Integrin {alpha}8ß1 mediates adhesion to LAP-TGFß1

Min Lu1, John S. Munger2, Melissa Steadele3, Christina Busald3, Marinka Tellier1 and Lynn M. Schnapp1,3,*

1 Pulmonary and Critical Care Medicine, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA
2 Departments of Medicine and Cell Biology, NYU School of Medicine, New York, NY 10016, USA
3 Pulmonary and Critical Care Medicine, Harborview Medical Center, University of Washington, Seattle, WA 98104, USA

* Author for correspondence (e-mail: lschnapp{at}u.washington.edu)

Accepted 3 September 2002


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 Materials and Methods
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The development of fibrosis is a common response to a variety of injuries and results in the net accumulation of matrix proteins and impairment of normal organ function. We previously reported that the integrin {alpha}8ß1 is expressed by alveolar interstitial cells in normal lung and is upregulated during the development of fibrosis. TGFß1 is an important mediator of the inflammatory response in pulmonary fibrosis. TGFß1 is secreted as a latent protein that is non-covalently associated with latency-associated peptide (LAP) and requires activation to exert its effects. LAP-TGFß1 and LAP-TGFß3 contain the tripeptide sequence, arginine-glycine-aspartic acid (RGD), a known integrin recognition motif. The integrin {alpha}8ß1 binds to several ligands such as fibronectin and vitronectin through the RGD sequence. Recent reports demonstrate that the integrins {alpha}vß1, {alpha}vß6 and {alpha}vß8 adhere to LAP-TGFß1 through the RGD site. Therefore, we asked whether LAP-TGFß1 might be a ligand for {alpha}8ß1 and whether this may be important in the development of fibrosis. We found that cell lines transfected with {alpha}8 subunit were able to spread on and adhere to recombinant LAP-TGFß1 significantly better than mock transfected cell lines. {alpha}8-transfected cells were also able to adhere to LAP-TGFß3 significantly better than mock transfected cells. Adhesion to LAP-TGFß1 was enhanced by activation of {alpha}8ß1 by Mn2+, or 8A2, an integrin ß1 activating antibody. Furthermore, cell adhesion was abolished when we used a recombinant LAP-TGFß1 protein in which the RGD site was mutated to RGE. {alpha}8ß1 binding to LAP-TGFß1 increased cell proliferation and phosphorylation of FAK and ERK, but did not activate of TGFß1. These data strongly suggest that LAP-TGFß1 is a ligand of {alpha}8ß1 and interaction of {alpha}8ß1 with LAP-TGFß1 may influence cell behavior.

Key words: Integrin, LAP-TGF-ß, {alpha}8ß1, Cell signaling


    Introduction
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 Introduction
 Materials and Methods
 Results
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 References
 
TGF-ß is a growth factor that was originally described by its ability to induce anchorage independent growth in fibroblasts. Three closely related isoforms exist (TGFß1, ß2 and ß3) which have a similar range of effects. Many of its effects are profibrotic: increased extracellular matrix synthesis, increased TIMP synthesis and decreased protease synthesis (Taipale et al., 1998Go). TGF-ß requires activation before it binds to its cognate receptors and exerts its effects. TGF-ß is synthesized as a proprotein. Proteolytic processing separates the N-terminal propeptide from TGFß. After processing, TGFß noncovalently associates with its propeptide. Because this interaction prevents TGFß from binding its receptors, the propeptide is termed latency-associated peptide (LAP). Within the secretory pathway, the complex of TGFß and LAP, referred to as the small latent complex, usually associates with another family of proteins, the latent TGF-ß-binding proteins (LTBP), to form large latent complex (LLC). LLC can become incorporated into the extracellular matrix. LAP-TGFß1 and LAP-TGFß3 contain a conserved tripeptide sequence, arginine-glycine-aspartic acid (RGD) that is found in many extracellular matrix proteins and is a known recognition sequence for integrins. Integrins are glycoproteins that consist of two non-covalently associated subunits, {alpha} and ß. Each integrin subunit has a unique cytoplasmic domain that elicits cellular responses by interacting with distinct signaling pathways. Interactions of integrins with their ligands result in alterations in many cellular activities such as migration, proliferation, apoptosis and matrix remodeling.

Recent work showed that LAP-TGFß1 binds to the integrins {alpha}vß1 (Munger et al., 1998Go) and {alpha}vß6 (Munger et al., 1999Go). Binding of {alpha}vß6 to LAP-TGFß1 activates TGFß1, independent of protease activity. Mice lacking the ß6 integrin subunit are protected from the development of pulmonary fibrosis due to the inability to activate TGFß1 (Munger et al., 1999Go).

