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Originally published In Press as doi:10.1074/jbc.M305880200 on October 31, 2003

J. Biol. Chem., Vol. 279, Issue 5, 3869-3876, January 30, 2004
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Suppression by p38 MAP Kinase Inhibitors (Pyridinyl Imidazole Compounds) of Ah Receptor Target Gene Activation by 2,3,7,8-Tetrachlorodibenzo-p-dioxin and the Possible Mechanism*

Masahiko Shibazaki{ddagger}, Takashi Takeuchi, Sohel Ahmed, and Hideaki Kikuchi§

From the Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan

Received for publication, June 4, 2003 , and in revised form, October 31, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytochrome P-450 1A1 (CYP1A1) is known to be induced by aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), through activation of the aryl hydrocarbon receptor (AhR). We found that p38 MAP kinase inhibitors (SB203580 and SB202190; 40 µM each; pyridinyl imidazole compounds) suppressed CYP1A1-mRNA induction by TCDD (2 nM) in mouse hepatoma Hepa-1 cells and in human hepatoma HepG2 cells, and also suppressed CYP1B1-mRNA induction by TCDD (2 nM) in human breast adenocarcinoma MCF7 cells. An analogue compound, SB202474, which does not inhibit p38 MAP kinase, also suppressed CYP1A1-mRNA induction by TCDD. Moreover, overexpression of a dominant-negative gene for p38 MAP kinase in Hepa-1 cells did not suppress Cyp1a1 reporter gene induction by TCDD. Therefore, the suppression of Cyp1a1 transcription by pyridinyl imidazole compounds is not because of their inhibition of p38 MAP kinase activity. Because SB203580 did not inhibit in vitro AhR transformation by TCDD, this compound was not acting as a simple AhR antagonist. SB203580 decreased TCDD-induced histone acetylation levels in the region of the Cyp1a1 gene promoter, especially around the TATA box sequence. This result suggests the possibility that pyridinyl imidazole compounds suppress the recruitment of some co-activator that has the histone acetyltransferase activity necessary for CYP1A1-mRNA transcription.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polycyclic aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)1 and 3-methylcholanthrene, are widespread environmental pollutants with many cytotoxic and biological effects. Immunosuppression, tumor promotion, and teratogenesis are well known toxic effects of these compounds in vertebrates (1-4).

The aryl hydrocarbon receptor (AhR) is a ligand-activated cytosolic protein that is a member of the family of the basic-helix-loop-helix-Per/ARNT/Sim transcription factors, and it regulates the induction of the Cyp1a1 gene (5). In the unstimulated state, AhR forms a complex with two molecules of heat shock protein 90 and the immunophilin homologue protein, XAP-2 (6, 7). When ligand (for example, TCDD) binds to the ligand-binding domain of AhR, a chaperone protein is dissociated from the complex, and AhR can then translocate into the nucleus. Once in the nucleus, AhR forms a heterodimer with AhR nuclear translocator (ARNT), and recognizes its target sequence, a xenobiotic responsive element (XRE) was found upstream of the CYP1A1, 1A2, 1B1, and NQO1 genes (5, 8). The involvement of AhR in cytotoxicity is evidenced by AhR-null mice being resistant to the toxic effects of TCDD and other aromatic hydrocarbons (9). Because AhR is a central molecule in the action process of TCDD toxicity, the induction of cytochrome P-450 1A1 (CYP1A1) has been used as a model system for the study of the molecular mechanisms underlying the toxicity of TCDD.

The induction of CYP1A1 by TCDD is suppressed by pretreatment of immune cells with lipopolysaccharide (LPS), interleukin-1, or tumor necrosis factor (10, 11). The mechanisms involved in the intracellular signaling cascades induced by LPS have been analyzed in detail, and are known to induce activation of the MAP kinase (extracellular signal-regulated kinase, JNKs, p38s) pathway through its membrane-bound receptor, Toll-like receptor (12). LPS also activates the NF-{kappa}B pathway. Tian and co-workers (10) showed that activated AhR can interact with NF-{kappa}B subunit p65 (RelA), and that this interaction results in a suppression of the transcription of its target gene. Introduction of the oncogene ras into cultured human breast cancer cells suppresses the transcriptional activation by TCDD of several members of the AhR battery of genes (13). Puga and co-workers (14) showed that TCDD treatment induced a transient activation of transcription factors, especially AP-1, in Hepa-1 cells, and that this activation was AhR-dependent (14). They also reported that TCDD affected the expression level of various genes, including those for members of Ras/MAPK-related signaling pathways in HepG2 cells (15). These data suggest that the AhR pathway and the MAP kinase pathway may cross-talk with each other, but the detailed mechanism remains controversial (for example, how LPS suppresses CYP1A1 and the extent of the contribution of the MAP kinase pathway to induction by TCDD).

In the light of the background described above, we decided to focus on the p38 MAP kinase pathway, which is strongly activated by LPS through its Toll-like receptor 4, to assess its involvement in the inhibition of CYP1A1 by LPS. The MAP kinase pathway of p38 ultimately activates transcription factor ATF-2, and regulates the transcription of target genes by binding to its regulatory sequence, AP-1 (12). This cascade can be blocked by a series of pyridinyl imidazole compounds, exemplified by SB203580, which were originally identified by their ability to suppress the synthesis of inflammatory cytokines (16). In the course of experiments with such chemical inhibitors, we found that CYP1A1-mRNA induction by TCDD was potently suppressed by pre-treatment of Hepa-1 cells with pyridinyl imidazole compounds.

