Valproic Acid–Induced Gene Expression Through Production of Reactive Oxygen Species Yumiko Kawai and Ifeanyi J

Research Article

Valproic Acid–Induced Gene Expression through Production of Reactive Oxygen Species Yumiko Kawai and Ifeanyi J. Arinze

Department of Biomedical Sciences, Meharry Medical College, Nashville, Tennessee

Abstract (HC toxin), also exhibit antiproliferative properties, an effect that Valproic acid (VPA) is a widely used anticonvulsive agent that has garnered increasing attention for HDAC inhibitors in cancer has profound antiproliferative effects in many cell types, as research (4, 7, 14–16). well as inductive effects on a number of genes. The mechanism Although not much has been published on the molecular of its gene-inducing effect has been reported to involve mechanism of the various actions of VPA, the few reports that VPA transcription factors, Sp1and activator protein-1.Using increases the expression of genes regulated by the transcription two well-characterized antioxidant response element (ARE)– factor, activator protein-1 (AP-1;refs. 17, 18), seem to be the basis driven gene promoters, i.e., mouse heme oxygenase-1and for the conclusion that the molecular mechanism of VPA-induced human NAD(P)H:quinone oxidoreductase 1genes as tools to gene expression is via DNA-binding activity of AP-1 transcription monitor the transcriptional response to VPA, we show here factors to the AP-1 response element (5, 19). We have shown that a that VPA-induced gene transcription was abrogated by the G i2 gene promoter, which does not contain the canonical AP-1-binding motif, could be transactivated by VPA through antioxidants. With the human GAi2 gene promoter, which was previously used to establish the involvement of Sp1in the Sp1-binding sites in the promoter (11). Mutations at the relevant transcriptional action of VPA, we found that VPA-induced Sp1-binding sites did not completely abolish VPA-induced gene transcription was also blocked by antioxidants. Mutation promoter activity, suggesting that there might be other mecha- of the ARE (5¶-TGACtggGC-3¶) in this promoter abrogated the nisms contributing to VPA-induced gene transcription. transcriptional response to VPA. With such mutants, the VPA (20–22) and other class I/II HDAC inhibitors, i.e., NADPH oxidase inhibitor, diphenyleneiodonium, had no effect suberoylanilide hydroxamic acid (SAHA;ref. 23), MS-275 (24, 25), on VPA-induced transcription. In gel mobility shift assays, and sodium butyrate (26), have been reported to elevate cellular VPA-induced binding of nuclear proteins to a DNA probe levels of reactive oxygen species (ROS). Neural tube defects containing the relevant ARE sequence in the GAi2 gene induced by VPA are prevented by vitamin E (27), a potent promoter was decreased in nuclear extracts from cells antioxidant;this effect is consistent with the idea that ROS pretreated with antioxidants. Chromatin immunoprecipita- production is part of the biological action of VPA. Although at tion assays showed that the prototype redox-sensitive tran- high levels, ROS induce toxicity to cells, at physiologic levels, they scription factors, Nrf2, small Maf protein(s), and c-Fos, were function as part of normal cell signaling. There has been recent recruited to this promoter in VPA-treated cells. Overall, this interest in unraveling molecular mechanisms of ROS-mediated study reveals that the mechanism of the transcriptional gene expression. For example, Das et al. (28) showed that ROS response to VPA includes VPA-induced production of reactive generated by the exposure of HCT116 human colon carcinoma oxygen species which induce the activation of redox-sensitive cells to glucose oxidase resulted in the induction of a mammalian transcription factors that interact with the ARE. (Cancer Res DNA endonuclease through CRE/AP-1 binding sites in the 2006;66(13): 6563-9) promoter that binds the transcription factors c-Jun and CREB/ ATF2. Others have shown that the electrophile tert-butylhydro- quinone (tBHQ), which also elicits the generation of ROS, Introduction activates transcription through the interaction of the transcrip- Valproic acid (2-propylpentanoic acid;VPA) is widely used tion factor Nrf2 (nuclear factor erythroid 2–related factor 2) at clinically as an anticonvulsant agent for suppressing epileptic antioxidant response elements (ARE) on target gene promoters seizures as well as a mood stabilizer in the treatment of manic (for review, see refs. 29, 30). We showed recently that tBHQ- depression (bipolar affective disorder;refs. 1–3). VPA induces induced transcription from the human gene promoter for the histone acetylation (4–7), DNA demethylation (8), chromatin signal transduction protein Gai2 occurred through redox-sensitive decondensation (9), and the expression of a variety of genes transcription factor interaction with the core ARE in the (10–12). It has antiproliferative effect on cells, and is known to promoter (31). In the present study, we have used prototypical induce differentiation of cell lines derived from myeloid leukemia ARE-containing gene promoters, i.e., mouse heme oxygenase-1 (4, 7), teratocarcinoma, glioma, and neuroblastoma (13). Other gene, human NAD(P)H:quinone oxidoreductase 1 gene, as well as inhibitors of histone deacetylases (HDAC), such as sodium the human Gai2 gene promoter, as tools to show that VPA can butyrate, trichostatin A, and Helminthosporium carbonum toxin induce gene transcription through the ARE. This effect was independent of VPA-induced promoter activity that results from Sp1 interaction at Sp1-binding sites, as evident from studies with a Requests for reprints: Ifeanyi J. Arinze, Department of Biomedical Sciences, the human G i2 gene promoter. Overall, this work reveals that the Meharry Medical College, 1005 Dr. David B. Todd, Jr. Boulevard, Nashville, TN 37208- gene expression–inducing effect of VPA can be accounted for, in 3599. Phone: 615-327-6586;Fax: 615-327-6442;E-mail: [email protected]. I2006 American Association for Cancer Research. part, by the interaction of redox-sensitive transcription factors doi:10.1158/0008-5472.CAN-06-0814 with the ARE. www.aacrjournals.org 6563 Cancer Res 2006; 66: (13).July 1, 2006

