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Activated thyroid hormone receptor modulates dioxin-inducible aryl hydrocarbon receptor-mediated CYP1A1 induction in human hepatocytes but not in human hepatocarcinoma HepG2 cells
Toxicology Letters 275 (2017) 77–82
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Activated thyroid hormone receptor modulates dioxin-inducible aryl hydrocarbon receptor-mediated CYP1A1 induction in human hepatocytes but not in human hepatocarcinoma HepG2 cells
MARK
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Radim Vrzal , Aneta Vrzalova, Aneta Grycova, Zdenek Dvorak Department of Cell Biology and Genetics, Faculty of Science, Palacky University in Olomouc, Slechtitelu 27, Olomouc, CZ-783 71, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords: Triiodothyronine CYP1A2 S14 Receptors cross-talk Dioxin receptor
Aryl hydrocarbon receptor (AhR) is a transcription factor, the activity of which is modulated by hormones including glucocorticoids and estrogens. In this study, we examined the effects of triiodothyronine (T3), a ligand and activator of thyroid hormone receptor (TR), on transcriptional activity of AhR and the expression of its target gene CYP1A1. Study was carried out in human hepatocellular carcinoma cells HepG2 and primary cultures of human hepatocytes (HH). Gene reporter assay in stably transfected AZ-AhR cells revealed that T3 dosedependently augmented 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AhR-dependent luciferase activity. In contrast, T3 had no effect on TCDD-inducible expression of CYP1A1 mRNA, protein and catalytic activity. Incubation of human hepatocytes with T3 had modulatory and inter-individual (7 cell cultures from 7 different liver donors) effects on both basal and dioxin-inducible CYP1A1/2. Since there was no correlation between T3 effects on CYP1A expression and T3-dependent expression of Spot14 mRNA, the involvement of additional factors besides TR is supposed. Overall, the co-incubation of normal and cancer human hepatic cells with TCDD and T3 suggested transcriptional cross-talk between AhR and TR, which may have physiological and toxicological implications.
1. Introduction Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which resides in cytoplasm together with co-chaperones and upon ligand presence it translocates to the nucleus. Here it heterodimerizes with AhR nuclear translocator (ARNT, also known as HIF-1β) and binds to dioxin responsive elements (DREs) localized in promoters of target genes, such as those for cytochromes P450 1A1/2 (CYP1A1/2). Besides the regulation of genes involved in metabolism of xenobiotics, AhR also regulates cell cycle, cell differentiation or the immune response since it is abundantly expressed in regulatory TH lymphocytes (Veldhoen et al., 2008). Thyroid hormone receptors (TRs) are the ligand -activated transcription factors which mediate the biological action of thyroid hormones, in particular triiodothyronine (T3). There are two genes (α1 and β1), which encode several isoforms capable as well as incapable of T3 binding (Flamant et al., 2006). They belong to the nuclear receptor superfamily with centrally located highly conserved DNA-binding domain, which interacts with thyroid hormone response elements (TREs). The ligand-binding domain not only binds T3 but it is
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Corresponding author. E-mail address: [email protected] (R. Vrzal).
http://dx.doi.org/10.1016/j.toxlet.2017.05.001 Received 13 January 2017; Received in revised form 29 March 2017; Accepted 2 May 2017 Available online 03 May 2017 0378-4274/ © 2017 Elsevier B.V. All rights reserved.
