A Critical Role For MAP Kinases in the Control of Ah Receptor Complex Activity

Zongqing Tan, Mingya Huang, Alvaro Puga and Ying Xia1

Center for Environmental Genetics and Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056

Received May 20, 2004; accepted July 14, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The heterodimeric complex of aromatic hydrocarbon receptor (AHR) and Ah receptor nuclear translocator (ARNT) plays a pivotal role in controlling the expression of drug metabolism genes, such as the cytochromes p450 (Cyp) 1a1 and 1b1, believed to be responsible for most toxic effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we show that activation of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) modulates ARNT transcription activity and potentiates the transcriptional activity of AHR/ARNT complexes. Inhibition of ERK by chemical compounds and ablation of JNK caused significant decreases in CYP1A1 induction by TCDD. Compared to wild type, JNK2 ablation significantly reduced TCDD-stimulated CYP1A1 expression in mouse thymus and testis, but not in liver. In contrast, CYP1B1 expression was unaffected in all three tissues of the knockout mice. These data suggest that JNK and ERK modulate ARNT activity and AHR/ARNT–dependent gene expression, contributing to the gene-specific and tissue-specific toxicity of environmental contaminants.

Key Words: Jun N-terminal kinase (JNK); extracellular signal-regulated kinase (ERK); aromatic hydrocarbon receptor (AHR); Ah receptor nuclear translocator (ARNT); 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); cytochrome p450 (CYP).


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Ah receptor is one of the best-studied cellular targets of TCDD and other dioxin-like environmental contaminants. Ligand-binding both induces the conformational changes that dissociate the AHR from the cytosolic complex consisting of HSP90 and accessory proteins, and promotes its nuclear translocation. In the nucleus, the Ah receptor heterodimerizes with the ARNT protein and the complex functions as a transcription factor, binding to AhRE/DRE/XRE motifs and activating transcription of target genes, including those encoding the detoxification enzymes CYP1A1, CYP1A2, and CYP1B1 (Delescluse et al., 2000Go; Nebert et al., 2000Go). Both AHR and ARNT contain a N-terminal basic region-helix-loop-helix motif and a PAS homology domain, characteristic of the large family of bHLH-PAS proteins (Jain et al., 1994Go; Sogawa et al., 1995aGo; Swanson and Bradfield, 1993Go). Ligand-induced AHR complex activation plays a critical role in mediating the biological effects of TCDD in higher organisms (Hahn, 1998Go; Whitlock, 1999Go), and Ahr-null mice are resistant to most toxic and pathologic effects of TCDD (Fernandez-Salguero et al., 1996Go; Shimizu et al., 2000Go).

It is important to note that TCDD exerts diverse species-specific toxic effects in animals and humans, including chloracne, immune, reproductive, and developmental toxicities, carcinogenicity, wasting syndrome, and death. Activation of the Ah receptor by its ligands alone cannot fully explain the diversity of TCDD toxic effects, to which other TCDD-induced molecular pathways also contribute (Enan et al., 1998Go; Tannheimer et al., 1998Go). In addition to its role in AHR binding, TCDD activates various intracellular signal transduction pathways. Recent studies have shown that, independent of the AHR, TCDD activates the ERKs and the JNKs (Tan et al., 2002Go), which, along with the p38s, constitute the family of the mitogen-activated protein kinases (MAPK) (Cobb and Goldsmith, 1995Go). The MAPKs function as critical intracellular signaling mediators whose activities are controlled by the MAPKKK-MAPKK signaling cascades. The activated MAPKs may in turn phosphorylate transcription factors and modulate the function of these factors, thus participating in the regulation of gene expression responsible for a wide array of biological responses (Hunter and Karin, 1992Go). We have shown that TCDD-stimulated MAPKs contribute to Ah receptor activity and receptor-dependent gene expression (Tan et al., 2002Go). The MAPKs might be additional cellular targets through which TCDD modulates the function of AHR complexes.

