HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 29, 22550-22557, July 21, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/29/22550    most recent M909785199v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, Y.-Z. Articles by Bonner, J. C. Search for Related Content PubMed PubMed Citation Articles by Wang, Y.-Z. Articles by Bonner, J. C. Social Bookmarking                      What's this?

Regulation of Interleukin-1-induced Platelet-derived Growth Factor Receptor- Expression in Rat Pulmonary Myofibroblasts by p38 Mitogen-activated Protein Kinase^*

Yi-Zhe Wang, Ping Zhang, Annette B. Rice, and James C. Bonner

From the Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709

Received for publication, December 10, 1999, and in revised form, March 31, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The potential role of p38 mitogen-activated protein (MAP) kinase in platelet-derived growth factor receptor- (PDGF-R) gene expression was investigated using cultured rat pulmonary myofibroblasts. p38 MAP kinase was constitutively expressed in myofibroblasts and activated by interleukin (IL)-1. A pyridinylimidazole compound, SB203580, completely inhibited the ability of p38 MAP kinase activity to phosphorylate PHAS-1 substrate. SB203580 inhibited IL-1-induced up-regulation of PDGF-R 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-R. The IL-1-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-1 pretreatment was blocked >70% by SB203580. SB203580 did not affect IL-1-induced ERK activation, yet enhanced IL-1-induced JNK activation approximately 2-fold. Treatment of cells with SB203580 after inhibition of transcription by actinomycin D decreased the half-life of IL-1-induced PDGF-R mRNA from >4 to ~1.5 h. Moreover, pretreatment of cells with cycloheximide blocked induction of PDGF-R mRNA by IL-1, suggesting that de novo protein synthesis was required for PDGF-R mRNA stabilization. These data indicate that p38 MAP kinase regulates PDGF-R expression at the translational level by signaling the synthesis of an mRNA-stabilizing protein.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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). Two PDGF receptor subtypes bind the three isoforms of PDGF differentially; -PDGF receptor (PDGF-R) can interact only with B-chain containing isoforms while -PDGF receptor (PDGF-R) can bind all three isoforms (2). PDGF binding results in receptor dimerization to form , , or 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- (reviewed in Ref. 3). The biologic activity of PDGF isoforms on rat pulmonary myofibroblasts is modulated in the extracellular microenvironment through interaction with its binding protein, ₂-macroglobulin (4, 5), and by regulation of cell-surface PDGF-R (6, 7).

The PDGF-R and its ligand, PDGF-AA, are essential to lung development (8), yet induction of the PDGF-R also occurs in adult tissues during the pathogenesis of certain fibroproliferative diseases. For example, human fibroblasts isolated from dermal keloids express elevated PDGF-R (9). We and others have reported that PDGF-R is up-regulated during the progression of pulmonary fibrosis in rats, while the PDGF-R is constitutively expressed (10, 11). Interleukin (IL)-1 is a potent inducer of the PDGF-R on cultured myofibroblasts isolated from rat lung and PDGF-R up-regulation enhances the mitogenic and chemotactic responses to PDGF isoforms (6, 12). The maximal responses of connective tissue cells to PDGF isoforms require PDGF-R in addition to the normally abundant PDGF-R (7, 13), and this could be due to unique signal transduction events stimulated by - receptor dimerization, as compared with - receptor dimerization (14). Other mediators, including transforming growth factor-1 (15) and prostaglandin E₂ (16) suppress PDGF-R expression and counteract the up-regulatory effect of IL-1.

It is becoming increasingly clear that IL-1 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 (17-20). p38 MAP kinase is activated upon stimulation of cells with cytokines, bacterial lipopolysaccharide, and stress (21, 22). Several transcription factors are substrates for p38 MAP kinase isozymes, including MAP-KAP kinase-2 (23), ATF-2 (24), CHOP/GADD153 (25), MAX (26), myocyte enhancer factor 2C (27), and ternary complex factor (28). In addition to the original p38 (also termed p38, 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), Mxi2 (26), p38 (also known as SAPK2B), p38-2 (also known as p382) (30), p38 (also known as ERK6 or SAPK3) (31), and p38 (also known as SAPK4) (32). A pyridinylimidazole compound, SB203580, is a highly specific inhibitor of p38 MAP kinase (33), and has been reported to inhibit cyokine production either at the translational level (18, 34) or the transcriptional level (35, 36).

