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Originally published In Press as doi:10.1074/jbc.M708226200 on March 18, 2008

J. Biol. Chem., Vol. 283, Issue 19, 12898-12908, May 9, 2008
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Transforming Growth Factor β1 Induces {alpha}vβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway*Formula

Dmitri V. Pechkovsky{ddagger}, Amelia K. Scaffidi§, Tillie L. Hackett{ddagger}, Joanne Ballard§, Furquan Shaheen{ddagger}, Philip J. Thompson§, Victor J. Thannickal||, and Darryl A. Knight{ddagger}1

From the {ddagger}James Hogg iCAPTURE Centre for Cardiovascular and Respiratory Research, Department of Anaesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia V6Z1Y6, Canada, §Lung Institute of Western Australia and Centre for Asthma, Allergy and Respiratory Research, University of Western Australia, Nedlands 6009, Western Australia, and ||Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109

Received for publication, October 3, 2007 , and in revised form, March 17, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}v 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 {alpha}vβ3 in human lung fibroblasts. TGFβ1-induced {alpha}vβ3 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 {alpha}vβ3 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 {alpha}vβ3 and p38 MAPK phosphorylation but not β5 integrin expression and Smad3 activation. The TGFβ1-induced {alpha}vβ3 expression was blocked by pharmacologic and genetic inhibition of p38 MAPK- but not Smad2/3-, Sp1-, ERK-, phosphatidylinositol 3-kinase, and NF-{kappa}B-dependent pathways. Our results demonstrate that TGFβ1 induces {alpha}vβ3 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the key events in wound repair is the infiltration of fibroblasts from surrounding tissue to the extracellular matrix (ECM)2 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 (1). However, their retention, uncontrolled proliferation, and excessive synthesis of ECM proteins represents a pathologic process that ultimately results in fibrosis (2). 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 (3–6). Adhesion of cells to ECM is mediated by a family of transmembrane proteins known as integrins that are expressed on the cell surface as {alpha}/β heterodimers (7, 8). 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. 7, 8). Over the past few years a close relationship between {alpha}v integrins (recognizing RGD motif) and TGFβ signaling pathways has been identified (9). These include activation of latent TGFβ complexes by {alpha}vβ6 and {alpha}vβ8 integrins in airway epithelium (8, 10), augmented TGFβ signaling by {alpha}vβ3 and {alpha}vβ5 integrins in scleroderma fibroblasts (11), and TGFβ receptor type II (TGFβRII)-{alpha}vβ3 integrin interaction-dependent proliferation and differentiation of human lung fibroblasts (4). Previous studies have also identified several molecules as inducers of {alpha}v integrin expression in various tissue culture systems (12–14). 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 (15–17). Recently we demonstrated that TGFβ1 not only synergistically interacts with {alpha}vβ3 integrins but also induces their gene transcription in human lung fibroblasts (4). However, the mechanisms involved in this process are still unknown. Therefore, this study was undertaken to delineate the signaling mechanisms that mediate {alpha}vβ3 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 PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids 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-{kappa}B 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 {alpha}vβ3 integrin was analyzed using an anti-human {alpha}vβ3 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 x 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 {alpha}-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 (18). 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).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TGFβ1 Increases β3 Protein Expression and Enhances Cell Surface Expression of {alpha}vβ3 Integrin on Human Lung Fibroblasts—Recently we showed that TGFβ1 increased β3 steady-state mRNA expression (4). Consistent with the increased β3 transcription, fibroblasts also increased β3 subunit and {alpha}vβ3 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 {alpha}vβ3 heterodimer was also significantly elevated after cell stimulation with TGFβ1 (60% increase in MFI and 2-fold increase in the percentage of {alpha}vβ3-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 {alpha}vβ3 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 {alpha}vβ3 cell surface expression (Fig. 1, A and B). In parallel we examined the effect of TGFβ1 on another {alpha}v partner, the β5 integrin. We found that incubation of HFL-1 with TGFβ1 for 18 h induced robust expression of {alpha}vβ5 (supplemental Fig. 3S) by increasing β5 protein production (Fig. 1C) similar to the effects on {alpha}vβ3 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 {alpha}vβ3 and {alpha}vβ5 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 {alpha}vβ3 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 (19). 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).


