Oxalate Selectively Activates p38 Mitogen-activated Protein Kinase and c-Jun N-terminal Kinase Signal Transduction Pathways in Renal Epithelial Cells
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m108203200
ISSN1083-351X
AutoresLakshmi S. Chaturvedi, Sweaty Koul, Avtar Sekhon, Akshay Bhandari, Mani Menon, Hari K. Koul,
Tópico(s)Phytochemistry and Bioactivity Studies
ResumoOxalate, a metabolic end product, is an important factor in the pathogenesis of renal stone disease. Oxalate exposure to renal epithelial cells results in re-initiation of the DNA synthesis, altered gene expression, and apoptosis, but the signaling pathways involved in these diverse effects have not been evaluated. The effects of oxalate on mitogen- and stress-activated protein kinase signaling pathways were studied in LLC-PK1 cells. Exposure to oxalate (1 mm) rapidly stimulated robust phosphorylation and activation of p38 MAPK. Oxalate exposure also induced modest activation of JNK, as monitored by phosphorylation of c-Jun. In contrast, oxalate exposure had no effect on phosphorylation and enzyme activity of p42/44 MAPK. We also show that specific inhibition of p38 MAPK by 4(4-(fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazole (SB203580) or by overexpression of a kinase-dead dominant negative mutant of p38 MAPK abolishes oxalate induced re-initiation of DNA synthesis in LLC-PK1 cells. The inhibition is dose-dependent and correlates with in situactivity of native p38 MAP kinase, determined as MAPK-activated protein kinase-2 activity in cell extracts. Thus, this study not only provides the first demonstration of selective activation of p38 MAPK and JNK signaling pathways by oxalate but also suggests that p38 MAPK activity is essential for the effects of oxalate on re-initiation of DNA synthesis. Oxalate, a metabolic end product, is an important factor in the pathogenesis of renal stone disease. Oxalate exposure to renal epithelial cells results in re-initiation of the DNA synthesis, altered gene expression, and apoptosis, but the signaling pathways involved in these diverse effects have not been evaluated. The effects of oxalate on mitogen- and stress-activated protein kinase signaling pathways were studied in LLC-PK1 cells. Exposure to oxalate (1 mm) rapidly stimulated robust phosphorylation and activation of p38 MAPK. Oxalate exposure also induced modest activation of JNK, as monitored by phosphorylation of c-Jun. In contrast, oxalate exposure had no effect on phosphorylation and enzyme activity of p42/44 MAPK. We also show that specific inhibition of p38 MAPK by 4(4-(fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazole (SB203580) or by overexpression of a kinase-dead dominant negative mutant of p38 MAPK abolishes oxalate induced re-initiation of DNA synthesis in LLC-PK1 cells. The inhibition is dose-dependent and correlates with in situactivity of native p38 MAP kinase, determined as MAPK-activated protein kinase-2 activity in cell extracts. Thus, this study not only provides the first demonstration of selective activation of p38 MAPK and JNK signaling pathways by oxalate but also suggests that p38 MAPK activity is essential for the effects of oxalate on re-initiation of DNA synthesis. mitogen-activated protein extracellular signal-regulated kinase stress-activated protein kinase mitogen-activated protein kinase c-Jun N-terminal kinase 4(4-(fluorophenyl)-2-(4-methylsulfonyl-phenyl)-5-(4-pyridyl)imidazole) mitogen-activated protein kinase-activated protein myelin basic protein phosphate-buffered saline mitogen-activated protein kinase kinases Dulbecco's modified Eagle's medium 4-morpholinepropanesulfonic acid glutathione S-transferase epidermal growth factor Ala-Gly-Phe Oxalate, a metabolic end product, is excreted primarily by the kidney and is associated with several pathological conditions. This organic dicarboxylate is freely filtered at the glomerulus and undergoes bi-directional transport in the renal tubules (1.Knight T.F. Senekjian H.O. Taylor K. Steplock D.A. Weinman E.J. Kidney Int. 1979; 16: 572-576Abstract Full Text PDF PubMed Scopus (19) Google Scholar, 2.Koul H. Ebisuno S. Renzulli L. Yanagawa M. Menon M. Scheid C. Am. J. Physiol. 