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TGF-β1 increases proliferation of airway smooth muscle cells by phosphorylation of map kinases Chen, Gang; Khalil, Nasreen Jan 3, 2006

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ralssBioMed CentRespiratory ResearchOpen AcceResearchTGF-β1 increases proliferation of airway smooth muscle cells by phosphorylation of map kinasesGang Chen and Nasreen Khalil*Address: Division of Respiratory Medicine, Department of Medicine, The University of British Columbia and the Vancouver Coastal Health Research Institute, Vancouver, BC V6H 3Z6, CanadaEmail: Gang Chen - gang.chen@vch.ca; Nasreen Khalil* - nkhalil@interchange.ubc.ca* Corresponding author    AbstractBackground: Airway remodeling in asthma is the result of increased expression of connectivetissue proteins, airway smooth muscle cell (ASMC) hyperplasia and hypertrophy. TGF-β1 has beenfound to increase ASMC proliferation. The activation of mitogen-activated protein kinases(MAPKs), p38, ERK, and JNK, is critical to the signal transduction associated with cell proliferation.In the present study, we determined the role of phosphorylated MAPKs in TGF-β1 induced ASMCproliferation.Methods: Confluent and growth-arrested bovine ASMCs were treated with TGF-β1. Proliferationwas measured by [3H]-thymidine incorporation and cell counting. Expressions of phosphorylatedp38, ERK1/2, and JNK were determined by Western analysis.Results: In a concentration-dependent manner, TGF-β1 increased [3H]-thymidine incorporationand cell number of ASMCs. TGF-β1 also enhanced serum-induced ASMC proliferation. AlthoughASMCs cultured with TGF-β1 had a significant increase in phosphorylated p38, ERK1/2, and JNK,the maximal phosphorylation of each MAPK had a varied onset after incubation with TGF-β1. TGF-β1 induced DNA synthesis was inhibited by SB 203580 or PD 98059, selective inhibitors of p38 andMAP kinase kinase (MEK), respectively. Antibodies against EGF, FGF-2, IGF-I, and PDGF did notinhibit the TGF-β1 induced DNA synthesis.Conclusion: Our data indicate that ASMCs proliferate in response to TGF-β1, which is mediatedby phosphorylation of p38 and ERK1/2. These findings suggest that TGF-β1 which is expressed inairways of asthmatics may contribute to irreversible airway remodeling by enhancing ASMCproliferation.IntroductionAsthma is characterized by airway inflammation, hyperre-sponsiveness, and remodeling [1-3]. Severe asthmaticsdevelop irreversible airway obstruction, which may be aairway wall that may be due to frequent stimulation ofASMCs by contractile agonists, inflammatory mediators,and growth factors [2,4]. Based on observations made onthe pathogenesis of hyperproliferation at other sites, it isPublished: 03 January 2006Respiratory Research 2006, 7:2 doi:10.1186/1465-9921-7-2Received: 16 August 2005Accepted: 03 January 2006This article is available from: http://respiratory-research.com/content/7/1/2© 2006 Chen and Khalil; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 10(page number not for citation purposes)consequence of persistent structural changes includingincreased airway smooth muscle cell (ASMC) mass in thespeculated that a number of cytokines may be importantin regulating the proliferation of ASMCs. Of theseRespiratory Research 2006, 7:2 http://respiratory-research.com/content/7/1/2cytokines, transforming growth factor-beta1 (TGF-β1), amultifunctional polypeptide, is one of the most potentregulators of connective tissue synthesis and cell prolifer-ation [2,5-8].The source of TGF-β1 in the airways may be from theinflammatory cells recruited to the airways or from theresidential airway cells themselves such as bronchial epi-thelial cells and ASMCs [7,8]. We had previously demon-strated that all isoforms of TGF-β, as well as TGF-βreceptor (TβR) type I and II were expressed by ASMCs inhuman and rat lungs [9,10]. In addition, we had foundthat in models emulating airway injury, such as in vitrowounding of confluent monolayers [11,12], exposure toproteases [12,13], or cells in subconfluent conditions[12], ASMCs released biologically active TGF-β1, which inturn led to increase in connective tissue proteins such ascollagen I and fibronectin. Recently, we had reported thatgranulocyte macrophage-colony stimulating