Figure 1
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  		FIGURE 1.
TGFβ1 specifically induces {alpha}vβ3 and {alpha}vβ5 integrin expression in human lung fibroblasts (HFL-1). A, β3 protein expression level. The whole cell lysates were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with anti-β3 and anti-β-tubulin antibodies (as shown in representative immunoblot). β-Tubulin was used as a loading control and for densitometry normalization in all experiments performed. Protein concentrations were quantitated by scanning densitometry as described under "Experimental Procedures." The bar graph demonstrates an increase of β3 protein in cytokine-treated cells over controls (bars represent means ± S.E., n = 3; *, p < 0.05 compared with control and EGF-treated cells). B, {alpha}vβ3 cell surface expression. HFL-1 cell were treated with TGFβ1 or EGF (10 ng/ml) or in combination for 24 h following staining with anti-{alpha}vβ3 antibody (LM609, solid line histograms) or normal mouse IgG (isotype control, dashed line histogram) and analyzed by flow cytometry. Histogram plots are a representative experiment of data generated from at least three individual experiments (in parenthesis MFI ratios between the anti-{alpha}vβ3 and isotype control antibodies). Graph bars represent the means ± S.E. percentage of {alpha}vβ3 positive cells (n = 3; *, p < 0.05 compared with control and EGF-treated cells). C, effect of TGF-neutralizing antibody on β3 and β5 integrin expression in HFL-1 cells. Fibroblasts were serum-starved overnight, pulse-stimulated with TGFβ1 for 0.5, 1.5, and 3 h, washed with fresh medium, and incubated for a total of 18 h in the presence of either 10 µg of pan-TGFβ neutralizing antibody or normal mouse IgG as a negative control. β3 and β5 integrins were determined by immunoblotting (one representative immunoblot of two independent experiments is shown).

 
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 {alpha}-smooth muscle actin expression were performed. TGFβ1-induced increase in {alpha}-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 {alpha}vβ3 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 2
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  		FIGURE 2.
Effect of TGFβ1 on {alpha}vβ3 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 {alpha}vβ3 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.

 
Cooperation between the Smad proteins and the transcription factor Sp1 may represent a general mechanism for conferring TGFβ1 inducibility of several genes, including integrins (13, 20). We sought to determine whether Sp1 was involved in the up-regulation of {alpha}vβ3 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 {alpha}vβ3 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 (3, 4, 6). In preliminary experiments we found that TGFβ1 was unable to increase {alpha}vβ3 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 {alpha}vβ3 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.

TGFβ1-induced β3 Integrin Expression Is Dependent on {alpha}vβ3 Activation—To evaluate the role of integrin activation on enhanced {alpha}vβ3 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 (17, 21, 22). 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 {alpha}vβ3 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 3
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  		FIGURE 3.
TGFβ1-induced{alpha}vβ3 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).

 
Because echistatin may also influence other RGD integrins apart from {alpha}vβ3, we next aimed to confirm the identity of the integrin involved in TGFβ1-induced β3 expression. To do this, we incubated fibroblasts with 10 µg/ml monoclonal blocking antibodies to human {alpha}vβ3 integrin (clone LM609) and {alpha}vβ5 integrin (clone P5H9) 1 h before addition of TGFβ1. Similar to the effect seen with echistatin, LM609, but not P5H9 or control IgG, completely abrogated the effect of TGFβ1 on β3 integrin expression (Fig. 4C). These results provide further support for the concept that the effect of TGFβ1 on β3 subunit expression is dependent on the activation of {alpha}vβ3, but not {alpha}vβ5 integrin, on the cell surface and recruitment of nonreceptor protein tyrosine kinases, including FAK and c-Src kinase.


