Probing the Control Elements of the CYP1A1 Switching Module in H4IIE Hepatoma Cells

http://www.100md.com 《毒物学科学杂志》

     Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523-1680 and CIIT-Centers for Health Research, Six Davis Drive, P.O. Box 12137, Research Triangle Park, North Carolina 27709

    ABSTRACT

    Previous research from our laboratory has shown a switch-like response to PCB 126 mediated CYP1A1 induction in primary rat hepatocytes and in H4IIE rat hepatoma cells. On a single cell level, cells appear to be either "on" or "off" for CYP1A1 induction at a given dose; some cells never respond to PCB 126. These cells represent a non-responding population. Cells that are switched "on" by PCB 126 display varying levels of induction, much like the dimmer on a light switch. The goal of the present research is to begin to uncover the mechanism for this switch-like response to CYP1A1 induction in H4IIE rat hepatoma cells. The AhR pathway is modulated by multiple co-activators and by phosporylation. This research focuses on the phosphorylation cascades initiated by PCB 126 and the role they play in CYP1A1 induction. Our research reveals a likely role for protein kinase C (PKC) in this switch response. Inhibition of PKC by H-7 dramatically reduced the percent of cells that express CYP1A1 in response to PCB 126 treatment, as determined by flow cytometry. The effect of H-7 was concentration dependent, decreasing the number of cells expressing CYP1A1 rather than decreasing the level of CYP1A1 in all cells. This finding provides further evidence for the switch-like behavior of CYP1A1 induction and implicates PKC in this response to PCB126. The protein kinase inhibitor, HA-1004, had only a minor effect on CYP1A1 induction. A high-throughput immunoblot screen for 40 proteins revealed the regulation of several proteins/phosphoproteins by PCB 126. Most importantly, two proteins containing phosphoserine/phoshothreonine residues were increased by PCB126 treatment. However, PKC translocation studies and activity studies failed to verify that PCB126 activates PKC. It is possible that constitutive PKC activity is sufficient to maintain phosphorylation of critical components of the AhR pathway. Immunoblotting studies showed that MAP kinases ERK and JNK are not activated by PCB 126 in H4IIE cells and the ERK inhibitor U0126 did not impair CYP1A1 induction. Additional studies are planned to further investigate the role of PKC in the switch-like response to PCB 126.

    Key Words: PCB 126; CYP1A1; liver; transcriptional switch; PKC; phosphorylation.

    INTRODUCTION

    Halogenated aromatic hydrocarbons (HAHs) including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and various polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that induce the expression of a number of genes. These genes include the cytochrome P450 family member, CYP1A1. The induction of CYP1A1 is mediated by activation of the aryl hydrocarbon receptor (AhR) (Whitlock, 1999). AhR is a cytosolic receptor that, upon ligand binding, translocates into the nucleus and dimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) (Ma, 2001). The ligand-bound AhR-Arnt complex binds to dioxin response elements (DREs) located in the enhancer/promoter region of TCDD responsive genes such as CYP1A1. PCB 126 (3,3', 4,4', 5-pentachlorobiphenyl) is the most potent PCB AhR ligand (Hestermann et al., 2000) and it was used in the present studies. CYP1A1 displays low constitutive expression and is highly inducible by PCB 126 and TCDD (Whitlock, 1999). In rat hepatoma (H4IIE) cells, PCB 126 concentrations as low as 2.5 nM induced CYP1A1 by approximately 1000-fold as determined in real time polymerase chain reaction experiments and this cell line has been used to investigate CYP1A1 induction at the single cell level using immunohistochemistry and flow cytometry (Broccardo et al., 2004).

    Our previous studies in H4IIE cells as well as experiments using primary rat hepatocyte cultures (French et al., 2004) and rat liver in vivo (Andersen et al., 1995; Bars and Elcombe, 1991; Tritscher et al., 1992) all show a "switch" response for CYP1A1 induction. In the livers of animals exposed to CYP1A1 inducers, there is a clear boundary between responsive and non-responsive regions (Bars and Elcombe, 1991; Bars et al., 1989). Hence, individual hepatocytes appear as either uninduced or induced at any specific concentration of chemical. Primary rat hepatocyte cultures display similar behavior (French et al., 2004). On a single cell basis, adjacent cells appear induced/uninduced for CYP1A1 protein and mRNA after in vitro PCB 126 treatment as seen by in situ hybridization and immunocytochemistry. H4IIE cells displayed a similar switch-like response to PCB 126 treatment as seen using flow cytometry and immunocytochemistry for CYP1A1 protein (Broccardo et al., 2004). The concentration-dependent switching response was indicated by an increase in the proportion of cells that expressed CYP1A1. The cells that were switched "on" by PCB126 displayed varying degrees of induction intensity. These data support a hybrid switch response, where a switch works in concert with a rheostat, much like a dimmer on a light switch in a home. Thus, H4IIE cells have been established as a good model to further study the switch response.

    Recently it has been acknowledged that gene expression may exhibit either a graded or a "switch-like" response to a stimulus (Louis and Becskei, 2002). Single cell studies have further revealed that many enhancer linked genes are generally "on" or "off" in individual cells; the active enhancer increases the probability that the gene will be active in a given cell (Fiering et al., 2000). However, given identical stimuli, some cells will still remain in the "off" state in such a stochastic model of enhancer-gene interaction. Other factors that may contribute to this switch-like, binary response include protein kinase cascades (Ferrell, 1996; Ferrell and Machleder, 1998), transcriptional synergy between transcription factors and promoter elements (Carey, 1998), the interactions of repressors, activators, and co-activators (Blankenship and Matsumura, 1997; Gradin et al., 1999; Mimura et al., 1999), and chromatin remodeling (Okino and Whitlock, 1995). Switch-like behavior of gene induction could explain the observed threshold response of a cell to a particular chemical, and perhaps the phenomenon that some cells appear to be non-responders, even at the highest concentration.