In this work, we have further investigated the effects of MAPK on AHR and ARNT functions. We show that activation of either JNK or ERK is sufficient to potentiate the transcriptional activity of ARNT, leading to the enhancement of AHR/ARNT–dependent transcription. Both JNK and ERK activities are essentially required for the optimal induction of AHR-dependent gene expression by TCDD. Furthermore, JNK2 ablation in mice greatly reduces AHR-dependent CYP1A1 induction by TCDD in a tissue-specific manner. These results demonstrate a role for the MAPKs in the control of AHR/ARNT activity, which might contribute to the diverse and tissue-specific toxicity of TCDD.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and antibodies. Both TCDD and B[a]P were purchased from Acustandard (New Haven, CT). The laboratories where this work was performed are certified for use of carcinogenic and toxic chemicals by the University of Cincinnati Biohazardous Chemicals Committee. The chemical inhibitors for ERK, U0126 and PD98095, JNK, SP600125, and p38, SB202190, were from Calbiochem (San Diego, CA). Antibody for AHR was from BioMol (Plymouth Meeting, PA), that for CYP1A1 and ß-actin were from Santa Cruz Biotechnology (Santa Cruz, CA), and that for ARNT was raised by Alpha Diagnostic International (San Antonio, CA). Peptides for ARNT (aa630–644) was injected into rabbits, and the anti-serum was passed consecutively through a peptide affinity column and a protein A column.

Mice, cell lines, growth conditions, and transfections. Jnk2-knockout mice (Sabapathy et al., 1999Go) were backcrossed to C57BL6 mice for five generations. Wild type and Jnk2-knockout mice were injected intraperitoneally with various doses of TCDD dissolved in corn oil or with an equivalent volume of corn oil, and 48 h after injection the mice were sacrificed and thymus, liver, and testis were collected. All experimental procedures using these mice were approved by the Ethics Committee of the University of Cincinnati.

The mouse Hepa-1 hepatoma cell line, its derivative c4, having a mutant inactive ARNT (Reisz-Porszasz et al., 1994Go), and the African Green Monkey adult kidney CV-1 cells were cultured in D-MEM, supplemented with 10% fetal bovine serum and 1% antibiotics in a humidified 5% CO2 atmosphere. The Jnk1(–/–)Jnk2(–/–) (Sabapathy et al., 2001Go) and Ahr(–/–) mouse embryo fibroblasts were prepared as previously described (Tan et al., 2002Go). Cells were transfected using Lipofectamine (Introgen, Carlsbad, CA) according to the protocols recommended by the manufacturer. All the cell culture reagents were from Invitrogen. Twenty-four hours after transfection, the cells were starved in serum-free medium for 16 h in the presence of the chemicals to be tested. Cell lysates were prepared in Reporter Lysis Buffer (Promega, Madison, WI) to determine luciferase and ß-galactosidase activities in a Wallac plate reader luminometer. Relative luciferase activities were obtained after normalization to ß-galactosidase activity.

Plasmid constructs. The mammalian expression vectors for AHR, ARNT, and the luciferase reporter pAhRDTKLuc3, which contains the mouse Cyp1a1 AHR-responsive AhRD enhancer fused to the herpes simplex virus type 1 thymidine kinase minimum promoter, have been described elsewhere (Chang and Puga, 1998Go). To generate the pAd-luc expression vector, a 303 bp Pst I/PvuII fragment containing the adenovirus major late promoter (MLP) with the E-box core sequence CACGTG was isolated from pMLH100 (Hawley and Roeder, 1987Go) and cloned into pGL3 by standard molecular cloning procedures. The mammalian expression vectors for kinase-inactive MEKK1 and constitutively active MEKK1, Raf1, MEK1, and MEK6 have been described elsewhere (Baud et al., 1999Go). The GAL4 fusion vector for mammalian expression (Sadowski et al., 1992Go) was used to construct the expression vectors for fusion proteins containing the DNA-binding domain of the yeast GAL4 protein fused to mouse AHR or ARNT by standard molecular cloning techniques.