Figure 4
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  		FIGURE 4.
Blocking of {alpha}vβ3 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, {alpha}vβ3 blocking antibody (clone LM609, PBS solution without preservatives and carrier proteins, 10 µg/ml), but not {alpha}vβ5 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 protein induced by cytokine in human lung fibroblasts. Data are means ± S.E. of three independent experiments (*, p < 0.05 compared with controls and {alpha}vβ5 blocking antibody). Ctrl, control.

 
Adenovirally Mediated Overexpression of FRNK Inhibits TGFβ1-induced FAK Activation but Not {alpha}vβ3 Expression—Based on the observation that integrin activation and clustering results in activation of FAK, we determined the effect of TGFβ1 on FAK phosphorylation. Fig. 5A demonstrates that TGFβ1 induced time-dependent phosphorylation of FAK on Tyr397, its major autophosphorylation site. The enhanced Tyr397 phosphorylation of FAK was somewhat delayed, being first observed 3 h following TGFβ1 exposure and further increased at later time points (Fig. 5A, p < 0.05) coincidentally with the induction of β3 expression. To confirm whether FAK activation was involved in TGFβ1-induced {alpha}vβ3 expression, fibroblasts were infected by a replication-defective adenovirus encoding a GFP-FRNK fusion protein (Adv-GFP-FRNK) and exposed to TGFβ1. FRNK is the C-terminal noncatalytic domain of FAK and acts as a negative regulator of FAK autophosphorylation (23, 24). Western Blot for GFP-FRNK revealed abundant expression in Adv-GFP-FRNK-infected cells but not in control Adv-GFP-infected cells, and infection of fibroblasts with Adv-GFP-FRNK completely abrogated spontaneous and TGFβ1-induced Tyr397-FAK phosphorylation (supplemental Fig. 2SB). However, TGFβ1 induced cell surface expression of {alpha}vβ3 in both Adv-GFP-FRNK- and Adv-GFP-infected cells (Fig. 5, B and C). These data suggest that inhibition of FAK autophosphorylation on Tyr397 does not influence TGFβ1-induced {alpha}vβ3 expression.


Figure 5
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  		FIGURE 5.
Blocking of TGFβ1-induced FAK autophosphorylation does not prevent up-regulatory effect on {alpha}vβ3 integrin expression. A, TGFβ1-induced FAK activation was determined by immunoblotting. Following stimulation with TGFβ1 for the indicated times, HFL-1 cells were lysed, and cell-extracted proteins were separated by SDS-PAGE. Immunoblotting was performed with specific monoclonal antibody against a phosphorylation site (Tyr397) of FAK (pFAK-Tyr397) and rabbit polyclonal antibodies against C-terminal region of human FAK protein (FAK). Immunoblots were scanned for the band densities, and pFAK/FAK ratio was calculated for each individual experiment. Data are expressed as percent increase in comparison with nonstimulated cell, and graph bars are the mean ± S.E. of four independent experiments (*, p < 0.05 compared with nonstimulated cells). Ctrl, control. B, two overlaid histogram plots demonstrating MFI and percentage of {alpha}vβ3 expressing cells in Adv-GFP- and Adv-GFP-FRNK-infected populations of fibroblasts either nonstimulated (shaded histograms, M1 marked area) or TGFβ1-stimulated (unshaded histograms, M2 marked area) are representative data of three independent experiments (dashed line histogram demonstrates MFI of cells stained with isotype control antibody). C, graph bars demonstrate a percentage of {alpha}vβ3 expressing cells. Data are mean ± S.E. of three independent experiments (*, p < 0.05 compared with nonstimulated cells (open bars)).

