Piceatannol upregulates endothelial heme oxygenase-1 expression via novel protein kinase C and tyrosine kinase pathways

Pharmacological Research 53 (2006) 113–122

Piceatannol upregulates endothelial heme oxygenase-1 expression via novel protein kinase C and tyrosine kinase pathways Being-Sun Wung a,∗ , Ming-Chun Hsu b , Chun-Ching Wu b , Chia-Wen Hsieh a a

Department of Applied Microbiology, National Chiayi University, No. 300, Shiuefu Road, Chiayi, Taiwan b Institute of Biotechnology, National Chiayi University, Chiayi, Taiwan Accepted 22 September 2005

Abstract Piceatannol is an anti-inflammatory and anti-proliferative plant-derived stilbene. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme to activate by various phytochemicals. In this study, we examined the ability of piceatannol to upregulate HO-1 expression in endothelial cells. We found piceatannol at micromolar (10–50 ␮M) concentrations dramatically increased HO-1 protein levels in a time-dependent manner. Piceatannol was similarly potent in the induction of HO-1 as hemin, arsenate, and 15d-PGJ2, and was more potent than some other phytochemicals including curcumin, EGCG, baicalein, and quercetin. In contrast, the similar chemical structure compounds, trans-stilbene, stilbene oxide, and resveratrol had no HO-1-inducing effects, suggesting a critical role for the hydroxyl groups in HO-1 induction. No cytotoxicity and superoxide production was observed after 10–50 ␮M piceatannol treatments. Piceatannol-mediated HO-1 induction was abrogated in the presence of N-acetylcysteine and glutathione, but not by other antioxidants. Induction of HO-1 by piceatannol was further enhanced by using buthionine sulfoximine. In addition, we determined that tyrosine kinase was involved in the induction of HO-1 by using tyrosine kinase inhibitors, herbimycin A, erbstatin, and genistein; in contrast, no significant changes in the pretreatment of PI3 kinase or MAP kinase inhibitors was determined. HO-1 induction was blocked by the protein kinase C inhibitors calphostin C, rottlerin, and long PMA pretreatment, whereas conventional PKC inhibitors, Go6976, and Ca2+ chelator BAPTA/AM, had no effect. Elevated HO-1 protein levels were associated with the inhibition of tumor necrosis factor-␣ (TNF␣)-induced intercellular adhesion molecule-1 (ICAM-1) expression. Treating ECs with zinc protoporphyrin, an HO-1 inhibito blocked the anti-inflammatory effect of piceatannol. In summary, this study identified piceatannol as a novel phytochemical inducer of HO-1 expression and identified the mechanisms involved in this process. © 2005 Elsevier Ltd. All rights reserved. Keywords: Endothelial cells; Heme oxygenase-1; Piceatannol; Protein kinase C

1. Introduction Oxidative stress can lead to endothelial dysfunction and is associated with the development of cardiovascular diseases such as hypertension and atherosclerosis [1]. Increased intracellular reactive oxygen species (ROS) levels induce redox-sensitive genes that may be involved in such diseases involving vascular dysfunction [2]. Hence, mechanisms that protect the endothelium from oxidative injury are likely to play a key role in maintaining the integrity of the vascular bed. Plant-derived phenols exhibit strong antioxidant properties due to Michael reaction acceptor function. Recent studies suggest that plant-derived chemical substances could be used for

∗

Corresponding author. Tel.: +886 5 271 7784; fax: +886 5 271 7780. E-mail address: [email protected] (B.-S. Wung).

1043-6618/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2005.09.006

therapeutic intervention in several disease states, including inflammation, atherosclerosis, and cancer. In particular, some polyphenolic antioxidants, such as curcumin [3] and resveratrol [4] act as non-stressful and non-cytotoxic inducers of the response protein heme oxygenase-1 (HO-1) and can maximize the intrinsic antioxidant potential of cells. First isolated from the seeds of Euphorbia lagascae, piceatannol is a trans-3,4,3 ,5 -tetrahydroxystilbene that is structure homologous to resveratrol [5]. Our previous data found resveratrol suppresses IL-6-induced ICAM-1 gene expression via inhibition of STAT3 phosphorylation [6]. In addition, piceatannol can suppress lipopolysaccharide-induced inducible NO synthase (iNOS) induction [7] and interferon-induced STAT3 phosphorylation [8]. Owing to these properties, piceatannol exhibits anti-inflammatory effects. HO-1 is a critical protein in the response to oxidative injury whose main function is associated with the degradation of heme