In vivo competition assay. Hepa-1 cells at 90% confluence were pre-treated for 0.5 h with TCDD (200 nM), B[a]P (20 µM), and various MAPK inhibitors at 5 µM. 3H-TCDD was added at 2 nM for 0.5 h, and the cells were lysed 90 min thereafter. A total of 800 µm of the lysates were used for immunoprecipitation using anti-AHR antibodies. The amount of 3H-TCDD bound to the AHR in the immunoprecipitates was measured by scintillation counting.

Cell and tissue lysate preparation and Western blot analyses. To measure CYP1A1 induction, Hepa-1 cells were pretreated with 5 µM U0126 (MEK inhibitor) for 30 min. Hepa-1, wild type or Jnk1(–/–)Jnk2(–/–) MEFs were incubated in growth medium with 5 nM TCDD for the indicated times. Mouse tissues were homogenized in lysis buffer. Of the total cell and tissue lysates, 100 to 200 µm was resolved in 10% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane, followed by Western blot analyses with antibodies to CYP1A1, AHR, ARNT, and ß-actin, as described elsewhere (Tan et al., 2002Go).

RNA isolation and real-time RT-PCR. Total RNA was isolated from wild type and Jnk1(–/–) Jnk2(–/–) fibroblasts and from thymus, testis, and liver of wild type and Jnk2-knockout mice using Tri-reagent (Molecular Research Center, Cincinnati, OH) and purified by RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse transcription was performed using SuperScript II RNase H reverse transcriptase (Invitrogen). Real-time PCR was carried out with a Cepheid PCR Analyzer using SYBR Green I (Stratagene) as described elsewhere (Zhang et al., 2003Go), using primers for CYP1A1, CYP1B1, and ß-actin. The cycle threshold (CT) of each sample was automatically determined to be the first cycle at which a significant increase in optical signal above an arbitrary baseline set at 30 fluorescence units was detected. All determinations were done in triplicate and experiments were repeated at least twice. The values shown for fold-induction are calculated by raising 2 to the power of the CT ratios of experimental to control cells normalized to ß-actin mRNA in the same sample.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
JNK and ERK are critical for AHR/ARNT activation.Our previous studies showed that TCDD caused an immediate induction of JNK and ERK activities that might be involved in Ah receptor activation (Tan et al., 2002Go). If these MAPK activities were essential in that context, their inhibition would prevent AHR activity and AHR-dependent gene expression. Pharmacological inhibitors have been widely used to suppress MAPK activation, but many of these compounds are flavones that are structurally similar to AHR ligands, raising the possibility that they might bind to and affect AHR function independently of MAPK inhibition. We first tested whether the commonly used MAPK inhibitors interacted directly with the AHR in an in vivo competition assay. Hepa-1 cells were incubated with 3H-TCDD with or without prior treatment with competitors that included TCDD, B[a]P, and various MAPK inhibitors. The AHR was immunoprecipitated, and the amount of 3H-TCDD bound to AHR under the various experimental conditions was determined. TCDD and B[a]P blocked more than 90% of 3H-TCDD binding to endogenous AHR (Fig. 1A), whereas neither the p38 inhibitor SB202190 nor the ERK inhibitor U0126 affected the capacity of TCDD to bind to AHR. Other compounds, including SP600125, a JNK inhibitor, and PD98059, a second ERK inhibitor, inhibited up to 70% of 3H-TCDD binding, a finding in agreement with those reported by others (Fig. 1A; Joiakim et al., 2003Go; Reiners, et al., 1998Go). These results validate the use of U0126 and SB202190 to investigate the roles of ERK and p38, respectively, in TCDD-stimulated AHR activity, whereas SP600125 and PD98059 will not be suitable for such study, because they themselves might be AHR agonists or antagonists. This feature of SP600125 is of particular importance, because this compound has been used as a potential therapeutic agent to suppress inflammatory responses in diseases like arthritis (Han et al., 2001Go). Its AHR binding activity would, no doubt, complicate its clinical application.