Materials and Methods result in total loss of promoter activity (11). In further exploration, Cell cultures and transfection studies. K562 cells, obtained from the we have observed that antioxidants block the gene expression– American Type Culture Collection (Manassas, VA), were maintained in inducing action of VPA. For example, the classic antioxidants NAC culture as previously described (11, 32). For transfection experiments, and PDTC, which are potent scavengers of ROS (34), inhibited VPA- 5 the cells (1 Â 10 cells per well in 1 mL of medium) were seeded in induced expression of Gai2, as evidenced from the decreased level of 24-well plates;wells on the periphery of the plates were not used. After Gai2 protein, measured by Western blotting (Fig. 1). 24 hours, the cells were transfected with 0.5 Ag of plasmid DNA (containing VPA induces the generation of ROS. The inhibitory effect of the gene promoter or mutant) and 1.5 AL of FuGENE 6 reagent (Roche a antioxidants on G i2 levels suggests that the treatment with VPA Applied Science, Indianapolis, IN) 1 hour before the addition of sodium most likely resulted in the generation of ROS. We confirmed this by valproate (Sigma, St. Louis, MO) or other HDAC inhibitors. When chemical using the nonfluorescent compound, DCFDA, a cell-permeable dye inhibitors such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium (DPI), that turns into a highly fluorescent species (DCF) upon oxidation, and pyrrolidine dithiocarbamate (PDTC) were used, they were added 30 minutes or 1 hour prior to the addition of valproate or other HDAC to monitor VPA-induced changes in the cellular content of ROS. As inhibitors. The cells were harvested 20 to 24 hours later, by centrifugation seen in Fig. 2A, VPA treatment increased DCF fluorescence in a at 12,000 Â g (45 seconds) in 1.5 mL microcentrifuge tubes, and washed time-dependent manner, indicating increased ROS production in once with 1Â PBS. The cell pellets were then lysed with 150 ALof1Â these cells. The data are consistent with reports in other cell Cell Lysis Reagent (Promega, Madison, WI) and kept at room temperature systems that VPA induces the production of ROS (20–22). Other for 10 minutes. The lysed material was then centrifuged for 2 minutes at HDAC inhibitors that are of interest as cancer chemotherapeutic 12,000 Â g, and the luciferase activity and protein content of each lysate agents, i.e., SAHA (23), MS-275 (24, 25), and sodium butyrate (26) were measured as previously described (11, 31). have also been reported to increase the production of ROS, A a Immunoblotting. Using 10 g of protein, immunoblotting of G i2 in although in some cases, ROS production occurred only in whole-cell extracts was carried out as previously described (32, 33). The transformed cells (25). However, none of these studies explored blots were reprobed for h-tubulin, as a loading control. Measurement of ROS. The total content of ROS was measured by using how the ROS production results in HDAC inhibitor–induced the nonfluorescent probe, 2¶,7¶-dichlorodihydrofluorescein diacetate regulation of any gene. We addressed this question in this study. A (DCFDA), which upon oxidation, becomes a highly fluorescent species, Inhibition of class I/II HDACs induces G i2 gene promoter 2¶,7¶-dichlorofluorescein (DCF). The cells (5,000 cells in 0.5 mL/well in a activity in an antioxidant-sensitive manner. Based on the 24-well plate) were loaded with 10 Amol/L of DCFDA for 30 minutes prior to premise that two major sources of ROS generation in cells are the addition of VPA (5 mmol/L). DCF fluorescence was measured at various mitochondria and the activity of NADPH oxidase (35), we measured time points thereafter, using CytoFluor Series 4000 Fluorescence Multi-Well VPA-induced promoter activity in cells pretreated with DPI, which Plate Reader (Applied Biosystems, Inc., Foster City, CA), at excitation and is often used to implicate the activity of NADPH oxidase in various emission settings of 485 and 530 nm, respectively. processes (36, 37), but which also inhibits the production of oxygen Site-directed mutagenesis. Specific nucleotides in the Gai2 gene promoter were mutated or deleted by using QuikChange Mutagenesis kit from Stratagene, Inc., (La Jolla, CA). Mutations were confirmed by DNA sequencing at the Molecular Biology Core Facility, Meharry Medical College. Preparation of nuclear extracts and electrophoretic mobility shift assays. Nuclear extracts were prepared as described previously (11, 31). For electrophoretic mobility shift assays (EMSA), annealed 5¶-overhang oligonucleotide sequence, containing ARE-binding motif, was labeled with [a-32P]dCTP, using the Klenow fill-in reaction, and purified (33). The labeled probe was used for the EMSA reaction as described in the legend to Fig. 5. After electrophoresis, the gel was dried, and then exposed to Kodak XAR5 film at À80jC. The radiolabeled bands were detected by autoradiography. Chromatin immunoprecipitation. Chromatin immunoprecipitation (ChIP) analysis to monitor association of transcription factor(s) with the

