Thy-1-Integrin αvβ5 Interactions Inhibit Lung Fibroblast Contraction-induced Latent Transforming Growth Factor-β1 Activation and Myofibroblast Differentiation*

Myofibroblasts, key effector cells in tissue fibrosis, are specialized contractile cells. Lung myofibroblast contraction induces integrin αvβ5-dependent latent transforming growth factor (TGF)-β1 activation suggests that myofibroblast contractility may be a driving force for the persistent myofibroblast differentiation observed in fibrotic lungs. Understanding the mechanisms that regulate fibroblast contraction and mechanotransduction will add new insights into the pathogenesis of lung fibrosis and may lead to new therapeutic approaches for treating fibrotic lung diseases. We and others previously demonstrated that lung fibroblast expression of Thy-1 prevents lung fibrosis. The mechanisms underlying the anti-fibrotic effect of Thy-1 are not well understood. In this study, we showed that Thy-1 interacts with integrin αvβ5, both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin αvβ5 interactions are RLD-dependent because mutated Thy-1, in which RLD is replaced by RLE, loses the ability to bind the integrin. Furthermore, Thy-1 expression prevents fibroblast contraction-induced, integrin αvβ5-dependent latent TGF-β1 activation and TGF-β1-dependent lung myofibroblast differentiation. In contrast, lack of Thy-1 expression or disruption of Thy-1-αvβ5 interactions renders lung fibroblasts susceptible to contraction-induced latent TGF-β1 activation and myofibroblast differentiation. These data suggest that Thy-1-integrin αvβ5 interactions inhibit contraction-induced latent TGF-β1 activation, presumably by blocking the binding of extracellular matrix-bound latent TGF-β1 with integrin αvβ5. Our studies suggest that targeting key mechanotransducers to inhibit mechanotransduction might be an effective approach to inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.

inhibit the deleterious effects of myofibroblast contraction on lung fibrogenesis.
Myofibroblasts are specialized contractile cells that have characteristics of both fibroblasts and smooth muscle cells. These cells are key effector cells in the connective tissue remodeling that takes place in both normal wound healing and tissue fibrosis. In the process of normal wound healing, myofibroblasts undergo apoptosis upon wound closure and eventually disappear from the wound site. Alternatively, they can dedifferentiate into a quiescent state. Persistent myofibroblast differentiation results in tissue fibrosis (1,2).
Idiopathic pulmonary fibrosis (IPF) 2 is a progressive lethal fibrotic lung disease with unknown etiology. The pathogenesis of this disease remains elusive. IPF is characterized by persistent myofibroblast differentiation and excessive synthesis of extracellular matrix (ECM) in the lung, forming so-called fibroblastic foci. The extent of fibroblastic foci present on lung biopsy predicts survival in IPF (3). Currently, there are no effective drug therapies for patients with IPF.
Transforming growth factor (TGF)-␤1 is a profibrotic cytokine that plays a key role in lung myofibroblast differentiation and lung fibrosis (4 -6). TGF-␤1 is initially synthesized as a biologically inactive latent complex (termed the small latent TGF-␤1 complex (SLC)) consisting of an N-terminal latencyassociated peptide (LAP) and a C-terminal active TGF-␤1 peptide that needs to be activated to elicit its biological functions (7)(8)(9). Most cells, including lung fibroblasts, secrete TGF-␤1 as a large latent complex (LLC) in which the SLC binds to a second gene product named latent TGF-␤-binding protein (LTBP) (10,11). LTBPs (LTBP-1, -3, and -4) target TGF-␤1 to the ECM and regulate latent TGF-␤1 activation (12)(13)(14)(15)(16)(17)(18). Latent TGF-␤1 activation can occur either through protease-dependent cleavage of LAP that releases the C-terminal active TGF-␤1 domain from the latent complex or by induction of a conformational change in the latent complex that exposes the active TGF-␤1 domain to allow binding to its cell surface receptors (19 -21). Recent studies suggest that mechanical force is a factor that regulates latent TGF-␤1 activation (22,23). Induction of lung myofibroblast contraction or application of isometric stretch on cultured lung myofibroblasts results in latent TGF-␤1 activation from the ECM (22). In this process, fibroblast integrins, primarily integrin ␣ v ␤ 5 , transmit stress fiber-derived contractile forces to the ECM. Because the latent TGF-␤1 complex physically "sits" between the cell surface and the ECM, binding both cell surface integrins with an RGD motif in the LAP and the ECM by LTBP, the force transmission results in a conformational change of the SLC that releases/exposes the active TGF-␤1 domain from the latent complex. It is plausible that myofibroblast contraction-induced latent TGF-␤1 activation may be a driving force for the persistent myofibroblastic phenotype observed in IPF lungs.