The signal transduction pathway(s) activated by IL-1 that regulate PDGF-R 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-B (NF-B) do not mediate IL-1-induced up-regulation of PDGF-R mRNA or protein (37). In this study, we have investigated the role of p38 MAP kinase in IL-1-induced up-regulation of the PDGF-R. We report that p38 MAP kinase activation following IL-1 treatment results in the stabilization of PDGF-R mRNA and this requires de novo protein synthesis. These findings indicate that p38 MAP kinase regulates PDGF-R expression at the translational level via synthesis of an mRNA-stabilizing protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- 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-1 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-R and anti-PDGF-R (Santa Cruz, Santa Cruz, CA); TRI^TM reagent (Molecular Research Center, Cincinnati, OH); p38 MAP kinase kit (Stratagene, La Jolla, CA). The PDGF-R cDNA was a generous gift from Dr. Yutaka Kitami, Ehime University, Japan.

Cell Culture-- Primary passage rat pulmonary myofibroblasts were isolated from male Harlan Sprague-Dawley rats as described previously (12). These cells stain positively for vimentin, desmin, and -smooth muscle actin which indicated a myofibroblast phenotype (10). 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.

Western Blot Analysis-- 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 mM NaCl, 1 mM EGTA, 1 mM Na₃VO₄, 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 M Tris-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 Hybond^TM 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-R and rabbit anti-human PDGF-R 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 ECL^TM 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 mM -mercaptomethonal and re-blotted with an antibody that detects total (activated and unactivated) p38 MAP kinase (New England BioLabs).

MAP Kinase Assay-- 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 [-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.

Analysis of MAPKAP Kinase-2 Activity-- 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-1 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 [-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.

[³H]Thymidine Incorporation Assay-- 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 Ecolume^TM (Costa Mesa, CA) and radioactivity measured on a liquid scintillation counter.

Northern Blot Analysis-- Confluent, quiescent myofibroblasts were treated with the agent of interest and total RNA was isolated with TRI^TM reagent (Molecular Research Center, Cincinnati, OH). Twenty µg of each sample was electrophoresed in 1% agarose/formaldehyde gels and capillary transferred onto BrightStar-Plus^TM positively charged nylon membranes (Ambion Inc, Austin, TX). A rat cDNA probe for the PDGF-R (gift from Dr. Yutaka Kitami, Ehime University, Japan) was labeled with [-³²P]dCTP using a DECAprime II^TM 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.

¹²⁵I-PDGF-AA Binding Assay-- 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 -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).

Statistical Analysis-- Statistical analysis was performed by analysis of variance and two-sample t tests. A p value of <0.05 was considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Temporal Activation of p38 MAP Kinase and Up-regulation of PDGF-R mRNA following IL-1 Treatment-- Treatment of cells with IL-1-activated p38 MAP kinase within 30 min as detected by Western blotting for the phosphorylated form of p38 (Fig. 1A). 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-R mRNA within 2 h following IL-1 treatment, which continued to increase by 24 h (Fig. 1B). GAPDH mRNA was not significantly affected by IL-1 treatment during the course of the experiment. Densitometric evaluation of p38 MAP kinase activation and PDGF-R mRNA induction demonstrated that phosphorylation of p38 MAP kinase peaked prior to an increase in PDGF-R mRNA (Fig. 1C).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   p38 MAP kinase activation precedes induction of PDGF-R mRNA in cultured rat pulmonary myofibroblasts following treatment with IL-1. Confluent cell cultures were rendered quiescent in SFDM for 24 h prior to treatment with IL-1 (10 ng/ml) for the indicated time points prior to harvesting cell lysates for Western blot analysis or mRNA for Northern blot analysis. Panel A, representative Western blots using an antibody specific for the phosphorylated form of p38 MAP kinase (phospho-p38) showing transient activation of p38 that peaked at 30 min, or an antibody that recognized total (unactivated and activated) p38 MAP kinase. Panel B, representative Northern blots demonstrating induction of PDGF-R mRNA within 4 h following IL-1 stimulation and constitutive GAPDH expression. Panel C, relative levels of phospho-p38 MAP kinase protein or PDGF-R mRNA expression were determined by densitometric scanning of the autoradiographic bands and normalized to the unactivated p38 MAP kinase or GAPDH bands, respectively. Data are expressed as the mean ± S.E. of three experiments.