factor (GM-CSF), another cytokine found in asthmatic airways,increased connective tissue expression of bovine ASMCsin response to TGF-β1 by induction of TβRs [14]. TGF-β1is likely to play an important role in airway remodeling inasthmatics. For example, Minshall et al [5] demonstratedthat, as compared with the control subjects, both theexpression of TGF-β1 mRNA and TGF-β1 immunoreactiv-ity were increased in the airway submucous eosinophils,the cell that had been confirmed the presence of activeTGF-β1, and these increases were directly related to theseverity of the disorder. In a mouse model of airwayremodeling induced by OVA sensitization and challenge,increased TGF-β1 was demonstrated by ELISA and immu-nohistochemistry with increased peribronchial collagensynthesis, thickness of peribronchial smooth musclelayer, and α-smooth muscle actin immunostaining [15].Redington et al [6] found an increased TGF-β1 level in thebronchoalveolar lavage fluid from asthmatic patientscompared to normal controls. Recently, McMillan et al[16] demonstrated that anti-TGF-β antibody significantlyreduced peribronchiolar extracellular matrix deposition,ASMC proliferation, and mucus production in an allergeninduced murine asthma model.The effects of TGF-β1 on cell proliferation are more com-plex and context dependent [17,18]. For example, TGF-β1inhibits proliferation of epithelial and hematopoietic cells[19]; however, TGF-β1 induces proliferation of the mesen-chymal phenotype of cells such as fibroblasts, smoothmuscle cells, and myofibroblasts [20]. Even within mes-enchymal cells, the cell responses to TGF-β1 are highlyvariable. For example, TGF-β1 stimulates proliferation ofconfluent vascular and airway smooth muscle cells, butinhibits the proliferation of the same cells when they aresmooth muscle cells, but a high dose of TGF-β1 inhibitsthe proliferation of the same cells [20,25]. The duration ofTGF-β1 treatment also affects the cellular proliferativeresponse to TGF-β1. For example, Incubation of ASMCs orarticular chondrocytes for 24 hours with TGF-β1 inhibitedcell proliferation, whereas 48- or 72-hour incubationstimulates proliferation of the same cells [26,27].The proliferation of several phenotypes of cells is medi-ated by growth factor or cytokine induced mitogen-acti-vated protein kinases (MAPKs), a family of serine-threonine protein. MAPKs consist of extracellular signal-regulated kinase (ERK), p38 MAPK (p38), and c-Jun NH2-terminal kinase (JNK) [28]. The activation of MAPKs is akey component in signal transduction associated with cellproliferation [29]. Among the three MAPKs, ERK has beenwell studied and proven to play a major role in the signal-ling of ASMC proliferation [30-38]. The activation of ERKby various substances, such as epidermal growth factor(EGF), platelet-derived growth factor (PDGF), fibroblastgrowth factor-2 (FGF-2, also called basic fibroblast growthfactor, bFGF), insulin-like growth factor-I (IGF-I),thrombin, endothelin, phorbol esters, beta-hexosamini-dase A (an endogenous mannosyl-rich glycoprotein), and5-hydroxytryptamine (5-HT), increased ASMC prolifera-tion [30-38]. The inhibitors or antisense oligonucleotideof ERK blocked the proliferation induced by these sub-stances [30-37]. Activated ERK stimulates numerous tran-scription factors such as Elk-1, c-Jun, c-Fos, and c-Myc inthe nucleus. The transcription factors in turn regulate theexpression of genes required for DNA synthesis, such ascyclin D1. It has been demonstrated that active Ras andMAPK/ ERK kinase-1 (MEK1) (the upstream activator ofERK) each induced cyclin D1 promoter activity [36]. Elk-1 and activator protein-1 (c-Jun, c-Fos) reporter activationby mitogens was reduced by inhibition of MEK in humanASMCs [31]. In addition, inhibition of MEK attenuatesmitogen-induced increase in promoter activity, mRNA orprotein of cyclin D1 or c-Fos [30,32,38]. However, therole of p38 and JNK in mitogen-induced ASM prolifera-tion is not well known. In addition, little is known aboutthe role of MAPKs in TGF-β1 induced proliferation inASMCs.This study was designed to investigate the effect of TGF-β1on asmc proliferation and the role of mapks in the TGF-β1 induced changes of asmc proliferation. We found thatTGF-β1 increased asmc proliferation and the