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FIG. 1. Inhibition of ERK significantly suppresses AHR-dependent gene induction by TCDD. (A) Hepa-1 cells were either untreated or treated with various unlabeled competitors, including TCDD (200 nM), B[a]P (20 µM), and various MAPK inhibitors at 5 µM, followed by incubation with 3H-TCDD (2 nM) for 1 h, as described in Materials and Methods. Cell lysates were used for immunoprecipitation with anti-AHR, and the 3H-TCDD in the precipitates was measured and compared to that of samples in the absence of the competitors. (B) Hepa-1 cells were deprived of serum for 24 h and pretreated for 0.5 h with 5 µM U0126, followed by stimulation with 5 nM TCDD for the indicated times. Cell lysates were processed for Western blotting using anti-CYP1A1 and anti-ß-actin. Inhibition of ERK causes a significant reduction in AHR-dependent transcription and CYP1A1 expression. SP = SP600125 JNK inhibitor; SB = SB202190 ERK inhibitor; U = U0126 ERK inhibitor; PD = PD98059 ERK inhibitor.

 
To assess whether ERK is required for TCDD to induce endogenous AHR/ARNT-regulated genes, we measured CYP1A1 protein in Hepa-1 cells treated with TCDD in the presence of U0126. Basal expression of CYP1A1 was minimal in untreated Hepa-1 cells. TCDD treatment induced CYP1A1 18-fold at 4 h and 31-fold at 12 h (Fig. 1B). However, U0126, by itself, was a weak ligand that induced AHR nuclear translocation (data not shown) and a sevenfold increase of CYP1A1 expression, and it caused a marked suppression of CYP1A1 induction by TCDD (Fig. 1B).

The only available specific JNK inhibitor is SP600125; hence, a study similar to the one described above could not be done for JNK. To investigate the role of JNK on AHR/ARNT–dependent gene expression, we studied CYP1A1 induction in MEFs prepared from Jnk1(–/–)Jnk2(–/–) double-knockout fetuses, which lack JNK1 and JNK2 activities. In agreement with our previous observations (Tan et al., 2002Go), wild type MEFs exhibited very low, if any, basal CYP1A1 expression, and TCDD, by 4 h of treatment, caused an obvious induction of both CYP1A1 mRNA and protein. The induction was markedly reduced to almost basal levels in Jnk1(–/–)Jnk2(–/–) MEFs (Fig. 2A and 2B). Because wild type and Jnk1(–/–)Jnk2(–/–) fibroblasts have similar levels of AHR and ARNT (Fig. 2C), we conclude that lack of JNK is the major cause of CYP1A1 reduction in these cells. Together, these results indicate that JNK and ERK MAP kinases are essential for maximal induction of an AHR/ARNT–dependent gene by TCDD.



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FIG. 2. JNK plays a critical role in TCDD-stimulated CYP1A1 expression in MEFs. Wild type and Jnk1(–/–)Jnk2(–/–) MEFs were starved as described in the text, and were either left untreated or were treated with 2 nM TCDD or DMSO vehicle for the indicated times. (A) RNAs isolated from these cells were subjected to real-time RT-PCR for Cyp1a1 and ß-actin mRNA. Cell lysates were analyzed by Western blotting using antibodies against (B) CYP1A1 and ß-actin, and (C) AHR and ARNT, respectively.

 
JNK2 is involved in CYP1A1 induction by TCDD in thymus and testis, but not liver. To study the in vivo role of JNK in TCDD-stimulated AHR activity, we examined wild type and Jnk2-knockout mice for the expression of Cyp1a1 and Cyp1b1, two AHR-dependent genes that are known to be induced by TCDD in a tissue-specific manner (Gonzalez, 2001Go). Like wild type mice, Jnk2-null mice express AHR and ARNT, and they have undetectable levels of basal CYP1A1 in all tissues examined (Fig. 3A and 3B). CYP1A1 expression was induced by 5 µg/kg or 50 µg/kg TCDD, and induction levels were greater in liver than in thymus or testis (Fig. 3B and 3C). Ablation of JNK2 led to a slight increase of TCDD-dependent induction of CYP1A1 mRNA and protein in the liver (Fig. 3C); in contrast, induction was very significantly reduced (p < 0.001) in thymus and testis of the Jnk2 knockout below that of the wild type (Fig. 3A and 3B).