Integrin ␣ v ␤ 5 is the primary mechanotransducer that mediates lung myofibroblast contraction-induced latent TGF-␤1 activation because anti-integrin ␣ v ␤ 5 antibody completely abrogates contraction agonist-induced latent TGF-␤1 activation by lung myofibroblasts (22). In addition, increased integrin ␣ v ␤ 5 expression has been linked to myofibroblast differentiation (38). Although integrin ␣ v ␤ 5 contains an RGD-directed binding site (39), Thy-1-␣ v ␤ 5 interactions have not yet been reported. In this study, we show that Thy-1 binds integrin ␣ v ␤ 5 both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin ␣ v ␤ 5 interactions inhibit fibroblast contraction-induced latent TGF-␤1 activation and TGF-␤1-dependent lung myofibroblast differentiation. Our studies provide a mechanistic insight for Thy-1 regulation of lung myofibroblast phenotype and fibrosis and suggest that targeting key mechanotransducers, such as integrin ␣ v ␤ 5 , might be an effective approach for blockade of the deleterious effects of myofibroblast contractility on myofibroblast differentiation and lung fibrosis.
Plasmid expressing the full-length human integrin ␤ 5 was a gift from Dr. Yoshihide Asano (University of Tokyo, Tokyo, Japan). Plasmid expressing full-length mouse Thy-1.2 was constructed as described previously (40).
Lung fibroblasts were cultured for 7 days to allow accumulation of the ECM. Transformed mink lung TGF-␤ reporter cells (TMLC) stably expressing the firefly luciferase reporter gene under the control of the TGF-␤-response element of the PAI-1 (plasminogen activator inhibitor-1) promoter (42) were added to fibroblasts and allowed to attach for 4 h. Co-cultured cells were serum-starved for 24 h and then were treated with calyculin (10 nM) or ET-1 (100 nM) in the presence or absence of cytochalasin D (10 M), blebbistatin (100 M), anti-TGF-␤1 antibody (1 g/ml), anti-TGF-␤2 antibody (1 g/ml), anti-TGF-␤3 antibody (2 g/ml), or pan-TGF-␤ antibody (1 g/ml) for 24 h. For P1F6 antibody pretreatment, 0 -10 g/ml P1F6 was incubated with lung fibroblast/TMLC co-cultured cells for 1 h before the addition of calyculin and ET-1. For purified Thy-1 and mutated Thy-1(RLE) treatments, lung fibroblasts were cultured in the presence of purified human Thy-1-IgG Fc (1 g/ml), mutated human Thy-1(RLE)-IgG Fc (1 g/ml), or human IgG Fc (1 g/ml) for 7 days. Fresh purified proteins were added every other day. Cells were then topped with TMLC and made quiescent. Quiescent cells were treated with calyculin and ET-1 as described above.
Binding of Soluble Thy-1 to the Cell Surface of Lung Fibroblasts and Flow Cytometry-Single cell suspensions (1 ϫ 10 6 cells) were pelleted and washed three times with PBS and then incubated with 10 g/ml recombinant human Thy-1-IgG Fc fusion protein, 10 g/ml recombinant mutated human Thy-1(RLE)-IgG Fc fusion protein, or 10 g/ml human IgG Fc in PBS containing 1% BSA and 0.1% sodium azide for 60 min at 4°C. Cells were washed three times with PBS. Cell surface molecules were stained by incubation of cells with fluorescein isothiocyanate (FITC)-conjugated anti-human Thy-1 antibody diluted in blocking buffer at a final concentration of 10 g/ml for 60 min at 4°C. After washing with PBS three times, the stained cells were fixed in PBS containing 1% paraformaldehyde. Thy-1 flow cytometry was performed on an LSRII flow cytometer (BD Biosciences), and data were processed using FACSDiva software (BD Biosciences).