1994; 266: F266-F274PubMed Google Scholar, 3.Kuo S.M. Aronson P.S. J. Biol. Chem. 1996; 271: 15491-15497Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The most common pathological condition involving oxalate is the formation of calcium oxalate stones in the kidney (4.Menon M. Koul H. J. Clin. Endocrinol. Metab. 1992; 74: 703-707Crossref PubMed Scopus (33) Google Scholar). Besides renal stone formation oxalate deposits are also associated with hyperplasic thyroid glands (5.Hackett R.L. Khan S.R. Scanning Microsc. 1988; 2: 241-246PubMed Google Scholar), benign neoplasm of the breast (6.Radi M.J. Arch. Pathol. Lab. 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Commun. 1994; 205: 1632-1637Crossref PubMed Scopus (98) Google Scholar, 11.Koul H. Kennington L. Jonassen J. Honeyman T. Menon M. Scheid C. Kidney Int. 1996; 50: 1525-1530Abstract Full Text PDF PubMed Scopus (55) Google Scholar, 12.Scheid C. Koul H. Hill W.A.G. Luber-Narod J. Jonassen J. Honeyman T. Kennington L. Kholi R. Hodapp J. Ayvazian P. Menon M. J. Urol. 1996; 155: 1112-1116Crossref PubMed Scopus (77) Google Scholar, 13.Koul H.K. Koul S. Fu S. Santosham V. Seikhon A. Menon M. J. Am. Soc. Nephrol. 1999; 10: S417-S421PubMed Google Scholar, 14.Khan S.R. Byer K.J. Thamilselvan S. Hackett R.L. McCormack W.T. Benson N.A. Vaughn K.L. Erdos G.W. J. Am. Soc. Nephol. 1999; 10: S457-S463PubMed Google Scholar). Previously, we demonstrated that oxalate exposure to renal epithelial cells (LLC-PK1) in culture results in the re-initiation of DNA synthesis, cell proliferation, and cell death depending on the levels of oxalate (10.Koul H. Renzulli L. Nair G. Honeyman T. Menon M. Schied C. Biochem. Biophys. Res. Commun. 1994; 205: 1632-1637Crossref PubMed Scopus (98) Google Scholar). Moreover, the exact signal transduction pathways for diverse actions of oxalate are not understood. In this study we used this cell line to investigate the effects of oxalate on SAPK and MAPK signaling pathways. We show that oxalate selectively, rapidly, and robustly activates p38 MAP kinase, causes a mild activation of JNK/SAPK, and does not activate the ERK group of MAP kinases in renal epithelial cells. Furthermore, we demonstrate that p38 MAP kinase in particular is most strongly targeted by oxalate and that p38 MAP kinase activity is essential for the effects of oxalate on re-initiation of DNA synthesis. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, penicillin/streptomycin, and myelin basic protein (MBP) were purchased from Invitrogen. Antibodies against phospho- and total p38 MAP kinase and phospho- and total c-Jun were obtained from New England Biolabs (Beverly, MA). Antibodies against p42/44 MAPK and c-Jun N-terminal kinase (JNK) antibodies were obtained from BD Transduction Laboratories (Los Angeles, CA). GST c-Jun-(1–79) was purchased from Amersham Biosciences. MAPKAP kinase-2 assay kit was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Goat anti-rabbit IgGs and goat anti-mouse IgGs were obtained from Kodak Biolabs Scientific Imaging systems (Rochester, NY). Recombinant protein A-agarose, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, and SB203580 were purchased from Sigma. Anisomycin was purchased fromCalbiochem-Novabiochem. ATP, [γ-32P]ATP, and [3H]thymidine was obtained from ICN Radiochemicals (Costa Mesa, CA). ImmobilonTM-P membrane was obtained from Millipore (Bedford, MA). All cell culture reagents were obtained from Invitrogen. The expression vector pCMV-p38 (AGF) (dominant negative mutant of p38 MAPK) was a generous gift from Dr. Roger J. Davis (Howard Hughes Medical Institute, University of Massachusetts, Worcester, MA). All chemicals were analytical grade and were obtained from Sigma. LLC-PK1 cells (American Type Culture Collection, Manassas, VA), grown on polystyrene (Corning Glass) T-75 flasks, were used between passages 216 and 240. The cells were serially passaged in low glucose Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). For transfections, LLC-PK1 cells were grown in 6-well plates to 60% confluency and were transiently transfected with control vector (pCMV Tag 5) or kinase-dead dominant negative construct pCMV-p38 (AGF) using LipoTAXI transfection reagent (Stratagene), following the manufacturer's instructions. Transfected cells were allowed to grow to confluence prior to use in experiments. All cultures were maintained at 37 °C in a humidified atmosphere of 95% air, 5% CO2. Sodium oxalate was added at a concentration of 1 mm, wherever indicated. [3H]Thymidine incorporation was used as an index of cell proliferation and was carried out as described previously (10.Koul H. Renzulli L. Nair G. Honeyman T. Menon M. Schied C. Biochem. Biophys. Res. Commun. 