Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

Abstract

In response to transforming growth factor β1 (TGFβ) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFβ has been shown to increase the expression of specific αv integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFβ1 increased both β3 integrin subunit mRNA and protein levels as well as surface expression of αvβ3 in human lung fibroblasts. TGFβ1-induced αvβ3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of β3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or αvβ3 blocking antibody prevented the increase in β3 but not β5 integrin expression. In addition, echistatin inhibited TGFβ1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFβ1-induced expression of αvβ3 and p38 MAPK phosphorylation but not β5 integrin expression and Smad3 activation. The TGFβ1-induced αvβ3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-κB-dependent pathways. Our results demonstrate that TGFβ1 induces αvβ3 integrin expression via a β3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFβ1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing. In response to transforming growth factor β1 (TGFβ) stimulation, fibroblasts modify their integrin repertoire and adhesive capabilities to certain extracellular matrix proteins. Although TGFβ has been shown to increase the expression of specific αv integrins, the mechanisms underlying this are unknown. In this study we demonstrate that TGFβ1 increased both β3 integrin subunit mRNA and protein levels as well as surface expression of αvβ3 in human lung fibroblasts. TGFβ1-induced αvβ3 expression was strongly adhesion-dependent and associated with increased focal adhesion kinase and c-Src kinase phosphorylation. Inhibition of β3 integrin activation by the Arg-Gly-Asp tripeptide motif-specific disintegrin echistatin or αvβ3 blocking antibody prevented the increase in β3 but not β5 integrin expression. In addition, echistatin inhibited TGFβ1-induced p38 MAPK but not Smad3 activation. Furthermore, inhibition of the Src family kinases, but not focal adhesion kinase, completely abrogated TGFβ1-induced expression of αvβ3 and p38 MAPK phosphorylation but not β5 integrin expression and Smad3 activation. The TGFβ1-induced αvβ3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-κB-dependent pathways. Our results demonstrate that TGFβ1 induces αvβ3 integrin expression via a β3 integrin-, c-Src-, and p38 MAPK-dependent pathway. These data identify a novel mechanism for TGFβ1 signaling in human lung fibroblasts by which they may contribute to normal and pathological wound healing. One of the key events in wound repair is the infiltration of fibroblasts from surrounding tissue to the extracellular matrix (ECM) 2The abbreviations used are: ECM, extracellular matrix; TGFβ1, transforming growth factor β1; FAK, focal adhesion kinase; FRNK, FAK-related non-kinase; GFP, green fluorescent protein; SFK, Src family kinase; EGF, epidermal growth factor; RGD, Arg-Gly-Asp tripeptide motif; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; PDTC, pyrrolidine dithiocarbamate; mAb, monoclonal antibody; siRNA, short interfering RNA; Ab, antibody; FN, fibronectin; WB, Western blot; FBS, fetal bovine serum; PBS, phosphate-buffered saline; MFI, mean fluorescence intensity. 2The abbreviations used are: ECM, extracellular matrix; TGFβ1, transforming growth factor β1; FAK, focal adhesion kinase; FRNK, FAK-related non-kinase; GFP, green fluorescent protein; SFK, Src family kinase; EGF, epidermal growth factor; RGD, Arg-Gly-Asp tripeptide motif; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; PDTC, pyrrolidine dithiocarbamate; mAb, monoclonal antibody; siRNA, short interfering RNA; Ab, antibody; FN, fibronectin; WB, Western blot; FBS, fetal bovine serum; PBS, phosphate-buffered saline; MFI, mean fluorescence intensity. in which they proliferate and differentiate into myofibroblasts. Under normal conditions myofibroblasts play a crucial role in ECM deposition and subsequent wound contraction and then disappear as the fibrotic response diminishes and normal structure and function are achieved (1Hinz B. Phan S.H. Thannickal V.J. Galli A. Bochaton-Piallat M.L. Gabbiani G. Am. J. Pathol. 