phenobarbital induction of cytochrome p4501a1 is regulated by camp-dependent protein kinase-mediated...

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 225, 455–461 (1996)ARTICLE NO. 1194

Phenobarbital Induction of Cytochrome P4501A1 Is Regulatedby cAMP-Dependent Protein Kinase-Mediated Signaling

Pathways in Rainbow Trout Hepatocytes

Marianne D. Sadar,*,†,1 Fredrik Blomstrand,‡ and Tommy B. Andersson*,§

*Department of Zoophysiology, University of Goteborg, S 40031 Goteborg, Sweden; †Department of BiomedicalSciences, University of Bradford, Bradford, West Yorkshire, United Kingdom BD7 1DP; ‡Institute of Anatomy

and Cell Biology, University of Goteborg, S 413 90 Goteborg, Sweden; and §Department ofPharmacokinetics and Drug Metabolism, Astra Hassle AB, S 43183 Molndal, Sweden

Received June 24, 1996

Phenobarbital (PB) induces CYP1A1 at the transcriptional level and causes nuclear translocation of thearomatic hydrocarbon (Ah) receptor in primary cultures of rainbow trout hepatocytes (1). The results fromthis study suggest that PB induction of CYP1A1 in rainbow trout hepatocytes is regulated by cAMP-dependent pathways (PKA), whereas TCDD induction is not dependent upon PKA. Epinephrine, whichincreases cAMP levels and activates PKA-dependent pathways, was a potent inhibitor of PB induction,while having no effect on TCDD induction of CYP1A1 gene expression. When PKA-dependent pathwayswere inhibited, PB induction of CYP1A1 gene expression was greatly potentiated, whereas TCDD inductionwas affected to a lesser extent. Inhibitors of calcium-phospholipid-dependent protein kinase (PKC) hadmodest or no effect on PB and TCDD induction of CYP1A1, respectively. Whether the relatively weak-to-no inhibition of CYP1A1 in response to PKC inhibitors in fish is due to differences in the types andlevels of PKC isoenzymes, cell permeability, protocol, or the role of PKC in the mechanism of CYP1A1induction in fish remains to be established. PB induced persistent and transient increases in the intracellularcalcium concentration. This may be an important factor regulating PKC which may have a role in PB-mediated induction of CYP1A1 gene transcription. q 1996 Academic Press, Inc.

CYP1A1 induction is mediated by the aromatic hydrocarbon (Ah) receptor and has beenextensively studied in mammals. Ligands of the Ah receptor are planar lipophilic polycyclicaromatic hydrocarbons (PAHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (2). Inthe inactive form the Ah receptor is located in the cytosol in a heteromeric complex with the90-kDa heat shock protein (hsp90) (3), and possibly other proteins (4). Ligand binding to theAh receptor is thought to release hsp90 from the Ah receptor and induce translocation of theligand-receptor complex into the nucleus where it dimerizes with the Ah receptor nucleartranslocator (Arnt) protein and subsequently binds to the xenobiotic responsive elements(XREs) on the 5*-flanking region of the CYP1A1 gene to initiate transcription (5).

Teleost hepatic CYP1A1 is similar to its mammalian counterpart in its ability to be inducedby numerous PAHs (6). However, less is known about the molecular mechanism of CYP1A1induction in fish. To date, an Ah receptor has been detected in the hepatic cytosol of therainbow trout (7); TCDD induces translocation of the Ah receptor to the nucleus (1,8); andTCDD activates DNA-binding activity of the Ah receptor in the nucleus (1). DNA sequenceanalysis of the rainbow trout CYP1A1 gene has resulted in the detection of two XREs in the5* flanking region of the gene (9).

The induction of mammalian CYP1A1 transcription has been suggested to require the

1 Corresponding author: Marianne D. Sadar, Department of Cancer Endocrinology, B.C. Cancer Agency, 600 West10th Avenue, Vancouver, B.C., Canada V5Z 4E6. FAX: 604 877 6011.