Gai2 gene promoter in vivo was done (31), in principle, according to the protocols recommended by Upstate Biotechnology (Lake Placid, NY) and by the Farnham Laboratory (http://www/genomecenter.ucdavis.edu/ farnham/).

Results

Antioxidants inhibit VPA-induced expression of GAi2. We have previously shown that VPA increases mRNA and protein levels of the signal transduction protein Ga in K562 cells;the induction was i2 Figure 1. Antioxidants inhibit VPA-induced expression of Gai2. K562 cells were confirmed by reporter gene assay, using human Gai2 gene promoter preincubated with NAC (5 mmol/L) or PDTC (50 Amol/L) for 1 hour prior to the linked to luciferase reporter gene (11). In that study, we showed addition of VPA (5 mmol/L). The cells were harvested 24 hours later, and lysed as described under ‘‘Materials and Methods.’’ The whole-cell lysates (10 Ag that, like other HDAC inhibitors tested (i.e., sodium butyrate and protein per lane) were used for Western blotting (top)forGai2 and h-tubulin trichostatin A), the mechanism of this induction involved the (used as loading control). Using UN-SCAN-IT software (Silk Scientific, Inc., Oren, UT), band intensities for Gai2 were scanned, and normalized against the binding of the Sp family of transcription factors to three Sp1 corresponding band intensities of the loading control (h-tubulin). To calculate fold consensus sequence-binding sites in the promoter. However, the stimulation for each treatment, the normalized value obtained for the lane in possible involvement of other transcription factors binding at other the presence of VPA was divided by the normalized value obtained for the lane in the absence of VPA. For each treatment, the normalized value obtained in the response elements could not be ruled out because deletion or point absence of VPA was set as 1.0. Bottom, columns, mean; bars, FSD (n =3 mutation of the relevant Sp1-binding motifs in the promoter did not experiments); Con, appropriate controls not treated with VPA.