2007; 170: 1807-1816Abstract Full Text Full Text PDF PubMed Scopus (1564) Google Scholar). However, their retention, uncontrolled proliferation, and excessive synthesis of ECM proteins represents a pathologic process that ultimately results in fibrosis (2Paine III, R. Ward P.A. Am. J. Med. 1999; 107: 268-279Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Both fibroblast proliferation and differentiation, as well as ECM protein synthesis, are profoundly influenced by growth factors such as TGFβ as well as cell adhesion (3Scaffidi A.K. Moodley Y.P. Weichselbaum M. Thompson P.J. Knight D.A. J. Cell Sci. 2001; 114: 3507-3516Crossref PubMed Google Scholar, 4Scaffidi A.K. Petrovic N. Moodley Y.P. Fogel-Petrovic M. Kroeger K.M. Seeber R.M. Eidne K.A. Thompson P.J. Knight D.A. J. Biol. Chem. 2004; 279: 37726-37733Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 5Schoppet M. Chavakis T. Al-Fakhri N. Kanse S.M. Preissner K.T. Lab. Investig. 2002; 82: 37-46Crossref PubMed Scopus (47) Google Scholar, 6Thannickal V.J. Lee D.Y. White E.S. Cui Z. Larios J.M. Chacon R. Horowitz J.C. Day R.M. Thomas P.E. J. Biol. Chem. 2003; 278: 12384-12389Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). Adhesion of cells to ECM is mediated by a family of transmembrane proteins known as integrins that are expressed on the cell surface as α/β heterodimers (7Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6757) Google Scholar, 8Sheppard D. Physiol. Rev. 2003; 83: 673-686Crossref PubMed Scopus (119) Google Scholar). Importantly, integrins not only support cell attachment but also act in concert with receptors for several growth factors, including TGFβ, to regulate survival, migration, proliferation, and differentiation of fibroblastic, epithelial, and endothelial cells (reviewed in Refs. 7Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6757) Google Scholar, 8Sheppard D. Physiol. Rev. 2003; 83: 673-686Crossref PubMed Scopus (119) Google Scholar). Over the past few years a close relationship between αv integrins (recognizing RGD motif) and TGFβ signaling pathways has been identified (9Yang Z. Mu Z. Dabovic B. Jurukovski V. Yu D. Sung J. Xiong X. Munger J.S. J. Cell Biol. 2007; 176: 787-793Crossref PubMed Scopus (248) Google Scholar). These include activation of latent TGFβ complexes by αvβ6 and αvβ8 integrins in airway epithelium (8Sheppard D. Physiol. Rev. 2003; 83: 673-686Crossref PubMed Scopus (119) Google Scholar, 10Munger J.S. Huang X. Kawakatsu H. Griffiths M.J. Dalton S.L. Wu J. Pittet J.F. Kaminski N. Garat C. Matthay M.A. Rifkin D.B. Sheppard D. Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1620) Google Scholar), augmented TGFβ signaling by αvβ3 and αvβ5 integrins in scleroderma fibroblasts (11Asano Y. Ihn H. Yamane K. Jinnin M. Mimura Y. Tamaki K. J. Immunol. 2005; 175: 7708-7718Crossref PubMed Scopus (194) Google Scholar), and TGFβ receptor type II (TGFβRII)-αvβ3 integrin interaction-dependent proliferation and differentiation of human lung fibroblasts (4Scaffidi A.K. Petrovic N. Moodley Y.P. Fogel-Petrovic M. Kroeger K.M. Seeber R.M. Eidne K.A. Thompson P.J. Knight D.A. J. Biol. Chem. 2004; 279: 37726-37733Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Previous studies have also identified several molecules as inducers of αv integrin expression in various tissue culture systems (12Ignotz R.A. Massague J. Cell. 1987; 51: 189-197Abstract Full Text PDF PubMed Scopus (369) Google Scholar, 13Lai C.F. Feng X. Nishimura R. Teitelbaum S.L. Avioli L.V. Ross F.P. Cheng S.L. J. Biol. Chem. 2000; 275: 36400-36406Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 14Sheppard D. Cohen D.S. Wang A. Busk M. J. Biol. Chem. 1992; 267: 17409-17414Abstract Full Text PDF PubMed Google Scholar). It was also reported that growth factors are able to activate integrins and that this activation provided an additional mechanism for a growth factor to induce a broad spectrum of cellular responses (15Trusolino L. Serini G. Cecchini G. Besati C. Ambesi-Impiombato F.S. Marchisio P.C. De Filippi R. J. Cell Biol. 1998; 142: 1145-1156Crossref PubMed Scopus (97) Google Scholar, 16Byzova T.V. Goldman C.K. Pampori N. Thomas K.A. Bett A. Shattil S.J. Plow E.F. Mol. Cell. 2000; 6: 851-860Abstract Full Text Full Text PDF PubMed Google Scholar, 17Sekimoto H. Eipper-Mains J. Pond-Tor S. Boney C.M. Mol. Endocrinol. 