In this paper, we describe possible mechanisms by which pyridinyl imidazole compounds may suppress CYP1A1-mRNA induction by TCDD. Our data suggest that these widely used p38 MAP kinase-specific inhibitors may have another target molecule(s), besides p38 MAP kinase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—HepG2 (HB 8065) and MCF7 (HTB 22) cells were purchased from the American Type Culture Collection (Rockville, MD). The Hepa-1 cell line, derived from mouse hepatoma, was kindly provided by Dr. K. Sogawa (Tohoku University). The cells were maintained in Dulbecco's minimal essential medium (Invitrogen) containing 10% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin G in humidified 95% air, 5% CO2 at 37 °C. The toxicities of all the inhibitors were tested by examining their effects on the growth rate of Hepa-1 cells, and the chemicals were used within the range of nontoxic doses (in terms of cell viability).

Chemicals—TCDD was purchased from Cambridge Isotope Laboratories (Cambridge, MA). SB203580 (99.8% purity) was from Promega Corp. (Madison, WI), and SB202474 (99.6% purity) and SB202190 (99.1% purity) from Calbiochem-Novabiochem Corp. (San Diego, CA). {alpha}-Naphthoflavone and trichostatin A (TSA) were from Wako Pure Chemical Industries Ltd. (Osaka, Japan), and omeprazole from LTK Laboratories, Inc. (St. Paul, MN). Labeled compound [{alpha}-32P]dCTP (110 TBq/mmol) was purchased from Amersham Biosciences.

Plasmid Construction and Transfection into Cells—The 5' upstream sequence (from -1642 bp to +57 bp) of the Cyp1a1 gene was provided by Dr. Daniel W. Nebert (University of Cincinnati Medical Center) (17). The isolated fragment was ligated into pGL3-Basic (Promega). The wild-type p38 MAP kinase, dominant-negative p38 (both pCMV-Flag-p38MAPK, TGY and AGF), and the dominant-negative vectors of MKK3 and MKK6, were provided by Dr. Roger J. Davis (University of Massachusetts Medical School) (18, 19). The constitutively activated MKK6 was produced by replacing Ser-207 and Thr-211 with Glu using the PCR procedure (19). The activity of the Gal4 DNA binding domain, GAL4/ATF2 (20), was measured in a cotransfection assay with the reporter plasmid pFR-Luc (Stratagene, La Jolla, CA). Transfection of plasmid DNA into cells was performed by the lipofection method using FuGENE6 (Roche Diagnostics, Japan). Transfection efficiency was normalized with respect to {beta}-galactosidase activity, which was produced by co-transfection of pCAGGS-lacZ (provided by Dr. J. Miyazaki, Osaka University Medical School) (21).

The cells were harvested 22 h after transfection with expression plasmid, then lysed by a two times freeze-thaw in reporter lysis buffer (Promega). The cleared lysate obtained by centrifugation was saved, and luciferase activity was measured in 10 µl of the sample using a Fluoroscan ASCENT FL (Labosystems, Finland).

RNA Extraction and Northern Hybridization—Total RNA was extracted from cells by the acid guanidine phenol-chloroform extraction method (22). Then, 20 µg of total RNA was electrophoresed on 1.0% formaldehyde-agarose gel for 2 h at 80 V, and blotted on Hybond N+ (Amersham Biosciences) using 20x SSC overnight. The coding region of the Cyp1a1-cDNA fragment (1.7 kb) was used for the hybridization probe. A fragment of {beta}-actin cDNA (398 bp) was amplified by reverse transcriptase-PCR using 5'-TGGAATCCTGTGGCATCCATGAAAC-3' and 5'-TAAAACGCAGCTCAGTAACAGTCCG-3' primers, and used as an endogenous control. The hybridization probe was labeled with [{alpha}-32P]dCTP using Prime-It (Stratagene), and purified on a Sephadex G-50 DNA-grade column (Amersham Biosciences). The membrane was pre-hybridized with pre-hybridization buffer for 4 h at 68 °C, and then hybridized with a 32P-labeled probe (1 x 106 cpm/ml) using hybridization buffer overnight (more than 16 h) at 68 °C. Then, the membrane was washed twice in 2x SSC containing 0.5% SDS for 20 min. Signal was detected and quantified using a Bioimage analyzer BAS2000 (Fujifilm Co., Tokyo, Japan).

Cytosolic Protein Preparation—Livers were removed from 6-week-old male Sprague-Dawley rats, and homogenized with ice-cold HEDG buffer (25 mM K+-HEPES, pH 7.5, 1.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 1x protease inhibitor (CompleteTM; Roche Diagnostics)) using 3 ml of buffer for 1 g of liver, and then centrifuged at 10,000 x g for 10 min at 4 °C. The supernatant was centrifuged again at 105,000 x g for 30 min at 4 °C, and this supernatant fraction (cytosolic protein) was used in the experiments involving an in vitro transformation assay (23).

