Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors

Accepted Manuscript Title: Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors Author: Gerald Thiel Oliver G. R¨ossler PII: DOI: Reference:

S1043-6618(16)31066-0 http://dx.doi.org/doi:10.1016/j.phrs.2016.12.029 YPHRS 3452

To appear in:

Pharmacological Research

Received date: Revised date: Accepted date:

18-10-2016 16-12-2016 18-12-2016

Please cite this article as: Thiel Gerald, R¨ossler Oliver G.Resveratrol regulates gene transcription via activation of stimulus-responsive transcription factors.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2016.12.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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REVIEW

Resveratrol regulates gene transcription via activation of stimulusresponsive transcription factors

Gerald Thiel, Oliver G. Rössler

Department of Medical Biochemistry and Molecular Biology Saarland University D-66421 Homburg Germany

Correspondence: Gerald Thiel, Department of Medical Biochemistry and Molecular Biology, Saarland University, Medical Faculty, Building 44, D-66421 Homburg, Germany E-mail: [email protected] Fax: +49-6841-1626500 Graphical abstract

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Abstract Resveratrol (trans-3,4´,5-trihydroxystilbene), a polyphenolic phytoalexin of grapes and other fruits and plants, is a common constituent of our diet and of dietary supplements. Many health-promoting benefits have been connected with resveratrol in the treatment of cardiovascular diseases, cancer, diabetes, inflammation, neurodegeneration, and diseases connected with aging. To explain the pleiotropic effects of resveratrol, the molecular targets of this compound have to be identified on the cellular level. Resveratrol induces intracellular signal transduction pathways which ultimately lead to changes in the gene expression pattern of the cells. Here, we review the effect of resveratrol on the activation of the stimulusresponsive transcription factors CREB, AP-1, Egr-1, Elk-1, and Nrf2. Following activation, these transcription factors induce transcription of delayed response genes. The gene products of these delayed response genes are ultimately responsible for the changes in the biochemistry and physiology of resveratrol-treated cells. The activation of stimulusresponsive transcription factors may explain many of the intracellular activities of resveratrol. However, results obtained in vitro may not easily be transferred to in vivo systems. Abbreviations: AP-1, activator protein-1; ATF, activating transcription factor; bZIP, basic region leucine zipper; CRE, cyclic AMP response element; CREB; cAMP response element binding protein; Nrf2, NF-E2-related factor-2; SRE, serum response element; SRF, serum response factor; TCF, ternary complex factors; TRE, TPA-responsive element key words: Resveratrol; AP-1; CREB; Elk-1; c-Fos; c-Jun; Nrf2; SIRT1

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1.

Introduction

Resveratrol is a polyphenolic phytoalexin belonging to the stilbenes that is found in the skin of grapes, in peanuts, soy beans, pomegranates, and in other fruits and plants. Resveratrol is therefore a substantial ingredient of our diet. Moreover, many dietary supplements contain resveratrol, either as a single component, or together with other compounds such as other polyphenols, vitamins, fatty acids, or anti-oxidants. A 2012 report from the marketing and research firm Frost & Sullivan estimated a market potential of about 50 million USD. Resveratrol initially gained much attention as an explanation for the “French paradox“, the observation that people of France have a low incidence of suffering from cardiovascular diseases despite a high-fat diet. It was suggested that the consumption of resveratrolcontaining red wine afforded protection from cardiovascular disease. The health-promoting effects of resveratrol provoked the question: How much wine do you have to drink to stay healthy ? [1]. Much attention has also been put into the investigation of the chemopreventive activity of resveratrol, including the inhibition of carcinogen activation, the detoxification by phase II enzymes, as well as the regulation of cell cycle progession and apoptosis. Moreover, resveratrol shows anti-inflammatory activity by reducing the biosynthesis of proinflammatory mediators and stimulating the expression of anti-inflammatory proteins. Furthermore, an antidiabetic activity of resveratrol has been observed. Resveratrol protects against neurological diseases and promotes neurogenesis in the hippocampus [2-8]. Recently published review articles addressed the pharmacology of resveratrol, the effect of resveratrol on the human metabolism, toxicological effects of resveratrol, clinical trials performed with resveratrol, the effect of resveratrol supplementation in the treatment of human diseases, in particular in preventing neurological disorders, inflammation, or cardiovascular diseases [7, 9-15]. Surprisingly, the role of resveratrol in the regulation gene transcription has not been received much attention, with the exception of transcription factors connected to the anticancer effects of resveratrol [16, 17]. This review adds to that body of knowledge by focusing on the transciptional effects of resveratrol. To understand the health benefits of resveratrol, the molecular targets and interaction partners of this compound have to be identified on the cellular level. Resveratrol is a nutrient signaling molecule that stimulates intracellular signaling pathways. Generally, cells of a multicellular organism receive information from their environment that is important for their survival and for fulfilling their particular functions. This information is brought to the cells

