, attenuation of ERK activity with the MEK inhibitor PD098059 usually increases the apoptotic response to these stresses. Precisely how ERK rescues cells from death is unclear, although AP-1 and NFκB have been implicated in growth factor deprivation induced apoptosis (9). In contrast to ERKs, the JNK/SAPK and p38 MAPK subfamilies are poorly activated by growth factors and phorbol esters, but are more responsive to inflammatory cytokines, heat shock, and various cellular metabolic inhibitors (10). C-Jun and ATF-2 are the two main targets of activated JNK (11), whereas p38 MAPK usually stimulates the activity of the transcription factors ATF-2 (12), gadd153 (13), and kinases such as the MAPK-integrating kinases (14), MSK (15), and MAPKAP2/3 (16). The activation of JNK and p38 MAPK plays a pivotal role during apoptosis (17), possibly via activation of gadd153 (18).
10.1021/tx0200663 CCC: $22.00 © 2002 American Chemical Society Published on Web 11/27/2002
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AP-1 and NF-κB are MAPK-regulated targets activated by oxidative stress. MAPKs activate AP-1 by phosphorylation or transcriptional induction of c-jun and c-fos, and AP-1 activation is suppressed by inhibiting JNKs/ SAPK and p38 MAPKs (19). MEK kinase regulates the activation of NF-κB by activating IκB kinase (20). Although the role of NF-κB in response to cell stress remains debatable, activated NFκB attenuates apoptosis induced by nitric oxide (21) and platelet-derived growth factor (22). The ability of NF-κB to protect against oncotic cell death, which typically occurs in response to toxic injury, including that induced by chemical exposure and reactive oxygen species (ROS), is unknown. Mechanisms of cell death are usually classified into two pathways, apoptosis and necrosis. However, it has been proposed that the term oncosis, with its root (Greek) meaning of “swelling” be used as the alternate descriptor of cell death occurring by nonapoptotic pathways. Thus, in contrast to apoptosis, oncotic cell death is characterized by cell and organelle swelling that eventually leads to the loss of plasma membrane integrity (23). Necrosis more accurately describes the consequences of oncotic cell death, usually the death of a large number of cells that results in moderate to severe tissue injury. Therefore, in accordance with the recommendations of the American Society of Toxicologic Pathologists (23), we will use the terms oncosis and apoptosis to describe the cellular and molecular processes that lead to cell death by either mechanism. 2,3,5-tris-(Glutathion-S-yl)hydroquinone (TGHQ) and other quinol-thioethers are potent nephrotoxicants (24). Quinol-thioethers redox cycle with the concomitant generation of ROS, and the mutation spectra induced by TGHQ is consistent with the participation of hydroxyl radicals in this process (25). Quinol-thioethers also catalyze the formation of single strand breaks in DNA in renal proximal tubule epithelial cells (LLC-PK1) (26, 27) accompanied by a rapid growth arrest characterized by a rapid inhibition of DNA synthesis, coupled to increased expression of gadd153 mRNA (26, 27), decreases in histone mRNA (28) and ultimately oncotic/ necrotic cell death. ROS also activate mitogenic signaling pathways. Because factors that regulate the consequences (cell survival or cell death) of such contradictory signals are unknown, the effects of TGHQ generated ROS on MAPK activation and cell survival in LLC-PK1 cells was investigated. Interestingly, in contrast to their documented roles in cell survival, TGHQ-activated ERK1/2 and p38 MAPK appear to contribute to oncotic cell death, since inhibition of ERK1/2 or p38 MAPK with PD098059 or SB202190 attenuated TGHQ-mediated cell death.