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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phosphorylation of both the Ah receptor and Arnt protein for DNA binding activity of theligand-receptor complex (10-11). In various mammalian studies the calcium-phospholipid-dependent protein kinase (PKC) (10-12) and a tyrosine protein kinase (13) have been describedto be involved in events leading to CYP1A1 induction.

In mammals, CYP2B1/2B2 isozymes are classically induced by phenobarbital (PB). How-ever, induction of CYP1A1 by PB has been reported for various mammalian cell cultures (14-16), despite the fact that PB shows poor affinity for the Ah receptor (17). We have reportedthat PB causes nuclear translocation of the Ah receptor and increased transcription of CYP1A1in primary cultures of rainbow trout hepatocytes (1). To determine whether PB induction ofCYP1A1 gene expression in primary cultures of rainbow trout hepatocytes is regulated byphosphorylation states, we employed studies with various modulators of protein kinase/phos-phatase activities and examined alterations in intracellular calcium concentrations ([Ca2/]i).

MATERIALS AND METHODSCell culture and treatments. All chemicals were purchased from Sigma (St. Louis, MO), unless stated otherwise.

Hepatocytes were isolated and cultivated from juvenile, cultured rainbow trout (Oncorhynchus mykiss) as describedpreviously (1). Cells were counted in a Burker chamber, assessed for cell viability by the Trypan Blue exclusion test,seeded at 107 cells per plate (10 cm), and incubated at 127C for 24 hours before inducers were added. PB, epinephrine,and RP-cAMPS (BIOLOG Life Science Institute, Bremen, FRG) were added to cultures in Medium 199 (Gibco).TCDD (Cambridge Isotope Laboratories), staurosporine, calphostin C, okadaic acid, and Fura-2/AM were dissolvedin dimethyl sulfoxide (DMSO, final concentration of 0.1%). Control cultures for TCDD received DMSO only (0.1%).Calphostin C mediated inhibition of PKC activity is light dependent (18), therefore calphostin C-treated cells werepre-incubated under a 15-W light source. All experiments were performed at least three times using different cellculture preparations, and the data presented represents a single experiment.

Northern blots. Total RNA was extracted from cultured hepatocytes using the RNAzol (Cinna/Biotecx Lab. Inc.)solution method, which employs acid guanidium thiocyanate-phenol-chloroform, as described (19-20). Total RNA(10 mg) was fractionated by agarose gel electrophoresis with formaldehyde. Equal loading and the integrity of eachsample was assessed by staining the gel with ethidium bromide and quantification of 18S rRNA. Membranes werehybridized with a cDNA probe that was specific for rainbow trout CYP1A1 mRNA (pSG-15 plasmid with 2553 bpof the cDNA for rainbow trout CYP1A1). This probe amplified by polymerase chain reaction (PCR) with the upperprimer recognizing position 661 bp of the gene (5*-GGC TTG GTG AAC ATG AGT-3*) and the lower primerrecognizing the sequence at 1433 bp of the gene (5*-GCC AAG AGG AAG ACC-3*) and was labeled with digoxygeninfor detection of CYP1A1 mRNA with the DIG Luminescent Detection Kit (Boehringer Mannheim). After stringencywashes (21SSC/0.1% SDS and 0.51SSC/0.1% SDS at room temperature and 687C, respectively), the chemilumines-cent signal was recorded on x-ray film and the CYP1A1 mRNA bands were quantitated by scanning with a densitometer(LKB 2222-020 Ultroscan XL Laser Densitometer).

Intracellular calcium analyses. Cultured hepatocytes were loaded with 5 mM Fura-2/AM, in DMSO, at a temperatureof 127C for 1 hour. The cells were washed with salmon buffer (21) and fixed into a chamber mounted on the stageof an inverted epifluorescence microscope (Nikon Diaphot), equipped with a fluorescence 401 objective, interfacedwith a SPEX CM-X microspectrofluorimeter. [Ca2/]i in individual cells was determined from the ratio of two excitationwavelengths, 340 and 380 nm, with the emission wavelength centered at 505 nm. Calcium fluxes in individual cellswere detected by a shift occurring in the excitation spectrum of Fura-2/AM from 380 nm in its unbound state to 340nm when it is bound to calcium. [Ca2/]i (nM) was estimated by comparing the experimental 340/380 ratio with acalibration curve for free Ca2/, obtained by using Ca2/ calibration buffers with known concentrations of Ca2/ andFura-2/AM, as previously described (22).