    The purpose of the present studies was to use the H4IIE rat hepatoma cell model to elucidate the mechanism of the switch response. It is postulated that this response can be explained by nongenomic factors modulating the AhR pathway, such as mitogen activated protein kinases (MAPKs), protein kinase C (PKC), co-activators, or other associated proteins that are activated by PCB 126 or other AhR ligands (Ferrell and Machleder, 1998; Long et al., 1998, 1999; Minsavage et al., 2004; Tan et al., 2002, 2004; Tian et al., 2003; Torchia et al., 1998; Yim et al., 2004). The effects of such mediators appear to be highly cell-specific and not all mediators appear to be crucial for CYP1A1 induction in all tissues. The focus of the present studies was on the investigation of a number of potential signal transduction pathways in H4IIE cells, the recently established model for the switching phenomenon, with an emphasis on phosphorylation pathways. In particular, the effects of PCB 126 on MAPKs and PKC, previously reported to be involved in CYP1A1 induction in other cells, was investigated. The concentration of PCB 126 used in the present studies results in maximal induction of CYP1A1 in these cells (Broccardo et al., 2004).

    MATERIALS AND METHODS

    Cell culture.

    All cell culture products were obtained from Gibco (Carlsbad, CA) unless otherwise noted. Rat hepatoma H4IIE cells (ATCC) were cultured in DMEM supplemented with 10% FBS (Hyclone, Logan, UT) and 100 units/ml penicillin/100 μg/ml streptomycin and maintained at 37°C and 5% CO2. Cells were seeded at 2.5 x 106 or 6.5 x 106 cells in 60-mm or 100-mm culture dishes, respectively (Falcon).

    Cell treatments.

    PCB 126 was obtained from Accustandard (New Haven, CT) and confirmed by GC/MS to be 100% pure and free of other congeners. For treatment, PCB 126 was dissolved in DMSO; treatments contained less than 0.2% DMSO. The concentration of PCB 126 (2.5 x 10–7 or 2.5 x 10–8) was previously found to maximally induce CYP1A1 in these cells (Broccardo et al., 2004). 1-(5-isoquinolinesulfonyl-2-methylpiperazine (H-7) and N- (2-guanidinoethyl)-5-isoquinoline-sulfonamide (HA-1004) (Biomol, Plymouth Meeting, PA) were dissolved in PBS. PMA (phorbol-12-myristate-13-acetate) was dissolved in DMSO (Cell Signaling, Beverly, MA). U0126 (bis[amino[(2-aminophenyl)thio]methylene]butanedinitrile) (Biomol) was dissolved in DMSO. No substantial changes in growth rate or morphology were observed after treatment with DMSO, PCB 126, or the inhibitors as compared to nave cells. Toxicity was also assessed by measuring trypan blue exclusion in suspensions of trypsinized cells. Trypan blue exclusion was at least 80% after all treatments.

    CYP1A1 flow cytometry.

    Cells, 2.5 x 106, were plated on 60-mm dishes for 24 h, serum starved for 24 h, then treated with DMSO or 2.5 x 10–8 M PCB 126 for 16 h. The inhibitors H-7, HA-1004, and U0126 were applied 30 m prior to the addition of PCB 126. PMA was applied with PCB 126 treatment. Flow cytometry was conducted as previously described (Broccardo et al., 2004). Briefly, cells were trypsinized, fixed in formaldehyde, and permeabilized with saponin. Cells were blocked in 5% goat serum (Sigma) in PBS/1% BSA and then incubated with rabbit anti-rat CYP1A1 polyclonal antibody (Chemicon, Temecula, CA; 1:500) followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes, Eugene, OR; 1:200). The cells were analyzed on a Beckman Coulter EPICS 5 Flow Cytometer. Alexa Fluor 488 was excited by the 488 nm line of an argon ion laser and fluorescence was detected by a photomultiplier equipped with a 525 band pass filter. Light scatter was collected in both the forward and right angle directions. Data were processed and displayed on the Cyclops software (DakoCytomation, Ft. Collins, CO). Nonspecific binding of the antibodies was found to be negligible by incubation with rabbit IgG (Sigma) instead of the rabbit anti-rat CYP1A1 primary antibody or by incubation without the primary antibody.

    Aryl hydrocarbon receptor flow cytometry.

    Cells, 2.5 x 106, were plated on 60-mm dishes for 24 h, and then treated with DMSO or 2.5 x 10–8 M PCB 126 for 16 h or 24 h. The same protocol as for CYP1A1 was employed, except for the use of the rabbit anti-rat AhR polyclonal antibody (Biomol; 8 μg/ml). Rabbit IgG (Sigma; 8 μg/ml) was used for gating purposes. Samples were analyzed on a CyAn LX (DakoCytomation) instrument. Alexa Fluor 488 was excited by a 488 nm (20 mW semiconductor) laser. Fluorescence was detected by a photomultiplier tube equipped with a 530/40 bandpass filter. Light scatter was collected in both the forward and right angle directions. Data were analyzed using Summit software (DakoCytomation).

    Custom protein immunoblot.

