The Role of Protein Kinase C in Regulation of TCDD-Mediated CYP1A1 Gene Expression
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《毒物学科学杂志》
Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, California 92093–0722
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093–0722
ABSTRACT
Cytochrome P450 1A1 (CYP1A1) is induced by halogenated and polycyclic aromatic hydrocarbons following activation of the aryl hydrocarbon receptor (AhR). Protein kinase C (PKC) has been implicated in the regulation of this response. In tissue culture, induction of PKC activity with phorbol esters synergizes the actions of TCDD-induced CYP1A1, while PKC inhibitors block induction of CYP1A1 by TCDD. Here, the actions of specific PKC inhibitors on CYP1A1 induction were examined using a HepG2 human cell line (TV101L) that carries a stably integrated firefly luciferase gene under control of the human CYP1A1 promoter (–1612/+293). TV101 cells were treated with TCDD and either the kinase inhibitor staurosporine or one of the PKC inhibitors GF109203X, G6983, or G6976. Aryl hydrocarbon receptor-dependent activation of CYP1A1-luciferase and cellular PKC activity were measured. TCDD treatment induced CYP1A1-luciferase activity in an AhR-dependent manner, as determined by binding of nuclear AhR to xenobiotic response elements (XREs). Dose-dependent inhibition of PKC activity by staurosporine was concordant with inhibition of TCDD-induced CYP1A1-luciferase activity. However, the PKC inhibitors GF109203X, G6983, and G6976 blocked PKC activity at concentrations independent of those necessary to block TCDD induction of CYP1A1-luciferase activity. For all inhibitors, reduction in CYP1A1-luciferase activity was independent of AhR activation, as determined by electrophoretic mobility shift analysis of TCDD-activated nuclear AhR. The specific PKC inhibitors did not significantly alter cytosolic or nuclear levels of AhR protein, whether alone or in combination with TCDD. These results suggested that PKC was not the sole factor responsible for regulation of CYP1A1.
Key Words: protein kinase C; cytochrome P450 1A1; regulation; gene expression; signal transduction.
INTRODUCTION
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon (HAH), commonly produced by incineration of municipal waste (Viel et al., 2000), and as a byproduct of pulp and paper bleaching (Schweer and Jennings, 1990). It is an endocrine disrupter (Safe et al., 1998) as well as a potent carcinogen, co-carcinogen, and teratogen (Poland and Kende, 1976; IARC, 1997; Steenland et al., 2004). TCDD induces cytochrome P450 1A1 (CYP1A1) through a ligand-dependent process mediated by the aryl hydrocarbon receptor (AhR). The AhR is a cytosolic phosphoprotein that is part of the basic helix-loop-helix (bHLH) protein family (Wilson and Safe, 1998). In the absence of ligand, the AhR is complexed with heat shock protein-90 (hsp90) and X-associated protein 2 (XAP2) in the cytosol (Meyer and Perdew, 1999). Upon ligand binding, hsp90 and XAP2 are released, and the liganded AhR is translocated to the nucleus, where it forms a heterodimer with the nuclear Arnt protein (Hoffman et al., 1991). The liganded AhR-Arnt heterodimer then binds to xenobiotic response elements (XREs) and initiates transcription of CYP1A1 (Reyes et al., 1992).
Activation of the AhR to a DNA-binding transcription factor is highly dependent on the phosphorylation state of the AhR complex, and AhR activation is linked to protein kinase C (PKC) activity. In vitro exposure of cytosol or nuclear extracts to acid or alkaline phosphatase renders the AhR unable to bind to XREs and activate transcription of CYP1A1 (Berghard et al., 1993; Carrier et al., 1992; Mahon and Gasiewicz, 1995; Pongratz et al., 1991). Short-term treatment of cells with phorbol esters such as PMA, which mimic diacylglycerol, leads to PKC activation (Castagna et al., 1982). Limited treatment with phorbol-12-myristate-13-acetate (PMA) plus TCDD causes synergistic activation of the CYP1A1 gene, as determined by increases in a human CYP1A1 promoter–luciferase reporter construct stably integrated into HepG2 cells (Chen and Tukey, 1996). Treatment with TCDD plus the kinase inhibitor staurosporine causes a nearly complete block in TCDD-initiated induction of CYP1A1-luciferase transcription (Chen and Tukey, 1996). However, at levels of staurosporine that inhibit cytosolic PKC activity, translocation of the liganded AhR to the nucleus and AhR binding to DNA are not inhibited (Schafer et al., 1993). Other studies indicate that the synergy in TCDD-initiated induction of CYP1A1 by phorbol esters does not require the transactivation domains of the AhR or Arnt (Long and Perdew, 1999; Safe et al., 1998). Together, these results indicate that PKC activity is required for nuclear events in the CYP1A1 transcriptional pathway (Chen and Tukey, 1996).
Protein kinase C–mediated signal transduction takes place through a family of PKC isoforms that exhibit differences in substrate specificity and cellular components necessary for activation. Activation of both conventional and novel PKC isoforms is dependent on diacylglycerol and phosphatidylserine, while conventional isoforms also require intracellular Ca2+ for activation (Kishimoto et al., 1980; Konno et al., 1989; Ohno et al., 1988;). Atypical PKC isoforms can be stimulated by phosphatidylserine (Akimoto et al., 1994; Ways et al., 1992). Staurosporine, a potent inhibitor of PKC activity, also inhibits many other protein kinases, such as tyrosine kinases, protein kinase A, protein kinase G, and calcium-calmodulin kinase (Fujita-Yamaguchi and Kathuria, 1988; Gadbois et al., 1992; Niggli and Keller, 1991). Recent advances in the characterization of the individual PKC isoforms have led to the identification of more selective chemical inhibitors. To study the role of PKC in regulation of CYP1A1, we examined AhR activation and CYP1A1 transcription in the presence of various pharmacological inhibitors known to be more selective for PKC than staurosporine.
MATERIALS AND METHODS
Chemicals.
2,3,7,8-tetrachlorodibenzo-p-dioxin was obtained from Wellington Laboratories (Guelph, Ontario, Canada). G6976, G6983, and GF109203X were obtained from Calbiochem (La Jolla, CA). Phorbol-12-myristate-13-acetate and staurosporine were purchased from Sigma Chemical Company (St. Louis, MO). D-luciferin, potassium salt, was purchased from Analytical Luminescence Laboratories (Ann Arbor, MI) and from Pharmingen (San Diego, CA). Bradford reagent dye concentrate was obtained from Bio-Rad (Hercules, CA). Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Cellgro (Herndon, VA). Fetal bovine serum was purchased from Omega Scientific (Tarzana, CA). Other tissue culture reagents were purchased from GIBCO/BRL (Grand Island, NY and Bethesda, MD). -32P-dCTP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Phosphatidylserine, diacylglycerol, rat brain lysate, and the PKC substrate peptide Ac-FKKSFKL-NH2 were kind gifts of Dr. Alexandra Newton (University of California San Diego, La Jolla, CA). Klenow fragment of DNA polymerase was obtained from Invitrogen (Carlsbad, CA). Poly(dI-dC) was obtained from Roche Molecular Biochemicals (Indianapolis, IN). Salmon sperm DNA was obtained from Life Technologies (Bethesda, MD). The rabbit anti-AhR antibody was a kind gift of Dr. Christopher Bradfield (McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI). Mouse anti--actin was obtained from Sigma. Goat anti-rabbit IgG-horseradish peroxidase and anti-mouse IgG-horseradish peroxidase were obtained from Cell Signaling Technologies (Beverly, MA). Western Lightning enhanced chemiluminescent substrate kit was obtained from PerkinElmer/NEN (Boston, MA). All other chemicals and reagents were of the highest purity available and were purchased from Sigma and Fisher Scientific.
Tissue culture and cell lines.
TV101L is a human hepatoma cell line derived from HepG2. It has been stably transfected with 1612 bp of 5' sequence of the human CYP1A1 gene, including the full promoter sequence and 5' flanking sequences, linked to the firefly luciferase gene, as previously reported (Postlind et al., 1993). TV101L cells were obtained from liquid nitrogen stocks in our laboratory and grown in monolayers on six-well, 10-cm, and 15-cm tissue culture plates. They were maintained at 37°C in 95% air and 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 20 mM HEPES, and 0.8 mg/ml G418. Only cells passaged 25 times or fewer which were 60% to 90% confluent were used for experiments.
Luciferase assay.
TV101L cells were maintained on 10-cm plates until the monolayer covered 60% to 90% of the plate surface area (60% to 90% confluent), split to six-well tissue culture plates, and used in experiments 18–24 h later. Chemical solutions were prepared by dissolving compounds in dimethyl sulfoxide (DMSO). Compounds were added to the culture media, and DMSO concentration in the culture media never exceeded 0.3% v/v. Compounds did not cause changes in cellular morphology or cell death. At the end of the treatment period, culture media was removed by aspiration, and cells were rinsed twice with 37°C phosphate-buffered saline (PBS). Cells were incubated with a lysis buffer containing 1% Triton X-100, 25 mM Tricine pH 7.8, 15 mM MgSO4, 4 mM EDTA, and 1 mM DTT at 37°C for 20 min for maximum luciferase recovery. Cells were then collected by scraping, and cells and lysis buffer were transferred to microcentrifuge tubes and centrifuged at 14,000 rpm for 12 min at 4°C. Supernatants were collected into separate microcentrifuge tubes and frozen at –70°C until assay. Protein concentration was determined by the method of Bradford (1976). Luciferase activity was determined by mixing 300 μl of sample buffer (25 mM Tricine pH 7.8, 15 mM MgSO4, 4 mM EDTA, 1 mM DTT, 2 mM ATP) with 10 μl of supernatant. Reactions were initiated by adding 100 μl of 1 mM D-luciferin (potassium salt) in 15 mM potassium phosphate buffer, pH 7.8. Light output was measured for 10 s at 25°C in a Monolight 2001 luminometer (Analytical Luminescence Laboratories, Ann Arbor, MI). Luciferase activity was normalized to the protein concentration of each sample.