5 by means of nutrients and a variety of specialized extracellular signaling molecules such as hormones, neurotransmitters, growth factors, and cytokines. These molecules transmit information to the cells via interaction with receptor proteins or ion channels. Nutrients function as signaling molecules, as exemplified by the role of glucose in triggering insulin secretion or of fatty acids in activating G protein-coupled receptors. In the cytoplasm, protein kinases are often activated as a result of signal transduction. Long-term changes are accomplished by changing the transcriptional pattern of the cells as a result of cellular signaling. Accordingly, resveratrol triggers the activation of protein kinases in the cells and regulate gene transcription. In this review article, we summarize and discuss the data regarding the effect of resveratrol on the activities of the stimulus-responsive transcription factors. These proteins are transiently activated by different means. The activation of Egr-1 and c-Fos is coupled to their biosynthesis. Both proteins are encoded by immediate-early genes that are rapidly transcribed following cellular stimulation. The stimulus-responsive transcription factors ATF2, CREB, and c-Jun are regulated by phosphorylation following stimulation-dependent activation of protein kinases. The transcription factor Nrf2 is regulated by a cytoplasmic inhibitor (Keap1) that immobilizes Nrf2 in the cytoplasm in the absence of stimulation. The stimulus-regulated transcription factors bind to the regulatory region of delayed response genes and stimulate transcription of these genes (Fig. 1). The gene products of the delayed response genes are ultimately responsible for the biochemical and physiological changes observed as a result of cellular stimulation. We propose that the pleiotropic activities of resveratrol may, at least in part, be explained by the change of the gene expression pattern of the cells following resveratrol treatment. 2. Methodological challenges Nakata et al. [19] stated that “it is no exaggeration to say that the literature on resveratrol is contradictory and confusing“. Accordingly, resveratrol stimulation has been described to either activate or inhibit stimulus-responsive transcription factors. These differences may, in part, be explained by the use of different cell types or concentrations of resveratrol. However, we think that an additional reason for these discrepancies is the use of different assay conditions to measure transcriptional factor activation. Some of the potential pitfalls are as follows: The biosynthesis of the transcription factors Egr-1 and c-Fos is upregulated as a result of cellular stimulation. Thus, immunohistochemistry or Western blot techniques are

6 often used to detect stimulus-induced expression of these proteins in tissues and cells. However, an upregulation of Egr-1 expression may not always imply higher Egr-1 activity, because the activity of Egr-1 depends on the concentrations of the proteins NAB1 and NAB2 that block transcriptional activation of Egr-1 [20-22]. Often, the expression of the c-Fos protein is monitored as a measure for AP-1 activation. c-Fos needs a dimerization parter, often c-Jun, for generation of a functional AP-1 transcription factor complex. Thus, the upregulation of c-Fos expression does not necessary mean that the cellular AP-1 activity is increased. The transcription factors CREB is phosphorylated following stimulation of the cells with appropriate ligands. Therefore, phosphorylation can be monitored with phosphospecific antibodies. However, phosphorylation of CREB is not a reliable predictor of CREB target gene transcription [23]. A rise in intracellular Ca2+, for instance, failed to activate CREB-dependent transcription in astrocytes, although CREB was phosphorylated on a critical serine residue Ser-133 [24]. Immunocytochemisty is not usable to measure AP-1 activity, because AP-1 exists either as a homodimer or as a heterodimer composed of various transcription factors of the Fos, Jun and ATF protein families. Often, protein-DNA binding assays, either in vitro or in vivo, are used as a measure for the activity of a transcription factor. However, the binding of AP-1 to DNA does not always mirror the transcriptional activity of the AP-1 transcriptional complex [25]. Although DNA binding is mostly a prerequisite for the activation of stimulus-induced transcription factors, it is necessary to emphasize that DNA binding, measured by EMSA or ChIP techniques, is not linearily translated into transcriptional activation. DNA binding can also be replaced by proteinprotein interactions, as shown for the Elk-1 transcription factor [26, 27]. Furthermore, binding of stimulus-responsive transcription factors, including CREB, serum response factor (SRF), and Elk-1, to DNA in the absence of stimulation has been observed [28, 29]. Thus, to analyze the activities of stimulus-responsive transcription factors, it is essential to measure their abilities to stimulate transcription of specific transcription factor-responsive reporter genes. This reporter genes need to be implanted into the chromatin of the cells to ensure that the reporter genes are packed into an ordered nucleosomal structure. Using transient transfections to introduce reporter genes into cultured cells may result in reporter genes that are incompletely organized in nucleosomes [30, 31]. The structure of these genes resemble rather a prokaryotic than an eukaryotic gene organisation with a nonrestrictive transcriptional ground state. In eukaryotes, the chromatin gene organisation is restrictive and does not allow an unrestrictive binding of RNA polymerases and transcriptional regulators to DNA. Hence, the conclusions discussed in this review article are mainly based on

7 experiments with promoter/reporter genes integrated into the chromatin for the investigation of transcriptional regulatory mechanisms. 3.