SB203580 Inhibits IL-1-induced p38 MAP Kinase Activity-- A specific inhibitor of p38 MAP kinase, SB203580, was used to inhibit activation of p38 MAP kinase in cells stimulated with IL-1. SB203580 does not inhibit the phosphorylation of p38 MAP kinase, but instead inhibits the kinase activity of p38 for phosphorylating substrates (33). First, we utilized a kinase assay wherein cells were pretreated with SB203580 for 1 h prior to stimulation with IL-1, then p38 MAP kinase was immunoprecipitated from cell lysates and assayed for its ability to phosphorylate the PHAS-1 substrate (39). IL-1 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). As shown in Fig. 2C, IL-1 clearly induced MAPKAP kinase 2 activity, which was significantly inhibited by SB203580.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Inhibition of p38 MAP kinase activity by SB203580. Confluent cultures of rat pulmonary myofibroblasts were rendered quiescent for 24 h in SFDM and then treated for 30 min with IL-1 in the absence or presence of 50 µM SB203580. p38 MAP kinase was immunoprecipitated from whole cell lysates and a kinase assay performed using PHAS-1 as the substrate. Panel A, a representative autoradiograph showing phosphorylation of PHAS-1 by p38 MAP kinase immunoprecipated from IL-1-treated cells and inhibition of p38 MAP kinase activation by SB203580. Panel B, relative expression of p38 MAP kinase activity in the absence or presence of SB203580 or IL-1 were determined by densitometric scanning of PHAS-1 bands. Panel C, induction of MAPKAP kinase 2 activity by IL-1 and inhibition by SB203580. Data are expressed as the mean ± S.E. of three experiments. **, p < 0.01 as compared with the value for IL-1 alone.

SB203580 Inhibits IL-1-induced Up-regulation of PDGF-R mRNA and Protein-- Pretreatment of cells with SB203580 (50 µM) reduced the basal expression of PDGF-R mRNA and blocked IL-1-induced up-regulation of PDGF-R mRNA by >70% (Fig. 3). IL-1-induced up-regulation of PDGF-R protein was also prevented by pretreatment with SB203580 as determined by Western blot analysis using an antibody specific for the PDGF-R (Fig. 4). In these Western blotting experiments, the level of PDGF-R was not changed by IL-1 treatment or by treatment with SB203580 (Fig. 4). An ¹²⁵I-PDGF-AA binding assay was used to quantitate cell surface PDGF-R, since PDGF-AA binds selectively to PDGF-R and not PDGF-R (1). SB203580 inhibited IL-1-induced up-regulation of cell surface ¹²⁵I-PDGF-AA binding to cultured cells in a concentration-dependent manner with an IC₅₀ between 5 and 10 µM SB203580 (Table I). IL-1 up-regulated ¹²⁵I-PDGF-AA specific binding in a dose-dependent manner that was maximal at 1 ng/ml and pretreatment with 50 µM SB203580 inhibited IL-1-stimulated up-regulation of ¹²⁵I-PDGF-AA by >70% (Fig. 5A). 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. 5B). 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-1-stimulated PDGF-R up-regulation (Table II).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   SB203580 blocks up-regulation of PDGF-R mRNA expression by IL-1. Confluent, quiescent rat pulmonary myofibroblasts were pretreated for 1 h with 50 µM SB203580 or Me2SO vehicle alone then stimulated for 4 h with 10 ng/ml IL-1 prior to collecting RNA for Northern blot analysis. Panel A, autoradiograph of PDGF-R and GAPDH Northern blots. Panel B, relative levels of PDGF-R mRNA expression were determined by densitometric scanning of the autoradiographic bands and normalized to the GAPDH bands, respectively. Data are expressed as the mean ± S.E. of three experiments. **, p < 0.01 as compared with the value for IL-1 alone.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   SB203580 prevents IL-1-induced up-regulation of PDGF-R 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-1 prior to collecting cell lysates for Western blot analysis as described under "Experimental Procedures" using antibodies specific for either PDGF-R or PDGF-R. Panel A, IL-1 pretreatment up-regulated PDGF-R protein 2-3-fold and SB203580 blocked the increase in PDGF-R levels by ~50% at 5 µM or 100% at 50 µM. PDGF-R was not affected by IL-1 or SB203580. Panel B, quantitative densitometry of PDGF-R (gray bars) and PDGF-R (black bars) levels. Data are representative of three separate experiments.

