The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inf lammatory, anti-platelet, and anticarcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (Ϸ3-10 M) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.Resveratrol (trans-3, 4Ј, 5-trihydroxystilbene) occurs naturally in grapes and a variety of medicinal plants. In plants, resveratrol functions as a phytoalexin that protects against fungal infections (1). Because of its high concentration in grape skin, significant amounts of resveratrol are present in wine (2, 3), and it has been proposed to explain, at least in part, the apparent ability of moderate consumption of red wine to reduce the risk of cardiovascular disease (4 -7). Resveratrol also has been reported to have cancer chemopreventive activity (8). The similarity in structure between resveratrol and the synthetic estrogen diethylstilbestrol (DES; 4, 4Ј-dihydroxy-trans-␣, ␤-diethylstilbene) prompted us to investigate whether resveratrol might exhibit estrogenic activity, a property that is known to produce a cardioprotective benefit (9, 10).Estrogens, including phytoestrogens, act via the estrogen receptor, a member of the nuclear receptor superfamily. Estrogen binding to the receptor activates the transcription of estrogen-responsive target genes. We report here that resveratrol binds to and activates transcription by the estrogen receptor at concentrations that are comparable to those required for its other biological effects. EXPERIMENTAL PROCEDURESCell Culture. MCF-7 cells, subclone WS8 (estrogen receptor-positive), MDA-MB-231 cells, subclone 10A (estrogen receptor-negative), and T47D cells, subclone A18 (estrogen receptor-positive) are derived from human breast adenocarcinomas and were provided by V. Craig Jordan (Northwestern University Medical Sch...

In the classical signaling pathway, the estrogen receptor (ER) binds directly to estrogen response elements (EREs) to regulate gene transcription. To test the hypothesis that the nonclassical pathway involves ER interactions with other proteins rather than direct binding to DNA, mutations were introduced into the DNA binding domain (DBD) of the mouse ER␣. The effects of these DBD mutations were examined in DNA binding assays using reporter constructs containing either EREs (classical) or AP1 (nonclassical) response elements. Using the AP1 reporter, there was a reversal of ER action relative to that seen with the ERE reporter. Estradiol induced suppression, and the antiestrogen ICI 182,780 stimulated transcription of the AP1 reporter. DBD mutations in the proximal (P-box) of the first zinc finger of the ER (E207A/G208A and E207G/G208S) eliminated ERE binding. These mutants were inactive using the ERE reporter but retained partial or full activity with the AP1 reporter. The DBD mutant ERs interacted with Jun when tested in mammalian cell two-hybrid assays. Two mutations (K366D and I362R) in the ER ligand binding domain known to alter coactivator interactions impaired transcriptional responses using either the ERE or AP1 reporters. We concluded that ER action through the AP1 response element involves interactions with other promoter-bound proteins instead of, or in addition to, direct binding to DNA. Interactions with coactivators were required for both pathways. These data supported a model in which ER-mediated transcriptional activation or repression is dependent on the ligand and the nature of the response element in the target gene.Estrogen has a wide range of physiologic activities, including the control of development, reproduction, and metabolism as well as effects on cell growth and differentiation. Most, if not all, actions of estrogen occur through its receptors, ER␣ 1 and ER␤. The functional domains of the ER are relatively well In the traditional model of ER action, the receptor binds as homodimers (3) or heterodimers (4 -7) to estrogen response elements (EREs) in the promoters of many, though not all, estrogen-responsive genes. Similar to other nuclear receptors, the ER recruits an array of transcriptional cofactors (coactivators and corepressors) that bind to the receptor and also interact with other transcription factors, including components of the general transcription factor apparatus. Some of the cofactors also possess chromatin-remodeling activities or recruit additional proteins to the complex to mediate transcription (reviewed in Ref. 8).It is now recognized that the type of ligand bound to the ER influences its interaction with cofactors. The crystal structures of the ER LBD when bound to an agonist (estradiol) or an antagonist (raloxifene) have been solved. Comparison of these structures suggests a molecular basis for the differential liganddependent cofactor binding (9). The binding of 17␤-estradiol induces a major shift in the position of helix 12, one of several helices that form the coac...

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We have previously identified and characterized a metalloproteinase from Drosophila that cleaves insulin and transforming growth factor-alpha, but not epidermal growth factor, at physiological concentrations. On the basis of enzymatic properties and substrate specificity, this enzyme was identified as the Drosophila homolog of the mammalian insulin-degrading enzyme (IDE). We now report the cloning and sequencing of the cDNA coding for the Drosophila IDE (dIDE). Northern blot analysis indicates that the dIDE is translated from a 3.6-kilobase transcript similar in size to one of the two human IDE transcripts. The gene for the dIDE has been mapped to chromosome 3L (77B). The sequence of the dIDE is very similar to that of the human IDE, and both enzymes share limited but significant identity with the bacterial metalloproteinase protease III. Indirect studies based upon inhibitors, degradation products, and microinjected antibodies have suggested that the IDE can initiate cellular insulin degradation in mammalian cells. To determine whether dIDE expressed in mammalian cells can also degrade insulin, we transfected the cDNA into murine NIH3T3 cells. Extracts of the transfected cells showed increased insulin-degrading activity, demonstrating that the dIDE can be functionally expressed in mammalian cells. These results indicate that the properties of the IDE are evolutionarily conserved.