We previously characterized the human integrin subunit, {alpha}8, which pairs exclusively with ß1 to form the heterodimer {alpha}8ß1 (Schnapp et al., 1995aGo). The integrin {alpha}8ß1 interacts with the RGD sequences in several matrix proteins including fibronectin, vitronectin, tenascin, osteopontin (Denda et al., 1998Go; Muller et al., 1995Go; Schnapp et al., 1995bGo) and nephronectin (Brandenberger et al., 2001Go). {alpha}8ß1 is expressed in alveolar interstitial cells and is upregulated during pulmonary and hepatic fibrosis (Levine et al., 2000Go). We now report that LAP-TGFß1 is a ligand for {alpha}8ß1 and that binding of LAP-TGFß1 to {alpha}8ß1 increases spreading and proliferation of cells and increases phosphorylation of the proteins FAK and ERK.


    Materials and Methods
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 Materials and Methods
 Results
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Cells and reagents
Human embryonic kidney 293 and human colon carcinoma SW480 cell lines and CHO cells were obtained from American Type Culture Collection and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and penicillin/streptomycin. AtT20 cells were a gift from Jean Schwarzbauer (Princeton University, NJ) and maintained in DMEM/Ham's F12, 10% FCS, 10% Nu-serum (Sigma), 200 µm HEPES, penicillin/streptomycin. ß6-transfected SW480 cells were a gift from Dean Sheppard (UCSF, CA). Cells were transfected with pCDNAIneo{alpha}8 ({alpha}8-transfected cells) or pCDNAIneo alone (mock-transfected cells) using the Lipofectin reagent (Gibco-BRL) according to the manufacturer's instructions. Stably transfected cell lines were selected in medium containing the neomycin analog G418 (0.4 mg/ml). Surface expression of {alpha}8ß1 was confirmed by immunoprecipitation of surface biotinylated proteins.

Mink lung epithelial cells (Mv1Lu) stably transfected with a portion of the plasminogen activator inhibitor 1 (PAI-1) promoter upstream of a luciferase reporter gene were used as previously described (Abe et al., 1994Go). Recombinant LAP-TGFß1 and RGE-LAP-TGFß1 were produced in a baculovirus system as described (Munger et al., 1998Go). Production of TGFß3 cDNA expression construct was previously reported (Annes et al., 2002Go). TGFß3 was cloned into pCDNA-Fc vector and used for protein purification as previously described (Annes et al., 2002Go).

Fibronectin (FN) was purchased from Boerhinger Mannheim and poly-L-lysine was purchased from Sigma. Rabbit polyclonal antibody to FAK and HRP-conjugated anti-phosphotyrosine antibody (clone 4G10) were obtained from Upstate Biotechnology. Antibody to phosphorylated ERK was obtained from Santa Cruz. Polyclonal antibody to recombinant human LAP-TGFß1 (AF-246-NA) was obtained from R&D Systems. The integrin-activating antibody 8A2 and the ß1 integrin blocking antibody 5D1 were a generous gift from John Harlan, University of Washington (Seattle, WA). The {alpha}v integrin blocking antibody L230 was prepared from hybridoma cells obtained from American Type Culture Collection (ATCC). Working dilutions for antibodies were determined for each application to optimize the results.

Adhesion assays
The assays were performed as previously described (Schnapp et al., 1995bGo). Briefly, untreated polystyrene 96-well flat bottom microtiter plates (Evergreen) were coated with increasing concentrations (0.3, 1, 3, 10, 20 µg/ml) of protein (LAP-TGFß1, LAP-TGFß3, FN) or 0.01% poly-L-lysine. As a negative control, wells were coated with 1% BSA. Wells coated at 37°C for 1 hour were washed with phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl and 10 mM Na2HPO4, pH 7.4) and non-specific protein binding sites were saturated with 1% BSA for 30 minutes at 37°C. Cells were detached with 2 mM EDTA, washed with PBS and resuspended in serum-free DMEM (pH 7.4) with or without 5 mM Mn2+. In some experiments, cells were preincubated with integrin blocking antibodies L230 or 5D1, or integrin-activating antibody 8A2 for 15 minutes on ice, prior to addition to wells. 50,000 cells were added to each well, centrifuged at 10 g for 3 minutes to ensure uniform settling of cells and incubated for 1 hour at 37°C. Non-adherent cells were then removed by centrifugation (top-side down) at 10 g for 5 minutes. The attached cells were fixed and stained with 1% formaldehyde/0.5% crystal violet/20% methanol for 30 minutes at RT. After washing with PBS, adherence was determined by absorption at 595 nm in a Microplate Reader (Bio-Rad, Richmond, CA). The data were reported as the mean absorbance of triplicate wells±s.e., minus the mean absorbance of BSA-coated wells.

