COM Crystals Activate the p38 Mitogen-activated Protein Kinase Signal Transduction Pathway in Renal Epithelial Cells
2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês
10.1074/jbc.m200832200
ISSN1083-351X
AutoresHari K. Koul, Mani Menon, Lakshmi S. Chaturvedi, Sweaty Koul, Avtar Sekhon, Akshay Bhandari, Meiyi Huang,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoInteraction of calcium oxalate monohydrate (COM) crystals with renal cells has been shown to result in altered gene expression, DNA synthesis, and cell death. In the current study the role of a stress-specific p38 MAP kinase-signaling pathway in mediating these effects of COM crystals was investigated. Exposure of cells to COM crystals (20 μg/cm2) rapidly stimulated strong phosphorylation and activation of p38 mitogen-activated protein kinase (p38 MAP kinase) and re-initiation of DNA synthesis. Inhibition of COM crystal binding to the cells by heparin blocked the effects of COM crystals on p38 MAPK activation. 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 dominant negative mutant of p38 MAP kinase abolishes COM crystal-induced re-initiation of DNA synthesis. The inhibition is dose-dependent and correlates with in situ activity of native p38 MAP kinase, determined as mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) activity in cell extracts. In summary, inhibiting activation of p38 MAPK pathway abrogated the DNA synthesis in response to COM crystals. These data are the first demonstrations of activation of the p38 MAPK signaling pathway by COM crystals and suggest that, in response to COM crystals, this pathway transduces critical signals governing the re-initiation of DNA synthesis in renal epithelial cells. Interaction of calcium oxalate monohydrate (COM) crystals with renal cells has been shown to result in altered gene expression, DNA synthesis, and cell death. In the current study the role of a stress-specific p38 MAP kinase-signaling pathway in mediating these effects of COM crystals was investigated. Exposure of cells to COM crystals (20 μg/cm2) rapidly stimulated strong phosphorylation and activation of p38 mitogen-activated protein kinase (p38 MAP kinase) and re-initiation of DNA synthesis. Inhibition of COM crystal binding to the cells by heparin blocked the effects of COM crystals on p38 MAPK activation. 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 dominant negative mutant of p38 MAP kinase abolishes COM crystal-induced re-initiation of DNA synthesis. The inhibition is dose-dependent and correlates with in situ activity of native p38 MAP kinase, determined as mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) activity in cell extracts. In summary, inhibiting activation of p38 MAPK pathway abrogated the DNA synthesis in response to COM crystals. These data are the first demonstrations of activation of the p38 MAPK signaling pathway by COM crystals and suggest that, in response to COM crystals, this pathway transduces critical signals governing the re-initiation of DNA synthesis in renal epithelial cells. calcium oxalate monohydrate stress-activated protein kinase mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun N-terminal kinases mitogen-activated protein kinase-activated protein myelin basic protein activating transcription factor myocyte enhancer factor 2C C/EBP homologous protein 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole phosphate-buffered saline Dulbecco-Vogt modified Eagle's medium phenylmethylsulfonyl fluoride mitogen-activated protein Renal tubular fluid is commonly supersaturated with calcium and oxalate ions, which nucleate to form crystals of calcium oxalate monohydrate (COM),1 the most common constituent of kidney stones. The majority of people do not form renal stones despite crystalluria (1Menon M. Koul H. J. Clin. Endocrinol. Metab. 1992; 74: 703-707Crossref PubMed Scopus (33) Google Scholar). Up to 1.1 × 107 crystals are excreted daily by normal individuals without any evidence of stone disease (2Robertson W.G. Peacock M. Clin. Sci. (Lond.). 