1994; 205: 1632-1637Crossref PubMed Scopus (98) Google Scholar). Briefly, LLC-PK1 cells were plated at a high density in 6-well plates and grown to confluence. These cells were serum-starved for 12–18 h and pre-exposed to various concentrations of SB203580 (0.5-100 μm) for 1 h before addition of oxalate (1 mm) for 24 h. In some experiments, cells were transfected with control vector (pCMV Tag 5) or kinase-dead dominant negative expression vector pCMV-p38 (AGF) and were grown to confluence. These cells were serum-starved for 12–18 h before exposure to oxalate (1 mm) for 24 h. During last 6 h of exposure 2–3 μCi of [3H]thymidine was added per well. At the end of experimental period, cells were washed with two changes of ice-cold phosphate-buffered saline (PBS) and trypsinized for 30–45 min at 37 °C. Two ml of cell suspension was combined with 2 ml of 10% trichloroacetic acid, and the acid-insoluble material was collected on Whatman glass fiber filters. Filters were then air-dried, and the radioactivity was counted using a Beckman Liquid Scintillation Counter (LS 6500). Cells were grown to confluence in 6-well plates and were serum-starved for 12–18 h. These growth-arrested cells were exposed for various time points (0–240 min) to oxalate (1 mm). Where indicated, cells were exposed to anisomycin (10 μg/ml), UV, or EGF (50 ng/ml) for appropriate positive controls. At the end of experimental periods, cells were washed with ice-cold PBS and solubilized with lysis buffer (20 mm Tris, pH 7.4, 1% Triton X-100, 1 mm sodium orthovanadate, 10 mm NaF, 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin). Lysates were sonicated for 1 s with a micro-ultrasonic cell disrupter and centrifuged at 14,000 × g at 4 °C for 15 min to remove insoluble material. Protein concentrations were determined by using the Bradford (Bio-Rad) method, and gel samples were prepared by adding 2× sample buffer (50 mm Tris, pH 6.7, 2% SDS, 2% β-mercaptoethanol, and bromphenol blue) and boiling for 3–5 min. Samples containing 50–100 μg of proteins were separated by SDS-PAGE and then transferred to an Immobilon-P membrane using standard electroblotting procedures. The membranes were blocked with 2% blocking solution (Eastman Kodak) in TBST (Tris, pH 7.2, 140 mm NaCl, 0.1% Tween 20). Blots were immunolabeled overnight at 4 °C with monoclonal antibodies that equally recognize phosphorylated and non-phosphorylated p42/p44 MAPK (1:1000) or with an antibody that specifically recognizes phospho-MAPK (1:1000). The phosphorylation state of JNK/SAPK was evaluated using an antibody that specifically recognizes phospho-Ser63–Ser73c-Jun or an antibody that equally recognizes phosphorylated and unphosphorylated c-Jun. Similarly, the phosphorylation state of p38 MAP kinase was evaluated using an antibody that specifically recognizes dual phosphorylation motif at Thr180 and Tyr182 of p38 MAP kinase (1:1500) or with an antibody that equally recognizes phosphorylated and dephosphorylated p38 MAP kinase (1:3000). Immunoblots were washed with several changes of TBST at room temperature and then incubated with anti-mouse or anti-rabbit IgG linked to horseradish peroxidase (Kodak). Immunoreactivity was detected with enhanced chemiluminescence detection system (Kodak) according to the manufacturer's recommended protocol and quantified using densitometric analysis (Stratagene Eagles EyeTM II). For these assays, cells were grown to confluence in 6-well plates and were serum-starved for 12–18 h. Cells were exposed for various time points (0–60 min) to oxalate (1 mm). Where indicated, cells were exposed to anisomycin (10 μg/ml), UV, or EGF (50 ng/ml) for appropriate positive controls. These cells were then solubilized with ice-cold lysis buffer (20 mm Tris, pH 7.4, 137 mm NaCl, 2 mmEDTA, 1% Triton X-100, 10% glycerol, 25 mmβ-glycerophosphate, 1 mm sodium orthovanadate, 2 mm pyrophosphate, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin) and centrifuged at 14,000 × g for 15 min at 4 °C. Immunoprecipitation of ERK, JNK, or p38 MAP kinase was achieved by adding 0.5 μg of anti-ERK-2, 1 μg of