Electromobility Shift Assay (EMSA)—Rat XRE oligomers (5'-GATCCGGCTCTTCTCACGCAACTCCGAGCTCA-3' and 5'-GATCTGAGCCTGGAGTTGCGTGAGAAGAGCC-3', with underlining representing the XRE core sequence) (24) were annealed and labeled with [{alpha}-32P]dCTP by means of Klenow fragment of Escherichia coli DNA polymerase I (Roche Molecular Biochemicals). Then, the 32P-labeled XRE was purified on a Sephadex G-50 spin column (Amersham Biosciences). In the experiment involving in vitro transformation of rat cytosolic protein, 800 µg of protein was incubated with 10 nM TCDD, either alone or together with 20-160 µM SB203580 or 40 µM PD98059, for 3 h at 22 °C. One-tenth of the incubated protein (80 µg protein) was then incubated for 20 min at 22 °C with 225 ng of poly(dI-dC) containing 95 mM NaCl and 1x protease inhibitor (CompleteTM; Roche Diagnostics) in HEDG buffer in a total volume of 25 µl (23), followed by the addition of 1 ng of 32P-labeled XRE oligonucleotide (~200,000 cpm). Then, the sample was incubated for an additional 20 min at 22 °C. To confirm the specific binding of XRE to AhR, a 250 times excess concentration of cold XRE was added. Then, samples were electrophoresed on 4% polyacrylamide in 0.5x TBE buffer for 90 min at 150 V. After the gel had been dried, the radioactive signal was detected and quantified using a Bioimage analyzer, BAS2000.

Western Blotting—Cells were transfected with expression vector, incubated for 48 h, then extracted with 50 µl of cell lysis buffer (1% Nonidet P-40, 0.1% sodium deoxycholate, 20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 1x protease inhibitor (CompleteTM; Roche Diagnostics), 1 mM dithiothreitol). Protein (50 µg) was electrophoresed on 8% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membrane. The membrane was immunostained with anti-FLAG antibody (1000-fold dilution), and developed using an enhanced chemiluminescence kit (Amersham Biosciences).

Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay was carried out essentially by the method of Wang and Hankinson (25). Hepa-1 cells were grown to 80% confluence in two plates in a 10-cm dish. The cells were washed twice with phosphate-buffered saline (-), cross-linked with 1% formaldehyde at 37 °C for 10 min, then collected using a cell scraper. They were then rinsed twice with ice-cold washing buffer (phosphate-buffered saline(-), 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml pepstatin), suspended in 1 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, 1x protease inhibitor (CompleteTM; Roche Diagnostics)), and incubated on ice for 10 min. Then, the cell suspension was sonicated for 15 s using a Branson sonifier (model W185 with micro-probe) at power setting 4, followed by centrifugation for 10 min at 4 °C, so that the DNA fragment size was 200-600 bp. Supernatant (120 µl) was collected and diluted in ChIP dilution buffer (1080 µl of 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1x protease inhibitor (CompleteTM; Roche Diagnostics)) followed by immunoclearing with 2.5 µg of shared salmon sperm DNA and Protein A buffer (60 µl of 50% slurry Protein A-agarose (Amersham Biosciences) in ChIP dilution buffer for 2 h at 4 °C. Immunoprecipitation was performed for 16 h at 4 °C with 6 µl (200-fold dilution) of anti-acetylated histone H4 antibody (Upstate Biotechnology, Charlottesville, VA) or anti-AhR antibody (SA-210; BIOMOL, Plymouth Meeting, PA). After immunoprecipitation, 60 µl of Protein A buffer was added, and the incubation was continued for another 1 h at 4 °C. Precipitates were washed sequentially in low-salt buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), high-salt buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), and LiCl detergent buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA). Precipitates were then washed twice in TE buffer (1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and extracted with 500 µl of elution buffer (1% SDS, 0.1 M NaHCO3). Eluates were pooled and incubated at 65 °C for 12 h to reverse the formaldehyde cross-linking. After incubation for 1 h at 45 °C with 10 µl of 0.5 M EDTA, 20 µl of 1 M Tris-HCl, pH 6.5, and 2 µl of Proteinase A (10 mg/ml), DNA fragments were purified by phenol-chloroform extraction, and precipitated using 2 volumes of ethanol. Precipitates were then suspended in 20 µl of TE buffer. For PCR, a 1-µl sample from a 20-µl DNA extraction was used, with 30 cycles of amplification. For detection of the Cyp1a1 TATA box (-285 to +66), the primers used were 5'-TTTCCTCAAACCCCTCCCTC-3' and 5'-GAAGTGAAGAGTGTTCTCTAGGAC-3' (11). For detection of Cyp1a1 XRE (-1141 to -784), the primers used were 5'-CTATCTCTTAAACCCCACCCCAA-3' and 5'-CTAAGTATGGTGGAGGAAAGGGTG-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of the p38 MAP Kinase Inhibitor, SB203580, on CYP1A1-mRNA Induction by TCDD—To clarify the relationship between the p38 MAP kinase pathway and CYP1A1-mRNA induction by TCDD, we used the p38 MAP kinase inhibitor, SB203580 (Fig. 1). Fig. 2 shows the results of Northern hybridization. Pretreatment with SB203580 for 1 h before the addition of 2 nM TCDD significantly suppressed CYP1A1-mRNA induction by TCDD in a dose-dependent manner in both Hepa-1 cells and HepG2 cells (Fig. 2, A and C). Generally, around 10 µM SB203580 is used for the inhibition of p38 MAP kinase activity. At such a dose, about a 50% suppression of CYP1A1-mRNA induction was observed. At 40 µM SB203580, suppression was by about 75-90%. This suppression was confirmed at a higher dose (20 nM) of TCDD (Fig. 2B). Induction of the mRNA for another AhR battery gene, CYP1B1, was also suppressed by SB203580 (Fig. 2D). On treatment of the cells with SB203580 alone, no CYP1A1-mRNA induction was detected in any of the cultured cells. At a concentration of 80 µM, SB203580 had no effect on cell survival or viability, as assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (data not shown).