proliferativeeffects were mediated by phosphorylation of ERK1/2 andp38.Materials and methodsCell culturePage 2 of 10(page number not for citation purposes)subconfluent [21-24]. A low dose of TGF-β1 stimulatesproliferation of fibroblasts, chondrocytes, and arterialBovine trachea was obtained from a local slaughterhouse.An explanted culture of the smooth muscle tissue wasRespiratory Research 2006, 7:2 http://respiratory-research.com/content/7/1/2established as described previously with some modifica-tion [14]. Briefly, the associated fat and connective tissueswere removed in cold phosphate buffered saline (PBS)with antibiotic reagents (penicillin G 100 U/ml, strepto-mycin 100 µg/ml) and antimycotic reagent (amphotericinB 0.25 µg /ml). Then, the smooth muscle was isolated, cutinto 1–2 mm cubic size, and placed on culture dishes withDulbecco's modified Eagle's Medium (DMEM) supple-mented with 10% fetal bovine serum (FBS) and antibi-otic-antimycotic reagents. In an incubator at 37°C with ahumidified atmosphere (5% CO2-balanced air), ASMCsmigrated from the tissue explants and approached conflu-ence around the explants. The explanted tissue wasremoved, and the ASMCs remaining in the culture werepassaged with 0.05% trypsin/0.53 mM EDTA. Smoothmuscle cell identity was verified by phase contrast micro-scopy for appearance of "hill and valley formation" andby immunocytochemistry staining for α-smooth muscleactin and smooth muscle-specific myosin heavy chain(SM1 and SM2). For the experiments, the ASMCs in pas-sage 1–5 were plated at density of 10000 cells/cm2 inDMEM with 10% FBS and antibiotic reagents. All reagentsabove were from GIBCO BRL (Burlington, ON, Canada).The cell viability was determined with trypan blue (Sigma,St. Louis, Missouri) exclusion.Since previous studies reported varied responses of TGF-β1 on ASMCs, we first determined an optimal culture con-dition for conducting the experiments. ASMCs were cul-tured in 24-well plates in DMEM with 10% FBS tomedia: DMEM with 0.2% bovine serum albumin (BSA,from Fisher Scientific, Fair Lawn, NJ), DMEM with 0.5%FBS, and DMEM with 10% FBS. Then, the cells weretreated with 5 ng/ml of TGF-β1 (R&D Systems, Minneap-olis, MN) or 10% FBS in the same fresh medium for 1 dayfollowed by [3H]-thymidine incorporation and cell count-ing. As shown in Figure 1, increases in [3H]-thymidineincorporation occurred in all three conditions, but TGF-β1 and 10% FBS induced the strongest response in ASMCscultured in 0.2% BSA/DMEM. Similar results were alsoseen in the number of cells (data not shown). Therefore,we chose 0.2% BSA/DMEM as the serum-free mediumculture condition in which all further experiments wereperformed.Cell proliferation studyThis study was performed by [3H]-thymidine incorpora-tion and cell counting. Growth-arrested ASMCs weretreated in serum-free medium in 24-well plates. Then, forsome plates, [3H]-thymidine (1 µCi/ml, from ICN, Irvine,CA) was added for the final 4 hours and the incorporationwas terminated by washing the cells with PBS twice. Thecells were lysed with 0.2 N NaOH and the radioactivitywas counted with a scintillation counter (BeckmanLS5000CE). For other plates, the cells were washed withPBS, trypsinized and counted with a hemacytometer. Toconfirm the involvement of MAPKs in TGF-β1 inducedproliferation of ASMCs, the cells were pretreated for onehour with 10 µM of SB 203580, 50 µM of PD 98059, or10 µM of SP 600125, selective inhibitors of p38, MAPkinase kinase (MEK, which is upstream from ERK) andJNK, respectively (all from Calbiochem, San Diego, CA).Then 1 ng/ml of TGF-β1 was added to the medium andthe cells were cultured for 24 hours, followed by [3H]-thy-midine incorporation assay.Western blotting and immune detectionAfter treatment, ASMCs were washed with cold PBS anddetached by trypsin. Whole cell protein was extracted onice with lysis buffer (50 mM Tris-HCl pH 8.0, 0.15 MNaCl, 1% Triton-X-100, 0.1% SDS, 5 mg/ml sodiumdeoxycholate) in the presence of the protease inhibitors(as mentioned above) as well as phosphatase inhibitorsincluding 1 mM NaF and 1 mM Na3VO4 (Sigma). Proteinconcentration was measured using the Bradford methodwith a BioRad Protein Assay Reagent (BioRad; Hercules,CA). Protein extracts were separated by SDS-PAGE onpolyacrylamide SDS gels