 
Inhibition of c-Src Kinase Activity Blocks TGFβ1-induced {alpha}vβ3 Integrin Expression—To confirm the involvement of c-Src kinase in TGFβ1-induced {alpha}vβ3 integrin expression, we exposed cells to the Src kinase inhibitor, PP2, and measured {alpha}vβ3 expression following TGFβ1 stimulation. As shown in Fig. 6A, exposure of cells to PP2 completely abrogated the stimulatory effect of TGFβ1 on {alpha}vβ3 surface expression. Furthermore, incubation with PP2 suppressed the effects of TGFβ1 on β3 gene expression over a 6-h time period (Fig. 6B). It has been demonstrated that Src kinases regulate signaling-dependent adhesion as well as FAK-mediated signaling events by regulating FAK kinase activity. As expected, PP2, but not the inactive PP3, almost completely inhibited both Tyr416-Src and Tyr397-FAK phosphorylation induced by TGFβ1 (data not shown). TGFβ1-induced β3 protein increase was also strongly inhibited by PP2 but not PP3 (Fig. 6C). Recently, it has been demonstrated that PP2 can also inhibit TGFβ receptor I and II kinase activity (25). Therefore, we also tested the more specific SFK inhibitor SU6656, which does not inhibit TGFβ receptor function. As shown in Fig. 6C, SU6656 inhibited TGFβ1-induced β3 protein in a dose-dependent manner. Moreover, SU6656 inhibited TGFβ1-induced p38 MAPK activation but not Smad3 phosphorylation (Fig. 6D). These results further support a role for autocrine integrin signaling in TGFβ1-induced β3 expression and suggest that c-Src kinase and p38 MAPK activation, but not Smad3, is a key element in the process.

p38 MAPK Inhibition Blocks TGFβ1-induced {alpha}vβ3 Integrin Expression—Besides Smad-mediated transcription, TGFβ1 activates other signaling cascades, including MAPK and NF-{kappa}B pathways (reviewed in Ref. 26). Importantly, Src kinases can interfere with these pathways directly or indirectly, and our results demonstrate that p38 MAPK activation by TGFβ1 is dependent on integrin activation and SFK recruitment. Therefore, we assessed whether inhibition of MAPK, including p38 and p42/p44, NF-{kappa}B, and PI3K affected TGFβ1-induced {alpha}vβ3 integrin expression. As seen in Fig. 7A, no changes in the cell surface expression of {alpha}vβ3 integrin were observed after treatment with the MEK/ERK inhibitor U0126, the NF-{kappa}B inhibitor PDTC, and the PI3K inhibitor wortmannin. Similar effects were seen for TGFβ1-induced β3 protein (data not shown). However, the p38 MAPK inhibitor SB203580 (10 µM), and the more potent inhibitor SB202129 at a concentration of 0.2 µM and higher, decreased β3 subunit and {alpha}vβ3 integrin expression induced by TGFβ1 (Fig. 7, A and B). To further validate the role of p38 MAPK, we transiently overexpressed a kinase mutant p38 MAPK (p38-KM) construct in fibroblasts (20). As expected, an increased level of p38 MAPK protein was seen in p38-KM-transfected cells but not in cells transfected with empty pcDNA3 vector (supplemental Fig. 2SA). As seen in Fig. 7C, TGFβ1 failed to induce β3 integrin expression in p38-KM-transfected cells. Importantly, the Smad signaling pathway was completely preserved and TGFβ1 efficiently induced β5 integrin expression in p38-KM-transfected cells (supplemental Fig. 2SA and Fig. 7C). Induction of p38 MAPK phosphorylation by treatment with recombinant tumor necrosis factor-{alpha} did not up-regulate β3 integrin in HFL-1 cells (data not shown). Thus, limiting TGFβ1-induced p38 MAPK activity did not alter Smad-dependent induction of β5 integrin, but it substantially inhibited β3 protein and {alpha}vβ3 cell surface expression.