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FIG. 3. JNK2 is required for TCDD to stimulate CYP1A1 expression in thymus and testis, but not liver. The indicated tissues from wild type and Jnk2(–/–) mice were examined for the expression of (A) AHR and ARNT, (B) CYP1A1 and ß-actin by Western blot analyses, and (C) mRNA of Cyp1a1, Cyp1b1, and ß-actin by real-time RT-PCR. The RNA levels are calculated as described in the Materials and Methods, and the results represent fold induction relative to untreated cells normalized to ß-actin levels.

 
Cytochrome P450 1B1 was abundantly expressed in the testis of both wild type and Jnk2-null mice, where its mRNA levels could not be induced further by TCDD treatment (Fig. 3C and data not shown; Mandal et al., 2001Go). In addition, CYP1B1 mRNA was present in liver and thymus at levels much lower than those in testis. In liver, CYP1B1 induction by TCDD was also higher in Jnk2(–/–) than in wild type mice, whereas in thymus, its induction was unaffected by JNK2 ablation (Fig. 3C). We conclude from these studies that JNK2 plays a tissue-specific role in the regulation of AHR/ARNT activity, contributing significantly to the elevation of the TCDD-stimulated expression of CYP1A1, but probably not CYP1B1.

MAPK activation potentiates the activity of AHR/ARNT complexes in gene expression. Next, we asked how the MAPKs affect the activity of AHR/ARNT complexes. As the MAPKs are known for their functions in the nucleus to modulate the activity of transcription factors, we hypothesized that the MAPKs might be involved in the induction of the transcriptional activity of AHR and ARNT in the nucleus. To investigate the possible cross-talk between the MAPKs and AHR complexes, we chose to use the African Green Monkey kidney cell line CV-1, which does not express endogenous AHR, but expresses ARNT. Ectopic expression of exogenous AHR in these cells results in ligand-independent constitutive AHR nuclear localization (Chang and Puga, 1998Go). This is an important experimental characteristic that makes these cells most suitable for studying the direct effects of MAPKs on nuclear AHR/ARNT complexes, bypassing the requirement for TCDD treatment to induce receptor transformation and translocation.

In agreement with previous reports (Chang and Puga, 1998Go), ectopic expression of exogenous AHR and ARNT in CV-1 cells in the absence of ligand resulted in high basal levels of luciferase expression from an AHR-dependent luciferase reporter. Previous studies have identified MAPKKKs and MAPKKs that activate specific MAPKs: Raf1 and MEK1(EE) are activators of the ERK pathway (Cobb and Goldsmith, 1995Go), MEKK1 preferentially activates the JNK MAPK (Zhang et al., 2003Go), and MEK6 (DD) is an upstream activator for p38 (Raingeaud et al., 1996Go). We expressed individual MAPK activators, together with AHR and ARNT. The AHR/ARNT-dependent luciferase activities were significantly increased in cells that co-expressed MEK1(EE), a mutant form of MEK1 that constitutively activates the ERK pathway (Fig. 4A). In contrast, the expression of MEK6 (DD), a constitutively active kinase specific for the p38 pathway, did not have an overt effect, confirming our previous observation that p38 did not play a role in TCDD-stimulated Ah receptor activation (Tan et al., 2002Go). In the absence of AHR, MEK1(EE) by itself did not activate luciferase expression (data not shown). MEK1(EE)-stimulated AHR activity depended on the activation of ERK, because U0126 prevented nearly 80% of the MEK(EE)-induced and AHR-dependent luciferase activity, whereas SB202190, an inhibitor of the p38 pathways, did not affect it significantly (Fig. 4A).