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production by these compounds could trigger the activation of redox-sensitive transcription factors that mediate response to oxidative stress in cells. VPA stimulates ARE-driven gene transcription. The transcription factor Nrf2, in conjunction with Keap 1 (Kelch-like ECH- associating protein 1), has been characterized to be intimately involved in redox sensing in oxidatively stressed cells (29, 30). Electrophiles, such as tBHQ, or ROS are known to promote the inactivation of the Nrf2-Keap 1 complex, leading to Nrf2 binding to ARE on target gene promoters. The ARE originally was defined as TGACnnnGC (39). This definition has been extended (TMAnnRTGAYnnnGCRwwww) to include sequences flanking the core sequence (RTGACnnnGCR;refs. 40, 41). Variants in flanking sequences have been reported (42). Mutations in the core sequence (39, 41–43) as well as in the flanking sequences (42, 43) impair the ability of the ARE to function optimally. To test the idea that the ARE is involved in VPA-induced transcription, we used two reporter gene constructs (see schematics in Fig. 3A) that are driven by well-characterized prototypical ARE, and have been previously shown to contain sufficient information to modulate Nrf2-dependent gene transcription (37, 44, 45): (a) a luciferase reporter construct of the heme oxygenase-1 gene (designated HO-1-ARE-Luc) in which three copies of the ARE sequence were cloned upstream of the mouse heme oxygenase-1 minimal promoter (37);and ( b) hNQO1-ARE-Luc [human NAD(P)H:quinone oxidoreductase 1-ARE-Luc] reporter construct that contains a single copy of ARE, derived from the human NQO1 promoter, placed upstream of a minimal promoter containing a TATA box fused to the luciferase gene (45). We found that VPA increased the ARE-driven promoter activity of HO- 1-ARE-Luc (Fig. 3B) as well as that of hNQO1-ARE-Luc (Fig. 3C). In both cases, the VPA effect was abrogated by DPI as well as by Figure 2. VPA-induced generation of ROS, and effects of DPI and PDTC on PDTC. In addition, other HDAC inhibitors tested (sodium butyrate, transcription fromthe G ai2 gene promoter induced by various HDAC inhibitors. A, detection of VPA-induced generation of ROS by DCF fluorescence. K562 HC toxin, and SAHA) also induced transcription from these cells (5,000 cells in 0.5 mL/well in 24-well plates) were loaded with 10 Amol/L of reporter gene constructs in an antioxidant-sensitive manner. These DCFDA for 30 minutes prior to the addition of VPA (5 mmol/L). DCF fluorescence results (data not shown) resembled the data obtained with the Ga was measured at various time points thereafter, up to 60 minutes, using i2 CytoFluor Series 4000 Fluorescence Multi-Well Plate Reader (Applied gene promoter, which is shown in Fig. 2B. Therefore, we confirmed Biosystems), at excitation and emission settings of 485 and 530 nm, the involvement of the ARE by extending the transcriptional respectively. VPA-induced DCF fluorescence at each time point is expressed as a the fold increase, relative to cells that were not treated with valproate (defined as analysis to the G i2 gene promoter which we have previously 1.0). Basal DCF fluorescence was relatively constant over the 60-minute shown (31) to be induced by Nrf2, and to be unresponsive to this measurement period. Points, mean for duplicate assays from four different transcription factor in the absence of the ARE sequence (5¶-TGA- experiments; bars, FSE. B, PDTC and the NADPH oxidase inhibitor, DPI, inhibit ¶ À À transcriptional response to various HDAC inhibitors (valproate, sodiumbutyrate, CtggGC-3 ) that maps at 84/ 76 in that promoter. This sequence HC toxin, and SAHA). PDTC (10 Amol/L) or DPI (10 Amol/L) was added to is exactly identical to that described as short ARE by Katsuoka et al. the cell cultures 1 hour after transfection, followed by the addition of either VPA (43), or as core sequence by Nguyen et al. (29) and was shown to be (2 mmol/L), sodium butyrate (2 mmol/L), HC toxin (40 nmol/L), or SAHA (10 Amol/L) 1 hour later. The cells were harvested 20 hours after the addition of essential for both basal and/or inducible activity (39). the HDAC inhibitors, and processed for luciferase assay as described under Using site-directed mutagenesis, we deleted the ARE in the Gai2 ‘‘Materials and Methods.’’ Promoter activity is expressed as fold stimulation, ¶ GACA ¶ relative to cells that were not treated with HDAC inhibitors. Columns, mean for gene promoter or mutated it to 5 - tggGC-3 , and then duplicate assays fromfour to six different experiments; bars, FSE. Con, compared promoter activity of the mutants to that of the wild-type appropriate controls not treated with any HDAC inhibitor. promoter, not only in cells treated with VPA but also in cells treated with tBHQ, which we used as a positive control because it radical by complex I (NADH dehydrogenase) of mitochondria (38). is known to activate Nrf2 (29), and also because it enhances the The results show that DPI (used at 10 Amol/L) inhibited VPA- Nrf2-induced transcription from this promoter (31). Additionally, f A a induced promoter activity by 80% (Fig. 2B);PDTC (10 mol/L) we monitored VPA-induced G i2 promoter activity of the mutant also inhibited VPA-induced Gai2 gene promoter activity (Fig. 2B). in cells pretreated with DPI, the rationale being that DPI, which Sensitivity of the VPA-induced gene expression to antioxidants inhibited VPA-induced promoter activity (Figs. 2B and 3B) should seen in K562 cells is not unique to transformed cells, as similar have no effect in the absence of the ARE. As seen in Fig. 4A, results were also obtained in endothelial cells isolated from mouse substitution mutation of this short ARE to 5¶-GACAtggGC-3¶ a aorta (data not shown). We found that the G i2 gene promoter- resulted in 55% loss of promoter activity. As expected, loss (65%) inducing activities of three other HDAC inhibitors (sodium of promoter activity was also evident when Sp1-binding sites butyrate, SAHA, and HC toxin) were also abrogated by DPI or were mutated (see also ref. 11). Double mutants in which both the PDTC (Fig. 2B). We speculated that the induction of ROS Sp1-binding sites and the ARE site were mutated, resulted in loss of www.aacrjournals.org 6565 Cancer Res 2006; 66: (13).July 1, 2006