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to biliverdin, iron, and carbon monoxide [9]. Several pathological states, including hypoxia, atherosclerosis, and inflammation, are accompanied by an overexpression of the HO-1 gene and increased HO-1 enzyme activity [10–12]. Recent studies have demonstrated that HO-1 functions as part of a cytoprotective mechanism derived from the antioxidant activities [13] and has anti-inflammatory [14], anti-proliferative [15], and antiapoptotic properties [16]. These primary physiological functions of HO-1 have been identified through observations of HO-1-overexpressed cultured cells and in HO-1 knockout mice [17,18]. Many stress-induced antioxidant proteins, including HO-1, glutathione S-transferase, glutamylcysteine synthetase, and phase II detoxifying enzymes, are regulated at the same transcriptional level by consensus sequences for the antioxidant response element (ARE) found in their promoter regions [19]. Thus, in light of the cytoprotective role of HO-1, the specific activation of HO-1 gene expression by pharmacological modulation may represent a novel target for therapeutic treatment of vascular diseases. The electrophile-induced signaling mechanisms used to activate HO-1 expression are poorly understood. Several studies have focused on the activation of the mitogen-activated protein kinases (MAPKs) that contribute to the induction of HO-1 [20–22] report that carnosol, a phytochemical derived from the rosemary herb, increases HO-1 protein via the activation of tyrosine kinase PI3 kinase/Akt cascade pathways. On the other hand, electrophile-induced glutathione (GSH) depletion could induce HO-1 expression via protein kinase C (PKC) pathways [23]. For these reasons, we used specific pharmacological inhibitors to test signaling pathways in order to elucidate the important components in piceatannol-mediated HO-1 expression. Thus, in the current study we not only demonstrate for the first time that piceatannol induces HO-1 expression, but we also identify its activation signaling pathways. Our results indicate that piceatannol activates HO-1 expression probably in a novel protein kinase C (nPKC)- and tyrosine kinase-dependent manner. In addition to its cytoprotective nature, elevated HO-1 protein levels were associated with the inhibition of TNF␣-induced ICAM-1 expression. These results provide new insights into the anti-inflammatory mechanisms of piceatannol. 2. Materials and methods 2.1. Materials Piceatannol (trans-3,4,3 ,5 -tetrahydroxystilbene), PD98059, SB203580, SP600125, LY294002, Go6976, actinomycin D, cycloheximin, herbimycin, erbstatin, calphostin-C, rottlerin, and genistein were purchased from Calbiochem (San Diego, CA, USA). 15-Deoxy-
12,14 -prostaglandin J2 , was purchased from Cayman Chemical (Ann Arbor, MI, USA). ECL reagents were purchased from Pierce (Rockford, IL, USA). HO-1 primary antibody was obtained from Stressgen Biotechnologies (SB, San Diego, CA). Antibody against ICAM-1 was purchased from Zymed (San Francisco, CA, USA). Peroxidase-conjugated anti-rabbit and anti-mouse antibodies were purchased from Amersham (Arlington Heights, IL, USA). Nitrocellulose was

