Oxalate Selectively Activates p38 Mitogen-activated Protein Kinase and c-Jun N-terminal Kinase Signal Transduction Pathways in Renal Epithelial Cells*

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.

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)(2)(3). The most common pathological condition involving oxalate is the formation of calcium oxalate stones in the kidney (4). Besides renal stone formation oxalate deposits are also associated with hyperplasic thyroid glands (5), benign neoplasm of the breast (6,7), renal cysts in acquired renal cystic disease, and proliferating cells in the kidney (8). Many of these conditions are associated with aberrant cell proliferation and cell death. Previous studies from our laboratory and those of others (9 -14) have demonstrated that oxalate interaction with renal epithelial cells results in a program of events, including immediate early gene expression, re-initiation of DNA synthesis, cell growth, and apoptosis. However, the specific cellular pathways activated in renal cells following oxalate exposure are not well delineated.
Five homologous subfamilies of MAPKs and SAPKs have been identified thus far. The three major families include p42/p44 MAPKs/ERKs, JNK/SAPK1, and p38 MAPK/SAPK2 kinase. The p38 MAP kinase is a mammalian homologue of yeast HOG kinase and participates in a cascade controlling cellular responses to stress and cytokines (23)(24)(25). The prototypical MAP kinases ERK-1 and ERK-2 are defined by motif TEY in the activation domain (26). The second subtype JNK/SAPK1 is characterized by the sequence TPY in the activation domain (27), whereas the third, p38/SAPK2/ reactivating kinase, has the motif TGY in the activation domain (23). These subtypes may be activated in parallel but to a distinct extent by different stimuli (28 -30). In general stress-activated protein kinases are activated primarily when cells are exposed to various kinds of stresses such as osmotic stress, inflammatory cytokines, ultraviolet light, and high temperature shock (27,31,32). However, increasing evidence suggests that, at least under certain conditions, these pathways can also be activated by mitogenic factors (33). In contrast, p42/p44 MAP kinases are primarily stimulated by mitogenic factors, although these kinases can also be activated under certain stress conditions (34,35).
LLC-PK1 cells, a line of porcine kidney epithelial cells with characteristics of the S1-S3 segment of proximal tubular epithelium, have been used widely as an in vitro model of renal epithelial cells (36 -39). These cells express transport systems for oxalate and other ions (2,38,40). It has been shown that LLC-PK1 cells are sensitive to oxalate and provide a useful system to study the effects of oxalate on re-initiation of the DNA synthesis, early growth-responsive gene expression, cell growth, and death (10 -14). 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). 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.

EXPERIMENTAL PROCEDURES
Materials-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 from Calbiochem-Novabiochem. ATP, [␥-32 P]ATP, and [ 3 H]thymidine was obtained from ICN Radiochemicals (Costa Mesa, CA). Immobilon TM -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.
Culture and Transfection of LLC-PK1 Cells-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 g/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 Lipo-TAXI 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% CO 2 . Sodium oxalate was added at a concentration of 1 mM, wherever indicated.
Initiation of the DNA Synthesis-[ 3 H]Thymidine incorporation was used as an index of cell proliferation and was carried out as described previously (10). 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 M) 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 Ci of [ 3 H]thymidine was added per well. At the end of experimental period, cells were washed with two changes of ice-cold phosphatebuffered 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).
Western Blot Analysis-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 g/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 mM phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, 2 g/ml aprotinin). Lysates were sonicated for 1 s with a micro-ultrasonic cell disrupter and centrifuged at 14,000 ϫ 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 2ϫ sample buffer (50 mM Tris, pH 6.7, 2% SDS, 2% ␤-mercaptoethanol, and bromphenol blue) and boiling for 3-5 min. Samples containing 50 -100 g 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-Ser 63 -Ser 73 c-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 Thr 180 and Tyr 182 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 antimouse 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 Eye TM II).