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  		FIG. 1.
Chemical structures of p38 MAP kinase inhibitors, pyridinyl imidazole compounds. Thin line squares show the common structure of pyridinyl imidazole. The IC50 values for p38 MAP kinase are from previous reports (40, 41).

 



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  		FIG. 2.
Effects of the p38 MAP kinase inhibitors on TCDD-induced CYP1A1- and CYP1B1-mRNAs. A and C, dose-dependent effect of SB203580 (SB) on the induction of CYP1A1-mRNA by TCDD. Hepa-1 (A) or HepG2 (C) cells were pretreated with various doses of SB203580 for 1 h before the addition of 2 nM TCDD. B, dose-dependent effect of TCDD on CYP1A1-mRNA, and its suppression by SB203580. Hepa-1 cells were pretreated with 40 µM SB203580 for 1 h before the addition of various doses of TCDD. D, effect of SB203580 on the induction of CYP1B1-mRNA by TCDD. MCF7 cells were pretreated with 40 µM SB203580 or 10 µM PD98059 (PD) for 1 h before the addition of 2 nM TCDD or solvent (DMSO). The membrane was hybridized with a CYP1B1-mRNA specific probe. In all experiments, cells were harvested 22 h after TCDD treatment. E, effect of SB202474 and SB202190 on CYP1A1 induction. Hepa-1 cells were pretreated with various doses of SB202474 and SB202190, analogues of pyridinyl imidazole, for 1 h before the addition of 2 nM TCDD. After incubation with TCDD for 22 h, cells were harvested, and Northern hybridization was performed using a Cyp1a1-cDNA probe. Upper panel of A shows quantification of the CYP1A1-mRNA signal using a Bioimage analyzer, BAS-2000 (Fujifilm Co.). Each data point indicates the mean from three independent experiments, with the bar showing the standard deviation. The intensity of CYP1A1-mRNA was normalized with respect to that of {beta}-actin.

 
SB203580 Suppresses CYP1A1-mRNA Induction by TCDD at the Level of Transcription—AhR recognizes the XRE sequence in the regulatory region upstream of the Cyp1a1 gene. Next, we examined whether the above suppression is exerted at the transcriptional level, using a luciferase reporter gene inserted into the regulatory region of the Cyp1a1 gene (-1642 bp to +57 bp) in a pGL3 Basic vector (Fig. 3A). Treatment with 2 nM TCDD induced about a 7-fold increase in luciferase activity in transfected Hepa-1 cells. On the other hand, in an experiment involving pretreatment with various doses of SB203580 1 h before the addition of 2 nM TCDD, a significant dose-dependent suppression of luciferase activity was observed (Fig. 3B). This trend was in good agreement with the results of Northern hybridization (Fig. 2). This result implied that SB203580 suppressed the transcription of the Cyp1a1 gene, rather than inhibiting of CYP1A1-mRNA degradation.



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  		FIG. 3.
Suppression of Cyp1a1 reporter gene by SB203580. Hepa-1 cells were transfected with 0.5 µg of a reporter gene, the Cyp1a1-promoter region (-1642 to +57) ligated with pGL3Basic vector, together with 0.2 µg of pCAGGS-lacZ vector. After 16 h incubation, cells were treated with various doses of SB203580 for 1 h before the addition of 2 nM TCDD. Cells were incubated for a further 22 h, then harvested and luciferase activity was measured. Luciferase activity was normalized with respect to {beta}-galactosidase activity. Asterisk (*) indicates p < 0.01 versus TCDD-treated sample (Student's t test).

 
Analogue Compounds of Pyridinyl Imidazole Suppress CYP1A1-mRNA Induction by TCDD—A series of pyridinyl imidazole compounds (Fig. 1) was developed, and their individual abilities to suppress the synthesis of inflammatory cytokines were assessed. The IC50 concentration of SB203580 for p38 MAP kinase is below 1 µM and that of SB202190 is below 0.5 µM, whereas SB202474, which has no ability to inhibit p38 MAP kinase activity, is generally used as a negative control compound in p38 MAP kinase studies (16). The effects of these compounds on CYP1A1-mRNA induction by TCDD were tested by Northern hybridization (Fig. 2E). SB202190 and SB202474, analogues of the p38 MAP kinase inhibitor SB203580, suppressed CYP1A1-mRNA induction by TCDD in the same way as SB203580 itself (lanes 3-16). Furthermore, it shows that this effect is not because of inhibition of the p38 MAP kinase activity, because SB202474, which does not inhibit p38 MAP kinase activity at all, suppressed CYP1A1-mRNA induction by TCDD (lanes 6-8).