PKC activity assay.
Lipid solution containing 1.4 mM phosphatidylserine and 38 μM diacylglycerol in 20 mM HEPES, pH 7.4 was prepared from CHCl3 stocks. Solvent was evaporated under a stream of N2, and the remaining lipid film was hydrated with 20 mM HEPES, pH 7.4, vortexed, and sonicated briefly in a bath sonicator. TV101L cells were grown on six-well tissue-culture–treated plates and treated as shown in Results. Treatments did not result in abnormal cellular morphology or cell death. Media were removed by aspiration, and cells were rinsed twice with ice-cold PBS. Cells were lysed in an ice-cold lysis buffer containing 1% Triton X-100, 20 mM HEPES pH 7.4, 85 μM leupeptin, 2 mM benzamidine, 1 μM microcystin, 200 μM PMSF, 1 mM DTT, 1 mM EDTA pH 7.4, and 1 mM EGTA pH 7.4, and scraped into microcentrifuge tubes. Cell lysates were centrifuged at 14,000 rpm for 12 min at 4°C, and supernatants were mixed with glycerol to a final concentration of 50% (v/v) and frozen at –20°C until assayed. Samples were prepared for assay under PKC activating conditions by diluting 4 μl of each supernatant with 8 μl of 5 mM CaCl2, 8 μl lipid solution, and 44 μl enzyme buffer (2 mM DTT, 20 mM HEPES, pH 7.4). A parallel set of samples was prepared for assay under non-activating conditions by diluting 4 μl of each supernatant with 8 μl of 5 mM EGTA and 52 μl enzyme buffer. The reaction was immediately initiated by adding 16 μL GO buffer containing 20 mM HEPES pH 7.4, 100 μM ATP, 5 mM MgCl2, 100 μg/ml substrate peptide Ac-FKKSFKL-NH2, and 1 μCi -32P-ATP. The reaction was allowed to proceed for 8 min at 30°C, and was stopped by adding 25 μl STOP buffer containing 0.1 M ATP and 0.1 M EDTA, pH 8. 85 μl from each stopped reaction was spotted onto rectangular strips of Whatman P81 cation exchange paper (Fisher Scientific) approximately 1.5 in. x 0.75 in. in size. Strips were then washed four times for 5 min each in 500 ml 0.4% H3PO4 to remove unincorporated -32P-ATP, followed by one 5-min wash with 100% EtOH. Strips were then transferred to scintillation vials and counted in 5-ml Ecolite scintillation fluid (ICN, Costa Mesa, CA) in an LS-6800 liquid scintillation counter (Beckman Coulter, Fullerton, CA). Protein kinase C activity was defined as the ratio of nmol phosphate transferred to the substrate per minute under activating conditions to the nmol of phosphate transferred to the substrate per minute under non-activating conditions.
Preparation of nuclear protein.
The method of preparation of nuclear protein was adapted from Miller and colleagues and others (Harper et al., 1991; Miller et al., 1983). TV101L cells were grown on 15-cm tissue culture plates. Compounds were added to culture medium, and DMSO concentration in the culture medium never exceeded 0.2% v/v. Compounds did not cause abnormal cellular morphology or cell death. At the end of the treatment period, culture medium was removed by aspiration, and cells were rinsed twice with 20 ml 10 mM HEPES at 4°C. Cells were incubated with 10 ml 10 mM HEPES at 4°C for 15 min. HEPES was removed by aspiration, and 2 ml MDH (3 mM MgCl2, 1 mM DTT, 25 mM HEPES, pH 7.5, plus protease inhibitors) was added at 4°C. Cells were collected by scraping and transferred to separate 15-ml centrifuge tubes on ice. Each group of cells was then transferred to a 2-ml Dounce homogenizer, homogenized with 30 strokes at 4°C, and transferred to a fresh 15-ml centrifuge tube. After homogenization, samples were centrifuged at 2500 rpm for 5 min at 4°C in a Sorvall swinging-bucket tabletop centrifuge. MDH was removed, and samples were resuspended in 1 ml ice-cold MDHK (3 mM MgCl2, 1 mM DTT, 25 mM HEPES, 0.1 M KCl, pH 7.5, plus protease inhibitors), centrifuged at 2500 rpm for 5 min at 4°C, and this process was repeated once. Samples were resuspended in 1 ml ice-cold MDHK, transferred to separate microcentrifuge tubes, and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was removed by aspiration, and samples were resuspended in 100 μl ice-cold HDK (25 mM HEPES, 1 mM DTT, 0.4 M KCl, pH 7.5, plus protease inhibitors). Samples were incubated on ice for 30 min, and were mixed during the incubation by gently inverting each tube 10 times once every 5 min. After this incubation, samples were centrifuged in a microcentrifuge at 14,000 rpm for 15 min at 4°C, and supernatants were transferred to polycarbonate tubes. Glycerol was added to a final concentration of 10% v/v, and samples were centrifuged in a TLA100.3 rotor at 50,000 rpm (105,000 x g) in a Beckman ultracentrifuge for 1 h at 4°C. Supernatants were immediately quick frozen in a dry ice / methanol bath and stored at –70°C until use. Protein concentration was determined by the method of Bradford (1976).
Preparation of cytosolic protein.
This method for preparing cytosolic protein was adapted from Harper (Harper et al., 1991). TV101L cells were washed twice with phosphate-buffered saline, and incubated for 15 min in HED buffer (25 mM HEPES pH 7.5, 1 mM EDTA, 1 mM DTT, plus protease inhibitors). Cells were collected by scraping and transferred to separate 15-ml centrifuge tubes on ice. Each group of cells was then transferred to a 2-ml Dounce homogenizer, homogenized with 30 strokes at 4°C, and transferred to a polycarbonate centrifuge tube. An equal volume of HED2G buffer (25 mM HEPES, 1 mM EDTA, 1 mM DTT, 20% glycerol, plus protease inhibitors) was added, and samples were centrifuged in a TLA100.3 rotor at 50,000 rpm (105,000 x g) in a Beckman ultracentrifuge for 1 h at 4°C. Supernatants were immediately quick frozen in a dry ice/ethanol bath and stored at –70°C until use. Protein concentration was determined by the method of Bradford (1976).
Oligonucleotide preparation and electrophoretic mobility shift assay (EMSA).
Two complementary DNA oligonucleotides with the sequence 5'-GATCTGAGCTCGGAGTTGCGTGAGAAGAGCCG-3' and 5'-GATCCGGCTCTTCTCACGCAACTCCGAGCTCA-3', containing the 27 bp AhR binding site of DRE3, designated here as DRE (Denison et al., 1988b), were synthesized commercially (GenBase, San Diego, CA). 2 μg of each oligonucleotide were annealed in a total volume of 20 μl by heating the mixture to 75°C for 5 min, then allowing the mixture to cool to 37°C for 90 min. The concentration of the double-stranded oligonucleotide (dsDRE) was determined using a spectrophotometer. 10 pmol of the dsDRE was radiolabeled in a total volume of 20 μl using the Klenow fragment of DNA polymerase (Invitrogen, Carlsbad, CA) and 5 μl -32P-dCTP (3000 Ci/mmol, 10 mCi/ml) according to the manufacturer's instructions. The labeling reaction was allowed to proceed for 3 h at room temperature and was stopped by the addition of 1 μl 0.1 M NaCl. Unincorporated radiolabel was removed using a Qiagen Nucleotide Removal Kit (Qiagen, Valencia, CA). Nuclear protein binding to dsDRE was measured by electrophoretic mobility shift assay (Denison et al., 1988a). Binding reactions were performed by preincubating 10 μg nuclear protein, 2.4 μg poly(dI-dC), 1 μg salmon sperm DNA, and binding buffer (25 mM HEPES, 1.5 mM EDTA, 1 mM DTT, and 10% glycerol, pH 7.5) for 15 min at room temperature. After preincubation, 1 x 106 cpm 32P-labeled dsDRE was added, and the reaction was allowed to proceed for an additional 15 min. Sufficient binding buffer was added to make the final reaction volume equivalent for all samples within a given EMSA. To ensure that the binding observed was specific for dsDRE, in each EMSA performed an excess of unlabeled dsDRE was added to one sample in which binding to dsDRE was expected to be observed. DNA–protein complexes were separated under nondenaturing conditions on a 6% polyacrylamide gel using 1x TBE (89 mM Tris borate, 89 mM boric acid, 2 mM EDTA, pH 7.5) as the running buffer. Gels were dried, and complexes were visualized by autoradiography.
Western blotting.
Protein samples were mixed with the appropriate amount of LDS sample buffer and reducing agent (Invitrogen, Carlsbad, CA), heated to 70°C for 10 min, cooled, pulse spun, and loaded on a 4–12% NuPAGE Bis-Tris pre-cast polyacrylamide gel (Invitrogen), and electrophoresed at 208 V for 40 min. Proteins from the gel were transferred to a polyvinylidine difluoride membrane (Millipore, Bedford, MA) for 70 min at 30 V in an XCell II blot module (Invitrogen) according to the manufacturer's instructions. Membranes were blocked overnight on a rotary shaker at 4°C in 100 ml TTBS (10 mM Tris-Cl pH 8, 15 mM NaCl, 0.1% Tween 20) with 5% dry milk. Between all incubations, membranes were washed three times for 10 min in TTBS. Membranes were incubated with primary antibody for 1 h on a rotary shaker at room temperature, and with secondary antibody for 1 h on a rotary shaker at room temperature. Bands were visualized using a enhanced chemiluminescent substrate kit according to the manufacturer's instructions (PerkinElmer/NEN, Boston, MA).
