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*

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) 1 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 (1)(2)(3). Integrins are membrane-spanning glycoproteins consisting of a heterodimer of non-covalently linked ␣ and ␤ subunits (4,5) which consist of a short cytoplasmic domain, a transmembrane domain, and a large extracellular domain that binds to ECM proteins via specific peptide sequences (6).
Considerable attention has focused on the ␣ v ␤ 3 integrin, because it appears to play a major role in several processes relevant to remodeling, such as binding and activation of matrix metalloproteinases (7) and growth factors (8), as well as cell proliferation (9), migration, and differentiation (10). We have shown that vitronectin (VN) and its receptors, including ␣ v ␤ 3 , can dramatically modulate the phenotype and function of human lung fibroblasts via integrin-specific intracellular signaling pathways (11). Clinical studies have also shown that ligands for ␣ v ␤ 3 are increased in inflamed and remodeled lungs (12)(13)(14).
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. TGF␤1 is produced by a variety of cells and controls multiple processes involved in wound repair (15)(16)(17), including the regulation of cell growth and differentiation as well as stimulating the net accumulation of ECM proteins (18,19). Its role in inducing proliferation is unclear, although it is thought to be a relatively weak mitogen for fibroblasts (20). TGF␤1 elicits its biological effects by interacting with the constitutively active serine/threonine kinase TGF␤ type II receptor (TGF␤IIR), which recruits and activates TGF␤ type I receptor (21,22). TGF␤1 has been shown to modulate the expression of integrins, although these effects are particularly dependent on cell type (23,24). Whether TGF␤1 effects ␣ v ␤ 3 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, ␣ v ␤ 3 has been shown to associate with receptor tyrosine kinases such as platelet-derived growth factor ␤ receptor (25) 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 (26). However, the potential for integrins to interact with non-receptor tyrosine kinases such as those for TGF␤ has not been investigated. Although there is evidence to suggest that integrinmediated signaling may converge with TGF␤ signaling pathways (27,28), data showing interactions involving TGF␤IIR and ␣ v ␤ 3 have not been described.
In this study we have examined the interaction between TGF␤IIR and ␣ v ␤ 3 integrins in normal human lung fibroblasts. We found that TGF␤1 induces transcription of the ␤ 3 subunit and cell surface expression of ␣ v ␤ 3 integrins. Immunofluorescence and co-immunoprecipitation studies suggest that ␣ v ␤ 3 associates with TGF␤IIR following exposure to TGF␤1. Using a novel biophysical method, BRET, we have shown that these receptors cluster and functionally interact in living cells in a TGF␤1-specific manner. As a consequence of this interaction, fibroblast proliferation and adhesion induced by TGF␤1 was significantly amplified when cells were co-exposed with the ␣ v ␤ 3 ligands, TN and VN.
Cell Culture-HFL-1 cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, penicillin, and gentamicin. Cells were maintained at 37°C in 5% CO 2. Immunoprecipitation and Western Blotting-Immunoprecipitation and Western blotting was performed as previously described (11). Briefly, cells were stimulated with TGF␤1 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 MgCl 2 , 1 mM CaCl 2 , 1 mM Na 3 VO 4 , 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-TGF␤IIR antibody. This was followed by detection of bound antibodies with peroxidase-conjugated goat antirabbit 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-TGF␤IIR 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 TGF␤1 (10 ng/ml) exposure for 5 min.
Transient Transfection-Lung fibroblasts were seeded in 6-well tissue culture plates, and transient transfections were performed the following day using 4 g 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 ϳ5 ϫ 10 4 cells were distributed to each well of 96-well white Optiplates and incubated in the presence or absence of TGF␤1 or ECM protein for the specified times at 37°C. Coelenterazine (h form) 488 was added to a final concentration of 5 M, 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 TGF␤IIR/Rluc and ␤ 3 /EYFP constructs were normalized against the BRET ratio for the TGF␤IIR/Rluc expression construct alone. The BRET ratio is defined as: [(emission at 510 -590) Ϫ (emission at 440 -500) ϫ 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 TGF␤1 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 TGF␤1 and ECM protein stimulation. A BrdUrd incorporation proliferation assay was utilized as per the manufacturer's protocol.