1972; 43: 499-506Crossref PubMed Scopus (167) Google Scholar). It is therefore believed that uncomplicated crystalluria does not indicate kidney stone disease. The mechanisms by which urinary crystals are retained in the kidney and grow into kidney stones are not known. Finlayson (3Finlayson B. Urol. Clin. North Am. 1974; 1: 181-212PubMed Google Scholar) calculated that, given the most favorable conditions, it would take 10 h for 1-μm COM crystals to grow large enough to block the duct of Bellini and become the nidus of a urinary stone. Because urinary transit time from glomerulus to the renal pelvis is ∼3 min, crystalline particles formed in the urine flowing freely through the renal tubule do not stay in the lumen long enough to attain the dimensions required to block a collecting duct and form a urinary microlith. Therefore, the attachment of crystals to the renal epithelial cells and the cellular responses to crystal interaction are critical in understanding the pathogenesis of renal calcification. Previous studies (4Koul H.K. Koul S. Fu S. Santosham V. Seikhon A. Menon M. J. Am. Soc. Nephrol. 1999; 14: S417-S421Google Scholar, 5Lieske J.C. Toback F.G. Am. J. Physiol. 1993; 264: F800-F807PubMed Google Scholar, 6Riese R.J. Riese J.W. Kleinman J.G. Wiessner J.H. Mandel G.S. Mandel N.S. Am. J. Physiol. 1988; 255: F1025-F1032PubMed Google Scholar) in several different cell lines of renal tubular origin suggest that COM crystals bind to specific receptors on the cell surface. Furthermore, the receptors that interact with COM crystals in tubular cells may be only minimally exposed under normal circumstances and increase in number under a variety of conditions that lead to cellular stress and injury (4Koul H.K. Koul S. Fu S. Santosham V. Seikhon A. Menon M. J. Am. Soc. Nephrol. 1999; 14: S417-S421Google Scholar, 7Thamilselvan S. Khan S.R. J. Nephrol. 1998; 11 suppl. 1: 66-69PubMed Google Scholar, 8Verkoelen C.F. van der Boom B.G. Houtsmuller A.B. Schroder F.H. Romijn J.C. Am. J. Physiol. 1998; 274: F958-F965Crossref PubMed Google Scholar). Nonetheless, COM crystal interaction with renal epithelial cells results in a program of events, including alterations in gene expression, initiation of DNA synthesis, cell growth, and death (9Hammes M.S. Lieske J.C. Pawar S. Spargo B.H. Toback F.G. Kidney Int. 1995; 48: 501-509Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 10Khan S.R. Urol. 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These regulatory cascades not only convey information to the target effectors but also coordinate incoming information from parallel signaling pathways, which allow for signal amplification, generation of a threshold, and a sigmoidal activation profile and is subject to multiple activation mechanisms (20Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (765) Google Scholar). These pathways include the extracellular signal-regulated kinase (ERK, also known as p42/44 MAP kinase), the c-Jun N-terminal kinase (JNK, also known as SAPK1), and p38 mitogen-activated protein kinase (p38 MAP kinase, also known as SAPK2/reactivating kinase). In general, ERK1 and ERK2 are key transducers of proliferation signals and are often activated by mitogens. In contrast, SAPKs/JNKs and p38 are poorly activated by mitogens but strongly activated by cellular stress inducers (21Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (1007) Google Scholar). The family of p38 MAP kinases is activated in response to diverse extracellular stimuli including osmotic stress, UV irradiation, heat shock, ionizing radiation, high osmotic stress, shear stress, proinflammatory cytokines, thrombin, epidermal growth factor, and hemopoietic growth factors with the exception of interleukin-4 (21Cano E. Mahadevan L.C. Trends Biochem. Sci. 1995; 20: 117-122Abstract Full Text PDF PubMed Scopus (1007) Google Scholar). The majority, but not all, of these stimuli are associated with cellular stress. The p38 MAP kinase pathway has been shown to mediate signals for the generation of important biological responses, such as phosphorylation of transcription factors involved in transcriptional regulation, platelet aggregation, induction of cytokine production, and apoptosis in neuronal cells and fibroblasts (22Kramer R.M. Roberts E.F. Strifler B.A. Johnstone E.M. J. Biol. 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In this study, we investigated the effect of COM crystals on LLC-PK1 cells, a line of renal epithelial cells. We used SB203580, a specific inhibitor of p38 MAP kinase. The kinase-dead dominant negative expression vector pCMV-p38 (AGF), which functions as a dominant inhibitor of p38 MAPK activation (27Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2061) Google Scholar), was also used to determine the role of p38 MAP kinase pathway in mediating the cellular response to COM crystals. We provide the first evidence that p38 MAP kinase is rapidly and robustly phosphorylated and activated in response to COM crystal interaction with renal epithelial cells. In addition, we demonstrate that p38 MAP kinase activity is essential for the effects of COM crystals on re-initiation of DNA synthesis. Dulbecco-Vogt modified Eagle's medium (DMEM), fetal bovine serum, penicillin/streptomycin, and myelin basic protein (MBP) were purchased from Invitrogen. Antibodies against phosphokinase and total p38 MAP kinase was obtained from New England Biolabs (Beverly, MA). 