RESULTS
The treatment of TV101L cells with AhR ligands led to a dose-dependent increase in luciferase activity. Co-treatment with 5 nM TCDD and varying concentrations of staurosporine for 6 h led to a dose-dependent inhibition of CYP1A1-luciferase activity. In comparing the ability of staurosporine to inhibit cellular PKC activity and CYP1A1-luciferase, the IC50 values for PKC (IC50 = 20 nM) (Table 1, Fig. 1) were similar to those observed for CYP1A1-luciferase (IC50 = 57 nM).
Three PKC inhibitors were chosen for dose–response experiments on the basis of their reported ability to inhibit only specific isoforms of PKC. GF109203X and G6983 were bisindolylmaleimide analogs derived from staurosporine, but with modifications to their functional groups which increased their selectivity for PKC (Gschwendt et al., 1996; Martiny-Baron et al., 1993). G6976 was also derived from staurosporine, but it was a structurally dissimilar methyl and cyanoalkyl substituted nonglycosidic indolocarbazole (Martiny-Baron et al., 1993). All three compounds worked primarily by competitive inhibition of ATP binding to PKC, although G6976 also exhibited noncompetitive substrate inhibition of PKC and mixed inhibition with respect to lipid cofactors of PKC (Gschwendt et al., 1996; Martiny-Baron et al., 1993). Reported IC50 values for the PKC isoforms are shown in Table 2, and structures of each of these inhibitors are shown in Figure 2.
It is important to note that these more specific PKC inhibitors did not block the activity of one and only one PKC isoform. Each inhibitor acted against more than one isoform of PKC, but exhibited a unique pattern of which isoforms were blocked, and at what inhibitor concentration those particular PKC isoforms were blocked. It is only by comparison of the CYP1A1-luciferase and PKC activities observed with each of the three inhibitors tested, across a broad range of inhibitor doses, that meaningful conclusions about the actions of particular PKC isoforms could be deduced.
Dose–response experiments were conducted with these more specific PKC inhibitors to ascertain the role of PKC in regulation of CYP1A1-luciferase transcription. When TV101L cells were treated with any of these PKC inhibitors alone, CYP1A1-luciferase activity was the same as untreated TV101L cells (insets in Figs. 3–5). Thus, the PKC inhibitors themselves did not stimulate CYP1A1-luciferase transcription. Co-treatment with PKC inhibitors and TCDD led to dose-dependent decreases in TCDD-mediated CYP1A1-luciferase transcription, as well as to dose-dependent decreases in cellular PKC activity. However, these dose–response profiles were different than the dose–response profiles observed for staurosporine. For example, increasing concentrations of GF109203X led to a dose-dependent, gradual decrease in both CYP1A1-luciferase activity and PKC activity. When G6983 was examined, CYP1A1-luciferase activity was largely unaffected across a range of G6983 concentrations, but dropped sharply at higher G6983 concentrations (Fig. 4). Only higher concentrations of GF109203X and G6983 led to a comparable inhibition of PKC and CYP1A1-luciferase activity. Taken alone, these results suggest that a fraction of total cellular PKC activity was required to maximally stimulate CYP1A1-luciferase transcription. Thus, a large fraction of PKC activity could be abolished without affecting CYP1A1 transcription, but once PKC activity fell below a threshold value, induction of CYP1A1 transcription would decline.
If one or more isoforms of PKC were more important to regulation of CYP1A1 transcription than others, then PKC inhibitors with different isoform specificities would be expected to have different effects on CYP1A1-luciferase transcription. Taken together, the differences observed in the dose–response curves of each PKC inhibitor supported this hypothesis. Co-treatment with TCDD and G6976 (Fig. 5) yielded very different results from co-treatment with TCDD and GF109203X (Fig. 3) or with TCDD and G6983 (Fig. 4). Both CYP1A1-luciferase activity and cellular PKC activity were unaltered over a range of G6976 concentrations, but activity dropped sharply at higher G6976 concentrations. However, there was a range of G6976 concentrations at which there were no significant effects on cellular PKC activity, but CYP1A1-luciferase activity was inhibited by 75%. Cellular PKC activity was reduced only at high G6976 concentrations (1.5 μM), and 60% inhibition of cellular PKC activity was the maximum achieved using G6976. This finding was in contrast to experiments with GF109203X and G6983, which showed a range of inhibitor concentrations at which CYP1A1-luciferase activity was not significantly affected, but PKC activity was suppressed. This suggested that G6976 inhibited one or more isoforms of PKC that were more involved in regulation of CYP1A1 transcription than isoforms inhibited by GF109203X and G6983.
G6976 exhibited a unique dose–response profile compared to other PKC inhibitors tested, which suggested that G6976 acted against isoforms important to regulation of CYP1A1 transcription. Indeed, G6976 was the only inhibitor tested to have activity against PKCμ. Dose–response curves indicated that G6976 caused inhibition of the CYP1A1-luciferase response at lower concentrations than G6983. Given that conventional PKC isoform IC50 values for G6976 and G6983 were similar, yet only G6976 strongly inhibited PKCμ (Gschwendt et al., 1996; Martiny-Baron et al., 1993;), these results suggested that PKCμ played an important role in CYP1A1 transcriptional regulation. Because PKCμ accounted for a small amount of the total PKC present in most cell types (Johannes et al., 1994), a specific inhibitor such as G6976 could block a large proportion of PKCμ activity and CYP1A1 transcription, while not dramatically altering other PKC isoforms or the total cellular PKC activity.
Combined with comparisons of dose–response curves obtained and reported PKC isoform IC50 values, it appeared that more than one PKC isoform was involved in regulation of CYP1A1. All inhibitors tested could block conventional PKC isoforms, and conventional PKC isoforms were the most abundant PKC isoforms in most types of cells. Thus, conventional PKC isoforms most likely accounted for at least a fraction of CYP1A1 regulation. However, other PKC isoforms appeared to be less involved in regulation of CYP1A1. G6983 and GF109203X, but not G6976, inhibited PKC. Because G6976 did not inhibit PKC, but did block CYP1A1-luciferase activity effectively, this suggested that PKC was not linked to regulation of CYP1A1 transcription. Conversely, because G6983 and GF109203X inhibited PKC activity at a number of concentrations where CYP1A1-luciferase activity was not blocked, it can be inferred that PKC was not involved in the control of CYP1A1. Similar inferences could be made regarding other PKC isoforms based on dose–response data. GF109203X was the only inhibitor of the three tested that was reported to have activity against PKC (Gschwendt et al., 1996). However, a range of concentrations was observed at which GF109203X blocked PKC activity but did not affect CYP1A1-luciferase activity. This suggested that PKC was not linked to regulation of CYP1A1 transcription. Similarly, G6983 was the only compound tested that has been shown to inhibit PKC. There was also a range of concentrations observed at which G6983 blocked PKC activity but did not affect CYP1A1-luciferase activity. This suggested that PKC was also not linked to regulation of CYP1A1 transcription.
To determine if inhibition of the CYP1A1-luciferase response was linked to Ah receptor DNA binding, electrophoretic mobility shift assays were conducted on nuclear proteins isolated from TV101L cells co-treated with TCDD and concentrations of PKC inhibitor shown to block luciferase activity (Figs. 6 and 7). After 5 nM TCDD treatment for 6 h, nuclear accumulation of the Ah receptor was observed, as shown by nuclear protein binding to radiolabeled human XRE sequences. This binding was not observed when an excess of unlabeled XRE sequence was included in the binding reaction, which confirmed the specificity of the assay. No Ah receptor DNA binding was observed when nuclear proteins from untreated cells, or from cells treated with PKC inhibitor alone, were tested. Aryl hydrocarbon receptor DNA binding observed with nuclear proteins from cells co-treated for 6 h with the various PKC inhibitors and 5 nM TCDD was the same as observed with nuclear proteins from cells treated with TCDD alone. These results demonstrated that TCDD-initiated transcriptional activation of CYP1A1-luciferase could be dramatically inhibited without affecting the ability of the Ah receptor to accumulate in the nucleus and bind to DNA. This also suggested that the PKC inhibitors tested were not AhR ligands.
A previous report indicated that staurosporine reduced intracellular levels of the AhR (Singh and Perdew, 1993). To determine whether the specific PKC inhibitors used in this study altered intracellular levels of the AhR, cytosolic and nuclear proteins were isolated from TV101L cells treated with the maximum concentration of each inhibitor used in this work, in the presence or absence of TCDD. Aryl hydrocarbon receptor protein was measured by Western blotting with a rabbit anti-AhR antibody (Fig. 8). Treatment with any of the three specific PKC inhibitors alone did not significantly alter cytosolic levels of the AhR (Fig. 8a). Nuclear levels of the AhR were slightly increased (Fig. 8b), but this increase did not coincide with any impact on AhR binding to radiolabeled XREs (Figs. 6 and 7). Additionally, treatment with any of the three specific PKC inhibitors alone did not alter CYP1A1-luciferase transcriptional activity. Thus, the observed increase in nuclear AhR in response to these PKC inhibitors did not appear to have a functional impact on regulation of CYP1A1 induction. As expected, TCDD treatment reduced cytosolic levels of the AhR and strongly induced nuclear accumulation of the AhR. However, both cytosolic and nuclear levels of the AhR in TV101L cells co-treated with TCDD and any of the specific PKC inhibitors were similar to AhR levels observed in TV101L cells treated with TCDD alone (Figs. 8a, 8b). This also suggested that the PKC inhibitors' actions on PKC, and not any possible actions on the AhR, were the key determinants of their actions in influencing CYP1A1-luciferase transcription.