Immunocomplex Kinase Assays-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 g/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 mM EDTA, 1% Triton X-100, 10% glycerol, 25 mM ␤-glycerophosphate, 1 mM sodium orthovanadate, 2 mM pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml aprotinin) and centrifuged at 14,000 ϫ g for 15 min at 4°C. Immunoprecipitation of ERK, JNK, or p38 MAP kinase was achieved by adding 0.5 g of anti-ERK-2, 1 g of anti-JNK, or 0.5 g of anti-p38 MAP kinase antibody, respectively, to cell lysate containing 500 g of total cellular protein and rocking at 4°C for 2-4 h. 50 l 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 mM HEPES, pH 7.4, 25 mM ␤-glycerophosphate, 25 mM MgCl 2 , 0.1 mM sodium orthovanadate, and 1 mM dithiothreitol). The kinase assay reactions consisted of kinase buffer supplemented with 20 M ATP containing 10 Ci of [␥-32 P]ATP and either 10 g of MBP for p38 MAP kinase and p42/p44 MAP kinase or 1 g of GST c-Jun-(1-79) for JNK assay, in a final volume of 50 l. The reactions were carried out at 30°C for 20 min with shaking. Reactions were stopped by 2 min of centrifugation at 14,000 ϫ g, and supernatant was suspended in 2ϫ Laemmli SDS-sample buffer containing ␤-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 32 P incorporation into specific substrate proteins.
MAPKAP Kinase-2 Enzyme Assays-These enzyme assays were carried out as described previously (41) 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 M) 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 mM sodium orthovanadate, 0.1% 2-mercaptoethanol, 1% Triton X-100, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 10 mM NaF, 0.1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml aprotinin. Lysates were incubated for 20 min at 4°C and then centrifuged for 20 min at 14,000 ϫ g to remove Triton-insoluble material. Aliquots containing 0.5 mg of total protein were immunoprecipitated for 2 h at 4°C with 5 g 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 l of non-denaturing lysis buffer A containing 0.5 M NaCl followed by 500 l of non-denaturing lysis buffer A and then finally with 100 l FIG. 1. Oxalate activates p38 MAP kinase rapidly and robustly. Confluent, growth-arrested, and serum-starved LLC-PK1 cells were exposed to oxalate (1 mM) between 0 and 60 min or to anisomycin (10 g/ml) for 30 min, as indicated. The cells were solubilized, separated on FIG. 2. Oxalate stimulates p38 MAP kinase in situ as determined by MAPKAP kinase-2 activity. Confluent, growth-arrested, and serum-starved LLC-PK1 cells exposed to DMEM alone or with oxalate (1 mM) for 30 and 60 min. The cells were lysed in ice-cold non-denaturing lysis buffer A, and cell lysates (0.5 mg of protein) were immunoprecipitated with 5 g of anti-MAPKAP kinase-2 polyclonal antibody coupled to protein A-agarose beads and tested for its ability to phosphorylate a specific MAPKAP kinase-2 substrate peptide (KKLN-RTLSVA). MAPKAP kinase-2 substrate peptide (200 M) and [␥-32 P]ATP (10 Ci/sample) were added to the MAPKAP kinase-2 and protein A-agarose complex and spotted onto p81 cellulose discs. The discs were counted by liquid scintillation counting, and radioactivity associated with the discs was used as an index of 32 P incorporated into the MAPKAP kinase-2 substrate peptide (KKLNRTLSVA). The data shown were obtained in one experiment, with similar results obtained in a separate experiment.
10% SDS-PAGE, and blotted on Immobilon-P membrane. Blots were probed with antibodies specific for either phosphorylated p38 MAP kinase or total (phospho-and dephosphorylated) p38 MAP kinase. A, representative Western blot illustrating the effects of oxalate and anisomycin on phospho-p38 MAP kinase immunoreactivity. B, the blot shown in A was stripped and reprobed with an antibody that recognizes total p38 (phospho-and dephosphorylated) MAP kinase. C, levels of phospho-p38 immunoreactivity as quantitated by densitometric analysis using Eagles Eye TM II (Stratagene). Data are expressed as the average percentage of control Ϯ S.D. and represent six individual dishes in each group, each performed in three separate experiments (*, p Ͻ 0.01; **, p Ͻ 0.001 compared with untreated control). D, the activity of p38 MAP kinase was measured in immunocomplex protein kinase assay using [␥-32 P]ATP and MBP as substrate. The cell lysates (0.5 mg of protein) immunoprecipitated with 0.5 g of anti-p38 antibody and protein A-agarose and tested for its ability to phosphorylate MBP. MBP (10 g/sample) and [␥-32 P]ATP (10 Ci/sample) were added to the p38 and protein A-agarose complex, and the immune complexes were resolved on 12% SDS-PAGE and visualized by autoradiography. E, densitometric analysis of phosphorylated MBP (p38) shown in D. Data are expressed as average percentage of change from control Ϯ S.D., and each point represents three separate independent experiments (*, p Ͻ 0.01; **, p Ͻ 0.001 compared with untreated control). of ice-cold assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM ␤-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol) 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 l of ice-cold assay dilution buffer solution, 10 l of 1 mM MAPKAP kinase-2 substrate peptide solution, and 10 l of [␥-32 P]ATP (10 Ci/assay, PerkinElmer Life Sciences) diluted to 1 Ci/l with magnesium and ATP mixture (75 mM magnesium chloride and 500 M ATP) in each tube containing immunocomplex-formed beads, in a final volume of 50 l. 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 l of the supernatant were spotted onto the center of 2 ϫ 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 32 P 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).