and then transferred onto aPVDF membrane (BioRad) as per Laemmli's method.After blockade with 5% milk in Tris-buffered saline con-taining 0.05% Tween-20, the membranes were incubatedovernight at 4°C with following primary antibodies (fromCell Signaling, Beverly, MA): anti-total or anti-phosphor-ASMC responses to TGF-β1 and serum in different culture conditionFigure 1ASMC responses to TGF-β1 and serum in different culture conditions. ASMCs were cultured with DMEM/10% FBS to confluence and then changed to DMEM/0.2% BSA, DMEM/0.5% FBS, or DMEM/10% FBS for 72 hours, fol-lowed by treatment with 5 ng/ml of TGF-β1 or 10% FBS for 24 hours prior to [3H]-thymidine incorporation assay. * p < 0.05, *** p < 0.001 compared to control of the same condi-tion. n = 4–6.******0500001000001500002000002500000.2%  BSA 0.5%  FBS 10%  FBS[3H]-TdR Incorporation (DPM) control TGF-ȕ1 10%  FCS********Page 3 of 10(page number not for citation purposes)confluence. After being washed with DMEM, the ASMCswere cultured for three days in one of following threeylated p38, ERK1/2 (which recognizes p42 and p44MAPK), and JNK (which recognizes p46 and p54 JNK).Respiratory Research 2006, 7:2 http://respiratory-research.com/content/7/1/2This was followed by incubating the blot with a HRP-con-jugated secondary antibody (Santa Cruz) for 1 hour atroom temperature. The target proteins on the membranewere then immunodetected by the ECL system (Amer-sham, Arlington Heights, IL) according to the manufac-turer's instruction. The equal loading of proteins wasconfirmed by immunodetecting the blots with anti-β-actin antibody (Sigma). Relative absorbance of the result-ant bands was determined using the Quantity One imag-ing system (BioRad).Statistical analysisThe results were expressed as mean ± standard error of themean (SEM). Student's t test and Kruskal-Wallis test com-bined with Dwass-Steel-Chritchlow-Fligner test were usedResultsTGF-β1 increased ASMC proliferationAll concentrations of TGF-β1 (0.1, 1 and 5 ng/ml)induced significant increase in [3H]-thymidine incorpora-tion by the ASMCs. Incubation of ASMCs with TGF-β1 for48 hours induced more proliferation than 24 hours ofincubation (Figure 2A). The TGF-β1 induced DNA synthe-sis was blocked by the addition of anti-TGF-β1 antibody(data not shown). TGF-β1 also induced a significant con-centration-dependent increase in cell numbers (Figure2B); however, the magnitude of the increased cell numberwas lower than the increased [3H]-thymidine incorpora-tion, suggesting that as a parameter of cell proliferation,[3H]-thymidine incorporation is more sensitive than cellnumber.TGF-β1 augmented serum-induced proliferationSerum contains a variety of mitogenic substances that mayenter the airways as protein exudates during airwayinflammation. ASMCs can respond synergistically to awide variety of mitogen combinations [39]. TGF-β mayinteract with these substances and affect ASMC prolifera-tion. To determine this, we treated confluent, serum-freeASMCs with 10% FBS in the absence or presence of TGF-β1 (1 ng/ml) for 48 hours and measured the changes ofthymidine incorporation and cell number. DNA synthesisand cell number were significantly increased after treat-ment with 10% FBS compared to the cells cultured inserum-free medium (Figure 3). The serum-inducedincreases in thymidine incorporation and cell numberwere further enhanced by addition of 1 ng/ml TGF-β1(Figure 3). Similar changes, to a lesser extent, wereobserved when 1% FBS was used (data not shown).Roles of MAPKs in TGF-β1 induced proliferationNext, we determined if MAPKs play any role in TGF-β1induced increase in proliferation. ASMCs were treatedwith 1 ng/ml of TGF-β1 for 1, 5, 30 minutes, 24 and 48hours, followed by extraction of the cellular protein. Theexpressions of total and phosphorylated p38, ERK1/2,and JNK were determined by Western analysis. TGF-β1induced rapid increases in phospho-p38 (Figure 4A) andphospho-JNK (Figure 4C), beginning as early as 1 minuteafter addition of TGF-β1 and lasting up to 24 hours forphospho-p38. However, the phosphorylation of JNK wasearly and brief in duration (Figure 4C). Longer treatment(48 hours) with TGF-β1 led to a decrease in both phos-pho-p38 and phospho-JNK. The TGF-β1 inducedincreases in phospho-ERK1/2 occurred only