DISCUSSION
Previous work indicated that when PKC was stimulated using phorbol esters, a synergistic activation of TCDD-mediated induction of CYP1A1 occurred, and when cells were treated with the kinase inhibitor staurosporine, a corresponding suppression of TCDD-mediated induction of CYP1A1 occurred (Chen and Tukey, 1996). Thus, it was concluded previously that PKC played a role in the regulation of TCDD-mediated CYP1A1 induction. This was not surprising, as PKC was known to play an important role in cellular communication by relaying signaling events upstream of phospholipid hydrolysis to downstream kinases such as the MAP kinases and the AP-1 complex (Buchner, 2000). However, we tested three inhibitors that are more specific for PKC than staurosporine. Our findings indicated that inhibition of cellular PKC activity did not correspond with inhibition of TCDD-mediated CYP1A1-luciferase activity.
In these experiments, luciferase and PKC activities were measured in whole-cell lysates. This approach would yield a more physiologically relevant IC50 value, as it would more closely replicate the cellular environment in which these signaling pathways are found. Previous experiments to establish the published IC50 values of the inhibitors for PKC isoforms were biochemical and pharmacological experiments conducted with isolated, purified components. The IC50 values measured in a reaction of purified components would be expected to be much lower than those measured in a more physiological context, where an abundance of other proteins existed. For example, the PKC IC50 of staurosporine in rat anterior pituitary was measured to be over 100 times weaker than published PKC IC50 values of staurosporine in biochemical experiments (Simpson et al., 1993). Thus, IC50 values for CYP1A1-luciferase activity could not be directly compared to published IC50 values from biochemical experiments to establish the PKC isoform specificity involved in regulation of CYP1A1. However, relative dose–response trends between inhibitors used in the same experimental system could be compared, and useful data could be extracted from such comparisons nonetheless.
In theory, it was possible that the PKC inhibitors used in these experiments did not block the isoform(s) of PKC responsible for regulation of CYP1A1 induction, but they did block other PKC isoforms. In this case, total PKC activity would decrease, whereas CYP1A1-luciferase activity would decrease only when the particular PKC isoform(s) involved in regulation of CYP1A1 were blocked. However, dose–response results from G6976 contradicted this possibility. There was a wide range of G6976 concentrations tested at which CYP1A1-luciferase activity was strongly inhibited, and PKC activity was largely unaffected. This suggested that either G6976 was not an effective inhibitor of PKC, a claim that is strongly refuted by the body of PKC literature, or that G6976 was a more effective inhibitor of one or more low-abundance PKC isoforms involved in regulation of CYP1A1 than other inhibitors tested.
In fact, G6976 was the most effective inhibitor of CYP1A1-luciferase activity among the three more selective inhibitors tested. Both G6976 and staurosporine were effective against PKCμ at low nanomolar concentrations, whereas G6983 was a very weak inhibitor of PKCμ, and GF109203X did not block PKCμ at all. Staurosporine and the specific PKC inhibitors had comparable effectiveness against conventional PKC isoforms as well (Gschwendt et al., 1996; Martiny-Baron et al., 1993). However, staurosporine has been shown to inhibit a variety of other kinases in addition to PKC, including tyrosine kinases, protein kinase A, protein kinase G, and calcium-calmodulin kinase (Fujita-Yamaguchi and Kathuria, 1988; Gadbois et al., 1992; Niggli and Keller, 1991). Thus, one or more of the kinases inhibited by staurosporine, but not blocked by the more specific PKC inhibitors, could account for the remainder of CYP1A1 transcriptional regulation. Taken together, these experiments indicated that PKC activity may be responsible for only a fraction of CYP1A1 induction.
Electrophoretic mobility shift assays indicated that PKC inhibitors did not alter AhR-Arnt heterodimer binding to radiolabeled XRE sequences, whether used alone or in combination with TCDD. This was not unexpected, given previous results from Chen and Tukey (1996) and Long et al. (Long et al., 1998). Chen and Tukey demonstrated that staurosporine did not alter in vivo or in vitro TCDD-induced AhR-Arnt heterodimer binding to DNA, and that kinase inhibition by staurosporine was able to block transactivation of CYP1A1. Long et al. demonstrated that the more specific PKC inhibitors chelerythrine chloride and GF109203X did not alter AhR or Arnt levels in the cytoplasm or in the nucleus, nor did they alter TCDD-induced AhR-Arnt binding to radiolabeled XRE sequences. Long and Perdew (1999) also showed that the transactivation domains of AhR and Arnt were not required for the observed PMA synergy with TCDD in activation of CYP1A1. This suggested that the influence of PKC activity on TCDD-mediated induction of CYP1A1 took place at another level of the AhR signaling pathway, or that the influence of PKC was not a direct interaction with the AhR signaling pathway. Together with these previous results, our data suggest that the actions of PKC on activation of CYP1A1 transcription by TCDD more likely take place through the influence of PKC on other signaling pathways, which in turn regulate CYP1A1 transcription.
Western blots with an anti-AhR antibody indicated that the specific PKC inhibitors used in this study did not alter the levels of cytosolic AhR, whether used alone or in combination with TCDD. This suggested that the inhibition of CYP1A1-luciferase transcription observed was not due to a reduction in the amount of AhR available for ligand binding. The specific PKC inhibitors also had no effect the nuclear accumulation of AhR protein in response to TCDD. Although the PKC inhibitors alone did slightly increase nuclear AhR levels, this effect was shown to have no functional bearing on regulation of CYP1A1. This also suggested that nuclear translocation of the liganded AhR-Arnt complex was not affected by these inhibitors.
A 6-h time point was chosen to assess both CYP1A1-luciferase induction and PKC inhibition. Although PKC could be activated in a matter of minutes, and these inhibitors acted in a similar time frame, time points shorter than approximately 3 h would be insufficient for functional induction of CYP1A1-luciferase (Postlind et al., 1993). Additionally, Singh and Perdew used an 8-h staurosporine treatment to establish that staurosporine directly reduced intracellular levels of the AhR (Singh and Perdew, 1993). To effectively ensure that these PKC inhibitors did not affect the AhR in a similar manner, a similarly long treatment time needed to be used.
Although these experiments established that PKC was not as directly linked to regulation of TCDD-mediated induction of CYP1A1 as once thought, a full explanation of the mechanism of interaction between PKC and CYP1A1 remains elusive. These and other experiments with chemical inhibitors established the involvement of PKC at some level of the pathway, but it is not known whether this was a direct interaction (i.e., PKC phosphorylated the AhR and/or CYP1A1 directly), or whether PKC-mediated phosphorylation of another cellular component led to an indirect effect on CYP1A1. Ikuta et al. (2004) located two serine residues in the nuclear localization signal of the AhR that were phosphorylated by PKC, serines 12 and 36. Phosphorylation at these sites was shown to be necessary for nuclear import of the liganded AhR. Nuclear and cytosolic levels of the AhR, as well as binding of the liganded AhR to radiolabeled XREs, were unaffected by the PKC inhibitors tested, whereas PKC activity and CYP1A1-luciferase activity were greatly reduced with the inhibitors tested. It is possible that only a small fraction of PKC activity is necessary for phosphorylation of these serine residues and nuclear import of the liganded AhR, whereas a different PKC-mediated phosphorylation event necessary for CYP1A1 transcription will require a greater fraction of PKC activity.
It is also possible that PKC accounted for a portion of the AhR-mediated regulation of CYP1A1 transcription, but another kinase downstream of PKC, such as one of the MAP kinases, or the AP-1 complex, also regulated CYP1A1 transcription directly. AhR ligands induced AP-1 activity (Weber et al., 1994), and several other signal transduction pathways have been shown to affect AP-1 (Wisdom, 1999). Interactions between MAP kinase activity and regulation of the AhR-Arnt heterodimer complex and of CYP1A1 have been described (Andrieux et al., 2004; Tan et al., 2004) Both overexpression of a constitutively active form of PKCμ- and PKC-mediated phosphorylation of PKCμ led to stimulation of the ERK1/2 MAP kinase cascade, and activation of p42 MAP kinase (Brandlin et al., 2002; Hausser et al., 2001). A recent report demonstrated that TCDD increased phosphorylation of p44/p42 MAP kinases, and inhibition of p42 MAP kinase by overexpression of a dominant negative resulted in a dramatic reduction in activity of a Cyp1a1 promoter–luciferase reporter construct (Yim et al., 2004). In this manner, PKC could feed forward into this mechanism, causing increased CYP1A1-luciferase transcription through PKCμ. This is consistent with results obtained by inhibition of PKCμ by G6976. Additional signaling directly to AP-1 or to MAP kinases such as p42 independent of PKC may provide the remainder of the regulatory influence on CYP1A1 transcription.
ACKNOWLEDGMENTS
Conflict of interest: The authors acknowledge that they have a grant from National Institute of Environmental Health Sciences (NIEHS; grant ES10337) to do research in this area; the funding organization does not have control over the resulting publication.