also involved in interactions with co-repressors (N-CoR – nuclear receptor co-repressor; SMRT – silencing mediator of retinoid and thyroid hormone receptor), co-activators (SRC-1–steroid receptor coactivator 1) and heterodimerization partner, retinoid X receptor (RXR). The expression of TR isoforms is tissue-dependent and developmentally regulated (Cheng, 2000). TRβ1 isoform is expressed predominantly in the kidneys, liver, brain, heart and thyroid (Williams, 2000). TRα1 is expressed at highest level in the brain, at lower levels in the kidneys, lungs, heart and liver (Williams, 2000). Based on studies in mice lacking particular isoforms, it was suggested that TRβ1 and TRα1 can substitute for each other to mediate T3 action as well as they possess isoforms-specific functions (Forrest et al., 1996a,b; Gothe et al., 1999; Wikstrom et al., 1998). Due to the important role of TRs in the development, the inadequate activation/inhibition might have severe impact on human health. Many compounds that are released into the environment disrupt endocrine system by interfering with TR signaling. It was reported that babies in America are born in average with 287 foreign chemicals in their blood and pediatric endocrinologist are advised to check for endocrinedisrupting chemicals when assessing endocrine clinical problems of
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unknown etiology (Skakkebaek et al., 2011). The emphasis is placed on chemicals which are persistent, such as polychlorinated biphenyls (PCBs), polybrominated diethyl esters (PBDEs) or dioxins. These compounds bind to thyroid hormone transport proteins with consequent disruption of thyroid hormone homeostasis (Montano et al., 2012). In addition, they are also well known ligands and activators of the AhR (Murk et al., 1996). There are numerous evidences for existence of the mutual crosstalk between AhR and TR signaling pathways, in particular: a) the presence of SMRT which inhibits AhR/ARNT binding to DRE (Nguyen et al., 1999); b) the downregulation of dioxin-inducible AhR-target gene expression (CYP1A1) in the presence of all-trans-retinoic acid (ATRA) in human keratinocyte cell line HaCat (Wanner et al., 1995, 1996). ATRA binds to retinoid acid receptor (RAR), which heterodimerizes with RXR similarly like TR; c) intraperitoneal administration of PCB118 to Wistar rats which resulted in the generation of various cytokines (e.g. IL-6) with subsequent inhibition of natrium/iodide symporter (NIS) (Xu et al., 2016). NIS is important for T3 formation as it catches iodine from serum. The presence of AhR antagonist α-naphthoflavone or AhR small interfering RNA restored the NIS expression. This inhibition was suggested to lead to the thyroid disruption. While there are studies demonstrating endocrine disrupting effects of persistent environmental pollutants (AhR activators) on TR-mediated signaling, the opposite, i.e. the effects of thyroid hormones on AhR signaling were neglected. In the current paper we investigated whether T3 affects basal and TCDD-inducible expression of AhR-target gene, CYP1A1. We used two in vitro models, human hepatocellular carcinoma (HepG2) and seven primary cultures of human hepatocytes. We measured the expression of CYP1A1 mRNA and protein, and also the catalytic activity of CYP1A1.
Table 1 Health and demographic data on human hepatocytes donors. Culture abbreviation
Gender
Age
Smoking
Alcohol use
LH37 LH38 LH42 LH50 LH51 LH59 Hep220770
male male female female female female female
65 58 60 55 58 42 35
unknown unknown unknown positive negative negative negative
unknown unknown unknown positive negative negative negative
were maintained at 37 °C and 5% CO2 in a humidified incubator. Hepatocytes were incubated with the tested compounds, inducers and/ or vehicle (DMSO; 0.1% v/v) for 24 h (RNA) and 48 h (protein, catalytic activity). 2.4. Quantitative reverse transcriptase polymerase chain reaction (qRTPCR) Total RNA was isolated using TRI Reagent® (Molecular Research Center, USA). cDNA was synthesized from 1000 ng of total RNA using M-MuLV Reverse Transcriptase (M0253, New England Biolabs) at 42 °C for 60 min in the presence of random hexamers (3801, Clontech). qRTPCR was carried out on Light Cycler 480 apparatus (Roche Diagnostic Corporation, Prague, Czech Republic). The levels of CYP1A1/1A2/ GAPDH mRNAs were determined using primers and Universal Probes Library (UPL; Roche Diagnostic Corporation, Prague, Czech Republic) technology described earlier (Vrzal et al., 2015). The primers and UPL probe for Spot14 were as follows, forward: Spot14, 5-CATGCACCTCACCGAGAA-3, reverse: 5-TGTCTTCTATCATGTGAAGGGATCT-3, UPL 79. The following program was used for monitoring the expression of all genes: an activation step at 95 °C for 10 min was followed by 45 cycles of PCR (denaturation at 95 °C for 10 s; annealing with elongation at 60 °C for 30 s). The measurements were performed in duplicates. Gene expression was normalized per glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. Data were processed by delta-delta method (Pfaffl, 2001). Results are expressed as fold induction over DMSO-treated cells.