RESULTS

Northern blots show PB to induce CYP1A1 mRNA accumulation (Fig. 1) as previouslyreported (1). Okadaic acid is an inhibitor of protein phosphatases. No effects on cell viability(data not shown) nor CYP1A1 induction (Fig. 1) was observed at 300 nM okadaic acid. PBinduction of CYP1A1 mRNA levels was increased by 1.4-fold at both 50 nM and 100 nMokadaic acid, as compared to PB CYP1A1 mRNA levels (Fig. 1). Okadaic acid (100 nM)enhanced TCDD induction of CYP1A1 mRNA 1.4-fold over TCDD levels. At 300 nM okadaicacid, PB- and TCDD-induced CYP1A1 mRNA levels decreased as compared with the levelswith 100 nM okadaic acid.

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FIG. 1. The effects of okadaic acid upon the induction of CYP1A1 by phenobarbital and TCDD. Primary culturesof rainbow trout hepatocytes were seeded for 24 hours before changing the medium and adding inducers with variousconcentrations of okadaic acid (OkA). Cells were incubated an additional 24 hours before harvesting and determiningCYP1A1 mRNA levels. CYP1A1 mRNA levels were measured by Northern blot analysis using a cDNA, pSG-15probe from rainbow trout and loading 10 mg of total RNA to each lane. Variation between two samples was not morethan 10% in CYP1A1 mRNA quantifying. Dimethyl sulfoxide (DMSO), 0.1%; phenobarbital (PB), 2 mM; 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 100 pM.

Epinephrine, which increases cAMP levels in isolated fish hepatocytes (23), caused a 74%decrease in PB induction of CYP1A1 mRNA while not affecting TCDD induction (Fig.2A). The addition of Rp-cAMPS, an inhibitor of PKA activity, potentiated CYP1A1 mRNAaccumulation by both PB (2.8-fold), and to a lesser extent TCDD (1.4-fold), as compared withindividual responses for PB and TCDD, respectively (Fig. 2B).

Staurosporine is an inhibitor of PKC, and to a lesser extent PKA, and numerous otherprotein kinases. Cell viability was not affected by the addition of 250 nM staurosporineto primary cultures of hepatocytes (data not shown). The levels of CYP1A1 mRNA wereenhanced in cells treated with 250 nM staurosporine, as compared to control levels (Fig.2A). This concentration of staurosporine caused a 17% decrease in the accumulation ofCYP1A1 mRNA in PB-treated cells, and a 9% decrease (considered negligible due to the10% margin of variation) in TCDD-treated cells. Calphostin C, a specific inhibitor ofPKC, caused a 19% decrease in CYP1A1 mRNA accumulation in PB-treated cells, and nosignificant change in TCDD-treated cells, as compared to PB and TCDD individual re-sponses, respectively (Fig. 2B).

FIG. 2. The effects of protein kinase activators and inhibitors on PB and TCDD induction of CYP1A1 mRNA.Primary cultures of rainbow trout hepatocytes were seeded for 24 hours before changing the medium and addinginducers with epinephrine or staurosporine (A), or RP-cAMPs or calphostin C (B). Cells were incubated an additional24 hours before harvesting and determining CYP1A1 mRNA levels. CYP1A1 mRNA levels were measured byNorthern blot analysis using a cDNA, pSG-15 probe from rainbow trout and loading 10 mg of total RNA to eachlane. Variation between two samples was not more than 10% in CYP1A1 mRNA quantifying. Dimethyl sulfoxide(DMSO), 0.1%; phenobarbital (PB), 2 mM; 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 100 pM; epinephrine (E),100 mM; staurosporine (St), 250 nM; RP-cAMPs (RPcAMPs), 200 mM; calphostin C (Calc), 1 mM.

