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Piceatannol Inhibits TNF-Induced NF-B Activation and NF-B-Mediated Gene Expression Through Suppression of IB Kinase and p65 Phosphorylation¹

Cytokine Research Laboratory, Department of Bioimmunotherapy, University of Texas, M. D. Anderson Cancer Center, Houston, TX 77030

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Piceatannol is an anti-inflammatory, immunomodulatory, and anti-proliferative stilbene that has been shown to interfere with the cytokine signaling pathway. Previously, we have shown that resveratrol suppresses the activation of the nuclear transcription factor NF-B. Piceatannol, previously reported as a selective inhibitor of protein tyrosine kinase Syk, is structurally homologous to resveratrol. Whether piceatannol can also suppress NF-B activation was investigated. The treatment of human myeloid cells with piceatannol suppressed TNF-induced DNA binding activity of NF-B. In contrast, stilbene or rhaponticin (another analog of piceatannol) had no effect, suggesting the critical role of hydroxyl groups. The effect of piceatannol was not restricted to myeloid cells, as TNF-induced NF-B activation was also suppressed in lymphocyte and epithelial cells. Piceatannol also inhibited NF-B activated by H₂O₂, PMA, LPS, okadaic acid, and ceramide. Piceatannol abrogated the expression of TNF-induced NF-B-dependent reporter gene and of matrix metalloprotease-9, cyclooxygenase-2, and cyclin D1. When examined for the mechanism, we found that piceatannol inhibited TNF-induced IB phosphorylation, p65 phosphorylation, p65 nuclear translocation, and IB kinase activation, but had no significant effect on IB degradation. Piceatannol inhibited NF-B in cells with deleted Syk, indicating the lack of involvement of this kinase. Overall, our results clearly demonstrate that hydroxyl groups of stilbenes are critical and that piceatannol, a tetrahydroxystilbene, suppresses NF-B activation induced by various inflammatory agents through inhibition of IB kinase and p65 phosphorylation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Trans-3,4',5-trihydroxystilbene (commonly called resveratrol), primarily derived from red grapes, exhibits anti-inflammatory activity, anti-platelet activity, and anti-proliferative activity; inhibits the expression of cyclooxygenase-2 (COX-2)³ and lipid peroxidation; and suppresses tumor initiation, promotion, and metastasis (for references, see Ref. 1). Naturally occurring hydroxystilbenes consist of two benzene rings connected through olefin (see Fig. 1). Piceatannol is a trans-3,4,3',5'-tetrahydroxystilbene first isolated from the seeds of Euphorbia lagascae (2). This stilbene has been shown to be a potent inducer of apoptosis in lymphoma cells and in primary leukemic lymphoblasts derived from childhood acute lymphoblastic leukemia patients through activation of caspase-3 (3). Other studies suggested that piceatannol is a potent inhibitor of protein tyrosine kinases (p56^lck and Syk) with an inhibitory constant (K_i) of 15 µM (4, 5) and of the following serine/threonine protein kinases: the catalytic subunit of the cAMP-dependent protein kinase, phospholipid-dependent protein kinase C, Ca²⁺-calmodulin-dependent myosin light chain kinase, and Ca²⁺-dependent protein kinase with K_i values of 3, 8, 12, and 19 µM, respectively (6). Piceatannol has also been shown to block FcR1-mediated signaling in mast cells through the suppression of Syk activity (7). Furthermore, piceatannol has been shown to suppress LPS-induced inducible NO synthase (iNOS) induction in macrophages (8). Inhibition of STAT3 and STAT3 phosphorylation activated by IFNs has been found to be suppressed by piceatannol (9). Thus, this stilbene exhibits anti-proliferative and anti-inflammatory effects that are most likely mediated through the suppression of protein kinases.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Structure of stilbene, rhaponticin, resveratrol, and piceatannol.