Figure 6
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  		FIGURE 6.
TGFβ1-induced and {alpha}vβ3 integrin-mediated c-Src activation is required for the increase in β3 integrin and {alpha}vβ3 cell surface expression. Quiescent, adherent fibroblasts were stimulated with TGFβ1 (10 ng/ml) in the absence/presence of the Src inhibitor PP2 (10 µM) for 18 h. Cell surface expression of {alpha}vβ3 was assessed by flow cytometry (A) and β3 subunit mRNA expression by real time reverse transcription-PCR (B). Graph bars are the mean ± S.E. of three independent experiments (*, p < 0.05 compared with TGFβ1-stimulated cells without inhibitor). C, PP2 and the more specific Src inhibitor SU6656 almost completely abrogated TGFβ1-induced β3 integrin expression in human lung fibroblasts. PP3, pharmacologically inactive form of PP2, used at the same concentrations and experimental conditions as PP2 did not influence TGFβ1-induced β3 protein (*, p < 0.05 compared with PP2 and SU6656 inhibitors, n = 3). D, HFL-1 cells treated with the Src inhibitor SU6656 for 45 min and then stimulated with TGFβ1 for additional 45 min. Phosphorylation of p38 MAPK and Smad3 was determined by immunoblotting and quantified by densitometry. The Src inhibitor SU6656 abrogated TGFβ1-induced p38 MAPK phosphorylation but increased phosphorylation of Smad3 in HFL-1 cells (*, p < 0.05 compared with controls (Ctrl); #, p < 0.05 compared with TGFβ1 alone, n = 3).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Adhesion of fibroblasts to the ECM via specific integrins can alter cell responses to growth factors, including TGFβ (3–6), which are often produced in excess and may promote specific cell-ECM interactions giving a rise to several pathological cycles and abnormal behavior of the cells in the settings of fibrotic diseases (2, 11).

In this study, we demonstrate that TGFβ1 increases β3 integrin expression and subsequent {alpha}vβ3 surface expression on human lung fibroblasts via an autocrine pathway involving c-Src- and p38 MAPK-dependent mechanisms. Significantly, this effect is independent of the conventional Smad/Sp1 signaling pathway. Similarly, although the effect was dependent on cell adhesion and FAK was phosphorylated by TGFβ1, inhibiting FAK activation did not influence the effect of TGFβ1 on {alpha}vβ3 expression. We show that the effect of TGFβ1 is dependent on {alpha}vβ3-mediated cell adhesion and activation of c-Src kinase and p38 MAPK pathway. Blockade of c-Src activity also completely abrogated the stimulatory effect of TGFβ1 on activation of p38 MAPK, suggesting that c-Src is upstream of this signaling kinase.


Figure 7
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  		FIGURE 7.
Inhibition of p38 MAPK signaling pathway abrogates TGFβ1-induced β3 integrin expression in HFL-1 cells. A, HFL-1 cells were pretreated with pharmacological inhibitors of MEK/ERK (2 µM U0126), NF-{kappa}B (100 µM PDTC), PI3K (100 nM wortmannin), and p38 MAPK (10 µM SB203580) signaling pathways for 40 min prior to the addition of TGFβ1. Following 18-h exposure of TGFβ1 (10 ng/ml), cells were harvested and washed in PBS, and {alpha}vβ3 integrin cell surface expression was analyzed by flow cytometry. Values are expressed as a percentage above control MFI of three independent experiments ± S.E. B, HFL-1 cells were pretreated with SB202190, pharmacological inhibitor of p38 MAPK at different concentrations for 45 min, then stimulated with TGFβ1 (10 ng/ml) for 18 h, and protein expression was analyzed by immunoblotting. The p38 MAPK inhibitor, SB202190, prevented β3 subunit expression induced by TGFβ1 (*, p < 0.05 compared with control and SB202190; n = 3). C, cells transiently expressing p38-KM did not up-regulate β3 integrin expression in response to TGFβ1 stimulation. TGFβ1-induced β5 integrin expression was similar in p38-KM- and pcDNA3-transfected cells as shown by a representative immunoblot of two independent experiments (*, p < 0.05 compared with pcDNA3-transfected cells).

 
To date there have been several reports investigating the effects of TGFβ1 in modulating expression and function of {alpha}vβ3 integrins on different cell types. For instance, TGFβ1 has been shown to have no effect on {alpha}vβ3 expression in human airway epithelial cells (27). In contrast, in human vascular smooth muscle cells (28), human WI-38 fibroblasts (12), and a variety of malignant cell lines, TGFβ1 increases {alpha}vβ3 expression. Although these studies focused on how the temporal expression of {alpha}vβ3 integrins is influenced by exposure to TGFβ1, they highlight that the effects of TGFβ1 on integrin expression may be cell type-specific, and little is known about the underlying signal transduction pathways involved.

