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αvβ3 Integrin Interacts with the Transforming Growth Factor β (TGFβ) Type II Receptor to Potentiate the Proliferative Effects of TGFβ1 in Living Human Lung Fibroblasts

2004; Elsevier BV; Volume: 279; Issue: 36 Linguagem: Inglês

10.1074/jbc.m403010200

1083-351X

Amelia K. Scaffidi, Nenad Petrović, Yuben Moodley, Mirjana Fogel‐Petrovic, Karen M. Kroeger, Ruth M. Seeber, Karin A. Eidne, Philip J. Thompson, Darryl A. Knight,

Protease and Inhibitor Mechanisms

The αvβ3 integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether αvβ3 regulates transforming growth factor β (TGFβ) 1-induced responses, we investigated the interaction between αvβ3 and TGFβ type II receptor (TGFβIIR) in primary human lung fibroblasts. We report that TGFβ1 up-regulates cell surface and mRNA expression of αvβ3 in a time- and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFβRII associates and clusters with αvβ3, following TGFβ1 exposure. This association was not observed with αvβ5 or α5β1. We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGFβ1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds αvβ3, did not induce a substantial BRET signal. Co-exposure to tenascin and TGFβ1 produced no further increases in BRET than TGFβ1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGFβ1 and tenascin, and as a consequence proliferation induced by TGFβ1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFβRII and αvβ3 and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGDSPCA, which blocks αvβ3, also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the αvβ3 integrin, the TGFβIIR, in which TGFβ1-induced responses are potentiated in the presence αvβ3 ligands. Our data provide a novel mechanism by which TGFβ1 may contribute to abnormal wound healing and tissue fibrosis. The αvβ3 integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether αvβ3 regulates transforming growth factor β (TGFβ) 1-induced responses, we investigated the interaction between αvβ3 and TGFβ type II receptor (TGFβIIR) in primary human lung fibroblasts. We report that TGFβ1 up-regulates cell surface and mRNA expression of αvβ3 in a time- and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFβRII associates and clusters with αvβ3, following TGFβ1 exposure. This association was not observed with αvβ5 or α5β1. We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGFβ1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds αvβ3, did not induce a substantial BRET signal. Co-exposure to tenascin and TGFβ1 produced no further increases in BRET than TGFβ1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGFβ1 and tenascin, and as a consequence proliferation induced by TGFβ1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFβRII and αvβ3 and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGDSPCA, which blocks αvβ3, also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the αvβ3 integrin, the TGFβIIR, in which TGFβ1-induced responses are potentiated in the presence αvβ3 ligands. Our data provide a novel mechanism by which TGFβ1 may contribute to abnormal wound healing and tissue fibrosis. Acute inflammation is a beneficial response of tissue injury and generally results in repair and restoration of normal tissue architecture and function. These processes are spatially and temporally controlled by local signals generated by a plethora of growth factors as well as the immediate extracellular micro-environment. However, chronic airway inflammation and associated aberrant repair, which often occurs in asthma and pulmonary fibrosis can lead to abnormal airway structure and function. The composition of the extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; TGFβ1, transforming growth factor β1; TGFβIIR, transforming growth factor type II receptor; BRET, bioluminescence resonance energy transfer; TN, tenascin; VN, vitronectin; PBS, phosphate-buffered saline; HPRT, hypoxanthine phosphoribosyltransferase; OSM, oncostatin M; Rluc, Renilla luciferase; EYFP, enhanced yellow fluorescent protein; CN, collagen; LM, laminin; MCD, methyl-β-cyclodextrine; BrdUrd, bromodeoxyuridine. 