2. Materials and methods 2.1. Compounds and reagents Dimethylsulfoxide (DMSO; purity ≥ 99.9%), 3,3′,5-Triiodo-L-thyronine sodium salt (T3; purity ≥ 95%), charcoal-stripped fetal serum (FBS) were purchased from Sigma-Aldrich (Prague, Czech Republic). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was from Ultra Scientific (Cincinnati, Ohio, USA). Oligonucleotide primers used in RT-PCR reactions were synthesized by Generi Biotech (Hradec Kralove, Czech Republic). LightCycler 480 Probes Master was from Roche Diagnostic Corporation (Intes Bohemia, Czech Republic). All other chemicals were of the highest quality commercially available.
2.5. Western blotting Total protein extracts for each sample were prepared from 1 well of a six-well plate dish. Cells were washed twice with ice-cold phosphate buffered saline (PBS) and scraped into 1 ml of PBS. The suspension was centrifuged (5000 × rpm at 4 °C for 2 min), and the pellet was resuspended in 150 μL of ice-cold lysis buffer (150 mM NaCl; 10 mM Tris pH 7.2; 0.1% (w/v) SDS; anti-protease cocktail, 1% (v/v) Triton X100; anti-phosphatase cocktail, 1% (v/v) sodium deoxycholate; 5 mM ethylenediaminetetraacetic acid [EDTA]). The mixture was vortexed and incubated for 10 min on ice and then centrifuged (13,000 × rpm at 4 °C for 13 min). Supernatant was collected and the protein content was determined with the Bradford reagent. For CYP1A1 protein detection in HepG2 cells, SDS–PAGE gels (8%) were run on a BioRad apparatus according to the general procedure followed by the protein transfer onto the polyvinylidene fluoride (PVDF) membrane. The membrane was saturated with 5% non-fat dried milk for 2 h at room temperature. Blots were probed with primary antibodies against CYP1A1 (goat polyclonal; sc-9828, G-18), CYP1A2 (mouse monoclonal; sc-53614, 3B8C1) and actin (goat polyclonal; sc-1616, 1–19), all purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Chemiluminescence detection was performed using horseradish peroxidase-conjugated secondary antibodies and Western blotting Luminol kit (both from Santa Cruz Biotechnology). Detection of CYP1A1, CYP1A2 and actin in human hepatocytes was performed with Sally Sue Simple Western System according to the
2.2. Cell cultures Human Caucasian hepatocellular carcinoma cells HepG2 (Public Health England, ECACC No. 85011430) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of fetal calf serum, 100 U/mL streptomycin, 100 μg/mL penicillin, 4 mM L-glutamine, 1% non-essential amino acids, and 1 mM sodium pyruvate. Cells were maintained at 37 °C and 5% CO2 in a humidified incubator. HepG2-derived AZ-AhR cell line was incubated under the same condition as parental HepG2. All treatments took place in medium supplemented with charcoal-stripped fetal serum (CS-FBS). 2.3. Human hepatocytes Human liver tissue was obtained from two sources: (i) from multiorgan donors or (ii) long-term human hepatocytes in monolayer (Biopredic International, Rennes, France) (Table 1). Tissue acquisition protocol was in accordance with the requirements issued by local ethical commission in the Czech Republic. Cells were plated into collagen-coated dishes in a hormonally and chemically defined medium consisting of a mixture of William’s E and Ham’s F-12 [1:1 (v/v)] and 78
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We found that neither basal nor TCDD-inducible mRNA level of CYP1A1 was significantly affected by T3 (Fig. 1D). Further, we monitored the effect of T3 on CYP1A1 protein and catalytic activity. The level of CYP1A1 protein (Fig. 1E) or CYP1A1-mediated catalytic activity (Fig. 1F) was not affected by the presence of T3 alone or in combination with TCDD.
manufacturer's instructions (ProteinSimple™). In brief, samples were diluted to adjust protein concentration to 1 μg/μl in 5 μl with sample buffer and further diluted by adding 1.25 μl of the 5× master mix (Protein Simple) to a final concentration of 1× sample buffer, 1× fluorescent molecular weight markers, and 40 mM dithiothreitol (DTT) and then heated at 95 °C for 5 min. The samples, blocking reagent, wash buffer, primary antibodies, secondary antibodies, and chemiluminescent substrate were dispensed into designated wells in the manufacturer provided microplate. After plate loading, the separation electrophoresis and immunedetection steps took place in the capillary system and were fully automated. Simple Western analysis was carried out at room temperature, and instrument default settings were used. The data were analyzed with inbuilt Compass software (ProteinSimple).