Oxalate Stimulates p38 MAP Kinase Activity Rapidly and
Robustly-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 g/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 situ activity. Thus in order to examine the specificity of oxalate exposure on the activity of p38 MAP kinase, LLC-PK1 cells were exposed to FIG. 3. Oxalate moderately activates JNK. Confluent, growtharrested, and serum-starved LLC-PK1 cells exposed to oxalate (1 mM) for various times between 0 and 60 min or to UV for 30 min, as indicated. The cells were solubilized in lysis buffer, and total proteins were separated on 10% SDS-PAGE and immunoblotted with antibodies specific for either phosphorylated c-Jun or total (phospho-and dephosphorylated) c-Jun. Antibody binding was detected using ECL detection system. A, representative immunoblot illustrating the effect of oxalate (1 mM) on total (phospho-and dephosphorylated) c-Jun immunoreactivity. B, the blots shown in A was stripped and reprobed with an antibody that recognizes only phospho-c-Jun. C, levels of phospho-c-Jun immunoreactivity as quantitated by densitometric analysis using Eagles Eye TM II (Stratagene). Data are expressed as the average percentage of control Ϯ S.D. and represent three separate independent experiments (*, p Ͻ 0.05; **, p Ͻ 0.001 compared with untreated control). D, confluent, growth-arrested, and serum-starved LLC-PK1 cells were solubi-lized in lysis buffer after exposure to oxalate (1 mM) for 0 -30 min, as indicated. The activity of JNK was measured in immunocomplex protein kinase assay using [␥- 32  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). Therefore, in vitro activity of immunoprecipitated MAPKAP kinase-2 reflects the in situ activity 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 situ activity of MAPKAP kinase-2.
Oxalate Causes Mild Stimulation of Jun N-terminal Kinase Activity-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 Ser 73 of c-Jun during the times tested (Fig. 3, B and C). However, we could not detect phosphorylation at Ser 63 (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 (ϳ4.5fold over control).
Oxalate Does Not Activate p42/44 Mitogen-activated Protein Kinase Activity-To determine the effects of oxalate on p42/p44 MAPK, LLC-PK1 cells were exposed to either oxalate (1 mM) or EGF (50 ng/ml) or a combination with both for various times (5-240 min). Samples of the whole cell extract were immunoblotted with an antibody that equally recognizes phosphorylated as well as dephosphorylated p42/p44 MAPK (total MAPK). Phosphorylated p42/p44 was identified by slower electrophoretic mobility. Oxalate exposure had no significant effect on either total p42/p44 MAPK or phosphorylated p42/p44 MAPK (Fig. 4, A and D) as compared with control. Please note that these cells retain a modest p42/p44 MAPK activity even prior to stimulation by oxalate. In contrast, exposure of cells to EGF, a prototypical activator of p42/p44 MAP kinase pathway, caused rapid and robust increase in tyrosine phosphorylation of p42/ p44 MAP kinase (Fig. 4, B and E). The exposure of oxalate and EGF together did not either increase or decrease the phosphorylation of p42/p44 MAP kinase compared with EGF alone (Fig.  4, C and F).
To characterize further the effects of oxalate on p42/p44 MAP kinase enzyme activity, LLC-PK1 cells were exposed to oxalate (1 mM) or EGF (50 ng/ml) or a combination of oxalate and EGF for 15 min. p44 MAP kinase (ERK-2) was then immunoprecipitated with an antibody that recognizes total p44 MAP kinase (phosphorylated as well as unphosphorylated), and immunocomplex kinase assay was performed as described under "Experimental Procedures." The results, shown in Fig. 4, G and H, confirm that oxalate had no effect on p44 MAP kinase activity (1.1-1.2-fold versus control). In contrast, exposure of cells to EGF caused significant increase in p44 MAP kinase activity (3-3.2-fold over control) as determined by MBP phosphorylation. Moreover, the exposure of oxalate and EGF together did not significantly increase or decrease the activity of p44 MAP kinase compared with EGF alone (3-3.4-fold over control versus 3-3.2-fold over control).