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TABLE IIntracellular Cytosolic Free-Calcium Concentrations ([Ca2/]i) in Fura-

2/AM-Loaded Rainbow Trout Hepatocytes Chronically Exposed toPhenobarbital or 2,3,7,8-Tetrachlorodibenzo-p-dioxin for 48 Hours

Control TCDD-treated PB-treated

[Ca2/]i nM 74.1 { 4.9a 74.1 { 8.5 110 { 15*m 30 17 10n 3 2 1

[Ca2/]i was measured in a single hepatocyte in salmon buffer con-taining 1.5 mM CaCl2 . Phenobarbital (PB), 2 mM; 2,3,7,8-tetrachlorodi-benzo-p-dioxin (TCDD), 100 pM. aValues represent the mean { SD.*Significantly different (p õ 0.01) from control values; m, number ofcells; n, individual cell culture preparations.

Basal [Ca2/]i in primary cultures of rainbow trout hepatocytes was calculated to be 74.1 {4.9 nM (Table I) which is in agreement with previous reports for rainbow trout hepatocytes(24). Hepatocytes treated for 48 hours with TCDD did not show any change in levels of [Ca2/]i

as compared to the basal levels. Hepatocytes treated for 48 hours with PB showed a significantincrease (1.5-fold) in [Ca2/]i over basal levels.

Transient increases in [Ca2/]i were measured in hepatocytes in the presence extracellularcalcium. Phenobarbital (2 mM) caused a significant rise in [Ca2/]i after a lag period rangingbetween 2 to 3 minutes (Fig. 3). Generally, hepatocytes responded to PB by symmetricaltransient increases, or spikes in [Ca2/]i , with [Ca2/]i returning to basal levels upon terminationof the spike. However, biphasic increases in [Ca2/]i did occur in response to PB. The [Ca2/]i

profile for the latter responses showed the basal levels of [Ca2/]i not to be re-established upontermination of the calcium peak.

DISCUSSION

The induction of CYP1A1 by PB shows a remarkable dependency upon cAMP and/or PKAactivity providing a clear cut difference between PB and TCDD in respect to cross-couplingof the regulation of CYP1A1 induction and cAMP/PKA pathways. Thus, PB effects uponCYP1A1 may include cAMP/PKA-dependent mechanisms, while TCDD induction of CYP1A1may be cAMP/PKA-independent. We reported previously that PB induction of CYP1A1 genetranscription is probably mediated by activation of the cytosolic Ah receptor which is translo-cated to the nucleus where it binds to the XRE on the gene (1). The effects of cAMP/PKAsignaling pathways on PB-mediated induction of CYP1A1 gene expression may be throughmodulation (phosphorylation) of the Ah receptor or other protein factors important for genetranscription. PB and TCDD exposure have both been shown to increase PKA levels in therat liver (25-26). Our results showed no effect of PKA activation upon TCDD induction ofCYP1A1 and thus are consistent with studies employing the mouse Hepa-1 cell line (27).Furthermore, our results are in agreement with an in vivo study showing PB induction of AHHactivity in the rat to be decreased by cAMP (28). Consistent with the epinephrine (cAMP and/or PKA-modulated) reduction of PB induction of CYP1A1, we show enhanced induction ofCYP1A1 mRNA when the PKA inhibitor, Rp-cAMPs, was employed. Corresponding treatmenthad modest effects on TCDD induction which is contrary to mammalian studies showing theinhibition of PKA to have no effect on TCDD induction of CYP1A1 mRNA (10). Rainbowtrout hepatocytes therefore appear to respond differently than mammals in regard to cAMPand/or PKA modulation of TCDD induction of CYP1A1.