View this table: [in this window] [in a new window]   Table I Concentration-dependent inhibition of IL-1-induced up-regulation of 125I-PDGF-AA specific binding by SB203580 in rat lung myofibroblasts Confluent, quiescent cells were treated with an increasing concentration of SB203580 or Me2SO vehicle for 1 h, then stimulated with IL-1 (10 ng/ml) for 24 h prior to performing an 125I-PDGF-AA binding assay as described under "Experimental Procedures." Data are expressed as the mean ± S.E. of three experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   SB203580 inhibits IL-1-induced up-regulation of 125I-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-1 for 24 h prior to performing 125I-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-1 treatment that was prevented by pretreatment with 50 µM SB203580. Data are representative of three separate experiments.

View this table: [in this window] [in a new window]   Table II Effect of various kinase inhibitors on IL-1-induced up-regulation of 125I-PDGF-AA specific binding to cultured rat lung myofibroblasts Confluent, quiescent cells were treated with the indicated concentration of inhibitor or Me2SO vehicle for 1 h, then stimulated with IL-1 (10 ng/ml) for 24 h prior to performing an 125I-PDGF-AA binding assay as described under "Experimental Procedures." Data are expressed as the mean ± S.E. of three experiments.

SB203580 Inhibits the Enhanced Mitogenic Response to PDGF-AA following IL-1-induced Up-regulation of PDGF-R-- Rat pulmonary myofibroblasts had a poor mitogenic response to PDGF-AA due to the low number of constitutively expressed PDGF-R at the cell surface, yet pretreatment with IL-1 for 24 h enhanced the concentration-dependent PDGF-AA mitogenic response severalfold. SB203580 (50 µM) alone had no effect on [³H]thymidine uptake by rat pulmonary myofibroblasts, but pretreatment of cells inhibited the IL-1-enhanced mitogenic response to PDGF-AA by 60-70% (Fig. 6). IL-1 caused a 3-fold increase in [³H]thymidine uptake in the absence of PDGF-AA and this increased mitogenesis was also blocked by SB203580.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   SB203580 inhibits the enhanced mitogenic response of rat pulmonary myofibroblasts to PDGF-AA following IL-1 stimulation. Confluent, quiescent cells were pretreated with 50 µM SB203580 or Me2SO vehicle for 1 h, then stimulated for 24 h with 10 ng/ml IL-1. PDGF-AA was then spiked into the medium in the presence of 5 µCi/ml [3H]thymidine for another 24 h prior to trichloroacetic acid precipitation and measurement of radioactivity. Data are the means of three separate experiments (S.E. <5% of the mean).

