From the Department of Environmental Health, University of Washington, Seattle, Washington 98195
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ABSTRACT |
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Previous investigations have indicated that de novo protein synthesis is a critical requirement for phenobarbital (PB) induction. We reexamined this issue in PB-responsive primary rat hepatocyte cultures using a broader array of protein synthesis inhibitors and experimental end points. Anisomycin, cycloheximide, emetine, puromycin, and puromycin aminonucleoside, a negative analog, were evaluated for their respective effects on protein synthesis and the PB-induction process. All of the inhibitors effectively repressed de novo protein synthesis in the cells in a concentration-dependent manner. However, anisomycin only minimally effected PB induction, ascertained though the measure of CYP2B1, CYP2B2, and CYP3A1 mRNA levels. The inactive agent, puromycin aminonucleoside, produced marked repression of the PB-induction response. Results from further experiments demonstrated that these protein synthesis inhibitors stimulated rapid and differential phosphorylation of the stress-activated protein kinase/c-Jun kinase (SAPK/JNK) pathway, indicating nonselective actions on cellular processes. Puromycin aminonucleoside was without effect on these pathways, despite its efficacy as an inhibitor of PB induction. These results demonstrate that de novo protein synthesis is not a requirement for PB induction, nor is activation of the SAPK/JNK kinase cascade responsible for down-regulating PB responsiveness in primary hepatocytes.
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INTRODUCTION |
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Phenobarbital (PB)1 is well recognized for its pleiotropic effects on mammalian cells, including its ability to transcriptionally activate a variety of genes (1, 2). However, identification of the molecular mechanisms controlling these inductive responses has proven difficult. Certain cytochome P450s (CYP), including the rat CYP2B1 and CYP2B2 genes, are highly responsive to PB in liver hepatocytes, and provide excellent models for mechanistic studies of the induction process (1, 3). PB and other PB-like inducers do not share obvious structural homology or chemical enantioselectivity (4), as typically associated with classical receptor-mediated gene activation responses.
Using a highly differentiated hepatocyte culture system, we previously provided evidence for involvement of distinct intracellular signaling pathways that act in concert to modulate PB induction. Elevation of intracellular cAMP/protein kinase A associated activity by physiological hormones, or other protein kinase A activators, completely repressed induction (5), implying a negative modulatory role for this pathway. Recently, we demonstrated that inhibition of a PP1/PP2A protein phosphatase pathway also effectively suppressed the PB-induction response (6). Thus, the latter pathway may serve as a positive signaling intermediate in the induction process. These results led us to hypothesize that a dephosphorylation event is a required trigger, upstream of PB-mediated transcriptional activation.
In potential conflict with this line of reasoning, results of earlier studies have led some to conclude that another, distinct control mechanism is involved in PB induction. For example, Burger et al. (7) proposed a requirement for de novo protein synthesis in the induction of rat CYP2B gene expression. This conclusion was based on the 46% inhibition of PB-inducible CYP2B1/2 mRNA expression following treatment of primary hepatocytes with 10 µM cycloheximide. In contrast, PB-inducible CYP3A1 gene expression was enhanced 2-19-fold in this study by cycloheximide. These results were interpreted to indicate that PB-mediated intracellular signaling displayed divergence in its mode of induction of the CYP2B versus the CYP3A gene family members. A report by Dogra et al. (8) indicated that in vivo injections of cycloheximide treatment alone activated the levels of PB-inducible CYP2H1 mRNA in chick embryo livers. In fact, these authors demonstrated that coadministration of cycloheximide and PB resulted in a superinduction of the CYP2H1 gene, similar effects to that observed with the dioxin-inducible CYP1A1 gene (9, 10). These findings imply that a labile repressor protein is involved in modulating PB-inducible gene expression in the chicken. Honkakoski et al. (11) used 10 µM cycloheximide treatment of primary mouse hepatocyte cultures and reported that this regimen was ineffective on the Cyp2b10 PB induction status. The latter results suggest that the PB-induction process in the mouse involves a preexisting pathway, and does not require de novo protein synthesis (12).
In the current investigation, we attempted to more fully characterize the potential role of de novo protein synthesis in PB induction. Using a well characterized primary rat hepatocyte culture model, we conducted concentration-effect studies for a broad range of protein synthesis inhibitors, and for an inactive analog, puromycin aminonucleoside. Despite its lack of effect on de novo protein synthesis, the latter agent produced dose-dependent repression of PB induction. Dose-response analyses with the active protein synthesis inhibitors revealed the lack of consistent correlation between extent of protein synthesis inhibition and repression of PB induction. With the exception of puromycin aminonucleoside, all of the protein synthesis inhibitors tested were potent activators of the stress-activated signal transduction pathway (SAPK/JNK) pathway, demonstrating a lack of specificity of these commonly used inhibitors. We conclude that, in rat hepatocytes, there is no requirement for de novo protein synthesis in the PB induction of CYP mRNA expression.