Wrinkle Assay-Flexible silicone substrates were generated with a modification of the previous protocols (43)(44)(45). Briefly, ϳ15 l of silicone monomer were applied onto 18-mm glass coverslips and allowed to spread for 30 min. The upper layer of silicone was polymerized by exposure of the coverslip to an open flame for 1.5 s. The silicone coverslips were placed into a 12-well plate and were equilibrated with 10 g/ml collagen type I in serum-free F12K medium, sterilized by UV light exposure, and left overnight in the incubator at 37°C. Lung fibroblasts (10 4 cells/well) were plated onto silicone coverslips in F12K containing 0.5% fetal bovine serum in the presence or absence of 10 nM calyculin and 100 nM ET-1. Cells were cultured for 24 h. Contractility of lung fibroblasts on deformable silicone substrates was assessed by formation of wrinkles seen with a ϫ20 objective on a Nikon Eclipse TE 300 microscope. Wrinkling fibroblasts were calculated as a percentage of the total cells. Mean values were calculated from 10 random regions, and at least three independent experiments were performed.
Bioassay of TGF-␤ Activity (PAIL Assay)-TGF-␤ activity was determined by the PAIL assay as described previously (42). Cell lysates were prepared using reporter lysis buffer (Promega, Madison, WI). Luciferase activity was measured as relative light units using an Orion Microplate Luminometer from Berthold (Pforzheim, Germany).
Isolation of Membrane-bound Proteins and ECM Proteins-Membrane proteins were isolated by using Mem-PER eukaryotic membrane protein extraction reagent kit (Pierce) according to the manufacturer's recommendation. Membrane fraction was dialyzed with Slide-A-Lyzer dialysis cassettes (Pierce) to reduce the detergent. Protein concentrations in membrane fraction were determined by using a Micro BCA TM protein assay (Pierce).
ECM proteins were isolated as described previously (46,47). Briefly, cell cultures were washed once with PBS and then treated three times with 0.5% sodium deoxycholate in 10 mM Tris-HCl buffer, pH 8.0, on ice for 10 min. The plates were then washed again with PBS and allowed briefly to dry, and the ECM samples were collected by extraction with Laemmli sample buffer and heat-treated at 95°C for 5 min. Protein concentrations in Laemmli sample buffer were determined as described previously (48).
Western Blotting-Cell lysates containing 40 g of total proteins were loaded onto SDS-polyacrylamide gels under reducing conditions. After electrophoresis, proteins were electrophoretically transferred from the gels to nitrocellulose at 100 V for 1.5 h at 4°C. Membranes were blocked in casein solution (1% casein, 25 mM Na 2 HPO 4 , pH 7.1) for 1 h at room temperature. Primary antibodies were diluted in TBS-T and casein solution (1:1) at a working concentration recommended by the manufacturers. Membranes were incubated with primary antibodies at room temperature for 1 h. After extensive washing, membranes were incubated with peroxidase-conjugated secondary antibodies (0.1 g/ml) diluted in TBS-T for 1 h at room temperature. Immunodetection was carried out by chemiluminescence.
Site-directed Mutagenesis-Site-directed mutagenesis of mouse Thy-1.2 to create the non-integrin-binding Thy-1(RLE) mutant was carried out using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The oligonucleotide primers used for introducing a single mutation (C to G at nucleotide 111) to change Asp (D) to Glu (E) at amino acid position 37 were 5Ј-AC CAA AAC CTT CGC CTG GAG(37E) TGC CGC CAT G-3Ј and 5Ј-C ATG GTG GCA CTC(37E) CAG GCG AAG GTT TTG GT-3Ј. The desired site of mutation was confirmed by automated DNA sequencing. The mutated plasmid was transformed into DH5␣-competent cells (Invitrogen) and amplified according to the manufacturer's recommendation.
Statistical Analysis-Statistical differences among treatment conditions were determined using one-way analysis of variance (Newman-Keuls method for multiple comparisons). The analysis was performed with SigmaStat 3.0 software (SPSS Inc., Chicago, IL). Values of p Ͻ 0.01 were considered significant.