LAP TGFß1 ELISA
Lungs were extracted from C57BL/6 mice (n=3) after perfusion with PBS/ heparin through the RV outflow tract until the lungs blanched, to remove blood. Lungs were weighed, placed in 2 ml PBS and homogenized 30 seconds with tissue homogenizer. Samples were filtered through a 0.45 micron filter to remove debris. Ninety-six well plates (Nunc-immunoplate, maxisorp surface) were coated overnight at 4°C with serial dilutions of lung homogenate in duplicate. Wells were coated with BSA alone as negative controls. To generate a standard curve, wells were coated with serial dilutions of recombinant LAP-TGFß1 protein. Non-specific binding sites were then saturated with 3% BSA /PBS for 1 hour at 37°C. Wells were washed with PBS-0.05% Tween and then incubated with 50 µl of anti-LAP-TGFß1 IgG antibody (0.5 µg/ml) (R&D Systems) at RT for 2 hours. Unbound protein was removed by washing with PBS/0.05% Tween. Biotinylated rabbit anti-goat IgG (0.15 µg/ml) was added to wells for 1 hour at RT, followed by addition of streptavidin AH-Biotin complex solution (Zymed SABC kit). Color development was performed using TMB Microwell Peroxidase Substrate system (KPL) and read at 450 nm after addition of stop solution (1 M phosphoric acid). The detection limit was approximately 60 pg/well. The concentration of LAP-TGFß1 in the samples was determined by interpolation from the standard curve.

TGFß bioassays
TGF-ß1 bioassay was performed as previously described (Munger et al., 1999Go). Briefly, 100 µl of Mv1Lu reporter cells were plated at a density of 105 cells/ml and allowed to adhere for 1 hour at 37°C in DMEM containing 10% FCS. Equal number of test cells were added to wells and cultured for 16 hours. In some experiments, test cells were incubated with the ß1 integrin-activating antibody 8A2 for 15 minutes prior to addition to Mv1Lu reporter cells. Lysates were assayed for luciferase activity using Luciferase Assay System (Promega). As a positive control, Mv1Lu reporter cells were cultured with recombinant TGFß1 (gift of Dan Rifkin). To determine whether {alpha}8ß1 expression affected the activation of TGFß1 by {alpha}vß6, we incubated Mv1Lu reporter cells with ß6-transfected SW480 (0.5x105 cells) and either {alpha}8-transfected SW480 or mock-transfected SW480 cells, and assayed for luciferase activity as described above.

To measure TGF-ß3 activation, we transfected mock or {alpha}8 transfectants with TGFß3 cDNA expression vector or control vector using Lipofectamine Plus (Life Technology) (Annes et al., 2002Go). After 16 hours, cells were added to reporter cells for 24 hours and luciferase activity was measured as above. When high amounts of TGFß3 cDNA were used for transfection, autoactivation of TGF-ß3 occurred. Therefore, we titered the amount of cDNA and found that transfection with 100 ng of TGFß3 cDNA eliminated autoactivation and resulted in detectable amounts of TGFß3 in supernatants.

Immunoprecipitation and western blot analysis
For FAK and ERK phosphorylation, cells were plated on ligands for 30 minutes, and then lysed in buffer containing 50 mM Tri-HCl (pH 7.4), 150 mM NaCl, 0.25% sodium deoxychloate, 1% IGEPAL CA-630 (non-ionic, non-denaturing detergent, Sigma), 1 mM EGTA, 1 mM PMSF, 1 mM NaVO3, 1 mM NaF, 1 mg/ml each of aprotonin, leupeptin, pepstatin. Samples were incubated with antibodies for 1-2 hours at 4°C. Immune complexes were captured with Protein A sepharose (Pharmacia). Beads were washed 5 times, boiled for 5 minutes in Laemli sample buffer and then proteins were separated by SDS-PAGE. Gels were transferred to Immobilon and non-specific binding sites were saturated with 3% BSA for 1 hour. Blots were incubated with primary antibody for 1 hour, followed by peroxidase conjugated secondary antibody for 1 hour and then developed with ECL (Amersham).

Proliferation assays
5x103 AtT20 or AtT20 {alpha}8-transfected cells were plated in serum-free media in 96 well plates coated with 5 µg/ml LAP-TGFß1, FN, or 0.01% poly-L-lysine. Proliferation was assayed at indicated times using the Roche Cell Proliferation Kit (MTT) per manufacturer's instructions. Four independent clones of AtT20{alpha}8 were tested. All experiments were performed in triplicate and presented as the mean±s.e.