Experimental Section Caution: TGHQ is nephrotoxic and nephrocarcinogenic in rats and therefore must be handled with protective clothing and in a well ventilated hood. Chemicals. Unless specified otherwise, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). TGHQ was synthesized according to established methodology (24). [γ-33P]ATP (specific activity, 2000-4000 Ci/mmol) and [γ-32P]ATP (specific activity, 5000 Ci/mmol) were products of NEN Life Science Products (Boston, MA). PD098059, SB202190, and SP600125 were obtained from Calbiochem (San Diego, CA). SN50, SN50M, and AG1478 were purchased from Biomol Biomolecules (Plymouth Meeting, PA). Epidermal growth factor (EGF) was obtained from Upstate biotechnology (Lake Placid,
Ramachandiran et al. NY). All reagents were of the highest grade commercially available. Cell Culture Conditions. LLC-PK1 cells, a renal proximal tubule epithelial cell line derived from the New Hampshire minipig (used between passage numbers 210-240), were obtained from the American Type Culture Collection (Rockville, MD). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (high glucose without pyruvate) containing 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA) at 37 °C in a humidified incubator containing 5% CO2. Cells at 70-80% confluency were used for all experiments. In Situ Terminal Deoxynucleotidyltransferase-Mediated Deoxy-UTP Nick End Labeling Assay (TUNEL). LLC-PK1 cells were plated on sterilized square cover slides in 6-well plates at a density of 3 × 105 cells/well and grown for 48 h to reach 60-70% confluency. The cells were then washed once and incubated with TGHQ (200 µM) for 2 h or gliotoxin (100 ng/mL) for 30 min followed by TNF-R (30 ng/mL) for 2 h in DMEM 25 mM with HEPES (pH 7.4). Apoptosis was detected by using the ApopTag kit (Intergen, Purchase, NY). Cells were fixed in 10% neutral buffered formalin for 10 min at room temperature, and ethanol-acetic acid (2:1) for 5 min at -20 °C, and then incubated in 2% hydrogen peroxide for 5 min followed by equilibration buffer for 2 min. The TdT enzyme solution (50 µL) was incubated with the cells at 37 °C for 60 min in a humid environment, and then washed in stop-wash buffer at 37 °C for 30 min, with gentle agitation. Cells were subsequently incubated with 50 µL anti-digoxigenin peroxidase enzyme at 37 °C for 45 min followed by 50 µL of DAB solution for 10 min in dark. The cover slides were then counterstained in methyl green for 1-5 min and dehydrated in 100% butanol and Hemo-de fixative solutions. Finally the cover slides were mounted on rectangular slides for microscopic examination. Images were taken with a Kodak DC120 digital camera and processed with Adobe Photoshop 6.0 software. Caspase 3 Activity Assay. LLC-PK1 cells were plated in 6-well plates at a density of 3 × 105 cells/well and treated as described above. Apoptosis was assessed by measuring caspase 3 activity (Caspase 3 Activity Assay Kit, Roche Molecular Biochemicals, Indianapolis, IN). Briefly, cells were lysed in lysis buffer containing 10 mM dithiothreitol (DTT) on ice for 30 min. Microtiter plates were coated with anti-caspase 3 coating buffer for 1 h at 37 °C and blocked in blocking solution for 30 min at room temperature. After three washings of the plate with incubation buffer, the lysates were added to the plates and incubated at 37 °C for 1 h. Plates were then washed carefully and incubated with substrate solution containing 50 mM AcDEVD-AFC at 37 °C for 2 h. Ac-DEVD-AFC is cleaved by caspase 3 in the cell lysates, and the liberated fluorescent AFC was quantified with a Bio-Tek FL600 microplate fluorescence reader and KC4 software (V2.5) (Bio-Tek Instruments, Inc., Winooski, Vermont) at 505 nm. Phosphorylation Status of ERK1/ERK2, JNKs/SAPKs, and