Suppression of activation of the nuclear transcription factor NF-B may explain several of the effects of piceatannol. A multisubunit factor known to play a role in inflammation and immune modulation (12), NF-B is primarily composed of proteins with molecular masses of 50 kDa (p50) and 65 kDa (p65) and is retained in the cytoplasm by an inhibitory subunit, IB. In its unstimulated form, NF-B is activated by a wide variety of inflammatory stimuli, including TNF, IL-1, okadaic acid, PMA, H₂O₂, ceramide, endotoxin, and gamma irradiation. Most of these agents induce the phosphorylation-dependent degradation of IB proteins, allowing active NF-B to translocate to the nucleus, where it regulates gene expression. The activation of NF-B has been shown to mediate inflammation and suppress apoptosis. Activated NF-B has been found in various inflammatory diseases, including rheumatoid arthritis, septic shock, and myocardial ischemia (13, 14, 15, 16, 17).

For several reasons we postulated that piceatannol would suppress NF-B activation induced by various inflammatory agents. First, piceatannol induces apoptosis (2, 3) and inhibits iNOS expression (8), both known to be regulated by NF-B. Second, activation of NF-B by certain inflammatory agents requires activation of p56^lck protein tyrosine kinase (18, 19, 20, 21), and piceatannol is known to suppress p56^lck (4), suggesting that this stilbene may suppress NF-B activation. Third, recently we (22) and others (23) have shown that resveratrol, a trihydroxystilbene, suppresses NF-B activation induced by a variety of inflammatory agents, suggesting that piceatannol, a tetrahydroxystilbene, may also mediate its effects through suppression of NF-B. Rhaponticin is a dihydroxylated stilbene that has anti-inflammatory activity and inhibits LPS-induced NO production, both regulated by NF-B (8, 10, 11). Thus, in the present report we investigated whether piceatannol, rhaponticin, and stilbene (lacking hydroxyl groups) suppress the NF-B activation induced by various inflammatory stimuli and whether they do so in different cell types. We also attempted to identify the pathway employed to suppress NF-B activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

Piceatannol and resveratrol were purchased from Calbiochem (San Diego, CA.). Trans-stilbene and rhaponticin were obtained from Sigma-Aldrich (St. Louis, MO). They were dissolved in ethanol as a 10-mM stock solution and stored at 4°C. Bacteria-derived human rTNF, purified to homogeneity with a sp. act. of 5 x 10⁷ U/mg, was provided by Genentech (South San Francisco, CA). Penicillin, streptomycin, RPMI 1640 medium, IMEM, and FBS were obtained from Life Technologies (Grand Island, NY). Tris, glycine, NaCl, SDS, BSA, LPS, and PMA were obtained from Sigma-Aldrich. We used the following polyclonal Abs: anti-p65, against the epitope corresponding to amino acids mapping within the amino-terminal domain of human NF-B p65; anti-p50, against a peptide 15 aa long mapping at the nuclear localization sequence region of NF-B p50; anti-IB-, against aa 297–317 mapping at the C terminus of IB-/MAD-3; and anti-c-Rel and anti-cyclin D1 against aa 1–295, which represents full-length cyclin D1 of human origin. All were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Phospho-IB (Ser³²) Ab was purchased from New England Biolabs (Beverly, MA). Anti-COX-2 and anti-matrix metalloprotease-9 (anti-MMP-9) Abs were purchased from Transduction Laboratories (now called BD Biosciences, Lexington, KY) and Cell Sciences (Norwood, MA), respectively. Anti-IB kinase (anti-IKK) and anti-IKK Abs were provided by Imgenex (San Diego, CA). Polyclonal Ab that recognizes the serine 529 phosphorylated form of p65 was obtained from Rockland Laboratories (Gilbertsville, PA).

Cell lines

For most experiments we used the leukemic cell line KBM-5, which is phenotypically myeloid with monocytic differentiation. The other cell lines used in this study were 293 (human embryonic kidney), p56^lck-deficient Jurkat T cells (JCaM1) cells, MCF-7 and HeLa (human epithelial cells), and H1299 (non-small cell lung carcinoma) cells, obtained from American Type Culture Collection (Manassas, VA). HeLa cells were maintained in MEM, and the other cell lines were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin.