Immunohistochemistry
Lungs were obtained from 8-week-old C57BL/6 mice as previously described (Madtes et al., 2001Go). Briefly, the lungs was inflated with 4% neutral buffered paraformaldehyde instilled at 30 cm H2O pressure through the trachea for 120 minutes. The trachea was then tied and the lung immersed in the RNAse-free, 4% buffered paraformaldehyde for 24 hours before embedding in paraffin. 5-µm sections of lung fixed with 4% (wt/vol) paraformaldehyde were deparaffinized and rehydrated. Endogenous peroxidase and biotin activity was saturated by incubation of the sections in Peroxoblock (Zymed), followed by Avidin-Biotin Blocking Reagent (Zymed). The sections were incubated overnight at 4°C with affinity purified goat anti-human LAP-TGFß1 IgG antibody (2.5 µg/ml) (R&D Systems). Primary antibody was detected with biotinylated rabbit anti-goat IgG antibody (Zymed Laboratories) (0.15 µg/ml). Bound antibody was visualized with ABC peroxidase (Vector Laboratories). The sections were counterstained with hematoxylin. As a negative control, adjacent serial sections were stained in the absence of primary antibody.


    Results
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 Materials and Methods
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 References
 
{alpha}8ß1 mediates adhesion to LAP-TGFß1
To determine whether {alpha}8ß1 binds to LAP-TGFß1, we examined the adhesion of {alpha}8-transfected cells to recombinant LAP-TGFß1 in 4 different cell lines. We found that {alpha}8-transfected cells adhered to 5 µg/ml LAP-TGFß1 significantly better than mock transfected in all cell lines tested (Fig. 1A). Adhesion of 293 cells to LAP-TGFß1 was inhibited with an anti-{alpha}v antibody, consistent with previous reports showing 293 cells adhere to LAP-TGFß1 through the integrin {alpha}vß1 (Munger et al., 1998Go). Adhesion of 293{alpha}8 and SW480{alpha}8 cells to LAP-TGFß1 was inhibited by an anti-ß1 integrin blocking antibody, 5D1. When adhesion to increasing concentrations of LAP-TGFß1 was tested, there was a significant difference in the adhesion of SW480{alpha}8 cells and SW480ß6 cells (Fig. 1B). SW480{alpha}8 cells adhered to LAP-TGFß1 only at concentrations of 5 µg/ml or higher, whereas SW480ß6 cells adhered to LAP-TGFß1 at much lower concentrations (0.3 µg/ml) consistent with previous reports (Munger et al., 1999Go). When {alpha}8ß1 was activated with either an integrin ß1 activating antibody, 8A2, or 1 mM Mn2+, we observed enhanced adhesion of SW480{alpha}8 to LAP-TGFß1, at levels equivalent to or greater than ß6-mediated adhesion (Fig. 1B and data not shown).



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Fig. 1. (A) {alpha}8ß1 adhesion to LAP-TGFß1. Mock transfected or {alpha}8-transfected cells (50,000/well) were allowed to attach to wells precoated with 5 µg/ml of recombinant LAP-TGFß1. In some cases, cells were incubated with {alpha}v integrin antibody (L230) or ß1 integrin antibody (5D1) prior to adhesion assay. After 1 hour, non-adherent cells were removed by brief centrifugation and adherent cells were fixed and stained with formaldehyde/crystal violet. Adherent cells were quantitated by measuring absorbance of wells at OD595. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well. (B) Adhesion of SW480{alpha}8 or SW480ß6-transfected cells to increasing concentrations of LAP-TGFß1 with or without 1mM Mn2+. After 1 hour, non-adherent cells were removed by brief centrifugation and adherent cells were fixed and stained with formaldehyde/crystal violet. Adherent cells were quantitated by measuring absorbance of wells at OD595. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well. (C) Adhesion of {alpha}8 or ß6-transfected cells to fibronectin. AtT20{alpha}8, SW480{alpha}8 or SW480ß6 cells were allowed to attach to wells precoated with the indicated concentration of fibronectin. After 1 hour, non-adherent cells were removed by brief centrifugation and adherent cells were fixed and stained with formaldehyde/crystal violet. Adherent cells were quantitated by measuring absorbance of wells at OD595. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well. (D) Mutation of RGD site in LAP TGFß1 eliminates {alpha}8ß1 adhesion. {alpha}8-transfected cells were allowed to attach to wells coated with 5 µg/ml of authentic recombinant LAP-TGFß1 (RGD-LAP-TGFß1) or recombinant LAP-TGFß1 containing a single glutamic acid for aspartic acid substitution mutation in the RGD site (RGE-LAP-TGFß1) for 1 hour; adhesion was then assessed by absorbance. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of BSA-coated well. (E) Adhesion of AtT20{alpha}8 cells to LAP-TGFß3 is similar to adhesion to LAP-TGFß1. AtT20 or AtT20{alpha}8 cells were allowed to adhere to increasing concentrations of recombinant LAP-TGFß1 or LAP-TGFß3 for 1 hour; adhesion was then assessed by absorbance. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well.