RESULTS
Purified Thy-1 Binds Integrin ␣ v ␤ 5 in a Cell-free System-We first performed an in vitro ligand-receptor interaction assay to determine whether Thy-1 interacts with integrin ␣ v ␤ 5 in a cellfree system. Purified, biotin-labeled Thy-1-IgG Fc fusion proteins and biotin-labeled IgG Fc fragments were added to immobilized integrin ␣ v ␤ 5 on a 96-well microtiter plate. Incubation of biotinylated Thy-1-IgG Fc to immobilized integrin ␣ v ␤ 3 and immobilized integrin ␤ 1 was used as positive and negative control, respectively (32,33). Data show that purified Thy-1-IgG Fc fusion proteins bound immobilized integrin ␣ v ␤ 5 , whereas purified IgG Fc fragments did not. The binding of human Thy-1-IgG Fc to integrin ␣ v ␤ 5 was completely inhibited by an ␣ v ␤ 5specific antibody, P1F6 (Fig. 1). Consistent with previously published data, purified Thy-1-IgG Fc bound immobilized integrin ␣ v ␤ 3 but not integrin ␤ 1 (32,33). LM609, an ␣ v ␤ 3 -specific antibody, blocked the binding of purified Thy-1 to integrin ␣ v ␤ 3 . In addition, we observed that Thy-1 binding to integrin ␣ v ␤ 5 was ϳ2-fold greater than Thy-1 binding to integrin ␣ v ␤ 3 . These results provide the first evidence that Thy-1 interacts with integrin ␣ v ␤ 5 .
Purified Thy-1 Binds Integrin ␣ v ␤ 5 on the Cell Surface of Lung Fibroblasts-To determine whether Thy-1 interacts with integrin ␣ v ␤ 5 on the cell surface of lung fibroblasts, we incubated purified Thy-1-IgG Fc fusion proteins with Thy-1(Ϫ) rat lung fibroblasts in the presence or absence of P1F6 antibody. Incubation of the cells with IgG Fc fragments was used as a negative control. After incubation, cells were stained with FITC-conjugated anti-Thy-1 antibody to detect Thy-1 binding on the cell surface of Thy-1(Ϫ) lung fibroblasts. Flow cytometry analyses showed that Thy-1(Ϫ) lung fibroblasts incubated with Thy-1-IgG Fc fusion proteins had an overall increase in fluorescent signal on the cell surface as compared with cells incubated with IgG Fc control ( Fig. 2A, c versus a). The increased Thy-1 staining was further enhanced by forced expression of human ␤ 5 integrin in Thy-1(Ϫ) cells ( Fig. 2A, d versus c) and was inhibited by pretreatment of Thy-1(Ϫ) cells with P1F6 ( Fig. 2A, b versus  c). These data suggest that purified Thy-1 binds to the cell surface of lung fibroblasts by interacting with integrin ␣ v ␤ 5 . The finding that P1F6 pretreatment did not completely abrogate Thy-1 binding to the cell surface ( Fig. 2A, b versus a) suggests that there are additional molecules that interact with Thy-1 on the cell surface and mediate Thy-1 binding to lung fibroblasts.

JOURNAL OF BIOLOGICAL CHEMISTRY 22385
Thy-1-Integrin ␣ v ␤ 5 Interactions Are RLD-dependent-Previous studies have shown that Thy-1 interacts with integrin ␣ v ␤ 3 through the Thy-1 RLD motif. Mutated Thy-1 in which the RLD motif has been replaced by RLE loses the ability to bind integrin ␣ v ␤ 3 (32). In this study, we determined whether RLD is required for Thy-1-integrin ␣ v ␤ 5 interactions. The in vitro ligand-receptor interaction assay showed that biotin-labeled, mutated Thy-1(RLE)-IgG Fc fusion proteins failed to bind immobilized ␣ v ␤ 5 and ␣ v ␤ 3 in a cell-free system, whereas control experiments showed that biotinylated Thy-1(wild-type)-IgG Fc fusion proteins consistently bound both immobilized ␣ v ␤ 3 and immobilized ␣ v ␤ 5 (Fig. 3A). Consistent with the previous observations, neither wild type Thy-1-IgG Fc nor mutated Thy-1(RLE)-IgG Fc fusion proteins bound integrin ␤ 1 . IgG Fc did not bind ␣ v ␤ 5 either.