Regulation of stimulus-responsive transcription factors by resveratrol

3.1. AP-1 The activator protein-1 (AP-1) transcription factor was identified as a heterodimer of the bZIP transcription factors c-Jun and c-Fos [32]. Today, AP-1 is viewed as a homodimeric or heterodimeric transcription factor complex that is built by proteins of the Fos, Jun and ATF families of basic region leucine zipper (bZIP) transcription factors. The leucine zipper domains are used for dimerization, whereas the basic regions function as DNA binding domain. Many signaling molecules, e.g. cytokines, growth factors, and hormones, activate AP-1, indicating that AP-1 functions in cells as a convergence point for intracellular signaling cascades. Many biological functions have been attributed to AP-1, including proliferation, transformation, differentiation, and programmed cell death [33]. The c-Fos protein contains a central bZIP domain and a C-terminal transactivation domain (Fig. 2A). The inactivation of the c-Fos gene resulted in growth-retarded mice that lacked osteoclasts [34]. Neuronal activity stimulates c-Fos expression and genetic experiments showed the importance of c-Fos for neuronal excitability and survival [35]. The c-Fos gene is the prototype immediate-early gene. Stimulation of the cells with Gs and Gq-coupled receptor ligands, receptor tyrosine kinase ligands, phorbol esters, cytokines, or calcium ionophores activates c-Fos expression. The c-Fos gene promoter contains binding sites for the transcription factors CREB, STAT, AP-1, serum response factor (SRF), and ternary complex factors (TCFs) that mediate stimulus-induced c-Fos expression. A central component of the AP-1 complex is the bZIP protein c-Jun (Fig. 2A). Phosphorylation of serine residues 63 and 73 of c-Jun by c-Jun N-terminal kinase (JNK) is necessary to activate [36]. Moreover, AP-1 triggers the expression of c-Jun [37]. Homozygous c-Jun-deficient transgenic mice die at mid-gestation [38], indicating that c-Jun is necessary for normal mouse development. The bZIP protein ATF2 is also found in AP-1 transcription factor complexes. ATF2 has a phosphorylation-regulated

N-terminal

transcriptional

activation

domain

that

is

8 phosphorylated by the protein kinases JNK and p38 [39] (Fig. 2A). ATF2-deficient mice die shortly after birth showing symptoms of the Meconium Aspiration Syndrome [40]. Recent studies of the biological function of ATF2 have emphasized its tumor suppressive function [41, 42]. Conflicting results have been published concerning the effect of resveratrol on cellular AP-1 activity. Several studies investigated the effect of resveratrol on phorbol ester or UV-induced AP-1 activation [43-45]. Thus, it was not the effect of resveratrol on basal AP-1 activity that was assessed, but rather the effect of resveratrol on already activated AP-1. Protein-DNA binding assays have frequently been used to measure the effect of resveratrol on AP-1, although DNA binding activity cannot be used as a measure for the activation of the AP-1 transcriptional complex [25]. Likewise, c-Fos expression or c-Fos nuclear translocation have been used as a measure for AP-1 activation [46], ignoring the fact that the AP-1 transcription factor complex can also be constituted by other proteins of the Fos family, together with cJun and ATF proteins. Using an integrated AP-1-responsive reporter gene, it has been shown that resveratrol significantly increased the cellular AP-1 activity (Fig. 2B). The AP-1 motif encompassing the sequence 5´-TGAGTCA-3´ was identified as the resveratrol responsive element [47]. The activation of AP-1 by resveratrol was corroborated by the finding that resveratrol treatment results in an upregulation of c-Jun and c-Fos biosynthesis (Fig. 2C). cJun has been identified as an important regulator of AP-1 activity in resveratrol-treated HEK293 cells [47]. The biosynthesis of c-Jun and c-Fos is also upregulated in cells following stimulation of Gq-coupled receptors [37, 48-50]. We would like to emphasize that resveratrol signaling requires 8 to 24 hours for maximal expression of c-Jun and c-Fos, while GPCR signaling activates c-Jun and c-Fos biosynthesis within 1 to 3 hours. This observation indicates that c-Jun and c-Fos expression is not "immediate-early" in resveratrol-treated cells. Likewise, an upregulation of c-Jun expression and phosphorylation has been observed in resveratrol-treated renal epithelial cells, that contributes to the stimulation of the Klotho gene, an aging suppressor gene [51]. The fact that expression of a dominant-negative mutant of cJun attenuated resveratrol-induced Klotho expression confirms the link between resveratrol stimulation, c-Jun activation, and Klotho expression. In addition, resveratrol stimulation upregulates the transcriptional activation potential of ATF2 (Fig. 2D) [52]. Given the identification of ATF2 as a tumor suppressor, we propose that the anti-cancerogenic activity of resveratrol may rely on the transcription of ATF2-controlled target genes. 3.2

CREB

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Cyclic AMP-response element (CRE)-binding protein (CREB) is a member of the bZIP family of transcription factors (Fig. 3A), like ATF2, c-Fos and c-Jun. CREB is activated following stimulation of the cells with hormones, neurotransmitters, neurotrophins, cytokines, or growth factors [53-55]. CREB was discovered as a cAMP-regulated transcription factor that binds to the cyclic AMP-response element (CRE), encompassing the palindromic sequence 5´-TGACGTCA-3´. The discovery that resveratrol stimulation increased the cellular cAMP concentration by inhibiting cAMP phosphodiesterases [56, 57] suggests that resveratrol may also stimulate cAMP-regulated gene transcription via CREB. The transcriptional activity of CREB depends on its phosphorylation state. CREB is phosphorylated by cAMP-dependent protein kinase PKA, Ca2+/calmodulin regulated protein kinase CaMKIV and mitogen-induced protein kinase MSK [53-55]. CREB activity and the CRE-controlled gene expression pattern have been connected with many biological functions, including cellular proliferation and cell death, intermediary metabolism, neuronal plasticity, neuroprotection, neuronal survival and inflammation [53-55, 58]. Concerning the role of resveratrol in activating CREB and inducing CRE-regulated gene transcription, conflicting results have been published. Administration of resveratrol for 14 days reduced the phosphorylation of CREB in the hippocampus of mice [59]. In contrast, elevated phospho-CREB levels were found in the hippocampal CA1 region of resveratroltreated rats [60, 61]. The protection of CA1 neurons against focal cerebral ischemic reperfusion-induced damage by resveratrol was attributed to the activation of the CREB signaling pathway [60, 61], although phospho-CREB levels increased only about 1.3-fold following resveratrol treatment. Surprisingly, high levels of phosphorylated CREB were already detected under control conditions [61]. It is necessary to emphasize that CREB phosphorylation does not necessary mean that CREB-regulated gene transcription is induced [23, 24]. Using a chromatin-embedded, CRE-controlled reporter gene, resveratrol stimulation was shown to induce CRE-mediated gene transcription (Fig. 3B), indicating that the CRE is a functional resveratrol-response element. These data are supported by the observation that resveratrol stimulation increased the transcriptional activation potential of CREB (Fig. 3C). Thus, the biological activity of CREB is higher in resveratrol-treated cells [52]. Given the fact that CREB activity and CRE-controlled gene expression is connected with neuroprotection and neuronal survival, it is likely that resveratrol is neuroprotective by activating CREB and stimulating CRE-mediated gene transcription. However, in the model of ischemic injury of the brain, a causal involvement of CREB and CREB-regulated gene

10 transcription in the resveratrol-mediated neuroprotection requires experimental proof. In human bronchial epithelial cells and in the lung of C57BL/6 mice, resveratrol treatment increased expression of MAP kinase phosphatase-1 (MKP-1) via the cAMP/PKA signaling pathway [57]. These data are supported by the fact that the MKP-1 gene regulatory region contains two CREs, suggesting that these motifs connect resveratrol treatment with MKP-1 expression. The analysis of a chromatin-embedded MKP-1 promoter/luciferase reporter gene confirmed that transcription of the reporter gene is induced following stimulation of the cells with resveratrol (G. Thiel, unpublished observations). Furthermore, the discovery that resveratrol treatment suppressed Haemophilus influenzae-induced inflammation via the activation of MKP-1 gene as a delayed response gene provides a molecular explanation for the anti-inflammatory activity of resveratrol. 3.3

Egr-1

Egr-1 is a member of a family of zinc finger transcription factors that encompass, in addition to Egr-1, the proteins Egr-2, Egr-3, and Egr-4. All Egr proteins have a homologous domain structure with a DNA binding domain that contains three zinc finger motifs for binding to a GC-rich DNA recognition site. The N-terminus contains an extensive transcriptional activation domain, while an inhibitory domain is located between the activation domain and the DNA binding domain that functions as a binding site for the two transcriptional co-factors NAB1 and NAB2 (Fig. 4A). As higher concentrations of NAB1/2 block Egr-1 activity [2022], elevated Egr-1 mRNA or protein levels alone do not indicate that the newly synthesized Egr-1 is biologically active. The Egr-1 gene is a typical immediate-early gene. Signaling molecules including growth factors, hormones, neurotransmitters and metabolites stimulate Egr-1 expression. Many biological functions have been connected to the Egr proteins, including control of neuronal cell death, neuronal plasticity, proliferation, reproduction, glucose homeostasis and inflammation [62-67]. Stimulation of human pancreatic adenocarcinoma cells, rat and human colon carcinoma cells, and lung cancer cells with resveratrol leads to an upregulation of Egr-1 expression [68-70]. In erythroleukemic cells and in neuroblastoma, resveratrol stimulation increased Egr-1 mRNA levels [71]. However, other researchers stated that resveratrol stimulation decreased Egr-1 mRNA levels in PC12 cells [72]. Fig. 4B shows that resveratrol stimulation induced the expression of Egr-1 in HEK293 cells, reaching highest levels after 24 hours. In contrast, EGF stimulation induced a transient upregulation of Egr-1 biosynthesis, where highest levels could