Binding of TGFβ1 to TGFβIIR causes the recruitment and activation of TGFβIR and subsequently Smad2/3 phosphorylation, which then associates with Smad4. This complex then translocates to the nucleus where it modulates transcription of a large number of genes. In contrast, Smad7 inhibits the TGFβ1-mediated phosphorylation of Smad2/3 through competition for binding to the TGFβ1 receptor (26, 29). Using several independent approaches, we determined that the Smad2/3 pathway is not required for the TGFβ stimulation of β3 integrin. Specifically, we have shown that in contrast to β5 expression, which was strongly dependent on Smad3 activation, TGFβ1-induced β3 integrin was maintained in the cells transfected with either Smad3 siRNA or a dominant Smad7 expression construct. Interestingly, in silico analysis of the mouse and human β3 integrin gene promoters (30, 31) (GenBankTM accession numbers AF026510 [GenBank] and AF02055, respectively) revealed considerable sequence homology across a 1.3-kb region upstream of the transcription start site and several conserved binding elements for Sp1 but not for Smad proteins. Recently, it has been demonstrated that cooperation between Sp1 and Smad proteins may represent a general regulatory mechanism for conferring the TGFβ1 inducibility of several genes, including β5, {alpha}5, and {alpha}11 integrins (13, 20, 32) and different types of collagen (33, 34). In these studies the antibiotic mithramycin A selectively and efficiently reduced TGFβ-induced collagen and {alpha}11 integrin expression in mesenchymal cells via inhibition of Sp1 binding to the gene promoter. In this study, we also demonstrated that mithramycin A significantly inhibited TGFβ1-induced expression of cellular FN, but it had no effect on β3 integrin expression. These paradoxical observations indicate that the effects of TGFβ1 on integrin expression are dependent on cell type and specific regulatory elements of the individual integrin gene promoters. Significantly, our data also suggest that TGFβ1 is able to activate signaling pathways in human lung fibroblasts to promote the expression of integrin complexes independently of Smad2/3 and Sp1.

In addition to Smad-mediated transcription, it has been shown that TGFβ also activates several specific signaling cascades, including MAPK, NF-{kappa}B, and PI3K (19, 35, 36). However, in this study pharmacological inhibition of NF-{kappa}B, MEK-ERK, and PI3K pathways did not significantly influence the induction of {alpha}vβ3 by TGFβ1. In contrast, blocking of p38 MAPK activation by either selective inhibitors or overexpression of a dominant p38-kinase dead mutant significantly attenuated TGFβ1-induced β3 transcription and {alpha}vβ3 surface expression. Furthermore, we found that p38 MAPK phosphorylation induced by TGFβ1 also could be inhibited by blocking of {alpha}vβ3-mediated c-Src activation. During the submission process of this manuscript, Galliher and Schiemann (37) showed that c-Src phosphorylates Tyr284 on the TGFβRII receptor and by that it regulates TGFβ induction of p38 MAPK signaling pathway in malignant mammary epithelial cells. It would be of interest to determine whether the same mechanisms operate in primary human fibroblasts or, alternatively, if there are any defects of TGFβ-induced p38 MAPK phosphorylation in β3–/– mouse fibroblasts. Nevertheless, our study provides evidence for the existence of an alternative c-Src- and p38 MAPK-dependent, Smad2/3-independent signaling pathway induced by TGFβ in human lung fibroblasts that may operate during a fibrotic process in the lung (Fig. 8).