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research promoter activity that was somewhat higher (f70%) than the single mutants. As expected (31), tBHQ-induced transcription was substantially decreased (88%) by deletion of the ARE site (ARE mtd), or by substitution mutation (ARE mts) at that site (75%; Fig. 4B). The latter compares favorably with results obtained with VPA (Fig. 4A), using ARE mts. Mutations at Sp1 sites had no effect on tBHQ-induced transcription (Fig. 4B), indicating that Sp1 does not mediate the effect of tBHQ. Consistent with our prediction, DPI had no effect on VPA-induced promoter activity if the promoter was mutated at the ARE (data not shown). Overall, these results

Figure 4. Mutations at ARE or Sp1-binding elements inhibit VPA-induced transcription fromthe G ai2 gene promoter. The ARE-binding site (5¶-TGACTGGGC-3¶)atÀ84/À76 in the promoter was mutated by either deletion (ARE mtd) or by substitution (ARE mts)to5¶-GACATGGGC-3¶ (31). Mutated bases are indicated in boldface, and are italicized. The Sp1 mutant is a triple mutant in which all three relevant Sp1-binding sites (À92/À85, À50/À36, and +68/+75) were mutated (11, 33). The promoter construct (0.5 Ag) was transfected 1 hour before the addition of VPA (2 mmol/L) or tBHQ (20 Amol/L), and the cells were harvested 24 hours later, and processed for luciferase assay as described under ‘‘Materials and Methods.’’ A, substitution mutations at Sp1 sites (Sp1 mt) or the ARE (ARE mts ) inhibit VPA-induced transcription fromthe Gai2 gene promoter. DMT, promoter mutated at both Sp1-binding sites and ARE-binding site. Columns, mean for duplicate assays from six different experiments; bars, FSE. B, tBHQ-induced Gai2 gene promoter activity is inhibited by the deletion of, or substitution mutations at the ARE, but not by mutation at Sp1 sites (Sp1 mt). Columns, mean for duplicate assays from four different experiments; bars, FSE. Con, appropriate control in the absence of inducer (tBHQ or VPA).