2005; 19: 1859-1867Crossref PubMed Scopus (32) Google Scholar). Recently we demonstrated that TGFβ1 not only synergistically interacts with αvβ3 integrins but also induces their gene transcription in human lung fibroblasts (4Scaffidi A.K. Petrovic N. Moodley Y.P. Fogel-Petrovic M. Kroeger K.M. Seeber R.M. Eidne K.A. Thompson P.J. Knight D.A. J. Biol. Chem. 2004; 279: 37726-37733Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). However, the mechanisms involved in this process are still unknown. Therefore, this study was undertaken to delineate the signaling mechanisms that mediate αvβ3 up-regulation in response to TGFβ1 stimulation. The results of this study demonstrate that TGFβ1-dependent induction of β3 integrin expression does not involve Smad2/3 or Sp1 transcription factors, but it is mediated by selective and specific activation of the integrin itself and c-Src and p38 MAPK pathways.EXPERIMENTAL PROCEDURESPlasmids and Pharmacological Inhibitors—The pcDNA3-Smad7 plasmid was provided by Dr. Steven Mutsaers (University of Western Australia), and the pcDNA3-p38-KM (kinase mutant) plasmid was provided by Dr. Kun Liang Guan (University of Michigan). The GFP-FRNK (FAK-related non-kinase)-expressing adenovirus (Adv-GFP-FRNK) and adenovirus expressing GFP alone (Adv-GFP) were kindly provided by Dr. Allen M. Samarel (the Cardiovascular Institute, Loyola University Medical Center). The pharmacological inhibitors SB203580, UO126, the Src inhibitor, PP2, and its inactive isomer PP3 were purchased from Calbiochem, and SB202190, wortmannin, the β3 selective disintegrin echistatin from Echis carinatus, Src inhibitor SU6656, Sp1 inhibitor mithramycin A from Streptomyces plicatus, and the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma.Cell Culture—Normal human diploid lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured at 37 °C and 5% CO2 in F-12K Nutrient Mixture medium (F-12K) supplemented with 10% FBS (Invitrogen) and antibiotics before they reached 90% confluence. Prior to the experiments cells were washed extensively in PBS and quiesced in serum-free F-12K for 24 h.Flow Cytometry—HFL-1 cells were cultured to 90% confluence and stimulated with recombinant human TGFβ1 (Pepro-Tech, Rocky Hill, NJ) or epidermal growth factor (EGF) (Sigma) in serum-free conditions for the indicated time periods. For experiments examining signaling pathways, cells were pretreated with pharmacological inhibitors of specific signaling molecules for 40 min prior to the addition of TGFβ1. Adherent cells were collected after trypsin/EDTA immersion, and surface expression of integrins was allowed to recover for 30 min in PBS with 10% FBS at room temperature. This step also served to block nonspecific antibody binding. Cells were then fixed with 2% paraformaldehyde for 10 min on ice. Cell surface expression of αvβ3 integrin was analyzed using an anti-human αvβ3 integrin antibody (mouse IgG1, clone LM609, Chemicon, Temecula, CA) and normal mouse IgG1 (Santa Cruz Biotechnology, Santa Cruz, CA) as an isotype control, followed by incubation with phycoerythrin-conjugated goat anti-mouse F(ab′)2 (Cedarlane Laboratories, Ontario, Canada). Cell-associated fluorescence was acquired by a Coulter EPICS XL flow cytometer (Beckman Coulter, Ontario, Canada) and analyzed using Win-MDI 2.8 software.RNA Extraction and Real Time Reverse Transcription-PCR— See on-line supplemental material for details.Transfection—HFL-1 were seeded at 1 × 105 cells per well in 6-well tissue culture plates for 24 h prior to transfection. The cells were transiently transfected with 2 μg of mouse