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Integrin Vßbeta;3 contains a receptor site for resveratrol

Hung-Yun Lin^*, Lawrence Lansing, Jean-Michel Merillon, Faith B. Davis^,1, Heng-Yuan Tang^*, Ai Shih, Xavier Vitrac, Stephanie Krisa, Travis Keating^*, H. James Cao, Joel Bergh, Steven Quackenbush and Paul J. Davis^*,,,||

^* Stratton Veterans Affairs Medical Center and

Ordway Research Institute, Albany, New York, USA;

Laboratoire de Mycologie et Biotechnologie Vegetale, Université de Bordeaux, Bordeaux, France; and

Albany Medical College and

^|| Wadsworth Center, New York State Department of Health, Albany, New York, USA

¹Correspondence: Ordway Research Institute, 150 New Scotland Ave., Albany, NY 12208, USA. E-mail: fdavis{at}ordwayresearch.org

SPECIFIC AIMS

The stilbene resveratrol (RV) exhibits properties of cancer prevention and apoptosis induction in human cancer cells. RV induces extracellular-regulated kinase (ERK) 1 and 2 activation and p53-dependent apoptosis in several cancer cell lines. However, the initial steps in the cellular action of RV as an antitumor agent are incompletely understood and a cellular receptor site at which the resveratrol signal is initiated has not been described. Because of the rapid actions of RV on signal transduction, we reasoned that RV might initiate its cellular effects at an integrin in the plasma membrane of cancer cells. Integrins are heterodimeric structural plasma membrane glycoproteins, and the intracellular domain of certain integrins, such as integrin Vßbeta;3, may activate ERK1/2. Integrin antagonist peptides, designed to mimic the integrin adhesion recognition sequence RGD (Arg-Gly-Asp), have displayed efficacy in the treatment of cancer. We therefore examined the possibility that resveratrol may initiate its ERK1/2-mediated cellular effects at a plasma membrane integrin.

PRINCIPAL FINDINGS

1. Resveratrol-induced p53 and ERK1/2 activation and apoptosis are blocked by an RGD recognition site peptide
To examine the role of integrin Vßbeta;3 in the action of RV, estrogen receptor (ER)-positive breast cancer MCF-7 cells were treated with 10 µM RV in the presence or absence of integrin Vßbeta;3 RGD recognition site peptide, 5–500 nM. Serine-15 phosphorylated p53 (pSer15-p53) and phosphorylated ERK1/2 accumulation increased in nuclei of RV-treated cells and apoptosis was induced (Fig. 1 A). The RGD peptide inhibited RV-induced ERK1/2 activation, phosphorylation of Ser-15-p53, and apoptosis in a concentration-dependent manner. Incubation with an RGE peptide, which does not bind to integrin Vßbeta;3, caused no inhibitory effect.

View larger version (21K): [in this window] [in a new window]   Figure 1. A) Evidence for integrin Vßbeta;3 as a binding site for resveratrol. MCF-7 cells were treated for 4 h with resveratrol (RV, 10 µM) in the presence or absence of RGD peptide (5–500 nM) or RGE peptide (500 nM). Nucleoproteins of treated cell samples were separated by gel electrophoresis and immunoblotted with antibodies to serine-15-phosphorylated 53 (pSer15-p53) or phosphorylated ERK1/2 (pERK1/2). Immunoblots shown in the figures are representative of three experiments, and the upper graph shows the mean ± SEM of change from control image intensities normalized to a value of 1. A lamin (Lamin B) immunoblot provides a loading control. MCF-7 cells treated for 24 h in the same manner were studied by nucleosome ELISA as a measure of apoptosis. The lower graph summarizes the results of three experiments. B) Effect of Ab to integrin Vßbeta;3 on RV action. MCF-7 cells were treated with either anti-Vßbeta;3 (1–20 µg/ml) or anti-Vßbeta;5 (20 µg/ml) for 24 h with or without RV (10 µM) added for the last 4 h. Anti-Vßbeta;3 and anti-Vßbeta;5, each alone, was used as a negative control. C) MDA-MB231 cells were treated with either anti-Vßbeta;3 (10–3–0.1 µg/ml) or anti-Vßbeta;5 (0.1 µg/ml) for 24 h with or without RV (10 µM) for the last 4 h. Anti-Vßbeta;3 and anti-Vßbeta;5, in the absence of RV, were used as negative controls. Results are presented as in B).