Effect of SB203580 on PDGF-R mRNA Stability-- To determine the effect of SB203580 on the stability of PDGF-R mRNA, rat pulmonary myofibroblasts were stimulated with IL-1 for 4 h to up-regulate PDGF-R mRNA. Cells were then treated with actinomycin D, a transcriptional inhibitor, or actinomycin D plus SB203580 was added. Total cellular RNA was isolated following various time periods after the addition of actinomycin D and examined for the presence of PDGF-R mRNA or GAPDH mRNA by Northern blot analysis. A representative result is shown in Fig. 7A. For correction for differences in loading, the densitometric signal of each RNA sample hybridized to the PDGF-R probe was divided by a GAPDH signal (Fig. 7B). IL-1-induced PDGF-R mRNA had a calculated half-life of >4 h in pulmonary myofibroblasts treated with actinomycin D alone. Treatment of cells with a combination of actinomycin D and SB203580 reduced the half-life of PDGF-R mRNA to ~1.5 h.

View larger version (33K): [in this window] [in a new window]   Fig. 7.   Effect of SB203580 and actinomycin D (Act D) on IL-1-induced PDGF-R mRNA expression. Rat pulmonary myofibroblasts were treated with IL-1 (10 ng/ml) for 4 h and subsequently incubated with actinomycin D (10 µg/ml) alone or actinomycin D plus SB203580 (50 µM) for the time periods indicated. Northern blot analysis was performed as described under "Experimental Procedures." Panel A, PDGF-R and GAPDH Northern blot autoradiographs representative of three experiments with similar results. Panel B, relative levels of PDGF-R mRNA expression were determined by densitometric scanning of the autoradiographic bands and normalized to the GAPDH signal. Data are expressed as the mean ± S.E. of three experiments. *, p < 0.05; or **, p < 0.01 as compared with Act D treatment.

Requirement of de Novo Protein Synthesis for IL-1-induced Up-regulation of PDGF-R mRNA-- The experiments described above using actinomycin D indicated that p38 MAP kinase plays in a role in the stabilization of PDGF-R mRNA. However, it was unclear whether p38 MAP kinase caused mRNA stabilization by mediating the synthesis of a new protein(s). In order to determine if de novo protein synthesis was required, cells were pretreated for 1 h with 5 µg/ml cycloheximide to block protein synthesis and then treated for 4 h with IL-1 to up-regulate PDGF-R mRNA. Cycloheximide treatment abolished the induction of PDGF-R mRNA caused by IL-1 (Fig. 8).

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effect of cycloheximide on IL-1-induced PDGF-R mRNA expression. Rat pulmonary myofibroblasts were treated with 5 µg/ml cycloheximide for 1 h, then 10 ng/ml IL-1 was spiked into the medium for an additional 4 h prior to collecting total RNA. Panel A, representative Northern blot autoradiographs of PDGF-R mRNA and GAPDH mRNA expression. Panel B, relative levels of PDGF-R mRNA expression were determined by densitometric scanning of the autoradiographic bands and normalized to the GAPDH signal. Data are expressed as the mean ± range of two experiments. **, p < 0.01, IL-1 treatment compared with IL-1 plus cycloheximide (CHX).

SB203580 Does Not Affect IL-1-induced ERK Activation but Enhances IL-1-induced JNK Activation-- To test whether SB203580 might have effects on the activity of other MAP kinases, we preincubated cells with increasing concentrations of SB203580 (1-100 µM) and then stimulated the cells with IL-1 for 30 min prior to collecting cell lysates. In kinase assays, SB203580 completely inhibited p38 MAP kinase activity (Fig. 9). However, 10 µM SB203580 inhibited IL-1-induced up-regulation of ¹²⁵I-PDGF-AA binding 60-70%, and higher concentrations of SB203580 (50 and 100 µM) were required to completely inhibit ¹²⁵I-PDGF-AA up-regulation in response to IL-1 (Table I). These data suggested that another signaling mechanism might be required to compliment p38 MAP kinase to facilitate up-regulation of PDGF-R. ERK activation induced by IL-1 was not affected by concentrations of SB203580 as high as 100 µM, while IL-1-induced JNK activation was enhanced approximately 2-fold by SB203580 (Fig. 9).