1The abbreviations used are: ECM, extracellular matrix; TGFβ1, transforming growth factor β1; TGFβIIR, transforming growth factor type II receptor; BRET, bioluminescence resonance energy transfer; TN, tenascin; VN, vitronectin; PBS, phosphate-buffered saline; HPRT, hypoxanthine phosphoribosyltransferase; OSM, oncostatin M; Rluc, Renilla luciferase; EYFP, enhanced yellow fluorescent protein; CN, collagen; LM, laminin; MCD, methyl-β-cyclodextrine; BrdUrd, bromodeoxyuridine. is critical to the regulation of normal tissue function, because it is capable of regulating a variety of cellular responses such as proliferation, differentiation, migration, and apoptosis via binding to specific cell surface integrins (1Clark R.A. Tonnesen M.G. Gailit J. Cheresh D.A. Am. J. Pathol. 1996; 148: 1407-1421PubMed Google Scholar, 2Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3754) Google Scholar, 3Critchley D.R. Curr. Opin. Cell Biol. 2000; 12: 133-139Crossref PubMed Scopus (489) Google Scholar). Integrins are membrane-spanning glycoproteins consisting of a heterodimer of non-covalently linked α and β subunits (4Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8935) Google Scholar, 5Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1455) Google Scholar) which consist of a short cytoplasmic domain, a transmembrane domain, and a large extracellular domain that binds to ECM proteins via specific peptide sequences (6Ruoslahti E. Annu. Rev. Cell Dev. Biol. 1996; 12: 697-715Crossref PubMed Scopus (2485) Google Scholar). Considerable attention has focused on the αvβ3 integrin, because it appears to play a major role in several processes relevant to remodeling, such as binding and activation of matrix metalloproteinases (7Meerovitch K. Bergeron F. Leblond L. Grouix B. Poirier C. Bubenik M. Chan L. Gourdeau H. Bowlin T. Attardo G. Vasc. Pharmacol. 2003; 40: 77-89Crossref PubMed Scopus (73) Google Scholar) and growth factors (8Trusolino L. Serini G. Cecchini G. Besati C. Ambesi-Impiombato F.S. Marchisio P.C. De Filippi R. J. Cell Biol. 1998; 142: 1145-1156Crossref PubMed Scopus (97) Google Scholar), as well as cell proliferation (9Cruet-Hennequart S. Maubant S. Luis J. Gauduchon P. Staedel C. Dedhar S. Oncogene. 2003; 22: 1688-1702Crossref PubMed Scopus (126) Google Scholar), migration, and differentiation (10Levinson H. Hopper J.E. Ehrlich H.P. J. Cell. Physiol. 2002; 193: 219-224Crossref PubMed Scopus (14) Google Scholar). We have shown that vitronectin (VN) and its receptors, including αvβ3, can dramatically modulate the phenotype and function of human lung fibroblasts via integrin-specific intracellular signaling pathways (11Scaffidi A.K. Moodley Y.P. Weichselbaum M. Thompson P.J. Knight D.A. J. Cell Sci. 2001; 114: 3507-3516Crossref PubMed Google Scholar). Clinical studies have also shown that ligands for αvβ3 are increased in inflamed and remodeled lungs (12Pohl W.R. Conlan M.G. Thompson A.B. Ertl R.F. Romberger D.J. Mosher D.F. Rennard S.I. Am. Rev. Respir. Dis. 1991; 143: 1369-1375Crossref PubMed Scopus (35) Google Scholar, 13Teschler H. Pohl W.R. Thompson A.B. Konietzko N. Mosher D.F. Costabel U. Rennard S.I. Am. Rev. Respir. Dis. 1993; 147: 332-337Crossref PubMed Scopus (24) Google Scholar, 14Kaminski N. Allard J.D. Pittet J.F. Zuo F. Griffiths M.J. Morris D. Huang X. Sheppard D. Heller R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1778-1783Crossref PubMed Scopus (359) Google Scholar). Whereas it is clear that interactions between integrins and the ECM play fundamental roles in the response of a tissue to injury, it is apparent that expression of integrins is dynamically regulated throughout the various stages of repair by a variety of cytokines and growth factors. TGFβ1 is produced by a variety of cells and controls multiple processes involved in wound repair (15Riedy M.C. Brown M.C. Molloy C.J. Turner C.E. Exp. Cell Res. 1999; 251: 194-202Crossref PubMed Scopus (42) Google Scholar, 16Hashimoto S. Gon Y. Takeshita I. Matsumoto K. Maruoka S. Horie T. Am. J. Respir. Crit. Care Med. 2001; 163: 152-157Crossref PubMed Scopus (192) Google Scholar, 17Pittet J.F. Griffiths M.J. Geiser T. Kaminski N. Dalton S.L. Huang X. Brown L.A. Gotwals P.J. Koteliansky V.E. Matthay M.A. Sheppard D. J. Clin. Invest. 