after 24-hourtreatment and this was not decreased by 48-hour treat-ment (Figure 4B). There was no change in the expressionof total p38, ERK1/2, and JNK. In addition, to confirmthat the TGF-β1 induced induction of phosphorylatedTGF-β1 concentration-dependently increased proliferation of ASMCsFigure 2TGF-β1 concentration-dependently increased prolif-eration of ASMCs. Confluent and growth-arrested ASMCs were incubated with various concentrations of TGF-β1 for 24 or 48 hours prior to [3H]-thymidine incorporation assay (A) or cell counting (B). Significant differences were detected at all concentrations of TGF-β1 treatment compared to the untreated control, p < 0.05 to p < 0.0001, n = 4–18.01002003004005000 0.1 1 5TGF-ȕ1 (ng/ml)[3H]-TdR Incorporation(% of control)24-hr treatment48-hr treatment501001502000 0.1 1 5TGF-ȕ1 (ng/ml)Cell number (% of control) ABPage 4 of 10(page number not for citation purposes)for the data analysis. Differences were considered statisti-cally significant when p < 0.05.p38, JNK, or ERK1/2 regulated cell proliferation, ASMCswere pretreated for one hour with SB 203580, PD 98059,Respiratory Research 2006, 7:2 http://respiratory-research.com/content/7/1/2or SP 600125, followed by 24-hour TGF-β1 treatment and[3H]-thymidine incorporation assay. The TGF-β1 inducedDNA synthesis was attenuated by SB 203580 or PD98059, but not SP 600125 (Figure 5). Furthermore, totaland phosphorylated p38, ERK1/2, and JNK were deter-mined using the cellular protein of ASMCs treated withTGF-β1 for 24 hours in the presence or absence of SB203580, PD 98059, or SP 600125. Western analysisrevealed that TGF-β1 induced phosphorylation of p38and ERK1/2 were inhibited by SB 203580, PD 98059,respectively (Figure 6). There were no changes in phos-phorylation of JNK between cells of control, TGF-β1, andSP 600125 plus TGF-β1 treatment (Figure 6). These datasuggest that TGF-β1 induced increase in proliferation maybe mediated by the activation of p38 and ERK1/2.Roles of FGF-2, PDGF, EGF and IGF-I in TGF-β1 induced proliferationTo examine if the TGF-β1 induced proliferation of ASMCsis a secondary effect mediated by other growth factors thathad been reported to be induced by TGF-β1 [20,23,40-42], ASMCs were treated with TGF-β1 in the absence orpresence of neutralizing antibodies against FGF-2, PDGF,EGF, and IGF-I (all from R&D Systems). [3H]-thymidineincorporation was performed after 48-hour treatmentwith TGF-β1. As shown in Figure 7, there were no signifi-cant differences in the DNA synthesis between TGF-β1treated ASMCs with and without these antibodies. Thedata suggest that TGF-β1 induced ASMC proliferation mayDiscussionIn this study we have demonstrated that TGF-β1 increasesproliferation in serum-free condition and enhancesserum-induced proliferation of confluent ASMCs. Thisobservation is consistent with the reports of others inwhich confluent ASMCs were treated with TGF-β1 in thepresence of 0.5 – 5% FBS [24,26,43]. These findings haveimportant clinical significance, because over expression ofTGF-β1 mRNA and protein was found in bronchial biop-sies from severe and moderate asthmatics [5,7,44,45]. Inaddition, it was reported that basal TGF-β1 levels in theairways were elevated in atopic asthma and that these lev-els increased further in response to allergen exposure [6].Most recently, it was found that C-509T SNP of the TGF-β1 gene is an important susceptibility locus for asthma[46]. Our previous data also demonstrated that woundedASMCs released biologically active TGF-β1, which in turninduced collagen and fibronectin synthesis [11,12].Therefore, it is conceivable that in chronic asthmatics withrepeated episode of injury and inflammation, TGF-β1 issynthesized and released into the airways or within thesmooth muscle cells of the airways. The release and per-sistent presence of TGF-β1 in asthmatic airways may grad-ually induce airway smooth muscle hypertrophy andhyperplasia. Moreover, our finding that TGF-β1 enhancesserum-induced ASMC proliferation may occur in asth-matic airways where there is inflammation leading toincrease in vascular permeability and leakage of plasmathat contains cytokines mitogenic for ASMCs. Our resultssuggest that the mitogenic effects of the cytokines wouldbe enhanced by TGF-β1, and augment the