NF-B activation assay

To determine NF-B activation, we conducted EMSA essentially as previously described (24). Briefly, nuclear extracts prepared from TNF-treated cells (2 x 10⁶/ml) were incubated with ³²P end-labeled 45-mer double-stranded NF-B oligonucleotide (4 µg of protein with 16 fmol of DNA) from the HIV long terminal repeat, 5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' (underline indicates NF-B binding sites) for 15 min at 37°C, and the DNA-protein complex formed was separated from free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5'-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3', was used to examine the specificity of binding of NF-B to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotide. For supershift assays, nuclear extracts prepared from TNF-treated cells were incubated with Abs against either p50 or p65 of NF-B for 30 min at room temperature before the complex was analyzed by EMSA. Abs against cyclin D1 and preimmune serum were included as negative controls. The dried gels were visualized, and radioactive bands were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) with ImageQuant software.

Oct-1 and cAMP response element binding protein (CREB) binding

The effect of piceatannol on the binding of Oct-1 and CREB was determined by incubating 8 µg of nuclear extracts with 16 fmol of ³²P end labeled with either the octamer-binding protein (Oct-1) consensus oligonucleotide 5'-TGTCGAATGCAAATCACTAGAA-3' (underline indicates Oct-1 binding site) or the CREB consensus oligonucleotide 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3' for 15 min at 37°C and was analyzed using 6% native polyacrylamide gel. Visualization and quantitation of radioactive bands were performed as indicated above.

Degradation of IB

To determine the levels of IB, postnuclear (cytoplasmic) extracts were prepared (25) from TNF-treated cells and resolved on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were electrotransferred to nitrocellulose filters, probed with rabbit polyclonal Abs against IB, and detected by chemiluminescence (ECL, Amersham, Arlington Heights, IL). The bands obtained were quantitated with Personal Densitometer Scan version 1.30 using ImageQuant software version 3.3 (Molecular Dynamics).

IB phosphorylation

To determine the effect of piceatannol on IB phosphorylation, cytoplasmic extracts were prepared from KBM-5 cells (2 x 10⁶ cells/ml) treated with 50 µM piceatannol for 8 h and then treated with 0.1 nM TNF for the indicated times. The extracts were resolved on 10% SDS-PAGE and analyzed by Western blot using Ab against phosphorylated IB. After electrophoresis, the proteins were detected by chemiluminescence (Amersham).

IKK assay

The IKK assay was performed by a method described previously (26). Briefly, IKK complex from cytoplasm was precipitated with Ab to IKK and IKK, followed by treatment with 20 µl of protein A/G-Sepharose (Pierce, Rockford, IL). After 2 h the beads were washed with lysis buffer and then assayed in kinase assay mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl₂, 2 mM DTT, 20 µCi [-³²P]ATP, 10 µM unlabeled ATP, and 2 µg of substrate GST-IB_1–54. After incubation at 30°C for 30 min, the reaction was terminated by boiling with 5 µl of 5x SDS sample buffer for 5 min. Finally, the protein was resolved on 10% polyacrylamide gel under reducing conditions, the gel was dried, and the radioactive bands were visualized by PhosphorImager. To determine the total amounts of IKK and IKK in each sample, 30 µg of the cytoplasmic protein was resolved on a 7.5% acrylamide gel and then electrotransferred to a nitrocellulose membrane; the membrane was blocked with 5% nonfat milk protein for 1 h and then incubated with either anti-IKK or anti-IKK (1/1000 dilution) for 1 h. The membrane was washed and treated with HRP-conjugated secondary anti-mouse IgG Ab and was finally detected by chemiluminescence (Amersham).

NF-B-dependent reporter gene transcription

The effect of piceatannol on TNF-induced NF-B-dependent reporter gene transcription was measured as previously described (27). Briefly, human embryonic 293 cells (0.5 million cells/well) were plated in six-well plates and transiently transfected the next day by the calcium phosphate method with pNF-B-secretory alkaline phosphatase (SEAP; 0.5 µg). To examine TNF-induced reporter gene expression, we transfected the cells with 0.5 µg of the SEAP expression plasmid and 2 µg of the control plasmid pCMVFLAG1 DNA for 18 h. Thereafter, cells were treated for 8 h with 50 µM piceatannol and then with TNF. The cell culture medium was harvested after 24 h of TNF treatment and analyzed for SEAP activity essentially according to the protocol described by the manufacturer (Clontech, Palo Alto, CA) using a 96-well fluorescence plate reader (Fluoroscan II; Labsystems, Chicago, IL) with excitation set at 360 nm and emission set at 460 nm.

