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• W2041081365 abstract "The potential role of p38 mitogen-activated protein (MAP) kinase in platelet-derived growth factor receptor-α (PDGF-Rα) gene expression was investigated using cultured rat pulmonary myofibroblasts. p38 MAP kinase was constitutively expressed in myofibroblasts and activated by interleukin (IL)-1β. A pyridinylimidazole compound, SB203580, completely inhibited the ability of p38 MAP kinase activity to phosphorylate PHAS-1 substrate. SB203580 inhibited IL-1β-induced up-regulation of PDGF-Rα mRNA and protein in a concentration-dependent manner. Other kinase inhibitors, including the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor PD98059, did not block up-regulation of PDGF-Rα. The IL-1β-induced increase in the number of 125I-PDGF-AA-binding sites at the cell surface was reduced >70% by pretreatment with SB203580. Accordingly, an enhancement of PDGF-AA-stimulated DNA synthesis following IL-1β pretreatment was blocked >70% by SB203580. SB203580 did not affect IL-1β-induced ERK activation, yet enhanced IL-1β-induced JNK activation approximately 2-fold. Treatment of cells with SB203580 after inhibition of transcription by actinomycin D decreased the half-life of IL-1β-induced PDGF-Rα mRNA from >4 to ∼1.5 h. Moreover, pretreatment of cells with cycloheximide blocked induction of PDGF-Rα mRNA by IL-1β, suggesting that de novo protein synthesis was required for PDGF-Rα mRNA stabilization. These data indicate that p38 MAP kinase regulates PDGF-Rα expression at the translational level by signaling the synthesis of an mRNA-stabilizing protein. The potential role of p38 mitogen-activated protein (MAP) kinase in platelet-derived growth factor receptor-α (PDGF-Rα) gene expression was investigated using cultured rat pulmonary myofibroblasts. p38 MAP kinase was constitutively expressed in myofibroblasts and activated by interleukin (IL)-1β. A pyridinylimidazole compound, SB203580, completely inhibited the ability of p38 MAP kinase activity to phosphorylate PHAS-1 substrate. SB203580 inhibited IL-1β-induced up-regulation of PDGF-Rα mRNA and protein in a concentration-dependent manner. Other kinase inhibitors, including the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor PD98059, did not block up-regulation of PDGF-Rα. The IL-1β-induced increase in the number of 125I-PDGF-AA-binding sites at the cell surface was reduced >70% by pretreatment with SB203580. Accordingly, an enhancement of PDGF-AA-stimulated DNA synthesis following IL-1β pretreatment was blocked >70% by SB203580. SB203580 did not affect IL-1β-induced ERK activation, yet enhanced IL-1β-induced JNK activation approximately 2-fold. Treatment of cells with SB203580 after inhibition of transcription by actinomycin D decreased the half-life of IL-1β-induced PDGF-Rα mRNA from >4 to ∼1.5 h. Moreover, pretreatment of cells with cycloheximide blocked induction of PDGF-Rα mRNA by IL-1β, suggesting that de novo protein synthesis was required for PDGF-Rα mRNA stabilization. These data indicate that p38 MAP kinase regulates PDGF-Rα expression at the translational level by signaling the synthesis of an mRNA-stabilizing protein. platelet-derived growth factor α-PDGF receptor β-PDGF receptor interleukin mitogen-activated protein extracellular signal-regulated kinase c-Jun NH2-terminal kinase MAP kinase kinase nuclear factor-κB activator protein-1 serum-free defined medium fetal bovine serum Dulbecco's modified Eagle's medium glyceraldehyde-3-phosphate dehydrogenase lipopolysaccharide tumor necrosis factor-α CAAT/enhancer-binding protein phosphorylatedheat- and acid-stable protein Platelet-derived growth factor (PDGF)1 is a potent mesenchymal cell mitogen and chemoattractant that exists as a disulfide-linked dimer of two polypeptide chains, A or B, that form functional PDGF-AA, PDGF-BB, or