2001; 107: 1537-1544Crossref PubMed Scopus (394) Google Scholar), including the regulation of cell growth and differentiation as well as stimulating the net accumulation of ECM proteins (18Moustakas A. Stournaras C. J. Cell Sci. 1999; 112: 1169-1179Crossref PubMed Google Scholar, 19Linnala A. Kinnula V. Laitinen L.A. Lehto V.P. Virtanen I. Am. J. Respir. Cell Mol. Biol. 1995; 13: 578-585Crossref PubMed Scopus (41) Google Scholar). Its role in inducing proliferation is unclear, although it is thought to be a relatively weak mitogen for fibroblasts (20McAnulty R.J. Hernandez-Rodriguez N.A. Mutsaers S.E. Coker R.K. Laurent G.J. Biochem. J. 1997; 321: 639-643Crossref PubMed Scopus (111) Google Scholar). TGFβ1 elicits its biological effects by interacting with the constitutively active serine/threonine kinase TGFβ type II receptor (TGFβIIR), which recruits and activates TGFβ type I receptor (21Weis-Garcia F. Massague J. EMBO J. 1996; 15: 276-289Crossref PubMed Scopus (133) Google Scholar, 22Chen R.H. Derynck R. J. Biol. Chem. 1994; 269: 22868-22874Abstract Full Text PDF PubMed Google Scholar). TGFβ1 has been shown to modulate the expression of integrins, although these effects are particularly dependent on cell type (23Sheppard D. Cohen D.S. Wang A. Busk M. J. Biol. Chem. 1992; 267: 17409-17414Abstract Full Text PDF PubMed Google Scholar, 24Kumar N.M. Sigurdson S.L. Sheppard D. Lwebuga-Mukasa J.S. Exp. Cell Res. 1995; 221: 385-394Crossref PubMed Scopus (69) Google Scholar). Whether TGFβ1 effects αvβ3 transcription and synthesis in normal human lung fibroblasts is yet to be determined. Several reports indicate that cell adhesion to the ECM influences growth factor-induced responses, which suggests the existence of coordinated mechanisms between integrin and growth factors in the control of cellular functions. Of these studies, αvβ3 has been shown to associate with receptor tyrosine kinases such as platelet-derived growth factor β receptor (25Schneller M. Vuori K. Ruoslahti E. EMBO J. 1997; 16: 5600-5607Crossref PubMed Scopus (423) Google Scholar) and insulin-like growth factor receptor, and its cognate ligand, VN, enhanced the mitogenic responses of platelet-derived growth factor and insulin-like growth factor (26Vuori K. Ruoslahti E. Science. 1994; 266: 1576-1578Crossref PubMed Scopus (338) Google Scholar). However, the potential for integrins to interact with non-receptor tyrosine kinases such as those for TGFβ has not been investigated. Although there is evidence to suggest that integrin-mediated signaling may converge with TGFβ signaling pathways (27Bhowmick N.A. Zent R. Ghiassi M. McDonnell M. Moses H.L. J. Biol. Chem. 2001; 276: 46707-46713Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 28Thannickal V.J. Lee D.Y. White E.S. Cui Z. Larios J.M. Chacon R. Horowitz J.C. Day R.M. Thomas P.E. J. Biol. Chem. 2003; 278: 12384-12389Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar), data showing interactions involving TGFβIIR and αvβ3 have not been described. In this study we have examined the interaction between TGFβIIR and αvβ3 integrins in normal human lung fibroblasts. We found that TGFβ1 induces transcription of the β3 subunit and cell surface expression of αvβ3 integrins. Immunofluorescence and co-immunoprecipitation studies suggest that αvβ3 associates with TGFβIIR following exposure to TGFβ1. Using a novel biophysical method, BRET, we have shown that these receptors cluster and functionally interact in living cells in a TGFβ1-specific manner. As a consequence of this interaction, fibroblast proliferation and adhesion induced by TGFβ1 was significantly amplified when cells were co-exposed with the αvβ3 ligands, TN and VN. Materials—Normal diploid human fetal lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Manassas, VA). Culture media, l-glutamine, penicillin, gentamicin, anti-αvβ5 (clone P1F6), and LipofectAMINE Plus were purchased from Invitrogen (Victoria, Australia). TGFβ1, VN, CN IV, laminin (LM), methyl-β-cyclodextrine (MCD), and protease inhibitor mixture were obtained from Sigma (New South Wales, Australia). The rabbit polyclonal anti-TGFβIIR antibody, anti-TGFβIIR