Aryl hydrocarbon receptor-dependent cell cycle arrest in isolated mouse oval cells

Toxicology Letters 223 (2013) 73–80

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Aryl hydrocarbon receptor-dependent cell cycle arrest in isolated mouse oval cells Dagmar Faust a , Stephanie Kletting a,1 , Elke Ueberham b,2 , Cornelia Dietrich a,∗ a b

Institute of Toxicology, University Medical Center of the Johannes Gutenberg-University, Obere Zahlbacherstr. 67, 55131 Mainz, Germany Institute of Biochemistry, University of Leipzig, Medical Faculty, Johannisallee 30, 04103 Leipzig, Germany

h i g h l i g h t s • • • •

Oval cells derived from mouse liver show an intact canonical AhR signaling. TCDD induces cell cycle arrest in mouse oval cells. TCDD-induced cell cycle arrest is mediated by the aryl hydrocarbon receptor. TCDD-treatment leads to downregulation of cyclin D1 and A and upregulation of p27.

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Article history: Received 15 January 2013 Received in revised form 26 August 2013 Accepted 28 August 2013 Available online 5 September 2013 Keywords: Mouse oval cells TCDD Aryl hydrocarbon receptor Cell cycle

a b s t r a c t The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, which mediates toxic responses to environmental pollutants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Besides its well known role in induction of xenobiotic metabolizing enzymes, for instance CYP1A1, the AhR is also involved in tumor promotion in rodents although the underlying mechanisms are still poorly understood. Additionally, the AhR is known to regulate cellular proliferation, which might result in either inhibition or stimulation of proliferation depending on the cell-type studied. Potential targets in hepatocarcinogenesis are liver oval (stem/progenitor) cells. In the present work we analyzed the effect of TCDD on proliferation in oval cells derived from mouse liver. We show that TCDD inhibits proliferation in these cells. In line, the amount of G0/G1 cells increases in response to TCDD. We further show that the expression of cyclin D1 and cyclin A is decreased, while p27 is increased. As a result, the retinoblastoma protein is not phosphorylated thereby inducing G0/G1 arrest. Pharmacological inhibition of the AhR and knock-down of AhR expression by RNA interference decreased the inhibitory effect on cell cycle and protein expression, indicating that the AhR at least partially mediates cell cycle arrest. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The AhR was originally discovered due to its stimulation by a variety of planar aromatic hydrocarbons, such as benzo[a]pyrene (B[a]P), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and polychlorinated biphenyls. It is generally accepted that the toxic responses of these environmental pollutants are the direct consequence of AhR activation. Binding of the ligand leads to nuclear translocation of the AhR, heterodimerization with aryl hydrocarbon receptor nuclear translocator (ARNT), and subsequent binding of the

∗ Corresponding author. Tel.: +49 6131 179141; fax: +49 6131 178499. E-mail address: [email protected] (C. Dietrich). 1 Present address: Helmholtz Center for Infection Research, University of the Saarland, 66123 Saarbrücken, Germany. 2 Present address: Fraunhofer Institute for Cell Therapy and Immunology (IZI), Perlickstr. 1, 04103 Leipzig, Germany. 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.08.022

AhR/ARNT heterodimer to dioxin responsive elements leading to transactivation of several genes encoding phase I and II xenobiotic metabolizing enzymes, such as cytochrome P450s (CYP1A1, CYP1A2, CYP1B1) (for review see Nebert et al., 2004; Barouki et al., 2012). This canonical AhR-dependent pathway at least partially explains the carcinogenicity of polycyclic aromatic hydrocarbons, which are metabolized to genotoxic compounds by these enzymes. However, it does not help to understand the molecular mechanisms of toxic effects induced by non-genotoxic AhR-ligands, such as TCDD, which is not metabolized. Although still poorly understood, this points to AhR functions beyond xenobiotic metabolism, and novel non-canonical AhR-driven pathways have been identified (Barouki et al., 2007; Dietrich and Kaina, 2010). Our full understanding of the AhR is further hampered by the well-known species-, tissue-, cell type-, gender- and age-specificity of AhR-mediated effects. Studies in mice expressing a constitutively active AhR indicate that the AhR plays a pivotal role in tumor promotion during