To determine whether Thy-1-integrin ␣ v ␤ 5 interactions on the cell surface of lung fibroblasts are RLDdependent, we incubated mutated Thy-1(RLE)-IgG Fc with ␤ 5 -overexpressing Thy-1(Ϫ) lung fibroblasts. Cells incubated with purified Thy-1-IgG Fc fusion proteins and IgG Fc fragments were used as positive and negative controls, respectively. ␤ 5 -overexpressing Thy-1(Ϫ) lung fibroblasts incubated with mutated Thy-1(RLE)-IgG Fc fusion proteins showed fluorescent signal on the cell surface at a level nearly equivalent to Thy-1(Ϫ) cells incubated with the IgG Fc negative control (Fig. 3B, b versus a). In contrast, cells incubated with Thy-1-IgG Fc fusion proteins had increased fluorescent signal on the cell surface as compared with cells incubated with mutated Thy-1(RLE)-IgG Fc or IgG Fc control (Fig. 3B, c versus b and c  versus a). These data suggest that the RLD motif is required for Thy-1-integrin ␣ v ␤ 5 interactions both in a cell-free system and on the cell surface of lung fibroblasts.
Calyculin and ET-1 Treatments Induce Fibroblast Contraction by Both Thy-1(Ϫ) Lung Fibroblasts and Thy-1(ϩ) Lung Fibroblasts-Calyculin and ET-1 are both cell contraction agonists. Calyculin, a type 1 phosphatase inhibitor, induces contraction of both fibroblasts and granulation tissues by elevation of myosin light chain phosphorylation (49,50).  (c and d). Some Thy-1(Ϫ) rat lung fibroblasts were treated with P1F6 for 30 min prior to incubation with Thy-1-IgG Fc (b). Thy-1(Ϫ) rat lung fibroblasts incubated with human IgG Fc were used as a negative control (a). The binding of human Thy-1 on the cell surface of rat lung fibroblasts was analyzed by flow cytometry with FITC-conjugated anti-human Thy-1 antibody. Thy-1(Ϫ) rat lung fibroblasts overexpressing human ␤ 5 integrin subunit were generated by transient transfection. Expression of human ␤ 5 was determined by immunoblot analysis with antibody specific to human ␤ 5 integrin. Cells transfected with empty vector were used as a control. B, Thy-1(ϩ) rat lung fibroblasts were transiently transfected with human ␤ 5 integrin-expressing plasmid or empty vector. Overexpression of human ␤ 5 integrin was determined by immunoblot analysis (IB) as described above. ␤ 5 -overexpressing Thy-1(ϩ) lung fibroblasts and Thy-1(ϩ) lung fibroblasts were lysed. Cell lysates with equal amounts of protein were immunoprecipitated (IP) with 300 g of anti-integrin ␤ 5 antibody. Proteins were separated by SDS-PAGE under reducing conditions, and Thy-1 was detected by immunoblot analysis with anti-Thy-1.2 antibody.
To determine whether Thy-1(Ϫ) and Thy-1(ϩ) lung fibroblasts differentially express integrin ␣ v ␤ 5 on the cell surface and deposit latent TGF-␤1 in the ECM, we performed immunoblot analyses to compare protein levels of integrin ␣ v and ␤ 5 from cell membrane fractions and LAP(TGF-␤1) from the ECM fractions of Thy-1(Ϫ) and Thy-1(ϩ) lung fibroblasts. Data show that Thy-1(Ϫ) and Thy-1(ϩ) lung fibroblasts expressed comparable levels of integrin ␣ v and ␤ 5 subunits and deposited equal amounts of LAP(TGF-␤1) in the ECM. Calyculin and ET-1 treatments did not significantly change ␣ v and ␤ 5 expression on the cell surface and deposition of latent TGF-␤1 in the ECM (Fig. 5D). The data rule out that the differential susceptibility of Thy-1(Ϫ) and Thy-1(ϩ) lung fibroblasts to contrac- . Cells were lysed, and TGF-␤ activity was determined with a PAI-1-luciferase-based bioassay, as described previously (42). All TGF-␤ assays were performed in triplicate. *, p Ͻ 0.01 for comparisons as indicated. D, 7-day cultured Thy-1(Ϫ) and Thy-1(ϩ) lung fibroblasts were made quiescent and treated with calyculin (caly) and ET-1. Membranebound protein and ECM protein were prepared as described under "Experimental Procedures." 10 g of membrane-bound protein and 20 g of ECM protein were separated by SDS-PAGE. Protein levels of integrin ␣ v and ␤ 5 subunits from membrane fraction and LAP(TGF-␤1) from ECM fraction were detected by immunoblot analyses.