antibody conjugated to agarose beads, and protein A-Sepharose were purchased from Santa Cruz Bio-technology Inc. (Santa Cruz, CA). Enhanced chemiluminescence and the BrdUrd incorporation proliferation assay were purchased from Amersham Biosciences. The β3 plasmid was kindly provided by Dr. D. A. Cheresh (Scripps Research Institute, La Jolla, CA), and the TGFβIIR plasmid was generously provided by Dr. J. Freeman (University of Texas Health Science Centre, San Antonio, TX). The RNeasy mini columns and OneStep RT-PCR kit were purchased from Qiagen (Victoria, Australia). Coelenterazine (h form) 488, anti-mouse IgGs conjugated to Alexa-546, and goat anti-rabbit IgGs conjugated to Alexa-488 were from Molecular Probes (Leiden, Netherlands). 96-well white optiplates were obtained from Packard instruments (Berthold, Australia). The peroxidase-conjugated goat anti-rabbit IgG was obtained from DAKO (New South Wales, Australia). The cyclic RGD peptide, Gpen-GRGDSPCA was purchased from Bachem (Bubendorf, Switzerland). The RGD peptide, GRGDNP, and TN were obtained from Calbiochem. Polyclonal antibodies against cyclin D1, α5β1, and monoclonal αvβ3 (clone LM609) were obtained from Chemicon (Temecula, CA). Cell Culture—HFL-1 cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mml-glutamine, penicillin, and gentamicin. Cells were maintained at 37 °C in 5% CO2. Immunoprecipitation and Western Blotting—Immunoprecipitation and Western blotting was performed as previously described (11Scaffidi A.K. Moodley Y.P. Weichselbaum M. Thompson P.J. Knight D.A. J. Cell Sci. 2001; 114: 3507-3516Crossref PubMed Google Scholar). Briefly, cells were stimulated with TGFβ1 for various periods of time and lysed for 20 min in Nonidet P-40 buffer (1% Nonidet P-40, 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 1 mm Na3VO4, 10% glycerol) supplemented with protease inhibitor mixture. One milligram of protein was immunoprecipitated for 12 h at 4 °C with integrin-specific antibodies. The protein-antibody complex were then precipitated with protein A-agarose, separated on a 4-15% gradient SDS-polyacrylamide gel, electroblotted onto a polyvinylidene difluoride membrane, and probed with anti-TGFβIIR antibody. This was followed by detection of bound antibodies with peroxidase-conjugated goat anti-rabbit IgG and enhanced chemiluminescence (ECL). In some experiments, reverse immunoprecipitation was performed. For these studies, 1 mg of total cell protein was immunoprecipitated for 12 h at 4 °C with anti-TGFβIIR antibodies conjugated to agarose beads. For experiments examining the effects of cholesterol depletion, cells were treated with 2% solution of MCD for 1 h and then washed twice with PBS, prior to TGFβ1 (10 ng/ml) exposure for 5 min. β3/EYFP, β3/Rluc, TGFβIIR/EYFP, and TGFβIIR/Rluc Constructs—The human β3/Rluc, β3/EYFP, TGFβIIR/Rluc, and TGFβIIR/EYFP cDNA constructs in pcDNA3 were generated by PCR amplification from the human β3 or TGFβIIR cDNA without their stop codons using sense and antisense primers containing HindIII and EcoRV sites, respectively. The fragment was then cloned in-frame into the Rluc or EYFP vector constructed by insertion of the Rluc or EYFP coding region into pcDNA3 as previously described (29Kroeger K.M. Hanyaloglu A.C. Seeber R.M. Miles L.E. Eidne K.A. J. Biol. Chem. 2001; 276: 12736-12743Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Transient Transfection—Lung fibroblasts were seeded in 6-well tissue culture plates, and transient transfections were performed the following day using 4 μg of DNA and LipofectAMINE plus as per the manufacturer's instructions. Flow cytometry was used to measure expression level of EYFP-tagged receptors. BRET Assay—Forty-two hours post-transfection cells were detached with 0.05% trypsin/EDTA and washed twice in PBS. Cells were resuspended in PBS, and ∼5 × 104 cells were distributed to each well of 96-well white Optiplates and incubated in the presence or absence of TGFβ1 or ECM protein for the specified times at 37 °C. Coelenterazine (h form) 488 was added to a final concentration of 5 μm, and readings were taken immediately, unless otherwise specified. Repeated readings were taken for