Cyclin D 1 Flow Cytometry-Fibroblasts were placed in serum-free medium and stimulated with TGF␤ (0.5 ng/ml) and/or TN (5 g/ml) for 8 h. Cells were harvested and permeabilized in 0.25% Triton X-100 for 3 min followed by incubation with anti-cyclin D 1 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 TGF␤1 (10 ng/ml) or left unstimulated. Cells were fixed and incubated with anti-␣ v ␤ 3 and anti-TGF␤IIR antibodies. Z-series projections of fluorescent images of ␣ v ␤ 3 or TGF␤IIR were obtained using a Bio-Rad MRC 1000 confocal laser-scanning microscope using COMOS software, as previously described (11).
RNA Extraction and Real-time PCR-Fibroblasts were treated with TGF␤1 (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 ␤ 3 and hypoxanthine phosphoribosyltransferase (HPRT) was performed using the icycler I Q Multi-Color real-time PCR detection system. After determination of the C T (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 C T 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 Ϯ 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 Ͻ0.05 was regarded as statistically significant.

TGF␤1 Enhances Cell Surface Expression
␣ v ␤ 3 -Flow cytometry was used to determine whether TGF␤1 altered cell surface expression of ␣ v ␤ 3 on human lung fibroblasts. Cells were treated with varying concentrations of TGF␤1 for 24 or 48 h. Fibroblasts stimulated with TGF␤1 at concentrations greater than 500 pg/ml demonstrated increased cell surface expression of ␣ v ␤ 3 by 24 h (Table I). Maximal increases in integrin expression were observed with 100 ng/ml TGF␤1, which produced a 40% increase in mean fluorescent intensity over control levels (19.6 Ϯ 5.6 versus 13.0 Ϯ 4.4 for unstimulated fibroblasts; Fig.  1A). Increased ␣ v ␤ 3 expression was also observed at 48 h, although levels of expression at this time were lower than seen at 24 h (Table I).
TGF␤1 Increases ␤ 3 mRNA Expression in Lung Fibroblasts-Because TGF␤1 increased cell surface expression of ␣ v ␤ 3 , we determined whether this effect correlated with enhanced transcription of ␤ 3 mRNA. Real-time PCR was conducted on RNA harvested from fibroblasts after incubation with TGF␤1 (10 ng/ml), for varying lengths of time (0, 3, 6, and 24 h). Fig. 1B illustrates that TGF␤1 significantly increased transcription of ␤ 3 mRNA to a maximum of 2.4-fold over untreated cells after 6-h incubation. This effect of TGF␤1 was maintained for up to 24 h.
TGF␤IIR and ␣ v ␤ 3 Co-localize in Lung Fibroblasts-To determine whether TGF␤IIR and ␣ v ␤ 3 co-localize, we performed confocal microscopy on cells stimulated by TGF␤1 or left untreated. Fig. 2A shows that TGF␤IIR and ␣ v ␤ 3 exhibit distinct staining patterns and only weakly associate in the absence of TGF␤1. However as shown in Fig. 2B, a 5-min exposure to TGF␤1 (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 TGF␤1 for various periods of time. Isolated proteins were immunoprecipitated with anti-␣ v ␤ 3 antibodies and immunoblotted with antibodies against TGF␤IIR. As shown in Fig. 3A, TGF␤IIR co-immunoprecipitates with ␣ v ␤ 3 , indicating that these receptors form a complex. Moreover, this association was enhanced when fibroblasts were stimulated with TGF␤1 (10 ng/ml) with maximal effects seen following 5-min exposure. Immunocomplex formation remained elevated over basal levels with TGF␤1 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 TGF␤RII and ␣ v ␤ 3 . Fig. 3B shows that cholesterol depletion from fibroblasts significantly impaired the TGF␤1-induced interaction between TGF␤RII and ␣ v ␤ 3 .
The specificity of the interaction between the TGF␤RII and ␣ v ␤ 3 was next investigated. We examined whether ␣ v ␤ 5 or ␣ 5 ␤ 1 , known to be expressed on fibroblasts, also partner TGF␤RII. Fig. 3C shows that these integrins do not co-immunoprecipitate with TGF␤RII following exposure to TGF␤1, suggesting the interaction is specific for ␣ v ␤ 3 .