3.2. The effect of activated TR on the signaling of AhR in primary cultures of human hepatocytes We further decided to investigate the effect of activated TRs on AhRmediated induction of CYP1A1/2 mRNA, protein and catalytic activity in primary cultures of human hepatocytes. In most cultures, T3 increased basal CYP1A1 mRNA only weakly (Fig. 2A), an exception being the culture LH51. The inducible CYP1A1 mRNA was affected in culture-specific way. In three cultures (LH37, LH38, Hep220770) there was an upregulation with 2 nM T3 while for other cultures there was no effect (LH42, LH51) or decrease in mRNA (LH50, LH59) (Fig. 2B). The basal CYP1A2 mRNA level was increased with T3 to higher extent than CYP1A1 but no reproducible trend was observed (Fig. 2C). The inducible CYP1A2 mRNA was decreased (cultures LH37, LH50, LH51), unaffected (cultures LH42, LH59) or increased (LH38, Hep220770) in the presence of T3 (Fig. 2D). After this step, we monitored the CYP1A proteins level. In cultures, where both mRNA and protein were determined, the protein level displayed similar trend like mRNA, as it can be seen on representative virtual western blot from culture LH50 and quantification for these data (Fig. 2E and F). Moreover, in cultures where CYP1A catalytic activity was measured (Fig. 2G), the level of activity followed the level of protein. However, we observed approx. 50% increase in TCDD-inducible CYP1A enzymatic activity in the presence of T3 in 3 cultures (LH51, LH59, LH64).
2.6. CYP1A enzymatic activity assay 7-Ethoxyresorufin-O-deethylase (EROD) activity was determined as described elsewhere (Vrzal et al., 2013). 2.7. Gene reporter assay Stably transfected cell line derived from human hepatoma HepG2 cells was used for assessment of AhR transcriptional activity (Novotna et al., 2011). Cells were incubated for 24 h with increased concentrations of T3 and/or vehicle (DMSO; 0.1% v/v), in the presence (“Antagonist settings”) or absence (“Agonist settings”) of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD; 5 nM). After the treatment, cells were lysed and luciferase activity was measured with Infinite M200 (TECAN, Austria). 2.8. Statistical analysis Data were analyzed by using GraphPad Prism Version 6.05 (GraphPad Software, San Diego, CA, USA). The differences in luciferase assay, mRNA, protein expression and catalytic aktivity following incubation with the compounds investigated compared with the respective vehicle controls were tested by using ANOVA with Dunnett’s post hoc test; P ≤ 0.05 was considered significant. p < 0.05 was considered statistically significant.
3.3. Functional verification of activated thyroid hormone receptor (TR) It is known, that TR is functional in HepG2 cells as it was demonstrated in some studies (Prieur et al., 2005) and our recently established stably transfected HepG2-derived TR-dependent cell line (Illes et al., 2015). Nevertheless, the inducibility of TR-target gene, Spot14 was verified. The mRNA level of Spot14 was induced in average 1.5-times with 2 nM of T3 only, the higher or lower concentrations were without the effect (Fig. 3A). Due to the atypical induction profile in HepG2 cells, we verified the Spot14 induction in human hepatocytes as well. In each hepatocyte culture, Spot14 was induced concentrationdependently with culture-specific magnitude (Fig. 3B). This demonstrates functional thyroid hormone receptor.
