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J. Biol. Chem., Vol. 279, Issue 36, 37726-37733, September 3, 2004
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v
3 Integrin Interacts with the Transforming Growth Factor (TGF) Type II Receptor to Potentiate the Proliferative Effects of TGF1 in Living Human Lung Fibroblasts*
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From the
Asthma and Allergy Research Institute and Centre for Asthma, Allergy and Respiratory Research, University of Western Australia, and the Western Australian Institute for Medical Research and University of Western Australia Centre for Medical Research, Nedlands, Western Australia, 6009
Received for publication, March 18, 2004 , and in revised form, May 25, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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v
3 integrin is known to cooperate with receptor tyrosine kinases to enhance cellular responses. To determine whether v3 regulates transforming growth factor (TGF) 1-induced responses, we investigated the interaction between v3 and TGF type II receptor (TGFIIR) in primary human lung fibroblasts. We report that TGF1 up-regulates cell surface and mRNA expression of v3 in a time- and dose-dependent manner. Co-immunoprecipitation and confocal microscopy showed that TGFRII associates and clusters with v3, following TGF1 exposure. This association was not observed with v5 or 51. We also used a novel molecular proximity assay, bioluminescence resonance energy transfer (BRET), to quantify this dynamic interaction in living cells. TGF1 stimulation resulted in a BRET signal within 5 min, whereas tenascin, which binds v3, did not induce a substantial BRET signal. Co-exposure to tenascin and TGF1 produced no further increases in BRET than TGF1 alone. Cyclin D1 was rapidly induced in cells co-exposed to TGF1 and tenascin, and as a consequence proliferation induced by TGF1 was dramatically enhanced in cells co-exposed to tenascin or vitronectin. Cholesterol depletion inhibited the interaction between TGFRII and v3 and abrogated the proliferative effect. The cyclic RGD peptide, GpenGRGDSPCA, which blocks v3, also abolished the synergistic proliferative effect seen. These results indicate a new interaction partner for the v3 integrin, the TGFIIR, in which TGF1-induced responses are potentiated in the presence v3 ligands. Our data provide a novel mechanism by which TGF1 may contribute to abnormal wound healing and tissue fibrosis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 v3 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 v3, 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 v3 are increased in inflamed and remodeled lungs (12-14).
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. TGF1 is produced by a variety of cells and controls multiple processes involved in wound repair (15-17), 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). TGF1 elicits its biological effects by interacting with the constitutively active serine/threonine kinase TGF type II receptor (TGFIIR), which recruits and activates TGF type I receptor (21, 22). TGF1 has been shown to modulate the expression of integrins, although these effects are particularly dependent on cell type (23, 24). Whether TGF1 effects v3 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, v3 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 integrin-mediated signaling may converge with TGF signaling pathways (27, 28), data showing interactions involving TGFIIR and v3 have not been described.
In this study we have examined the interaction between TGFIIR and v3 integrins in normal human lung fibroblasts. We found that TGF1 induces transcription of the 3 subunit and cell surface expression of v3 integrins. Immunofluorescence and co-immunoprecipitation studies suggest that v3 associates with TGFIIR following exposure to TGF1. Using a novel biophysical method, BRET, we have shown that these receptors cluster and functionally interact in living cells in a TGF1-specific manner. As a consequence of this interaction, fibroblast proliferation and adhesion induced by TGF1 was significantly amplified when cells were co-exposed with the v3 ligands, TN and VN.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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v5 (clone P1F6), and LipofectAMINE Plus were purchased from Invitrogen (Victoria, Australia). TGF1, VN, CN IV, laminin (LM), methyl--cyclodextrine (MCD), and protease inhibitor mixture were obtained from Sigma (New South Wales, Australia). The rabbit polyclonal anti-TGFIIR antibody, anti-TGFIIR 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 3 plasmid was kindly provided by Dr. D. A. Cheresh (Scripps Research Institute, La Jolla, CA), and the TGFIIR 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, 51, and monoclonal v3 (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 mM L-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 (11). Briefly, cells were stimulated with TGF1 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-TGFIIR 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-TGFIIR 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 TGF1 (10 ng/ml) exposure for 5 min.
3/EYFP, 3/Rluc, TGFIIR/EYFP, and TGFIIR/Rluc Constructs—The human 3/Rluc, 3/EYFP, TGFIIR/Rluc, and TGFIIR/EYFP cDNA constructs in pcDNA3 were generated by PCR amplification from the human 3 or TGFIIR 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 (29).