    Cells were seeded at 6.5 x 106 cells in 100-mm dishes, allowed to plate down for 24 h, then serum starved for 24 h. Cells were exposed to 2.5 x 10–7 M PCB 126 or DMSO for 30 m or 6 h. Cells were rinsed with ice cold PBS and 1 ml of boiling lysis buffer was added (10 mM Tris HCl, pH 7.4, 1 mM sodium ortho-vanadate, 1% SDS). Lysate was removed with a cell scraper, and 3 100-mm dishes per treatment were combined into a 50 ml conical tube, microwaved for 5–10 s, and sonicated for 30 s. Protein concentration was determined using a BCA protein assay using bovine serum albumin as a standard (Pierce, Rockford, IL). We contracted with BD Biosciences Pharmingen (San Diego, CA) to perform a custom "PowerBlot" 40-antibody miniscreen via large scale Western blotting. This analysis utilized 200 μg of cellular protein that was run on a 4–15% gradient SDS-polyacrylamide gel. The proteins were transferred to Immobilon-P membrane (Millipore, Billerica, MA), the membrane was blocked, and then clamped with a Western blotting manifold that isolates 41 channels across the membrane. In each channel a complex antibody cocktail was added. Two different molecular weight standard cocktails were included on the gel. Proteins were detected using fluorescent secondary antibodies (goat anti-mouse Alexa 680 for monoclonal primary antibodies and goat anti-rabbit IR Dye 800 [Rockland, Gilbertsville, PA] for polyclonal antibodies). The membrane was scanned at 700 nm (for monoclonal antibody target detection) and 800 nm (for polyclonal antibody target detection) using an Odyssey Infrared Imaging System (LI-COR). Blots were performed in triplicate for each treatment.

    Data analysis of custom protein immunoblot.

    Fluorescent intensities of the spots on the membrane were normalized to the sum intensity of all valid spots on a blot and then multiplied by 1,000,000. The normalized quantity for experimental spots (PCB 126) was expressed as a ratio of the normalized quantity for the corresponding control spots (DMSO). This ratio was used to determine changes in protein expression. Triplicate blots were analyzed using a 3 x 3 matrix comparison method. For example, runs 1, 2, 3 of the control were compared independently to runs 1, 2, 3 of the experimental samples. Results are finally expressed as a fold change, a semi-quantitative value that represents the general trend of protein changes, either increasing or decreasing, for the experimental sample relative to control.

    P-ERK, ERK, P-JNK, JNK Western blotting.

    Cells were allowed to plate down with serum at 6.5 x 106 cells per 100-mm dish for 24 h and then serum starved for 24 h. Cells were treated with 120 nM PMA, 2.5 x 10–7 M PCB 126 or DMSO for 5 min, 15 min, 30 min, or 60 min. Cells were rinsed with ice cold PBS and 0.6 ml of ice cold RIPA buffer (1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, protease inhibitor cocktail tablet [Complete Mini, Roche, Indianapolis, IN] in phosphate buffered saline) was added. Cells were incubated on ice for 5 min, removed with a cell scraper, and transferred to a microcentrifuge tube. Plates were rinsed with 0.3 ml cold RIPA buffer, combined with the first lysate, and cells/DNA were sheared using a 21- and 24-gauge needle, successively and then incubated for 30–60 min on ice. Lysates were centrifuged at 10,000 x g for 10 min at 4°C and the supernatant was removed and frozen at –80°C. A BCA protein assay was performed (Pierce) using albumin standards in RIPA buffer. 10 μg protein (phosphorylated extracellular regulated kinase, P-ERK), 30 μg protein (phosphorylated c-Jun N-terminal kinase, P-JNK), and purchased positive control PC12 cell extracts treated with nerve growth factor (for P-ERK) or sorbitol (for P-JNK) (Promega, Madison, WI) were diluted with 2x Laemmli buffer (Bio-Rad) and boiled for 3 min. An unstained Precision Plus Protein standard (Bio-Rad, 1:60), and a prestained Precision Plus Protein Kaleidescope standard were loaded (Bio-Rad) for chemiluminescent detection of band size, and to monitor transfer, respectively. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% Tris-HCl polyacrylamide gels, 50 μl/well, 10 wells, (Bio-Rad) in a Mini-Protean II Electrophoresis Cell (Bio-Rad). The gel was run for 1.5–2 h at 80 V, and then proteins were transferred to a nitrocellulose membrane of 0.45 μm pore size (Bio-Rad). Membranes were blocked for 60 min in Tris-buffered saline pH 7.6 with 1% Tween 20 (TBS-T) containing 5% nonfat dried milk. Thereafter, polyclonal rabbit P-ERK or monoclonal mouse P-JNK (Cell Signaling; 1:2000) antibodies were incubated in TBS-T containing 5% bovine serum albumin (BSA) overnight on a shaker at 4°C. Membranes were then washed six times for 10 min on a shaker in TBS-T. Horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse for P-JNK, Bio-Rad; goat anti-rabbit for P-ERK, Santa Cruz; 1:2000) were incubated on a shaker for 60 min in TBS-T containing 5% nonfat dried milk. The Precision StrepTactin-HRP conjugate (Bio-Rad) secondary antibody against the unstained Precision Plus Protein standard was simultaneously added at 1:15,000. Membranes were then washed six times for 10 min on a shaker in TBS-T. Bands were visualized after a 3 min incubation in Immun-Star HRP (Bio-Rad) and viewed using the UVP BioImaging Systems Epi Chemi II Darkroom. Membranes were then stripped in boiling buffer (62.5 mM Tris pH 6.7, 2% SDS, 100 mM -mercaptoethanol) twice for 30–45 s, rinsed in copious volumes of TBS-T, and blocked in 5% nonfat dried milk in TBS-T for 60 min on a shaker. Polyclonal rabbit ERK and JNK antibodies (Cell Signaling, 1:2000) were incubated in 5% BSA in TBS-T overnight at 4°C on a shaker. Membranes were washed and a secondary goat anti-rabbit HRP conjugate (Santa Cruz, 1:2000) and the Precision StrepTactin-HRP conjugate were added as above. Membranes were washed and visualized as with P-ERK and P-JNK.

    Protein kinase C subcellular fractionation.