anti-JNK, or 0.5 μg of anti-p38 MAP kinase antibody, respectively, to cell lysate containing 500 μg of total cellular protein and rocking at 4 °C for 2–4 h. 50 μl of a 10% (w/v) suspension of recombinant protein A-agarose beads was then added, and the reaction slurry was allowed to rock at 4 °C for 8–12 h. The immunoprecipitation complexes were washed twice with 0.5 ml of ice-cold lysis buffer and five times with kinase assay buffer (25 mmHEPES, pH 7.4, 25 mm β-glycerophosphate, 25 mm MgCl2, 0.1 mm sodium orthovanadate, and 1 mm dithiothreitol). The kinase assay reactions consisted of kinase buffer supplemented with 20 μm ATP containing 10 μCi of [γ-32P]ATP and either 10 μg of MBP for p38 MAP kinase and p42/p44 MAP kinase or 1 μg of GST c-Jun-(1–79) for JNK assay, in a final volume of 50 μl. The reactions were carried out at 30 °C for 20 min with shaking. Reactions were stopped by 2 min of centrifugation at 14,000 × g, and supernatant was suspended in 2× Laemmli SDS-sample buffer containing β-mercaptoethanol and bromphenol blue. Samples were boiled for 2 min and run on 12% SDS-polyacrylamide gels for p38 MAP kinase and p42/44 MAPK or on 15% SDS-polyacrylamide gels for JNK enzyme activity. Kinase activity was measured as the amount of 32P incorporation into specific substrate proteins. These enzyme assays were carried out as described previously (41.Beitner-Johnson D. Millhorn D.E. J. Biol. Chem. 1998; 273: 19834-19839Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) with slight modifications. Briefly, cells were grown to confluence in 6-well plates. Serum-starved cells were exposed to oxalate (1 mm) for various time points between 0 and 60 min. In some experiments, cells were pretreated for 1 h with various concentrations of SB203580 (0.5–30 μm) or the cells were transfected with control vector (pCMV Tag 5) or kinase-dead dominant negative expression vector pCMV-p38 (AGF) before exposure to oxalate. At the end of the treatment period, cells were harvested by scraping in 0.5 ml of an ice-cold non-denaturing lysis buffer A containing 50 mm Tris, pH 7.5, 1 mm EDTA, 1 mm EGTA, 0.5 mmsodium orthovanadate, 0.1% 2-mercaptoethanol, 1% Triton X-100, 5 mm sodium pyrophosphate, 10 mm sodium glycerophosphate, 10 mm NaF, 0.1 mmphenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin. Lysates were incubated for 20 min at 4 °C and then centrifuged for 20 min at 14,000 × g to remove Triton-insoluble material. Aliquots containing 0.5 mg of total protein were immunoprecipitated for 2 h at 4 °C with 5 μg of anti-MAPKAP kinase-2 polyclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY) coupled to recombinant protein A-agarose beads (Sigma). The protein A-agarose enzyme immunocomplex was washed with 500 μl of non-denaturing lysis buffer A containing 0.5 m NaCl followed by 500 μl of non-denaturing lysis buffer A and then finally with 100 μl of ice-cold assay dilution buffer (20 mmMOPS, pH 7.2, 25 mm β-glycerophosphate, 5 mmEGTA, 1 mm sodium orthovanadate, 1 mmdithiothreitol) solution. The MAPKAP kinase-2 enzyme activity was then assayed in an immunocomplex kinase assay using a specific MAPKAP kinase-2 substrate peptide (KKLNRTLSVA) from a MAPKAP kinase-2 immunoprecipitation kinase kit, as described in the manufacturer's recommended protocol (Upstate Biotechnology). Briefly, beads were supplemented with 10 μl of ice-cold assay dilution buffer solution, 10 μl of 1 mm MAPKAP kinase-2 substrate peptide solution, and 10 μl of [γ-32P]ATP (10 μCi/assay, PerkinElmer Life Sciences) diluted to 1 μCi/μl with magnesium and ATP mixture (75 mm magnesium chloride and 500 μm ATP) in each tube containing immunocomplex-formed beads, in a final volume of 50 μl. The mixtures were incubated for 30 min at 30 °C in a shaking incubator. At the end of the experimental period, tubes were spun down, and 25 μl of the supernatant were spotted onto the center of 2 × 2 cm p81 phosphocellulose discs. The discs were washed three times in 0.75% phosphoric acid (v/v) and once with acetone. Radioactivity associated with the discs was counted by liquid scintillation counting and used as an index of 32P incorporated into the substrate peptide (KKLNRTLSVA). The counts/min of enzyme samples was compared with counts/min of control samples that contained no enzyme (background control). For these studies, LLC-PK1 cells