at least 5-30 min using a custom-designed BRET instrument, which allows sequential integration of the signals detected in the 440- to 500-nm (bioluminescence signal-Rluc) and 510- to 590-nm (fluorescence signal-EYFP) windows. The BRET ratios for the co-expression of the TGFβIIR/Rluc and β3/EYFP constructs were normalized against the BRET ratio for the TGFβIIR/Rluc expression construct alone. The BRET ratio is defined as: [(emission at 510-590) - (emission at 440-500) × cf]/(emission at 440-500), where cf corresponds to (emission at 510-590)/(emission at 440-500) for the Rluc construct expressed alone in the same experiment. Proliferation Assay—Cells were seeded onto 96-well tissue culture plates at a density of 7000 cells/well. Cells were stimulated with TGFβ1 and/or ECM proteins for 24 and 48 h. For experiments using RGD peptides, cells were exposed to either the RGD peptide GRGDNP, or the cyclic RGD peptide GpenGRGDSPCA 30 min prior to TGFβ1 and ECM protein stimulation. A BrdUrd incorporation proliferation assay was utilized as per the manufacturer's protocol. Cyclin D1 Flow Cytometry—Fibroblasts were placed in serum-free medium and stimulated with TGFβ (0.5 ng/ml) and/or TN (5 μg/ml) for 8 h. Cells were harvested and permeabilized in 0.25% Triton X-100 for 3 min followed by incubation with anti-cyclin D1 antibodies for 30 min at 4 °C. Following multiple washings, cells were incubated with a rabbit anti-mouse phyco-erythrin-conjugated secondary antibody. Labeled cells were analyzed by flow cytometry as described above. 10,000-30,000 events were collected for analysis. Positive fluorescence was expressed as channel number mean fluorescence intensity. For experiments examining the effects of cholesterol depletion, cells were treated with 2% MCD for 30 min and then washed twice with PBS, prior to stimulation. Confocal Microscopy—Transfected fibroblasts were fixed in 1% paraformaldehyde and mounted using fade resistance aqueous mounting medium 48 h post-transfection. For co-localization experiments utilizing non-transfected cells, fibroblasts were incubated with TGFβ1 (10 ng/ml) or left unstimulated. Cells were fixed and incubated with anti-αvβ3 and anti-TGFβIIR antibodies. Z-series projections of fluorescent images of αvβ3 or TGFβIIR were obtained using a Bio-Rad MRC 1000 confocal laser-scanning microscope using COMOS software, as previously described (11Scaffidi A.K. Moodley Y.P. Weichselbaum M. Thompson P.J. Knight D.A. J. Cell Sci. 2001; 114: 3507-3516Crossref PubMed Google Scholar). RNA Extraction and Real-time PCR—Fibroblasts were treated with TGFβ1 (10 ng/ml) for the indicated times and lysed using RNeasy mini columns. PCR reactions were carried out using OneStep RT-PCR kit, including the SYBR Green reporter molecule. Quantitative real-time PCR analysis for β3 and hypoxanthine phosphoribosyltransferase (HPRT) was performed using the icycler I Q Multi-Color real-time PCR detection system. After determination of the CT (defined as the number of cycles for the amplification of a sample to reach a point where fluorescent intensity exceeded the threshold) the amount of mRNA in the sample was calculated from the CT of the sample relative to the standard curve, which correlates with the amount of starting material present. The obtained quantity was normalized to the amount of HPRT, and all the values for experimental samples are expressed as -fold differences between the stimulated mRNA sample and the stimulated mRNA sample. Statistical Analysis—Data are expressed as mean ± S.E. of at least four experiments. Statistical comparisons of mean data were performed using one-way analysis of variance and Student's t test with Bonferroni correction performed post-hoc to correct for multiple comparisons. A p value of <0.05 was regarded as statistically significant. TGFβ1 Enhances Cell Surface Expression αvβ3—Flow cytometry was used to determine whether TGFβ1 altered cell surface expression of αvβ3 on human lung fibroblasts. Cells were treated with varying concentrations of TGFβ1 for 24 or 48 h. Fibroblasts stimulated with TGFβ1 at concentrations greater than 500 pg/ml demonstrated increased cell surface expression of αvβ3 by 24 h (Table I). Maximal increases in integrin expression were observed with 100 ng/ml TGFβ1, which produced a 40% increase in mean fluorescent intensity over control levels (19.6 ± 5.6 versus 13.0 ± 4.4 for unstimulated fibroblasts; Fig. 1A). Increased αvβ3 expression was also observed at 48 h, although levels of expression at this time were lower than seen at 24 h (Table I).Table IUp-regulation of αv β3 integrin expression on lung fibroblasts following TGFβ 1 treatmentTreatmentControlTGFβ11 pg/ml5 pg/ml500 pg/ml10 ng/ml100 ng/ml24-h13.0 ± 4.414.6 ± 5.814.4 ± 6.016.8 ± 6.2aValues are expressed as mean fluorescence intensity of four separate experiments ± S.E.; *p < 0.05 compared with control.18.2 ± 6.2aValues are expressed as mean fluorescence intensity of four separate experiments ± S.E.; *p < 0.05 compared with control.19.6 ± 5.6aValues are expressed as mean fluorescence intensity of four separate experiments ± S.E.; *p < 0.05 compared with control.48-h13.3 ± 4.212.8 ± 5.011.2 ± 3.214.9 ± 4.617.0 ± 4.517.1 ± 4.3a Values are expressed as mean fluorescence intensity of four separate experiments ± S.E.; *p < 0.05 compared with control. Open table in a new tab TGFβ1 Increases β3 mRNA Expression in Lung Fibroblasts—Because TGFβ1 increased cell surface expression of αvβ3, we determined whether this effect correlated with enhanced transcription of β3 mRNA. Real-time PCR was conducted on RNA harvested from fibroblasts after incubation with TGFβ1 (10 ng/ml), for varying lengths of time (0, 3, 6, and 24 h). Fig. 1B illustrates that TGFβ1 significantly increased transcription of β3 mRNA to a maximum of 2.4-fold over untreated cells after 6-h incubation. This effect of TGFβ1 was maintained for up to 24 h. TGFβIIR and αvβ3 Co-localize in Lung Fibroblasts—To determine whether TGFβIIR and αvβ3 co-localize, we performed confocal microscopy on cells stimulated by TGFβ1 or left untreated. Fig. 2A shows that TGFβIIR and αvβ3 exhibit distinct staining patterns and only weakly associate in the absence of TGFβ1. However as shown in Fig. 2B, a 5-min exposure to TGFβ1 (10 ng/ml) results in an overlapping staining pattern of the receptors (intense yellow staining), which indicates co-localization. This was particularly apparent along the cell membrane and at sites of focal contacts. The results obtained by confocal microscopy were confirmed by co-immunoprecipitation. Cell lysates were obtained from fibroblasts treated with TGFβ1 for various periods of time. Isolated proteins were immunoprecipitated with anti-αvβ3 antibodies and immunoblotted with antibodies against TGFβIIR. As shown in Fig. 3A, TGFβIIR co-immunoprecipitates with αvβ3, indicating that these receptors form a complex. Moreover, this association was enhanced when fibroblasts were stimulated with TGFβ1 (10 ng/ml) with maximal effects seen following 5-min exposure. Immunocomplex formation remained elevated over basal levels with TGFβ1 stimulation for 40 min, and by 60 min the interaction began to diminish. Because cholesterol is essential for the maintenance of lipid rafts and integrin signaling complexes, we investigated the involvement of lipid rafts in the interaction between TGFβRII and αvβ3. Fig. 3B shows that cholesterol depletion from fibroblasts significantly impaired the TGFβ1-induced interaction between TGFβRII and αvβ3. The specificity of the interaction between the TGFβRII and αvβ3 was next investigated. We examined whether αvβ5 or α5β1, known to be expressed on fibroblasts, also partner TGFβRII. Fig. 3C shows that these integrins do not co-immunoprecipitate with TGFβRII following exposure to TGFβ1, suggesting the interaction is specific for αvβ3. BRET Occurs between TGFβIIR and αvβ3 Integrins in an Agonist-dependent Manner—Visualization of αvβ3-TGFβIIR co-localization by confocal microscopy does not measure direct association of the two receptor complexes, nor does it allow for the detection of low level protein interaction, and even though co-immunoprecipitation does have the ability to measure direct receptor interactions, it does not quantify this dynamic interaction in living cells. Therefore, we used the novel technique of BRET to investigate whether a biophysical interaction