obtained from Schleicher & Schuell (Dassel, Germany). Zinc protoporphyrin (ZnPP) was obtained from Frontier Scientific Inc. All other reagents, including resveratrol (3,5,4-trihydroxytrans-stilbene), stilbene, trans-stilbenes, stilbene oxides, and quercetin (3,3,4,5,7-pentahydroxyflavone) were purchased from Sigma (St. Louis, MO, USA). 2.2. Endothelial cell cultures Bovine aortic endothelial cells (BAECs) were cultured in Dulbecco modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U mL−1 penicillin, and 100 ␮g mL−1 streptomycin. Cells were kept at 37 ◦ C in a humidified atmosphere of air and 5% CO2 . Cells were grown in Petri dishes for 3 days and allowed to reach confluence [24]. The culture medium was then replaced with serum-free DMEM and the cells were incubated for 12 h prior to experimental treatments. 2.3. Western blotting A total of 1 × 106 cells were lysed on ice in lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor mixture) and whole-cell extracts were boiled for 5 min prior to separation on 10% SDS–PAGE, in which protein samples were evenly loaded. The proteins were then transferred to a nitrocellulose filter (Millipore) in Tris–glycine buffer at 100 V for 1.5 h. Membranes were blocked with PBS buffer containing 5% non-fat milk and incubated with HO-1 antibodies for 2 h at 4 ◦ C, with gentle shaking. The results were visualized by chemiluminescence using ECL (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Relative protein levels were determined by scanning densitometry analysis using the Alphainnotech Image software. Values given below each panel in figures represent the mean (in relative units), normalized to control values, of at least three experiments performed with similar results. 2.4. RT-PCR analysis of HO-1 mRNA levels Total cellular RNA was extracted using the phenol– guanidinium isothiocyanate method [24]. Equal amounts (5 ␮g) of RNA from the different treatments were reverse-transcribed for 50 min at 42 ◦ C using 50 units of superScript II (Invitrogen, Carlsbad, CA, USA). Amplification of cDNA was performed in 25 ␮L of PCR buffer (10 mM Tris–HCl, 50 mM KCl, 5 mM MgCl2 , and 0.1% Triton X-100, pH 9.0) containing 0.6 units of Taq DNA polymerase (Promega, Madison, WI, USA) and 30 pmol of synthetic specific primers for bovine HO-1 (size of PCR product, 575 bp): sense 5 -CAA GGA GGT GCA CAC GG-3 ; antisense 5 -GCT GGA TGT TGA GCA GGA AGG-3 or GAPDH (size of PCR product, 470 bp): sense 5 -ATG GGC GTG AAC CAC GAG AA-3 ; antisense 5 -TCG CTG TTG AAG TCG CAG GA-3 . The first cDNA synthesis was performed following the manufacturer’s instructions. PCR was performed after a 10-min denaturation at 94 ◦ C, and repeating the cycles of 94, 55, and 72 ◦ C each for 40 s; the number of cycles was

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specific for each primer set. PCR products were electrophoresed in a 1.5% agarose gel containing ethidium bromide.

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ences between groups were considered to be significant at p < 0.05.

2.5. Cell viability assay 3. Results Cell viability was performed by an Alamar blue assay according to the manufacturer’s instructions (Serotec, Oxford, UK) [3]. The assay is based on detection of metabolic activity of living cells using a redox indicator that changes from oxidized (blue) to reduced (red) form. The intensity of the red color is proportional to the viability of cells, which is calculated as the difference in absorbance between 570 and 600 nm and expressed as percentage of control. 2.6. Chemiluminescence assay of superoxide production Superoxide levels were measured by lucigenin-amplified chemiluminescence. BAECs were lysed with a buffer containing lucigenin (200 ␮mol L−1 ) as previously described [25]. Measurements were initiated upon addition of lysis buffer and recorded with a luminometer (Berthord, Pforzheim, Germany). 2.7. Statistical analysis Differences among the groups were analyzed using the t-test. Values were expressed as mean ± S.E.M., and differ-

3.1. Piceatannol induction of HO-1 in ECs To test whether piceatannol could induce the antioxidant enzyme HO-1, ECs were treated with various concentrations of piceatannol for 3, 6, 12, and 24 h. As shown in Fig. 1A and B, HO-1 protein increased with time at a concentration of 10 ␮mol L−1 . The induction of HO-1 by piceatannol was evident as early as 3 h, and the augmentation lasted for at least 12 h and then decreased after 24 h treatment. To further investigate piceatannol-mediated HO-1 induction, confluent ECs were co-treated with piceatannol and actinomycin D (AD), a transcriptional inhibitor, or cycloheximin (CHX), a general translational inhibitor. Both AD and CHX completely blocked HO-1 induction (Fig. 1C). To further study the effect of piceatannol on induction of HO-1 expression in transcriptional level, ECs were treated with 10 ␮M piceatannol for 1, 3, 6, 12, or 24 h. RTPCR (Fig. 1D) indicated that piceatannol induced an increase in HO-1 mRNA at 1 h and last for 24 h. These results suggest that piceatannol-mediated HO-1 induction may require de novo RNA and protein synthesis.