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FIG. 4. Induction of ERK and JNK by their upstream activators potentiates AHR/ARNT–dependent transcription. CV-1 cells were transfected with pAhRDTKLuc3, together with a mammalian expression vector for ß-galactosidase, AHR, and ARNT. Co-transfection was carried out using expression vectors for (A) constitutively active MEK1 [MEK1(EE)], and MEK6 [MEK6(DD)] or (B) Raf1BXB and {Delta}MEKK1. (C) Transfected cells were treated with EGF (10 ng/ml), TPA (10 nM), and bovine serum (10 %), without or with TCDD (5 nM) for 16 h. Some of the transfected cells were also pretreated with either DMSO vehicle or 5 µM U0126 or SB202190 for 16 h before harvesting. The cell lysates were prepared 40–48 h after transfection and luciferase, and ß-galactosidase activities were measured. Luciferase activities were normalized to ß-galactosidase activities. SEM was calculated from four independent experiments. Statistical analyses were done by t-tests, and p < 0.01 was denoted as significant (*).

 
A role for the MAPKs in AHR/ARNT activity was further investigated by overexpression of the Raf1 kinase domain (Raf1BXB) and of an active MEKK1 ({Delta}MEKK1), the two constitutively active MAPK kinase kinases for the ERK and the JNK pathways, respectively. Co-expression of Raf1BXB or of {Delta}MEKK1 with AHR and ARNT stimulated AHR-dependent luciferase activity fourfold and sevenfold, respectively (Fig. 4B). When the transfected cells were treated with U0126 and SB202190 at concentrations specific for their designated pathways (Favata et al., 1998Go; Manthey et al., 1998Go), the increase of AHR-dependent luciferase activity by active Raf1 was suppressed by 44% upon inhibition of the ERK. The p38 pathway did not appear to have a role in Raf1 signaling, as SB202190 failed to exert an effect on Raf1-induced AHR-ARNT-dependent luciferase activity. Neither the ERK nor the p38 inhibitors suppressed the MEKK1-stimulated increase of AHR/ARNT transcriptional activity (Fig. 4B), in agreement with the known role of MEKK1 as a specific activator of the JNK pathway (Zhang et al., 2003Go). Together, these data suggest that activation of ERK and JNK, but not p38, enhances the activity of the nuclear AHR/ARNT complex in gene expression.

The extracellular factors that activate the MAPKs might activate transcription directed by AHR/ARNT complexes. Indeed, treatment of the AHR/ARNT transfected CV-1 cells with EGF or with serum, both known extracellular stimuli that activate JNK and ERK (Assefa et al., 1997Go), resulted in a fourfold to fivefold induction of AHR-dependent luciferase activity (Fig. 4C). The enhancement of AHR-dependent luciferase activity by TCDD was further elevated 2.5-fold to threefold after treatment with EGF and serum (Fig. 4C). Not all stimuli that induce MAPK activity, however, lead to the activation of the AHR/ARNT complex. For example, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC that activates ERK (She et al., 2002Go), failed to exert an effect on AHR-dependent luciferase expression (Fig. 4C). Hence, activation of the PKC-ERK pathway is not sufficient, and EGF and serum must activate additional cellular events to potentiate AHR/ARNT–dependent transcription.

The MAPKs enhance the transcriptional activity of ARNT, but not AHR. To examine the roles of MAPKs in the transcription activity of AHR and ARNT, we tested fusion proteins containing the GAL4 DNA binding domain fused to AHR or ARNT for their ability to stimulate the expression of a luciferase reporter driven by a GAL4 promoter. We examined the activation of GAL4-AHR in CV-1 cells, which possess normal ARNT levels but are negative for AHR. Expression of GAL4-AHR led to a fivefold induction of luciferase activity over GAL4 alone, suggesting that GAL4-AHR is already transcriptionally active (data not shown). Active MEKK1 and Raf1 increased luciferase activity 2.5-fold and 4.5-fold, respectively, whereas expression of kinase-inactive MEKK1 did not cause any changes in luciferase activity (Fig. 5A). However, when we looked at GAL4-AHR activation in c4 cells, a Hepa-1 derivative lacking functional ARNT, co-expression of active MEKK1 or Raf1 did not further increase luciferase activity over that induced by GAL4-AHR alone. As the active MAPKKKs potentiate AHR transcriptional activity only in ARNT-positive cells, but not in ARNT-negative cells, these results favor the idea that the MAPKs function through the activation of ARNT, the heterodimerization partner of AHR.