The p38 MAP Kinase Pathway Is Not Involved in the Induction of CYP1A1-mRNA by TCDD—To evaluate the contribution made by the p38 MAP kinase pathway to the induction of CYP1A1-mRNA by TCDD, we used dominant-negative constructs of p38 MAP kinases. MKK3 and MKK6 are upstream molecules of p38 MAP kinase. MKK3/6 dominant-negative constructs were transfected into Hepa-1 cells, and their effects were assessed on the activation of the Cyp1a1 reporter gene by TCDD (Fig. 4A). As shown in Fig. 4A, no significant suppression of reporter gene expression was observed with any combination. Furthermore, a p38 MAP kinase dominant-negative construct was also tested in the same way as MKK3/6 (Tables I). The "-fold induction" (that is, the luciferase activity of the reporter gene in the presence of TCDD divided by that in the absence of TCDD) was not significantly different between the wild-type p38 MAP kinase and the dominant-negative p38 MAP kinase construct at 2 µg of plasmid DNA. The inhibitory function of dominant negative p38 MAP kinase in Hepa-1 cells was confirmed using the constitutively activated MKK6 and ATF-2 systems (Table II) (19). The left and right panels in Fig. 4B show the expression levels (detected by Western blotting) of the dominant-negative constructs for MKK3/6 and p38 MAP kinase, respectively. These results, like those described above, imply that the p38 MAP kinase pathway is not involved in the induction of CYP1A1-mRNA by TCDD.



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  		FIG. 4.
Effect of MKK3/6 or p38 MAPK dominant-negative constructs on TCDD-induced Cyp1a1 reporter gene activity. A, Hepa-1 cells were transfected with MKK3 or MKK6 dominant-negative constructs (MKK3-D.N. or MKK6-D.N.) together with 0.5 µg of Cyp1a1 reporter gene and 0.2 µg of pCAGGS-lacZ. Then, 16 h after transfection cells were treated for 22 h with 2 nM TCDD. Cells were harvested and luciferase activity was measured. Luciferase activity was normalized with respect to {beta}-galactosidase activity. B, the expression levels of transfected MKK3-D.N. and MKK6-D.N. (left panel) and p38 MAPK-D.N. (right panel) were determined by Western blotting using anti-FLAG antibody.

 


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  		TABLE I
Effect of p38 MAP kinase dominant-negative constructs on TCDD-induced Cypla1 reporter-gene activity

Hepa-1 cells were transfected with p38 MAPK wild-type construct (Wild) or a dominant-negative construct (p38MAPK-D.N.) together with 0.5 µg of Cypla1 reporter gene and 0.2 µg of pCAGGS-lacZ. Then, 16 h after the transfection cells were treated for 22 h with 2 nM TCDD (+TCDD) or vehicle (-TCDD). Cells were harvested and luciferase activity was measured. Luciferase activity was normalized with respect to {beta}-galactosidase activity. The values given for old increase are the luciferase activity in the presence of TCDD divided by that in the absence of TCDD. The expression levels of transfected p38 MAPK (Wild) and p38MAPK.D.N (D.N.) were determined by Western blotting using anti-FLAG antibody.

 


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  		TABLE II
Hepa-1 cells were transfected with constitutively activated MKK6 (C.A.)(0.2 µg) and pFA-ATF2 (0.2 µg) together with 0.2 µg of pFR-Luc reporter gene and 0.2 µg of pCAGGS-lacZ

The effect of p38 MAPK dominant-negative (D.N.) on the transactivation function of ATF-2 was compared with p38 MAPK (wild). The values were expressed in arbitrary units (luciferase/{beta}-galactosidase).

 
SB203580 Does Not Suppress in Vitro AhR Transformation by TCDD—The flavonoid derivative, PD98059 (which was developed as a specific MEK inhibitor), strongly suppresses CYP1A1 induction by TCDD. Reiners and co-workers (26) revealed that this suppression was because of an antagonistic effect of PD98059 on AhR. This compound strongly suppresses in vitro AhR transformation by TCDD. We performed an in vitro AhR transformation assay using SB203580, with PD98059 as a positive control. As shown in Fig. 5 (lane 3) a rat cytosolic AhR activated by 10 nM TCDD formed a complex with a 32P-labeled XRE sequence, and the complex formation was completely suppressed by the addition of a 200 times excess of cold XRE (lane 2). Co-incubation with PD98059 completely inhibited AhR activation by TCDD (lane 13), but co-incubation with SB203580 did not (lanes 8-11). Although at a concentration of 160 µM SB203580, AhR transformation was slightly decreased (about 13%, lane 11), this concentration was 4 times higher than that needed to suppress CYP1A1-mRNA induction by 75-90% (Fig. 2A). On treatment with 160 µM SB203580 alone, no AhR transformation was observed (lane 7). These results suggest that SB203580 does not act as a simple AhR antagonist, as PD98059 does.



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  		FIG. 5.
Effect of SB203580 on in vitro AhR activation by TCDD. Rat liver extracts (800 µg) were transformed in vitro for 3 h using 10 nM TCDD in the presence of various concentrations of either SB203580 (20, 40, 80, or 160 µM) or 40 µM PD98059. Subsequently, the extracts were incubated for 20 min at 22 °C with 1 ng of 32P-labeled XRE probe, and analyzed on 4% polyacrylamide native gel. To confirm the specificity of XRE binding to AhR, a 250-fold excess of cold XRE was added (lane 2). The data shown are the most representative of those obtained in three independent experiments.