Figure 8
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  		FIGURE 8.
Schematic model of the proposed synergistic interaction between {alpha}vβ3-mediated c-Src and TGFβ1-induced p38 MAPK activation in the β3 subunit expression by human lung fibroblasts. {alpha}vβ3 is activated upon interaction with the TGFβ type II receptors and its ligand TGFβ1 (4, 37). Following {alpha}vβ3 activation c-Src, associated with the β3 integrin subunit, is phosphorylated at Tyr416 and then recruited to the kinase domain of TGFβRII, which results in phosphorylation of the receptor at Tyr284 (37). In addition, {alpha}vβ3 binding to ECM proteins also promotes the activation of SFK. Once activated c-Src coordinates the activation of TGFβ1-induced p38 MAPK signaling pathway and thereby the transcriptional regulation of β3 integrin subunit expression. We propose the rapid increase in β3 integrin leads to high {alpha}vβ3 cell surface expression giving rise to a positive feedback loop affecting several fibroblast functions such as adhesion, migration, ECM remodeling, and autocrine TGFβ expression and signaling (zigzag indicates phosphorylation sites).

 
Several studies have demonstrated physical or functional relationships between integrins and growth factor receptors (reviewed in Refs. 7, 38). Previously we demonstrated that TGFβIIR functionally interacts with {alpha}vβ3, but not with {alpha}vβ5 integrins in human lung fibroblasts, and this interaction results in substantial enhancement in TGFβ1-induced proliferation (4). However, whether TGFβ1 alters the affinity or activity of {alpha}vβ3 integrins and their signal transduction pathways is unknown. It is likely that the activation of growth factor receptors induced by their specific ligands has an important function in organizing the signaling pathways associated with the attachment of integrins to ECM. For example, vascular endothelial growth factor is able to enhance the affinity of {alpha}vβ1 and {alpha}vβ3 integrins in human endothelial cells (16). In addition, platelet-derived growth factor or EGF-induced activation of FAK through integrins was proposed as an important proximal link between growth factor receptor and integrin signaling pathways (24). In agreement with these findings, we showed that TGFβ1 significantly increases affinity of {alpha}v integrins on lung fibroblasts to their respective ligands and also activates FAK.

Traditionally FAK activation has been associated with integrin signaling, because it is recruited and phosphorylated at sites of focal adhesions upon integrin aggregation, and it serves as a bridge between growth factor receptors and β integrin cytoplasmic tails (24). In this study we found that TGFβ1 induced FAK autophosphorylation on Tyr397 (Tyr(P)397-FAK), and FAK protein paralleled the time course of β3 integrin mRNA expression (4). Critically, TGFβ1 did not influence {alpha}vβ3 expression on fibroblasts in suspension, but adhesion of cells on ECM proteins significantly enhanced the effect of TGFβ1 on β3 integrin expression. However, although overexpression of the FAK inhibitor, FRNK, completely inhibited basal and TGFβ1-induced Tyr(P)397-FAK, it did not significantly influence TGFβ1-induced {alpha}vβ3 expression. In contrast, inhibition of c-Src kinase, which also has been shown to play an important role in downstream signaling of integrin receptors, dramatically blunted TGFβ1-induced {alpha}vβ3 expression and Tyr(P)397-FAK.

Although c-Src kinase activity is clearly required for rapid and full phosphorylation of Tyr(P)397-FAK (39), c-Src kinase activity is not affected in FAK–/–cells (40), suggesting that c-Src can be activated independently of FAK. Furthermore, activated Src, unlike FAK, is concentrated in perinuclear and plasma membrane but not in focal adhesions after integrin ligation (39), suggesting some divergence in their signaling. These findings corroborate our results showing that activation of c-Src kinase, but not FAK autophosphorylation, is the critical downstream pathway in TGFβ1-induced {alpha}vβ3 expression.

Recently, it has been shown that Src associates with {alpha}vβ3 and is activated following integrin ligation (39, 41). Moreover, the cytoplasmic tail of the β3 integrin, but not other integrins, directly interacts with Src kinase through the β3-specific domain (39, 41, 42). These findings may explain the specificity of {alpha}vβ3 integrins for TGFβ1-induced β3 subunit expression in human lung fibroblasts. Indeed, our data demonstrate that either disintegrin or monoclonal antibody-mediated blockade of {alpha}vβ3 activation (but not {alpha}vβ5), prior to TGFβ1 stimulation, efficiently abrogated the increases of both c-Src activation and β3 integrin expression.