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 x 104 cells were distributed to each well of 96-well white Optiplates and incubated in the presence or absence of TGF1 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 TGFIIR/Rluc and 3/EYFP constructs were normalized against the BRET ratio for the TGFIIR/Rluc expression construct alone. The BRET ratio is defined as: [(emission at 510-590) - (emission at 440-500) x 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 TGF1 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 TGF1 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 TGF (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 TGF1 (10 ng/ml) or left unstimulated. Cells were fixed and incubated with anti-v3 and anti-TGFIIR antibodies. Z-series projections of fluorescent images of v3 or TGFIIR 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 TGF1 (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 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 Enhances Cell Surface Expression v3—Flow cytometry was used to determine whether TGF1 altered cell surface expression of v3 on human lung fibroblasts. Cells were treated with varying concentrations of TGF1 for 24 or 48 h. Fibroblasts stimulated with TGF1 at concentrations greater than 500 pg/ml demonstrated increased cell surface expression of v3 by 24 h (Table I). Maximal increases in integrin expression were observed with 100 ng/ml TGF1, 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 v3 expression was also observed at 48 h, although levels of expression at this time were lower than seen at 24 h (Table I).
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1 Increases 3 mRNA Expression in Lung Fibroblasts—Because TGF1 increased cell surface expression of v3, 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 TGF1 (10 ng/ml), for varying lengths of time (0, 3, 6, and 24 h). Fig. 1B illustrates that TGF1 significantly increased transcription of 3 mRNA to a maximum of 2.4-fold over untreated cells after 6-h incubation. This effect of TGF1 was maintained for up to 24 h.
TGFIIR and v3 Co-localize in Lung Fibroblasts—To determine whether TGFIIR and v3 co-localize, we performed confocal microscopy on cells stimulated by TGF1 or left untreated. Fig. 2A shows that TGFIIR and v3 exhibit distinct staining patterns and only weakly associate in the absence of TGF1. However as shown in Fig. 2B, a 5-min exposure to TGF1 (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.
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1 for various periods of time. Isolated proteins were immunoprecipitated with anti-v3 antibodies and immunoblotted with antibodies against TGFIIR. As shown in Fig. 3A, TGFIIR co-immunoprecipitates with v3, indicating that these receptors form a complex. Moreover, this association was enhanced when fibroblasts were stimulated with TGF1 (10 ng/ml) with maximal effects seen following 5-min exposure. Immunocomplex formation remained elevated over basal levels with TGF1 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 TGFRII and v3. Fig. 3B shows that cholesterol depletion from fibroblasts significantly impaired the TGF1-induced interaction between TGFRII and v3.
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RII and v3 was next investigated. We examined whether v5 or 51, known to be expressed on fibroblasts, also partner TGFRII. Fig. 3C shows that these integrins do not co-immunoprecipitate with TGFRII following exposure to TGF1, suggesting the interaction is specific for v3.
BRET Occurs between TGFIIR and v3 Integrins in an Agonist-dependent Manner—Visualization of v3-TGFIIR 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 TGFIIR and v3 in fibroblasts in real-time. The TGFIIR and v3 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 3/EYFP or TGFIIR/EYFP fusion protein with immunofluorescence staining of cells following binding of antibodies against 3 or TGFIIR. TGFIIR 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 TGFIIR/EYFP and 3/EYFP constructs (Fig. 4, A-D). BRET measurements performed on cells co-transfected with the TGFIIR/Rluc and 3/EYFP showed that TGF1 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 TGF1. The basal BRET signal (cells treated with PBS) was not different from the signal generated by cells transfected with the TGFIIR/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 TGFIIR/Rluc and the EYFP vector (at similar expression levels to that of the TGFIIR/Rluc and 3/EYFP co-transfected cells), which had been stimulated with TGF1 (Fig. 5A). To determine whether TN enhanced the interactions between TGFIIR and v3 above that seen with TGF1, fibroblasts were exposed to either TN alone or TGF1 and TN. Following exposure to TN, a slight increase in the BRET ratio was seen. When cells were co-exposed to TGF1 and TN the BRET signal was not greater than that seen with TGF1 alone (Fig. 5B).
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1 Potentiates the Proliferative Effects of Fibroblasts Exposed to v3 Ligands, VN and TN—Having established that TGFIIR interacts with v3 following exposure to TGF1, we next examined the functional consequences of this complex formation. We first determined whether this interaction influenced the mitogenic potential of TGF1. TGF1 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 the cells to the v3 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 TGF1, the proliferative response was synergistically increased. The greatest effect was observed when cells were exposed to TGF1 (10 ng/ml) for 24 h in the presence of TN (5 µg/ml), which resulted in an 82% increase in proliferation above control levels. Similarly, fibroblasts exposed to TGF1 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 the specificity of this effect, we co-exposed cells to collagen IV and laminin as examples of ECM proteins that do not normally recognize v3. Fig. 6D shows that co-exposure of fibroblasts to TGF1 and either CN IV or LM does not result in augmented proliferation.