Smad7 DNA using Lipofectamine Plus (Invitrogen) for 24 h according to the manufacturer's instructions. Confirmation of function was determined by Western blot (WB) analysis of α-smooth muscle actin expression in TGFβ1-stimulated cells (see supplemental Fig. 1SA). Human Smad3-specific chimera-RNA interference (Smad3 siRNA) (Abnova Corp, Taipei City, Taiwan) and nonsilencing control siRNA (Qiagen, Ontario, Canada) were transfected into HFL-1 cells by using HiPerFect transfection reagent (Qiagen) as instructed by the manufacturer. Forty eight hours after transfection cells were washed with fresh culture medium and further stimulated with TGFβ1 for 24 h. siRNA transfection efficiency was determined by WB (supplemental Fig. 1SB). The mammalian expression plasmids, pcDNA3-p38-KM, pcDNA3 (empty vector control), and pcDNA3-GFP (transfection efficiency control) were transfected into lung fibroblasts using FuGENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's instructions. The optimal ratio of FuGENE 6 (μl) to DNA (μg) was determined to be 6:1 for HFL-1 cells (supplemental Fig. 2SA). Expression of plasmids was monitored by WB for p38 MAPK expression and by fluorescence microscopy for GFP (data not shown). Following transfection, cells were incubated with 10% FBS in media for 24 h and then with 1% FBS for the next 24 h. Medium was changed, and cells were cultured for an additional 18 h in the presence or absence of TGFβ1. Replication-defective adenoviruses encoding GFP-FRNK fusion protein and GFP alone were amplified and purified using HEK-293 cells as described (18Heidkamp M.C. Bayer A.L. Kalina J.A. Eble D.M. Samarel A.M. Circ. Res. 2002; 90: 1282-1289Crossref PubMed Scopus (111) Google Scholar). Preliminary experiments determined that a concentration of 50–100 particles of Adv-GFP-FRNK and Adv-GFP per cell strongly induced the expression of these proteins (supplemental Fig. 2SB) and infected virtually every fibroblast (∼90% of GFP positive cells by flow cytometry) after 48 h of exposure (data not shown). After infection with Adv-GFP-FRNK and Adv-GFP, cells were cultured in the presence of serum for 24 h, then serum-starved for additional 24 h, and stimulated with TGFβ1 for the indicated time periods.Western Blotting—After TGFβ1 stimulation, control or cell signaling inhibitor-pretreated cell monolayers were lysed in protein extraction buffer with protease and phosphatase inhibitor cocktails (Sigma). Equal concentrations of protein lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with a mixture of primary antibodies. p38 MAPK phosphorylation was determined using mouse mAb against human phosphorylated p38 MAPK (Thr180/Tyr182) and rabbit polyclonal antibodies against human p38 MAPK (both from Cell Signaling Technology, Danvers, MA). FAK phosphorylation was determined using mouse mAb against human phosphorylated FAK (pY397) (BD Biosciences) and rabbit polyclonal Ab against C-terminal region of human FAK (pp125FAK, Sigma). c-Src and Smad3 phosphorylation was determined using rabbit mAb against phospho-Src (Tyr-416) (Cell Signaling Technology), rabbit polyclonal Ab against c-Src (Santa Cruz Biotechnology), and rabbit mAb against phospho-Smad3 (Ser423/425) (Epitomics, Burlingame, CA), respectively, and anti-β-tubulin mAb (Upstate Biotechnology Inc., Lake Placid, NY) to control equal protein loading. Expression of β3 integrin chain, β5 integrin chain, and fibronectin was detected with mouse mAb (BD Biosciences) or rabbit polyclonal Ab (Cell Signaling Technology) against human β3 integrin, polyclonal rabbit Ab against human β5 integrin (Abcam, Cambridge, MA), and cellular fibronectin (Chemicon), respectively. Detection was performed with IR700 and IR800 anti-mouse and anti-rabbit antibodies (Cell Signaling Technology) and the Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE) using the manufacturer's protocol. Density of the bands was analyzed with Odyssey software 1.1 (LI-COR Biotechnology) using two infrared channels independently. The results are expressed as a phosphorylated protein/nonphosphorylated