Inhibition of p38 MAP Kinase Activity Blocks Oxalate-induced Re-initiation of the DNA Synthesis-Previous studies (10) demonstrated that oxalate exposure resulted in re-initiation of the DNA synthesis in growth-arrested LLC-PK1 cells. However, little is known about the specific signaling pathways involved in this process. Because the present studies demonstrated that p38 MAP kinase signaling pathway is selectively activated by oxalate, additional studies were designed to test whether oxalate-induced re-initiation of the DNA synthesis in LLC-PK1 cells was mediated by activation of p38 MAP kinase pathway. These studies utilized SB203580, a selective inhibitor of p38 MAP kinase pathway as well as kinase-dead dominant negative expression of p38 MAP kinase. For these studies, confluent growth-arrested LLC-PK1 cells were exposed to oxalate (1 mM) for various times (0 min to 24 h) in the absence or presence of increasing concentrations of SB203580, a potent inhibitor of p38 MAP kinase (43,44). Alternatively cells were transfected with control vector (pCMV Tag 5) or kinase-dead dominant negative expression vector pCMV-p38 (AGF) as described under "Experimental Procedures." DNA synthesis was measured as described under "Experimental Procedures." Exposure to oxalate resulted in DNA synthesis 3-fold above that of control cells. Pretreatment of cells with SB203580, a selective inhibitor of p38, was able to attenuate the oxalate-induced DNA synthesis in a dose-dependent manner with complete inhibition at 20 M (Fig. 5A). SB203580 belongs to the group of pyridinyl imidazole compounds and has been shown to demonstrate a highly specific and potent inhibitory activity against p38 MAP kinase. Previous studies (24,45) have also shown that SB203580 had no inhibitory activity against ERK-1, ERK-2, or JNK activity. These data suggested involvement of p38 MAP kinase pathway in oxalate-induced re-initiation of the DNA synthesis.
These results were further confirmed by overexpression of kinase-dead dominant negative p38 MAP kinase. Exposure of cells transfected with control vector (pCMV Tag 5) to oxalate resulted in ϳ3-fold increase in DNA synthesis over untreated cells (315.9 Ϯ 13.37 versus 100 Ϯ 7). These results are similar to the effects of oxalate on non-transfected cells. The transfection of cells with kinase-dead dominant negative expression vector pCMV-p38 (AGF) greatly attenuated the oxalate-induced DNA synthesis (oxalate ϩ pCMV-p38 (AGF) versus pCMV-p38 (AGF): 156 Ϯ 11 versus 98 Ϯ 8.9) (Fig. 5B). Taken together these data implicate p38 MAP-kinase pathway in oxalate-induced re-initiation of the DNA synthesis.
It has been shown that the inhibitory effects of imidazole compounds on p38 MAP kinase are reversible (46). This raises the possibility that in vitro activity of p38 MAP kinase may not reflect its native in situ activity following addition of SB203580 to the cells. Thus, additional studies evaluated the specificity of SB203580 on the in situ activity of p38 MAP kinase in our system. We also evaluated the effects of transfection of cells with kinase-dead dominant negative expression vector pCMV-p38 (AGF) on the in situ activity of p38 MAP kinase. For these studies cells were exposed to oxalate (1 mM) in the absence and the presence of SB203580 (0.5 to 30 M). Alternatively, cells transfected with control vector (pCMV Tag 5) or kinase-dead FIG. 4. Lack of the effect of oxalate on ERK1/2 activation. Confluent, growth-arrested, and serum-starved LLC-PK1 cells were exposed to DMEM alone or oxalate (1 mM) or EGF (50 ng/ml) or in combination of both for 0 -240 min, as indicated. The cells were solubilized in lysis buffer, and total proteins were subjected to 10% SDS-PAGE and immunoblotted with an antibody that recognizes ERK1 and ERK2. The antibody binding was detected using ECL detection system. A, representative immunoblot illustrating the effect of oxalate (1 mM) on mobility of ERK1/2 MAP kinase. B, representative immunoblot illustrating the effect of EGF (50 ng/ml) on ERK1/2 MAP kinase mobility. C, representative immunoblot illustrating the effect of oxalate (1 mM) and EGF (50 ng/ml) in combination on ERK1/2 MAP kinase mobility. D-F, the levels of immunoreactivity of phospho-ERK2 (mobility shift) following exposure to oxalate (1 mM), EGF (50 ng/ml), or a combination of oxalate and EGF, respectively, were quantitated by densitometric analysis using Eagles Eye TM II (Stratagene). The data shown are expressed as average percentage of change from dominant negative expression vector pCMV-p38 (AGF) were exposed to oxalate (1 mM). Cell lysates were prepared for the determination of native MAPKAP kinase-2 activity. As stated earlier MAPKAP kinase-2 is a protein kinase that is phosphorylated and activated only by the p38 MAP