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FIG. 5. The MAPKKKs stimulate the transcription activity of ARNT, but not AHR. CV-1, c4 cells and Ahr–/– MEFs were transfected with a plasmid for ß-galactosidase, together with those for (A) GAL4-luciferase reporter and GAL4-AHR, (B) GAL4-luciferase reporter and GAL4-ARNT, and (C) ARNT with pAd-luc or AHR and ARNT with pAhRDTKLUC3. Also co-transfected were either an empty vector, {Delta}MEKK1, MEKK1 (KM), Raf1(BXB), or MEK1(EE), as indicated. Then, 24 h after transfection, the cells were serum-starved for an additional 24 h and harvested; the lysates were measured for luciferase and ß-galactosidase activities. Fold induction by the MAPKKKs versus control was calculated based on the relative luciferase activities normalized for transfection efficiency to the ß-galactosidase activity. Results represent the average of 4–6 experiments.

 
To test directly the effects of MAPKs on ARNT activity, we used GAL4-ARNT to measure ARNT-dependent luciferase activity in CV-1 and AHR-null MEFs, both of which are negative for endogenous AHR. Ectopic coexpression of GAL4-ARNT with MEKK1 led to an enhancement of luciferase activity over control of threefold and fivefold in CV-1 and AHR (–/–) MEFs, respectively, whereas Raf1 caused sevenfold and fourfold increases (Fig. 5B).

In addition to being a partner of AHR and HIF-1{alpha}, ARNT also forms homodimers that bind to the E box core sequence CACGTG within the adenovirus major late promoter (Sogawa et al., 1995bGo). This feature allowed us to measure ARNT/ARNT complex activity directly, using the adenovirus promoter-driven luciferase reporter pAd-luc. In AHR-negative CV-1 cells, induction of pAd-luc by ARNT overexpression was further induced eightfold and fourfold, respectively, by {Delta}MEKK1-activated JNK and MEK(EE)-activated ERK. Similar induction of luciferase activities was observed on pAhRDTKLuc3, the expression of which was regulated by the AHR/ARNT complexes (Fig. 5C). These data confirm that MAPK-mediated ARNT activation can lead to the transcriptional activation of either ARNT-ARNT homodimers or ARNT-AHR heterodimers.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Based on our previous findings that TCDD activates ERK and JNK (Tan et al., 2002Go), we now focus on developing an understanding of the mechanisms through which the MAPKs regulate AHR/ARNT functions. Our results show that the MAPKs function in the nucleus to potentiate ARNT transcriptional activity and AHR/ARNT–dependent gene expression. The induction of AHR/ARNT activity by the MAPKs is fivefold to tenfold less than the induction by TCDD. Possibly, TCDD makes major contribution to AHR/ARNT activity by binding to and inducing the conformational changes necessary for the cytosolic Ah receptor to translocate to the nucleus and activate. Once the AHR is in the nucleus, the MAP kinases enhance the transcriptional activity of the AHR/ARNT complex and increase the final levels of gene induction. Within the complexes, the MAPKs appear to modulate the transcriptional activity of ARNT, but not of AHR. It is known that AHR is phosphorylated at a tyrosine residue, important for its ability to bind to DNA (Minsavage et al., 2003Go); this phosphorylation is obviously not directly catalyzed by the serine/threonine MAPKs. Similar to their roles in modifying many other transcription factors, the MAPKs might regulate ARNT activity through post-translational modification mechanisms (Hunter and Karin, 1992Go). Emerging evidence indicates that, in vivo, the AHR/ARNT complex is associated with numerous transcription coactivators and/or corepressors, many of which, including p300, p160, RB, and SMRT, are regulated or modified by MAP kinases. Modulation of co-factors might also be a mechanism through which the MAPKs influence the transcriptional activity of the AHR/ARNT complex (Carlson and Perdew, 2002Go).