    H4IIE cells were plated onto 100-mm dishes at a density of 6.5 x 106 cells. Cells were allowed to adhere for 24 h then serum was removed for 24 h. 6 100-mm dishes per treatment were exposed to 2.5 x 10–8 M PCB 126 or equivolume DMSO equivalent to 0.2% for 10 min, 30 min, 2 h, 6 h, or 16 h. 120 nM PMA (phorbol-12-myristate-13-acetate; Cell Signaling) in DMSO was applied for 10 min. After treatment, the media was removed and the plates were rinsed twice in ice cold PBS and plates were kept on ice. 350 μl of ice-cold homogenization buffer (20 mM Tris-HCl pH 7.5, 0.25 M sucrose, 5 mM EGTA pH 8.0, 20 mM -mercaptoethanol, 1 mM sodium orthovanadate, protease inhibitor cocktail tablet in phosphate buffered saline) was added to each dish and cells were scraped into a tube and kept on ice. The 6 100-mm dishes per treatment were combined into one tube. Cells were sonicated for three 10 s pulses at 25 Hz with intervals of 15 s on ice. Cells were centrifuged at 100,000 x g at 4°C for 45 min to separate the soluble (cytosolic) fraction from the particulate (membrane) fraction. The supernatant was then removed and saved as the cytosolic fraction at –80°C. The pellet was resuspended in homogenization buffer containing 1% Triton X-100 and 2 mM EDTA, pH 8.0 and incubated on ice for 10 min. The pellets were then sonicated for 3 10 s pulses at 25 Hz with intervals of 15 s on ice. The samples were then centrifuged at 100,000 x g at 4°C for 45 min. The supernatant was saved as the soluble membrane fraction and frozen at –80°C and the pellet of insoluble protein was discarded. A BCA protein assay was performed (Pierce) using albumin standards in homogenization buffer.

    Protein kinase C Western blotting.

    25 μg protein was diluted with 2x Laemmli buffer (Bio-Rad) and boiled for 3 min. An unstained Precision Plus Protein standard (Bio-Rad, 1:60), and a prestained Precision Plus Protein Kaleidescope standard were loaded (Bio-Rad) for chemiluminescent detection of band size, and to monitor transfer, respectively. 10 μg rat cerebrum lysate (BD Transduction Laboratories) was loaded as a positive control. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 7.5% Tris-HCl polyacrylamide gels, 50 μl/well, 10 wells, (Bio-Rad) in a Mini-Protean II Electrophoresis Cell (Bio-Rad). The gel was run for 1.5 at 80 V, and then proteins were transferred to a nitrocellulose membrane of 0.45 μm pore size (Bio-Rad). Membranes were blocked for 60 m in Tris-buffered saline pH 7.6 with 1% Tween 20 (TBS-T) containing 5% BSA. Thereafter, the PKC lambda, delta and epsilon mouse monoclonal antibodies (1:1000, BD Transduction Laboratories) and mouse monoclonal PKC alpha (1:100, Santa Cruz) were incubated in TBS-T containing 5% bovine serum albumin (BSA) overnight on a shaker at 4°C. Membranes were then washed five times for 10 min on a shaker in TBS-T. Horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:5000, Bio-Rad) was incubated on a shaker for 60 min in TBS-T containing 5% nonfat dried milk. The Precision StrepTactin-HRP conjugate (Bio-Rad) secondary antibody against the unstained Precision Plus Protein standard was simultaneously added at 1:15,000. Membranes were then washed five times for 10 min on a shaker in TBS-T. Bands were visualized after a 3 min incubation in Immun-Star HRP (Bio-Rad) and viewed using the UVP BioImaging Systems Epi Chemi II Darkroom.

    PKC kinase activity assay.

    H4IIE cells were plated onto 100-mm dishes at a density of 6.5 x 106 cells. Cells were allowed to adhere for 24 h then serum was removed for 24 h. One 100-mm dish per treatment was exposed to 2.5 x 10–8 M PCB 126 or equivolume DMSO equivalent to 0.2% for 5 min, 10 min, 30 min, and 1 h. A media only control was also used. After treatments media was removed and cells were rinsed with ice cold PBS. 1 ml of lysis buffer (20 mM MOPS, 50 mM -glycerolphosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM EGTA, 2 mM EDTA, 1% NP40, 1 mM dithiothreitol (DTT), 1 mM benzamidine, and protease inhibitor cocktail tablet [Roche]) was then added per 100-mm dish and allowed to stand for 10 m on ice. Cells were then removed with a cell scraper and the lysate was collected in a pre-chilled microcentrifuge tube. Samples were then centrifuged at 13,000 rpm for 15 min at 4°C. The supernatants were then transferred to new microcentrifuge tubes, aliquoted into 100 μl samples, and frozen at –80°C. Protein was measured using the RC-DC Protein Assay kit and the protocol was modified to be read on a plate reader set to 750 nm (BioRad). PKC kinase activity was measured using the ELISA-based PKC kinase activity assay kit (Stressgen, Victoria, BC, Canada) according to company directions. Each treatment was run on triplicate wells. Briefly, 0.1 μg protein was used per well; the reaction was initiated by the addition of ATP, and then a phosphospecific antibody against the substrate CREB was added. Anti-rabbit IgG:HRP conjugate was then applied to each well, followed by the addition of the TMB (tetramethylbenzidine) substrate. Acid stop solution was added and absorbances were read on a plate reader set to 450 nm. Active PKC was used as a positive control and a blank well without antibodies was used to measure the background fluorescence of the buffers. For data analysis, the triplicate wells for each treatment were averaged, the background fluorescence of the blank well was subtracted, and the PCB treatment value was divided by the respective control value to determine a relative percent kinase activity. ANOVA analysis was used to determine significant difference between the PCB treatment at each time point and the DMSO control at each time point.