View larger version (27K): [in this window] [in a new window]   Fig. 9.   Effect of SB203580 on IL-1-induced activation of ERK and JNK. Rat pulmonary myofibroblasts were pretreated with increasing concentrations of SB203580 for 1 h, then treated with 10 ng/ml IL-1 for 30 min. Kinase assays were performed as described under "Experimental Procedures." SB203580 caused complete inhibition of p38 MAP kinase activation at 10 µM, yet concentrations as high as 100 µM did not affect ERK activation. SB203580 pretreatment enhanced IL-1-induced JNK activation approximately 2-fold.

p38 MAP Kinase Is Necessary yet Alone May Not Be Sufficient to Cause Up-regulation of PDGF-R-- The experiment described above in Fig. 9 suggested that activation of p38 MAP kinase alone might not be sufficient to up-regulate PDGF-R. Therefore we compared LPS, another known inducer of PDGF-R (12), and TNF-, which has been reported to have no effect on induction of PDGF-R (37), for their ability to activate p38 MAP kinase, ERK, or JNK. IL-1 activated all three MAP kinases, while LPS and TNF- activated only p38 MAP kinase (Fig. 10A). Both IL-1 and LPS, but not TNF-, up-regulated ¹²⁵I-PDGF-AA specific binding to cultured myofibroblasts (Fig. 10B). Since TNF- activates p38 MAP kinase but does not up-regulate PDGF-R, these data indicate that another signaling mechanism compliments p38 MAP kinase to facilitate up-regulation of PDGF-R in response to IL-1.

View larger version (22K): [in this window] [in a new window]   Fig. 10.   Differential activation of MAP kinases and induction of PDGF-R by various inflammatory mediators. A, activation of MAP kinases by IL-1, LPS, or TNF-. Rat pulmonary myofibroblasts were treated with IL-1 (10 ng/ml), LPS (10 µg/ml), or TNF- (10 ng/ml) for 30 min prior to collecting cell lysates. JNK, ERK, or p38 MAP kinase were immunoprecpitated from cell lysates and kinase activity was measured as described under "Experimental Procedures." B, up-regulation of PDGF-R by IL-1 and LPS, but not TNF-. Cells were treated for 24 h with the same concentrations of inflammatory mediators used in A and levels of cell-surface PDGF-R were measured by 125I-PDGF-AA radioligand binding assay.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

IL-1 is the major factor produced by activated pulmonary macrophages that up-regulates the PDGF-R on lung myofibroblasts (6, 12). In this study we report that p38 MAP kinase is a required signaling intermediate for IL-1-induced up-regulation of the PDGF-R, as SB203580 blocked the increase in PDGF-R mRNA expression (Fig. 3) and appearance of functional cell-surface PDGF-R protein following IL-1 treatment (Figs. 4 and 5). Moreover, pretreatment of cells with SB203580 significantly reduced IL-1-induced enhancement of PDGF-AA-stimulated mitogenesis (Fig. 6). We clearly showed that inhibition of p38 MAP kinase activation by SB203580 resulted in accelerated degradation of PDGF-R mRNA (Fig. 7), which proved that p38 MAP kinase plays a role in the stabilization of PDGF-R mRNA. IL-1-induced up-regulation of PDGF-R mRNA was abolished by pretreatment with cycloheximide (Fig. 8), which showed that de novo protein synthesis was required for the IL-1-stimulated increase in PDGF-R mRNA. Taken together, these data support the idea that IL-1 activates p38 MAP kinase, which then signals downstream events that culminate in the synthesis of a protein that stabilizes PDGF-R mRNA.