DISCUSSION
The major findings in this study are that Thy-1 interacts with integrin ␣ v ␤ 5 through an RGD-like motif, both in a cell-free system and on the cell surface of rat lung fibroblasts. Thy-1-integrin ␣ v ␤ 5 interactions inhibit cell contraction-induced latent TGF-␤1 activation and TGF-␤1-dependent myofibroblast differentiation by lung fibroblasts. Because wild-type Thy-1 inhibits LAP(TGF-␤1) binding to immobilized integrin ␣ v ␤ 5 in vitro, whereas the non-integrin-binding Thy-1(RLE) mutant does not, we propose that Thy-1-integrin ␣ v ␤ 5 interactions interfere with the binding of the latent TGF-␤1 complex to integrin ␣ v ␤ 5 , resulting in the abrogation of cell contraction-initiated mechanotransduction that is required for activation of the latent TGF-␤1 complex from the ECM.
Myofibroblasts acquire contractile activity by forming ␣-SMA-containing stress fibers. It is known that myofibroblast contractility is important for wound closure in normal wound healing. However, the role of myofibroblast contractility in fibrosis is not clear. Previous studies have shown that fibroblast contraction induces the unfolding of cryptic sites of fibronectin, resulting in autofibrillogenesis and long matrix fibril formation (53). Similarly, mechanical deformation may render the ED-A segment available for specific integrins, which in turn, promotes myofibroblast differentiation (54). In a recent study, Wipff et al. (22) showed that increased myofibroblast contraction promotes activation of latent TGF-␤1 that is predeposited in the ECM. In contrast, attenuation of myofibroblast contrac-tility by stress release induces myofibroblast apoptosis both in vitro and in vivo (55)(56)(57). These studies suggest that (myo)fibroblast contraction may actively participate in the establishment and progression of fibrosis.
Previous studies showed that attenuation of myofibroblast contractile force reduces type I collagen synthesis (58,59). Intracellular delivery of AcEEED, an N-terminal sequence of ␣-SMA that is crucial for ␣-SMA polymerization (60), reduces the tension exerted by cultured myofibroblasts on their substrates and the contractile activity of granulation tissue strips after ET-1 stimulation. This results in a decrease in type I collagen synthesis by myofibroblasts and a delay of wound contraction of splinted rat wounds (58). Rho/Rho kinase plays an important role in regulation of fibroblast and myofibroblast contractility (61)(62)(63). Inhibition of Rho/Rho kinase dramatically reduces the amount of force generated by fibroblasts and myofibroblasts cultured in three-dimensional collagen lattices (59). In the present study, we showed that Thy-1 interacts with integrin ␣ v ␤ 5 , a key mechanotransducer that mediates mechano-induced latent TGF-␤1 activation by lung fibroblasts. Thy-1-integrin ␣ v ␤ 5 interactions block fibroblast contractioninduced latent TGF-␤1 activation and TGF-␤1-dependent lung myofibroblast differentiation. Our study suggests that blockade of mechanotransduction by targeting key mechanotransducers may be an effective new strategy for preventing myofibroblast contraction and tissue fibrosis.