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liver carcinogenesis (Moennikes et al., 2004). In addition, these mice show an increased incidence in the development of stomach tumors (Andersson et al., 2002). In vivo studies in two genetically different rat strains indicate that AhR-driven CYP1A1 induction and tumor promotion can be uncoupled from each other (Tuomisto, 2005) again indicating AhR functions beyond metabolism. Indeed, a number of studies using different cell culture and animal models have shown that the AhR regulates and deregulates cellular proliferation. However, the observed effects on proliferation are diverse: depending on the cell system studied, AhR-activation may either lead to inhibition or stimulation of proliferation (for review see Dietrich, 2012). For instance, TCDD was shown to inhibit proliferation in rat primary hepatocytes (Hushka and Greenlee, 1995) as well as in the rat hepatoma cell line 5L (Göttlicher et al., 1990; Weiss et al., 1996). In contrast, TCDD and other AhR ligands enhance proliferation in serum-depleted rat and mouse primary hepatocytes when co-treated with serum, growth factors or ethinyl estradiol (Wölfle et al., 1988, 1993; Schrenk et al., 1992, 1994; Münzel et al., 1996). Liver oval (stem/progenitor) cells are assumed to be involved in liver tumorigenesis in rodents and humans (Roskams, 2006), and they also might be targets in TCDD-mediated hepatocarcinogenesis in rat (Hailey et al., 2005). We have recently shown that TCDD induces a release from contact inhibition in the rat liver oval cell line WB-F344 (Münzel et al., 1996; Dietrich et al., 2002, 2003; Chramostová et al., 2004; Vondráˇcek et al., 2005) which might help to explain the tumor promoting effects of TCDD in rat liver. At the molecular level, TCDD induces an increased abundance of the AP-1 transcription factor JunD, which in turn activates transcription of the cyclin A gene and thereby loss of contact inhibition (Weiss et al., 2008). Whether control of cell cycle is also regulated by the AhR in oval cells derived from mouse liver is not known so far. We therefore investigated the effect of TCDD on proliferation in oval cells isolated from mouse liver and potential involvement of the AhR in cell cycle regulation. In contrast to our observations in rat liver oval cells, TCDD inhibits proliferation in oval cells derived from mouse liver by inducing G0/G1-arrest. By using pharmacological inhibitors of the AhR and RNA interference techniques we show that the AhR at least partially mediates cell cycle arrest. 2. Materials and methods 2.1. Cell culture Mouse oval cells (OVUE265) were isolated from transgenic mice with hepatocyte-specific expression of p16INK4a (Ueberham et al., 2008). These cells show proliferative capacity in contrast to cells from wt mice prepared according to the same experimental procedure. Lack of p16 expression was controlled in a luciferase assay (data not shown). Passages from 10 to 20 were used since they showed similar response to TCDD on cell cycle. OVUE cells were routinely cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal calf serum (FCS) (PAA, Pasching, Austria), 4 mM glutamine, penicillin and streptomycin (each 100 U/ml). For the experiments, cells were seeded to a density of 1.4 × 104 /cm2 and cultured and treated as described in the figure legends. 3 -methoxy-4 nitroflavone (MNF) (10 ␮M, kindly provided by J. Abel, Institute for Environmental Medical Research, Düsseldorf, Germany) or CH223191 (1 ␮M, Calbiochem, Darmstadt, Germany) were added 1 h before cells were exposed to TCDD (Amchro, Hattersheim, Germany). Indolo[3,2-b]carbazole (synthesized by the Biochemical Institute for Environmental Carcinogens, Grosshansdorf, Germany), or benzo[b]fluoranthene (kindly provided by Jan Vondracek and Miroslav Machala, Brno, Czech Republic) were added at the indicated concentrations. Controls were treated with 0.25% DMSO. 2.2. Determination of cell number and cytotoxicity Cells were washed, trypsinized, stained with trypan blue and counted in a hemocytometer. 2.3. Flow cytometric analysis Cells were trypsinized and washed twice with phosphate-buffered saline (PBS). 1–2 × 106 cells were vortexed in 200 ␮l of PBS and fixed with 2 ml of ice-cold 70%