3. Results 3.1. The effect of 3,3′,5-triiodo-L-thyronine on AhR signaling in HepG2 cells At the beginning, we used stably transfected AZ-AhR cell line (Novotna et al., 2011) to investigate the effect of T3 on AhR-mediated luciferase activity. Triiodothyronine did not have any effect on luciferase activity, while positive control dioxin (TCDD) induced the activity in average 1500-fold over negative control, DMSO (Fig. 1A), providing the evidence of the functionality of the system. In the presence of TCDD, the luciferase activity was stimulated concentration-dependently by T3 and the effect was significant for 2 highest concentrations (Fig. 1B). However, since additive or synergistic effects are sometimes better to study with non-saturating concentrations, we tried to verify if the effect would be different for lower concentrations of TCDD. Therefore, we calculated the EC10, EC25 and EC50 from doseresponse curve for TCDD on AZ-AhR cell line. From calculated 95% confidence interval we chose 0.3, 0.6 and 1.2 nM concentrations of TCDD representing EC10, EC25 and EC50, respectively. We found that there is synergistic effect between T3 and TCDD no matter of TCDD concentration (Fig. 1C). Triiodothyronine-stimulated increase reached in average 22% above TCDD alone in its maximum. This is not different from saturating concentration 5 nM of TCDD for AhR (Fig. 1B). Therefore, we further used this concentration only. Due to the synergistic effect of T3 on TCDD-inducible AhR-dependent luciferase activity, we decided to investigate the expression level of AhR-target gene, CYP1A1.
4. Discussion In the current work, we investigated if T3-activated thyroid hormone receptor can affect the activity of AhR and its target gene CYP1A1 expression in human hepatocellular carcinoma HepG2 cells or in primary cultures of human hepatocytes. In HepG2 cells, there was no effect of T3 on basal or TCDD-inducible level of CYP1A1 at mRNA, protein or activity levels. However, we recorded significant stimulation of TCDD-inducible AhR-mediated luciferase activity in stably transfected HepG2 cells. The monitoring of AhR activity via CYP1A1/2 induction in human hepatocytes revealed irreproducible effect of T3, which differed among cultures. In some cultures there was stimulation of TCDD-inducible CYP1A1/2 mRNA, in others no effect or mild inhibition. This was more or less followed at the protein and the activity level of CYP1A enzymes. Our data from human hepatocytes provide the evidence that even with increasing induction of Spot14 and anticipated increasing activation of TRs, the effects on the AhR signaling, monitored as CYP1A1/2 expression, are unlikely to be directly connected with TRs. While we always observed induction of Spot14, the effects especially on TCDD79
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Fig. 1. Cells were treated with 3,3′, 5-Triiodo-L-thyronine sodium salt (T3; 0.2–20 nM) in the absence or presence of TCDD (5 nM) and/or DMSO for 24 h (A–D) or 48 h (E and F). Thereafter, gene reporter assay (A–C), qRT-PCR (D), western blotting (E) or enzymatic activity determination (F) were performed as described in Materials and methods section. The data are the mean ± SD from 3 to 4 independent experiments and are expressed as fold activation/induction over DMSO-treated cells (A, B, D, F) or as % of positive control (TCDD) (B) or as relative luminescence units (RLU) (C). *—value is significantly different from untreated cells (DMSO; p < 0.05) (A, D, F) or TCDD-treated cells (B and C).
activation with delayed dissociation of N-CoR and SMRT, which in turn may more easily associate with CYP1A1 promoter (Nguyen et al., 1999). In addition, at least one TRα splicing variant, c-erbA α-2, inhibits of T3 action in hormone-independent fashion (Koenig et al., 1989). Another thing that may also contribute to heterogenous effects in hepatocytes is the fact that TRs induce hypoxia-inducible factor 1 alpha (HIF-1α), among many other genes (Otto and Fandrey, 2008). As it is known that this protein associates with HIF-1β (ARNT), it is likely that the response in some hepatocyte culture may be affected by tiny shift in equilibrium between these two proteins. Consequently, CYP1A1/2 expression is affected. Concerning the results, we obtained in HepG2 cells, we may ask the
inducible CYP1A1/2 expression level was inconsistent among cultures with all 3 possible outputs, i.e. potentiation, inhibition, no effect. Thus, we can expect that CYP1A1/2 expression and activity can be modulated by donor-specific manner and among many, genetic polymorphisms of components of TR signaling pathway may play a significant part. This is likely the most plausible explanation when looking at the data. Moreover, in contrast to HepG2 cells, which should be uniform in their TRs expression ratios, each hepatocyte donor may have different level of expression of these isoforms and consequently the results may be affected this way. Thus, diverse results could have been obtained due to the fact that some TR-regulated genes display differential response to T3 with TRα and TRβ, where higher levels of T3 are needed for optimal induction with TRβ (Lin et al., 2013). This may lead to delayed TRβ 80
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Fig. 2. Primary cultures of human hepatocytes were treated with 3,3′,5-Triiodo-L-thyronine sodium salt (T3; 0.2–20 nM) in the absence or presence of TCDD (5 nM) and/or DMSO for 24 h (A–D) or 48 h (E–G). Thereafter, qRT-PCR (A–D), capillary electrophoresis with immunedetection (E and F) or enzymatic activity determination (G) were performed as described in Materials and methods section. Results are expressed as fold induction over DMSO-treated cells ± SD. A representative virtual western blot from culture LH50 is shown (E) together with quantification data from the same culture (F).