PDGF-AB isoforms (reviewed in Ref. 1.Heldin C-H. Westermark B. J. Cell Sci. 1990; 96: 193-196Crossref PubMed Google Scholar). Two PDGF receptor subtypes bind the three isoforms of PDGF differentially; β-PDGF receptor (PDGF-Rβ) can interact only with B-chain containing isoforms while α-PDGF receptor (PDGF-Rα) can bind all three isoforms (2.Seifert R.A. Hart C.E. Phillips P.E. Forstrom J.W. Ross R. Murray M.J. Bowen-Pope D.F. J. Biol. Chem. 1989; 264: 8771-8778Abstract Full Text PDF PubMed Google Scholar). PDGF binding results in receptor dimerization to form αα, αβ, or ββ combinations, followed by tyrosine kinase phosphorylation of the intracellular receptor domain and activation of a vast array of signal transduction molecules including Src family kinases, Grb2, Shc, phosphatidylinositol 3-kinase, GAP, Shb, PTP 1D, and phospholipase C-γ (reviewed in Ref. 3.Claesson-Welsh L. J. Biol. Chem. 1994; 269: 32023-32026Abstract Full Text PDF PubMed Google Scholar). The biologic activity of PDGF isoforms on rat pulmonary myofibroblasts is modulated in the extracellular microenvironment through interaction with its binding protein, α2-macroglobulin (4.Bonner J.C. Lindroos P.M. Hoffman M.R. Badgett A. J. Biol. Chem. 1995; 270: 6389-6395Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 5.Bonner J.C. Osornio-Vargas A.R. J. Biol. Chem. 1995; 270: 16236-16242Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), and by regulation of cell-surface PDGF-Rα (6.Lindroos P.M. Coin P.G. Osornio-Vargas A.R. Bonner J.C. Am. J. Respir. Cell Mol. Biol. 1995; 13: 455-465Crossref PubMed Scopus (50) Google Scholar, 7.Osornio-Vargas A.R. Lindroos P.M. Coin P.G. Badgett A. Hernandez-Rodriguez N.A. Bonner J.C. Am. J. Physiol. 1996; 271: L93-L99Crossref PubMed Google Scholar). The PDGF-Rα and its ligand, PDGF-AA, are essential to lung development (8.Bostrom H. Willetts K. Pekny M. Leveen P. Lindahl P. Hedstrand H. Pekna M. Hellstrom M. Gebre-Medhin S. Schalling M. Nilsson M. Kurland S. Tornell J. Heath J.K. Betsholtz C. Cell. 1996; 85: 863-873Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar), yet induction of the PDGF-Rα also occurs in adult tissues during the pathogenesis of certain fibroproliferative diseases. For example, human fibroblasts isolated from dermal keloids express elevated PDGF-Rα (9.Haisa M. Okochi H. Grotendorst G.R. J. Invest. Dermatol. 1994; 103: 560-563Abstract Full Text PDF PubMed Scopus (116) Google Scholar). We and others have reported that PDGF-Rα is up-regulated during the progression of pulmonary fibrosis in rats, while the PDGF-Rβ is constitutively expressed (10.Bonner J.C. Lindroos P.M. Rice A.B. Moomaw C.R. Morgan D.L. Am. J. Physiol. 1998; 274: L72-L80Crossref PubMed Google Scholar, 11.Lasky J.A. Tonthat B. Liu J.-Y. Friedman M. Brody A.R. Am. J. Respir. Crit. Care Med. 1998; 157: 1652-1657Crossref PubMed Scopus (44) Google Scholar). Interleukin (IL)-1β is a potent inducer of the PDGF-Rα on cultured myofibroblasts isolated from rat lung and PDGF-Rα up-regulation enhances the mitogenic and chemotactic responses to PDGF isoforms (6.Lindroos P.M. Coin P.G. Osornio-Vargas A.R. Bonner J.C. Am. J. Respir. Cell Mol. Biol. 1995; 13: 455-465Crossref PubMed Scopus (50) Google Scholar,12.Coin P.G. Lindroos P.M. Bird G.S.J. Osornio-Vargas A.R. Roggli V.L. Bonner J.C. J. Immunol. 1996; 156: 4797-4806PubMed Google Scholar). The maximal responses of connective tissue cells to PDGF isoforms require PDGF-Rα in addition to the normally abundant PDGF-Rβ (7.Osornio-Vargas A.R. Lindroos P.M. Coin P.G. Badgett A. Hernandez-Rodriguez N.A. Bonner J.C. Am. J. Physiol. 1996; 271: L93-L99Crossref PubMed Google Scholar,13.Seifert R.A. van Koppen A. Bowen-Pope D.F. J. Biol. Chem. 1993; 268: 4473-4480Abstract Full Text PDF PubMed Google Scholar), and this could be due to unique signal transduction events stimulated by α-β receptor dimerization, as compared with β-β receptor dimerization (14.Rupp E. Seigbahn A. Ronnstrand L. Wernstedt C. Claesson-Welsh L. Heldin C.