Fig. 1. Piceatannol induction of HO-1 in ECs. (A) EC cultures were incubated with 10 ␮M piceatannol treatment for 3, 6, 12, and 24 h or with 25 ␮M piceatannol treatment for 3, 6, and 12 h. Cell lysates (30 ␮g) were prepared and subjected to Western blot analysis with antibodies against HO-1 or tubulin as indicated. The tubulin band intensities indicate equal loading of each well. (B) EC cultures were incubated with 25 ␮M piceatannol treatment for 6, 12, and 24 h or with 50 ␮M piceatannol treatment for 3, 6, 12, and 24 h. Cell lysates were then subjected to Western blot analysis. (C) EC cultures were pretreated with 2 ␮g mL−1 actinomycin D (AD) and 3.5 ␮M cycloheximin (CHX) for 30 min, prior to a 6 h piceatannol (10 ␮M) treatment, and analyzed by Western blotting. (D) EC cultures were incubated with 10 ␮M piceatannol for 1, 3, 6, 12, or 24 h and analyzed by RT-PCR.

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Fig. 2. Piceatannol is a potent inducer of HO-1. Chemical structure of piceatannol, resveratrol, trans-stilbenes, and stilbene oxides share a common basic structure characterized by two carbons bound to two aromatic rings. The side chains on the aromatic rings diversify the compounds and influence their ability to induce heme oxygenase. (A) EC cultures were incubated with 15d-PGJ2 (10 ␮M), hemin (10 ␮M), sodium arsenite (10 ␮M), curcumin (15 ␮M), EGCG (50 ␮M), baicalein (100 ␮M), and piceatannol (10 ␮M) for 6 h. Cell lysates were then subjected to Western blot analysis. (B) EC cultures were incubated with trans-stilbene treatment (5, 25, and 50 ␮M) for 6 h, or piceatannol treatment (1, 5, 10, and 25 ␮M) for 6 h, and analyzed by Western blotting. (C) EC cultures were incubated with stilbene oxide treatment (5, 25, and 50 ␮M) for 6 h or piceatannol treatment (1, 5, 10, and 25 ␮M) for 6 h, and analyzed by Western blotting. (D) EC cultures were incubated with piceatannol (25 and 50 ␮M), resveratrol (25 and 50 ␮M), and quercetin (25 and 50 ␮M) for 12 h, and analyzed by Western blotting.

3.2. Piceatannol is a potent inducer of HO-1 To assess relative potency of piceatannol to induce HO-1, we compared its induction capacity with other known inducers, including 15d-PGJ2 [26], hemin [27], sodium arsenite [20], curcumin [10], epigallocatechin-3-gallate (EGCG), and baicalein [28]. As shown in Fig. 2A, piceatannol was the most potent HO-1 inducer among the phytochemicals and was comparable in potency to the other highly potent inducers 15d-PGJ2 , hemin, and sodium arsenite. In various cell types, many stilbene compounds are known to be inducers of HO-1. Oguro et al. [29] demonstrated that trans-stilbenes and stilbene oxides increased HO-1 activity in rat livers. We wondered whether the structure of different stilbenes would affect the induction of HO-1 in ECs. To test this, we compared the performance of piceatannol to various stilbenes at different concentrations using a Western blot assay. Interestingly, trans-stilbene and stilbene oxide had little effect on HO-1 expression after incubation for 6 h (Fig. 2B and C).

In another study, a hydroxy-stilbene known as resveratrol was shown to induce HO-1 expression in smooth muscle cells [4], and Lin et al. [28] found that quercetin, a flavonoid compound, was a HO-1 inducer in macrophages. When compared with piceatannol, resveratrol and quercetin had little effect on HO-1 induction in ECs (Fig. 2D). These results indicate that the structure of piceatannol is critical for its HO-1 induction activity. 3.3. Endothelial cell viability and superoxide production after exposure to piceatannol Considering those stressful events such as sodium arsenite, H2 O2 are usually responsible for HO-1 upregulation [30], we assessed the viability of endothelial cells after 12 h incubation with 5–50 ␮M concentration of piceatannol. No significant difference has been found between treated and non-treated ECs (Fig. 3A). HO-1 expression has been shown to become upregulated in response to cellular stress or changes in the intracellu-

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Fig. 3. Endothelial cell viability and superoxide production after exposure to piceatannol. (A) Endothelial cells were incubated piceatannol treatment (5, 10, 25, and 50 ␮M) for 12 h, and cell viability was measured spectrophotometrically using an Alamar blue assay according to the manufacturer’s instructions. Data are expressed as the mean ± S.E.M. of five independent experiments. * P < 0.05 vs. control (0 ␮M). (B) ECs subjected to sodium arsenite (10 ␮M) and piceatannol (10, 25, and 50 ␮M) for 30 min. ECs were lysed and immediately followed with superoxide measurements using the lucigenin-amplified chemiluminescence method. Results are shown as mean ± S.E.M. from at least three separate experiments. * P < 0.05 vs. control ECs.