kinase family of protein kinases, and the activity of MAPKAP kinase-2 is dependent on its phosphorylation by p38 kinase (42). Therefore, in vitro activity of immunoprecipitated MAPKAP kinase-2 reflects the in situ activity of p38 MAP kinase. Results shown in Fig. 5C show that oxalate-induced MAPKAP kinase-2 activity was inhibited by SB203580 in a dose-dependent fashion. These data demonstrate that SB203580 inhibits in situ activity of p38 MAP kinase. The inhibitory effect of SB203580 on oxalateactivated MAPKAP kinase-2 shows complete inhibition at 20 M. Thus the inhibitory effects of SB203580 on oxalate-induced DNA synthesis correlates with the inhibitory effects of SB203580 on oxalate-activated MAPKAP kinase-2 in a dosedependent fashion. Similarly, transfection of cells with kinasedead dominant negative expression vector pCMV-p38 (AGF), but not with control vector (pCMV Tag 5), greatly attenuated the effects of oxalate on MAPKAP kinase-2 activity (oxalate ϩ pCMV-p38 (AGF) versus pCMV-p38 (AGF), 5.74 pmol/min versus 2.5 pmol/min; oxalate ϩ control (pCMV Tag 5) vector versus control (pCMV Tag 5) vector, 17.59 pmol/min versus 3.47 pmol/ min) (Fig. 5D). Thus the inhibitory effect of transfection of cells with kinase-dead dominant negative expression vector pCMV-p38 (AGF) on oxalate-induced DNA synthesis correlates with its inhibitory effects on oxalate-activated MAPKAP kinase-2.
Taken together the effects of specific p38 MAP kinase inhibitor SB203580 and overexpression of kinase-dead dominant negative p38 MAP kinase clearly demonstrate that p38 MAP kinase is essential for oxalate-induced re-initiation of the DNA synthesis.

DISCUSSION
Oxalate exposure to renal epithelial cells has been shown to result in cell growth as well as apoptosis. The present studies were aimed at identification of specific intracellular signaling pathways that are targeted by oxalate. Various stimuli that control cell growth and apoptosis use kinase cascades to transmit signals to the nucleus. Activation of MAP kinase cascades is believed to be critical for the response of the cells to the growth factors as well as environmental stress. Our findings demonstrate that oxalate stimulates selective phosphorylation and enzymatic activation of p38 MAP kinase. Oxalate exposure also caused a slight activation of JNK. In contrast, p42/p44 MAP kinase/ERK was not affected by oxalate exposure. These findings suggest involvement of these protein kinases in cellular actions and response to oxalate.
Diverse extracellular stimuli including osmotic stress, UV light, heat shock, ionizing radiation, high osmotic stress, shear stress, proinflammatory cytokines, EGF, and hemopoietic growth factors with the exception of interleukin-4 have been shown to trigger the p38 MAP kinase pathway through phosphorylation on a TGY motif within the kinase activation loop (26). The majority, but not all, of these stimuli are associated with cellular stress. p38 MAP kinase appears to play a major role in apoptosis, cytokine production, transcriptional regula-tion, and cytoskeletal reorganization and has been implicated in sepsis, ischemic heart disease, arthritis, human immunodeficiency virus infection, and Alzheimer's disease (23,27,32,47). Activation of the p38 pathway results in a plethora of changes in transcription, protein synthesis, cell surface receptor expression, and cytoskeletal structure, ultimately affecting cell survival or leading to programmed cell death (apoptosis) (47). In addition, the p38 MAP kinase pathway has been suggested to play important roles in embryonic development and organogenesis (48,49). Upon exposure to oxalate, renal cells have been shown to display a program of events, including early growthresponsive gene expression, re-initiation of the DNA synthesis, and apoptosis (10 -14), consistent with cellular stress. Our results demonstrate that oxalate exposure to renal epithelial cells stimulates tyrosine phosphorylation and enzymatic activation of p38 MAP kinase (Fig. 1). The observed correlation between phosphorylation and activation of p38 MAP kinase (Fig. 1, A and D) is consistent with the evidence that phosphorylation of tyrosine at TGY activation motif is required for enzymatic activity (50,51). The activation of p38 MAP kinase by oxalate in LLC-PK1 was rapid and robust. Our results also demonstrate that oxalate exposure resulted in rapid activation of MAPKAP kinase-2 (Fig. 2). MAPKAP kinase-2 is known to be an in vivo substrate for p38 MAP kinase and that p38 MAP kinase is the only known activator of MAPKAP kinase-2. Thus these data demonstrate in situ activation of p38 MAP kinase by oxalate. The activation of p38 MAP kinase cascade is suggestive of the functional role of this kinase cascade in mediating cellular actions of oxalate.