BRET Occurs between TGF␤IIR and ␣ v ␤ 3 Integrins in an Agonist-dependent Manner-Visualization of ␣ v ␤ 3 -TGF␤IIR colocalization 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 TGF␤IIR and ␣ v ␤ 3 in fibroblasts in real-time. The TGF␤IIR and ␣ v ␤ 3 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 Ͻ100 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  1. TGF␤1 regulates integrin expression. A, fibroblasts were cultured in the presence (unshaded curve) or absence (shaded curve) of TGF␤1 (10 ng/ml) for 24 h. Cells were stained with anti-␣ v ␤ 3 antibody followed by incubation with anti-mouse conjugated to Alexa 488 and then assayed by flow cytometry. Histogram plots are a representative experiment of data generated from at least four individual experiments. B, serum-starved fibroblasts were treated with TGF␤1 (10 ng/ml) over 24 h. Total RNA was extracted from cells at various time points and real-time PCR with SYBR Green detection was conducted. Results are expressed as -fold change over the housekeeping gene HPRT. Data represent the mean Ϯ S.E. of four independent experiments. *, p Ͻ 0.05 compared with cells in serum-free medium.
receptors with endogenous forms of the receptors by using confocal microscopy. Comparison was made between fibroblasts expressing either the ␤ 3 /EYFP or TGF␤IIR/EYFP fusion protein with immunofluorescence staining of cells following binding of antibodies against ␤ 3 or TGF␤IIR. TGF␤IIR and ␤ 3 appeared to be diffusely expressed on the cell surface with some staining also appearing intracellularly. A similar distribution pattern was evident with the TGF␤IIR/EYFP and ␤ 3 /EYFP constructs (Fig. 4, A-D). BRET measurements performed on cells co-transfected with the TGF␤IIR/Rluc and ␤ 3 /EYFP showed that TGF␤1 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 TGF␤1. The basal BRET signal (cells treated with PBS) was not different from the signal generated by cells transfected with the TGF␤IIR/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 (30), 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 TGF␤IIR/ Rluc and the EYFP vector (at similar expression levels to that of the TGF␤IIR/Rluc and ␤ 3 /EYFP co-transfected cells), which had been stimulated with TGF␤1 (Fig. 5A). To determine whether TN enhanced the interactions between TGF␤IIR and ␣ v ␤ 3 above that seen with TGF␤1, fibroblasts were exposed to either TN alone or TGF␤1 and TN. Following exposure to TN, a slight increase in the BRET ratio was seen. When cells were co-exposed to TGF␤1 and TN the BRET signal was not greater than that seen with TGF␤1 alone (Fig. 5B).
TGF␤1 Potentiates the Proliferative Effects of Fibroblasts Exposed to ␣ v ␤ 3 Ligands, VN and TN-Having established that TGF␤IIR interacts with ␣ v ␤ 3 following exposure to TGF␤1, we next examined the functional consequences of this complex formation. We first determined whether this interaction influenced the mitogenic potential of TGF␤1. TGF␤1 had little effect on fibroblast proliferation over a 48-h period, increasing Br-dUrd incorporation to a maximum of 20% above unstimulated levels (Fig. 6A). Similarly exposing the cells to the ␣ v ␤ 3 ligands VN and TN individually produced only weak mitogenic responses. For example, cells treated with TN alone exhibited a 12% increase in proliferation over unstimulated cells (Fig. 6B). However, when fibroblasts were exposed to a combination of these ECM proteins and TGF␤1, the proliferative response was synergistically increased. The greatest effect was observed when cells were exposed to TGF␤1 (10 ng/ml) for 24 h in the presence of TN (5 g/ml), which resulted in an 82% increase in proliferation above control levels. Similarly, fibroblasts exposed to TGF␤1 in the presence of VN exhibited a 37% increase in proliferation, whereas incubation with VN alone resulted in a 12% increase in proliferation (Fig. 6C). To further investigate FIG. 2. ␣ v ␤ 3 and TGF␤IIR are co-localized following stimulation with TGF␤1. Fibroblasts were left unstimulated (A) or treated with TGF␤1 (10 ng/ml, B) for 5 min. Dual-label images of cells were obtained by confocal microscopy of fixed cells stained with monoclonal anti-␣ v ␤ 3 antibody and polyclonal anti-TGF␤IIR antibody followed with rabbit anti-mouse IgG conjugated to Alexa-546 (red) and goat anti-rabbit IgGs conjugated to Alexa-488 (green). Yellow/orange staining represents co-localization. Magnification, ϫ60.