-H. Eur. J. Biochem. 1994; 225: 29-41Crossref PubMed Scopus (47) Google Scholar). Other mediators, including transforming growth factor-β1 (15.Bonner J.C. Badgett A. Lindroos P.M. Osornio-Vargas A.R. Am. J. Respir. Cell Mol. Biol. 1995; 13: 496-505Crossref PubMed Scopus (58) Google Scholar) and prostaglandin E2 (16.Boyle J.E. Lindroos P.M. Rice A.B. Zhang L. Zeldin D.C. Bonner J.C. Am. J. Respir. Cell Mol. Biol. 1999; 20: 433-440Crossref PubMed Scopus (33) Google Scholar) suppress PDGF-Rα expression and counteract the up-regulatory effect of IL-1β. It is becoming increasingly clear that IL-1β signals the production of a variety of different mediators (e.g. cytokines, metalloproteinases, prostaglandin H synthase 2, nitric oxide, and inducible nitric-oxide synthase) via the activation of p38 mitogen-activated protein (MAP) kinases (17.Ridley S.H. Sarsfield S.J. Lee J.C. Bigg H.F. Cawston T.E. Taylor D.J. DeWitt D.L. Saklatvala J. J. Immunol. 1997; 158: 3165-3173PubMed Google Scholar, 18.Miyazawa K. Mori A. Miyata H. Akahane M. Ajisawa Y. Hirokazu O. J. Biol. Chem. 1998; 273: 24832-24838Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 19.Badger A.M. Cook M.N. Lark M.W. Newmann-Tarr T.M. Swift B.A. Nelson A.H. Barone F.C. Kumar S. J. Immunol. 1998; 161: 467-473PubMed Google Scholar, 20.Foey A.D. Parry S.L. Williams L.M. Feldmann M. Foxwell B.M.J. Brennan F.M. J. Immunol. 1998; 160: 920-928PubMed Google Scholar). p38 MAP kinase is activated upon stimulation of cells with cytokines, bacterial lipopolysaccharide, and stress (21.Raingeaud 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 (2038) Google Scholar, 22.Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2407) Google Scholar). Several transcription factors are substrates for p38 MAP kinase isozymes, including MAP-KAP kinase-2 (23.Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (774) Google Scholar), ATF-2 (24.Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1336) Google Scholar), CHOP/GADD153 (25.Wang X. Ron D. Science. 1996; 272: 1347-1349Crossref PubMed Scopus (741) Google Scholar), MAX (26.Zervos A.S. Faccio L. Gatto J.P. Kyriakis J.M. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10531-10534Crossref PubMed Scopus (138) Google Scholar), myocyte enhancer factor 2C (27.Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Nature. 1997; 386: 296-299Crossref PubMed Scopus (682) Google Scholar), and ternary complex factor (28.Price M.A. Cruzalegui F.H. Treisman R. EMBO J. 1996; 15: 6552-6563Crossref PubMed Scopus (301) Google Scholar). In addition to the original p38 (also termed p38α, cytokine-suppressive, anti-inflammatory drug-binding protein-2, or SAPK2A), the p38 subgroup of MAP kinases now consists of cytokine-suppressive, anti-inflammatory drug-binding protein 1 (29.Lee J.C. Young P.R. J. Leukocyte Biol. 1996; 59: 152-157Crossref PubMed Scopus (373) Google Scholar), Mxi2 (26.Zervos A.S. Faccio L. Gatto J.P. Kyriakis J.M. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10531-10534Crossref PubMed Scopus (138) Google Scholar), p38β (also known as SAPK2B), p38-2 (also known as p38β2) (30.Stein B. Yang M.X. Young D.B. Janknecht R. Hunter T. Murray B.W. Barbosa M.S. J. Biol. Chem. 1997; 272: 19509-19517Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), p38γ (also known as ERK6 or SAPK3) (31.Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (314) Google Scholar), and p38δ (also known as SAPK4) (32.Kumar S. McDonnell P.C. Gum R.J. Hand A.T. Lee J.C. Young P.R. Biochem. Biophys. Res. Commun. 1997; 235: 533-538Crossref PubMed Scopus (448) Google Scholar). A pyridinylimidazole compound, SB203580, is a highly specific inhibitor of p38 MAP kinase (33.Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1973) Google Scholar), and has been reported to inhibit cyokine production either at the translational level (18.Miyazawa K. Mori A. Miyata H. Akahane M. Ajisawa Y. Hirokazu O. J. Biol. Chem. 1998; 273: 24832-24838Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 34.Lee J.C. Lydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McMulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3128) Google Scholar) or the transcriptional level (35.Bayaert R. Cuenda A. Berghe W.V. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (599) Google Scholar, 36.Vanden Berghe W. Plaisance S. Boone E. De Bosscher K. Schmitz M.L. Fiers W. Haegeman G. J. Biol. Chem. 1998; 273: 3285-3290Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). The signal transduction pathway(s) activated by IL-1β that regulate PDGF-Rα expression are not well understood. Our previous studies have shown that the extracellular signal-regulated kinases (ERK-1 and -2), c-Jun NH2-terminal kinase (JNK), and nuclear factor-κB (NF-κB) do not mediate IL-1β-induced up-regulation of PDGF-Rα mRNA or protein (37.Lindroos P.M. Rice A.B. Wang Y.Z. Bonner J.C. J. Immunol. 1998; 161: 3464-3468PubMed Google Scholar). In this study, we have investigated the role of p38 MAP kinase in IL-1β-induced up-regulation of the PDGF-Rα. We report that p38 MAP kinase activation following IL-1β treatment results in the stabilization of PDGF-Rα mRNA and this requiresde novo protein synthesis. These findings indicate that p38 MAP kinase regulates PDGF-Rα expression at the translational level via synthesis of an mRNA-stabilizing protein. Reagents were from the indicated sources, SB203580 (Calbiochem, La Jolla, CA); PD98059 (New England Biolabs Inc., Beverly, MA); genistein (Roche Molecular Biochemicals, Indianapolis, IN); phorbol 12-myristate 13-acetate (Sigma); recombinant murine IL-1β and recombinant human PDGF-AA (Upstate Biotechnologies, Lake Placid, NY). Actinomycin D (Roche Molecular Biochemicals); cycloheximide (Sigma);125I-PDGF-AA (Biomedical Technologies, Stoughton, MA); [3H]thymidine (Amersham Pharmacia Biotech); anti-phospho-p38 MAP kinase and anti-p38 (total) MAP kinase (New England Biolabs); anti-PDGF-Rα and anti-PDGF-Rβ (Santa Cruz, Santa Cruz, CA); TRITM reagent (Molecular Research Center, Cincinnati, OH); p38 MAP kinase kit (Stratagene, La Jolla, CA). The PDGF-Rα cDNA was a generous gift from Dr. Yutaka Kitami, Ehime University, Japan. Primary passage rat pulmonary myofibroblasts were isolated from male Harlan Sprague-Dawley rats as described previously (12.Coin P.G. Lindroos P.M. Bird G.S.J. Osornio-Vargas A.R. Roggli V.L. Bonner J.C. J. Immunol. 1996; 156: 4797-4806PubMed Google Scholar). These cells stain positively for vimentin, desmin, and α-smooth muscle actin which indicated a myofibroblast phenotype (10.Bonner J.C. Lindroos P.M. Rice A.B. Moomaw C.R. Morgan D.L. Am. J. Physiol. 1998; 274: L72-L80Crossref PubMed Google Scholar). In addition, examination of glutaraldehyde-fixed cell pellets by transmission electron microscopy showed ultrastructural features consistent with a myofibroblast phenotype (abundant intermediate filaments and rough endoplasmic reticulum, and lack of Weibel-Palade bodies characteristic of endothelial cells). Cells were grown to confluence in 10% FBS/DMEM before being seeded for the assays described below. Cells were grown to a confluent state in 10% FBS/DMEM in 75-cm2 tissue culture dishes, then rendered quiescent for 24 h with serum-free defined medium (SFDM) consisting of Ham's F-12 medium supplemented with 0.25% bovine serum albumin and an insulin/transferrin/selenium mixture (Roche Molecular Biochemicals). After treating with the agent of interest, The cultures were washed with ice-cold phosphate-buffered saline and cell lysates collected by incubation with 250 μl of lysis buffer consisting of 50 mm Tris-HCl (pH 7.4), 1% Triton X-100, 150 mmNaCl, 1 mm EGTA, 1 mmNa3VO4, 1 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 0.25% sodium deoxycholate, and 20 μg/ml of each of the following proteinase inhibitors (aprotinin, leupeptin, and pepstatin). Twenty μl of each sample were mixed with 5 μl of sample buffer (0.5 mTris-HCl, pH 6.8, 10% SDS, 0.1% bromphenol blue, 20% glycerol, and 50 mm 2-mercaptoethanol and separated by SDS-PAGE in a 10–20% Tris glycine gel for p38 MAP kinase blots or a 8–16% Tris glycine gel for PDGF-R blots (Novex, San Diego, CA). The proteins were transferred to HybondTM nitrocellulose membrane (Amersham Pharmacia Biotech). The membrane was blocked for 2 h at room temperature with 5% non-fat milk in TBS-Tween buffer (20 mm Tris, 500 mm NaCl, 0.01% Tween 20). The membranes were incubated with primary p38 MAP kinase and PDGF-R antibodies overnight at 4 °C. Anti-phospho-p38 antibody (New England BioLab) was used at a dilution of 1:1,000. Rabbit anti-mouse PDGF-Rα and rabbit anti-human PDGF-Rβ antibodies (Upstate Biotechnologies) were used at a 1:500 dilution. The membranes were washed 3 times with phosphate-buffered saline-Tween prior to a 90-min incubation with a 1:2,000 dilution of horseradish peroxidase-swine anti-rabbit IgG (Dakopatts, Carpenteria, CA). After thoroughly washing in phosphate-buffered saline-Tween, the horseradish peroxidase-labeled proteins were visualized with an ECLTM kit (Amersham Pharmacia Biotech). Phospho-p38 MAP kinase blots were subsequently stripped at 50 °C for 30 min in a buffer containing 62.5 mm Tris (pH 6.7), 2% SDS, and 100 mmβ-mercaptomethonal and re-blotted with an antibody that detects total (activated and unactivated) p38 MAP kinase (New England BioLabs). Confluent, quiescent cells were treated with the agent of interest and cell lysates collected as described above for “Western blotting” were immunoprecipited with total p38 MAP kinase antibody (Santa Cruz). Kinase activity was measured using a p38 MAP kinase Kit (Stratagene) according to the manufacturer's instructions. Briefly, the immune complex was resuspended in Stratagene reaction buffer containing 120 μg of PHAS-1 substrate along with 3-μCi of [γ-32P]ATP in a final volume of 190 μl. Kinase reactions took place for 30 min at room temperature and were stopped by adding 4 × SDS-PAGE reducing sample buffer and boiling for 10 min. The reaction samples were resolved on 10 to 20% PAGE gels, dried, and autoradiographed. A similar procedure was used to assay JNK and ERK kinase activities, using c-Jun and PHAS-1 as substrates, respectively. For determination of the effect of SB203580 on the activity of p38 MAP kinase, MAPKAP kinase 2 activity in rat lung myofibroblasts was measured by a MAPKAP kinase-2 immunoprecipitation assay kit according to the manufacturer's instructions (Upstate Biotechnologies). Briefly, confluent cells were rendered quiescent for 24 h in SFDM and then incubated with or without 50 μm SB203580 for 1 h prior to stimulation with 10 ng/ml IL-1β for 2 h. Cells were placed on ice and lysates scraped off the dish with 250 μl of ice-cold lysis buffer. Lysates were clarified by centrifugation to pellet cellular debris, then incubated with 2 μg of sheep anti-MAPKAP kinase 2 antibody adsorbed to protein G-agarose beads (Santa Cruz) for 2 h at 4 °C. The immunoprecipitates were washed twice with lysis buffer, then twice with kinase buffer and resuspended in 30 μl of kinase assay buffer containing 100 μm substrate peptide KKLNRTLSVA, 50 μm ATP, and 10 μCi of [γ-32P]ATP. The reactions were incubated at 30 °C for 30 min and blotted onto p81 phosphocellulose paper. The papers were washed twice the 0.75% phosphoric acid, one with acetone and radioactivity measured on a liquid scintillation counter. Cells were grown to confluence with 10% FBS/DMEM in 24-well tissue culture plates (2 cm2 wells) and then