ASMC hyper-plasia and remodeling changes. The proliferative changes,combined with TGF-β1 induced connective tissue synthe-sis in ASMCs [11,12,14], would thicken the airway wall,reduce baseline airway caliber and exaggerate airway nar-rowing. Unlike Black and co-workers' finding that TGF-β1treatment for 24 hours and 48 hours led to inhibition andpromotion, respectively, of ASMC growth, in our presentstudy, both 24-hour and 48-hour treatment with TGF-β1induced increases in ASMC proliferation. The differencefor the cell response after 24-hour TGF-β1 treatment maybe due to the different culture condition. Black et altreated ASMCs in the presence of 2% serum in the culturemedium, while we did not use any serum when we treatedthe cells. Therefore, the different extent of serum-depriva-tion may affect the cell response to mitogens.Little is known about the mechanisms by which TGF-β1affects ASMC proliferation. In human ASMCs, it wasfound that TGF-β1 induced a 10–20 fold increase in insu-lin-like growth factor binding protein-3 (IGFBP-3) mRNAand protein and a 2-fold increase in cell proliferation,which was blocked by IGFBP-3 antisense or IGFBP-3 neu-TGF-β1 enhanced serum-induced proliferation of ASMCsFigure 3TGF-β1 enhanced serum-induced proliferation of ASMCs. Confluent and growth-arrested ASMCs were treated with 10% FBS in the absence or presence of TGF-β1 (1 ng/ml) for 48 hours and the changes of [3H]-thymidine incorporation (n = 9) and cell number (n = 6) were deter-mined. All values are % of untreated control cultured in 0.2% BSA/DMEM. p values indicated were compared to control (10% FBS only).0200400600800100012003H-TdR Cell numberProliferation (% of control)10% FBS10% FBS+TGF-ȕ1P=0.006P=0.0002 Page 5 of 10(page number not for citation purposes)not be mediated by these previously described TGF-β1inducible growth factors.tralizing antibody, suggesting IGFBP-3 mediates TGF-β1induced proliferation [43]. In cells other than ASMCs, itRespiratory Research 2006, 7:2 http://respiratory-research.com/content/7/1/2was suggested that release of PDGF mediated by TGF-β1induces mesenchymal cells proliferation [20,42,23]. Forarterial smooth muscle cell proliferation at low concentra-tions by stimulating autocrine PDGF-AA secretion [20].TGF-β1 increased expression of phosphorylated MAPKs in ASMCsFigure 4TGF-β1 increased expression of phosphorylated MAPKs in ASMCs. Confluent and growth-arrested ASMCs were incubated with 1 ng/ml of TGF-β1 for 1, 5, 30 minutes, 24 or 48 hours prior to protein extraction and Western analysis for phosphorylated or total p38 (Panel A), ERK1/2 (Panel B), and JNK (Panel C). * p < 0.05, ** p < 0.01, ** p < 0.001 compared to control, n = 4–10, C = control.Time of TGF-E1 treatment 0501001502002503003500 1' 5' 30' 24h 48hPhospho-p38(% of control)**** ****A0501001502002503003500 1' 5' 30' 24h 48hPhospho-ERK 1/2(% of control)*B0501001502002503003500 1' 5' 30' 24h 48hPhospho-JNK(% of control)*** **C䣣-p38total-p38C    1’   5’  30’    C   24h   C   48h 䣣-JNK total-JNK C    1’   5’  30’    C   24h    C  48h 䣣-ERK1/2 total-ERK1/2 C    1’   5’  30’    C   24h   C  48h Page 6 of 10(page number not for citation purposes)example, Battegay and co-workers found that TGF-β1induced human dermal fibroblasts, chondrocytes, andOther studies showed that TGF-β1 induced markedgrowth responses, alone or in combination with EGF,Respiratory Research 2006, 7:2 http://respiratory-research.com/content/7/1/2FGF-2, or PDGF-BB, that were largely independent ofPDGF-AA [41]. We had recently demonstrated that treat-ment of primary interstitial pulmonary fibroblasts withTGF-β1 released large quantity of FGF-2, which led to pro-liferation. This TGF-β1 induced proliferation of thefibroblasts was mediated by FGF-2, but not EGF, IGF-I orPDGF [[47] and our unpublished data). In our presentstudy, we used neutralizing antibodies against EGF, FGF-2, IGF-I or PDGF to examine the possible role of thesegrowth factors in TGF-β1 induced ASMC proliferation.However, these antibodies did not block the TGF-β1induced DNA synthesis. Our data suggest that the TGF-β1induced proliferation of ASMCs in our model might beindependent of the growth factors previously reported tomediate the proliferative effects of TGF-β1 in mesenchy-mal cells.Phosphorylation