Immunocytochemistry for NF-B p65 localization

TNF-treated cells were plated on a glass slide by centrifugation using a cytospin 4 (Thermoshendon, Pittsburgh, PA), air-dried for 1 h at room temperature, and fixed with cold acetone. After a brief washing in PBS, slides were blocked with 5% normal goat serum for 1 h and then incubated with rabbit polyclonal anti-human p65 Ab (dilution, 1/100). After overnight incubation, the slides were washed and then incubated with goat anti-rabbit IgG-Alexa 594 (1/100) for 1 h and counterstained for nuclei with Hoechst (50 ng/ml) for 5 min. Stained slides were mounted with mounting medium (Sigma-Aldrich) and were analyzed under an epifluorescence microscope (Labophot-2; Nikon, Tokyo, Japan). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) and MetaMorph version 4.6.5 software (Universal Imaging, Downingtown PA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study we examined the effect of piceatannol on NF-B activation induced by TNF and various other inflammatory stimuli. We also compared the effect of piceatannol with those of stilbene, resveratrol, and rhaponticin (see Fig. 1). For most studies KBM-5 cells were used because they express both types of TNF receptors. The concentrations of the various stilbenes used and their times of exposure had no effect on the viability of these cells (data not shown).

Hydroxyl groups of stilbene are required for suppression of TNF-induced NF-B activity

To investigate the role of hydroxyl groups, we examined the effects of stilbene (no hydroxyl group), rhaponticin (dihydroxy), resveratrol (trihydroxy), and piceatannol (tetrahydroxy) on TNF-induced NF-B activation. As shown in Fig. 2, both resveratrol and piceatannol at 50 µM blocked TNF-induced NF-B activation, but stilbene under these conditions had no effect (Fig. 2A). Rhaponticin also did not inhibit TNF-induced NF-B activation even at 200 µM (Fig. 2B). These results indicate that hydroxyl groups of piceatannol are critical for its activity.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. A, Effect of stilbene, resveratrol, and piceatannol on TNF-induced NF-B activation. KBM-5 cells (2 x 106/ml) were pretreated with stilbene (50 µM), resveratrol (50 µM), and piceatannol (50 µM) at 37°C for 8 h, then activated with TNF (0.1 nM) for 30 min and assayed for NF-B activation as described. B, Effect of rhaponticin on TNF-induced NF-B activation. KBM-5 cells (2 x 106/ml) were pretreated with different concentrations of rhaponticin at 37°C for 8 h, then activated with TNF (0.1 nM) for 30 min and assayed for NF-B activation as described in Materials and Methods.

Piceatannol inhibits TNF-dependent NF-B activation in a dose- and time-dependent manner

KBM-5 cells were preincubated for 8 h with different concentrations of piceatannol and treated with TNF (0.1 nM) for 30 min at 37°C, and then nuclear extracts were prepared and assayed for NF-B activation by EMSA. As shown in Fig. 3A, piceatannol inhibited TNF-mediated NF-B activation in a dose-dependent manner, with maximum inhibition occurring at 50 µM. Piceatannol by itself did not activate NF-B. We next tested the length of incubation required for piceatannol to block TNF-induced NF-B activation. The cells were preincubated with piceatannol for different times before the addition of TNF and then were treated with TNF for 30 min. Only when the cells were pretreated for 8 h with piceatannol (50 µM) was maximum inhibition of NF-B activation observed (Fig. 3B).