 

In contrast to LAP-TGFß1, fibronectin (a known RGD-containing ligand for {alpha}vß6 and {alpha}8ß1) supported adhesion of unactivated SW480ß6 and SW480{alpha}8 cells equally well (Fig. 1C). Cell surface expression of {alpha}8 and ß6 was similar in SW480 cells (data not shown). To confirm that the RGD site in LAP-TGFß1 was involved in binding to {alpha}8ß1, we examined adhesion of {alpha}8-transfected cells to a recombinant LAP-TGFß1 in which the RGD site was mutated to RGE. Mutation of RGD to RGE eliminated {alpha}8ß1-mediated adhesion to LAP-TGFß1 in all cell lines tested (Fig. 1D).

LAP-TGFß1 and LAP-TGFß3 support {alpha}8ß1 adhesion with similar efficacy
Because LAP-TGFß3 contains an RGD sequence in a similar location as LAP-TGFß1, we asked whether LAP-TGFß3 was also a ligand for {alpha}8ß1. We examined the adhesion of AtT20{alpha}8 cells and AtT20 mock cells to recombinant LAP-TGFß3. We found that AtT20{alpha}8 cells adhered to LAP-TGFß3 significantly better than mock transfected cells (Fig. 1E). The adhesion of {alpha}8-transfected cells to LAP-TGFß3 was similar to adhesion to LAP-TGFß1 (Fig. 1E).

Adhesion to LAP-TGFß1 results in cell signaling
Integrin mediated signaling results in alterations in the cytoskeleton, leading to cell shape changes. When AtT20 cells were plated on LAP-TGFß1, cells became flat and spread on the substrate, and developed long extensions (Fig. 2i). Focal adhesion kinase (FAK) is present at focal contacts and becomes phosphorylated after integrin-mediated cell adhesion and plays a role as an adapter protein for integrin-mediated cell signaling. We hypothesized that if LAP-TGFß1 is a ligand for {alpha}8ß1, adhesion of {alpha}8ß1-expressing cells to LAP-TGFß1 would lead to FAK phosphorylation. We plated mock-transfected or {alpha}8-transfected AtT20 cells on plates coated with poly-L-lysine (which allows non-integrin mediated adhesion), fibronectin (a known ligand for {alpha}8ß1) and LAP-TGFß1 and RGE-LAP-TGFß1 for 30 minutes in serum-free media. Cells were lysed in the presence of phosphatase and protease inhibitors, and immunoprecipitated with anti-FAK antibody, followed by blotting with anti-phosphotyrosine antibody or FAK antibody. We found that interaction of LAP-TGFß1 with {alpha}8ß1 leads to tyrosine phosphorylation of FAK comparable to phosphorylation seen after fibronectin adhesion (Fig. 2ii). Furthermore, mutation of the LAP-TGFß1 RGD site to RGE eliminated FAK phosphorylation. Mock transfected cells did not show FAK phosphorylation when grown on LAP-TGFß1 or fibronectin.



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Fig. 2. (i) {alpha}8ß1 mediates spreading on LAP-TGFß1. {alpha}8-transfected or mock-transfected AtT20 cells were plated on 5 µg/ml of recombinant LAP-TGFß1 in serum-free media for 24 or 48 hours. At 24 hours, mock transfected AtT20 cells remain rounded and unattached on LAP-TGFß1 (A), whereas {alpha}8-transfected cells attach and spread on LAP-TGFß1 (B). Further spreading is seen at 48 hours (C). (ii) Adhesion of {alpha}8ß1 to LAP-TGFß1 results in FAK phosphorylation. {alpha}8-transfected or mock-transfected AtT20 cells were plated on 0.01% poly-L-lysine (PLL), 5 µg/ml fibronectin (FN), 5 µg/ml LAP-TGFß1 (LAP1) or 5 µg/ml RGE-LAP-TGFß1 (RGE) for 30 minutes in serum-free media. Cells were lysed in buffer containing phosphatase inhibitors, immunoprecipitated with anti-FAK antibody followed by western blotting with either anti-phosphotyrosine antibody PY20 (top panel) or anti-FAK antibody (bottom panel).

 

LAP-TGFß1-{alpha}8ß1 mediates cell proliferation
We then asked whether cell behaviors such as proliferation were affected by adhesion to LAP-TGFß1. We found that {alpha}8-transfected cells proliferated significantly better when grown on LAP-TGFß1 compared to mock transfected cells grown on LAP-TGFß1 in serum-free media (Fig. 3A). The degree of proliferation was similar to that of cells grown on fibronectin. To insure that the enhanced proliferation was not due to clonal variation, we tested four independent clones of AtT20{alpha}8 transfectants. All showed a significant increase in proliferation compared to mock transfected or wild type cells (Fig. 3A and data not shown) The average fold increase in proliferation was 1.9 compared to mock transfected cells. We examined whether ERK was phosphorylated in response to {alpha}8ß1-LAP-TGFß1 binding. We found an increase in phospho-ERK levels in {alpha}8-transfected cells adherent to LAP-TGFß1, compared to {alpha}8-transfected cells adherent to poly-L-lysine, or mock-transfected cells (Fig. 3B).