ethanol for 30 min at 4 ◦ C. Cells were then permeabilized by incubation with 1 ml of 0.2% Tween 20/PBS for 15 min at 37 ◦ C. Cells were resuspended in 2% FCS/PBS in the presence of RNAse A (11.25 kU/sample) and incubated with propidium iodide (50 ␮g/sample, Applichem, Darmstadt, Germany) for 30 min at room temperature in the dark. Finally, the cells were resuspended in 800 ␮l of PBS, and flow cytometric analysis was performed by a FACSCalibur (BD Becton Dickinson, Heidelberg, Germany). 2.4. Western blot analysis Total cell extracts were prepared by lysing the cells in hot Laemmli sample buffer (Laemmli, 1970). Separation of nuclear and cytoplasmic proteins was performed by incubating the cells in a hypotonic buffer (10 mM Hepes, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, DTT and protease inhibitors) and lysing the cells by addition of 0.3% NP40 (v/v). Nuclei were separated by centrifugation and equal volumes of Laemmli sample buffer were added to cytosolic and nuclear fractions. Protein concentration was determined according to Smith et al. (1985). Equal amounts of protein (20–50 ␮g protein/lane) were separated by SDS-PAGE (7.5–15%) and electroblotted overnight onto Immobilon membrane (Merck Millipore, Darmstadt, Germany). The membranes were blocked for 1 h with 5% low-fat milk-powder in TBS (50 mM Tris–HCl, pH 7.5, 150 mM NaCl) containing 0.05% Tween 20 and then incubated overnight at 4 ◦ C with anti-cyclinD1-, anti-cyclin E- (1:200, Santa Cruz, CA, USA), anti-cyclin A-, anti-p27-, anti-Cdk2-, anti-Cyp1A1- (1:1000, Santa Cruz), anti-Cyp1B1- (1:500, Santa Cruz), anti-Cdk4- (1:2000, Santa Cruz), anti-pRb(1:1000, BD Biosciences, Heidelberg, Germany), anti-AhR- (1:2000, Biomol, PA USA) or anti-ARNT-antibody (1:500, Santa Cruz) followed by incubation with horseradishperoxidase-conjugated secondary antibody (Cell Signaling, Danvers, MA, USA) and ECL-detection (Cell Signaling) according to the manufacturer’s instructions. To control equal loading, the blots were stripped and reprobed with anti-p38- (1:1000, Santa Cruz, Figs. 1D–G, 4 and 5), anti-HSP90-(1:1000, Santa Cruz, Fig. 1A), antitubulin- (1:1500, Santa Cruz, Fig. 1B) as a cytosolic marker, or anti-PARP-1-antibody (1:1000, kindly provided by Alexander Bürkle, Konstanz, Germany, Fig. 1B) as a nuclear marker. Chemiluminescence signals were quantified using ImageJ software. 2.5. Immunocytochemistry Cells were seeded on glass coverslips, cultured for 24 h and treated with 5 nM TCDD for 2 h. Cells were fixed and permeabilized with ice-cold acetone for 2 min, air dried, washed with PBS and blocked for 20 min with 10% goat serum/PBS at room temperature. Cells were incubated for 1.5 h with anti-AhR-antibody (1:200, Biomol, PA, USA) in 5% goat serum/PBS at room temperature followed by incubation with anti-rabbit Alexa488-antibody (1:300, Life Technologies, CA USA) in 1% goat serum/PBS for 1 h at room temperature. Cells were washed with PBS, with PBS/0.4 M NaCl for 2 min and finally with PBS for 5 min. For nuclear staining, cells were then incubated with To-Pro 3 (1 ␮M, Life Technologies) for 15 min at room temperature. Cells were washed and mounted on glass slides in Vectashield mounting medium (Vector Laboratories, CA, USA). Cells were visualized by a LSM 710 (Zeiss, Oberkochen, Germany). 2.6. Transfection with siRNA For transient transfection of AhR siRNA or control siRNA, 4 × 104 cells/well (24 well plate) were seeded and cultured for 24 h to reach 80–90% confluence. Transfections were performed in a total volume of 600 ␮l containing 40 pmol siRNA and 1.5 ␮l of Lipofectamine 2000 (Life Technologies, CA, USA) according to the manufacturer’s instructions. After 24 h, cells were exposed for 48 h to 5 nM TCDD. AhR siRNA (directed against rat and mouse AhR mRNA sequence [gi:142369296] CGUUAGAUGUUCCUCUGUGtt (sense) and CACAGAGGAACAUCUAACGtt (antisense) (Weiss et al., 2008). The sequence of control siRNA (directed against mRNA encoding the red fluorescence protein DsRed from the coral Discosoma) has been published previously (Weiss et al., 2005). 2.7. Statistical analyses The results are expressed as means ± SD. Comparisons between treatments were made by one-way analysis of variance (ANOVA) followed by multiple t-test. A pvalue of less than 0.05 was considered to be significant.