FIG. 3. TGF␤1 enhances association of ␣ v ␤ 3 with TGF␤IIR in lung fibroblasts. Quiescent cells were either incubated with 10 ng/ml TGF␤1 for various times as indicated (A) or were treated with 2% MCD for 1 h, washed, and stimulated with TGF␤1 for 5 min (B). Cells were lysed with a solution containing 1% Nonidet P-40, and the lysate (1 mg) was immunoprecipitated with a monoclonal anti-␣ v ␤ 3 antibody separated by SDS-PAGE and immunoblotted with a polyclonal anti-TGF␤IIR antibody. C, fibroblasts were stimulated with TGF␤1 for 5 min, and following lysis samples were immunoprecipitated with anti-TGF␤IIR antibodies conjugated to agarose beads. Protein-antibody complex was subjected to SDS-PAGE and immunoblotted with a variety of antibodies as indicated. Results are representative of three independent experiments. the specificity of this effect, we co-exposed cells to collagen IV and laminin as examples of ECM proteins that do not normally recognize ␣ v ␤ 3 . Fig. 6D shows that co-exposure of fibroblasts to TGF␤1 and either CN IV or LM does not result in augmented proliferation.
TGF␤1 Enhanced Proliferation on TN Is Dependent on ␣ v ␤ 3 Avidity-To confirm that the enhanced proliferative response observed was mediated by ␣ v ␤ 3 , fibroblasts were exposed to the RGD peptide GRGDNP, which blocks ␣v␤1, or the cyclic RGD peptide GpenGRGDSPCA, which specifically blocks ␣ v ␤ 3 . Fig.  7A shows that exposure of cells to either peptide alone did not influence the proliferative response of fibroblasts. Pretreatment of fibroblasts with the cyclic RGD peptide abolished TGF␤1-induced proliferation in the presence of TN. In contrast, pretreatment with the GRGDNP peptide did not influence proliferation, suggesting that the proliferative effects of TGF␤1 exposed to TN are dependent on activation of ␣ v ␤ 3 .
Because the proliferative effects induced by co-exposure to TGF␤1 and TN occurred at times after increased integrin ex-pression was observed (Fig. 1), we next investigated whether the interaction between TGF␤RII and ␣ v ␤ 3 influenced cell cycle dynamics independently of integrin expression. Fig. 7B shows that cells co-exposed to TGF␤1 and TN induce a significant increase in cyclin D 1 protein after 8 h. This increase is completely inhibited by pretreating the cells with MCD. Thus, the interaction of TGF␤RII and ␣ v ␤ 3 is sufficient for the induction of cyclin D 1 .

DISCUSSION
The results of this study provide evidence of a direct interaction between TGF␤IIR and the ␣ v ␤ 3 integrin, whereby the effects of TGF␤1 become amplified in human lung fibroblasts. We show that TGF␤1 increased gene transcription and cell surface expression of ␣ v ␤ 3 . Independently of this, TGF␤1 induced co-localization and immunocomplex formation between TGF␤IIR and ␣ v ␤ 3 on the cell surface. We have also used a novel biophysical method, BRET, to study the dynamics of the intermolecular interaction between TGF␤IIR and ␣ v ␤ 3 in real- FIG. 4. Visualization of TGF␤IIR/ EYFP, ␤ 3 /EYFP constructs, and immunofluorescence of TGF␤IIR and ␤ 3 . A, ␤ 3 EYFP; B, TGF␤IIR/EYFP constructs were transiently transfected into fibroblasts and 48 h post-transfection were fixed in 1% paraformaldehyde, and images were obtained by confocal microscopy. C, anti-␤3 antibody; D, anti-TGF␤IIR antibody immunostaining was performed on fixed cells, and confocal microscopy was performed. Magnification, ϫ60.