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Fig. 3. (A) Proliferation of cells adherent to LAP-TGFß1. Equal numbers of {alpha}8-transfected or mock-transfected AtT20 cells were plated on 5 µg/ml LAP-TGFß1 or 5 µg/ml fibronectin (FN) in serum-free media. After 3 days, proliferation was assayed using the Roche Cell Proliferation kit (MTT). Results for four independent clones of AtT20{alpha}8 cells are shown. Data is reported as the mean absorbance of triplicate wells±s.d. (B) ERK phosphorylation on LAP-TGFß1. {alpha}8-transfected or mock-transfected AtT20 cells were plated on 0.01% poly-L-lysine (PLL) 5 µg/ml fibronectin (FN), 5 µg/ml LAP-TGFß1 (LAP1), or 5 µg/ml RGE-LAP-TGFß1 (RGE) for 30 minutes in serum-free media and then lysed in buffer containing phosphatase inhibitors. Equal amounts of protein were loaded and probed with an antibody to phospho-ERK (top) or ERK (bottom).

 

{alpha}8ß1 binding to LAP-TGFß1 does not affect activation of TGFß1
We asked whether binding of {alpha}8ß1 to LAP-TGFß1 activated TGFß1, as described for {alpha}vß6 (Munger et al., 1999Go). As an indicator of TGFß1 activation, we used Mv1Lu reporter cells transfected with a portion of the plasminogen activator inhibitor-1 (PAI-1) promoter upstream of a luciferase reporter gene. The PAI-1 promoter contains a well-characterized TGFß-responsive element. Therefore, if active TGFß1 is present, an increase in luciferase activity will be detected. Mv1Lu reporter cells were co-cultured with mock-transfected SW480 cells or {alpha}8-transfected SW480 cells. As a positive control, Mv1Lu reporter cells were cultured with TGFß1, or with SW480ß6 cells. Luciferase activity did not increase when {alpha}8-transfected cells were cultured with MLEC, suggesting that adhesion of LAP-TGFß1 to {alpha}8ß1 was not sufficient to activate TGFß1 (Fig. 4A). Addition of 8A2 or Mn2+, which enhanced adhesion of {alpha}8 cells to LAP-TGFß1, did not affect TGF-ß activation (Fig. 4A, data not shown).



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Fig. 4. (A) Adhesion of LAP-TGFß1 to {alpha}8ß1 does not activate TGFß1. Equal number of Mv1Lu reporter cells and test cells (SW480{alpha}8 or SW480ß6) were co-cultured for 16 hours with indicated concentrations of the integrin-activating antibody 8A2 and lysed for measurement of luciferase activity. Results are the means of at least two experiments done in duplicate. (B) {alpha}8-transfected cells do not affect TGF-ß1 activation by SW480ß6 cells. Mv1Lu reporter cells were cultured with SW480{alpha}8 cells and SW480ß6 cells, or mock-transfected SW480 cells and SW480ß6 cells, for 16 hours and lysed for measurement of luciferase activity. Results are the means of duplicate experiments±s.d. (C) Adhesion of LAP-TGFß3 to {alpha}8ß1 does not activate TGFß3. CHO or CHO{alpha}8 cells were transiently transfected with full-length expression construct of TGFß3. After 16 hours, transfected cells were cultured for 24 hours with Mv1Lu reporter cells. Cells were lysed and luciferase activity was measured. Results are the mean of triplicate experiments±s.d.

 

We then asked whether adhesion of {alpha}8ß1 to LAP-TGFß1 affected the activation of TGFß1 mediated by {alpha}vß6, by competing for LAP-TGFß1. We set up a triple co-culture system using Mv1Lu reporter cells cultured with SW480ß6 and either {alpha}8-transfected SW480 cells or mock-transfected SW480 cells. No difference in {alpha}vß6-mediated activation of TGF-ß1 was found between {alpha}8-transfected SW480 cells and mock-transfected SW480 cells (Fig. 4B). Since we found adhesion of {alpha}8-transfected cells to LAP-TGFß3, we asked whether that interaction led to activation of LAP-TGFß3. Recent reports showed that {alpha}vß6, which activated TGFß1, also binds and activates TGFß3 (Annes et al., 2002Go). However, we found no difference in activation of LAP-TGFß3 when we compared CHO and CHO-{alpha}8 cells transfected with LAP-TGFß3 (Fig. 4C). Similar negative results were observed with 293 and 293{alpha}8 cells (data not shown). These results suggest that {alpha}8ß1 does not activate LAP-TGFß3.