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Department of Pharmacology, University of California, San Diego, La Jolla, California 92093–0722
ABSTRACT
Cytochrome P450 1A1 (CYP1A1) is induced by halogenated and polycyclic aromatic hydrocarbons following activation of the aryl hydrocarbon receptor (AhR). Protein kinase C (PKC) has been implicated in the regulation of this response. In tissue culture, induction of PKC activity with phorbol esters synergizes the actions of TCDD-induced CYP1A1, while PKC inhibitors block induction of CYP1A1 by TCDD. Here, the actions of specific PKC inhibitors on CYP1A1 induction were examined using a HepG2 human cell line (TV101L) that carries a stably integrated firefly luciferase gene under control of the human CYP1A1 promoter (–1612/+293). TV101 cells were treated with TCDD and either the kinase inhibitor staurosporine or one of the PKC inhibitors GF109203X, G6983, or G6976. Aryl hydrocarbon receptor-dependent activation of CYP1A1-luciferase and cellular PKC activity were measured. TCDD treatment induced CYP1A1-luciferase activity in an AhR-dependent manner, as determined by binding of nuclear AhR to xenobiotic response elements (XREs). Dose-dependent inhibition of PKC activity by staurosporine was concordant with inhibition of TCDD-induced CYP1A1-luciferase activity. However, the PKC inhibitors GF109203X, G6983, and G6976 blocked PKC activity at concentrations independent of those necessary to block TCDD induction of CYP1A1-luciferase activity. For all inhibitors, reduction in CYP1A1-luciferase activity was independent of AhR activation, as determined by electrophoretic mobility shift analysis of TCDD-activated nuclear AhR. The specific PKC inhibitors did not significantly alter cytosolic or nuclear levels of AhR protein, whether alone or in combination with TCDD. These results suggested that PKC was not the sole factor responsible for regulation of CYP1A1.
Key Words: protein kinase C; cytochrome P450 1A1; regulation; gene expression; signal transduction.
INTRODUCTION
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a halogenated aromatic hydrocarbon (HAH), commonly produced by incineration of municipal waste (Viel et al., 2000), and as a byproduct of pulp and paper bleaching (Schweer and Jennings, 1990). It is an endocrine disrupter (Safe et al., 1998) as well as a potent carcinogen, co-carcinogen, and teratogen (Poland and Kende, 1976; IARC, 1997; Steenland et al., 2004). TCDD induces cytochrome P450 1A1 (CYP1A1) through a ligand-dependent process mediated by the aryl hydrocarbon receptor (AhR). The AhR is a cytosolic phosphoprotein that is part of the basic helix-loop-helix (bHLH) protein family (Wilson and Safe, 1998). In the absence of ligand, the AhR is complexed with heat shock protein-90 (hsp90) and X-associated protein 2 (XAP2) in the cytosol (Meyer and Perdew, 1999). Upon ligand binding, hsp90 and XAP2 are released, and the liganded AhR is translocated to the nucleus, where it forms a heterodimer with the nuclear Arnt protein (Hoffman et al., 1991). The liganded AhR-Arnt heterodimer then binds to xenobiotic response elements (XREs) and initiates transcription of CYP1A1 (Reyes et al., 1992).
Activation of the AhR to a DNA-binding transcription factor is highly dependent on the phosphorylation state of the AhR complex, and AhR activation is linked to protein kinase C (PKC) activity. In vitro exposure of cytosol or nuclear extracts to acid or alkaline phosphatase renders the AhR unable to bind to XREs and activate transcription of CYP1A1 (Berghard et al., 1993; Carrier et al., 1992; Mahon and Gasiewicz, 1995; Pongratz et al., 1991). Short-term treatment of cells with phorbol esters such as PMA, which mimic diacylglycerol, leads to PKC activation (Castagna et al., 1982). Limited treatment with phorbol-12-myristate-13-acetate (PMA) plus TCDD causes synergistic activation of the CYP1A1 gene, as determined by increases in a human CYP1A1 promoter–luciferase reporter construct stably integrated into HepG2 cells (Chen and Tukey, 1996). Treatment with TCDD plus the kinase inhibitor staurosporine causes a nearly complete block in TCDD-initiated induction of CYP1A1-luciferase transcription (Chen and Tukey, 1996). However, at levels of staurosporine that inhibit cytosolic PKC activity, translocation of the liganded AhR to the nucleus and AhR binding to DNA are not inhibited (Schafer et al., 1993). Other studies indicate that the synergy in TCDD-initiated induction of CYP1A1 by phorbol esters does not require the transactivation domains of the AhR or Arnt (Long and Perdew, 1999; Safe et al., 1998). Together, these results indicate that PKC activity is required for nuclear events in the CYP1A1 transcriptional pathway (Chen and Tukey, 1996).
Protein kinase C–mediated signal transduction takes place through a family of PKC isoforms that exhibit differences in substrate specificity and cellular components necessary for activation. Activation of both conventional and novel PKC isoforms is dependent on diacylglycerol and phosphatidylserine, while conventional isoforms also require intracellular Ca2+ for activation (Kishimoto et al., 1980; Konno et al., 1989; Ohno et al., 1988;). Atypical PKC isoforms can be stimulated by phosphatidylserine (Akimoto et al., 1994; Ways et al., 1992). Staurosporine, a potent inhibitor of PKC activity, also inhibits many other protein kinases, such as tyrosine kinases, protein kinase A, protein kinase G, and calcium-calmodulin kinase (Fujita-Yamaguchi and Kathuria, 1988; Gadbois et al., 1992; Niggli and Keller, 1991). Recent advances in the characterization of the individual PKC isoforms have led to the identification of more selective chemical inhibitors. To study the role of PKC in regulation of CYP1A1, we examined AhR activation and CYP1A1 transcription in the presence of various pharmacological inhibitors known to be more selective for PKC than staurosporine.
MATERIALS AND METHODS
Chemicals.
2,3,7,8-tetrachlorodibenzo-p-dioxin was obtained from Wellington Laboratories (Guelph, Ontario, Canada). G6976, G6983, and GF109203X were obtained from Calbiochem (La Jolla, CA). Phorbol-12-myristate-13-acetate and staurosporine were purchased from Sigma Chemical Company (St. Louis, MO). D-luciferin, potassium salt, was purchased from Analytical Luminescence Laboratories (Ann Arbor, MI) and from Pharmingen (San Diego, CA). Bradford reagent dye concentrate was obtained from Bio-Rad (Hercules, CA). Dulbecco's Modified Eagle's Medium (DMEM) was purchased from Cellgro (Herndon, VA). Fetal bovine serum was purchased from Omega Scientific (Tarzana, CA). Other tissue culture reagents were purchased from GIBCO/BRL (Grand Island, NY and Bethesda, MD). -32P-dCTP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Phosphatidylserine, diacylglycerol, rat brain lysate, and the PKC substrate peptide Ac-FKKSFKL-NH2 were kind gifts of Dr. Alexandra Newton (University of California San Diego, La Jolla, CA). Klenow fragment of DNA polymerase was obtained from Invitrogen (Carlsbad, CA). Poly(dI-dC) was obtained from Roche Molecular Biochemicals (Indianapolis, IN). Salmon sperm DNA was obtained from Life Technologies (Bethesda, MD). The rabbit anti-AhR antibody was a kind gift of Dr. Christopher Bradfield (McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI). Mouse anti--actin was obtained from Sigma. Goat anti-rabbit IgG-horseradish peroxidase and anti-mouse IgG-horseradish peroxidase were obtained from Cell Signaling Technologies (Beverly, MA). Western Lightning enhanced chemiluminescent substrate kit was obtained from PerkinElmer/NEN (Boston, MA). All other chemicals and reagents were of the highest purity available and were purchased from Sigma and Fisher Scientific.
Tissue culture and cell lines.
TV101L is a human hepatoma cell line derived from HepG2. It has been stably transfected with 1612 bp of 5' sequence of the human CYP1A1 gene, including the full promoter sequence and 5' flanking sequences, linked to the firefly luciferase gene, as previously reported (Postlind et al., 1993). TV101L cells were obtained from liquid nitrogen stocks in our laboratory and grown in monolayers on six-well, 10-cm, and 15-cm tissue culture plates. They were maintained at 37°C in 95% air and 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 20 mM HEPES, and 0.8 mg/ml G418. Only cells passaged 25 times or fewer which were 60% to 90% confluent were used for experiments.
Luciferase assay.
TV101L cells were maintained on 10-cm plates until the monolayer covered 60% to 90% of the plate surface area (60% to 90% confluent), split to six-well tissue culture plates, and used in experiments 18–24 h later. Chemical solutions were prepared by dissolving compounds in dimethyl sulfoxide (DMSO). Compounds were added to the culture media, and DMSO concentration in the culture media never exceeded 0.3% v/v. Compounds did not cause changes in cellular morphology or cell death. At the end of the treatment period, culture media was removed by aspiration, and cells were rinsed twice with 37°C phosphate-buffered saline (PBS). Cells were incubated with a lysis buffer containing 1% Triton X-100, 25 mM Tricine pH 7.8, 15 mM MgSO4, 4 mM EDTA, and 1 mM DTT at 37°C for 20 min for maximum luciferase recovery. Cells were then collected by scraping, and cells and lysis buffer were transferred to microcentrifuge tubes and centrifuged at 14,000 rpm for 12 min at 4°C. Supernatants were collected into separate microcentrifuge tubes and frozen at –70°C until assay. Protein concentration was determined by the method of Bradford (1976). Luciferase activity was determined by mixing 300 μl of sample buffer (25 mM Tricine pH 7.8, 15 mM MgSO4, 4 mM EDTA, 1 mM DTT, 2 mM ATP) with 10 μl of supernatant. Reactions were initiated by adding 100 μl of 1 mM D-luciferin (potassium salt) in 15 mM potassium phosphate buffer, pH 7.8. Light output was measured for 10 s at 25°C in a Monolight 2001 luminometer (Analytical Luminescence Laboratories, Ann Arbor, MI). Luciferase activity was normalized to the protein concentration of each sample.