    Statistical analysis.

    Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison tests. A p value of 0.05 was used for significance between groups. All data are expressed as mean ± SEM.

    RESULTS

    Effects of Protein Kinase Inhibitors on CYP1A1 Expression in Single Cells

    The protein kinase inhibitors H-7 and HA-1004 were used to assess the roles of PKC and other protein kinases in PCB 126 mediated induction of CYP1A1 in H4IIE cells. H-7 and HA-1004 are broad spectrum protein kinase inhibitors that have similar Ki values for most kinases, including protein kinase A (PKA) and protein kinase G, but H-7 is approximately seven times more potent in inhibiting PKC (Hidaka et al., 1984). Therefore, this pair of chemicals can be used to implicate specific effects on PKC. We selected this approach because of problems reported with more specific inhibitors of PKC. For example, it has been found that the effects of several inhibitors of PKC cannot be separated from cytotoxic effects (Reiners et al., 1993), there are interactions with the AhR (Schafer et al., 1993) or they have non-specific effects on MAPK (Yu et al., 2000). Cells were pretreated for 30 min with various concentrations of either H-7 or HA-1004 then treated for 16 h with 2.5 x 10–8 M PCB 126. CYP1A1 expression decreased significantly (p < 0.05) from 62 ± 3.6% of the cells to 18 ± 0.33% after the use of H-7 at a concentration of 50 μM (Fig. 1a). Fifty μM H-7 plus DMSO treatment resulted in no significant difference between DMSO treatment alone (p > 0.05). If H-7 turns off the switch, intermediate concentrations would be expected to decrease the percent of cells expressing CYP1A1 rather than decreasing the level of CYP1A1 in all cells, in a concentration-dependent fashion. Figure 1b shows the H-7 concentration response relationship. Concentrations as low as 20 μM decreased the percent of cells expressing CYP1A1; the IC50 was 35 μM. The histogram overlay distinctly shows the decrease in cells expressing CYP1A1 due to H-7 treatment (Fig. 1c). Cells within region 1 represent the "off" cells for CYP1A1 expression and general background fluorescence, and cells showing an increase in log intensity green fluorescence in region 2 are positive for CYP1A1 expression. The shift of the curve from region 2 to region 1 for the PCB 126 plus H-7 treatment highlights the movement of cells from "on" to "off." At the concentration of 30 μM it can be seen that H-7 has decreased the number of "on" cells rather than decreasing the level of CYP1A1 in all cells which would have been indicated by a shift in the peak in region 2 to the left. Consistent with previous results (Broccardo et al., 2004), the population of "on" cells displays variable levels of CYP1A1 expression.

    Only at the highest concentration of HA-1004 (50 μM) was there a significant decrease in CYP1A1 expression from 57 ± 3.6% to 43 ± 0.88% of the cells (p < 0.05) (Fig. 2a). As seen in the histogram overlay, there is a subtle decrease in the percent of cells expressing CYP1A1 due to HA-1004 treatment as compared to PCB 126 treatment alone (Fig. 2b). Taken together, the data show that the effects of H-7 are primarily due to PKC inhibition.

    To further study the role of PKC in CYP1A1 induction the effects of phorbol 12-myristate 13-acetate (PMA) were investigated. Phorbol esters have been shown to either increase (Chen and Tukey, 1996; Long et al., 1998, 1999; Moore et al., 1993) or decrease (Berghard et al., 1993; Guo et al., 2001; Reiners et al., 1993) CYP1A1 induction, in a cell dependent and time dependent fashion. In vivo liver induction of CYP1A1 is rapidly decreased by phorbol esters (Okino et al., 1992). PMA (10, 40, 80, 120 nM) was applied in conjunction with 2.5 x 10–8 M PCB 126 for 4, 8, and 16 h and compared to PCB alone, DMSO alone, and 120 nM PMA alone (Fig. 3). At the earliest time point, 4 h, PMA decreased the percent of cells expressing CYP1A1 from 27 to 17% at the lowest concentration used. There was little further change with increasing PMA concentration. With time, the inhibitory effects of PMA were diminished.

    It has been reported that MAPKs are involved in the induction of CYP1A1 (Tan et al., 2002, 2004; Yim et al., 2004). These reports utilized the specific inhibitor of MEK, U0126. In the present studies, cells were treated for 16 h with U0126 (10 μM) along with PCB126 (2.5 x 10–8 M). There was a small increase in CYP1A1 expression in cells treated with U0126 plus PCB126 (64 ± 0.33% vs. 72 ± 1.2%, n = 3, p < 0.05), suggesting that ERK is not required for CYP1A1 induction in these cells (data not shown).

    Expression of Aryl Hydrocarbon Receptor in Single Cells

    Flow cytometry was employed to determine the distribution of the AhR in H4IIE cells. Cells in region 2 of the histogram contain the AhR, whereas the cells within region 1 represent background fluorescence of the cells and any nonspecific binding of the rabbit IgG antibody used for gating purposes (Fig. 4). The distinct separation of these curves signifies the presence of the AhR in the majority of the cell population after PCB 126 treatment as well as in the control cells (DMSO treatment). Approximately 87% of the cells expressed the AhR after treatment with 2.5 x 10–8 M PCB 126 for 16 h. Although this represents a small difference in percent of cells expressing the AhR compared to DMSO treated cells (95%) it is apparent that PCB treatment primarily caused a shift of the distribution to the left, most likely reflecting a decrease in the level of the AhR in most cells. It is well established that ligands enhance degradation of the AhR (Ma and Baldwin, 2000). These results show that lack of the AhR, even after PCB treatment, cannot account for the non-responding cell population and the apparent switch response.