lar redox status in ECs [10]. Upon reaction of a Michael acceptor with a nucleophile, a stable product will be formed. This product could undergo phenol oxidation and successively give rise to thiyl radicals, which would propagate the oxidative stress reactions. Several reports have described the stimulating effects of polyphenolic compounds exert its electrophilic reactivity to enhance the oxidative stress, which could induce HO-1 expression [22,31] To investigate this with respect to piceatannol, we measured the intracellular ROS levels of superoxides in ECs before and after treatment with piceatannol for 1 h using lucigenin-amplified chemiluminescence. We did not detect any significant increase of intracellular ROS levels with treatment of 10–50 ␮M piceatannol (Fig. 3B).

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Fig. 4. The effects of antioxidants on piceatannol induction of HO-1. (A) Western blot analysis of ECs pretreated, where indicated, with catalase (1000 U mL−1 ), NAC (20 mM), glutathione (GSH, 20 mM), or superoxide dismutase (SOD, 500 U mL−1 ) for 30 min, then incubated with 10 ␮M piceatannol for 6 h. (B) ECs were pretreated with the NADPH oxidase inhibitors apocynine (Apo, 100 ␮M); the xanthine oxidase inhibitor, oxypurinol (Oxy, 100 ␮M) and allopurinol (Allo, 1 mM); the mitochondrial complex I inhibitor, rotenone (Rot, 5 ␮M); the hydroxyl radical scavenger, 1,3-dimethylthio-2-thiourea (DMTU, 5 mM); and the ferrous iron chelator, o-phenanthroline (Phe, 20 ␮M) followed by 10 ␮M piceatannol for 6 h. Western blot analysis was then performed. (C) ECs were pretreated with buthionine sulfoximine (BSO) 100 ␮M for 4 and 6 h or 10 mM for 2, 4, and 6 h, followed by 5 ␮M piceatannol for 6 h. Western blot analysis was then performed.

3.4. Effects of antioxidants on piceatannol induction of HO-1 To further rule out the role of oxidative stress in piceatannolinduced HO-1, we used various antioxidants to identify the effects of piceatannol on HO-1 induction. ECs were treated with the antioxidants catalase, NAC, reduced glutathione (GSH), or super oxide dismutase (SOD) prior to the addition of piceatannol to the medium. We observed that although 20 mM NAC and GSH inhibited piceatannol-induced HO-1 expression, there was no measurable inhibiting effect following pretreatment with catalase or SOD (Fig. 4A). ECs were also tested with pretreatments of the NADPH oxidase inhibitor, apocynine; the xanthine oxidase inhibitors, oxypurinol and allopurinol; the mitochondrial complex I inhibitor, rotenone; the hydroxyl radical scavenger, 1,3-dimethylthio-2-thiourea (DMTU); and the ferrous iron chelator, o-phenanthroline, followed by piceatannol treatment for 30 min. Except for NAC and GSH, none of the tested antioxidants were found to inhibit piceatannol-induced HO-1 expression (Fig. 4B). Together, these results suggest