Other studies have shown that p38 MAP kinase may play a role in stabilizing mRNA or by increasing transcription. For example, Miyazawa and co-workers (18) reported that IL-1 induced IL-6 gene expression in human fibroblast-like synoviocytes was blocked by SB203580 (18). Similar to our observation in the present study, they observed that SB203580 increased the IL-6 mRNA degradation rate in the presence of actinomycin D and concluded that p38 MAP kinase controlled IL-6 expression at the translational level by stabilization of IL-6 mRNA (18). However, they observed that cycloheximide had no effect on the increase in IL-6 mRNA after IL-1 stimulation (18). In our hands, cycloheximide abolished the increase in PDGF-R mRNA following IL-1 treatment, suggesting that de novo protein synthesis was required for PDGF-R mRNA stabilization. Other investigators have reported that IL-6 mRNA expression and NF-B reporter gene activation by TNF- in murine fibrosarcoma L929 cells was completely inhibited by SB203580, leading to the conclusion that p38 MAP kinase controlled TNF--induced IL-6 expression at the transcriptional level (35, 36).

Our data in the present study support the concept that p38 MAP kinase signals the de novo synthesis of a protein(s) that stabilizes PDGF-R mRNA. Several proteins such as AUF1 (40) and TTP (41) have been reported to reduce mRNA stability, whereas other proteins including AUBF (42) and the -globulin complex (43) increase mRNA stability. All of these factors, whether they function to stabilize or destabilize mRNAs, bind AU-rich sequences in the 3'-untranslated region of the mRNA to cause either mRNA stability or degradation. In particular, repeated AUUUA sequences in the 3'-untranslated region of many proto-oncogenes and cytokine mRNAs are the target for RNA-binding proteins (44-46). PDGF-R mRNA contains 10 copies of the AUUUA sequence in its 3'-untranslated region (47). Thus, it is not unexpected that PDGF-R mRNA would be the target for RNA-binding proteins that would influence mRNA stability.

IL-1 activates other MAP kinases in pulmonary myofibroblasts including JNK and ERK, yet activation of these kinases apparently does not result in PDGF-R up-regulation. For example, treatment of cells with the MEK inhibitor, PD98059, enhanced IL-1-induced up-regulation of PDGF-R 2-3-fold (Ref. 37 and Table II). Thus, activation of ERK has the opposite effect of p38 MAP kinase activation on IL-1-induced expression of PDGF-R. Nevertheless, we investigated the possibility that SB203580 might be affecting the activity of ERK. However, concentrations of SB203580 as high as 100 µM did not affect IL-1-induced ERK activation (Fig. 9). We also investigated JNK as a possible signaling intermediate that might mediate the increase in PDGF-R following IL-1 treatment. In the present study, SB203580 enhanced IL-1-induced JNK activity approximately 2-fold (Fig. 9). Alone these data suggest the possibility that the effect of SB203580 on IL-1-induced up-regulation of PDGF-R was mediated in part by JNK activation. However, LPS strongly up-regulates PDGF-R in myofibroblasts without activating JNK (Fig. 10). Additionally, pyrrolidine dithiocarbamate activates JNK in myofibroblasts but does not up-regulate PDGF-R (37). Collectively, these findings indicate that JNK does not play a role in induction of PDGF-R. Finally, we excluded a role for receptor tyrosine kinases or protein kinase C, as genistein or phorbol 12-myristate 13-acetate had no effect on IL-1-induced PDGF-R expression, respectively.

While p38 MAP kinase appears to be necessary for IL-1-induced up-regulation of the PDGF-R, the possibility exists that p38 MAP kinase activation alone might not be sufficient to cause up-regulation of PDGF-R. Indeed, we found that TNF- activates p38 MAP kinase in rat pulmonary myofibroblasts, yet TNF- did not up-regulate PDGF-R (Fig. 10). These data suggest that IL-1 and other agents that cause up-regulation of PDGF-R following activation of p38 MAP kinase (e.g. LPS) might also activate a signaling pathway that is required to compliment p38 MAP kinase to facilitate up-regulation of PDGF-R. Alternatively, TNF- could activate a signaling pathway that suppresses expression of PDGF-R in addition to activating p38 MAP kinase. In any case, our comparison of various inflammatory mediators in Fig. 10 suggest that p38 MAP kinase activation is necessary yet alone is not sufficient to cause up-regulation of PDGF-R.