    Effects of PCB 126 on Protein Expression

    To explore the effects of PCB 126 on the expression of proteins primarily involved in phosphorylation pathways, a custom protein immunoblot was performed. Cells were treated with 2.5 x 10–7 M PCB 126 or DMSO for 30 min and 6 h. Table 1 shows the proteins that were probed for and indicates those proteins that were not detected on the blots. It is of interest that P-ERK was not detected at either time point. P-JNK and P-p38 were detected but the levels were very low, based upon the densitometry values, and PCB treatment did not affect these low levels. These results are reported in the Supplementary Data. Table 2 shows the proteins that were altered by the PCB126 treatment compared to the DMSO control. At 30 min, the antibody against CREB/CREM (46/26 kDa) detected an increase of 1.25–1.9 fold of the smaller 26 kDa band. This band represents an alternatively spliced product of the gene CREM known as the cAMP-inducible repressor isoform or ICER (Inducible cAMP Early Repressor). At 6 h, two unknown proteins (109 kDa, 233 kDa) showed an increase in phosphoserine/phosphoserine/threonine residues by 1.25–1.9 fold. Protein kinase A regulatory subunit IIb (PKARIIb, pS114), phospho-specific was decreased by more than 2 fold at 6 h. Jun was increased by more than 1.5 fold at 6 h. It is noteworthy that there was no effect of the PCB treatment on any of the PKC isoforms detected, which includes PKC alpha, delta, epsilon, and lambda. PKC beta, gamma, and theta, and phospho-PKC alpha were not detected. Based upon the densitometry values, PKC alpha and delta are the most abundant isoforms in these cells, followed by PKC epsilon. There was also no significant effect of PCB treatment on the phosphorylated MAPKs at either of these time points.

    Lack of Effect of PCB 126 on Phosphorylation of ERK and JNK

    The following experiments were conducted in order to extend the custom immunoblot results to additional time points. In these experiments, PMA was used as a positive control. It was found to stimulate both ERK and JNK phosphorylation after 30 min of treatment of the cells with 120 nM PMA or 40 nM PMA as can be seen from P-ERK bands at 44/42 kDa and the P-JNK bands at 54/46 kDa (Fig. 5). In contrast, P-ERK and P-JNK bands were not observed in cells treated with 2.5 x 10–7 M PCB 126 or DMSO for 5 min, 15 min, 30 min, and 60 min. The positive control PC12 cell extracts for P-ERK and P-JNK displayed bands at 44/42 kDa and 54/46 kDa, respectively, allowing for confirmation of band sizes. All extracts stained for ERK and JNK. Treatments had no effect on levels of ERK and JNK (Fig. 5).

    Lack of Effect of PCB 126 on Translocation of PKC Isoforms

    The purpose of these experiments was to determine if PCB 126 treatment was capable of activating PKC translocation from the cytosolic to the membrane fraction. Cells were treated with 2.5 x 10–8 M PCB 126 or DMSO for 10 min, 30 min, 2 h, 6 h, or 16 h. Figure 6a shows a typical response at the 10 min time point. PMA (120 nM) was applied for 10 min and represents a positive control. The , , and PKC isoforms responded to PMA treatment, displaying translocation from the cytosol to the membrane (Fig. 6b). PKC , an atypical PKC, does not respond to PMA. In contrast with PMA, there were no striking differences between PCB 126 treatment and DMSO control at any of the time points measured; PCB 126 did not induce membrane translocation of any of the four isoforms tested. PKC remained primarily in the cytosolic fraction at all times tested. PKC , , and did not redistribute to the membrane fraction in response to PCB 126, and were present in both the cytosolic and membrane fractions at all times measured. The media only control showed that, constitutively, PKC remains mostly in the cytosolic fraction. In contrast, the , , and isoforms display constitutive localization to both the cytoplasmic and membrane fractions.

    Lack of Effect of PCB 126 on PKC Kinase Activity

    To explore the effect of PCB 126 on PKC kinase activity, an ELISA-based assay was conducted. H4IIE cells were treated with media only, DMSO or 2.5 x 10–8 M PCB 126 for 5 min, 10 min, 30 min, and 1 h. No significant difference between the PCB treatment and the DMSO vehicle control at any time point was found as determined by ANOVA (Table 3). The media only samples had similar absorbances to all treatments. Despite the lack of increased PKC kinase activity at any time point measured, there was a trend of increased kinase activity at the 1 h time point.

    DISCUSSION

    This research stems from previous studies that documented a switch-like response to CYP1A1 induction in H4IIE cells (Broccardo et al., 2004) as well as in cultured rat hepatocytes (French et al., 2004) and in vivo (Bars and Elcombe, 1991; Bars et al., 1989). Biologically, this behavior represents a hybrid switch model, and is comparable to a dimmer on a light switch in a home, where a switch works in concert with a rheostat. Experimentally, such a hybrid switch model on the single cell level represents an initial induction threshold to turn on the switch then a graded response thereafter (Broccardo et al., 2004; French et al., 2004).

    The present studies aim to explore mechanisms of PCB 126-mediated switch-like behavior in H4IIE cells. We have investigated several signal transduction pathways in H4IIE cells, focusing on phosphorylation, and the response to PCB 126. The data presented in this article specifically implicate PKC in the switch response to PCB126. This is in stark contrast to the lack of effect on a number of other pathways investigated in the custom 40-antibody immunoblot, including MAPKs. The results show that inhibition of PKC with H-7 dramatically impairs PCB 126-mediated induction of CYP1A1 at the single cell level in these cells. Furthermore, H-7 appears to turn off the switch instead of inhibiting induction in a graded fashion in all cells.