SMRT (Chan and Privalsky, 2006), which may lead to higher, i.e. repressive manner on CYP1A1 promoter in HepG2 cells. This may explain why we observed significantly potentiated TCDD-inducible AhR-dependent luciferase activity (Fig. 1B and C) in our stably transfected AZ-AhR cell line with DREs only (Novotna et al., 2011)
question why there was no effect in contrast to hepatocytes, where actually all possibilities (i.e. increase, decrease, no effect) were observed. One thing that might contribute to the explanation is the fact that many hepatocellular carcinomas have mutated TRs (Lin et al., 1999). Some of these mutants have decreased binding to N-CoR or
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Fig. 3. HepG2 cells (A) or primary cultures of human hepatocytes (B) were treated with 3,3′,5-Triiodo-L-thyronine sodium salt (T3; 0.2–20 nM) for 24 h. Thereafter, qRT-PCR was performed as described in Materials and methods section. Results are expressed as fold induction over DMSO-treated cells ± SD. *—value is significantly different from untreated cells (DMSO; p < 0.05).
but no effect on CYP1A1 mRNA level (Fig. 1D), the expression of which comes from the full promoter. In conclusion, there are inconsistent and rather modulatory effects of activated TRs on basal or TCDD-inducible CYP1A1/2 expression or activity in human hepatocytes. The resulting output is likely a mix of several factors, like the level of expression of each isoform of TRs, polymorphisms of components in TR signaling cascade or the shift in the release/recruitment of sharing transcription factors between AhR and TR transcription machinery. Conflict of interest Authors declare no conflict of interest Acknowledgement This work was supported by a grant from the Czech Science Foundation P303/12/G163. References Chan, I.H., Privalsky, M.L., 2006. Thyroid hormone receptors mutated in liver cancer function as distorted antimorphs. Oncogene 25, 3576–3588. Cheng, S.Y., 2000. Multiple mechanisms for regulation of the transcriptional activity of
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Activated thyroid hormone receptor modulates dioxin-inducible aryl hydrocarbon receptor-mediated CYP1A1 induction in human hepatocytes but not in human hepatocarcinoma HepG2 cells
MARK
⁎
Radim Vrzal , Aneta Vrzalova, Aneta Grycova, Zdenek Dvorak Department of Cell Biology and Genetics, Faculty of Science, Palacky University in Olomouc, Slechtitelu 27, Olomouc, CZ-783 71, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords: Triiodothyronine CYP1A2 S14 Receptors cross-talk Dioxin receptor
Aryl hydrocarbon receptor (AhR) is a transcription factor, the activity of which is modulated by hormones including glucocorticoids and estrogens. In this study, we examined the effects of triiodothyronine (T3), a ligand and activator of thyroid hormone receptor (TR), on transcriptional activity of AhR and the expression of its target gene CYP1A1. Study was carried out in human hepatocellular carcinoma cells HepG2 and primary cultures of human hepatocytes (HH). Gene reporter assay in stably transfected AZ-AhR cells revealed that T3 dosedependently augmented 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible AhR-dependent luciferase activity. In contrast, T3 had no effect on TCDD-inducible expression of CYP1A1 mRNA, protein and catalytic activity. Incubation of human hepatocytes with T3 had modulatory and inter-individual (7 cell cultures from 7 different liver donors) effects on both basal and dioxin-inducible CYP1A1/2. Since there was no correlation between T3 effects on CYP1A expression and T3-dependent expression of Spot14 mRNA, the involvement of additional factors besides TR is supposed. Overall, the co-incubation of normal and cancer human hepatic cells with TCDD and T3 suggested transcriptional cross-talk between AhR and TR, which may have physiological and toxicological implications.