PKC activity assay.
Lipid solution containing 1.4 mM phosphatidylserine and 38 μM diacylglycerol in 20 mM HEPES, pH 7.4 was prepared from CHCl3 stocks. Solvent was evaporated under a stream of N2, and the remaining lipid film was hydrated with 20 mM HEPES, pH 7.4, vortexed, and sonicated briefly in a bath sonicator. TV101L cells were grown on six-well tissue-culture–treated plates and treated as shown in Results. Treatments did not result in abnormal cellular morphology or cell death. Media were removed by aspiration, and cells were rinsed twice with ice-cold PBS. Cells were lysed in an ice-cold lysis buffer containing 1% Triton X-100, 20 mM HEPES pH 7.4, 85 μM leupeptin, 2 mM benzamidine, 1 μM microcystin, 200 μM PMSF, 1 mM DTT, 1 mM EDTA pH 7.4, and 1 mM EGTA pH 7.4, and scraped into microcentrifuge tubes. Cell lysates were centrifuged at 14,000 rpm for 12 min at 4°C, and supernatants were mixed with glycerol to a final concentration of 50% (v/v) and frozen at –20°C until assayed. Samples were prepared for assay under PKC activating conditions by diluting 4 μl of each supernatant with 8 μl of 5 mM CaCl2, 8 μl lipid solution, and 44 μl enzyme buffer (2 mM DTT, 20 mM HEPES, pH 7.4). A parallel set of samples was prepared for assay under non-activating conditions by diluting 4 μl of each supernatant with 8 μl of 5 mM EGTA and 52 μl enzyme buffer. The reaction was immediately initiated by adding 16 μL GO buffer containing 20 mM HEPES pH 7.4, 100 μM ATP, 5 mM MgCl2, 100 μg/ml substrate peptide Ac-FKKSFKL-NH2, and 1 μCi -32P-ATP. The reaction was allowed to proceed for 8 min at 30°C, and was stopped by adding 25 μl STOP buffer containing 0.1 M ATP and 0.1 M EDTA, pH 8. 85 μl from each stopped reaction was spotted onto rectangular strips of Whatman P81 cation exchange paper (Fisher Scientific) approximately 1.5 in. x 0.75 in. in size. Strips were then washed four times for 5 min each in 500 ml 0.4% H3PO4 to remove unincorporated -32P-ATP, followed by one 5-min wash with 100% EtOH. Strips were then transferred to scintillation vials and counted in 5-ml Ecolite scintillation fluid (ICN, Costa Mesa, CA) in an LS-6800 liquid scintillation counter (Beckman Coulter, Fullerton, CA). Protein kinase C activity was defined as the ratio of nmol phosphate transferred to the substrate per minute under activating conditions to the nmol of phosphate transferred to the substrate per minute under non-activating conditions.
Preparation of nuclear protein.
The method of preparation of nuclear protein was adapted from Miller and colleagues and others (Harper et al., 1991; Miller et al., 1983). TV101L cells were grown on 15-cm tissue culture plates. Compounds were added to culture medium, and DMSO concentration in the culture medium never exceeded 0.2% v/v. Compounds did not cause abnormal cellular morphology or cell death. At the end of the treatment period, culture medium was removed by aspiration, and cells were rinsed twice with 20 ml 10 mM HEPES at 4°C. Cells were incubated with 10 ml 10 mM HEPES at 4°C for 15 min. HEPES was removed by aspiration, and 2 ml MDH (3 mM MgCl2, 1 mM DTT, 25 mM HEPES, pH 7.5, plus protease inhibitors) was added at 4°C. Cells were collected by scraping and transferred to separate 15-ml centrifuge tubes on ice. Each group of cells was then transferred to a 2-ml Dounce homogenizer, homogenized with 30 strokes at 4°C, and transferred to a fresh 15-ml centrifuge tube. After homogenization, samples were centrifuged at 2500 rpm for 5 min at 4°C in a Sorvall swinging-bucket tabletop centrifuge. MDH was removed, and samples were resuspended in 1 ml ice-cold MDHK (3 mM MgCl2, 1 mM DTT, 25 mM HEPES, 0.1 M KCl, pH 7.5, plus protease inhibitors), centrifuged at 2500 rpm for 5 min at 4°C, and this process was repeated once. Samples were resuspended in 1 ml ice-cold MDHK, transferred to separate microcentrifuge tubes, and centrifuged at 5000 rpm for 10 min at 4°C. The supernatant was removed by aspiration, and samples were resuspended in 100 μl ice-cold HDK (25 mM HEPES, 1 mM DTT, 0.4 M KCl, pH 7.5, plus protease inhibitors). Samples were incubated on ice for 30 min, and were mixed during the incubation by gently inverting each tube 10 times once every 5 min. After this incubation, samples were centrifuged in a microcentrifuge at 14,000 rpm for 15 min at 4°C, and supernatants were transferred to polycarbonate tubes. Glycerol was added to a final concentration of 10% v/v, and samples were centrifuged in a TLA100.3 rotor at 50,000 rpm (105,000 x g) in a Beckman ultracentrifuge for 1 h at 4°C. Supernatants were immediately quick frozen in a dry ice / methanol bath and stored at –70°C until use. Protein concentration was determined by the method of Bradford (1976).
Preparation of cytosolic protein.
This method for preparing cytosolic protein was adapted from Harper (Harper et al., 1991). TV101L cells were washed twice with phosphate-buffered saline, and incubated for 15 min in HED buffer (25 mM HEPES pH 7.5, 1 mM EDTA, 1 mM DTT, plus protease inhibitors). Cells were collected by scraping and transferred to separate 15-ml centrifuge tubes on ice. Each group of cells was then transferred to a 2-ml Dounce homogenizer, homogenized with 30 strokes at 4°C, and transferred to a polycarbonate centrifuge tube. An equal volume of HED2G buffer (25 mM HEPES, 1 mM EDTA, 1 mM DTT, 20% glycerol, plus protease inhibitors) was added, and samples were centrifuged in a TLA100.3 rotor at 50,000 rpm (105,000 x g) in a Beckman ultracentrifuge for 1 h at 4°C. Supernatants were immediately quick frozen in a dry ice/ethanol bath and stored at –70°C until use. Protein concentration was determined by the method of Bradford (1976).
Oligonucleotide preparation and electrophoretic mobility shift assay (EMSA).
Two complementary DNA oligonucleotides with the sequence 5'-GATCTGAGCTCGGAGTTGCGTGAGAAGAGCCG-3' and 5'-GATCCGGCTCTTCTCACGCAACTCCGAGCTCA-3', containing the 27 bp AhR binding site of DRE3, designated here as DRE (Denison et al., 1988b), were synthesized commercially (GenBase, San Diego, CA). 2 μg of each oligonucleotide were annealed in a total volume of 20 μl by heating the mixture to 75°C for 5 min, then allowing the mixture to cool to 37°C for 90 min. The concentration of the double-stranded oligonucleotide (dsDRE) was determined using a spectrophotometer. 10 pmol of the dsDRE was radiolabeled in a total volume of 20 μl using the Klenow fragment of DNA polymerase (Invitrogen, Carlsbad, CA) and 5 μl -32P-dCTP (3000 Ci/mmol, 10 mCi/ml) according to the manufacturer's instructions. The labeling reaction was allowed to proceed for 3 h at room temperature and was stopped by the addition of 1 μl 0.1 M NaCl. Unincorporated radiolabel was removed using a Qiagen Nucleotide Removal Kit (Qiagen, Valencia, CA). Nuclear protein binding to dsDRE was measured by electrophoretic mobility shift assay (Denison et al., 1988a). Binding reactions were performed by preincubating 10 μg nuclear protein, 2.4 μg poly(dI-dC), 1 μg salmon sperm DNA, and binding buffer (25 mM HEPES, 1.5 mM EDTA, 1 mM DTT, and 10% glycerol, pH 7.5) for 15 min at room temperature. After preincubation, 1 x 106 cpm 32P-labeled dsDRE was added, and the reaction was allowed to proceed for an additional 15 min. Sufficient binding buffer was added to make the final reaction volume equivalent for all samples within a given EMSA. To ensure that the binding observed was specific for dsDRE, in each EMSA performed an excess of unlabeled dsDRE was added to one sample in which binding to dsDRE was expected to be observed. DNA–protein complexes were separated under nondenaturing conditions on a 6% polyacrylamide gel using 1x TBE (89 mM Tris borate, 89 mM boric acid, 2 mM EDTA, pH 7.5) as the running buffer. Gels were dried, and complexes were visualized by autoradiography.
Western blotting.