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that piceatannol-induced HO-1 expression occurs via a GSHdependent pathway and that this is not due to any concomitant decrease in intracellular ROS levels. Intracellular GSH levels may lead to the abrogation of electrophilic reactivity by conjugation. Thus, to assess the role of GSH in HO-1 induction, ECs were treated with increasing concentrations of buthionine sulfoximine (BSO), a specific inhibitor of ␥-glutamyl cysteine synthetase used to reduce intracellular GSH levels, before piceatannol treatment for 6 h. As shown in Fig. 4C, the low concentration of piceatannol (5 ␮mol L−1 ) induced HO-1 expression and this was further enhanced by treatment with BSO. These data indicate that piceatannol-induced HO-1 expression is independent of oxidative stress, but dependent on cellular GSH level. 3.5. Piceatannol-induced HO-1 expression mediated by tyrosine kinase In order to evaluate the role of the tyrosine kinases in HO-1 induction, a pharmacological approach was utilized by pretreating ECs with three protein tyrosine kinase inhibitors, herbimycin A, erbstatin, and genistein. As shown in Fig. 5A, these tyrosine kinase inhibitors attenuated the piceatannol-induced HO-1 expression in a concentration-dependent manner. Martin et al. [22] reported that carnosol increases HO-1 expression via the activation of tyrosine kinase PI3 kinase/Akt cascade pathways. Thus, we tested two PI3-kinase inhibitors, wortmannin and LY294002 in piceatannol-treated ECs. Cells were pretreated with several concentrations of LY294002 and incubated with 10 ␮M piceatannol for 6 h. Fig. 5B illustrates that neither PI3-kinase inhibitor was able to block piceatannol induction of HO-1. Previous studies have demonstrated that the activation of the MAPKs pathway, which can be activated by tyrosine kinase pathways, contributes to the induction of HO-1 [20,21]. We tested whether piceatannol-induced HO-1 expression occurs through the MAPKs pathway by using the three specific pharmacological inhibitors, PD098059, an inhibitor of MEK1/2, the upstream activator of extracellular signal-regulated kinases 1 and 2 (ERK1/2); SB203580, p38 MAPK inhibitor; and SP600125, a JNK inhibitor. As can be seen in Fig. 5C, when these reagents were added at different concentrations to cultures prior to treatment with piceatannol, there was no effect on HO-1 induction. These results indicate that the ERK, p38, and JNK pathways do not participate in the induction of HO-1 in ECs, pointing instead to the involvement of other tyrosine kinase pathways. 3.6. Piceatannol-induced HO-1 expression mediated by novel protein kinase C (nPKC) To examine whether piceatannol induces the expression of HO-1 through the PKC pathway, we tested induction using specific PKC inhibitors. As shown in Fig. 6A, Calphostin C, an inhibitor of conventional PKC (cPKC) and novel PKC (nPKC), and rottlerin, a nPKC-selective inhibitor, both inhibited piceatannol-induced HO-1 expression in a concentration-

Fig. 5. Piceatannol-induced HO-1 expression mediated by tyrosine kinase. (A) Western blot analysis of ECs pretreated with protein tyrosine kinase inhibitors, erbstatin (Erb, 10 and 50 ␮M), herbimycin A (Herb, 1 and 5 ␮M), or genistein (Gen, 50 and 100 ␮M) for 30 min, then incubated with 10 ␮M piceatannol for 6 h. (B) ECs were pretreated with PI3-kinase inhibitors, wortmannin (Wor, 1 and 5 ␮M) and LY294002 (LY, 5 and 25 ␮M) followed by 10 ␮M piceatannol for 6 h. Western blot analysis was then performed. (C) ECs were pretreated with the MEK1/2 inhibitor PD098059 (PD, 25 and 50 ␮M), a p38 MAP kinase inhibitor SB203580 (SB, 10 and 25 ␮M), and a JNK inhibitor SP600125 (SP, 10 and 25 ␮M) followed by 10 ␮M piceatannol for 6 h. Western blot analysis was then performed.

dependent manner. In contrast, GO6976, a cPKC isozymespecific inhibitor, had no significant effect. The activation of cPKC and nPKC requires the second messenger diacylglycerol (DAG). To identify whether piceatannolinduced HO-1 expression requires DAG by activating the PKC pathways, ECs were pretreated with a DAG analogue, phorbol 12-myristate-13-acetate (PMA) for 6 or 24 h to cause desensitization of PKC, showing a significant inhibition in PMA pretreatment of 24 h (Fig. 6B). Because intracellular calcium is required for cPKC activation, we tested cells exposed to piceatannol plus two concentrations of BAPTA/AM, a membrane-permeable Ca2+ chelator. As seen in Fig. 6C, there was no significant inhibition of piceatannol-induced HO-1 expression, which further rules out the involvement of cPKC. Furthermore, the calcium ionophore A23187 was unable to induce HO-1 expression (Fig. 6B). These data indicate that piceatannol-induced HO-1 expression is mediated via the nPKC-dependent pathway.