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1 Enhanced Proliferation on TN Is Dependent on v3 Avidity—To confirm that the enhanced proliferative response observed was mediated by v3, fibroblasts were exposed to the RGD peptide GRGDNP, which blocks v1, or the cyclic RGD peptide GpenGRGDSPCA, which specifically blocks v3. 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 TGF1-induced proliferation in the presence of TN. In contrast, pretreatment with the GRGDNP peptide did not influence proliferation, suggesting that the proliferative effects of TGF1 exposed to TN are dependent on activation of v3.
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1 and TN occurred at times after increased integrin expression was observed (Fig. 1), we next investigated whether the interaction between TGFRII and v3 influenced cell cycle dynamics independently of integrin expression. Fig. 7B shows that cells co-exposed to TGF1 and TN induce a significant increase in cyclin D1 protein after 8 h. This increase is completely inhibited by pretreating the cells with MCD. Thus, the interaction of TGFRII and v3 is sufficient for the induction of cyclin D1. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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IIR and the v3 integrin, whereby the effects of TGF1 become amplified in human lung fibroblasts. We show that TGF1 increased gene transcription and cell surface expression of v3. Independently of this, TGF1 induced co-localization and immunocomplex formation between TGFIIR and v3 on the cell surface. We have also used a novel biophysical method, BRET, to study the dynamics of the intermolecular interaction between TGFIIR and v3 in real-time and in living cells. Our results show that these receptors are bought into close molecular proximity in response to TGF1. 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 TGF1 and v3 ligands. These results clearly show that TGFIIR functionally interact with v3 integrins and suggest that changing the extracellular environment may profoundly alter cellular responses to TGF1.
To date there have been a wide variety of reports investigating the effects of TGF1 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 (31-33). Ignotz et al. (34) demonstrated that treatment of WI-38 human lung fibroblasts with a single concentration of TGF1 increased cell surface expression of v3 by 3-fold. Our data support and extend these results, showing a dose- and time-dependent effect of TGF1on v3 expression. We have also shown that surface expression of v3 is increased 24 h after TGF1 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 TGF1. In contrast to v3, TGF1 reduced cell surface expression of v5.
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 (35-38). Our aim was to assess whether v3 interacts with the serine threonine kinase TGFIIR 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 v3 associates with TGFIIR following exposure to TGF1. In quiescent fibroblasts we observed a weak association between v3 and TGFIIR, 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 TGFIIR-v3 interaction was augmented when fibroblasts were exposed to TGF1, because immunocomplex formation increased within 5 min of the addition of the growth factor and was sustained for at least 40 min.
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 TGFRII and v3, suggesting that rafts are also involved in regulating this association.
Having identified that v3 and TGFRII 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 TGFRII and either the 51 or v5 integrin.
We also used a biophysical molecular proximity technique, BRET, to demonstrate a functional interaction between v3 and TGFIIR 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 R0 of <50 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 v3/EYFP and TGFIIR/Rluc produced a measurable BRET signal in the presence of TGF1, 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 TGF1. 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, TGF1 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 TGFIIR. This is further supported by data demonstrating that no further increases in BRET signal are observed when cells are co-exposed with TGF1 and TN, compared with TGF1 alone. However, we cannot exclude the possibility that co-stimulation with TN may further induce interactions between the receptors, although TGF1 may have induced an optimal configurative state between TGFIIR- 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 TGFIIR-v3 interaction. TGF1 is known to have differential effects on proliferation, depending on cell type and concentration. McAnulty et al. (20) demonstrated that TGF1 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 TGF1 at concentrations ranging from 5 pg/ml to 5 ng/ml did not significantly influence fibroblast proliferation. However, TGF1 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 v3 integrin. Importantly, we showed that the proliferative effects of TGF1 occurred at higher concentrations of TGF1 (0.5 ng/ml rather than 5 pg/ml) when co-exposed with TN or VN, suggesting that these interactions may occur under patho-physiological conditions, because increased levels of ECM proteins and TGF1 have been observed in several lung diseases, including pulmonary fibrosis and asthma (12-14).
This study demonstrates that TGF1 increases transcription and synthesis of the v3 integrin and enhances a functional, biophysical interaction between its cognate receptor TGFRII and the integrin, which results in enhanced cellular function. TGF1 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 v3 ligands, TGF1, 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.
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FOOTNOTES |
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¶ To whom correspondence should be addressed (present address): James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital, University of British Columbia, Vancouver, British Columbia V6Z 1Y6, Canada. Tel.: 604-682-2344 (ext. 62217); Fax: 604-806-9274; E-mail: dknight{at}mrl.ubc.ca.
1 The abbreviations used are: ECM, extracellular matrix; TGF1, transforming growth factor 1; TGFIIR, 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--cyclodextrine; BrdUrd, bromodeoxyuridine.
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ACKNOWLEDGMENTS |
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IIR cDNA and Dr. David Cheresh for kindly providing the 3 cDNA. We are grateful to Dr. Paul Rigby for helpful suggestions and Dr. Rachel Venables for technical assistance. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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