protein density ratio or protein/β-tubulin density ratio.Statistical Analysis—Data are expressed as mean ± S.E. of at least three independent experiments. Statistical comparisons were performed using ANOVA with post hoc Fisher's protected least significant difference. Probability values were considered significant if they were less than 0.05. All tests were done using StatView 5.0 software (SAS Institute Inc., Cary, NC).RESULTSTGFβ1 Increases β3 Protein Expression and Enhances Cell Surface Expression of αvβ3 Integrin on Human Lung Fibroblasts—Recently we showed that TGFβ1 increased β3 steady-state mRNA expression (4Scaffidi A.K. Petrovic N. Moodley Y.P. Fogel-Petrovic M. Kroeger K.M. Seeber R.M. Eidne K.A. Thompson P.J. Knight D.A. J. Biol. Chem. 2004; 279: 37726-37733Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Consistent with the increased β3 transcription, fibroblasts also increased β3 subunit and αvβ3 integrin expression on the cell surface after exposure to TGFβ1 at a concentration of 10 ng/ml and higher. As can be seen in Fig. 1A, β3 protein levels significantly increased after 24 h of exposure to TGFβ1 at a concentration of 10 ng/ml. As shown in Fig. 1B, surface expression of αvβ3 heterodimer was also significantly elevated after cell stimulation with TGFβ1 (60% increase in MFI and 2-fold increase in the percentage of αvβ3-positive cells). Increased integrin expression on the cell surface was observed for up to 48 h, although the magnitude of expression was not significantly different from that seen at 24 h (data not shown). In contrast, EGF treatment did not significantly modify β3 protein production or surface expression of αvβ3 after 24 h of exposure (Fig. 1, A and B). Moreover, when EGF was simultaneously added with TGFβ1, it abrogated both TGFβ1-induced β3 subunit and αvβ3 cell surface expression (Fig. 1, A and B). In parallel we examined the effect of TGFβ1 on another αv partner, the β5 integrin. We found that incubation of HFL-1 with TGFβ1 for 18 h induced robust expression of αvβ5 (supplemental Fig. 3S) by increasing β5 protein production (Fig. 1C) similar to the effects on αvβ3 expression. In addition, both removal of exogenously added TGFβ1 (washing) and neutralizing of endogenously produced TGFβ with a pan-TGFβ blocking antibody (clone 1D11), dramatically attenuated the effect of TGFβ1 on β3 and β5 integrin expression (Fig. 1C). These results demonstrate that the TGFβ1 effects on αvβ3 and αvβ5 integrin expression are: 1) associated with up-regulation of corresponding β integrin chain expression, and 2) highly specific and not mediated by a secondary mediator.TGFβ1-induced Expression of αvβ3 Integrin by Human Lung Fibroblasts Does Not Require Smad and Sp1—In our previous studies we have shown that both Smad2 and p38 MAPK were activated in human lung fibroblasts within 10 min of TGFβ1 stimulation, and peak activation was reached at 1 h (19Horowitz J.C. Lee D.Y. Waghray M. Keshamouni V.G. Thomas P.E. Zhang H. Cui Z. Thannickal V.J. J. Biol. Chem. 2004; 279: 1359-1367Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In pilot experiments we found that both TGFβ1-induced Smad3 and p38 MAPK phosphorylation was still detectable after 18 h and correlated with the increased levels of β3 and β5 integrins (supplemental Fig. 4S and Fig. 1C).We used two approaches to evaluate the role of Smad signaling in regulating the effect of TGFβ1 on integrin expression. In the first set of experiments, a murine Smad7-expressing construct was used. Although mouse Smad7 is 95% homologous with the human cDNA, experiments investigating the effect of this construct on TGFβ1-induced α-smooth muscle actin expression were performed. TGFβ1-induced increase in α-smooth muscle actin expression in fibroblasts was completely abolished by Smad7 overexpression, confirming that the mouse protein is functional in human fibroblasts (supplemental Fig. 1SA). However, transfection of the Smad7 construct did not significantly influence the effect of TGFβ1 on αvβ3 expression (Fig. 2A). To further prove that