reveal that the ARE is involved in the ROS-dependent, VPA-induced transcription from this gene promoter, and that the ARE response is independent of the Sp1 response. Together with the data in Fig. 2, these studies establish an interesting mechanistic link Figure 3. VPA stimulates ARE-mediated transcription from heme oxygenase-1 between ROS production induced by VPA or by other HDAC and NAD(P)H:quinone oxidoreductase 1 gene promoters. A, schematics of ARE-luc reporters used in the study. The HO-1-ARE-Luc contains three copies inhibitors, and transcription from ARE-containing target gene of ARE (5¶-CGGACCTTGACTCAGCAGAAAA-3¶) cloned upstreamof the mouse promoter(s). To our knowledge, this is the first evidence that the HO-1 minimal (À32 to +72 bp) promoter (37). The hNQO1-ARE-Luc [human NAD(P)H:quinone oxidoreductase 1-ARE-Luc] contains a single copy of ARE, ARE is involved in the transcriptional effect of VPA on any gene derived fromthe human NQO1 promoter, placed upstream of a minimal promoter. Taken together with our previous study implicating Sp1 promoter containing a TATA box fused to the luciferase gene (37, 45). B and C, in the transcriptional action of VPA (11), and the results by others effect of DPI and PDTC on VPA-induced reporter gene activities. K562 cells (1 Â 105 cells/well) were cultured for 24 hours, and then transfected with 0.5 Ag implicating canonical AP-1 motif in AP-1 consensus sequence- of the reporter construct [HO-1-ARE-Luc (B)orhNQO1-ARE-Luc (C)] or empty containing promoters (17, 18), it seems clear that VPA-induced vectors 1 hour before the addition of PDTC (10 Amol/L) or DPI (10 Amol/L). VPA (2 mmol/L) was added 40 minutes after the addition of the inhibitors. The gene transcription involves multiple mechanisms. cells were harvested 20 hours later, and luciferase assay was carried out as Binding activity of the ARE in VPA-treated cells. Because Nrf2 described under ‘‘Materials and Methods.’’ Columns, mean for duplicate assays fromfour different experiments; bars, FSE. Con, appropriate controls not has been widely documented as the prototype redox-sensitive treated with VPA. transcription factor that binds, in association with small Maf

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Valproic Acid–Induced Gene Expression proteins or other bZIP proteins, to the ARE (29, 30, 46–49), we did primer set that bracketed a 170 bp fragment (À234/À65) EMSA as well as ChIP analysis, to show that VPA induces binding containing the ARE site (see Fig. 6A). As shown in Fig. 6B,PCR of these transcription factors to the ARE region in the Gai2 gene analysis of the target region showed that, within 1 to 2 hours of promoter. In the in vitro binding assay (i.e., EMSA), using nuclear VPA treatment, there was increased association of a variety of extracts from VPA-treated cells and a double-stranded DNA probe redox-sensitive transcription factors with the promoter;these containing the corresponding ARE sequence in the Gai2 gene included c-Fos, Nrf2, and nuclear factor-nB (NF-nB), as well as promoter, the protein-DNA complex could be clearly detected. The small Maf proteins, which are known to be heterodimer partners of binding activity increased with time after treating cells with VPA, Nrf2 on the ARE on target gene promoters (29, 49). The PCR exhibited dose dependence, and was greater in VPA-treated cells analysis also indicated the association of Sp1 with the promoter; than in control cells. Binding was competitively abolished by an this is expected because the area amplified in the ChIP analysis unlabeled oligonucleotide (Fig. 5A, compare lane 3 with lane 2,or (see Fig. 6A) contained one (À92/À85) of the Sp1-binding sites that lane 10 with lane 9), but was not affected by an unrelated we previously identified to be involved in VPA-induced transcrip- oligonucleotide (consensus Sp1 oligonucleotide;Fig. 5 A, compare tion from this promoter (ref. 11;see also Fig. 4 A). No association lane 5 with lane 2,orlane 12 with lane 9) or a mutated was detected in the nontarget region (+1099/+1347) used as oligonucleotide probe (Fig. 5A, compare lane 4 with lane 2,orlane negative control (Fig. 6B, right) or in samples treated with normal 11 with lane 9). Furthermore, pretreatment of the cells with PDTC IgG (Fig. 6B, left and right). These data complement the gel shift or NAC decreased the binding to basal levels or less (Fig. 5B). The assays, and show that redox-sensitive transcription factors as sensitivity to antioxidants supports the idea that VPA-induced well as Sp1 are recruited to the Gai2 gene promoter in vivo during binding to this element required the activation of redox-sensitive VPA-induced transcription from this promoter. transcription factors. To verify that Nrf2 was contained in the protein-DNA complex, antibody to Nrf2 was added in the assay. The antibody depressed the band intensity of the complex, indicating that it prevented the interaction of Nrf2 with the ARE probe (Fig. 5C,comparelane 7 with lane 3). Interestingly, antibodies to small Maf proteins dramatically depressed the band density of the protein-DNA complex, not only in the basal condition (data not shown), but also in VPA-treated cells (Fig. 5C,comparelanes 4-6 with lane 3). Components of the AP-1 transcription factor complex (Fos and Jun) effectively form heterodimers with Maf through their leucine zipper structures (46). Therefore, it was not surprising that antibody to c-Fos also caused reduction in band intensity (Fig. 5C,comparelane 8 with lane 3). These results indicate the presence of Nrf2, small Maf(s), and c-Fos in the protein-DNA complex detected with the ARE probe. Next, we used ChIP analysis to confirm the association of these transcription factors with the Gai2 gene promoter, in the context of the intact cell. For the PCR analysis of the target region, we used a