11 already be detected after 1 hour [73]. Using higher concentrations of resveratrol, Egr-1 expression was already induced after four hours in lung cancer cells [70]. Resveratrol treatment increased the transcriptional Egr-1 activity in HEK293, measured with an Egr-1responsive, chromatin-embedded reporter gene [73]. Thus, resveratrol stimulation triggers the biosynthesis of biologically active Egr-1. These experiments identified the GC-rich Egr-1 binding site EBS (5'-GCGGGGGCG-3') as a resveratrol-responsive element. Interestingly, a resveratrol-responsive suicide gene therapy vector has been designed consisting of Egr-1 promoter, driving the expression of cell cycle arresting protein GADD45. Stimulation of lung cancer cells expressing this suicide gene with resveratrol resulted in proliferation arrest and programmed cell death [70]. 3.4

Elk-1

The transcription factor Elk-1 belongs to the family of ternary complex factors (TCF) that bind together with a serum response factor (SRF) dimer to the serum-response element (SRE) (Fig. 5A). The SRE has the consensus sequence CC[A/T]6GG, also known as CArG box. Many genes contain, adjacent to the CArG box, a binding site for TCFs with the Ets consensus core sequence GGAA/T. Fig. 5B shows the modular structure of Elk-1. The protein contains a DNA binding domain, localized on the N-terminus, and a transcriptional activation domain that is localized on the C-terminus. Between these domains lies the B domain that is required for the formation of the ternary Elk-1-SRF complex. The transcriptional activity of Elk-1 is regulated by MAP kinases via phosphorylation of serine residues S383 and S389 within the transcriptional activation domain. Resveratrol treatment of lung cancer cells was shown to stimulate Elk-1 expression in a time- and dose-dependent manner [70]. In addition, experiments with HEK293 cells showed that the transcriptional activation potential of Elk-1 was significantly elevated following stimulation of the cells with resveratrol (Fig. 5C) [73], indicating that intracellular signaling cascades, induced by resveratrol treatment, triggered the phosphorylation and activation of Elk-1. Moreover, resveratrol treatment stimulated transcription of a reporter gene that is solely controlled by a SRE (Fig. 5D). Thus, the SRE functions as a resveratrol-responsive element. Elk-1 is required for the resveratrol-induced transcription of SRE-controlled immediate-early genes cFos and Egr-1 [47, 70, 73], but resveratrol is also able to regulate transcription of genes with binding sites for Elk-1 that lack binding sites for SRF [73]. Elk-1 is additionally a regulator of AP-1, most likely via controlling c-Fos expression [47, 49, 50, 74, 75]. Accordingly, interference of Elk-1 function blocked the resveratrol-mediated upregulation of AP-1 [47].

12 Together, these results indicate that Elk-1 activation is a key step in connecting the intracellular signaling cascade elicited by resveratrol with enhanced c-Fos and Egr-1 expression and an increase in AP-1 and Egr-1 activities. 3.5

Nrf2

The bZIP transcription factor NF-E2-related factor-2 (Nrf2) (Fig. 6A) is activated by oxidative stress-generating agents, antioxidants, electrophilic, or xenobiotic compounds, and provides a key mechanism for efficiently detoxifying reactive metabolites [76]. Nrf2 is sequestered in the cytoplasm by the protein Keap1 (Fig. 6B) which functions as an adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Nrf2 is ubiquitinated and degraded, resulting in low Nrf2 levels in resting cells. Exposure to compounds that trigger oxidative stress in the cells leads to the modification of critical cysteine residues in the Keap1 molecule. These modifications induce a conformational change in the Keap1-Nrf2-Cul3 complex, attenuating ubiquitination and degradation of Nrf2. The Nrf2 concentration increases and Nrf2 translocates from the cytoplasm to the nucleus when the binding capacity of Keap1 is exhausted (Fig. 6C). In the nucleus, Nrf2 dimerizes with other bZIP transcription factors, and binds to a DNA motif known as either stress-response element (StRE), antioxidant response element (ARE), or electrophilic response element (EpRE). The genes encoding heme oxygenase (HO-1), NAD(P)H:quinone oxidoreductase I (NQO1), glutathione S-transferases, glutamate-cysteine ligase, and glutathione oxidases are regulated by Nrf2 via the StRE/ARE/EpRE. The gene products provide cytoprotection against multiple insults and coordinate the phase II response. The effect of resveratrol on activating Nrf2-regulated gene transcription has been discussed in the framework of the anticancer activity of resveratrol [17]. However, tumor suppression is not the only activity of Nrf2, and even an oncogenic role of Nrf2 has been discussed [77]. Additionally, Nrf2 regulates many other biological functions, including differentiation, inflammation, and protection against neurodegeneration and cardiovascular dysfunction. The fact that resveratrol stimulates Nrf2-mediated gene transcription connects resveratrol with the regulation of proliferation, cardiovascular functions, neuronal cell death, and stress response. Resveratrol has been shown to upregulate heme oxygenase-1 expression via Nrf2 in PC12 cells and to stimulate transcription from a StRE-controlled reporter gene [78]. Increased Nrf2 protein levels were observed in resveratrol-stimulated human bronchial epithelial cells. Resveratrol stimulated NAD(P)H:quinone oxidoreductase I (NQO1) expression to a similar level as the well-characterized activator of Nrf2, tert-butylhydrochinone [79]. Resveratrol