rendered quiescent for 24 h with SFDM containing 0.5% FBS. The cells were pretreated with fresh 0.5% FBS/SFDM containing SB203580 in Me2SO or Me2SO alone (vehicle control) for 1 h at 37 °C, then PDGF-AA (1 to 50 ng/ml) was spiked into the medium along with 5 μCi/ml [3H]thymidine (Amersham Pharmacia Biotech) for 36 h. The cells were washed with Ham's F-12 at 25 °C, placed on ice, and incubated with 0.5 ml/well 5% trichloroacetic acid for 10 min. After washing 3 times with ice-cold distilled water, solubilization was performed with 0.5 ml/well in 0.2 n NaOH containing 0.1% SDS for 30 min on an oscillating platform. 100 μl of each sample was added to 1 ml of EcolumeTM (Costa Mesa, CA) and radioactivity measured on a liquid scintillation counter. Confluent, quiescent myofibroblasts were treated with the agent of interest and total RNA was isolated with TRITM reagent (Molecular Research Center, Cincinnati, OH). Twenty μg of each sample was electrophoresed in 1% agarose/formaldehyde gels and capillary transferred onto BrightStar-PlusTM positively charged nylon membranes (Ambion Inc, Austin, TX). A rat cDNA probe for the PDGF-Rα (gift from Dr. Yutaka Kitami, Ehime University, Japan) was labeled with [α-32P]dCTP using a DECAprime IITM DNA labeling kit (Ambion). The hybridization and washing procedure for blotting was performed with Northern Max-Plus Kit according to the supplied protocol (Ambion). The autoradiographic signal was visualized by exposing the film at −70 °C for the appropriate time. Myofibroblasts in 24-well plates were grown to confluence in 10% FBS/DMEM and then rendered quiescent for 24 h in SFDM consisting of Ham's F-12 with HEPES, CaCl2, 0.25% bovine serum albumin supplemented with an insulin/transferrin/selenium mixture (Roche Molecular Biochemicals). Cells were then treated with an agent of interest for 24 h. Cultures were chilled to 4 °C, rinsed in cold binding buffer (Ham's F-12 with HEPES, CaCl2, and 0.25% bovine serum albumin), and exposed to 2 ng/ml 125I-PDGF-AA for 3–4 h at 4 °C on an oscillating platform in the absence or presence of 500 ng/ml nonradioactive PDGF-AA to measure total and nonspecific binding, respectively. For saturation binding analysis, cells were incubated with 0.5 to 20 ng/ml 125I-PDGF-AA in the absence or presence of 500 ng/ml PDGF-AA. Cells were then rinsed 3 times in ice-cold binding buffer, solubilized in 1% Triton X-100, 0.1% bovine serum albumin, and 0.1 m NaOH, and cell associated radioactivity measured with a γ-counter. Specific binding was defined as the difference between total and nonspecific binding. Saturation binding data were subjected to Scatchard analysis to obtain dissociation constants (K d) and maximum number of binding sites (B max) (38.Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17789) Google Scholar). Statistical analysis was performed by analysis of variance and two-sample t tests. A pvalue of <0.05 was considered to be significant. Treatment of cells with IL-1β-activated p38 MAP kinase within 30 min as detected by Western blotting for the phosphorylated form of p38 (Fig.1 A). Western blotting for total p38 protein demonstrated that the amount of unactivated p38 did not significantly change during the course of the experiment. Northern blot analysis showed up-regulation of PDGF-Rα mRNA within 2 h following IL-1β treatment, which continued to increase by 24 h (Fig. 1 B). GAPDH mRNA was not significantly affected by IL-1β treatment during the course of the experiment. Densitometric evaluation of p38 MAP kinase activation and PDGF-Rα mRNA induction demonstrated that phosphorylation of p38 MAP kinase peaked prior to an increase in PDGF-Rα mRNA (Fig. 1 C). A specific inhibitor of p38 MAP kinase, SB203580, was used to inhibit activation of p38 MAP kinase in cells stimulated with IL-1β. SB203580 does not inhibit the phosphorylation of p38 MAP kinase, but instead inhibits the kinase activity of p38 for phosphorylating substrates (33.Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1973) Google Scholar). First, we utilized a kinase assay wherein cells were pretreated with SB203580 for 1 h prior to stimulation with IL-1β, then p38 MAP kinase was immunoprecipitated from cell lysates and assayed for its ability to phosphorylate the PHAS-1 substrate (39.Lin T.A. Kong X. Haystead T.A. Pause A. Belsham G. Sonenberg N. Lawrence Jr., J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (599) Google Scholar). IL-1β strongly activated p38 kinase activity and SB203580 (50 μm) completely inhibited p38-induced phosphorylation of PHAS-1 (Fig.2, A and B). In addition, we used a MAPKAP kinase 2 assay to measure the inhibitory effect of SB203580, as MAPKAP kinase 2 is a downstream substrate of p38 MAP kinase (23.Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (774) Google Scholar). As shown in Fig. 2 C, IL-1β clearly induced MAPKAP kinase 2 activity, which was significantly inhibited by SB203580. Pretreatment of cells with SB203580 (50 μm) reduced the basal expression of PDGF-Rα mRNA and blocked IL-1β-induced up-regulation of PDGF-Rα mRNA by >70% (Fig. 3). IL-1β-induced up-regulation of PDGF-Rα protein was also prevented by pretreatment with SB203580 as determined by Western blot analysis using an antibody specific for the PDGF-Rα (Fig.4). In these Western blotting experiments, the level of PDGF-Rβ was not changed by IL-1β treatment or by treatment with SB203580 (Fig. 4). An125I-PDGF-AA binding assay was used to quantitate cell surface PDGF-Rα, since PDGF-AA binds selectively to PDGF-Rα and not PDGF-Rβ (1.Heldin C-H. Westermark B. J. Cell Sci. 1990; 96: 193-196Crossref PubMed Google Scholar). SB203580 inhibited IL-1β-induced up-regulation of cell surface 125I-PDGF-AA binding to cultured cells in a concentration-dependent manner with an IC50between 5 and 10 μm SB203580 (TableI). IL-1β up-regulated125I-PDGF-AA specific binding in a dose-dependent manner that was maximal at 1 ng/ml and pretreatment with 50 μm SB203580 inhibited IL-1β-stimulated up-regulation of 125I-PDGF-AA by >70% (Fig. 5 A). Scatchard analysis of 125I-PDGF-AA saturation binding data demonstrated that SB203580 prevented an increase in the number of binding sites without altering receptor affinity (Fig. 5 B). A variety of other kinase inhibitors, including those for MEK (PD98059), receptor tyrosine kinases (genistein), and protein kinase C (phorbol 12-myristate 13-acetate) had no inhibitory effect on IL-1β-stimulated PDGF-Rα up-regulation (Table II).Figure 4SB203580 prevents IL-1β-induced up-regulation of PDGF-Rα protein as determined by Western blot analysis. Confluent, quiescent rat pulmonary myofibroblasts were pretreated for 1 h with 5 or 50 μm SB203580 or Me2SO vehicle alone, then stimulated for 24 h with 10 ng/ml IL-1β prior to collecting cell lysates for Western blot analysis as described under “Experimental Procedures” using antibodies specific for either PDGF-Rα or PDGF-Rβ. Panel A, IL-1β pretreatment up-regulated PDGF-Rα protein 2–3-fold and SB203580 blocked the increase in PDGF-Rα levels by ∼50% at 5 μm or 100% at 50 μm. PDGF-Rβ was not affected by IL-1β or SB203580. Panel B, quantitative densitometry of PDGF-Rα (gray bars) and PDGF-Rβ (black bars) levels. Data are representative of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IConcentration-dependent inhibition of IL-1β-induced up-regulation of 125 I-PDGF-AA specific binding by SB203580 in rat lung myofibroblastsSB203580125I-PDGF-AA bound (cpm/culture)−IL-1β+IL-1βμm01296 ± 968880 ± 7661.01274 ± 1037670 ± 8015.01276 ± 686630 ± 55510.01286 ± 744440 ± 515 ap < 0.01 as compared with the corresponding value for IL-1β plus vehicle.50.01342 ± 803000 ± 362 ap < 0.01 as compared with the corresponding value for IL-1β plus