3. Results 3.1. AhR/ARNT signaling functions in mouse oval cells Since the AhR has not been characterized in mouse oval cells (OVUEs) so far, we started by analyzing expression and activation of the AhR. Using Western blot analysis we show that the AhR and ARNT are expressed in OVUE cells (Fig. 1A). Cellular fractionation and immunocytochemistry revealed translocation of the AhR to the nucleus upon stimulation with 5 nM TCDD (Fig. 1B and C). In line, we

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Fig. 1. The canonical AhR/ARNT pathway is functionally intact in mouse oval cells (OVUEs). (A) AhR and ARNT are expressed in OVUE cells. Western blot analysis of whole cell extracts was performed using anti-AhR- or anti-ARNT-antibody. The blots were stripped and reprobed with anti-HSP90-antibody to control equal loading. Cell extracts from Hepa1c1c7 cells were loaded as positive control. (B and C) AhR is translocated to the nucleus in response to TCDD. (B) Cells were treated for 1 or 2 h with TCDD (5 nM) and separated into cytosolic and nuclear fractions. Western blot analysis was performed using anti-AhR-antibody. To control for proper separation, the blot was stripped and reprobed with an antibody directed against the cytosolic marker protein tubulin. Proper nuclear fractionation was controlled by a separate Western blot using an antibody against the nuclear protein PARP-1. (C) Cells were not treated or treated with TCDD (5 nM) for 2 h. Immunocytochemistry was performed using anti-AhR-antibody and nuclei were subsequently stained with To-Pro3. (D–G) The AhR-target genes Cyp1A1 and Cyp1B1 are induced by TCDD-exposure. Cells were treated with TCDD for 24 h in the absence or presence of the AhR-inhibitor CH223191 (D and F) or in the absence or presence of the AhR-antagonist MNF (E and G). Western blot analysis of whole cell extracts was performed using anti-Cyp1A1-antibody (D and E) or anti-Cyp1B1-antibody (F and G). The blots were stripped and reprobed with anti-p38-antibody to control for equal loading.

demonstrated TCDD-mediated induction of the two well-known TCDD-target genes Cyp1A1 and Cyp1B1 (Fig. 1D–G). Induction of both, Cyp1A1 and Cyp1B1, was blocked by the AhR inhibitor CH223191 (Kim et al., 2006) and the AhR antagonist 3 -methoxy4 -nitroflavone (MNF) (Lu et al., 1995; Zhou and Gasiewicz, 2003), albeit to different degrees (Fig. 1D–G). While TCDD-mediated induction of Cyp1A1 was abolished by 10 ␮M CH223191 or 10 ␮M MNF, induction of Cyp1B1 could not be fully blocked by CH223191, even at the highest concentration tested. While MNF was not toxic at the concentrations used, 10 ␮M CH223191 exhibited some

cellular toxicity (data not shown). Hence, AhR/ARNT signaling is intact in OVUE cells. 3.2. TCDD induces inhibition of proliferation in mouse oval cells in an AhR-dependent manner When exponentially growing OVUE cells were treated with increasing concentrations of TCDD (0.1–5 nM), the cell number decreased in a concentration-dependent manner after 48 h compared to untreated cells (Fig. 2A). Similar results were obtained with