FIG. 5. Agonist-dependent interaction between TGF␤IIR and ␣ v ␤ 3 . A, BRET was measured in fibroblasts transfected with cDNAs encoding the TGF␤IIR and ␤ 3 receptors fused at the carboxyl termini to Rluc or EYFP as indicated. Energy transfer was initiated with the addition of coelenterazine (5 m) 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 TGF␤IIR/Rluc was expressed alone. Data represents the mean Ϯ S.E. of four to six independent experiments. B, BRET ratio measurements were performed in response to TN, TGF␤, and TN plus TGF␤1. Coelenterazine was added directly after treatment. Data represent the mean Ϯ S.E. generated from four independent experiments. time and in living cells. Our results show that these receptors are bought into close molecular proximity in response to TGF␤1. This interaction resulted in substantial alterations in cell cycle kinetics, such that cyclin D1 was up-regulated and, as a consequence, proliferation was synergistically enhanced in the presence of TGF␤1 and ␣ v ␤ 3 ligands. These results clearly show that TGF␤IIR functionally interact with ␣ v ␤ 3 integrins and suggest that changing the extracellular environment may profoundly alter cellular responses to TGF␤1.
To date there have been a wide variety of reports investigating the effects of TGF␤1 in modulating expression of integrins on an array of cell types. These studies have highlighted how differential expression of integrins by this growth factor are directly related to the cell type (31)(32)(33). Ignotz et al. (34) demonstrated that treatment of WI-38 human lung fibroblasts with a single concentration of TGF␤1 increased cell surface expression of ␣ v ␤ 3 by 3-fold. Our data support and extend these results, showing a dose-and time-dependent effect of TGF␤1 on ␣ v ␤ 3 expression. We have also shown that surface expression of ␣ v ␤ 3 is increased 24 h after TGF␤1 exposure and at concentrations ranging from 0.5 to 100 ng/ml. This increase in protein expression was due to enhanced gene transcription, because ␤ 3 mRNA expression was also significantly increased by TGF␤1.
There are a growing number of reports providing strong evidence to suggest that integrin-mediated signaling processes cooperate with receptor tyrosine kinase signaling pathways to influence cellular function (35)(36)(37)(38). Our aim was to assess whether ␣ v ␤ 3 interacts with the serine threonine kinase TGF␤IIR and to further examine the functional consequences of this interaction.
We used three independent methods to investigate this novel interaction. Confocal microscopy and co-immunoprecipitation demonstrated that ␣ v ␤ 3 associates with TGF␤IIR following exposure to TGF␤1. In quiescent fibroblasts we observed a weak association between ␣ v ␤ 3 and TGF␤IIR, suggesting that these receptors may cluster to a small degree without ligand exposure. However we could not exclude the possibility that these cells may be releasing growth factors or ECM proteins and in an autocrine manner may induce this moderate interaction. However, our results clearly demonstrate that the TGF␤IIR-␣ v ␤ 3 interaction was augmented when fibroblasts were exposed to TGF␤1, because immunocomplex formation increased within 5 min of the addition of the growth factor and was sustained for at least 40 min.
FIG. 6. Effect of ␣ v ␤ 3 ligands, TGF␤1, and RGD peptides on proliferation of lung fibroblasts. BrdUrd incorporation was used to assess proliferation of fibroblasts incubated with TGF␤1 at various concentrations as indicated for 24 or 48 h (A); fibroblasts treated with TN and VN for the indicated time points and doses (B); cells co-exposed to either TN (5 g) plus TGF␤1 (0.05 ng/ml or 0.5 ng/ml), VN (5 g) plus TGF␤1 (0.05 ng/ml or 0.5 ng/ml), or ECM proteins alone for 24 or 48 h (C); and fibroblasts incubated with either LM (5 g) plus TGF␤1 (0.5 ng/ml), CNIV (5 g) plus TGF␤1 (0.5 ng/ml), TN (5 g) plus TGF␤1 (0.5 ng/ml), or ECM proteins alone for 24 or 48 h (D). Values are expressed as the percentage of cells proliferating above unstimulated cells. Results are calculated from triplicate wells of at least three independent experiments and are expressed as mean Ϯ S.E. *, p Ͻ 0.05 between cells stimulated with TN and TGF␤1.
Lipid rafts have been shown to modulate a number of cellular functions and intracellular signaling events. Our data show that depletion of cholesterol from the cell membrane disrupts the interaction between TGF␤RII and ␣ v ␤ 3 , suggesting that rafts are also involved in regulating this association.
Having identified that ␣ v ␤ 3 and TGF␤RII interact, it was important to determine whether other integrins could interact with the growth factor receptor. Using co-immunoprecipitation we did not observe an interaction between TGF␤RII and either the ␣ 5 ␤ 1 or ␣ v ␤ 5 integrin.