    PKC represents a family of protein kinases that includes at least 12 isozymes. The regulatory region of PKCs contains one or two zinc finger-membrane motifs (C1 and C2 domains). The C1 domain is activated by 1,2 diacylglycerol (DAG), phorbol esters, and phosphatidylserine (PS). The C2 domain contains a Ca2+ binding motif. The conventional PKCs (, I, II, ) contain functional C1 and C2 domains. Novel/nonconventional PKCs (, , , μ, ) contain a functional C1 domain, but lack a functional C2 domain. The atypical PKCs (, , ) contain a non-ligand-binding C1 domain and no C2 domain (Ventura and Maioli, 2001). PKC localization is tightly regulated and subcellular targeting plays a major role in isoform activation (Newton, 2003; Ohmori et al., 1998, 2000; Rybin et al., 2004; Shirai and Saito, 2002; Ventura and Maioli, 2001). However, at the time points we tested, PCB 126 did not induce membrane translocation of PKC distinct from DMSO controls. These time points were selected based upon a large body of data in the literature with other activators, including PMA. However, it is possible that the translocation is so transient that the time points studied were not sufficient to observe the response. It has been reported that ATP induces membrane translocation of PKC within 30 s, and a subsequent return to the cytosol within 3 min (Ohmori et al., 1998). Alternatively, PCB 126 may activate PKC kinase activity without membrane translocation. H2O2 has been shown to increase tyrosine phosphorylation on PKC and concomitantly increase its enzymatic activity without inducing membrane translocation (Konishi et al., 2001). However, we did not observe a PCB 126-mediated increase in PKC activity, as determined by an ELISA-based PKC activity assay. This is in contrast to results reported with other HAHs, primarily TCDD, in some cells and in vivo (Bombick et al., 1985; Hanneman et al., 1996; Puga et al., 1992; Weber et al., 1994; Williams et al., 2004). It is possible that PCB 126 increases PKC-mediated phosphorylation of proteins involved in the AhR-mediated pathway leading to induction of CYP1A1 without stimulating overall PKC activity. It is known that both AhR and ARNT are phosphoproteins and phosphatase treatment impairs DNA binding (Berghard et al., 1993; Carrier et al., 1992; Long et al., 1999; Mahon and Gasiewicz, 1995; Pongratz et al., 1991). In support of this idea, it has recently been shown that a mixture of conventional PKC isoforms (, , ) are capable of phosphorylating serine/threonine residues on the full length AhR and that tyrosine 9 of the AhR greatly facilitates this phosphorylation (Minsavage et al., 2004) although tyrosine 9 is not phosphorylated. We did not detect any change with PCB 126 treatment in phosphotyrosines in the 40-antibody immunoblot experiments which is consistent with the lack of effect of protein tyrosine kinase inhibitors on CYP1A1 induction in H4IIE cells (Backlund et al., 1997). However, the immunoblot detected an increase in two phosphoserine/threonine proteins. One of these proteins (109 kDa) is similar to the size of the AhR, 106 kDa in Sprague Dawley rat liver (Franc et al., 2001). It is of interest to determine in future experiments if PCB 126 increases AhR phosphorylation in H4IIE cells. It is also possible, however, that the PKC inhibitor (H-7) turns off the transcriptional switch simply by shifting the balance between protein kinase activity and protein phosphatase activity in favor of removal of a critical phosphorylated residue such as recently observed (Minsavage et al., 2004).

    In the present studies, we investigated the effect of the phorbol ester, PMA, on PCB 126-mediated induction of CYP1A1 in H4IIE cells. The effect of phorbol esters on CYP1A1 induction is highly dependent upon the cell type and time of treatment. In some cases, phorbol esters potentiate CYP1A1 induction (Chen and Tukey, 1996; Long et al., 1998, 1999; Moore et al., 1993) and in other studies, phorbol esters block induction (Berghard et al., 1993; Guo et al., 2001; Okino et al., 1992; Reiners et al., 1993). The mechanisms involved have not been identified although it is clear that the "PMA effect" is due to effects on PKC, as determined by the use of PKC inhibitors. In these studies, a number of possible mechanisms whereby PKC activity modulates CYP1A1 induction have been discounted (Chen and Tukey, 1996; Long et al., 1998, 1999; Long and Perdew, 1999; Schafer et al., 1993). In the present studies, PMA had a transient effect (decrease) on the number of cells expressing CYP1A1 upon treatment with PCB 126; this was observed at 4 h but diminished by 8 h. It is interesting that PMA rapidly (10 min) caused a translocation of PKC isoforms from the cytosol to the membrane. It is possible that the reduced PKC activity in the cytosol results in decreased phosphorylation of cytosolic proteins such as the AhR that are critical to CYP1A1 induction. Further experiments are required to investigate this possible mechanism.

    The effect of PCB 126 on the activation of MAPK was investigated. This phosphorylation pathway has been implicated in transcriptional switch responses (Ferrell, 1996; Ferrell and Machleder, 1998; Ferrell and Xiong, 2001; Hazzalin and Mahadevan, 2002) and TCDD has been reported to activate ERK (Tan et al., 2002; Yim et al., 2004) and JNK (Tan et al., 2002) in some cells. In contrast, p38 was not activated by TCDD (Tan et al., 2002). Recently, it was reported that PCB 126 activates ERK and p38 in HepG2 cells (Song and Freedman, 2005). The concentration of PCB 126 used in these studies apparently caused an oxidative stress response but these concentrations are 100–1000 times higher than used in our studies. The concentrations of PCB 126 used in the present studies are known to maximally induce CYP1A1 (Broccardo et al., 2004). At these concentrations, PCB126 did not activate ERK and JNK at any of the time periods investigated. In addition, p38 phosphorylation was not increased by PCB 126 treatment at the 30 min and 6 h time points analyzed by the custom immunoblots. These results suggest that MAPKs are not the transcriptional switch in H4IIE cells that mediates CYP1A1 induction. The role of MAPKs in CYP1A1 induction is apparently dependent upon cell type. Treatments that reduce ERK and JNK activity impair TCDD induction of CYP1A1 in some cells (Tan et al., 2002, 2004; Yim et al., 2004) but induction is not impaired in other cells (Andrieux et al., 2004; Guo et al., 2001; Tsukumo et al., 2002). In vivo, the roles of MAPKs in AhR activity appear to be highly tissue specific. For example, JNK2 knockout mice demonstrated opposite roles of JNK2 in the liver versus the thymus and testes, showing reduced TCDD-stimulated CYP1A1 expression in thymus and testes but increased expression in liver (Tan et al., 2004). In our studies, the ERK inhibitor, U0126, did not decrease CYP1A1 induction by PCB126 in H4IIE cells, supporting the conclusion that MAPKs do not mediate the switch-like response in this model. The small increase in CYP1A1 induction that was observed may be explained by the observation that this inhibitor interacts with the AhR and induces CYP1A1 (Andrieux et al., 2004).