View larger version (65K): [in this window] [in a new window]   FIGURE 3. Piceatannol suppresses TNF-dependent NF-B activation. The effect of piceatannol on inhibition of TNF-dependent NF-B activation. A, KBM-5 cells (2 x 106/ml) were preincubated at 37°C for 8 h with different concentrations of piceatannol, followed by 30-min incubation with 0.1 nM TNF. After these treatments, nuclear extracts were prepared and then assayed for NF-B, as described in Materials and Methods. B, Cells were preincubated at 37°C with 50 µM piceatannol for the indicated times and then tested for NF-B activation after treatment with or without 0.1 nM TNF at 37°C for 30 min. After treatment, nuclear extracts were prepared and assayed for NF-B.

View larger version (51K): [in this window] [in a new window]   FIGURE 4. A, NF-B consists of p50 and p65 subunits, and its binding to DNA is specific. Nuclear extracts were prepared from untreated or TNF (0.1 nM)-treated KBM-5 cells (2 x 106/ml), incubated for 30 min with different Abs or unlabeled NF-B oligo probes, and then assayed for NF-B by EMSA. B and C, Piceatannol has no effect on Oct-1 and CREB in KBM-5 cells. KBM-5 cells (2 x 106/ml) were preincubated at 37°C for 8 h with different concentrations of piceatannol. After these treatments, nuclear extracts were prepared and assayed for Oct-1 (B) and CREB (C), as described in Materials and Methods.

Inhibition of NF-B activation by piceatannol is not cell type specific

That distinct signal transduction pathways could mediate NF-B induction in epithelial and lymphoid cells has been demonstrated (28). All the effects of piceatannol described to date were observed in the human monocytic cell line KBM-5, but we also wanted to establish whether piceatannol could block TNF-induced NF-B activation in lymphoid cells (Jurkat), breast adenocarcinoma cells (MCF-7), and epithelial (HeLa) cells. These cells were pretreated with different concentrations of piceatannol for 8 h, and NF-B was activated by treatment with TNF for 30 min. Piceatannol inhibited most TNF-induced NF-B activation in all cell types (Fig. 5).

View larger version (34K): [in this window] [in a new window]   FIGURE 5. Piceatannol suppresses TNF-induced NF-B in different cell types. Jurkat (A), MCF-7 (B), and HeLa (C) cells (2 x 106/ml)) were preincubated at 37°C for 8 h with or without 50 µM piceatannol and then treated with 0.1 nM TNF for 30 min. Nuclear extracts were prepared and tested for NF-B activation as described in Materials and Methods.

Piceatannol blocks NF-B activation induced by PMA, LPS, H₂O₂, okadaic acid, and ceramide

Besides TNF, NF-B is also activated by a wide variety of other agents, including PMA, LPS, H₂O₂, okadaic acid, and ceramide. However, the signal transduction pathways induced by these agents probably differ (29). We therefore examined the effect of piceatannol on the activation of NF-B by these agents. As shown in Fig. 6, piceatannol suppressed the activation of NF-B induced by all these agents, suggesting that the piceatannol acts at a step in the NF-B activation pathway that is common to all these agents.

View larger version (47K): [in this window] [in a new window]   FIGURE 6. Piceatannol blocks NF-B activation induced by PMA, LPS, H2O2, okadaic acid, and ceramide. KBM-5 cells (2 x 106/ml) were preincubated for 8 h at 37°C with piceatannol (50 µM); treated with TNF (0.1 nM), LPS (10 µg/ml), PMA (100 ng/ml), H2O2 (250 µM), okadaic acid (500 nM), or ceramide (10 µM) for 30 min; and tested for NF-B activation as described in Materials and Methods.

Piceatannol represses TNF-induced NF-B-dependent reporter gene expression

Although we have shown by EMSA that piceatannol blocks NF-B activation, DNA binding alone does not always correlate with NF-B-dependent gene transcription, suggesting that there are additional regulatory steps (30). To determine the effect of piceatannol on TNF-induced NF-B-dependent reporter gene expression, we transiently transfected piceatannol-pretreated or untreated cells with the NF-B-regulated SEAP reporter construct and then stimulated the cells with TNF. An almost 8-fold increase in SEAP activity over the vector control was noted upon stimulation with 1 nM TNF (Fig. 7A). Most TNF-induced SEAP activity was abolished by dominant negative IB, indicating specificity (data not shown). When the cells were pretreated with piceatannol, TNF-induced NF-B-dependent SEAP expression induced by either 0.1 or 1 nM TNF was inhibited. These results demonstrate that piceatannol also represses NF-B-dependent reporter gene expression induced by TNF.