occurs between TGFβIIR and αvβ3 in fibroblasts in real-time. The TGFβIIR and αvβ3 cDNA were fused at the carboxyl-terminal with either Renilla luciferase (Rluc) or enhanced yellow fluorescent protein (EYFP). When the Rluc and EYFP moieties are <100 Å apart and ceolenterazine is added, energy transfer by Rluc to EYFP results in emission of a fluorescent signal. We compared the distribution of exogenously administered tagged receptors with endogenous forms of the receptors by using confocal microscopy. Comparison was made between fibroblasts expressing either the β3/EYFP or TGFβIIR/EYFP fusion protein with immunofluorescence staining of cells following binding of antibodies against β3 or TGFβIIR. TGFβIIR and β3 appeared to be diffusely expressed on the cell surface with some staining also appearing intracellularly. A similar distribution pattern was evident with the TGFβIIR/EYFP and β3/EYFP constructs (Fig. 4, A-D). BRET measurements performed on cells co-transfected with the TGFβIIR/Rluc and β3/EYFP showed that TGFβ1 induced an increase in the relative BRET ratio compared with untreated cells when readings were taken from 0 to 10 min as well as from 20 to 30 min (Fig. 5A). However, the latter time point showed a decline in the BRET ratio compared with 0-10 min treatment with TGFβ1. The basal BRET signal (cells treated with PBS) was not different from the signal generated by cells transfected with the TGFβIIR/Rluc construct alone. These data demonstrate that the two receptors functionally interact in an agonist-dependent manner and that the interaction was sustained over at least a 30-min time frame. In addition, stimulation of fibroblasts with an irrelevant stimulus, oncostatin M (OSM), which binds to the gp130 receptor and induces proliferation (30Scaffidi A.K. Mutsaers S.E. Moodley Y.P. McAnulty R.J. Laurent G.J. Thompson P.J. Knight D.A. Br. J. Pharmacol. 2002; 136: 793-801Crossref PubMed Scopus (81) Google Scholar), did not induce an increase in the BRET ratio. Slight increases in BRET seen with OSM are likely to be due to small fluctuations of nonspecific interactions between Rluc and EYFP moieties. The specificity of this interaction was further confirmed by the lack of BRET signal detected when cells were co-transfected with TGFβIIR/Rluc and the EYFP vector (at similar expression levels to that of the TGFβIIR/Rluc and β3/EYFP co-transfected cells), which had been stimulated with TGFβ1 (Fig. 5A). To determine whether TN enhanced the interactions between TGFβIIR and αvβ3 above that seen with TGFβ1, fibroblasts were exposed to either TN alone or TGFβ1 and TN. Following exposure to TN, a slight increase in the BRET ratio was seen. When cells were co-exposed to TGFβ1 and TN the BRET signal was not greater than that seen with TGFβ1 alone (Fig. 5B).Fig. 5Agonist-dependent interaction between TGFβIIR and αvβ3.A, BRET was measured in fibroblasts transfected with cDNAs encoding the TGFβIIR and β3 receptors fused at the carboxyl termini to Rluc or EYFP as indicated. Energy transfer was initiated with the addition of coelenterazine (5 μm) either directly after treatment (white bars) or 20 min post-treatment (black bars), and BRET reading were taken immediately over a 10-min time frame. The values were corrected by subtracting the background signal detected when TGFβIIR/Rluc was expressed alone. Data represents the mean ± S.E. of four to six independent experiments. B, BRET ratio measurements were performed in response to TN, TGFβ, and TN plus TGFβ1. Coelenterazine was added directly after treatment. Data represent the mean ± S.E. generated from four independent experiments.View Large Image Figure ViewerDownload (PPT) TGFβ1 Potentiates the Proliferative Effects of Fibroblasts Exposed to αvβ3Ligands, VN and TN—Having established that TGFβIIR interacts with αvβ3 following exposure to TGFβ1, we next examined the functional consequences of this complex formation. We first determined whether this interaction influenced the mitogenic potential of TGFβ1. TGFβ1 had little effect on fibroblast proliferation over a 48-h period, increasing BrdUrd incorporation to a maximum of 20% above unstimulated levels (Fig. 6A). Similarly exposing