were exposed to DMEM alone or in combination with oxalate (1 mm) for various times (5–60 min) or to anisomycin (10 μg/ml) for 30 min. At the end of experimental periods, whole cell lysates were subjected to SDS-PAGE and then immunoblotted with an antibody specific for phosphorylated p38 MAP kinase. Phosphorylated p38 MAP kinase is considered essential for its enzyme activity. As can be seen from the Fig. 1A, exposure to oxalate progressively induced phospho-p38 MAP kinase immunoreactivity. These blots were then stripped and re-probed with an antibody that equally recognizes phosphorylated as well as unphosphorylated p38 MAP kinase,i.e. total p38 MAP kinase. As shown in Fig. 1B,oxalate exposure did not alter the total amount of p38 MAP kinase protein. Maximum activation of p38 occurred at about 30 min of oxalate exposure with an average 4.6-fold increase in p38 MAP kinase phospho-immunoreactivity (Fig. 1C). These results demonstrate that p38 MAP kinase is activated by oxalate. To characterize further the effects of oxalate on p38 MAP kinase enzyme activity, LLC-PK1 cells were exposed to DMEM alone or in combination with oxalate (1 mm) for various times (5–30 min). p38 MAP kinase was then immunoprecipitated with an antibody that recognizes total p38 MAP kinase (phosphorylated as well as unphosphorylated), and immunocomplex kinase assay was performed as described under "Experimental Procedures." As shown in Fig. 1D, oxalate robustly stimulated p38 MAP kinase activity within 30 min of exposure. It can be seen in Fig. 1E that the effect of oxalate on p38 MAP kinase is most robust at 30 min (average 7.4-fold activation over control). Some concerns have been raised that in vitro examination of p38 MAP kinase activity may not reflect its in situactivity. Thus in order to examine the specificity of oxalate exposure on the activity of p38 MAP kinase, LLC-PK1 cells were exposed to DMEM alone or in combination with oxalate (1 mm) for 30 and 60 min, and cell lysates were prepared for determination of native MAPKAP kinase-2 activity. It is important to point out here that p38 MAP kinase is the only known activator of MAPKAP kinase-2, and the activity of MAPKAP kinase-2 is dependent on its phosphorylation by p38 MAP kinase (42.Krump E. Sanghera J.S. Pelech S.L. Furuya W. Grinstein S. J. Biol. Chem. 1997; 272: 937-944Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Therefore, in vitro activity of immunoprecipitated MAPKAP kinase-2 reflects the in situactivity of p38 MAP kinase. Results presented in Fig. 2 demonstrate that oxalate-induced MAPKAP kinase-2 activity followed an activation pattern similar to that of p38 MAP kinase with maximal activation at 30 min following oxalate exposure. These data demonstrate that the rapid and robust activation of p38 MAP kinase by oxalate correlates with the in situactivity of MAPKAP kinase-2. Next we evaluated the effect of oxalate on JNK, the major SAPK. LLC-PK1 cells were exposed to oxalate for various times (0–60 min) or to UV for 30 min, and JNK enzyme activity was measured as described under "Experimental Procedures." Oxalate exposure resulted in time-dependent increase in immunoreactivity of c-Jun (Fig. 3A). We also observed a mild phosphorylation at Ser73 of c-Jun during the times tested (Fig. 3, B and C). However, we could not detect phosphorylation at Ser63 (data not shown). These data demonstrate that oxalate exposure results in increased immunoreactivity of the transcription factor c-Jun. However, unlike its effects on p38 MAP kinase, oxalate exposure resulted only in a modest increase in JNK activity as compared with UV exposure. To characterize further the effects of oxalate on JNK/SAPK1 enzyme activity, LLC-PK1 cells were exposed to oxalate (1 mm) for various times (5–30 min). JNK/SAPK1 was then immunoprecipitated with an antibody that recognizes total JNK/SAPK1 (phosphorylated as well as unphosphorylated), and immunocomplex kinase assay was performed as described under "Experimental Procedures." As shown in Fig. 3D oxalate had only modest effect on JNK/SAPK1 activity within these time points. It can be seen in Fig. 3E that the effect of oxalate on JNK/SAPK1 was mild (1.8–2.2-fold over control) as compared with strong stimulation of JNK/SAPK1 by UV exposure (∼4.5-fold over contro
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