We also used a biophysical molecular proximity technique, BRET, to demonstrate a functional interaction between ␣ v ␤ 3 and TGF␤IIR in living cells. This technique is a highly sensitive, quantitative approach used to measure protein-protein interactions in real-time, which involves the transfer of energy between the bioluminescent donor (Rluc) and fluorescent acceptor (EYFP). Because the transfer of energy between the bioluminescent donor luciferase and a fluorescent acceptor EYFP occurs with an R 0 of Ͻ50 Å and no transfer of energy is detected for distances above 100 Å (39,40), it is a powerful tool used to monitor changes in molecular proximity. BRET has been widely used to study oligomerization of G-protein-coupled receptors (29,41,42) and insulin receptors (43,44). However, to our knowledge this is the first study to utilize the BRET technology to demonstrate integrin-growth factor receptor interaction.
Co-expression of ␣ v ␤ 3 /EYFP and TGF␤IIR/Rluc produced a measurable BRET signal in the presence of TGF␤1, suggesting that the two receptors were able to efficiently interact in response to agonist challenge in living cells. Exposure to OSM did not induce a BRET signal, suggesting this interaction was specifically induced by TGF␤1. Agonist-induced signals that do not act directly on integrins can affect the integrin cytoplasmic domain, which in turn regulates integrin-ligand binding, by imposing conformational changes on the extracellular domain (45). In this context, membrane proximal sequences on the ␤ subunit of the integrin complex are involved in maintaining an inactive integrin state. Interactions of these regions with other molecules may be responsible for a conformational change that is transferred across the membrane switching the extracellular domain into the active state (46,47). Our results suggest that, upon binding to its receptor, TGF␤1 triggers signaling pathways that in turn affect ␤ 3 integrin function. In contrast, TN alone does not induce a substantial increase in BRET signal, which suggests that this protein does not augment clustering and interaction of ␤ 3 with TGF␤IIR. This is further supported by data demonstrating that no further increases in BRET signal are observed when cells are co-exposed with TGF␤1 and TN, compared with TGF␤1 alone. However, we cannot exclude the possibility that co-stimulation with TN may further induce interactions between the receptors, although TGF␤1 may have induced an optimal configurative state between TGF␤IIR-and ␤ 3 -tagged receptors, and hence small conformational changes that may occur with TN co-exposure will not be detected by BRET.
We have characterized some of the functional consequences of the TGF␤IIR-␣ v ␤ 3 interaction. TGF␤1 is known to have differential effects on proliferation, depending on cell type and concentration. McAnulty et al. (20) demonstrated that TGF␤1 has biphasic effects on fibroblast proliferation, with concentrations of 160 pg/ml and above inhibiting proliferation, whereas low concentrations (5 pg/ml) induced a modest proliferative response (20). Our data demonstrate that TGF␤1 at concentrations ranging from 5 pg/ml to 5 ng/ml did not significantly influence fibroblast proliferation. However, TGF␤1 induced a strong proliferative response when cells were co-exposed to TN or VN. Using RGD peptides we further showed that the augmented proliferative response was specifically mediated by the ␣ v ␤ 3 integrin. Importantly, we showed that the proliferative effects of TGF␤1 occurred at higher concentrations of TGF␤1 (0.5 ng/ml rather than 5 pg/ml) when co-exposed with TN or VN, suggesting that these interactions may occur under pathophysiological conditions, because increased levels of ECM proteins and TGF␤1 have been observed in several lung diseases, including pulmonary fibrosis and asthma (12)(13)(14).
This study demonstrates that TGF␤1 increases transcription and synthesis of the ␣ v ␤ 3 integrin and enhances a functional, biophysical interaction between its cognate receptor TGF␤RII and the integrin, which results in enhanced cellular function. TGF␤1 is known to play a major role in wound repair by affecting multiple facets of fibroblast behavior. Our data suggest that, in the presence of ␣ v ␤ 3 ligands, TGF␤1, which is normally a weak mitogen, is able to induce a marked proliferative response and enhance cell adhesion. Our data provide an additional mechanism by which this growth factor may contribute to fibroproliferative diseases such as pulmonary fibrosis and asthma.