p38. LLC-PK1 cells were seeded at a density of 1.0 × 106 cells/100 mm dish and allowed to grow for 36-40 h. After specified treatment, the cells were washed with cold PBS and homogenized in ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.8% Triton X-100) containing, 1 mM sodium fluoride, 5 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and one protease inhibitor tablet (Boehringer Mannheim, Indianapolis, IN) for 10 mL of lysis buffer. Homogenates were centrifuged at 14000g for 10 min at 4 °C. Supernatants were collected, and 50 µg of protein electrophoresed on 12% SDS-polyacrylamide gels and transferred to a PVDF membrane. After blocking in TBS-T containing 5% dry milk, membranes were incubated with primary antibodies (dilution1:500) directed against ERK1/2 or pERK1/2 (Cell Signaling Technology, Beverly, MA), JNKs and pJNKs (Santa Cruz Biotechnology, Santa Cruz, CA and Cell Signaling Technology, Beverly, MA) or p-38 or phospho-p38 pJNKs (Cell Signaling Technology, Beverly, MA). Washing was performed in TBS-T and membranes were then incubated with appropriate
MAPKs and Oncotic Cell Death horse-radish peroxidase-conjugated secondary antibodies (1: 2000) (Santa cruz Biotechnology). After thoroughly washing in TBS-T, bound antibodies were visualized using standard chemilumiscence on autoradiographic film. Eletrophoretic Mobility Shift Assays for AP-1 and NFKB DNA-Binding Activity. LLC-PK1 cells were seeded in 100 mm dishes and treated as described above. Cells were washed in cold PBS and homogenized in ice-cold HEGD buffer (25 mM HEPES, pH 7.6, 1.5 mM EDTA, 10% Glycerol, 1 mM DTT) containing freshly added protease inhibitors. Homogenates were centrifuged at 10000g for 5 min at 4 °C. Nuclear pellets were resuspended in HEGDK buffer (HEGD, 0.5 M KCl) containing protease inhibitors. High salt extraction was performed at 4 °C for 1 h, followed by centrifugation at 14000g for 5 min at 4 °C, and the supernatant collected. Nuclear extracts (10 µg) were incubated in a reaction mixture containing HEGD, KCl, 3.5 nM AP-1 or NF-κB probe (Santa Cruz Biotechnology, Santa Cruz, CA) labeled with [γ-32P]ATP, and 62.5 ng/µL polyD (I-C). Free probe was separated from that bound to transcription factor on a 5% polyacrylamide nondenaturing gel. Free probe was never rate-limiting for the binding reaction. Specificity for the binding reaction was confirmed by addition of unlabeled target DNA to competitively eliminate the inducible band. Gels were dried and exposed to Maxfilm (Eastman Kodak Company, Rochester, NY). Quantitation of autoradiograms was performed using Packard Instant Imager Electronic Autoradiography. Neutral Red Assay for Cell Viability. Cells were seeded at a density of 0.5 × 105 cells/well in 24-well plates and used following 36 h culture. Cells were washed and treated with TGHQ in DMEM containing 25 mM HEPES pH 7.4. Neutral red assays were then performed as previously described (29). Statistical Analysis. All data are expressed as the mean ( standard error. Mean values were compared by analysis of variance with a post-hoc Student Newman Kuel’s test. P < 0.05 was accepted as significant.
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Figure 1. TGHQ does not induce apoptosis in LLC-PK1 cells. (A) Biochemical assessment of apoptosis was performed with a caspase 3 assay kit as described in methods. LLC-PK1 cells were treated with TGHQ (200 µM; hatched bar) for 2 h, or gliotoxin (100 ng/mL) for 0.5 h followed by TNF-R (30 ng/mL) for 2 h (black bar). Results represent the average of three independent experiments. (B) Histological assessment of apoptosis was monitored by the TUNEL assay as described in the Experimental Procedures. Apoptotic cells stain dark brown, whereas normal cells counter stain with methyl green.