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EXPERIMENTAL PROCEDURES |
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Cell Culture Materials and Chemicals--
Unless otherwise
stated, all cell culture media and TrizolTM (RNA isolation
reagent) were obtained from Life Technologies Inc. MatrigelTM, ITS+ (insulin, transferrin, selenium, bovine
serum albumin and linoleic acid) and Nu-SerumTM were
obtained from Collaborative Biomedical Products (Bedford, MA).
Leucine-free Dulbecco's modified Ealge's medium/F-12 medium was
purchased from Sigma. Tissue culture-treated plastic flasks (75 cm2) were from Falcon (Franklin Lakes, NJ).
3H-Labeled leucine was purchased from NEN Life Science
Products. Anisomycin, cycloheximide, emetine, puromycin, puromycin
aminonucleoside, and dexamethasone
(9-fluoro-16
-methyl-11
,17
,21-trihydroxy-1,4-pregnadiene-3,20-dione) were obtained from Sigma, as were all other unspecified chemicals (of
the highest grade possible). The phospho-specific SAPK/JNK antibody kit
was from New England Biolabs (Beverly, MA).
Isolation and Culture of Hepatocytes-- Rat hepatocytes were isolated by a modification of the two-step collagenase perfusion in situ (13) and cultured with a modification (14, 15) of the protocol described previously (16).
Gene Induction Treatments--
Anisomycin and cycloheximide were
dissolved in Me2SO as stock solutions (20 mM) and
stored at 20 °C. Emetine, puromycin, and puromycin aminonucleoside
were dissolved in tissue culture-grade water as stock solutions (20 mM) and stored as aliquots at
20 °C. Unless otherwise
stated, cells were cultured for 48 h prior to addition of
inhibitor or vehicle (Me2SO or tissue culture-grade water)
alone. Cells were treated with increasing concentrations of the various
protein synthesis inhibitors for 30-60 min prior to the addition of PB
(500 µM). Unless otherwise stated, all inducer treatments
were conducted for 24 h, at which point total RNA was isolated.
Representative data are presented from multiple studies performed
independently with different hepatocyte preparations.
RNA Analysis-- Total RNA was isolated (17) using TrizolTM as described elsewhere (15) from cells pooled from one 75-cm2 flask for each treatment and analyzed as described previously (15, 18).
Assessment of Protein Synthesis in Primary Hepatocytes-- Primary hepatocytes were cultured for 48 h. Prior to measurement of protein synthesis, cells were washed with L-leucine-free Dulbecco's modified Eagle's medium/F-12 medium (Sigma) supplemented with L-glutamine, L-lysine, L-methionine, CaCl2, MgCl2, MgSO4, NaHCO3, phenol red, ITS+, penicillin, streptomycin, and 25 nM dexamethasone. Cells were subsequently returned to the incubator in this same medium for a further 60 min. Protein synthesis was assessed by the ability of the cells to incorporate L-[3H]leucine (0.5 µCi/ml) in the presence or absence of increasing concentrations (0.01-25 µM) of the various protein synthesis inhibitors. Unless otherwise stated, leucine incorporation was evaluated for a 1-h pulse at 37 °C. Following the pulse, medium was removed, and the cells were washed twice with ice-cold phosphate-buffered saline to remove unincorporated label. Cells were harvested by scraping into 5 ml of ice-cold phosphate-buffered saline with a cell scraper (Nunc, Naperville, IL). After centrifugation (50 × g for 5 min, at 4 °C), 1 ml of ice-cold 10% trichloroacetic acid was added to the pellets. The resulting lysate was placed on ice for a further 30 min. Subsequently, 300 µl of this total extract were spotted, under vacuum, onto a prewetted (10% trichloroacetic acid) glass filter (no. 32, Schleicher & Schuell). The filters were washed three times with 1 ml of 10% trichloroacetic acid followed by twice with 1 ml of 95% ethanol and then dried under a vacuum. Filter-bound radioactivity was measured by liquid scintillation spectroscopy (Beckman LS3801) after the addition of 5 ml of scintillation solution (Ecoscint A, National Diagnostics, Atlanta, GA).