View larger version (48K): [in this window] [in a new window]   FIGURE 7. A, Piceatannol inhibits TNF-induced NF-B-dependent reporter gene (SEAP) expression. 293 cells treated with 50 µM piceatannol were transiently transfected with an NF-B-containing plasmid linked to the SEAP gene. After 24 h in culture with 0.1 or 1 nM TNF, cell supernatants were collected and assayed for SEAP activity as described in Materials and Methods. Results are expressed as fold activity (actual values are indicated on top of the bars) over the nontransfected control value. B, Piceatannol inhibits TNF-induced expression of COX-2, MMP-9, and cyclin D1. H-1299 cells (2 x 106/ml) were treated with in the absence or the presence of piceatannol (50 µM) for 8 h, then treated with TNF (0.1 nM) for different times. Whole-cell extracts were prepared, and 80 µg of the whole cell lysate was analyzed by Western blot using Abs against COX-2, MMP-9, or cyclin D1.

Whether piceatannol also affects NF-B-dependent expression of COX-2, MMP-9, and cyclin D1 was investigated. To determine this, H1299 cells were pretreated with piceatannol for 8 h, then treated with TNF for different periods; whole-cell extracts were prepared and analyzed by Western blot for the expression of COX-2, MMP-9, and cyclin D1 (Fig. 7B). TNF-induced the expression of COX-2, MMP-9, and cyclin D1 in a time-dependent manner, and piceatannol completely blocked TNF-induced expression of all three gene products. These results further suggest that piceatannol inhibits TNF-induced NF-B-dependent gene expression.

Piceatannol inhibits TNF-dependent IB phosphorylation

The translocation of NF-B to the nucleus is preceded by the phosphorylation, ubiquitination, and proteolytic degradation of IB (12). To determine whether inhibition of TNF-induced NF-B activation was due to inhibition of IB degradation, we pretreated cells with piceatannol for 8 h, and then exposed them to 0.1 nM TNF for different times. We examined the cells for NF-B in the nucleus by EMSA and for IB in the cytoplasm by Western blot. As shown in Fig. 8A, TNF activated NF-B in the control cells in a time-dependent manner, but had little effect on piceatannol-pretreated cells. TNF induced IB degradation in control cells as early as 10 min, but in piceatannol-pretreated cells TNF-induced IB degradation was unaffected (Fig. 8B).

View larger version (51K): [in this window] [in a new window]   FIGURE 8. A and B, Effects of piceatannol on TNF-induced NF-B activation (A) and degradation of IB (B). KBM-5 cells were incubated at 37°C with piceatannol (50 µM) for 8 h, then treated with 0.1 nM TNF at 37°C for different times as indicated and tested for NF-B activation by EMSA (A) and for IB in cytosolic fractions by Western blot analysis (B). Equal protein loading was evaluated by -actin. C, Piceatannol inhibits TNF-induced phosphorylation of IB. KBM-5 cells (2 x 106/ml) were incubated first with piceatannol (50 µM) for 8 h, and then treated with 100 µg/ml ALLN for 1 h. Thereafter, cells were treated with TNF (0.1 nM) for different times and analyzed by Western blot using Abs against phosphorylated IB. D, Piceatannol inhibits TNF-induced phosphorylation of p65. KBM-5 cells (2 x 106/ml) were incubated first with piceatannol (50 µM) for 8 h and then treated with 100 µg/ml ALLN for 1 h. Thereafter, cells were treated with TNF (0.1 nM) for different times and analyzed by Western blot using Abs against the phosphorylated form of p65.

Piceatannol inhibits TNF-induced phosphorylation of p65

TNF has been shown to induce the phosphorylation of p65, which is required for its translocation to the nucleus. Therefore, we also tested the effect of piceatannol on TNF-induced phosphorylation of p65. As shown in Fig. 8D, TNF induced the phosphorylation of p65 in a time-dependent manner, and piceatannol treatment suppressed p65 phosphorylation almost completely.