13 also stimulated nuclear translocation of Nrf2 and triggered an increase in NQO1 activity in astrocytes [80]. In contrast, no statistically significant activation of an electrophilic response element-controlled reporter gene could be detected in resveratrol-treated HepG2 cells [81]. As Nrf2 induces expression of cytoprotective enzymes, resveratrol-induced activation of Nrf2 has been correlated with protection against oxidative stress [82, 83] and cancer [84]. Together, these data support the view that Nrf2 is a molecular target of resveratrol, leading to increased expression of Nrf2 target genes and protection against oxidative stress. Downregulation of Nrf2 expression via transfection of a Nrf2-specific siRNA decreased the resveratrol-mediated upregulation of the catalytic subunit of glutamate-cysteine ligase in lung cancer cells [82], providing the first link between resveratrol stimulation, Nrf2 activation and transcription of Nrf2 target genes. Moreover, Ungvari et al [85], showed that resveratrolinduced upregulation of NAD(P)H:quinone-oxidoreductase-1, -glutamylcysteine synthtase, and heme oxygenase-1 expression was attenuated by transfection of a Nrf2-specific siRNA, and by overexpressing Keap1, suggesting that resveratrol activates Nrf2 in a Keap1dependent manner. Further research should show whether the dissociation of Nrf2 from Keap1 is accomplished by oxidative or covalent modifications of reactive cysteine residues within the Keap1 molecule. In addition, it would be of interest to elucidate the role of Bach1, a nuclear transcriptional repressor that forms heterodimers with small Maf proteins in resveratrol-treated cells [86]. Bach1 competes with Nrf2 for DNA binding, indicating that an increase or a decrease in Bach1 expression by resveratrol would significantly influence Nrf2mediated gene transcription. 4.

Resveratrol and epigenetic gene control

Originally, resveratrol was described as an activator of SIRT1, a NAD+-dependent histone deacetylase, that uses NAD+ as a cosubstrate to catalyze the deacetylation of histones and non-histone proteins. Histone deacetylases catalyze the removal of the acetyl group from the -amino group of lysine residues of histones. As a result, ionic interactions between the negatively charged DNA phosphate backbone and the positively charged amino termini of the core histones are initiated, leading to a compact chromatin structure that is not accessible by transcription factors and the RNA polymerases. In contrast, net positive charges of the core histones are reduced by acetylation. The binding affinity of the histones to DNA is reduced, the nucleosomes unfold and the DNA is accessible for the transcriptional machinery. Thus, transcriptional activation requires the destabilization of the repressive histone-DNA interaction by histone acetylation.

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It has been hypothesized that resveratrol regulates transcription via activating SIRT1. However, while resveratrol treatment was able to stimulate the deacetylation of an artificial fluorophore-tagged SIRT1 substrate, it failed to deacetylate native SIRT1 substrates [87, 88]. These data raised questions about the function of resveratrol as a direct SIRT1 activator, despite the fact that the function of resveratrol in mice and rats were consistent with an activation of SIRT1 [89-91]. Further research showed that resveratrol activates SIRT1 indirectly, based on the activation of AMP-activated protein kinase (AMPK), which in turn increases the cellular NAD+/NADH ratio. Thus, SIRT1 is activated as a result of increasing concentrations of NAD+ [56, 92]. Accordingly, AMPK-deficient mice were shown to be resistant to the metabolic effects of resveratrol [93]. A coherent picture encompassing the biological activities of resveratrol and SIRT1 is currently not available. SIRT1 has been described to inhibit c-Jun and CREB regulated gene transcription [91, 94, 95]. SIRT1 also catalyzes the deacetylation Nrf2, leading to a decreased Nrf2-dependent gene transcription. Accordingly, pharmacological inhibition of SIRT1 stimulated Nrf2-mediated transcription, whereas resveratrol was inhibitory [96]. These contradictions indicate that there is currently no coherent picture describing the connection between stimulus-responsive transcription factor activation and histone acetylation/deacetylation. As resveratrol is a promiscuous molecule that influences many enzymes [97], the direct relationship between resveratrolinduced activation of stimulus-responsive transcription factors and the activation of SIRT1 and SIRT1-catalyzed deacetylation of histones and non-histone proteins remains unresolved and requires further studies involving specific SIRT1 gain-of-function and loss-of-function experiments. Resveratrol stimulation enhances expression of the transcriptional co-activator p300 [98]. p300 and its structural homologue CREB binding protein (CBP) are histone acetyltransferases that function as co-activators for many transcription factors, including CREB, c-Jun, c-Fos, Egr-1, Elk-1, and Nrf2 [99]. CBP/p300 catalyzes histone acetylation and thus generates an open chromatin structure. CBP also acetylates Nrf2, resulting in increased binding of Nrf2 to the stress/antioxidant response element and increased Nrf2dependent transcription [96]. Thus, the regulation of CBP/p300 by resveratrol may contribute to the activation of stimulus-responsive transcription factors by resveratrol. 5.