Immunolocalization and concentration of LAP-TGFß1 in lung
We previously showed that {alpha}8 is localized to lung interstitial cells and is upregulated during pulmonary fibrosis. Using an antibody specific for LAP-TGFß1, we examined the immunolocalization of LAP-TGFß1 in normal lung tissue (Fig. 5). Immunoreactivity for LAP was detected along the interstitial cells, in a patchy pattern similar to alpha 8 immunolocalization, as well as in macrophages. To estimate the relative concentration of LAP-TGFß1 in mouse lung, we developed an ELISA for LAP-TGFß1 for use on whole mouse lung homogenates. The measurements ranged from 0.5 to 8 µg/mg lung tissue with an average of 3.38 µg of LAP-TGFß1 per mg lung tissue.



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Fig. 5. LAP-TGFß1 localization in adult lung tissue. Paraffin-embedded, formalin fixed tissues were stained with an antibody specific for LAP-TGFß1 and counterstained with hematoxylin. Immunoreactivity for LAP-TGFß1 is seen on a macrophage (arrowhead) and interstitial cells (arrows). Magnification: A, 4x; B,C, 20x; D, 40x.

 


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We present evidence that LAP-TGFß1 is a ligand for the integrin {alpha}8ß1, and binding results in activation of cell signaling pathways associated with cell survival and proliferation. The main biological roles of active TGFß1 include growth inhibition of epithelial, endothelial and hematopoeitic cells, increase extracellular matrix (ECM) formation and immunomodulation (Taipale et al., 1998Go). Interestingly, LAP-TGFß1 may regulate cell behaviors such as cell proliferation in a manner distinct from active TGFß1. LAP-TGFß1 is targeted to the ECM by LTBP, where LAP-TGFß1 localizes to fibrillar structures of the ECM (Taipale et al., 1996Go). The half-life of TGFß1 in plasma is short; however, LAP-TGFß1 half-life is significantly longer (Wakefield et al., 1990Go) and may be increased by incorporation into the ECM. The extracellular matrix is a complex meshwork of proteins and proteoglycans. In addition to structural support, the ECM directly effects cell signaling through interactions with cell surface receptors such as integrins. The ECM also serves as a `sponge' for many growth factors and cytokines (Saharinen et al., 1999Go). LAP-TGFß1 incorporation into the ECM may increase its local concentration and facilitate signaling through {alpha}8-expressing cells. Normally, the ability of TGFß1 to interact with its receptor requires cleavage of LAP or activation of TGFß1 by other mechanisms. However, we show that the `latent form' of TGFß1 can signal independently from activation. Therefore, additional complexity of TGF signaling may be obtained by regulating the levels of LAP-TGFß vs. active TGFß.

Ligands for integrins include ECM proteins such as fibronectin, collagens, and laminin, and cell surface counter receptors such as immunoglobulin superfamily members. However, the ligand repertoire of integrins may be considerably greater, considering the number of proteins that contain potential integrin binding motifs. For example, several viruses contain conserved RGD sequences in their envelope, which interact with integrins and contribute to viral adhesion and entry (Chiu et al., 1999Go; Jackson et al., 2000Go; Neff et al., 1998Go). Disintegrins also contain RGD sites that are used to interact with integrins (Gould et al., 1990Go; McLane et al., 1998Go; Niewiarowski et al., 1994Go).