 
SB203580 Suppresses CYP1A1-mRNA Induction by Omeprazole—Kikuchi et al. (27) showed that omeprazole induced CYP1A1-mRNA in a ligand-independent manner, and that the gene(s) that mediate its induction are present on human chromosome 10p (27). The induction of CYP1A1-mRNA by omeprazole was not suppressed by the AhR antagonist PD98059, suggesting the existence of a ligand-independent signaling pathway (Fig. 6, lane 9). However, PD98059 did suppress the ligand-dependent induction of CYP1A1-mRNA by TCDD (Fig. 6, lane 7). Omeprazole indirectly activates AhR, and the downstream mechanism is the same as that for TCDD. Hence, if SB203580 could be shown to suppress CYP1A1-mRNA induction by omeprazole, we would be able to locate the target molecule(s) of SB203580 to a site downstream of AhR signal transduction. As expected, SB203580 did suppress CYP1A1-mRNA induction by omeprazole (Fig. 6, lane 8). Because the p38 MAP kinase pathway is not involved in CYP1A1-mRNA induction by omeprazole (data not shown), these data suggest that SB203580, and its group of pyridinyl imidazole compounds, may suppress some molecule(s) downstream of AhR signal transduction that are necessary for CYP1A1-mRNA transcription.



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  		FIG. 6.
Effect of SB203580 on CYP1A1-mRNA induction by omeprazole. HepG2 cells were pretreated for 1 h with 40 µM SB203580 (SB) or 10 µM PD98059 (PD), then treated for a further 22 h with 2 nM TCDD, 200 µM omeprazole (OP), or solvent (DMSO). Cells were harvested, and Northern hybridization was performed using a Cyp1a1-cDNA probe.

 
SB203580 Does Not Suppress Translocation of AhR to the Nucleus by TCDD—The activated AhR is translocated from the cytosol to the nucleus within a few hours after ligand treatment. To examine the possibility that SB203580 suppresses AhR translocation to the nucleus, we performed an EMSA using nuclear protein from Hepa-1 cells. As shown in Fig. 7, a specific shifted band was detected following treatment with 10 nM TCDD (lane 3), and this was completely suppressed by use of a 200-fold excess of cold XRE (lane 2). A shifted band of the same intensity was observed when Hepa-1 cells were pretreated with 40 µM SB203580 before the addition of 10 nM TCDD (lane 4). This result indicates that SB203580 did not influence the translocation to the nucleus of the AhR activated by TCDD.



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  		FIG. 7.
Effect of SB203580 on EMSA using nuclear protein. Hepa-1 cells were pretreated for 1 h with 40 µM SB203580, then treated for a further 1.5 h with 10 nM TCDD. Cells were harvested, and nuclear protein was prepared. Then, 5 µg of nuclear protein was incubated with 1 ng of 32P-labeled XRE probe, and analyzed on a 4% polyacrylamide native gel. To confirm the specificity of XRE binding to AhR, a 200-fold excess of cold XRE was added (lane 2). The positions of the nonspecific band (N.S.), specific band, and free probe are shown by thick bars at the side of lane 4.

 
SB203580 Suppresses the Chromatin Acetylation Induced by TCDD—Tian and co-workers (10) showed that TCDD induces histone acetylation in the Cyp1a1 promoter region, and that this acetylation is involved in CYP1A1-mRNA transcription (10). Fig. 8 shows the result of our ChIP assay using AhR- and histone H4-specific antibodies. Panel A shows the time course of the AhR recruitment to the XRE region after TCDD treatment. At 1.5 h after the treatment, AhR had accumulated in the XRE region, and this effect then declined. As expected, there was no signal in the TATA box region in the immunoprecipitates generated using anti-AhR antibody (Fig. 8A). With regard to the histone acetylation level, the TATA box region was significantly acetylated at 2 h after TCDD treatment (i.e. 0.5 h after the peak of the AhR accumulation in the XRE region) (Fig. 8B). The histone in the XRE region was also slightly acetylated at 2 h after the treatment (Fig. 8B). These data suggest that the histone acetylation state really is changed in the course of CYP1A1-mRNA induction by TCDD. Next, we examined the effect of SB203580 on the histone acetylation level induced by TCDD. Pretreatment with 40 µM



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  		FIG. 8.
Effect of SB203580 on TCDD-induced chromatin acetylation. Hepa-1 cells were treated with 10 nM TCDD, then harvested at the times indicated. A ChIP assay was performed using (A) an AhR- or (B) an acetylated histone H4 (AcH4)-specific antibody. To identify the immunoprecipitated sequence, XRE- and TATA-box-specific primers, as shown at the top of the panel, were used for semiquantitative PCR. C, Hepa-1 cells were pretreated for 1 h with 40 µM SB203580 (SB) or 10 µM PD98059, then treated for a further 1.5 h with either 10 nM TCDD or solvent (DMSO). Cells were harvested, and the ChIP assay was performed using AcH4-specific antibody.