vehicle.100.01361 ± 1131980 ± 209 bp < 0.001 as compared with the corresponding value for IL-1β plus vehicle.Confluent, quiescent cells were treated with an increasing concentration of SB203580 or Me2SO vehicle for 1 h, then stimulated with IL-1β (10 ng/ml) for 24 h prior to performing an125I-PDGF-AA binding assay as described under “Experimental Procedures.” Data are expressed as the mean ± S.E. of three experiments.a p < 0.01 as compared with the corresponding value for IL-1β plus vehicle.b p < 0.001 as compared with the corresponding value for IL-1β plus vehicle. Open table in a new tab Figure 5SB203580 inhibits IL-1β-induced up-regulation of125I-PDGF-AA-binding sites on rat pulmonary myofibroblasts. Confluent, quiescent cells were pretreated with 50 μm SB203580 or Me2SO vehicle for 1 h, then stimulated with 10 ng/ml IL-1β for 24 h prior to performing125I-PDGF-AA binding analysis as described under “Experimental Procedures.” Panel A, the dose-dependent up-regulation in 125I-PDGF-AA specific binding was inhibited >70% by pretreatment with SB203580. Data are the means of three separate experiments. Panel B, scatchard analysis of 125I-PDGF-AA saturation binding data demonstrated an increase in the number of PDGF-AA-binding sites following IL-1β treatment that was prevented by pretreatment with 50 μm SB203580. Data are representative of three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIEffect of various kinase inhibitors on IL-1β-induced up-regulation of 125 I-PDGF-AA specific binding to cultured rat lung myofibroblas" @default.
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• W2041081365 title "Regulation of Interleukin-1β-induced Platelet-derived Growth Factor Receptor-α Expression in Rat Pulmonary Myofibroblasts by p38 Mitogen-activated Protein Kinase" @default.
• W2041081365 cites W1493414055 @default.
• W2041081365 cites W1502860897 @default.
• W2041081365 cites W1519060218 @default.
• W2041081365 cites W1533808086 @default.
• W2041081365 cites W1549679997 @default.
• W2041081365 cites W1554558913 @default.
• W2041081365 cites W1567131741 @default.
• W2041081365 cites W1587892340 @default.
• W2041081365 cites W1660988713 @default.
• W2041081365 cites W1868769527 @default.
• W2041081365 cites W1968432271 @default.
• W2041081365 cites W1968483745 @default.
• W2041081365 cites W1972644785 @default.
• W2041081365 cites W1973766879 @default.
• W2041081365 cites W1975408686 @default.
• W2041081365 cites W1976415203 @default.
• W2041081365 cites W1980831535 @default.
• W2041081365 cites W1981124481 @default.
• W2041081365 cites W1983649348 @default.
• W2041081365 cites W1984665850 @default.
• W2041081365 cites W1993783742 @default.
• W2041081365 cites W2001399809 @default.
• W2041081365 cites W2004209406 @default.
• W2041081365 cites W2007986629 @default.
• W2041081365 cites W2011936496 @default.
• W2041081365 cites W2015313106 @default.
• W2041081365 cites W2020374796 @default.
• W2041081365 cites W2022914311 @default.
• W2041081365 cites W2029706883 @default.
• W2041081365 cites W2032731042 @default.
• W2041081365 cites W2035140776 @default.
• W2041081365 cites W2037079787 @default.
• W2041081365 cites W2069652470 @default.
• W2041081365 cites W2080532845 @default.
• W2041081365 cites W2083398588 @default.
• W2041081365 cites W2086525709 @default.
• W2041081365 cites W2096687508 @default.
• W2041081365 cites W2098775968 @default.
• W2041081365 cites W2103737508 @default.
• W2041081365 cites W2115018128 @default.
• W2041081365 cites W2124273066 @default.
• W2041081365 cites W2142638534 @default.
• W2041081365 cites W2149333426 @default.
• W2041081365 cites W2170361601 @default.
• W2041081365 cites W2341881834 @default.
• W2041081365 cites W2409414966 @default.
• W2041081365 cites W4313310048 @default.
• W2041081365 cites W4313377128 @default.
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