Fibroblast contractility is primarily regulated by myosin light chain (MLC) phosphorylation, a process that is controlled by the opposing activities of myosin light chain kinase and myosin light chain phosphatase (63). Consistent with this, calyculin, a myosin phosphatase inhibitor, induces fibroblast contraction by both Thy-1(Ϫ) and Thy-1(ϩ) lung fibroblasts, suggesting that inhibition of myosin phosphatase activity promotes lung fibroblast contraction. Previous studies suggest that MLC phosphorylation is regulated by various protein kinase networks, including integrin-linked kinase (64). It has been shown that integrin-linked kinase binds the cytoplasmic domains of integrin ␤ 5 , ␤ 3 , and ␤ 1 and regulates MLC phosphorylation by both activation of MLC and inactivation of myosin light chain phosphatase (65)(66)(67). Although Thy-1 interacts with integrin ␣ v ␤ 5 and probably other types of integrins on the cell surface of rat lung fibroblasts, it is unlikely that Thy-1 engagement of integrin-dependent integrin-linked kinase signaling plays a major role in Thy-1 regulation of calyculin-and ET-1-induced latent TGF-␤1 activation. This is because both Thy-1(Ϫ) and Thy-1(ϩ) lung fibroblasts respond to calyculin and ET-1 with increased contraction. It suggests that cell signaling involved in regulation of fibroblast contraction is preserved in both cells.
Our studies suggest that Thy-1 regulation of latent TGF-␤1 activation under fibrogenic conditions can occur through multiple mechanisms. Previously, we showed that Thy-1 regulates the ability of lung fibroblasts to activate latent TGF-␤ in response to fibrogenic stimuli (27). LTBP-4, a member of the LTBP/fibrillin family known to regulate latent TGF-␤1 bioavailability and activation, mediates latent TGF-␤1 activation by Thy-1(Ϫ) lung fibroblasts in response to bleomycin (18). Bleomycin treatment remarkably increases LTBP-4 expression, resulting in the accumulation of large amounts of soluble LTBP-4-bound LLC both in the conditioned medium of cultured Thy-1(Ϫ) lung fibroblasts and in the bronchoalveolar lavage fluids of Thy-1 knock-out mice. In contrast, bleomycininduced LTBP-4 expression is not evident in cultured Thy-1(ϩ) lung fibroblasts and is attenuated in the bronchoalveolar lavage fluids of wild-type littermates. The presentation of LTBP-4bound LLC in a soluble form facilitates MMP-mediated latent TGF-␤1 activation (18,27). These findings suggest that lung fibroblast expression of Thy-1 inhibits bleomycin-induced activation of soluble latent TGF-␤1 by suppression of LTBP-4 expression and the formation of soluble LTBP-4-bound LLC. Moreover, our current study shows that Thy-1 inhibits fibroblast contraction-induced activation of ECM-bound (insoluble) latent TGF-␤1 by interacting with the mechanotransducer, integrin ␣ v ␤ 5 . Together, these studies suggest that lung fibroblast expression of Thy-1 prevents both soluble and insoluble latent TGF-␤1 activation. Loss of Thy-1 expression renders lung fibroblasts susceptible to fibrogenic stimulation-induced LTBP-4 expression and LTBP-dependent soluble latent TGF-␤1 activation. Furthermore, the lack of Thy-1 protection allows integrin ␣ v ␤ 5 transmission of stress fiber-derived contractile force to the ECM-bound latent TGF-␤1, resulting in activation of the insoluble latent complex.
Previous studies suggest that a mechanically resistant (stiff) ECM is required for both myofibroblast contraction-induced latent TGF-␤1 activation and active TGF-␤1-dependent myofibroblast differentiation. Lung myofibroblast contraction-induced latent TGF-␤1 activation was observed in cells grown on stiff substrates with Young's moduli of 9 -47 kilopascals but not on compliant substrates with Young's modulus of 5 kilopascals (22). It has been reported that active TGF-␤1-induced ␣-SMA expression by lung fibroblasts and hepatic stellate cells requires collagen substrates with Young's moduli of at least 15-16 kilopascals (68,69), which is close to the stiffness of fibrotic tissues (70). Although it is not known whether Thy-1 may play a role in regulation of ECM stiffening, it is conceivable that changing the mechanical properties of the stiff ECM may be an approach for blocking myofibroblast contraction-induced latent TGF-␤1 activation and active TGF-␤1-dependent myofibroblast differentiation.
In summary, this study provides a mechanistic insight into Thy-1 expression by lung fibroblasts in regulation of myofibroblast differentiation and lung fibrosis. It suggests that blockade of mechanical force-induced latent TGF-␤1 activation due to increased fibroblast contractility might be an effective approach for inhibiting myofibroblast differentiation in persistent lung fibrosis.