Figure 5. Binding of nuclear extract proteins to DNA probe containing ARE sequence. EMSA was done with nuclear extracts fromcontrol (cells that were not treated with VPA) and VPA-treated K562 cells, using the protocol described in detail elsewhere (31). The EMSA reactions were carried out with 2 Ag of nuclear extract protein for each lane. When antibodies were used, the nuclear extract was incubated with the labeled probe for 20 minutes at 25jC prior to addition of each antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and then incubated for an additional 30 minutes, followed by electrophoresis. The EMSA was done with a labeled double-stranded DNA probe 5¶-GCCCGCCCCGGCCCAGTCACAGGCTTGGTTC-3¶, which contains the canonical ARE (underlined) that maps at À84/À76 in the Gai2 gene promoter. The sequence of the mutated oligonucleotide was 5¶-GCCCGCCCCGGCCCATGTCCAGGCTTGGTTC-3¶ (mutated bases are in boldface and italic). EMSA results shown are representative of three different nuclear extract preparations. A, DNA binding competition experiments with 20-fold excess of the following oligonucleotides: unlabeled oligonucleotide identical (in sequence) to the labeled oligonucleotide probe (lanes 3 and 10), mutated oligonucleotide (lanes 4 and 11), and Sp1 consensus oligonucleotide (lanes 5 and 12). Consensus Sp1 oligonucleotide was fromSanta Cruz Biotechnology. Antibodies to Nrf2 and c-Fos were used at 2 Ag(lanes 6, 7, 13, and 14). In VPA-treated cells, nuclear extracts were prepared 1 hour after the VPA (5 mmol/L) treatment. B, pretreatment of cells with antioxidants results in decreased binding to the ARE. K562 cells were incubated with PDTC (50 Amol/L) or NAC (5 mmol/L) for 1 hour before the addition of VPA (5 mmol/L). Nuclear extracts were prepared 1 hour after the addition of VPA. C, effect of increasing amounts of antibodies to small Maf proteins. Use of antibodies to Nrf2 and c-Fos was repeated in this assay, for reference purposes. The amounts of anti–small Maf antibodies used in lanes 4, 5, and 6 were 0.5, 1, and 2 Ag, respectively. Antibodies to Nrf2 and c-Fos were used at 2 Ag(lanes 7 and 8). Ab, antibody; NE, nuclear extract. www.aacrjournals.org 6567 Cancer Res 2006; 66: (13).July 1, 2006