Determination of SAPK/JNK Phosphorylation in Primary
Hepatocytes--
We examined the ability of the various protein
synthesis inhibitors to activate the SAPK/JNK kinase pathway. Cells
were cultured for 72 h prior to start of treatments with 10 µM of the various protein synthesis inhibitors for a
period of 5-60 min (short term) or 1-24 h (long term). Controlled
time points were taken as follows: cells were washed twice with 5 ml of
ice-cold phosphate-buffered saline followed by the addition of 1.5 ml
of ice-cold cell lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium
pyrophosphate, 1 mM -glycerophosphate, 1 mM
sodium ortho-vanadate, 1 µg/ml leupeptin, 47.9 µg/ml
4-(2-aminoethyl)benzenesulfonyl fluoride, and 15.7 µg/ml
benzamidine). Cells were harvested by scraping in cell lysis buffer and
then were placed on ice. Subsequently, all extracts were homogenized by
a brief sonication pulse and then centrifuged (16,000 × g, 10 min, 4 °C) to remove insoluble material. The
resulting supernatant (cell extract) was carefully removed, and total
protein was determined (BCA reagent kit, Pierce). Equal quantities of
cell extracts (20 µg) were separated via SDS-polyacrylamide gel
electrophoresis on a Mini-Protean II 10% Tris-HCl precast gel
(Bio-Rad) according to the manufacturer's protocol. Proteins were
subsequently transferred onto Immobilon-P membranes (Millipore, Bedford, MA) for immunoblot analyses. The membranes were probed (manufacturer's blotting instructions) with a phosphorylation-specific antibody (1:1000 dilution), which recognizes SAPK/JNK only when phosphorylated at the Thr-183/Tyr-185 residues and a control SAPK/JNK antibody (phosphorylation state-independent, 1:1000 dilution) to
normalize for protein loading. The membranes were then incubated with a
goat anti-rabbit-horseradish peroxidase conjugated secondary antibody
(1:2000 dilution) and an horseradish pereoxidase-conjugated anti-biotin
antibody (1:1000 dilution) to detect biotinylated protein markers. The
Phototope-HP (New England Biolabs) chemiluminescence system was used to
visualize specific immunoreactive proteins.
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RESULTS |
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Inhibition of de Novo Protein Synthesis in Primary Rat Hepatocytes-- Various inhibitors were examined for their ability to inhibit de novo protein synthesis in cultures of primary rat hepatocytes. Cells were treated with increasing concentrations of inhibitors for 30-60 min prior to the addition of [3H]leucine. Following an additional 60-min incubation, protein synthesis activity was assessed by the measure of label incorporation. With the exception of the inactive analog of puromycin, puromycin aminonucleoside, all the inhibitors effected a concentration-dependent inhibition of de novo protein synthesis (Fig. 1). The relative potency of inhibition is in the order: emetine > anisomycin > cycloheximide > puromycin, with the last named agent exhibiting only marginal inhibition at a concentration of 1 µM. In fact, 90-95% inhibition of protein synthesis was achieved only with emetine and anisomycin, at 10 µM concentrations. Cycloheximide and puromycin exerted only 80% and 60% inhibition, respectively, at a similar concentration. Puromycin aminonucleoside was without effect on de novo protein synthesis at all concentrations examined.
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Effect of Protein Synthesis Inhibitors on PB Induction-- After establishing the concentration-effect relationships for the various inhibitors and the inactive analog, we next examined the efficacy of these agents as modulators of PB-inducible CYP gene expression. Hepatocytes were treated for 60 min with the same concentration range of inhibitors and then continuously in the absence (control) or presence (500 µM) of PB for a total of 24 h before RNA harvest. The results of RNA slot-blot hybridization analyses are presented in Fig. 2, A and B.
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Effects of Protein Synthesis Inhibitors on the SAPK/JNK Pathway-- After noting the differential effects of protein synthesis inhibitors on PB-mediated CYP gene expression, we questioned the specificity of these agents. In particular, we hypothesized that these substances were effecting other intracellular pathways, independent of their inhibitory properties on de novo protein synthesis. We tested this hypothesis by examining the ability of the inhibitors to stimulate phosphorylation of the SAPK/JNK kinase (p46 and p54 kinases) cascade. Primary hepatocytes were treated with 10 µM concentrations of the protein synthesis inhibitors for time periods ranging from 5 min to 24 h, after which total cell extracts were prepared. The results of Western blot immunoanalyses of differential phosphorylation of the p46 and p54 kinases (SAPK/JNK) between 5 and 60 min are presented in Fig. 4A (panels a, c, and e). These results were normalized to the corresponding levels of immunoreactive p46 and p54 proteins (panels b, d, and f) and are graphically represented in Fig. 4B. Normalized signal values are expressed as relative phosphorylation (arbitrary units) in Fig. 4B.
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DISCUSSION |
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In this investigation we used four distinct inhibitors of de novo protein synthesis to more carefully ascertain the role of de novo synthesis on the PB-induction process. These analyses were conducted in a well characterized primary rat hepatocyte model, assessing PB-inducible CYP gene expression as a major end point. Our results demonstrated a potent concentration-dependent inhibition of de novo protein synthesis by these agents and a corresponding but differential inhibition of PB-inducible CYP gene expression. In the results obtained by Burger et al. (7), 10 µM cycloheximide treatment produced only 46% inhibition of PB-inducible CYP2B gene expression, while PB induction of CYP3A1 gene expression was actually elevated 2-19-fold. The authors postulated that PB exhibits a distinct divergence in its mode of induction of the CYP2B and CYP3A gene family members. In our system, 8 h of 10 µM cycloheximide exposure resulted in nearly complete inhibition of PB induction, at both the CYP2B and CYP3A locus. In addition, all of the additional protein synthesis inhibitors tested resulted in a parallel inhibition of PB-induced CYP2B and CYP3A mRNA expression.