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Fig. 2. Cell proliferation of OVUE cells is inhibited by AhR-activation. (A) Cells were sparsely seeded, cultured for 24 h and then treated with increasing concentrations of TCDD. Cell number was determined after 48 h by cell counting. (B) Cells were seeded and cultured as described in (A) and then treated with the AhR-agonists benzo[b]fluoranthene (BbF, 1 ␮M) or indolo[3,2-b]carbazole (ICZ, 1 ␮M). Cell number was determined after 48 h by cell counting. (C) Cells were sparsely seeded and treated after 24 h with TCDD (0 d). Cell number was determined after another 1, 2, or 3 days by cell counting. (D) Cells were seeded and treated with TCDD as described in (A) either in the absence or presence of CH223181 or MNF, respectively. Cell number was determined after 48 h by cell counting. (E) Cells were transiently transfected with siRNA targeted against the AhR or with control siRNA. After 24 h, cells were treated with TCDD for 48 h and cell number was determined. Knock-down of the AhR was shown by Western blot analysis. The results are expressed as means ± standard deviation (SD) with n = 4.

the AhR agonists benzo[b]fluoranthene and indolo[3,2-b]carbazole (Fig. 2B). Analysis of the growth curve of OVUE cells in the presence or absence of TCDD suggested that the cells stop growing in response to TCDD (Fig. 2C). TCDD was not toxic at any concentration tested as assessed by trypan blue exclusion assay (data not shown), nor was apoptosis detected as shown by analyzing the subG1 fraction (Fig. 3) and Annexin V/PI staining and subsequent flow cytometric analysis (data not shown). TCDD-mediated inhibition of proliferation could be diminished by cotreatment with CH223191 or MNF indicating that the AhR is at least partially involved (Fig. 2D). When given alone, CH223191 or MNF did not affect cellular proliferation (Supplementary Fig. S1). Our data were confirmed by knocking-down the AhR by transient transfection with siRNA targeted against the AhR. While transfection of control siRNA had no effect on

TCDD-induced inhibition of proliferation, it was partially reversed by AhR-knock-down (Fig. 2E). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.toxlet.2013.08.022. 3.3. TCDD increases the number of cells in G0/G1 in an AhR-dependent manner Our data of TCDD-mediated inhibition of cell proliferation in OVUE cells were confirmed by flow cytometric analysis. We revealed that after 24 h exposure to TCDD (5 nM), the number of cells in G0/G1 increased to 65% compared to 40% in untreated control cultures, while the amount of cells in S and G2/M decreased to 16% (versus 31% in untreated controls) and 19% (versus 29% in

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Expression levels of Cdk2 were slightly and those of Cdk4 were not altered. Although we have not measured kinase activities of Cdk4 and Cdk2, the decline in cyclin D1 generally results in decreased Cdk4 activity while the upregulation of p27 is known to inhibit Cdk2 activity which leads to decreased phosphorylation of the retinoblastoma protein and thereby inhibition of S-phase entry (Malumbres and Barbacid, 2005). This can be demonstrated by a decrease in the slower migrating species of pRB (Mittnacht and Weinberg, 1991). As expected, the slower migrating species of pRB disappeared at increasing cell densities which indicates low activity of Cdk4 and Cdk2 and clearly reflects G1-arrest (Fig. 4). Fig. 4 further shows that the expression of cyclin D1 and cyclin A decreased in response to TCDD already 24 h after exposure and hence independently of cell-density. The level of the Cdk inhibitor p27 was elevated by exposure to TCDD. As a result, the retinoblastoma protein (pRB) was not phosphorylated which was detected by disappearance of the slower migrating species of pRB after TCDD treatment (Fig. 4). These data explain the growth-inhibitory action of TCDD induced by cell cycle arrest in G1-phase. 3.5. TCDD deregulates cell cycle proteins in an AhR-dependent manner Since our data strongly indicated an involvement of the AhR in cell cycle arrest, we next investigated whether the deregulation of cyclin D1, cyclin A and p27 could be reversed by MNF (10 ␮M) or CH223191 (1 ␮M). We demonstrate that the decrease in protein expression of cyclin D1 and cyclin A as well as the increase in p27 expression could be abolished by cotreatment with MNF or CH223191 (Fig. 5). Hence, we conclude that activation of the AhR by TCDD results in downregulation of cyclin D1 and cyclin A as well as upregulation of p27 which leads to an accumulation of cells in G1 and thereby cell cycle arrest.