How then does TGFβ1 activate or promote activation and signaling of {alpha}vβ3 integrins? Our data suggest through a process of inside-out signaling whereby activated TGFβRI or TGFβRII directly interacts with the β3 cytoplasmic domain, which in turn induces a conformational change in the integrin resulting in activation of the downstream SFK (4, 37). A similar process of inside-out activation of {alpha}vβ3 integrin by ligation of insulin-like growth factor I receptor in the murine preadipocyte 3T3-L1 cell line has been described recently (17).

TGFβ1 could also induce synthesis of several {alpha}vβ3 ligands, including FN, vitronectin, and tenascin (3, 34, 43), and by enhancement of ligand-integrin interactions activate integrin-mediated signaling. Indeed, we demonstrated that seeding of fibroblasts on FN significantly enhanced the effect of TGFβ1on β3 subunit expression. However, these exogenously added ECM proteins did not induce or up-regulate β3 expression in the absence of TGFβ1. Furthermore, inhibition of FN production by mithramycin did not prevent TGFβ1-induced expression of the β3 integrin.

Finally, TGFβ1-induced activation of {alpha}vβ3 integrin could be modified by the expression level of the {alpha}v subunit. We showed that TGFβ1-induced expression of both β3 and β5 subunit is sufficient to up-regulate the cell surface of {alpha}vβ3 and {alpha}vβ5, respectively. This is consistent with the notion that the {alpha}v subunit is constitutively and excessively expressed in the cytoplasm of fibroblasts as a monomer, and the cell surface expression level of {alpha}v-containing integrins is controlled by the levels of β subunits, which are exclusively involved in the integrin-mediated signaling mechanisms (11, 14).

Given that TGFβ1 up-regulates β3 expression in a delayed manner, another possibility is the involvement of other factors induced by TGFβ1. EGF is a well known and potent activator of integrins (16, 24, 36). However, in this study, although EGF induced a robust FAK autophosphorylation it did not influence {alpha}vβ3 expression, either alone or in combination with TGFβ1. Moreover, TGFβ neutralization with blocking mAb and removal of recombinant cytokine after a short time exposure almost completely abrogated β3 integrin expression suggesting that activation of integrin/c-Src signaling as well as production of autocrine TGFβ is exclusively dependent on the initial TGFβR ligation by active TGFβ1. Altogether these data also indicate that the effect on {alpha}vβ3 expression is highly specific for TGFβ1.

In summary, our results suggest a model where TGFβ1 induces activation of {alpha}vβ3 integrin, which in turn leads to c-Src phosphorylation and the initiation of p38 MAPK signaling cascade that is essential for β3 chain expression (Fig. 8). Our study for the first time describes a mechanism for the regulation of the integrin β3 subunit expression in human fibroblasts, which involves TGFβ1 and the {alpha}vβ3 integrin itself. Significantly, this appears to be a Smad-independent process. Our data support the concept of a functional synergy between TGFβ1-dependent signaling pathways and {alpha}vβ3-mediated adhesion processes in normal wound healing and pathological phenomena such as fibrosis.


   FOOTNOTES
 
* This work was supported by the British Columbia Lung Association, the Canadian Institutes of Health Research, The Wolfe and Gita Churg Foundation, and the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Experimental Procedures and Figs. 1S–5S. Back

1 To whom correspondence should be addressed: St. Paul's Hospital, 1081 Burrard St., Vancouver, British Columbia V6Z 1Y6, Canada. Tel.: 604-682-2344 (Ext. 62217); Fax: 604-806-9274; E-mail: dknight{at}mrl.ubc.ca.

2 The 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. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Allen M. Samarel for the generous gift of GFP-FRNK-expressing adenovirus, Dr. Steven Mutsaers for kindly providing the Smad7 cDNA, Dr. Kun Liang Guan for p38-KM plasmid, and Dr. Xiaoning Si for anti-c-Src antibody.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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