TGFβ1 effect on β3 integrin expression is Smad-independent, fibroblasts were transfected with control or Smad3 siRNA, and the level of β3 and β5 integrin expression was determined by immunoblotting. The control experiments showed that total and Ser423/425-phosphorylated Smad3 protein was knocked down by 60% compared with control (supplemental Fig. 1SB). In keeping with the results from Smad7 overexpression, inhibition of Smad3 did not influence the expression of β3 integrin induced by TGFβ1. In contrast, Smad3 siRNA significantly attenuated β5 integrin expression induced by TGFβ1 (Fig. 2B).FIGURE 2Effect of TGFβ1 on αvβ3 expression is Smad2/3- and Sp1-independent. A, fibroblasts transiently transfected with Smad7 cDNA or transfection reagent alone were stimulated with TGFβ1 (10 ng/ml) for 24 h, and αvβ3 integrin cell surface expression was analyzed by flow cytometry as described under “Experimental Procedures.” Values are expressed as a percentage above control MFI of three independent experiments ± S.E. B, HFL-1 cells were transfected with human Smad3 inhibiting chimera-RNA interference or control siRNA, and stimulated with TGFβ1 (10 ng/ml) for 24 h. Treatment with Smad3 siRNA abrogated TGFβ1-induced β5 integrin but did not change expression of β3 integrin in HFL-1 cells. Protein levels were determined by densitometry, and data are expressed as a percent of protein compared with transfection reagent control (TrR) (mean ± S.E., n = 3; *, p < 0.05 compared with control siRNA and 1 nm Smad3 siRNA). C, HFL-1 cells were treated with different concentrations of the Sp1 inhibitor mithramycin A for 40 min and then stimulated with TGFβ1 (10 ng/ml) for 18 h. β3 integrin subunit, fibronectin, and β-tubulin expression were determined by immunoblotting. One representative immunoblot of two independent experiments demonstrates that mithramycin A in a concentration-dependent manner attenuates up-regulatory effect of TGFβ1 on fibronectin production, whereas it has no effects on spontaneous and TGFβ1-induced β3 integrin production of human lung fibroblasts.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cooperation between the Smad proteins and the transcription factor Sp1 may represent a general mechanism for conferring TGFβ1 inducibility of several genes, including integrins (13Lai C.F. Feng X. Nishimura R. Teitelbaum S.L. Avioli L.V. Ross F.P. Cheng S.L. J. Biol. Chem. 2000; 275: 36400-36406Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 20Larouche K. Leclerc S. Salesse C. Guerin S.L. J. Biol. Chem. 2000; 275: 39182-39192Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We sought to determine whether Sp1 was involved in the up-regulation of αvβ3 integrin induced by TGFβ1. As shown in Fig. 2C, the Sp1 inhibitor mithramycin had no effect on β3 subunit, but it efficiently prevented the increase in fibronectin expression induced by TGFβ1. Together these results indicate that the Smad/Sp1 pathway does not play a major role in TGFβ1-induced up-regulation of αvβ3 integrins on human lung fibroblasts.TGFβ1-induced β3 Expression Requires Cell Adhesion and Is Enhanced by Integrin Activation with ECM Proteins—Our previous studies have demonstrated that several TGFβ-mediated effects in human lung fibroblasts are adhesion- and integrin-dependent (3Scaffidi A.K. Moodley Y.P. Weichselbaum M. Thompson P.J. Knight D.A. J. Cell Sci. 2001; 114: 3507-3516Crossref PubMed Google Scholar, 4Scaffidi A.K. Petrovic N. Moodley Y.P. Fogel-Petrovic M. Kroeger K.M. Seeber R.M. Eidne K.A. Thompson P.J. Knight D.A. J. Biol. Chem. 2004; 279: 37726-37733Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 6Thannickal V.J. Lee D.Y. White E.S. Cui Z. Larios J.M. Chacon R. Horowitz J.C. Day R.M. Thomas P.E. J. Biol. Chem. 2003; 278: 12384-12389Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). In preliminary experiments we found that TGFβ1 was unable to increase αvβ3 integrin expression in cells in suspension but did so in cells adherent on a plastic surface (data not shown). Therefore, we determined whether integrin activation