In general, we noted differential effects with these agents between their relative percent inhibition of protein synthesis and PB-inducible gene expression. This was especially clear with anisomycin and puromycin, the most and least potent inhibitors of protein synthesis, respectively, while exhibiting converse effects on the relative degree of inhibition of PB induction. These results, coupled with the finding that 10 µM puromycin aminonucleoside, a puromycin analog devoid of any protein synthesis inhibitory activity (19) (data herein), produced largely complete inhibition of the PB-induction process, raises serious issue with the tenet that de novo protein synthesis is a requirement in the PB-mediated signaling process (7, 20).
Our results are consistent with those from Negishi and co-workers (11), who reported no observable effect of cycloheximide treatment on the accumulation of the PB-inducible mouse Cyp2b10 mRNA (21). However, the latter as well as previous investigations of this issue all failed to provide 1) any measures of concentration effect on the induction process, 2) measure of the relative efficacy of their test inhibitors on de novo protein synthesis, or 3) use of any negative analogs. Typically, past conclusions regarding the requirement of de novo protein synthesis for the PB-induction process were made on the basis of a single in vivo injection (20, 22), or single concentrations of an inhibitor in cultures of primary hepatocytes (7).
To assess the specificity of the inhibitors, and whether the inhibitors affected other intracellular signaling processes independent of their action on protein synthesis, we examined the SAPK/JNK cascade. Previous studies established that the SAPK/JNK cascade is potently activated by agents such as anisomycin and puromycin (23, 24). Our data establish, for the first time, the differential phosphorylation of the enzymes involved in this pathway consequent to hepatocyte exposure to protein synthesis inhibitors. Anisomycin was clearly the most potent SAPK/JNK activator. Puromycin, despite being the most effective inhibitor of PB induction, was the least effective modulator of SAPK/JNK phosphorylation. Puromycin aminonucleoside, although highly effective in inhibiting PB induction, was completely ineffective in activating SAPK/JNK phosphorylation. We conclude, therefore, that activation of the SAPK/JNK cascade is not a signaling pathway involved in PB induction. Rather, it appears that inhibitors of protein synthesis exert a nonspecific modulation of PB induction that is quite independent of their ability to disrupt protein synthesis or effect the SAPK/JNK cascade. The exact pathway(s) affected by these agents that modulate the PB-induction process remain to be determined.
One could speculate that if de novo protein synthesis was indeed a critical mode of regulating the PB-induction process, then striking differences would result in the relative DNA-binding patterns with nuclear proteins isolated from control and PB-induced extracts. Trottier et al. (25) examined electrophoretic mobility shift assay complexes and observed that signals from complexes C1 to C3 were similar with nuclear extracts from PB-treated than untreated rat liver. In a recent study (12), establishing the role of a 177-base pair activator region in the 5' region of the PB-inducible mouse Cyp2b10 gene, Negishi and co-workers detected no differences in protein binding patterns from untreated or PB-treated nuclear extracts with any of the sequences identified as important in PB regulation. We reported similar results using extracts from control and PB-induced nuclei and their interaction with the proximal promoter region of the CYP2B1 and CYP2B2 genes (26).
Our recent studies examining the role of cAMP/protein kinase A activators and PP1/PP2A inhibitors in the PB-signaling process indicate that an upstream phosphatase pathway may participate as an activator of PB induction. The dependence on critical phosphorylation/dephosphorylation events is a well characterized phenomenon regulating enzymatic activities as well as the transcriptional status of many genes (27-30). In summary, we interpret our data to indicate that PB induction of CYP mRNA expression in rat hepatocytes is likely to signal through preexisting phosphatase-regulated factors and therefore does not require a de novo protein synthesis event. The precise pathways involved in this process remain to be identified.
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ACKNOWLEDGEMENT |
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We gratefully acknowledge the technical assistance of Diane Wing.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Grant GM32281 (to C. J. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Burroughs Wellcome Toxicology Scholar. To whom correspondence
should be addressed: University of Washington, Environmental Health,
Roosevelt, 4225 Roosevelt Way NE #100, Seattle, WA 98105-6099. Tel.:
206-543-1700; Fax: 206-685-4696; E-mail: cjo{at}u.washington.edu.
1 The abbreviations used are: PB, phenobarbital; CYP, cytochome P450; SAPK, stress-activated protein kinase; JNK, c-Jun kinase.
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REFERENCES |
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