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Fig. 7. Protective effects of piceatannol against TNF␣-induced ICAM-1 expression. (A) Western blot analysis of ECs pretreated with10 ␮M piceatannol for 1, 3, and 6 h, then incubated with or without 100 U mL−1 TNF␣ for 6 h. (B) Cells were preincubated with 10 ␮M piceatannol with or without ZnPP (10 ␮M) for 6 h, and then treated with 100 U mL−1 TNF␣ for 6 h. Cell lysates were examined by Western blotting. Fig. 6. Piceatannol-induced HO-1 expression mediated by novel protein kinase C. (A) Western blot analysis of ECs pretreated with the cPKC and nPKC inhibitor, calphostin C (Cal, 0.1 and 0.5 ␮M); the nPKC-selective inhibitor, rottlerin (Rot, 1 and 10 ␮M); or the cPKC isozymes-specific inhibitor, GO6976 (GO, 0.1 and 5 ␮M) for 30 min, then incubated with 10 ␮M piceatannol for 6 h. (B) ECs were pretreated with PMA (100 ng mL−1 ) for 6 and 24 h, followed by 10 ␮M piceatannol for 6 h. In some of the reactions, EC cultures were incubated only with the calcium ionophore A23187 (10 or 25 ␮M) for 6 h. Western blot analysis was then performed. (C) ECs were pretreated with calcium chelator BAPTA/AM (0.5 and 10 ␮M) followed by 10 ␮M piceatannol for 6 h. Western blot analysis was then performed.

3.7. Protective effects of piceatannol against TNFα-induced ICAM-1 expression Previous studies have shown that endothelial HO-1 induction modulates ICAM-1, an inducible cell adhesion glycoprotein [32,33]. We have previously shown that ICAM-1 is upregulated when ECs are exposed to various inflammatory cytokines, including TNF␣ [34]. We tested whether piceatannolinduced HO-1 expression facilitates this anti-inflammatory effect. Piceatannol prevented TNF␣-induced ICAM-1 expression in ECs and at the same time piceatannol-induced HO1 expression increased (Fig. 7A). To further demonstrate the anti-inflammatory effect mediated by HO-1, ECs treated with piceatannol plus zinc protoporphyrin (ZnPP), an HO-1 competitive inhibitor, abolished the preventive effect of piceatannol on ICAM-1 induction (Fig. 7B). These data indicate that

piceatannol-induced HO-1 expression may be responsible for anti-inflammatory effects. 4. Discussion HO-1 can be upregulated by several plant constituents, including curcumin [3], baicalein [28], quercetin [28], and carnosol [22]. In this study, we identified piceatannol as a HO-1 protein expression enhancer in ECs and have linked its activation mechanism to nPKC and tyrosine kinase-dependent pathways. We demonstrated that piceatannol-induced HO-1 expression occurred in a time-dependent manner, and that it is a more potent HO-1 inducer than several other commercially available, highpurity phytochemicals including curcumin, EGCG, baicalein, quercetin, resveratrol, and some stilbene compounds. This relatively high-induction activity may indicate differing activation pathways of the phytochemical inducers. In addition, by using pharmacological inhibitors, we have demonstrated the involvement of the nPKC and tyrosine kinase pathways in the induction process. In various cell types, many stilbene compounds are known to be inducers of HO-1 [4,29]. Our results show that the pattern of inducibility differed for piceatannol, resveratrol, trans-stilbene, and stilbene oxide, indicating that even subtle changes of the chemical structure can significantly affect the potency to upregulate HO-1. As shown in Fig. 2B and C, when compared with