2. Antibody (Ab) to the ßbeta;3 monomer of Vßbeta;3 and siRNAßbeta;3 each block resveratrol action
Further evidence supporting the role of Vßbeta;3 in the action of RV is presented in Fig. 1B . RV-induced effects in MCF-7 cells were progressively inhibited by increasing concentrations of Ab to Vßbeta;3 but were not affected by anti-Vßbeta;5. In ER-negative MDA-MB231 breast cancer cells, RV-induced ERK1/2 activation, p53 phosphorylation, and apoptosis were inhibited by even lower concentrations of Vßbeta;3 Ab (Fig. 1C ) but were not inhibited by anti-Vßbeta;5.

That integrin Vßbeta;3, principally the ßbeta;3 component, contains the binding site or principal contact points for RV was further supported by experiments using small interfering RNA (siRNA) of integrin Vßbeta;3. Transfection of MCF-7 cells with siRNA of V or ßbeta;3 resulted in decreased cellular abundance of V or ßbeta;3 (Fig. 2 A). However, only siRNA knockdown of ßbeta;3 diminished RV-induced ERK1/2 activation and Ser-15 p53 phosphorylation. These results indicated that only the ßbeta;3 monomer of integrin Vßbeta;3 was essential for activation of ERK1/2, serine-15 phosphorylation of p53 and induction of apoptosis by RV.

View larger version (14K): [in this window] [in a new window]   Figure 2. A) Transfection of siRNA of the ßbeta;3 component of the integrin blocks resveratrol (RV) action in MCF-7 cells. Cells were exposed for 48 h to either scrambled RNA (scRNA) or small interfering RNA (siRNA) for integrin V or integrin ßbeta;3, and treated with or without resveratrol (RV, 10 µM) for the last 4 h. RV-induced ERK1/2 activation and p53 serine-15 phosphorylation were then examined. The representative immunoblots below the graph are of plasma membrane proteins and illustrate the effectiveness of the siRNA transfections for V and ßbeta;3. B) [14C]-Resveratrol (RV) binds to MCF-7 cell plasma membranes as well as to integrin monomers V and ßbeta;3. Two samples each of purified integrin Vßbeta;3 (Chemicon, Temecula, CA, USA) and of plasma membrane proteins from MCF-7 cells were separated by 5% native PAGE. Proteins in one-half of the gel, with one membrane protein and one integrin sample, were transferred to nitrocellulose and subjected to Western blotting with Vßbeta;3 Ab ( left panel). Immunoreactive proteins were detected by chemiluminescence. The remaining half of the gel, also containing one membrane protein and one purified integrin sample, was dried and exposed to Imaging Screen K (Bio-Rad, Hercules, CA, USA) and studied by laser densitometry. Identification of monomers in immunoblots was by MW. Additional studies were performed to determine the extent of displaceable binding and are shown in the graph below. Purified integrin Vßbeta;3 was incubated with 10 µM [ 14C]-RV in the absence or presence of 1 or 10 µM unlabeled RV for 30 min at room temperature. Samples were separated in 5% native gels, which were dried and analyzed by radioautography. Displacement by unlabeled resveratrol of [ 14C]-RV from the ßbeta;3 component of the integrin was then examined. Results shown are representative of three experiments.

3. Resveratrol binds primarily to the ßbeta;3 monomer of Vßbeta;3
To demonstrate direct interaction of RV with integrin Vßbeta;3, studies of [¹⁴C]-RV binding to plasma membranes or purified integrin were performed. MCF-7 cell membrane proteins were incubated with [¹⁴C]-RV and separated by nondenaturing gel electrophoresis. Purified integrin served as a positive control. Electrophoretic mobilities of the V and ßbeta;3 monomers, shown by the immunoblots in Fig. 2B , were similar to those of the membrane protein bands from MCF-7 cells. Results of labeled RV binding studies, indicate that labeled RV migrated with protein bands in both cell membrane and purified integrin preparations, that were consistent with the V and ßbeta;3 monomers.