of ERK1/2 has been reported to mediatemitogen-induced proliferation, while the phosphoryla-tion of JNK and p38 are activated by a variety of non-spe-cific stimuli such as changes in oxidation, osmolarity, andinflammatory cytokines [28,48]. The important roles ofMAPKs activation in ASMC proliferation induced byendothelin-1, thrombin, FGF-2, PDGF, EGF, IGF-I, 5-HTand so on have been reported [29,49,30-37]. However, itis not known if MAPKs mediate TGF-β1 induced ASMCproliferation. In this study, for the first time, we havedemonstrated that TGF-β1 induced proliferation ofof induction. Since the inhibitors of p38 and ERK blockedTGF-β1 induced proliferation, our data suggest that theactivation of p38 and ERK is important for the TGF-β1induced increase in ASMC proliferation. Our results arepartly supported by another study using tracheal smoothmuscle cells, which demonstrated that activation of p38pathway by TGF-β modulated smooth muscle migrationand remodeling [50]. In our study, there are some differ-ences in the time required for activation of MAPKs afterTGF-β1 stimulation amongst the 3 MAPKs. P38 and JNKwere rapidly activated by TGF-β1, which was as early as 1minute. However, the activation of ERK1/2 required pro-longed treatment with TGF-β1 (24 hours). The activationof JNK lasted only 5 min, and the blockade of JNK activa-tion failed to inhibit the ASMC proliferation induced by24-hour of TGF-β1 treatment, indicating that the activa-tion of JNK may not be important in mediating TGF-β1induced proliferation of ASMCs. Interestingly, our findingis similar to a previous report using human lung fibrob-lasts, in which TGF-β1 activated ERK and p38 but not JNK[40]. The authors used 30-minute, 2-, 6-, 16-, and 24-hourTGF-β1 treatment and found that phosphorylation of p38began within 30 minutes, while ERK1/2 activation beganat 2 hour with maximal induction by 16 hour. They alsofound that activator protein-1(AP-1) binding dependedon ERK1/2 but not p38 activation. However, using fibrob-lasts, we and others reported that TGF-β1 activated JNKand p38, but not ERK1/2 [47,51]. In another study, aninteraction between ERK and p38 in macrophages wasproposed in which TGF-β1 activated ERK, which in turnup-regulated MAPK phosphatase-1, thereby inactivatingp38 [52]. A recent study using selective inhibitors of thethree MAPKs [53] showed that inhibition of one of theintracellular pathway was sufficient to inhibit IL-1βinduced ASMC proliferation and simultaneous inhibitiondid not lead to further reduction in the proliferation, sug-gesting the three major MAPK pathways are independentregulators of IL-1β dependent proliferation of rat ASMCs.Taken together, the above data indicate that one or moreMAPK can be activated by TGF-β1 and the differentMAPKs may act through different pathways in TGF-β1induced proliferation of mesenchymal cells.Our findings differ from a study by Cohen et al [54] inwhich TGF-β1 alone had no effect on human ASMC pro-liferation, but TGF-β1 inhibited EGF- and thrombin-induced DNA synthesis, which was independent of ERKactivation. However, it is somewhat incomparable withour data, because in addition to the species difference, thecells they used had no proliferative response to TGF-β1alone, and they did not show whether TGF-β1 affected theactivation of MAPKs. In addition, they used 5 µg/ml ofinsulin in their serum-free medium, which may affect theEffects of MAPKs inhibitors on TGF-β1 induced increase of proliferation in ASMCsFigure 5Effects of MAPKs inhibitors on TGF-β1 induced increase of proliferation in ASMCs. Confluent and growth-arrested ASMCs were pretreated for 1 hour with SB 203580, PD 98059, or SP 600125, prior to 24-hour treat-ment with 1 ng/ml of TGF-β1 (T). DNA synthesis was meas-ured by [3H]-thymidine incorporation assay. Inhibition of phosphorylated p38 and ERK1/2 reduced TGF-β1 induced DNA synthesis. ## p < 0.01 compared to untreated control (C), ** p < 0.01, *** p < 0.001 compared to T, n = 7–8.050100150200250C T SB SB+T PD PD+T SP SP+T[3H]-TdR Incorporation(DPM % of control)##*****Page 7 of 10(page number not for citation purposes)ASMCs is associated with increased expression of phos-phorylated ERK1/2, p38, and JNK with different kineticscell's response to growth factors or downstream