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, M2 monoclonal antibody, leupeptin, aprotinin, phenylmethylsulfonyl fluoride, aphidicolin, and 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole (SB203580) were purchased from Sigma. Anisomycin was purchased fromCalbiochem-Novabiochem. [γ-32P]ATP (4500 Ci/mmol) and [methyl-3H]thymidine (83 Ci/mmol) were obtained from ICN Radiochemicals, Inc (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 Roger J. Davis (Howard Hughes Medical Institute, University of Massachusetts, Worcester, MA) (27Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2061) Google Scholar). All chemicals used were analytical grade and were obtained from Sigma. Calcium oxalate monohydrate (COM) crystals were prepared as described previously (28Wiessner J.H. Hasegawa A.T. Hung L.Y. Mandel G.S. Mandel N.S. Kidney Int. 2001; 59: 637-644Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) with slight modifications. Briefly, 5 ml each of 10 mmCaCl2 and 10 mm sodium oxalate were mixed simultaneously. Upon mixing the two solutions at room temperature, COM crystals formed immediately. The suspension was allowed to equilibrate for 3 days at 4 °C, and COM crystals were centrifuged at 1000 × g for 10 min. (For the preparation of [14C]COM crystals, 100 μCi of [14C]oxalate was added to 5 ml of 10 mmsodium oxalate solution prior to mixing with CaCl2solution). The supernatant was decanted, and the resulting pellet was washed six times with water. The pellet was then dried overnight at 60 °C under vacuum. Crystal morphology was examined by light microscopy and scanning electron microscopy and found to be primarily spherulitic, about 1–2 μm in diameter. A stock solution of 5 mg/ml was prepared in sterile PBS. LLC-PK1 cells (American Type Culture Collection, Manassas, VA), grown on polystyrene (Corning Glass) T-75 flasks and were used between passages 216 and 240. The cells were serially passaged in low glucose 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 appropriate control vector (pCMV Tag 5) or kinase-dead dominant negative expression vector pCMV-p38 (AGF) using Lipo TAXI transfection reagent (Stratagene), following the manufacturer's instructions. The transfection efficiency was 50–65% as determined by β-galactosidase expression in parallel experiments. For specific treatments, transfected cells were allowed to grow to confluence and serum-starved overnight prior to use in experiments. All cultures were maintained at 37 °C in a humidified atmosphere of 95% air, 5% CO2. [14C]COM crystal binding was carried out as described previously (4Koul H.K. Koul S. Fu S. Santosham V. Seikhon A. Menon M. J. Am. Soc. Nephrol. 1999; 14: S417-S421Google 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 prior to COM crystal binding. The cells were exposed to COM crystals (20 μg/cm2) in the presence or absence of heparin (a selective inhibitor of COM crystal binding to the cells) for 4 min at 37 °C. At end of the experimental period, media were aspirated and the cells washed twice with ice-cold PBS (2 ml/well/wash). The cells were lysed by the addition of 500 μl of lysis solution (0.1n NaOH, 1% SDS), and the amount of 14C label associated with the cells was used as an index of the amount of COM crystal binding. [3H]Thymidine incorporation was used as an index of cell proliferation and was carried out as described previously (29Koul H. Kennington 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 exposed to various doses of COM crystals (2–100 μg/cm2; 10–500 μg/ml). During the last 6 h of exposure 2–3 μCi of [3H]thymidine was added per well. At the end of the experimental period, the cells were washed with two changes of ice-cold phosphate-buffered saline and precipitated with 10% trichloroacetic acid. The trichloroacetic acid-insoluble material was digested with 0.1n NaOH and 1% SDS. Radioactivity was counted in a Beckman liquid scintillation counter (LS 6500). Cells were washed with ice-cold PBS and solubilized with ice-cold 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 the insoluble material. Samples containing equal amounts of protein (50 μg) were separated on 10% SDS-PAGE and then transferred to an Immobilon-P membrane using standard electroblotting procedures. Western analysis was carried out as described previously (30Koul S. Chaturvedi L.S. Sekhon A. Bhandari A. Menon M. Koul H. Kidney Int. 2002; 61: 525-533Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31Chaturvedi L. Koul S. Sekhon A. Bhandari A. Menon M. Koul H. J. Biol. Chem. 2002; 277: 13321-13330Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Briefly, blots were immunolabeled overnight at 4 °C with monoclonal antibodies that specifically recognize 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 (10 mm Tris, pH 7.2, 140 mm NaCl, 0.1% Tween 20) at room temperature and then incubated with anti-mouse or anti-rabbit IgG linked to horseradish peroxidase (Eastman Kodak Co.). Immunoreactivity was detected with enhanced chemiluminescence detection system (Kodak). For these assays, the cells were 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 p38 MAP kinase was achieved by adding 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 mm HEPES, 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 20 μCi of [γ-32P]ATP and 10 μg of MBP 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 the supernatant was suspended in 2× Laemmli SDS sample buffer containing β-mercaptoethanol and bromphenol blue. Samples were boiled for 2 min and run on 15% SDS-polyacrylamide gel. Kinase activity was measured as the amount of 32P incorporation into MPB substrate. These enzyme assays were carried out as described previously (31Chaturvedi L. Koul S. Sekhon A. Bhandari A. Menon M. Koul H. J. Biol. Chem. 2002; 277: 13321-13330Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Briefly, cells were harvested by scraping in 0.5 ml of 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% β-mercaptoethanol, 1% Triton X-100, 5 mm sodium pyrophosphate, 10 mm sodium glycerophosphate, 10 mm NaF, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin. Lysates were incubated 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. The recombinant 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 mm MOPS, pH 7.2, 25 mmβ-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, described in manufacturer's recommended protocol (Upstate Biotechnology, Inc.). The enzyme activity was expressed in pmol/min. As can be seen from Fig. 1 A, exposure to COM crystals 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.1 B, COM crystal exposure did not alter the total amount of p38 MAP kinase protein. The maximum activation of p38 was reached at 15 min of COM crystal exposure (4–6-fold over control) with an average 3.7-fold increase in p38 MAP kinase phospho-immunoreactivity (Fig.1 C). The COM crystal exposure-induced increase in phospho-p38 MAP kinase immunoreactivity was comparable with anisomycin, a known activator of p38 MAP kinase. These results suggest that COM crystals activate p38 MAP kinase. To characterize further the effects of COM crystals on p38 MAP kinase enzyme activity, p38 MAP kinase was 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." COM crystals rapidly stimulated p38 MAP kinase activity within 5 min of exposure as shown in Fig. 1 D. Densitometric analysis in Fig. 1 E showed that the effect of COM crystals on p38 MAP kinase was maximal at 15 min (∼9-fold activation over control), whereas anisomycin exposure resulted in about a 5-fold increase in p38 MAP activity as compared with untreated control. These data demonstrate that COM crystal exposure resulted in a rapid and robust increase in p38 MAP kinase activity. Some concerns have been raised that in vitro activity of p38 MAP kinase may not reflect its in situ activity. Thus in situactivity of p38 MAP kinase was determined by MAPKAP kinase-2 enzyme 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 (32Krump 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 (161) Google Scholar). 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 COM crystal-induced MAPKAP kinase-2 activity followed the activation pattern similar to that of p38 MAP kinase, with maximal activation 15 min following COM crystal exposure. These data demonstrate that the rapid and robust activation of p38 MAP kinase by COM crystals correlates with the in situ activity of MAPKAP kinase-2. Heparin has been shown to be the most effective natural polysaccharide inhibitor of COM crystal binding to renal epithelial cells (13Verkoelen C.F. Romijn J.C. Cao L.C. Boeve E.R. de Bruijn W.C. Schroder F.H. J. Urol. 1996; 155: 749-752Crossref PubMed Scopus (58) Google Scholar). We evaluated the effect of heparin on COM crystal binding to LLC-PK1 cells and on COM crystal-stimulated p38 MAP kinase activation. As shown in Fig. 3 A, exposure of LLC-PK1 cells to heparin (50 and 100 μg/ml) inhibited COM crystal binding to the LLC-PK1 cells (by 65.9 ± 3.5 and 75.8 ± 1.2, respectively). Data presented in Fig. 3 C show that inhibition of COM crystal binding by heparin (50 and 100 μg/ml) had no effect on total p38 MAP kinase protein levels, but inhibited COM crystal-stimulated p38 MAP kinase phosphorylation (Fig. 3 B) and p38 MAP kinase activation (Fig. 3 D). These data suggest that COM crystal-stimulated p38 MAP kinase activation requires COM crystal binding to the renal epithelial cells. Exposure of renal epithelial cells to COM crystals has been shown to result in the initiation of DNA synthesis (12Lieske J.C. Walsh-Reitz M.M. Toback F.G. Am. J. Physiol. 