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FIG. 3. Examples of phenobarbital-induced [Ca2/]i changes in Fura-2/AM-loaded rainbow trout hepatocytes. [Ca2/]i

was measured in a single hepatocyte in salmon buffer containing 1.5 mM CaCl2 . Phenobarbital (PB), 2 mM, wasadded where the arrow depicts, and the calcium peak occurred after two to three minutes. More than 75% of cellsresponded to PB by increases in [Ca2/]i (8 cells from 3 different cell culture preparations). Fluorescence ratio: 340nm/380 nm.

Various mammalian studies show TCDD induction of CYP1A1 requires a functional Ahreceptor and Arnt protein, both of which are phosphoproteins (10-11). The successful bindingof the activated ligand-receptor complex to the XREs requires the dimerization of the Ahreceptor with the Arnt protein, as well as phosphorylation (11,29) and PKC seems to beimportant for successful DNA binding activity in mammals (10-11). Additionally, numerousmammalian studies show TCDD induction to result in increases of both PKC levels (26-27,30), and various protein tyrosine kinases (30). Increases in PKC and protein tyrosine kinaseactivities also occur in the TCDD-induced rainbow trout hepatoma cell line, RTH-149 (31).Conversely, PB exposure in mammals results in decreases in PKC levels by displacing diacyl-glycerol from its binding site (32) and inhibiting the phorbol ester-inducing translocation ofPKC from the cytosol to the plasma membrane (33). Here we show the addition of PKC

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inhibitors to attenuate PB induction of CYP1A1. Staurosporine and calphostin C had similarinhibitory effects on PB induction of CYP1A1, while not affecting TCDD-induced levels. Asimilar study employing rainbow trout hepatocytes showed the induction of 7-ethoxyresorufin-O-deethylase activity by b-naphthoflavone to be inhibited by staurosporine (34), but unfortu-nately the effects on CYP1A1 mRNA were not examined. Similarly, it should be noted thatwhen comparing the percentage of inhibition of CYP1A1 mRNA by PKC inhibitors, theresponse in fish hepatocytes was often weaker than what is generally seen in mammaliancultures, but the response was always consistent and clear. Whether the relatively weak-to-noinhibition of CYP1A1 in response to PKC inhibitors in fish is due to differences in the typesand levels of PKC isoenzymes, cell permeability, protocol, or the role of PKC in the mechanismof CYP1A1 induction in fish remains to be established.

Mammalian studies show persistent increases in [Ca2/]i after TCDD-treatment in cell cultures(35-37) and rat liver (38), while a decrease in [Ca2/]i was observed in isolated rat hepatocytestreated with PB (39). Our results suggest that rainbow trout hepatocytes differ from rat hepato-cytes in their [Ca2/]i responses to PB and TCDD. We observed a persistent increase in [Ca2/]i

in response to PB, whereas TCDD had no effect. Persistent increases in [Ca2/]i induced byPB may involve its possible interaction with diacylglycerol (32) which is presently thought toparticipate in the re-establishment (down-phase of calcium peak) of baseline [Ca2/]i . PB alsoinduced transient increases in [Ca2/]i . This is similar to mammalian studies finding TCDD tocause a transient increase in [Ca2/]i when employing cultured mouse hepatoma cells (27) andchick myocytes (37). These transient increases in [Ca2/]i induced by PB may play an importantrole in the early events of CYP1A1 induction by activating PKC. The weak response of PKCinhibitors shown in this study may, as discussed earlier, be due to the protocol or the potencyof the inhibitors in fish cells. The role of PKC in both PB- and TCDD-mediated induction ofCYP1A1 transcription remains to be established.

ACKNOWLEDGMENTSThe authors wish to express thanks to E. Nilsson and J. Sundqvist for their excellent technical assistance, and

B.-E. Grøssvik (University of Bergen, Bergen, Norway) for the CYP1A1-cDNA plasmids. M. D. Sadar is grateful tothe European Science Foundation for supporting this research through a fellowship providing financial assistance.T. B. Andersson is grateful to the Swedish Natural Science Research Council for financial support.

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phenobarbital induction of cytochrome p4501a1 is regulated by camp-dependent protein kinase-mediated...

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