    The 40-antibody immunoblots were performed after 30 min and 6 h of PCB 126 treatments. These time points were designed to observe the typically fast response of phosphorylation, and also to observe any changes in immediate early genes. At the 30 min time point there was an increase in the transcriptional repressor ICER. ICER is involved in auto regulatory feedback loops that regulate the transcription of immediate early genes such as jun and all genes that contain a cAMP response element (CRE) (Servillo et al., 2002). ICER is induced in H35 hepatoma cells by cAMP as early as 30 min and peaks by 2 h; ICER is also induced by 2 h after a partial hepatectomy (Servillo et al., 1997). The down regulation of the phosphorylated form of PKA regulatory subunit IIb represents potential modulation of the PKA pathway by PCB 126. It has recently been reported that TCDD activates PKA (Vogel et al., 2004). The increased levels of the protooncogene Jun at 6 h is consistent with the literature and TCDD and PCB 126 have been shown to affect this pathway (Hoffer et al., 1996; Puga et al., 1992; Tanno and Aoki, 1996). TCDD has been shown to induce Jun and increase AP-1 transcription factor activity, and this response may be PKC dependent (Puga et al., 1992). In addition to AhR independent pathways, Jun contains DRE sequences in its promoter region, lending this gene to direct regulation by the AhR pathway (Hoffer et al., 1996). PCB 126 has also been shown to lead to increased phosphorylation of c-Jun (Tanno and Aoki, 1996). Taken together, the results of the protein blots suggest a possible dynamic cellular response to PCB 126 that involves cAMP, PKA, and Jun. It is plausible that PCB 126 increases cAMP thus activating PKA. ICER is then induced in an auto regulatory feedback loop down regulating the cAMP mediated transcriptional responses, including Jun (Vogel et al., 2004). It is unlikely, however, that PKA plays a major role in the switch response since the protein kinase inhibitor, HA-1004, had only a small effect on CYP1A1 induction in these cells. This result is consistent with the lack of effect of HA-1004 (Reiners et al., 1993) and the more specific PKA inhibitor, H89 on CYPA1 expression (Chen and Tukey, 1996). Interestingly, it has been reported that there is cross-talk between PKA and PKC in J774 macrophages (Chio et al., 2004). Additional experiments are necessary to further explore the role of PKA in CYP1A1 induction in H4IIE cells.

    An underlying question that must be answered is the distribution of the AhR on a single cell basis. If many cells have lost the AhR, this could potentially explain the lack of PCB 126 mediated CYP1A1 induction in the non-responding population of cells. Flow cytometry experiments designed to measure the distribution of the AhR reveal that the AhR is present in at least 87% of the population after 16 h PCB 126 treatment although there was a decrease in AhR levels after treatment, consistent with the known degradation of the AhR following ligand treatment (Ma and Baldwin, 2000). This was an important experiment because it has been reported that there is a heterogeneous distribution of the AhR in rat liver that might contribute to the switch-like response in vivo (Lindros et al., 1997). The histogram overlay for the rabbit IgG isotype control and AhR show two distinct curves, indicating that most of the cell population does, in fact, contain the AhR.

    The purpose of these experiments was to begin to uncover the mechanism for the switch-like response to PCB 126 mediated CYP1A1 induction. These results suggest that PKC plays a key role in this response. Other phosphorylation pathways, particularly MAPKs, do not appear to be involved in the response in this model. Additional experiments are planned to further study the role of PKC in this system, as the traditional membrane translocation of PKC did not occur at the time points measured.

    SUPPLEMENTARY DATA

    The supplementary data includes the results of the 30 min and 6 h custom immunoblots. Each Excel file contains four worksheets with tabs at the bottom for navigation. The "analysis" tab contains the raw and normalized densitometry values. The "summary of changes" tab shows the final results of the data analysis, listing the proteins with changes in the treatment (PCB 126) versus control (DMSO). Those results are categorized by confidence levels as outlined on that page. The tab called "proteins not detected" lists the proteins tested for but not detected. The tab entitled "proteins detected" clearly lists the proteins detected, regardless of whether there was a change in treatment versus control. The two folders entitled Grid Images contain the actual Western blot images for the three replicates of DMSO and three replicates for PCB 126 at each time point. They are overlaid with a grid/vertical lines allowing for easy lane determination. In addition to the proteins probed for, the blots included several standard proteins for determination of molecular size. Supplementary data are available online at www.toxsci.oupjournals.org.

    ACKNOWLEDGMENTS

    This research was supported by a contract from the American Chemistry Council and by an award from the Colorado State University College of Veterinary Medicine and Biomedical Sciences Research Fund. We thank Leslie Armstrong and Dr. Michael Fox for their assistance with the flow cytometry and Dr. Ronald Tsalkens for reviewing the manuscript.

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