Protein samples were mixed with the appropriate amount of LDS sample buffer and reducing agent (Invitrogen, Carlsbad, CA), heated to 70°C for 10 min, cooled, pulse spun, and loaded on a 4–12% NuPAGE Bis-Tris pre-cast polyacrylamide gel (Invitrogen), and electrophoresed at 208 V for 40 min. Proteins from the gel were transferred to a polyvinylidine difluoride membrane (Millipore, Bedford, MA) for 70 min at 30 V in an XCell II blot module (Invitrogen) according to the manufacturer's instructions. Membranes were blocked overnight on a rotary shaker at 4°C in 100 ml TTBS (10 mM Tris-Cl pH 8, 15 mM NaCl, 0.1% Tween 20) with 5% dry milk. Between all incubations, membranes were washed three times for 10 min in TTBS. Membranes were incubated with primary antibody for 1 h on a rotary shaker at room temperature, and with secondary antibody for 1 h on a rotary shaker at room temperature. Bands were visualized using a enhanced chemiluminescent substrate kit according to the manufacturer's instructions (PerkinElmer/NEN, Boston, MA).
RESULTS
The treatment of TV101L cells with AhR ligands led to a dose-dependent increase in luciferase activity. Co-treatment with 5 nM TCDD and varying concentrations of staurosporine for 6 h led to a dose-dependent inhibition of CYP1A1-luciferase activity. In comparing the ability of staurosporine to inhibit cellular PKC activity and CYP1A1-luciferase, the IC50 values for PKC (IC50 = 20 nM) (Table 1, Fig. 1) were similar to those observed for CYP1A1-luciferase (IC50 = 57 nM).
Three PKC inhibitors were chosen for dose–response experiments on the basis of their reported ability to inhibit only specific isoforms of PKC. GF109203X and G6983 were bisindolylmaleimide analogs derived from staurosporine, but with modifications to their functional groups which increased their selectivity for PKC (Gschwendt et al., 1996; Martiny-Baron et al., 1993). G6976 was also derived from staurosporine, but it was a structurally dissimilar methyl and cyanoalkyl substituted nonglycosidic indolocarbazole (Martiny-Baron et al., 1993). All three compounds worked primarily by competitive inhibition of ATP binding to PKC, although G6976 also exhibited noncompetitive substrate inhibition of PKC and mixed inhibition with respect to lipid cofactors of PKC (Gschwendt et al., 1996; Martiny-Baron et al., 1993). Reported IC50 values for the PKC isoforms are shown in Table 2, and structures of each of these inhibitors are shown in Figure 2.
It is important to note that these more specific PKC inhibitors did not block the activity of one and only one PKC isoform. Each inhibitor acted against more than one isoform of PKC, but exhibited a unique pattern of which isoforms were blocked, and at what inhibitor concentration those particular PKC isoforms were blocked. It is only by comparison of the CYP1A1-luciferase and PKC activities observed with each of the three inhibitors tested, across a broad range of inhibitor doses, that meaningful conclusions about the actions of particular PKC isoforms could be deduced.
Dose–response experiments were conducted with these more specific PKC inhibitors to ascertain the role of PKC in regulation of CYP1A1-luciferase transcription. When TV101L cells were treated with any of these PKC inhibitors alone, CYP1A1-luciferase activity was the same as untreated TV101L cells (insets in Figs. 3–5). Thus, the PKC inhibitors themselves did not stimulate CYP1A1-luciferase transcription. Co-treatment with PKC inhibitors and TCDD led to dose-dependent decreases in TCDD-mediated CYP1A1-luciferase transcription, as well as to dose-dependent decreases in cellular PKC activity. However, these dose–response profiles were different than the dose–response profiles observed for staurosporine. For example, increasing concentrations of GF109203X led to a dose-dependent, gradual decrease in both CYP1A1-luciferase activity and PKC activity. When G6983 was examined, CYP1A1-luciferase activity was largely unaffected across a range of G6983 concentrations, but dropped sharply at higher G6983 concentrations (Fig. 4). Only higher concentrations of GF109203X and G6983 led to a comparable inhibition of PKC and CYP1A1-luciferase activity. Taken alone, these results suggest that a fraction of total cellular PKC activity was required to maximally stimulate CYP1A1-luciferase transcription. Thus, a large fraction of PKC activity could be abolished without affecting CYP1A1 transcription, but once PKC activity fell below a threshold value, induction of CYP1A1 transcription would decline.
If one or more isoforms of PKC were more important to regulation of CYP1A1 transcription than others, then PKC inhibitors with different isoform specificities would be expected to have different effects on CYP1A1-luciferase transcription. Taken together, the differences observed in the dose–response curves of each PKC inhibitor supported this hypothesis. Co-treatment with TCDD and G6976 (Fig. 5) yielded very different results from co-treatment with TCDD and GF109203X (Fig. 3) or with TCDD and G6983 (Fig. 4). Both CYP1A1-luciferase activity and cellular PKC activity were unaltered over a range of G6976 concentrations, but activity dropped sharply at higher G6976 concentrations. However, there was a range of G6976 concentrations at which there were no significant effects on cellular PKC activity, but CYP1A1-luciferase activity was inhibited by 75%. Cellular PKC activity was reduced only at high G6976 concentrations (1.5 μM), and 60% inhibition of cellular PKC activity was the maximum achieved using G6976. This finding was in contrast to experiments with GF109203X and G6983, which showed a range of inhibitor concentrations at which CYP1A1-luciferase activity was not significantly affected, but PKC activity was suppressed. This suggested that G6976 inhibited one or more isoforms of PKC that were more involved in regulation of CYP1A1 transcription than isoforms inhibited by GF109203X and G6983.
G6976 exhibited a unique dose–response profile compared to other PKC inhibitors tested, which suggested that G6976 acted against isoforms important to regulation of CYP1A1 transcription. Indeed, G6976 was the only inhibitor tested to have activity against PKCμ. Dose–response curves indicated that G6976 caused inhibition of the CYP1A1-luciferase response at lower concentrations than G6983. Given that conventional PKC isoform IC50 values for G6976 and G6983 were similar, yet only G6976 strongly inhibited PKCμ (Gschwendt et al., 1996; Martiny-Baron et al., 1993;), these results suggested that PKCμ played an important role in CYP1A1 transcriptional regulation. Because PKCμ accounted for a small amount of the total PKC present in most cell types (Johannes et al., 1994), a specific inhibitor such as G6976 could block a large proportion of PKCμ activity and CYP1A1 transcription, while not dramatically altering other PKC isoforms or the total cellular PKC activity.
Combined with comparisons of dose–response curves obtained and reported PKC isoform IC50 values, it appeared that more than one PKC isoform was involved in regulation of CYP1A1. All inhibitors tested could block conventional PKC isoforms, and conventional PKC isoforms were the most abundant PKC isoforms in most types of cells. Thus, conventional PKC isoforms most likely accounted for at least a fraction of CYP1A1 regulation. However, other PKC isoforms appeared to be less involved in regulation of CYP1A1. G6983 and GF109203X, but not G6976, inhibited PKC. Because G6976 did not inhibit PKC, but did block CYP1A1-luciferase activity effectively, this suggested that PKC was not linked to regulation of CYP1A1 transcription. Conversely, because G6983 and GF109203X inhibited PKC activity at a number of concentrations where CYP1A1-luciferase activity was not blocked, it can be inferred that PKC was not involved in the control of CYP1A1. Similar inferences could be made regarding other PKC isoforms based on dose–response data. GF109203X was the only inhibitor of the three tested that was reported to have activity against PKC (Gschwendt et al., 1996). However, a range of concentrations was observed at which GF109203X blocked PKC activity but did not affect CYP1A1-luciferase activity. This suggested that PKC was not linked to regulation of CYP1A1 transcription. Similarly, G6983 was the only compound tested that has been shown to inhibit PKC. There was also a range of concentrations observed at which G6983 blocked PKC activity but did not affect CYP1A1-luciferase activity. This suggested that PKC was also not linked to regulation of CYP1A1 transcription.
To determine if inhibition of the CYP1A1-luciferase response was linked to Ah receptor DNA binding, electrophoretic mobility shift assays were conducted on nuclear proteins isolated from TV101L cells co-treated with TCDD and concentrations of PKC inhibitor shown to block luciferase activity (Figs. 6 and 7). After 5 nM TCDD treatment for 6 h, nuclear accumulation of the Ah receptor was observed, as shown by nuclear protein binding to radiolabeled human XRE sequences. This binding was not observed when an excess of unlabeled XRE sequence was included in the binding reaction, which confirmed the specificity of the assay. No Ah receptor DNA binding was observed when nuclear proteins from untreated cells, or from cells treated with PKC inhibitor alone, were tested. Aryl hydrocarbon receptor DNA binding observed with nuclear proteins from cells co-treated for 6 h with the various PKC inhibitors and 5 nM TCDD was the same as observed with nuclear proteins from cells treated with TCDD alone. These results demonstrated that TCDD-initiated transcriptional activation of CYP1A1-luciferase could be dramatically inhibited without affecting the ability of the Ah receptor to accumulate in the nucleus and bind to DNA. This also suggested that the PKC inhibitors tested were not AhR ligands.
A previous report indicated that staurosporine reduced intracellular levels of the AhR (Singh and Perdew, 1993). To determine whether the specific PKC inhibitors used in this study altered intracellular levels of the AhR, cytosolic and nuclear proteins were isolated from TV101L cells treated with the maximum concentration of each inhibitor used in this work, in the presence or absence of TCDD. Aryl hydrocarbon receptor protein was measured by Western blotting with a rabbit anti-AhR antibody (Fig. 8). Treatment with any of the three specific PKC inhibitors alone did not significantly alter cytosolic levels of the AhR (Fig. 8a). Nuclear levels of the AhR were slightly increased (Fig. 8b), but this increase did not coincide with any impact on AhR binding to radiolabeled XREs (Figs. 6 and 7). Additionally, treatment with any of the three specific PKC inhibitors alone did not alter CYP1A1-luciferase transcriptional activity. Thus, the observed increase in nuclear AhR in response to these PKC inhibitors did not appear to have a functional impact on regulation of CYP1A1 induction. As expected, TCDD treatment reduced cytosolic levels of the AhR and strongly induced nuclear accumulation of the AhR. However, both cytosolic and nuclear levels of the AhR in TV101L cells co-treated with TCDD and any of the specific PKC inhibitors were similar to AhR levels observed in TV101L cells treated with TCDD alone (Figs. 8a, 8b). This also suggested that the PKC inhibitors' actions on PKC, and not any possible actions on the AhR, were the key determinants of their actions in influencing CYP1A1-luciferase transcription.