1992; 262: F622-F630PubMed Google Scholar). We evaluated the effect of varying COM crystal concentrations on re-initiation of the DNA synthesis in LLC-PK1 cells. As shown in Fig.4 A, exposure of LLC-PK1 cells to COM crystals resulted in re-initiation of DNA synthesis. COM crystals induced DNA synthesis in a dose-dependent fashion in the range tested (from 2–100 μg/cm2 to 10–500 μg/ml). We observed significant stimulation (∼2-fold) at 20 μg/cm2 to 100 μg/ml COM crystal exposure (p < 0.01). Hence, 20 μg/cm2 COM crystal was used in all additional studies. Fig. 4 B shows that COM crystal-induced DNA synthesis was completely inhibited by aphidicolin (5 μg/ml), a specific inhibitor of DNA polymerase α. These data demonstrate the requirement of DNA polymerase α activity for COM crystal-induced re-initiation of the DNA synthesis. For these studies, confluent growth-arrested LLC-PK1 cells were exposed to COM crystals (20 μg/cm2) for various time points (0 min to 24 h) in the absence or presence of increasing concentrations of SB203580. The DNA synthesis was measured as described under "Experimental Procedures." Exposure to COM crystals resulted in a 2-fold increase of DNA synthesis above that of untreated (control) cells. Pre-treatment of cells with SB203580 attenuated the COM crystal-induced DNA synthesis in a dose-dependent manner with complete inhibition at 20 μm (Fig.5 A). (Please note that at concentrations above 50 μm, SB203580 was toxic to these cells and inhibition of the DNA synthesis above that of control may reflect that fact.) These data suggest involvement of p38 MAP kinase pathway in COM crystals-induced re-initiation of the DNA synthesis. It has been shown that the inhibitory effects of imidazole compounds on p38 MAP kinase are reversible (33Young P.R. McLaughlin M.M. Kumar S. Kassis S. Doyle M.L. McNulty D. Gallagher T.F. Fisher S. McDonnell P.C. Carr S.A. Huddleston M.J. Seibel G. Porter T.G. Livi G.P. Adams J.L. Lee J.C. J. Biol. Chem. 1997; 272: 12116-12121Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). This raises the possibility thatin 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. As shown in Fig. 5 B, SB203580 inhibited COM crystal-induced MAPKAP kinase-2 activity in a dose-dependent manner. The inhibitory effect of SB203580 on COM crystal-activated MAPKAP kinase-2 shows complete inhibition at 20 μm. Thus the inhibitory effects of SB203580 on COM crystal-induced DNA synthesis correlates with inhibitory effects of SB203580 on COM crystal-activated MAPKAP kinase-2 in dose-dependent fashion. These studies indicate that p38 MAP kinase is essential for COM crystal-induced re-initiation of the DNA synthesis. The role of p38 MAP kinase pathway in COM crystal-stimulated DNA synthesis was further confirmed by overexpression of kinase-dead dominant negative mutant of p38 MAP kinase. In these studies, we first evaluated the effects of transfection of cells with kinase-dead dominant negative expression vector pCMV-p38 (AGF) on COM crystal-stimulated activity of p38 MAP kinase. The expression of the kinase-dead mutant was confirmed by Western blot analysis using anti-M2 monoclonal antibody. As shown in Fig.6 A, only cells transfected with dominant negative expression vector pCMV-p38 (AGF), but not control cells or cells transfected with vector (pCMV Tag 5), showed immunoreactivity to M2 monoclonal antibody. As shown in Fig. 6,B and C, transfection of cells with kinase-dead dominant negative expression vector pCMV-p38 (AGF), but not with control vector (pCMV Tag 5), greatly attenuated the effects of COM crystals on p38 MAP kinase activity (COM + pCMV-p38 (AGF)versus pCMV-p38 (AGF), 188.6 ± 19.3 versus69.1 ± 14.4; COM + control (pCMV Tag 5) vector versuscontrol (pCMV Tag 5) vector, 448.6 ± 16.5 versus100 ± 6.6). 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, measured as in vitroMAPKAP
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