The major stress-activated signaling pathway acts through the JNK family of protein kinases (20,21). Like p38 MAP kinase, the JNK family is activated by a number of cellular stresses but is distinctive in its ability to phosphorylate transcription factor c-Jun (23,24). We found that oxalate caused only a modest activation of JNK in LLC-PK1 compared with the robust activation induced by UV exposure (Fig. 3). Previous studies (52) have shown that JNK is activated by osmotic stress and during ischemia/reperfusion of the kidney. Recent reports (53)(54)(55) suggest involvement of JNK in apoptotic signals. It has also been shown that ERK activation inhibits apoptosis, whereas JNK mediates apoptosis induced by cytokine (53). Oxalate exposure results in re-initiation of the DNA synthesis, cell growth, as well as cell death by apoptosis (10 -14). These findings are consistent with the existence of apoptotic signals during such stress. As renal cells are exposed to oxalate, the existence of an un-opposed apoptotic signal would obviously lead to cell death of all the cells immediately following oxalate exposure. Thus regulation of apoptotic pathway would ensure that all cells would not die in response to oxalate toxicity. It has been proposed that JNK activation mediates apoptotic signals, whereas ERK pathway mediates growth signals and opposes the apoptotic signals that are induced during osmotic stress. Our studies presented here demonstrate that only p38 MAP kinase and JNK are induced following oxalate exposure, and ERK is not activated at all. We also show that p38 MAP kinase activation is essential for re-initiation of the DNA synthesis following oxalate exposure. Whether JNK mediates these control Ϯ S.D. and represent three separate independent experiments (*, p Ͻ 0.01, and **, p Ͻ 0.001, compared with untreated control). G, confluent, growth-arrested, and serum-starved LLC-PK1 cells were solubilized in lysis buffer after exposing to oxalate (1 mM) and EGF (50 ng/ml) as indicated. The activity of ERK2 MAP kinase was measured in immunocomplex protein kinase assay using [␥-32 P]ATP and MBP as substrate. The cell lysates (0.5 mg of protein) were subjected to immunoprecipitation with 0.5 g of polyclonal anti-ERK2 antibody and protein A-agarose and tested for its ability to phosphorylate MBP. MBP (10 g/sample) and [␥- 32  apoptotic signals remains to be determined.
Oxalate exposure did not cause activation of p42/p44 MAP kinase in LLC-PK1 cells. Our results demonstrate that oxalate had no direct effect on p42/44 MAP kinase phosphorylation or p42/44 MAP kinase activity (Fig. 4, A, D, G, and H), whereas EGF increased the amount of the phosphorylated p42/44 MAP kinase with maximum stimulation within 15 min (Fig. 4, B and  E). Moreover, addition of oxalate to EGF-treated cells did not stimulate or inhibit p42/44 MAP kinase activity (Fig. 4, C and  F). These data demonstrate that p42/44 MAP kinase is not the target of activation following oxalate exposure. Interestingly, p42/44 MAP kinases are activated by mitogens, and a common view is that they are essentially shared elements in mitogenic signaling. However, there are several instances where DNA synthesis has been shown to be independent of p42/44 MAP kinase activation (56 -58). It is worthwhile to mention here that the previous study (10) from our laboratory has shown that the effects of oxalate and fetal bovine serum on the DNA synthesis were additive, suggesting involvement of independent signaling pathways in oxalate and fetal bovine serumstimulated DNA synthesis. These observations were further confirmed by evaluating the effects of various growth factors alone or in combination with oxalate on the re-initiation of DNA synthesis (73). The fact that oxalate induces re-initiation of the DNA synthesis without activating p42/44 MAP kinase suggests that oxalate-induced re-initiation of the DNA synthesis in renal epithelial cells follows signal transduction pathways that are distinct from serum-and growth factor-stimulated DNA synthesis.