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same concentration of piceatannol, resveratrol, trans-stilbene, and stilbene oxide had little effect on HO-1 induction in ECs. We found that stilbene and stilbene oxide, which lacks hydroxyl groups suggesting that hydroxyl groups are essential. Resveratrol, a tetrahydroxystilbene, also showed less effect in HO-1 induction. Thus, the position of the hydroxyl groups may also play an important role in the HO-1 induction. Recent reports have shown that some polyphenolic compounds, including resveratrol, EGCG, and cyanidin-3-glucoside activate endothelial nitric oxide synthase and then increase nitric oxide (NO) release from vascular endothelial cells [35–37]. NO has been found to increase HO-1 induction in various cell types [16], but whether NO is involved in piceatannol-induced HO1 expression remained unclear. Thus, we pretreated ECs with an endothelial NO synthase antagonist, nitro-l-arginine methyl ester (l-NAME) to rule out this possibility. l-NAME was unable to block HO-1 induction in piceatannol-treated ECs (data not shown). In some stress-related conditions, stimulation of HO-1 is directly associated with a change in intracellular ROS levels [38,34]. The substrate heme, which functions as a native inducer of HO-1, increases HO-1 expression by alteration of the cellular redox state. An imbalance in the redox state after a challenge with oxidants including ultraviolet A radiation [39], hypoxia [10], arsenite [39], as well as with NO [16] is known to promote activation of the HO-1 system in various cell types. Our data indicate that piceatannol is as highly potent in the induction of HO-1 as some of the stress-related inducers, such as sodium arsenite and heme. In addition, some studies have found that polyphenolic compounds can increase oxidative stress by electrophilic reactivity [22,31]. However, in this study, we did not find evidence for oxidative stress in piceatannol-treated ECs. The transcription factor Nrf2 plays an essential role in the ARE-mediated expression of phase II detoxifying and antioxidant enzymes and in the activation of other stressinducible genes in response to electrophilic stress [40,41]. These Nrf2 responsive antioxidant proteins include GCL, GSH-Stransferase (GST), quinone reductase (NQO1), and HO-1 [42]. Piceatannol is a highly electrophilic compound and a potent inducer of HO-1 expression. Whether piceatannol-induced HO1 expression is mediated by Nrf2 translocation needs to be further determined. We did not find significant inhibition in the induction of HO-1 by pretreatment with various antioxidants, except when testing with NAC and GSH. The suggestion that nucleophiles, such as GSH, can react with electrophilic compounds and block their biological activity is also corroborated by our data showing that piceatannol-induced HO-1 expression can be primarily regulated by GSH and thiol antioxidants. These results are consistent with the notion that activation of the HO-1 gene by electrophiles (such as acrolein, NO, and 15d-PGJ2 ) can be effectively abolished in the presence of thiol compounds [43–45]. However, Scapagnini et al. [46] found that the polyphenolic compounds, caffeic acid phenethyl ester- and curcumin-induced HO-1 expression failed to be blocked by NAC. The specific chemical structure may play a crucial role in preferential affinity toward selective cysteine residues of targeted proteins that

control the gene expression. Thus, we suggest that the ability of piceatannol to stimulate HO-1 expression is not defined by oxidative stress, but by its propensity to react with signal transduction protein. Our results suggest that the activation of tyrosine kinase pathways is involved in piceatannol-induced HO-1 expression. Martin et al. [22] reported that carnosol increases HO-1 expression via activation of tyrosine kinase PI3 kinase/Akt cascade pathways and several other studies on the regulation of HO-1 expression have focused on the role of the MAPKs pathways, which can be activated by tyrosine kinase pathways. Oxidative stress stimuli, including sodium arsenite, increases HO-1 protein in cell cultures via activation of the MAPK cascade pathways [20]. However, piceatannol-treated ECs pretreated with either PI3-kinase inhibitors or MAPK inhibitors did not exhibit blocked HO-1 induction, suggesting that the piceatannolactivated tyrosine kinase pathways are not ubiquitous. In this study, we demonstrated the involvement of the nPKC but not the cPKC and aPKC pathway in piceatannolmediated HO-1 induction. Evidence in support of the nPKC pathway was provided by several findings. The cPKC and nPKC inhibitor, calphostin C, and the nPKC selective inhibitor, rottlerin, inhibited piceatannol-induced HO-1 expression in a concentration-dependent manner. However, a specific cPKC inhibitor, Go6976, had no significant effect. Additionally, the investigation of DAG required for HO-1 induction is demonstrated by pretreatment with DAG analogue PMA to desensitize PKC activity. Our study also revealed that cells incubated in the membrane-permeable calcium chelator BAPTA/AM or the calcium ionophore, A23187, showed unaltered HO-1 expression, which further rules out the involvement of cPKC because intracellular calcium is required for cPKC activation. Together, these results indicate that piceatannol-induced HO-1 expression is mediated via the nPKC-dependent but cPKC- and aPKCindependent pathway. A previous study found that PKC isoforms in ECs cover PKC␣, PKC-␦, PKC-␧, and PKC-␰ [47]. Only the isoforms PKC-␦ and PKC-␧ belong to a Ca2+ -independent nPKC family. The specific PKC isoform involved in HO-1 induction requires further investigation. Nevertheless, we have observed that there is no induction of HO-1 in PMA treated alone, even though it is a powerful PKC-activating agent. Consequently, PKC activation in isolation is not sufficient for HO-1 gene induction. Although the key factors of signal transduction mechanisms and the specific chemical target required for induction of HO-1 remain to be fully identified, the piceatannol-induced enzyme can be regarded as a potential therapeutic target in a variety of oxidant- and inflammation-mediated vascular diseases. In this respect, the search for novel and more potent inducers of HO1 will facilitate the development of new applications for the prevention or treatment of cardiovascular diseases.

Acknowledgment This work was supported in part by grant from the National Science Council, Taiwan.

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