Results TGHQ Stimulates MAPK Phosphorylation in LLC-PK1 Cells. We have previously shown that TGHQ induces oncotic cell death in LLC-PK1 cells (30), with no evidence of apoptosis. To confirm our ability to monitor apoptosis in LLC-PK1 cells, we utilized a combination of gliotoxin and TNF-R to induce caspase 3 activity (Figure 1A). In contrast, TGHQ failed to increase caspase 3 activity (Figure 1A) under conditions that result in oncotic cell death. TUNEL staining (Figure 1B) confirmed the biochemical assessment of apoptosis, or lack thereof. Mechanisms that couple the generation of ROS and DNA damage to ROS-induced oncotic cell death are poorly understood. In addition to their roles in cell proliferation and differentiation, MAPKs are also activated by genotoxicants and agents that generate ROS, including UVC irradiation (1, 3) and hydrogen peroxide (4, 5). Since quinol-GSH conjugates cause DNA damage via the generation of ROS (25, 27), we investigated the effects of TGHQ on the activity of ERK1/2. Western blot analysis revealed that both ERK1 and ERK2 were rapidly phosphorylated following exposure of LLC-PK1 cells to TGHQ, in both a time and concentration dependent manner (Figures 2 and 3). Maximum ERK1/2 phosphorylation occurred at 1 h and subsequently declined but remained elevated even at 5 h (Figure 2). Increased ERK1/2 phosphorylation was not due to increases in overall ERK1/2 expression, since total ERK1/2 expression was initially unaffected by TGHQ treatment. In contrast, the decline in phosphorylated ERK1/2 at 5 h may in part be a consequence of decreases in total ERK1/2 protein levels (Figure 2).
Figure 2. Time-dependent activation of MAPKs by TGHQ in renal epithelial cells. LLC-PK1 cells were treated with TGHQ (200 µM) in DMEM for different periods of time. Total protein was separated by 12% SDS-PAGE. Western blot analyses for ERK, phospho-ERK, JNK1/2, phospho-JNK1/2, p38 MAPK, and phospho-p38 MAPK were performed as described in the Experimental Procedures. The experiment was repeated two times and produced similar results.
Phosphorylation of JNK1/2 and p38 MAPKs were also determined, in view of the role of these MAPK subfamilies in response to extracellular stresses. The phosphorylation of JNK1/2 and p38 MAPK increased following treatment with TGHQ (Figures 2 and 3). Interestingly, whereas little constitutive phosphorylation of ERK1/2 and p38 MAPK was observed in LLC-PK1 cells, pJNK1/2 were clearly present in untreated LLC-PK1 cells. The kinetics of MAPK phosphorylation were remarkably similar, with activation of all three subfamilies observed within 15 min of exposure of LLC-PK1 cells to TGHQ (Figure 3). However, pERK1/2 and p-p38 MAPK expres-
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Figure 3. Dose-dependent activation of MAPKs by TGHQ in renal epithelial cells. LLC-PK1 cells were treated with 100, 200, or 400 µM of TGHQ for 1 h. Total protein was separated by 12% SDS-PAGE. Western blot analyses for ERK, phosphoERK, JNK1/2, phospho-JNK1/2, p38 MAPK, and phospho-p38 MAPK were performed as described in the Experimental Procedures. The experiment was repeated two times and produced similar results.
sion persisted for a longer period than pJNK1/2. Moreover, although pERK1/2 and p-p38 MAPK phosphorylation remained maximal following exposure to the highest concentration of TGHQ (400 µM), this degree of stress suppresses the pJNK1/2 response (Figure 3). The combined data suggest that, as either the severity of the stress progresses (with time; Figure 2) or as the initial stress increases (with dose; Figure 3), the pJNK1/2 response was impaired. Moreover, decreases in pJNK2 at extended time points appeared to be a consequence of the selective loss of JNK2 protein (Figure 2) since JNK1 protein levels remained unaltered after TGHQ treatment. The basis for the selective loss of JNK2 protein is not known. ROS Are Required for MAPK Activation. The activation of MAPKs by TGHQ is rapid (