Our findings do not rule out the possibility that increased PDGF-R mRNA expression but is also controlled at the level of PDGF-R transcription. Kitami and co-workers (48) recently reported that members of the CAAT/enhancer-binding protein (C/EBP) family control expression of the PDGF-R. Specifically, they found that a high level of C/EBP- expression was a major determinant for elevated gene expression of the PDGF-R in vascular smooth muscle cells of genetically hypertensive rats (48). Whether or not C/EBP plays a role in IL-1-induced up-regulation of the PDGF-R, (i.e. transcriptional regulation) remains to be elucidated. To our knowledge, no transcription factors other than C/EBP have been linked to the regulation of the PDGF-R. A previous study from our laboratory addressed the possible role of NF-B in the regulation of PDGF-R by IL-1, yet IL-1-induced up-regulation of PDGF-R was independent of NF-B since other activators of NF-B (e.g. TNF-) did not up-regulate PDGF-R. Moreover, the PDGF-R is up-regulated by dexamethasone (49) and staurosporine,² yet these agents do not activate NF-B.

Several studies have shown that maximal mitogenic and chemotactic responses to PDGF isoforms require co-expression of both PDGF-R and PDGF-R (6, 7, 12, 13), yet expression of PDGF-R in many mesenchymal cell types is constitutively suppressed. However, the PDGF-R is up-regulated during the progression of several fibroproliferative diseases (9-11). During pulmonary fibrogenesis in rats, the temporal up-regulation of this receptor precedes myofibroblast hyperplasia (10, 11). Moveover, induction of PDGF-R in cultured myofibroblasts stimulated with IL-1 results in enhanced proliferative and chemotactic responses to all PDGF isoforms (6, 12). Collectively, these in vitro and in vivo observations indicate that induction of the PDGF-R is a mechanism that contributes to accelerated myofibroblast growth during pulmonary fibrogenesis. Overall, the PDGF receptor system appears to be important to the progression of lung fibrosis as this disease in rats is reduced by the administration of a PDGF-specific receptor tyrosine kinase inhibitor (50).

In summary, our findings support the idea that IL-1 induces PDGF-R expression in rat pulmonary myofibroblasts by activating p38 MAP kinase, which functions to stabilize PDGF-R mRNA by acting downstream to signal de novo synthesis of a protein(s) that stabilizes PDGF-R mRNA. Further investigation is warranted to identify the RNA-binding protein(s) that regulate PDGF-R mRNA stability. Expression of the PDGF-R appears to be a mechanism of fibroproliferative lung disease. Therefore, elucidation of the molecular mechanisms that control the expression of this receptor may lead to strategies for therapeutic intervention of the disease.

	ACKNOWLEDGEMENTS

We thank Dr. Perry Blackshear and Dr. John O'Bryan at the National Institute of Environmental Health Sciences for helpful comments during the preparation of this manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Pulmonary Pathobiology, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-0766; Fax: 919-541-4133; E-mail: bonnerj@niehs.nih.gov.

Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M909785199

² P. M. Lindroos and J. C. Bonner, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: PDGF, platelet-derived growth factor; PDGF-R, -PDGF receptor; PDGF-R, -PDGF receptor; IL, interleukin; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH₂-terminal kinase; MEK-1, MAP kinase kinase; NF-B, nuclear factor-B; AP-1, activator protein-1; SFDM, serum-free defined medium; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; TNF-, tumor necrosis factor-; C/EBP, CAAT/enhancer-binding protein; PHAS, phosphorylated heat- and acid-stable protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES