* Department of Chemistry & Biochemistry, University of California, San Diego, La Jolla, California 920930722; Department of Pharmacology, University of California, San Diego, La Jolla, California 920930722
1 To whom correspondence should be addressed at Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, Mail Code 0722, La Jolla, CA 920930722. Fax: 8588220363. E-mail: rtukey{at}ucsd.edu.
Received February 14, 2005; accepted June 2, 2005
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ABSTRACT |
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Key Words: protein kinase C; cytochrome P450 1A1; regulation; gene expression; signal transduction.
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INTRODUCTION |
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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 promoterluciferase 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 Cmediated 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.
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MATERIALS AND METHODS |
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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 1824 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-culturetreated 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. DNAprotein 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 412% 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).
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RESULTS |
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Doseresponse 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. 35). 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 doseresponse profiles were different than the doseresponse 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 Gö6983 was examined, CYP1A1-luciferase activity was largely unaffected across a range of Gö6983 concentrations, but dropped sharply at higher Gö6983 concentrations (Fig. 4). Only higher concentrations of GF109203X and Gö6983 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.
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Gö6976 exhibited a unique doseresponse profile compared to other PKC inhibitors tested, which suggested that Gö6976 acted against isoforms important to regulation of CYP1A1 transcription. Indeed, Gö6976 was the only inhibitor tested to have activity against PKCµ. Doseresponse curves indicated that Gö6976 caused inhibition of the CYP1A1-luciferase response at lower concentrations than Gö6983. Given that conventional PKC isoform IC50 values for Gö6976 and Gö6983 were similar, yet only Gö6976 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 Gö6976 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 doseresponse 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. Gö6983 and GF109203X, but not Gö6976, inhibited PKC. Because Gö6976 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 Gö6983 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 doseresponse 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, Gö6983 was the only compound tested that has been shown to inhibit PKC
. There was also a range of concentrations observed at which Gö6983 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.
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DISCUSSION |
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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 doseresponse 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, doseresponse results from Gö6976 contradicted this possibility. There was a wide range of Gö6976 concentrations tested at which CYP1A1-luciferase activity was strongly inhibited, and PKC activity was largely unaffected. This suggested that either Gö6976 was not an effective inhibitor of PKC, a claim that is strongly refuted by the body of PKC literature, or that Gö6976 was a more effective inhibitor of one or more low-abundance PKC isoforms involved in regulation of CYP1A1 than other inhibitors tested.
In fact, Gö6976 was the most effective inhibitor of CYP1A1-luciferase activity among the three more selective inhibitors tested. Both Gö6976 and staurosporine were effective against PKCµ at low nanomolar concentrations, whereas Gö6983 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 promoterluciferase 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 Gö6976. 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.
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ACKNOWLEDGMENTS |
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