How much the ARNT contributes to the transcriptional activity of the AHR/ARNT complex remains a controversial issue (Reisz-Porszasz et al., 1994Go; Sogawa et al., 1995aGo). Important in this context is that ERK inhibition and JNK1 and JNK2 depletion lead to suppression of TCDD-induced CYP1A1 expression. Furthermore, the Jnk2-null mice have reduced CYP1A1 induction in thymus and testis, but not in liver. Although in vitro, JNK2 displays very little, if any, cell-type specificity in activity and function, in vivo, it functions in a tissue-specific and stimulus-dependent manner, required for thymocyte development (Sabapathy et al., 1999Go; Conze et al., 2002Go). In fact, studies of genetically modified mice have revealed the tissue-specific roles for other MAPKs (Mazzucchelli et al., 2002Go; Adams et al., 2000Go). Hence, the MAPKs might determine or contribute to ARNT activation in a tissue-specific manner, with JNK2 being critical for some tissues and ERK for others. Our findings might be relevant to unexplained tissue-specific toxicity of TCDD, which, for example, causes hypertrophy of the liver, but atrophy of the thymus and testis (Staples et al., 1998Go; Whitlock, 1999Go). Atrophy of thymus and testis might be related to JNK2 stimulation by TCDD and a combination of ensuing CYP1A1 induction with other downstream events. Unlike CYP1A1, CYP1B1 is transcriptionally regulated through both AHR-dependent and cAMP-dependent mechanisms (Zheng et al., 2003Go). We find that CYP1B1 expression is unaffected by JNK ablation in all tissues examined. Perhaps in tissues where cAMP signals are intense, JNK2-regulated AHR/ARNT activity is less important for CYP1B1 transcription. The contrast between the effects on CYP1A1 and CYP1B1 expression suggests that the role of JNK in AHR activity is not only tissue-specific but also gene-specific. Jnk1(–/–)Jnk2(–/–) double knockout mice are not viable, and so it is unfeasible to test whether complete JNK ablation in mice would lead to a more profound effect on the expression of CYP1A1 and CYP1B1.

Growth promoter and MAPK activators, such as EGF and serum, further potentiate TCDD-induced AHR activities, suggesting that maximal AHR activation requires the participation of other environmental and extracellular factors. These observations are particularly intriguing because, in addition to being targets for TCDD, the JNK and ERK are controlled by many other growth and environmental signals. The MAPKs might serve as a centerpiece for signal integration in the regulation of AHR/ARNT–mediated gene expression. Activation of MAP kinases might be an alternative combinatorial mechanism by which TCDD and other environmental agents participate in the regulation of AHR functions. The involvement of MAP kinases in TCDD signaling may have a broad impact on the diversity and tissue-specific TCDD toxic, adaptive and biological effects.


    ACKNOWLEDGMENTS
 
We thank Drs. Timothy Dalton and Daniel W. Nebert for critical reading of the manuscript and for valuable suggestions. We also thank Shujie Shi and Xiaoqing Chang for making the GAL4-ARNT mammalian expression construct, and Maureen Mongan and Tong Shen for technical assistance. This research was supported in part by National Institute of Environmental Health Sciences grants P30 ES06096, P42 ES04908, ES11798, ES06273, and by a grant from Philip Morris USA.


    NOTES
 

1 To whom correspondences should be addressed at Department of Environmental Health, University of Cincinnati Medical Center, 123 E. Shields Street, Cincinnati, OH 45267-0056. Fax: (513) 558-0974. E-mail: xiay{at}email.uc.edu.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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