Piceatannol inhibits TNF-induced nuclear translocation of p65

Following phosphorylation TNF has been shown to induce the nuclear translocation of the p65 subunit. Therefore, we also tested the effect of piceatannol on TNF-induced nuclear translocation of p65 by immunocytochemistry. As shown in Fig. 9, TNF induced the nuclear translocation of p65, and piceatannol treatment abrogated p65 translocation.

View larger version (80K): [in this window] [in a new window]   FIGURE 9. Piceatannol inhibits TNF-induced nuclear translocation of p65. KBM-5 cells (1 x 106/ml) were untreated or were pretreated for 8 h with 50 µM piceatannol at 37°C, then treated with 1 nM TNF. After cytospin, immunocytochemistry was performed as described in the text.

It has been shown that IKK is required not only for TNF-induced phosphorylation of IB, but also for phosphorylation of p65 (32). Since piceatannol inhibits the phosphorylation of both IB and p65, we also tested the effect of piceatannol on TNF-induced IKK activation. As shown in Fig. 10, in immune complex kinase assays TNF activated IKK, and the activation could be seen as early as 2 min after TNF treatment. Piceatannol treatment almost completely suppressed TNF-induced activation of IKK.

View larger version (52K): [in this window] [in a new window]   FIGURE 10. Piceatannol inhibits TNF-induced IKK activity. KBM-5 cells (2 x 106 cells/ml) were treated with piceatannol (50 µM) for 8 h and then activated with TNF (0.1 nM) for different time intervals. A, Cytoplasmic extracts (CE) were prepared, and 200 µg of CE were immunoprecipitated with Abs against IKK and IKK. Thereafter an immune complex kinase assay was performed as described in Materials and Methods. To examine the effect of piceatannol on the level of expression of IKK proteins, 30 µg of CE protein was run on a 7.5% SDS-PAGE, electrotransferred, and immunoblotted with the indicated Abs.

Piceatannol suppresses TNF-mediated NF-B activation in Syk-deleted cells

Piceatannol has been shown to inhibit p56^lck and Syk protein tyrosine kinases (7). To determine whether these kinases play a role in the NF-B suppression activity of piceatannol, we used a Jurkat cell line (JCaM-1) that expresses neither p56^lck nor Syk kinases (33). TNF activated NF-B in these cells, and piceatannol suppressed TNF-induced NF-B activation in a dose-dependent manner (Fig. 5A). These results clearly suggest that Syk and p56^lck play no role in TNF-induced NF-B activation and that piceatannol does not suppress NF-B activation through inhibition of these kinases.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Piceatannol is an anti-inflammatory, immunomodulatory, and anti-proliferative stilbene that has been shown to interfere with the cytokine signaling pathway. Due to the central role of NF-B in the effects assigned to piceatannol and due to its strong structural homology to resveratrol, another NF-B blocker, we postulated that piceatannol must suppress NF-B activation. Our results indeed demonstrate that piceatannol suppresses the DNA binding activity of NF-B induced by TNF and various other inflammatory agents in a variety of cell types. Piceatannol also suppressed NF-B-dependent reporter gene expression, IB phosphorylation, p65 phosphorylation, and IKK activation. The suppression of NF-B activation by piceatannol was not due to inhibition of Syk protein kinase, and the hydroxyl groups in piceatannol were critical for its activity.

Our results indicate that piceatannol, a tetrahydroxystilbene, behaves similarly to resveratrol, a trihydroxystilbene, with respect to its ability to suppress NF-B activation. We found that stilbene, which lacks hydroxyl groups, had no effect on NF-B, suggesting that hydroxyl groups are essential. Rhaponticin, a dihydroxy stilbene, did not suppress NF-B activation. The position of the hydroxyl groups may also play an important role in the suppression of NF-B.

Our results demonstrate that piceatannol suppresses TNF-induced NF-B activation, as monitored by DNA binding and reporter gene expression. These results are similar to those reported for resveratrol by us and others (22, 23).

In investigating how piceatannol suppresses TNF-induced NF-B, we found that piceatannol inhibited TNF-induced IB phosphorylation and activation of IKK.