following adhesion to different ECM proteins influences the ability of TGFβ1 to induce αvβ3 expression. Fig. 3 demonstrates that cell adhesion to either fibronectin (FN) or collagen did not significantly modify basal β3 expression; however, FN strongly potentiated the effect of TGFβ1. These findings suggest that signals mediated by integrin activation following cell adhesion are necessary for TGFβ1 to elevate β3 integrin expression.FIGURE 3TGFβ1-inducedαvβ3 expression is dependent on integrin-mediated cell adhesion. Bar graph shows the synergistic positive effect of fibronectin on TGFβ1-induced β3 integrin expression in lung fibroblasts. HFL-1 cells were placed on the wells of a tissue culture plate pre-coated with fibronectin (1 μg/ml) or collagen type I (COL, 1 μg/ml) and stimulated with 10 ng/ml of TGFβ1 for 24 h. β3 integrin expression was determined by immunoblotting. Bars are mean ± S.E. (n = 3); *, p < 0.05 compared with FN without TGFβ1 stimulation (Non-stim).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TGFβ1-induced β3 Integrin Expression Is Dependent on αvβ3 Activation—To evaluate the role of integrin activation on enhanced αvβ3 expression, we treated adherent fibroblasts with the disintegrin echistatin, which has been shown to inhibit the activation of RGD-binding integrins in several cell types (17Sekimoto H. Eipper-Mains J. Pond-Tor S. Boney C.M. Mol. Endocrinol. 2005; 19: 1859-1867Crossref PubMed Scopus (32) Google Scholar, 21Della Morte R. Squillacioti C. Garbi C. Derkinderen P. Belisario M.A. Girault J.A. Di Natale P. Nitsch L. Staiano N. Eur. J. Biochem. 2000; 267: 5047-5054Crossref PubMed Scopus (25) Google Scholar, 22Marcinkiewicz C. Vijay-Kumar S. McLane M.A. Niewiarowski S. Blood. 1997; 90: 1565-1575Crossref PubMed Google Scholar). Echistatin had no effect on cell attachment but dose-dependently impaired the ability of fibroblasts to spread and support monolayer integrity (supplemental Fig. 5S). As shown in Fig. 4A, exposure of cells to echistatin abrogated the effect of TGFβ1 on β3, but not on β5 integrin expression. To show that echistatin prevents activation of αvβ3 and downstream integrin-mediated signaling, we determined levels of Tyr397-FAK and Tyr416-Src phosphorylation following exposure to the disintegrin. Echistatin dramatically inhibited TGFβ1-induced c-Src kinase (Fig. 4B) and FAK phosphorylation (data not shown) in a concentration-dependent manner. To demonstrate that echistatin inhibits β3 integrin expression by blocking integrin, but not TGFβ1 signaling, FN expression in response to TGFβ1 exposure was determined. In contrast to β3, FN expression induced by TGFβ1 was enhanced in the presence of echistatin indicating that TGFβ1 up-regulates β3 integrin expression directly by activation of the integrin on the cell surface rather than indirectly through enhanced production of ECM proteins. Echistatin also had no effect on Smad3 activation per se (Fig. 4B), but it inhibited TGFβ1-induced p38 MAPK in parallel with β3 integrin expression (Fig. 4, A and B).FIGURE 4Blocking of αvβ3 integrin activation by disintegrin echistatin or LM609 monoclonal antibodies completely abrogates up-regulatory effects of TGFβ1 on β3 integrin expression in lung fibroblasts. A, echistatin dose-dependently inhibits TGFβ1-induced β3 integrin but not β5 integrin expression (n = 3; *, p < 0.05 compared with TGFβ1 alone). B, echistatin dose-dependently inhibits c-Src phosphorylation at Tyr416 but promotes production of fibronectin (left panel, representative immunoblot for two independent experiments). Echistatin (2 μg/ml) did not change Smad3 phosphorylation but inhibited TGFβ1-induced p38 MAPK activation (right panel, representative immunoblot for two independent experiments). C, αvβ3 blocking antibody (clone LM609, PBS solution without preservatives and carrier proteins, 10 μg/ml), but not αvβ5 blocking antibody (clone P5H9, carrier proteins-free solution) and mouse IgG isotype control applied at the same concentration 1 h before stimulation with TGFβ1, prevents the increase in β3 pr