Resveratrol – only an in vitro drug ?

15 The results reviewed above clearly showed that resveratrol functions as a signaling molecule leading to changes in the gene expression pattern of the cells. In contrast, other polyphenols (e.g. quercetin, curcumin, naringenin) are less or only marginally active in stimulating transcription, highlighting the exceptional activity of resveratrol in comparison to other plant polyphenols. Many of these studies were performed with cultured cells, using M concentrations of resveratrol. In humans, resveratrol has a limited bioavailability, due to a rapid metabolism in the intestine and liver. Resveratrol is quickly absorbed and rapidly conjugated with sulfate or glucuronic acid. In the plasma, a maximal concentration of 2 M of free and conjugated resveratrol was detected in human that had received mg amounts of resveratrol [100-102]. It has been suggested that resveratrol may accumulate in tissues, e.g. in epithelial cells along the digestive tract or in the liver, leading to higher and biologically active concentrations. However, the effects of resveratrol in humans are often tiny and not always reproducible. These in vitro/in vivo discrepancies provoked the statement that resveratrol has no proven human activity [103]. In line with this, 21 resveratrol investigators stated that published evidence is not sufficiently strong to justify administration of resveratrol to humans [104]. Resveratrol can be used as a scaffold compound to design and synthesize resveratrol analogs. Several of these derivatives have already been tested [105, 106]. The aim would be to create resveratrol analogs that have an increased bioavailability, potency and efficacy in comparison with resveratrol so that lower concentrations could be used as therapeutics in vivo. As resveratrol is a highly promiscuous molecule, resveratrol analogs may also be more specific in their activities. In fact, thiazole analogues of resveratrol have been synthesized, showing high potency and selectivity towards activation of NAD(P)H:quinone reductase 1 [107]. Furthermore, the bioavailability of resveratrol or resveratrol analogs has been improved by administration of resveratrol together with another compounds or as a prodrug [108-110]. Several other delivery systems for resveratrol have been described, including nanoencapsulation of resveratrol, micelle formation with 3,7,12-triketocholic acid, resveratrolloaded

calcium

or

zinc-pectinate

microbeads,

resveratrol-loaded

beta-cyclodextrin

nanosponges and others [109, 111]. Thus, to overcome the physicochemical, pharmacokinetic and metabolic limitations of resveratrol, administration of resveratrol analogs exhibiting higher bioavailability, potency and selectivity than resveratrol should be combined with a

16 sophisticated delivery system. This approach may result in biological effects in humans that are comparable to the described activities observed in cell culture or in rodent model systems. 6.

Conclusion and future prospects

Chemopreventive, cardioprotective, antioxidant, anti-inflammatory, and anti-aging activities have been connected with resveratrol stimulation, resulting in the use of resveratrol as a dietary supplement. However, how resveratrol its analogs exert these effects in detail is still not yet clear and several hypotheses have been suggested to explain the mechanism of resveratrol function at the molecular level. We propose that activation of the stimulusresponsive transcription factors explains many of the diverse intracellular activities following resveratrol stimulation. In addition, resveratrol may influence the epigenome leading to either stimulation or repression of stimulus-responsive transcription factors. To explain the functional changes of the cells following treatment with resveratrol, the delayed response genes of stimulus-responsive transcription factors need to be identified and several resveratrol-regulated genes have already been suggested [47, 57, 98, 112, 113]. Although AP-1, CREB, Egr-1, Elk-1, and Nrf2 are expressed in all cell types, it is important to emphasize that stimulus-responsive transcription factors may activate target genes in a celltype specific manner [28]. Genome-wide analysis of transcription factor binding sites has revealed that only a portion of the ´consensus´ site containing target genes are bound by a particular transcription factor, indicating that epigenetic regulators, inducing an open or compact chromatin architecture, allow or inhibit the access of stimulus-responsive transcription factors to their target genes.