We show that {alpha}8ß1 recognizes LAP-TGFß1 via the RGD sequence. {alpha}8, along with {alpha}5, {alpha}v and {alpha}IIb, form a subfamily of integrin subunits that are related based on sequence homology, binding to RGD sequences and absence of I domain. Three other family members, {alpha}vß1, {alpha}vß6 and {alpha}vß8, also bind LAP-TGFß1. However, only {alpha}vß6 or {alpha}vß8 binding to LAP-TGFß1 activates TGFß1 (Mu et al., 2002Go; Munger et al., 1999Go). Thus, the adhesion of {alpha}8ß1 more closely resembles the adhesion of {alpha}vß1 to LAP-TGFß1. Although {alpha}8ß1 did not result in activation of TGFß1 by the assay performed, it may facilitate activation by another mechanism. Binding of LAP-TGFß1 to {alpha}8ß1 may localize LAP-TGFß1 to the cell surface and lead to activation by other pathways, such as proteolytic cleavage or by thrombospondin. The binding avidity of the integrin to LAP-TGFß1 may determine whether TGFß1 activation occurs. Both {alpha}8ß1 and {alpha}vß1 only bind to LAP-TGFß1 at higher concentrations than {alpha}vß6. However, we are able to increase adhesion of {alpha}8ß1 to LAP-TGFß1 to levels comparable to {alpha}vß6 by Mn2+ or integrin ß1-activating antibody and despite the increased adhesion, activation of TGFß1 did not occur. Another possibility is that a second binding determinant is required. For example, interaction of the disintegrin echistatin with ß1 and ß3 integrins involves a secondary binding determinant on the C-terminus in addition to the RGD site (Wierzbicka-Patynowski et al., 1999Go). Recently, the sequence DLXXL was reported as a ligand for {alpha}vß6 (Kraft et al., 1999Go). A similar sequence is found adjacent to the RGD site in LAP-TGFß1 (RGDLXXI), LAP-TGF-ß3 (RGDLXXL) and adjacent to the RGD site in Foot and Mouth Disease Virus, another recently described ligand for {alpha}vß6 (Jackson et al., 2000Go). Therefore, activation of TGFß1 by integrin binding may be determined by sequence adjacent to RGD sequence. Because LAP-TGFß3 also contains an RGD sequence, we examined adhesion of {alpha}8ß1 to LAP-TGFß3. We found {alpha}8-transfected cells adhered similarly to LAP-TGFß3 as to LAP-TGFß1. However, similar to {alpha}8ß1-LAP-TGFß1 interaction, {alpha}8ß1-LAP-TGFß3 interaction was not sufficient to activate LAP-TGFß3.

Integrins such as {alpha}8ß1 and {alpha}vß1 that bind but do not activate LAP-TGFß1 may negatively regulate TGFß1 activity by sequestering latent TGFß1 and preventing access to an activating integrin such as {alpha}vß6. Although we did not see this affect in vitro, excess TGFß1 is present in serum and may overcome sequestration by {alpha}8ß1. Another explanation for the lack of affect of {alpha}8ß1 on {alpha}vß6-mediated activation was that {alpha}8ß1 and {alpha}vß6 were expressed on different cells. However, this mimics the in vivo situation, where {alpha}vß6 is expressed on epithelial cells and {alpha}8ß1 is expressed on interstitial cells (Breuss et al., 1995Go; Levine et al., 2000Go).

Is the concentration of LAP-TGFß1 required to see an effect physiologically relevant? We have several lines of evidence to suggest it might be. Measurements of TGFß1 concentrations in bronchoalveolar lavage fluid (BALF) from 67 patients with persistent acute respiratory distress syndrome (ARDS) showed values as high as 973 pg/ml, with an average concentration of 124 pg/ml +/-182 pg/ml (Personal communication, Richard B. Goodman, University of Washington). Significant immunoreactivity was only detected after acid activation of BALF, indicating that the measured TGFß1 was present in the latent form. Because bronchoalveolar lavage in humans has been reported to dilute lung fluid by 100- fold (Miller et al., 1992Go), we estimate the lung fluid concentrations of LAP TGFß1 in these patients to be as high as 0.1 µg/ml, with an average concentration of 12 ng/ml. This value is likely to underestimate the LAP-TGFß1 concentration within the microenvironment of the lung parenchyma, as the distribution of LAP-TGFß1 is heterogeneous (Fig. 5). Next, we measured LAP-TGFß1 in mouse lung homogenates and detected a range of LAP-TGFß1 from 0.5 to 8 µg/mg lung tissue, with an average of 3.38 µg±4. Finally, in vitro interactions of the integrin {alpha}vß6 with LAP-TGFß1 occur with coating concentrations in the microgram range and this interaction has important physiological consequences in the regulation of lung inflammation (Munger et al., 1999Go). Thus, we conclude that coating concentrations of LAP-TGFß1 used in this report are in the range of concentrations potentially encountered in vivo.

LAP-TGFß1 distribution in the lung interstitium was similar to {alpha}8 distribution suggesting that {alpha}8ß1-LAP-TGFß1 interactions can occur in vivo. The ability of {alpha}8ß1-LAP-TGFß1 interactions to induce FAK and ERK phosphorylation and promote cell proliferation strongly argues that LAP may be a relevant biological ligand in vivo and that interactions of cells with ECM-bound LAP may result in alterations in cell behavior. LAP-TGFß1 ligation to {alpha}8ß1 resulted in cell spreading, adhesion, proliferation, and phosphorylation of FAK and ERK. LAP-TGFß3 is likely to have similar effects. Thus, independent of its role in regulating the amount of active TGFß1, LAP-TGFß1 may have a novel role in regulation of cell behavior via interaction with integrins such as {alpha}8ß1.


    Acknowledgments
 
This work was supported by AHA-Heritage Affiliate Grant-in-Aid, AHA Northwest Affiliate Grant-in-Aid and RO1 HL 57890 (to L.M.S.)


    References
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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