Several isoforms of p38 MAP kinase family of protein kinases have been identified (59). p38 and p38␤ are expressed in many tissues including the kidneys (43). These two isoforms are equally inhibited by pyridinyl imidazole compounds and display near identical response to tumor necrosis factor, phorbol serum-starved LLC-PK1 cells pre-exposed to DMEM alone or various concentrations of SB203580 (0.5, 1, 10, 20, 50, and 100 M) for 1 h prior to the addition of oxalate (1 mM) for 24 h. During the last 6 h of oxalate exposure [ 3 H]thymidine (2-3 Ci) was added per well. The radioactivity retained in the trichloroacetic acid precipitate was measured and used as an index of DNA synthesis. Data shown are representative of those obtained in three separate experiments. # (p Ͻ 0.001) indicates significant difference from control, and * (p Ͻ 0.001) indicates significant difference from oxalate treatment. B, transfected cells either with control vector (pCMV tag 5) or with dominant negative p38 expression vector pCMV-p38 (AGF) were exposed to oxalate (1 mM) for 24 h. During last 6 h of oxalate exposure [ 3 H]thymidine (2-3 Ci) was added per well. The radioactivity retained in the trichloroacetic acid precipitate was measured and used as an index of DNA synthesis. Data shown are representative of those obtained in three separate experiments. * (p Ͻ 0.001) indicates significant difference from control vector. C, inhibition of p38 kinase in situ by SB203580 as determined by examination of MAPKAP kinase-2 activity. Confluent, growth-arrested, and serumstarved LLC-PK1 cells pre-exposed to various concentrations of SB203580 (0.5, 1, 5, 10, 20, and 30 M) for 1 h prior to addition of oxalate (1 mM). The cells were lysed in ice-cold non-denaturing lysis buffer A, and cell lysates (0.5 mg of protein) were immunoprecipitated with anti-MAPKAP kinase-2 polyclonal antibody coupled to protein A-agarose beads and tested for its ability to phosphorylate a specific MAPKAP kinase-2 substrate peptide (KKLNRTLSVA) as described in Fig. 2. The data shown were obtained in one experiment, with similar results obtained in a separate experiment. D, LLC-PK1 cells were transiently transfected either with control vector (pCMV tag 5) or with dominant negative p38 expression vector pCMV-p38 (AGF). Transfected cells were exposed to 1 mM oxalate for 30 min, and in situ MAPKAP kinase-2 activity was analyzed as described under "Experimental Procedures." Briefly, cells were lysed in ice-cold non-denaturing lysis buffer A, and cell lysates (0.5 mg of protein) were immunoprecipitated with anti-MAPKAP kinase-2 polyclonal antibody coupled to protein A-agarose beads and tested for its ability to phosphorylate a specific MAPKAP kinase-2 substrate peptide (KKLNRTLSVA). Values represent mean Ϯ S.D. * (p Ͻ 0.001) indicates significant difference from control vector. 12-myristate 13-acetate, UV irradiation, H 2 O 2 , osmotic stress, and arsenate (60). It has therefore been suggested that of the known isoforms of p38 MAP kinases, p38 and p38␤ are the only p38 MAP kinases relevant to the study of kidney cells (45). Although both p38 and p38␤ are activated equally by stimuli, such as cytokines and environmental stresses, they differ in their upstream activators. p38 MAP kinase is activated in parallel by MKK3, MKK4, and MKK6, whereas p38␤ is activated predominantly by MKK6. Irrespective of the upstreamactivating pathway, SB203580 inhibits both p38 and p38␤. Our studies demonstrate that SB203580 inhibited oxalate-stimulated MAPKAP kinase-2 activity in LLC-PK1 cells in a concentration-dependent manner with complete inhibition at 20 M, indicating total inhibition of oxalate-stimulated p38 MAP kinase activation (Fig. 5C). Pretreatment of cells with SB203580 was also able to inhibit oxalate-stimulated DNA synthesis, indicating the requirement of p38 MAP kinase signal transduction in this process (Fig. 5A). Additionally, our studies demonstrate that overexpression of dominant negative p38 MAP kinase by transfecting the cells with kinase-dead dominant negative expression vector pCMV-p38 (AGF) (50) resulted in attenuation of oxalate-induced MAPKAP kinase-2 activity (Fig.  5D) as well as oxalate-induced re-initiation of the DNA synthesis (Fig. 5B), thus clearly establishing involvement of p38 MAP kinase signal transduction pathway in oxalate-induced DNA synthesis. In Schizosaccharomyces pombe the p38 MAP kinase homologue Spc1 is needed for cell cycle progression under stressful conditions and overexpression of Pyp-1, a tyrosine phosphatase that inactivates Spc1 resulting in slowing of growth (61). p38 MAP kinase has also been shown to play a critical role in DNA synthesis in response to hemopoietic growth factors with the exception of interleukin-4 (33). Similarly in fibroblasts and cultured mesangial cells, hypoxia-associated DNA synthesis but not serum-stimulated DNA synthesis has been shown to be dependent on the activation of p38 MAP kinase pathway (56,62,63). Taken together these data suggest that p38 MAP kinase activation may play a central role in DNA synthesis associated with cellular stress.