Fig. 3. OVUE cells are arrested in G1-phase in an AhR-dependent manner. Cells were sparsely seeded, cultured for 24 h and treated with TCDD for another 24 h in the absence (A) or in the presence of MNF (B) or CH223191 (C). Cell cycle distribution was determined by propidium iodide staining and flow cytometric analysis. The data show one representative example out of three independent experiments all leading to similar results.

untreated controls), respectively (Fig. 3A). No increase in the subG1 fraction, which would indicate apoptotic cells, was detected. In line with the above mentioned results on cell number, the effect of TCDD on cell cycle could be blocked by cotreatment with CH223191 or MNF (Fig. 3B and C), again strongly indicating involvement of the AhR. 3.4. TCDD deregulates expression of cell cycle proteins Our data suggested that cell cycle regulating proteins might be AhR targets. The eukaryotic cell cycle is regulated by cyclins and cyclin-dependent kinases (Malumbres and Barbacid, 2005). We therefore investigated the effect of TCDD on expression of cyclins and cdks and focussed our interest on those proteins which are important during G1- and early S-phase regulation. Exponentially growing cells were treated with TCDD (5 nM) for 24, 48 and 72 h, respectively, according to Fig. 2C, and Western blot analysis was then performed. In line with the observation that untreated cells reached confluence after 48 h (Fig. 2C) and therefore ceased growing, the levels of cyclin D1, cyclin E (G1-cyclins) and cyclin A (S-phase cyclin) gradually declined whereas the Cdk-inhibitor p27 accumulated at increasing cell-density (Fig. 4). This phenomenon is generally referred to as contact inhibition (Eagle and Levine, 1967; Polyak et al., 1994; Dietrich et al., 1997; Faust et al., 2005).

4. Discussion Oval (stem/progenitor) cells provide an important reservoir for liver regeneration under conditions when proliferation of mature hepatocytes is blocked, for instance after strong toxic insult, viral infection, or fibrosis (Roskams, 2006). They are located in the canal of Hering and are capable of differentiating into hepatocytes and cholangiocytes. Although not finally proven, oval cells are considered to be involved in liver tumorigenesis in rodents and humans (Roskams, 2006; Nejak-Bowen and Monga, 2011) and might be targets in TCDD-mediated hepatocarcinogenesis in rat (Hailey et al., 2005). Whether oval cells are also targets for TCDD-induced liver tumorigenesis in mouse, is not known so far. We and others have shown that TCDD induces a release from contact inhibition in the established rat liver oval cell line WB-F344 (Münzel et al., 1996; Dietrich et al., 2002, 2003; Chramostová et al., 2004; Vondráˇcek et al., 2005), which might help to explain the tumor promoting effects of TCDD in rat liver. At the molecular level, TCDD induces an increased abundance of the AP-1 transcription factor JunD, which in turn activates transcription of the cyclin A gene and thereby loss of contact inhibition (Weiss et al., 2008). Here we show that oval cells isolated from mouse liver (Ueberham et al., 2008) behave differently after TCDD-exposure although the canoncial AhR/ARNT pathway is functionally intact. In contrast to our previous results in rat oval cells, proliferation of mouse oval cells is inhibited by TCDD. Whether oval cell proliferation is also reduced in response to TCDD in mouse liver in vivo remains to be determined. However, the inhibition of proliferation of OVUE cells by TCDD presented here confirms other studies describing no apparent involvement of oval cells at least in TCDD promoted liver toxicity in mice (Kopec et al., 2013) and hence reconfirmes solid species-specific differences in