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LEGENDS FOR FIGURES FIGURE 1 Stimulus-responsive transcription factors and their delayed response genes Stimulation of the cells with extracellular signaling molecules induces the activation of stimulus-responsive transcription factors via activating of transcription factor expression (Egr-1, c-Fos, c-Jun), phosphorylation (ATF2, CREB, c-Fos, c-Jun, Nrf2), or nuclear translocation (Nrf2). These transcription factors bind to their delayed response genes, often in a tissue-specifc fashion. The gene products of these delayed response genes are then responsible for the stimulus-induced alterations in cellular biochemistry and physiology. Shut-off devices, including phosphatases (MAP kinase phosphatase-1 (MKP-1), calcineurin), negative cofactors (NAB1/2), and cytoplasmic inhibitors (Keap1), reduce the transcriptional activity of stimulus-responsive transcription factors (reproduced with modifications from ref. [18] with permission from the Nature Publishing Group). FIGURE 2 Resveratrol stimulation upregulates AP-1 activity, induces the biosynthesis of c-Jun and c-Fos, and increases the transcriptional activation potential of ATF2. (A) Schematic representation of the modular structure of the bZIP proteins c-Jun, c-Fos, and ATF2. These proteins contain phosphorylation-regulated activation domains as indicated. (B) Resveratrol increases cellular AP-1 activity. AP-1 transcriptional activity was measured in resveratrol-treated HEK293 and HepG2 cells with a collagenase promoter/luciferase reporter gene that was implanted into the chromatin of the cells to ensure that the reporter gene was embedded into a nucleosomal architecture. Cells were stimulated with resveratrol (20 M) for 24 hours. Cell extracts were prepared and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration (, P  0.001). (C) Induction of c-Jun and c-Fos biosynthesis in resveratrol-stimulated HEK293 cells. Nuclear extracts of resveratrol-treated HEK293 cells and control cells were prepared and subjected to Western blot analysis using an antibody directed against either c-Jun or c-Fos. The antibody directed against HDAC1 was used as a loading control. (D) Upregulation of the transcriptional activation potential of ATF2 in resveratrol-stimulated HEK293 cells (, P  0.001) (reproduced with modifications from ref. [47] and [52] with permission from the John Wiley and Sons Publisher). FIGURE 3

28 Resveratrol stimulates transcription via the cyclic AMP response element and upregulates the transcriptional activation potential of CREB. (A) Modular structure of CREB. The bZIP domain is C-terminal. There are two constitutively active glutamine-rich activation domains (Q1 and Q2). The kinase-inducible domain (KID) is the phosphorylation-regulated transcriptional activation domain. (B) HEK293 cells were infected with a recombinant lentivirus containing a reporter gene under the control of 4 copies the cAMP response element (CRE). Cells were stimulated with resveratrol (20 M) for 24 hours. Cell extracts were prepared and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration (, P  0.001). (C) Resveratrol stimulation increases the transcriptional activation potential of CREB in HEK293 and C17.2 neural stem cells. Serum-starved cells were stimulated with resveratrol (20 M) for 24 hours. Cell extracts were prepared and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration (, P  0.001) (reproduced with modifications from ref. [52] with permission from the John Wiley and Sons Publisher). FIGURE 4 Resveratrol upregulates Egr-1 expression and activity. (A) Schematic representation of the modular structure of Egr-1. (B) Egr-1 expression is regulated by resveratrol in HEK293 cells. Serum-starved HEK293 cells were stimulated with resveratrol (20 M) or EGF (10 ng/ml) as indicated. Nuclear proteins were isolated and analyzed using an antibody directed against Egr-1. As a loading control, an antibody against histone deacetylase-1 (HDAC1) was used. (C) Resveratrol stimulation increases Egr-1 activity in HEK293 cells. HEK293 cells were infected with a recombinant lentivirus encoding an Egr-1-responsive reporter gene. Serum-starved cells were stimulated with either resveratrol (20 M) for 24 hours. Cell extracts were prepared and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration (, P  0.001) (reproduced with modifications from ref. [73] with permission from Elsevier Inc.). FIGURE 5 Resveratrol upregulates the transcriptional activation potential of the ternary complex factor Elk-1 and stimulates Elk-1 regulated transcription. (A) The serum-response element (SRE) consists of the binding site for a SRF dimer and an adjacent Elk-1 binding site. (B) Modular structure of Elk-1. (C) Upregulation of the

29 transcriptional activation potential of Elk-1 in resveratrol-stimulated HEK293 cells. Cells were serum-starved for twenty-four hours and then stimulated with resveratrol (20 M) for 24 hours. Cell extracts were prepared and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration (, P  0.001). (D) Resveratrol stimulation induces transcription of an SRE-controlled reporter gene. Cell extracts were prepared and analyzed for luciferase activities. Luciferase activity was normalized to the protein concentration. (, P  0.001) (reproduced with modifications from ref. [73] with permission from Elsevier Inc.). FIGURE 6 Modular structure and signaling of Nrf2 (A) Schematic representation of the modular structure of the bZIP protein Nrf2. The transactivation domain, the bZIP DNA binding and heterodimerization domain, and the interaction site with Keap1 are depicted. (B) Schematic representation of the modular structure of Keap1. The binding sites for Cul3 and Nrf2 are depicted. (C) Schematic representation of the intracellular signaling pathway regulating stress/antioxidant/electrophile response element-regulated transcription. Keap1 retains Nrf2 in the cytoplasm, and regulates degradation of Nrf2 via recruiting Cul3 ubiquitin ligase to the Keap1-Nrf2 complex. Oxidative stress induces the oxidation or modification of critical cysteine residues of Keap1, resulting in an attenuation of Nrf2 degradation via the proteasome. Nrf2 translocates into the nucleus, heterodimerizes with other bZIP proteins, mainly the small Maf proteins, and activates transcription of genes that have stress/antioxidant/electrophile response elements in their regulatory regions. The DNA binding of Nrf2 is restricted by Bach1, a transcriptional repressor. Resveratrol treatment has been suggested to interfere with the Keap1/Nrf2 interaction.

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