 
SB203580 at 1 h before treatment with 10 nM TCDD significantly suppressed histone H4 acetylation around the TATA box region (Fig. 8C), whereas pretreatment with 10 µM PD98059, an AhR antagonist, completely suppressed it (Fig. 8C). Under the same conditions, CYP1A1-mRNA induction, too, was significantly suppressed by SB203580 and completely suppressed by PD98059 (Fig. 2D).

SB203580 Suppresses TSA Augmentation of CYP1A1-mRNA Induction by TCDD—Trichostatin A is known to inhibit the activity of histone deacetylase. Xu and co-workers (28) showed that the 7-ethoxyresorufin O-deethylation activity induced by TCDD in rat hepatocytes was augmented by TSA treatment. Using these drastic conditions, we examined the effect of SB203580 on CYP1A1-mRNA induction and on the histone acetylation state in Hepa-1 cells. As shown in Fig. 9A, the histone H4 acetylation level was significantly increased by treatment with either TCDD alone or TSA alone. The CYP1A1-mRNA level induced by TCDD was also augmented by TSA pretreatment (Fig. 9B, lanes 7-9), although at a high TSA concentration the augmentation was slightly weaker (possibly because of the cytotoxicity of TSA). Following pretreatment with 40 µM SB203580 for 1 h before treatment with TCDD, CYP1A1-mRNA induction was strongly suppressed (Fig. 9B, lanes 11-13). It is not clear whether the effect of pyridinyl imidazole (PI) compounds is to inhibit AhR activity in some way that would decrease acetylation or whether the compounds influence acetylation by some other method. This could be addressed in part by examining the effects of PI compounds on TSA-mediated acetylation that should allow an independent effect on acetylation to be distinguished from an inhibition of AhR activity. The right panel of Fig. 9A shows that SB203580 did not influence the acetylation of histone H4 in the region of Cyp1a1 gene mediated by TSA.



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  		FIG. 9.
Effect of SB203580 on TSA augmentation of CYP1A1-mRNA induction by TCDD. Hepa-1 cells were pretreated for 1 h with various concentrations of TSA (0.5, 1, or 2 µM), then treated for 1 h with 40 µM SB203580 (SB). Subsequently, cells were divided into two groups. A, cells were incubated for 1.5 h with 10 nM TCDD, then harvested for the ChIP assay using histone H4-specific antibody and TATA box-specific primers (-285 to +66 bp). B, the remainder of the cells were incubated for 22 h in the presence of either 2 nM TCDD or solvent. Cells were harvested, and Northern hybridization was performed using a Cyp1a1-cDNA probe. Upper panel shows quantification of CYP1A1 signals (performed as described in the legend to Fig. 2). The radioactivity of CYP1A1 was normalized with respect to that of {beta}-actin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PI compounds, exemplified by SB203580, are widely used as p38 MAPK chemical inhibitors. SB203580 has been shown to bind competitively to the ATP-binding pocket of p38 MAPK, and to inhibit its activity in a specific manner (29). We observed here that PI compounds inhibited CYP1A1-mRNA induction by TCDD (Fig. 2). At first, we suspected that the p38 MAPK pathway might be involved in CYP1A1-mRNA induction by TCDD. However, p38 MAPK dominant-negative and MKK3/6 dominant-negative constructs did not suppress the induction of Cyp1a1 reporter gene activity by TCDD (Tables I and II and Fig. 4). Furthermore, SB202474, which has no ability to inhibit p38 MAPK activity and is generally used as a negative control compound in p38 MAPK studies (16), also suppressed CYP1A1-mRNA induction by TCDD. From these results, we conclude that the p38 MAPK pathway is not involved in CYP1A1-mRNA induction by TCDD, and that PI compounds have another target molecule(s), which might be important for CYP1A1-mRNA transcription by TCDD, besides p38 MAPK.

Several compounds are known to suppress CYP1A1-mRNA induction by TCDD; for example, {alpha}-naphthoflavone (30, 31) and the MEK inhibitor PD98059 (2'-amino-3'-methoxyflavone) (26), which has a flavonoid structure, like the naphthoflavones. Previous studies have demonstrated that these compounds act as AhR antagonists. For instance, resveratrol, which is a member of the polyphenol compounds, suppresses CYP1A1-mRNA induction by TCDD by acting as a simple AhR antagonist (32). Therefore, there was a possibility that PI compounds also directly inhibit this induction by acting as an antagonist to AhR. However, the results of our in vitro AhR transformation and EMSA using nuclear extracts showed that SB203580 did not suppress either AhR activation (Fig. 5) or its nuclear translocation (Fig. 7) by TCDD. Therefore, PI compounds are not acting as simple AhR antagonists. Kikuchi et al. (27, 33) have shown that omeprazole, an inhibitor of H+/K+-ATPase, activates AhR in a ligand-independent way. Our Fig. 6 shows that PD98059, an AhR antagonist, potently suppressed CYP1A1-mRNA induction by TCDD, but did not suppress that by omeprazole. However, SB203580 did suppress CYP1A1-mRNA induction by omeprazole. Omeprazole is not a ligand for AhR (34), but instead activates AhR through a tyrosine kinase-dependent pathway, and it induces CYP1A1-mRNA in human HepG2 cells (33). Therefore, the above data again suggest that PI compounds are not simple AhR antagonists, and may instead act on the downstream process involved in AhR signal transduction.