The mechanisms by which p38 MAP kinase regulates oxalate-stimulated DNA synthesis are not understood. It is possible that p38 MAP kinase may regulate stability or transcription of early growth-responsive genes. p38 MAP kinase is known to phosphorylate and activate a number of transcription factors. In vivo activation of p38 increases the transcriptional activity of Elk-1 and ATF2 (60,64). p38 MAP kinase is known to phosphorylate and stimulate a number of transcription factors including cAMP-response element-binding protein and ATF1 through MAPKAP kinase-2 (65,66). These transcription factors control the expression of various genes (67), which have been demonstrated to play a key role in cell growth and cellular homeostasis. These observations suggest that the effects of p38 MAP kinase may be complex. Nevertheless, these studies demonstrate that oxalate-stimulated DNA synthesis depends on the p38 MAP kinase signal transduction pathway. Further studies are required to identify the mechanism by which p38 MAP kinase plays a central role in oxalate-stimulated DNA synthesis.
Several studies have implicated p38 MAP kinase in the induction of apoptosis (26). Withdrawal of nerve growth factor from PC-12 cells has been shown to stimulate apoptosis in p38 MAP kinase (68). Similarly, p38 MAP kinase-dependent apoptosis has been shown in transforming growth factor-␤1-induced apoptosis in murine hepatocytes and in cytokine-induced rat islet cell apoptosis (69,70). Whether or not p38 MAP kinase plays any role in renal epithelial cell apoptosis following oxa-late exposure has not been evaluated in this study and needs further study.
The exact profile and relative activation of each of the three MAP kinase subtypes is characteristic for the stimuli used. For instance, 12-O-tetradecanoylphorbol-13-acetate exclusively activates single MAP kinase subtype, the ERKs, whereas anisomycin and okadaic acid strongly activate JNK/SAPK and p38 MAP kinase but do not activate the ERK group of MAP kinases, and finally EFG and UV irradiation activate all three cascades, but to different extents. EGF activates ERKs robustly but JNK and p38 MAP kinase weakly, and UV irradiation activates JNK and p38 MAP kinase robustly but ERKs weakly (26,71). Based on these observations, it has been suggested that stimuli that activate MAP kinases could be divided into three broad categories. The first category involves those stimuli that activate only ERKs exclusively; the second category includes those stimuli that activate JNK and p38 MAP kinase but not ERKs, and the third category of stimuli involves those stimuli that activate all the three cascades. Recently, however, it has been shown that in PC12 cells hypoxia-activated p38 MAP kinase robustly and ERKs weakly (72). Thus a fourth class of stimuli is emerging, stimuli those activate p38 MAP kinase and ERK but not JNK. Our results demonstrate that oxalate selectively stimulates p38 MAP kinase rapidly and robustly and JNK mildly. The differential effects of oxalate on selective activation of p38 MAP kinase and mild activation of JNK contribute to a growing number of stimuli that selectively activate p38 MAP kinase and JNK but not ERKs. These stimuli include exposure to okadaic acid and anisomycin. Oxalate holds a distinction in this class in being the only known metabolic end product that selectively causes rapid and robust activation of p38 MAP kinase and a weak stimulation of JNK.
Taken together these studies demonstrate that oxalate, an end product of metabolism, selectively, rapidly, and robustly activates p38 MAP kinase, causes a mild activation of JNK/ SAPK, and does not activate an 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. This is the first demonstration of activation of MAP kinase family of protein kinases by oxalate.