When unlabeled RV (1–10 µM) was added to purified integrin Vßbeta;3 previously equilibrated with [¹⁴C]-RV for 30 min, it was shown to bind to both V and ßbeta;3 (Fig. 2B , graph). The addition of 10 µM unlabeled RV displaced 70% of the [¹⁴C]-RV from the ßbeta;3 band. There was no effect of added RV on binding of the stilbene by V. Thus, the binding of the stilbene to V was nonspecific in nature.

DISCUSSION

Dysregulation of the ßbeta;3 integrins has been implicated in cancer pathogenesis. Tumor growth and associated angiogenesis, particularly that mediated by vascular endothelial growth factor, are enhanced in ßbeta;3-null mice. Integrin ßbeta;3 overexpression, in contrast, suppresses tumor growth of a human glioma model in rats. Thus, promotion of integrin ßbeta;3 expression in cancer cells may be a therapeutic goal.

In the studies described here we show that integrin Vßbeta;3 satisfies the definition of a receptor in its binding of RV. That is, dissociable ligand-binding and functional consequences of binding—transduction of the RV signal into apoptosis—are demonstrated. The series of events initiated by RV binding to integrin Vßbeta;3 in the plasma membrane (Fig. 3 ) includes activation of the ERK1/2 pathway and consequent serine-15 phosphorylation of p53. Resulting p53-responsive gene transcription leads to cancer cell apoptosis.

View larger version (36K): [in this window] [in a new window]   Figure 3. Proposed signaling pathway by which interaction of resveratrol with integrin Vßbeta;3 in the plasma membrane of cancer cells leads to cancer cell apoptosis. Resveratrol (RV) binds primarily to the ßbeta;3 monomer of the integrin, activating the ERK1/2 pathway with consequent phosphorylation of serine-15 of p53. Nuclear phosphorylated p53 accumulates, leading to stimulation of p53-responsive gene transcription and apoptosis of breast cancer cells.

Both commercially purified integrin Vßbeta;3 and integrin Vßbeta;3 in MCF-7 cell plasma membranes bind RV and the binding of this ligand to monomeric ßbeta;3 is dissociable at a concentration of RV which we have shown to be biologically active. From these studies and those with siRNA ßbeta;3, we propose that integrin binding to the ßbeta;3 monomer constitutes the principal site of stilbene interaction with the receptor. In addition, occlusion of the RGD binding site in the integrin extracellular domain blocks the cellular actions of RV previously described in a variety of cancer cell lines, including ERK1/2 pathway activation and serine phosphorylation of p53, leading to cancer cell apoptosis. The effectiveness of an RGD peptide, but not a control RGE peptide, as an inhibitor of RV binding to the integrin suggests that the binding site for the stilbene on the receptor protein is at or near the RGD recognition site that is important to its interactions with extracellular matrix proteins. However, it is possible that the binding of RGD peptide by the integrin leads to allosteric changes in the protein that affect an RV binding site elsewhere on the heterodimer.

Appreciation that integrin Vßbeta;3 bears a receptor site for RV suggests a role for the dimer as a screening tool for activity of RV analogues and perhaps for estimating in vitro or in vivo responsiveness of tumor cells to the stilbene. The RGD domain of the integrin may also be a target for development of nonstilbene, small molecule pharmaceuticals with potential for induction of apoptosis in tumor cells. Translating RV from an interesting naturally occurring substance into a specific ligand will facilitate more critical studies of its mechanisms of action.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5743fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5743fjev1 20/10/1742    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lin, H.-Y. Articles by Davis, P. J. Search for Related Content PubMed PubMed Citation Articles by Lin, H.-Y. Articles by Davis, P. J.