media-tors.Respiratory Research 2006, 7:2 http://respiratory-research.com/content/7/1/2The effects of TGF-β are mediated by TβR I and TβR II,which phosphorylate Smad 2 and Smad 3. The phospho-rylated Smad 2 and Smad 3 bind Smad 4. The resultantcomplex translocates to the nucleus and activates theexpression of target genes. It was demonstrated that Ras/MEK/ERK pathway is partially required in order for TGF-βto activate Smad , and is also required for the Smad-medi-ated induction of connective tissue growth factor (CTGF)by TGF-β2 . In addition, it was reported that constitutiveactivation of p38 pathway-induced transcriptional activa-tion was enhanced synergistically by coexpression ofSmad2 and Smad 4, and was inhibited by expression of C-terminal truncated, dominant negative Smad 4 . ZhangFos) among the targets of the MARK pathways . Mostrecently, in cultured airway smooth muscle cells, Xie andcoworkers  found that TGF-β1 induced a significant acti-vation of Smad 2/3 and translocation of phospho-Smad2/3 and Smad 4 from cytosol to nucleus, as well as a time-and concentration-dependent expression of CTGF geneand protein. The TGF-β1 induced phosphorylation ofSmad 2/3 and the expression of CTGF mRNA and proteinwere all blocked by the inhibition of ERK and JNK, butnot by the inhibition of p38 and phosphatidylinositol 3-kinase (PI3K). The evidences given emphasize that there isa stimulatory interaction between MAPK pathway andSmad pathway in the context of TGF-β signaling. ThisEffects of MAPKs inhibitors on TGF-β1 induced activation of MAPKsFigure 6Effects of MAPKs inhibitors on TGF-β1 induced activation of MAPKs. Confluent and growth-arrested ASMCs were pretreated for 1 hour with SB 203580, PD 98059, or SP 600125, prior to 24-hour treatment with 1 ng/ml of TGF-β1 (T), fol-lowed by protein extraction and Western analysis for phosphorylated or total p38 (Panel A), ERK1/2 (Panel B), and JNK (Panel C). The blots are representatives of 3 independent experiments. C = control. ** p < 0.01 *** p < 0.001 compared to T.䣣-ERK1/2 total-ERK1/2 C          T PD   PD+T䣣-p38total-p38C         T       SB   SB+T䣣-JNKtotal-JNK C         T      SP   SP+TABC050100150200250C T SB SB+ Tphospho-p38(% of control)050100150200250C T PD PD + Tphospho-ERK1/2(% of control)050100150200250C T S P S P +Tphospho-JNK(% of control)*****Page 8 of 10(page number not for citation purposes)and coworkers demonstrated a direct interaction betweenSmad 3/4 and two transcriptional factors (c-Jun and c-interaction may play an important role in the airwayremodeling. For example, CTGF is a downstream media-Respiratory Research 2006, 7:2 http://respiratory-research.com/content/7/1/2tor of TGF-β fibrotic effects and is constitutively overex-pressed in fibrotic airways. It is not clear whether thisinteraction is involved in the ASMC proliferation, how-ever, it is possible in our present work that the TGF-β1induced expression of MAPKs cross-talks with Smad path-way, and they act together which results in proliferationand fibrosis.ConclusionIn conclusion, our results demonstrate that TGF-β1increases ASMC proliferation, and also enhances serum-induced ASMC proliferation. In addition, the activation ofp38 and ERK play an important role in mediating theTGF-β1 induced proliferation by ASMCs. These findingssuggest that TGF-β1 which is expressed in airways of asth-matics may contribute to irreversible airway remodelingby enhancing ASMC proliferation.Competing interestsThe author(s) declare that they have no competing inter-ests.Authors' contributionsGC carried out all the experiments, wrote the manuscriptand helped with the intellectual development of the work.NK obtained funding for the work, initiated and sup-ported the intellectual development of the work.AcknowledgementsReferences1. Bousquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM: Asthma.From bronchoconstriction to airways inflammation andremodeling.  Am J Respir Crit Care Med 2000, 161:1720-1745.2. Elias JA: Airway remodeling in asthma. Unanswered ques-tions.  Am J Respir Crit Care Med 2000, 161:S168-S171.3. Stewart AG: Airway wall remodelling and hyperresponsive-ness: modelling remodelling in vitro and in vivo.  Pulm Pharma-col Ther 2001, 14:255-265.4. 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