cellular levels of ROS induced by this compound. We show here that VPA increases gene transcription in an ARE-dependent manner, in at least three reporter gene constructs [i.e., human a G i2, mouse heme oxygenase-1, and human NAD(P)H:quinone oxidoreductase 1 gene promoters]. The ARE in mouse heme oxygenase-1 and human NAD(P)H:quinone oxidoreductase 1 gene promoters conforms to prototype ARE that contains core and a flanking sequences (39–43), whereas the ARE in the human G i2 gene promoter (5¶-TGACtggGC-3¶) matches the sequence referred to as short ARE by Katsuoka et al. (43). Whereas mutation at a both the ARE and Sp1-binding sites in the human G i2 gene promoter affected the promoter activity induced by VPA, only mutation at the ARE affected promoter activity induced by tBHQ. a This indicates that VPA-induced G i2 gene promoter activity resulting from redox-sensitive transcription factor interaction at the ARE was independent of VPA-induced promoter activity resulting from Sp1 interaction at Sp1-binding sites. These findings are novel because until now, no data were available to suggest that the ARE is a distinct target for VPA-induced gene transcription. Although some HDAC inhibitors, notably SAHA, sodium butyrate, and MS-275, have been shown to cause increased generation of ROS in other cell systems (23–26), those studies provide no information as to the relationship of the increased ROS with regulation of the expression of any particular gene. Furthermore, recent reports that VPA induces production of ROS (20–22) do not explore how the resultant elevated level of ROS influences expression of any gene. Our study clearly provides mechanistic insight into the disposition of VPA- or other HDAC inhibitor–induced ROS production vis-a`-vis gene expression;it provides a link between ROS-induced activation of redox-sensitive transcription factors (typified by Nrf2) and their subsequent a Figure 6. ChIP of the G i2 promoter DNA. ChIP analysis of DNA from K562 interaction with the ARE on specific gene promoters. In the cells cultured in the absence or presence of VPA (5 mmol/L) for 1 or 2 hours was carried out to determine the in vivo association of various transcription absence of the ARE sequence, VPA-induced transcription was not a factors with the G i2 gene promoter. Details of the ChIP analysis have been sensitive to antioxidants. previously described (31). A, sketch of the Gai2 gene indicating target and nontarget regions amplified in the ChIP analysis. B, PCR products amplified Although much remains to be learned about the ROS targets with primers (forward, 5¶-TTTGGAACCACCCCTATTGC-3¶; reverse, that are involved in signaling response(s) to HDAC inhibitor(s), 5¶-AACCAAGCCTGTGACTG-3¶) that bracketed the 170 bp (À234/À65) target it seems clear from the studies reported here that targeting region. Right, (nontarget region) depicts PCR products (249 bp) amplified with primers (forward, 5¶-CCCAAGGAGGCTCTATGTTTTC-3¶; reverse, through the ARE underlies the VPA-induced, ROS-mediated gene 5¶-TCAGAGGCTACATCCCAGG-3¶) to a 249 bp ‘‘nontarget region’’ (+1099/ transcription. There may be additional means by which ROS +1347) that maps in intron 1. Left and right, input DNA was diluted, and analyzed production may indirectly influence HDAC inhibitor–induced by using amounts equivalent to 1% of immunoprecipitated samples. gene transcription. For example, upon ROS-mediated activation of a redox-sensitive transcription factor, the transcription factor Discussion could become acetylated by acetylases such as PCAF/p300 to There is burgeoning interest in the use of VPA as an anticancer yield a transcriptionally active species. The transcriptionally agent because of its potent antiproliferative effects in various cell active and acetylated forms of such factors would then be types (13, 15, 16, 50). In fact, the findings by Takai et al. (50) that its maintained in the nucleus by inhibition of HDACs if the cells antiproliferative activity in HEC-1B endometrial cancer cells, as were treated with HDAC inhibitor(s). This seems to be the case well as its antitumor activity in nude mice, which occurs without with SAHA-induced NF-nB-mediated gene transcription in lung any major side effects, prompted the authors to suggest that VPA cell carcinoma (23). Whether this applies to VPA-induced may become a useful adjuvant therapy for endometrial cancers. In activation of Nrf2 or other redox-sensitive transcription factors addition to its antiproliferative effects, VPA has also been shown to is yet to be investigated. Taken together with the fact that induce the expression of a variety of genes in various cell types hyperacetylation (5), Sp1- (11), and AP-1-binding motifs (5, 17–19) (10–12). Although the mechanism of this effect has been elusive, it have already been implicated in VPA induction of gene expres- seems to involve the interaction of AP-1 transcription factors at sion, our present study highlighting the role of Nrf2 and the AP-1 response elements (17, 18) and/or Sp1 transcription factor ARE clearly indicates that multiple mechanisms underlie the interaction at Sp1-binding sites (11), depending on the gene transcriptional response to VPA, and perhaps other HDAC promoter. However, these conclusions did not consider the inhibitors. potential effects of ROS generated in response to VPA treatment. At the present time, the mechanism(s) by which VPA or any The present study provides an additional understanding of the other HDAC inhibitor induces generation of ROS are unknown. transcriptional mode of action of VPA, with regard to elevation of Nonetheless, ROS generation by these compounds should be an

Cancer Res 2006; 66: (13).July 1, 2006 6568 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Valproic Acid–Induced Gene Expression important consideration in their use as chemotherapeutic agents, Grant support: S06-GM08037 (NIH-NIGMS). The costs of publication of this article were defrayed in part by the payment of page especially because ROS are increasingly recognized as potent charges. This article must therefore be hereby marked advertisement in accordance activators of signaling cascades culminating in the up-regulation of with 18 U.S.C. Section 1734 solely to indicate this fact. stress-responsive genes. We thank Dr. Jianqi Yang and Melanie Theodore for excellent assistance in the electronic preparation of the figures in this article, Dr. ZhongMao Guo (Division of Cardiovascular Biology, Meharry Medical College) for providing us mouse aortic Acknowledgments endothelial cells, and Dr. Sekhar P. Reddy (Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, MD) for providing us HO-1-ARE-Luc and hNQO1- Received 3/3/2006;revised 4/11/2006;accepted 4/26/2006. ARE-Luc plasmid constructs and empty vectors.

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