DISCUSSION
Previous work indicated that when PKC was stimulated using phorbol esters, a synergistic activation of TCDD-mediated induction of CYP1A1 occurred, and when cells were treated with the kinase inhibitor staurosporine, a corresponding suppression of TCDD-mediated induction of CYP1A1 occurred (Chen and Tukey, 1996). Thus, it was concluded previously that PKC played a role in the regulation of TCDD-mediated CYP1A1 induction. This was not surprising, as PKC was known to play an important role in cellular communication by relaying signaling events upstream of phospholipid hydrolysis to downstream kinases such as the MAP kinases and the AP-1 complex (Buchner, 2000). However, we tested three inhibitors that are more specific for PKC than staurosporine. Our findings indicated that inhibition of cellular PKC activity did not correspond with inhibition of TCDD-mediated CYP1A1-luciferase activity.
In these experiments, luciferase and PKC activities were measured in whole-cell lysates. This approach would yield a more physiologically relevant IC50 value, as it would more closely replicate the cellular environment in which these signaling pathways are found. Previous experiments to establish the published IC50 values of the inhibitors for PKC isoforms were biochemical and pharmacological experiments conducted with isolated, purified components. The IC50 values measured in a reaction of purified components would be expected to be much lower than those measured in a more physiological context, where an abundance of other proteins existed. For example, the PKC IC50 of staurosporine in rat anterior pituitary was measured to be over 100 times weaker than published PKC IC50 values of staurosporine in biochemical experiments (Simpson et al., 1993). Thus, IC50 values for CYP1A1-luciferase activity could not be directly compared to published IC50 values from biochemical experiments to establish the PKC isoform specificity involved in regulation of CYP1A1. However, relative dose–response trends between inhibitors used in the same experimental system could be compared, and useful data could be extracted from such comparisons nonetheless.
In theory, it was possible that the PKC inhibitors used in these experiments did not block the isoform(s) of PKC responsible for regulation of CYP1A1 induction, but they did block other PKC isoforms. In this case, total PKC activity would decrease, whereas CYP1A1-luciferase activity would decrease only when the particular PKC isoform(s) involved in regulation of CYP1A1 were blocked. However, dose–response results from G6976 contradicted this possibility. There was a wide range of G6976 concentrations tested at which CYP1A1-luciferase activity was strongly inhibited, and PKC activity was largely unaffected. This suggested that either G6976 was not an effective inhibitor of PKC, a claim that is strongly refuted by the body of PKC literature, or that G6976 was a more effective inhibitor of one or more low-abundance PKC isoforms involved in regulation of CYP1A1 than other inhibitors tested.
In fact, G6976 was the most effective inhibitor of CYP1A1-luciferase activity among the three more selective inhibitors tested. Both G6976 and staurosporine were effective against PKCμ at low nanomolar concentrations, whereas G6983 was a very weak inhibitor of PKCμ, and GF109203X did not block PKCμ at all. Staurosporine and the specific PKC inhibitors had comparable effectiveness against conventional PKC isoforms as well (Gschwendt et al., 1996; Martiny-Baron et al., 1993). However, staurosporine has been shown to inhibit a variety of other kinases in addition to PKC, including tyrosine kinases, protein kinase A, protein kinase G, and calcium-calmodulin kinase (Fujita-Yamaguchi and Kathuria, 1988; Gadbois et al., 1992; Niggli and Keller, 1991). Thus, one or more of the kinases inhibited by staurosporine, but not blocked by the more specific PKC inhibitors, could account for the remainder of CYP1A1 transcriptional regulation. Taken together, these experiments indicated that PKC activity may be responsible for only a fraction of CYP1A1 induction.
Electrophoretic mobility shift assays indicated that PKC inhibitors did not alter AhR-Arnt heterodimer binding to radiolabeled XRE sequences, whether used alone or in combination with TCDD. This was not unexpected, given previous results from Chen and Tukey (1996) and Long et al. (Long et al., 1998). Chen and Tukey demonstrated that staurosporine did not alter in vivo or in vitro TCDD-induced AhR-Arnt heterodimer binding to DNA, and that kinase inhibition by staurosporine was able to block transactivation of CYP1A1. Long et al. demonstrated that the more specific PKC inhibitors chelerythrine chloride and GF109203X did not alter AhR or Arnt levels in the cytoplasm or in the nucleus, nor did they alter TCDD-induced AhR-Arnt binding to radiolabeled XRE sequences. Long and Perdew (1999) also showed that the transactivation domains of AhR and Arnt were not required for the observed PMA synergy with TCDD in activation of CYP1A1. This suggested that the influence of PKC activity on TCDD-mediated induction of CYP1A1 took place at another level of the AhR signaling pathway, or that the influence of PKC was not a direct interaction with the AhR signaling pathway. Together with these previous results, our data suggest that the actions of PKC on activation of CYP1A1 transcription by TCDD more likely take place through the influence of PKC on other signaling pathways, which in turn regulate CYP1A1 transcription.
Western blots with an anti-AhR antibody indicated that the specific PKC inhibitors used in this study did not alter the levels of cytosolic AhR, whether used alone or in combination with TCDD. This suggested that the inhibition of CYP1A1-luciferase transcription observed was not due to a reduction in the amount of AhR available for ligand binding. The specific PKC inhibitors also had no effect the nuclear accumulation of AhR protein in response to TCDD. Although the PKC inhibitors alone did slightly increase nuclear AhR levels, this effect was shown to have no functional bearing on regulation of CYP1A1. This also suggested that nuclear translocation of the liganded AhR-Arnt complex was not affected by these inhibitors.
A 6-h time point was chosen to assess both CYP1A1-luciferase induction and PKC inhibition. Although PKC could be activated in a matter of minutes, and these inhibitors acted in a similar time frame, time points shorter than approximately 3 h would be insufficient for functional induction of CYP1A1-luciferase (Postlind et al., 1993). Additionally, Singh and Perdew used an 8-h staurosporine treatment to establish that staurosporine directly reduced intracellular levels of the AhR (Singh and Perdew, 1993). To effectively ensure that these PKC inhibitors did not affect the AhR in a similar manner, a similarly long treatment time needed to be used.
Although these experiments established that PKC was not as directly linked to regulation of TCDD-mediated induction of CYP1A1 as once thought, a full explanation of the mechanism of interaction between PKC and CYP1A1 remains elusive. These and other experiments with chemical inhibitors established the involvement of PKC at some level of the pathway, but it is not known whether this was a direct interaction (i.e., PKC phosphorylated the AhR and/or CYP1A1 directly), or whether PKC-mediated phosphorylation of another cellular component led to an indirect effect on CYP1A1. Ikuta et al. (2004) located two serine residues in the nuclear localization signal of the AhR that were phosphorylated by PKC, serines 12 and 36. Phosphorylation at these sites was shown to be necessary for nuclear import of the liganded AhR. Nuclear and cytosolic levels of the AhR, as well as binding of the liganded AhR to radiolabeled XREs, were unaffected by the PKC inhibitors tested, whereas PKC activity and CYP1A1-luciferase activity were greatly reduced with the inhibitors tested. It is possible that only a small fraction of PKC activity is necessary for phosphorylation of these serine residues and nuclear import of the liganded AhR, whereas a different PKC-mediated phosphorylation event necessary for CYP1A1 transcription will require a greater fraction of PKC activity.
It is also possible that PKC accounted for a portion of the AhR-mediated regulation of CYP1A1 transcription, but another kinase downstream of PKC, such as one of the MAP kinases, or the AP-1 complex, also regulated CYP1A1 transcription directly. AhR ligands induced AP-1 activity (Weber et al., 1994), and several other signal transduction pathways have been shown to affect AP-1 (Wisdom, 1999). Interactions between MAP kinase activity and regulation of the AhR-Arnt heterodimer complex and of CYP1A1 have been described (Andrieux et al., 2004; Tan et al., 2004) Both overexpression of a constitutively active form of PKCμ- and PKC-mediated phosphorylation of PKCμ led to stimulation of the ERK1/2 MAP kinase cascade, and activation of p42 MAP kinase (Brandlin et al., 2002; Hausser et al., 2001). A recent report demonstrated that TCDD increased phosphorylation of p44/p42 MAP kinases, and inhibition of p42 MAP kinase by overexpression of a dominant negative resulted in a dramatic reduction in activity of a Cyp1a1 promoter–luciferase reporter construct (Yim et al., 2004). In this manner, PKC could feed forward into this mechanism, causing increased CYP1A1-luciferase transcription through PKCμ. This is consistent with results obtained by inhibition of PKCμ by G6976. Additional signaling directly to AP-1 or to MAP kinases such as p42 independent of PKC may provide the remainder of the regulatory influence on CYP1A1 transcription.
ACKNOWLEDGMENTS
Conflict of interest: The authors acknowledge that they have a grant from National Institute of Environmental Health Sciences (NIEHS; grant ES10337) to do research in this area; the funding organization does not have control over the resulting publication.
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