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Fig. 4. Expression of cell cycle regulatory proteins is altered by TCDD-treatment in OVUE cells. Cells were seeded, cultured and treated as described in Fig. 1C. Cells were harvested at day 1, 2, or 3, respectively. Western blot analysis of whole cell extracts was performed using the appropriate antibodies. The blots were stripped and reprobed with anti-p38-antibody to control for equal loading. The blots each show one representative example out of three independent experiments all leading to similar results.

response to TCDD. One might therefore speculate that oval cells are not targets of TCDD in mouse liver. The fact that the decrease in cell number (i) is not restricted to TCDD, but is detected in response to different AhR ligands and (ii) can be reversed by the AhR antagonists MNF and CH223191 as well as by knock-down via siRNA interference strongly points to an involvement of the AhR. However, reversal of TCDD-induced decrease in cell number and of increase in cells in G1 by all of our approaches was only partial. MNF is known to retain some agonist activites depending on the cellular context and gene studied (Zhou and Gasiewicz, 2003) thus providing a possible explanation for the

Fig. 5. The change in expression of cell cycle regulatory proteins is AhR-dependent. Cells were sparsely seeded, cultured for 24 h and then treated with TCDD for 48 h in the absence or presence of MNF or CH223191, respectively. Western blot analysis of whole cell extracts was performed using anti-cyclin D1-, anti-cyclin A-, or antip27-antibodies. The blots were stripped and reprobed with anti-p38-antibody to control for equal loading. The blots each show one representative example out of three independent experiments all leading to similar results.

partial effect. The use of CH223191 which is known to be a pure inhibitor of the AhR without any agonist activity (Kim et al., 2006) was limited to a concentration of 1 ␮M since higher concentrations appeared to be toxic in OVUE cells. This concentration was shown to be suboptimal as induction of Cyp1A1 and Cyp1B1 was only partially blocked. Transient transfection of siRNA targeted against the AhR leads to downregulation, but not complete loss of AhR function. Hence, only a partial reversal of TCDD-mediated decrease in proliferation was expected. Although we provide plausible technical explanations for the partial reversal by the approaches used in this study, additional AhR-independent effects are possible and cannot be excluded yet. We revealed that protein levels of cyclin D1 and cyclin A were decreased and p27 accumulated upon TCDD-exposure in an AhR-dependent manner. Cyclin D1 is a G1-phase specific regulatory protein which activates Cdk4 (cyclin-dependent kinase 4) and thereby initiates phosphorylation of pRB (PlanasSilva and Weinberg, 1997). Downstream, the cyclin E/Cdk2 (cyclin-dependent kinase 2) complex further contributes to pRB phosphorylation. The activity of the cyclin E/Cdk2 complex is regulated by association of small inhibitory proteins, such as p27KIP . Hence, downregulation of cyclin D1 and upregulation of p27 will lead to reduction of Cdk4 and Cdk2 activity, respectively, and as a result in hypophosphorylation of pRB (Matsushime et al., 1992; Toyoshima and Hunter, 1994; Reynisdottir et al., 1995). The different hypo/hyperphosphorylated species of pRB can be discriminated by a shift in the electrophoretic mobility. In line, we only detected the faster migrating species after TCDD-exposure reflecting hypophosphorylated pRB (Mittnacht and Weinberg, 1991). It is known that pRB associates with the transcription factor E2F in its hypophosphorylated form thereby inhibiting its function. As a result, transcription of S-phase-specific genes, such as cyclin A, and thereby S-phase entry are prevented (Malumbres and Barbacid,