We found that piceatannol inhibits TNF-induced phosphorylation of p65 subunit of NF-B. Recently it has been shown that the phosphorylation and acetylation of p65 play a major role in DNA binding and trans-activation of NF-B (34, 35, 36). Mesalamine has been shown to block IL-1-induced NF-B-dependent reporter activity through suppression of p65 phosphorylation (34). How piceatannol inhibits p65 phosphorylation is not clear. Because IKK has been shown to phosphorylate p65 (32, 36), it is possible that piceatannol inhibits p65 phosphorylation through inhibition of IKK. The latter was indeed inhibited by piceatannol in our studies. Resveratrol is also known to suppress the phosphorylation of p65 (22), but whether this is through IKK is not fully understood.

Previous reports indicate that piceatannol can suppress signaling mediated through FcR (7) and through STAT3 and STAT5 (9). Suppression of phosphorylation has been suggested as the major mechanism by which piceatannol mediates its effects. In particular, piceatannol has been shown to block protein tyrosine phosphorylation. Tyrosine phosphorylation has been implicated in NF-B activation induced by a wide variety of agents, including pervanadate, ceramide, HIV-tat, erythropoietin, TNF, nerve growth factor, and H₂O₂ (18, 19, 20, 37, 38, 39, 40). We found that besides TNF, piceatannol can abrogate NF-B activation induced by various other inflammatory agents, including PMA, LPS, H₂O₂, okadaic acid, and ceramide. Because the signal transduction pathway induced by these agents has been shown to differ (18, 19, 34), it suggests that piceatannol must act at a step in the NF-B activation pathway common to all these agents. Our results, however, indicate that the effects of piceatannol in our system are not mediated though inhibition of tyrosine phosphorylation, as the JCaM1 cell line that lacks both the Syk and p56^lck protein kinases (33) was fully functional in NF-B activation. We also found that the effects of piceatannol were specific to NF-B, as other transcription factors (Oct-1 or CREB) were not affected. Why cells need to be exposed to piceatannol for 8 h to observe optimum suppression of NF-B is not clear. It is possible that piceatannol regulates the expression of certain genes, which then subsequently inhibit NF-B activation. Such possibilities require further investigation.

We found that suppression of DNA binding of NF-B protein does lead to inhibition of NF-B-mediated reporter gene expression by piceatannol. In addition, we found that piceatannol blocked TNF-induced expression of COX2, MMP-9, and cyclin D1, all the genes known to be regulated through NF-B (for references, see Refs. 12 and 13). Piceatannol has also been reported to inhibit iNOS expression (8, 11), a gene whose expression is regulated by NF-B (41, 42). Thus suppression of iNOS by piceatannol could be due to inhibition of NF-B activation. Additionally there are genes involved in tumor cell proliferation that are also regulated by NF-B (12, 13). The inhibition of expression of these NF-B-regulated genes may explain the antitumor effects that were previously described (2, 3). Overall, our results suggest that piceatannol is an effective inhibitor of NF-B, which may explain its immunomodulatory, anti-inflammatory, and anti-proliferative effects.

	Acknowledgments

 
We thank Walter Pagel for a careful review of the manuscript.

	Footnotes

 
¹ This work was supported by The Clayton Foundation

² Address correspondence and reprint requests to Dr. Bharat B. Aggarwal, Cytokine Research Laboratory, Department of Bioimmunotherapy, University of Texas, M. D. Anderson Cancer Center, Box 143, 1515 Holcombe Boulevard, Houston, TX 77030. E-mail address: aggarwal{at}mdanderson.org

³ Abbreviations used in this paper: COX-2, cyclooxygenase-2; ALLN, N-acetyl leucyl leucyl nonleucinal; CREB, cAMP response element binding protein; IB, inhibitory subunit of NF-B; IKK, IB kinase; iNOS, inducible NO synthase; MMP-9, matrix metalloprotease 9; SEAP, secretory alkaline phosphatase; K_i, inhibitory constant; Oct-1, octamer-binding protein-1.

Received for publication August 5, 2002. Accepted for publication October 2, 2002.

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