Tan and co-workers (35) reported that SB202190 suppressed both JNK activation and the induced expression of CYP1A1 protein by TCDD. We used the method of reporter gene assay to examine the possible involvement of the JNK pathway in CYP1A1-mRNA induction by TCDD using a JNK dominant-negative expression vector. However, in our system there was no significant activity change in the reporter gene, which was driven by a Cyp1a1 regulatory sequence, between the sample obtained using control vector and that obtained using a dominant-negative JNK expression vector (data not shown). Therefore, the suppression effect on CYP1A1-mRNA exerted by SB202190 may not be because of a decrease in the activity of JNK.

Recently, it has been shown that nitrobenzylthioinosine-sensitive equilibrative nucleoside transporter 1 is another target molecule for PI compounds, and that it potently suppresses the differentiation of K562 cells induced by cytarabine (Ara C) (36). We checked the possibility that CYP1A1-mRNA suppression by PI compounds may be attributable to a suppression of equilibrative nucleoside transporter 1. However, nitrobenzylthioinosine, an equilibrative nucleoside transporter 1 inhibitor, did not suppress CYP1A1-mRNA induction by TCDD (data not shown). This implies that there is another target molecule(s) for PI compounds, besides equilibrative nucleoside transporter 1.

A previous study has shown that LPS, a bacterial cell-wall component, which can activate the immune system through its receptor (Toll-like receptor 4), suppresses CYP1A1-mRNA induction by TCDD (3). Recently, Ke and co-workers noted that histone acetylation of the Cyp1a1 promoter, especially the TATA box region, was suppressed by treatment with LPS (11). There is much evidence that histone molecules tend to be acetylated in the chromatin of the promoter region of the target gene before the transcription of its mRNA, and the subsequent alteration in the chromatin structure gives transcription factors easy access to the promoter region (37). In the case of CYP1A1-mRNA induction by TCDD, histone acetylation and chromatin remodeling are important. Basic transcription factors, such as p300, SRC-1, and cAMP-response element-binding protein, which have histone acetyltransferase activity, are necessary for CYP1A1-mRNA transcription (38, 39). Furthermore, Brahma/SWI2-related gene-1, which is an ATPase subunit of chromatin remodeling factor SWI-SNF complexes, is critical for CYP1A1-mRNA transcription by TCDD (25). For that reason, we thought that PI compounds might affect the chromatin modification process. The results of our ChIP assay showed that AhR binding to the XRE sequence (1.5 h after TCDD treatment) was followed by histone acetylation of TATA box chromatin (2-3 h after TCDD treatment) (Fig. 8). This histone acetylation by TCDD was suppressed by pretreatment with SB203580 (Fig. 8C). This result suggests that PI compounds may suppress the recruitment to the Cyp1a1 gene promoter region of some coactivator that is necessary for mRNA transcription.

The augmentation of CYP1A1-mRNA induction by pretreatment with TSA (Fig. 9) also indicated that histone acetylation is important in this process. The TSA treatment may increase the global acetylation of histone H4 in the region of Cyp1a1 gene, so that the activated Ah receptor can easily access to XRE and can activate transcription of the Cyp1a1 gene. However, the global acetylation of the gene by TSA may not be enough to accelerate the transcription. Fig. 9B, lane 13, shows the suppression of the amount of CYP1A1-mRNA in the presence of SB203580, even though the histone H4 of the TATA box nucleosomes is highly acetylated by TSA treatment (Fig. 9A, right panel). Furthermore, SB203580 did not inhibit for activated Ah receptor to bind with the XRE sequence, as shown in Fig. 7, lane 4. Therefore, we may speculate that some unknown factor participates in Cyp1a1 transcription and PI compounds inhibit the recruitment of this factor on this site.

Finally, PI compounds have been studied as anti-inflammatory drugs for clinical use because they have the ability to inhibit the secretion of inflammatory cytokines by inhibiting the p38 MAPK pathway. The results of our study should inform on the development of anti-inflammatory drugs that contain derivatives of the pyridinyl imidazole structure, and may also provide a good tool for investigating the mechanism of CYP1A1-mRNA transcription at the level of the chromatin structure.


   FOOTNOTES
 
* This work was supported in part by Grants-in-aid for Scientific Research (B) 11558068 and 12480153 and Exploratory Research 12878090 and 13878099 from the Ministry of Education, Culture, Sports, Science and Technology (Monbu Kagakusho). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by a grant from the Japan Society for the Promotion of Science (JSPS). Current address: Peptide Biosignal Engineering Research Unit, Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan. Back

§ To whom correspondence should be addressed: Dept. of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, 4-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi Japan. Tel.: 81-22-717-8469; Fax: 81-22-717-8470; E-mail: hkikuchi{at}idac.tohoku.ac.jp.

1 The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; CYP1A1, cytochrome P-450 1A1; AhR, aryl hydrocarbon receptor; TSA, trichostatin A; ARNT, Ah receptor nuclear translocator; XRE, xenobiotic responsive element; LPS, lipopolysaccharide; EMSA, electromobility shift assay; ChIP, chromatin immunoprecipitation; PI compounds, pyridinyl imidazole compounds; MAP kinase, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Roger J. Davis (University of Massachusetts Medical School) for generously providing the dominant negative p38 and MKK3/6.



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 DISCUSSION
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