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2005). We propose that in OVUE cells, AhR-activation leads to a downregulation of cyclin D1 and upregulation of p27 thereby inhibiting Cdk4 and Cdk2 function resulting in hypophosphorylation of pRB, which finally leads to G1 arrest. As a result, cyclin A is not expressed. Whether cyclin A is possibly also directly targeted by the AhR, has not been studied yet. OVUE cells used in this study were isolated from a transgenic mice expressing p16INK4a under the control of a hepatocyte specific promoter. p16INK4a is a small inhibitory protein known to inhibit cyclin D/Cdk4 activity (Wieser et al., 1999). Hence, cell cycle arrest in response to TCDD could also be the result of TCDD-mediated transgenic p16INK4a expression and a subsequent differentiation of oval cells into hepatocytes. However, we did not detect any activation of the transfected promoter as assessed by a luciferase assay neither during cultivation of the cells, nor in response to TCDD (unpublished observation). Accordingly, expression of transgenic p16INK4a and hence differentiation of the OVUE cells into hepatocytes can be excluded. Upregulation of p27 as one consequence of TCDD-treatment has been shown in various cell lines including thymocytes, 5L hepatoma, MCF-7 breast cancer, human neuronal cells and lipopolysaccharide-activated B-cells (Kolluri et al., 1999; Marlowe et al., 2004; Jin et al., 2004; Latchney et al., 2010; Crawford et al., 2003). It has been convincingly shown that p27 upregulation is dependent on AhR function, and a potential XRE has been found in the promoter of p27 (Kolluri et al., 1999; Marlowe et al., 2004; Pang et al., 2008). However, also posttranslational modification of p27 in response to TCDD has been reported (Crawford et al., 2003). In line with these observations, TCDD-mediated accumulation of p27 in OVUE cells can be reversed by the AhR inhibitors CH223191 and MNF. We therefore conclude, that induction of p27 is very likely mediated by the canonical AhR/ARNT pathway. It has also been described that pRB plays a coactivating role in AhR/ARNTdependent transcriptional activation of p27 (Marlowe et al., 2004). Whether pRB is involved in TCDD-mediated p27 induction in OVUE cells, has not been studied. The underlying mechanism of cyclin D1 deregulation has not been elucidated so far. However, a similar downregulation of cyclin D1 in response to TCDD has been detected in human prostate carcinoma and human neuronal precursor cells (Barnes-Ellerbe et al., 2004; Latchney et al., 2010). An alternative pathway of AhR-dependent cell cycle arrest is a direct interaction of the AhR with pRB thereby displacing the histone acetyl transferase p300 from E2F-dependent promoters which has been shown in 5L rat hepatoma, mouse Hepa-1 and human MCF-7 cells (Ge and Elferink, 1998; Marlowe et al., 2004). Whether a similar AhR/pRB interaction contributes to TCDD-mediated cell cycle arrest in OVUE cells has not been studied. Our observation that oval cells of rat and mice respond differently after TCDD-exposure is in accordance with the wellknown species- and cell type-specificity of AhR function (Dietrich, 2012). While AhR-dependent stimulation of proliferation has been described in different cell lines, such as HepG2, human medulloblastoma cells, ureteric epithelial cells and others (Pierre et al., 2011; Dever and Opanashuk, 2012; Abbott and Birnbaum, 1990), AhR-dependent inhibition of proliferation has been detected for instance in 5L rat hepatoma, rat primary hepatocytes and MCF7 cells (Göttlicher et al., 1990; Weiss et al., 1996; Hushka and Greenlee, 1995; Puga et al., 2000). Also, in vivo liver regeneration after 2/3 partial hepatectomy is impaired in mouse and rat in response to TCDD (Bauman et al., 1995; Mitchell et al., 2006). The molecular mechanisms of this cell-type-, species- and tissuespecificity of AhR function remain to be elucidated. Conflict of interest There are no conflicts of interest.

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Acknowledgements We are indebted to Anna Frumkina for expert technical assistance. The technical support by Julia Altmaier, FACS and Array Core Facility, is greatfully acknowledged. We thank Beate Köberle for critical reading of the manuscript. The work was financially supported by the Deutsche Forschungsgemeinschaft (Di793/3-1). This work is part of the diploma thesis of SK.

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