1. Introduction Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which resides in cytoplasm together with co-chaperones and upon ligand presence it translocates to the nucleus. Here it heterodimerizes with AhR nuclear translocator (ARNT, also known as HIF-1β) and binds to dioxin responsive elements (DREs) localized in promoters of target genes, such as those for cytochromes P450 1A1/2 (CYP1A1/2). Besides the regulation of genes involved in metabolism of xenobiotics, AhR also regulates cell cycle, cell differentiation or the immune response since it is abundantly expressed in regulatory TH lymphocytes (Veldhoen et al., 2008). Thyroid hormone receptors (TRs) are the ligand -activated transcription factors which mediate the biological action of thyroid hormones, in particular triiodothyronine (T3). There are two genes (α1 and β1), which encode several isoforms capable as well as incapable of T3 binding (Flamant et al., 2006). They belong to the nuclear receptor superfamily with centrally located highly conserved DNA-binding domain, which interacts with thyroid hormone response elements (TREs). The ligand-binding domain not only binds T3 but it is
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Corresponding author. E-mail address: [email protected] (R. Vrzal).
http://dx.doi.org/10.1016/j.toxlet.2017.05.001 Received 13 January 2017; Received in revised form 29 March 2017; Accepted 2 May 2017 Available online 03 May 2017 0378-4274/ © 2017 Elsevier B.V. All rights reserved.
also involved in interactions with co-repressors (N-CoR – nuclear receptor co-repressor; SMRT – silencing mediator of retinoid and thyroid hormone receptor), co-activators (SRC-1–steroid receptor coactivator 1) and heterodimerization partner, retinoid X receptor (RXR). The expression of TR isoforms is tissue-dependent and developmentally regulated (Cheng, 2000). TRβ1 isoform is expressed predominantly in the kidneys, liver, brain, heart and thyroid (Williams, 2000). TRα1 is expressed at highest level in the brain, at lower levels in the kidneys, lungs, heart and liver (Williams, 2000). Based on studies in mice lacking particular isoforms, it was suggested that TRβ1 and TRα1 can substitute for each other to mediate T3 action as well as they possess isoforms-specific functions (Forrest et al., 1996a,b; Gothe et al., 1999; Wikstrom et al., 1998). Due to the important role of TRs in the development, the inadequate activation/inhibition might have severe impact on human health. Many compounds that are released into the environment disrupt endocrine system by interfering with TR signaling. It was reported that babies in America are born in average with 287 foreign chemicals in their blood and pediatric endocrinologist are advised to check for endocrinedisrupting chemicals when assessing endocrine clinical problems of
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unknown etiology (Skakkebaek et al., 2011). The emphasis is placed on chemicals which are persistent, such as polychlorinated biphenyls (PCBs), polybrominated diethyl esters (PBDEs) or dioxins. These compounds bind to thyroid hormone transport proteins with consequent disruption of thyroid hormone homeostasis (Montano et al., 2012). In addition, they are also well known ligands and activators of the AhR (Murk et al., 1996). There are numerous evidences for existence of the mutual crosstalk between AhR and TR signaling pathways, in particular: a) the presence of SMRT which inhibits AhR/ARNT binding to DRE (Nguyen et al., 1999); b) the downregulation of dioxin-inducible AhR-target gene expression (CYP1A1) in the presence of all-trans-retinoic acid (ATRA) in human keratinocyte cell line HaCat (Wanner et al., 1995, 1996). ATRA binds to retinoid acid receptor (RAR), which heterodimerizes with RXR similarly like TR; c) intraperitoneal administration of PCB118 to Wistar rats which resulted in the generation of various cytokines (e.g. IL-6) with subsequent inhibition of natrium/iodide symporter (NIS) (Xu et al., 2016). NIS is important for T3 formation as it catches iodine from serum. The presence of AhR antagonist α-naphthoflavone or AhR small interfering RNA restored the NIS expression. This inhibition was suggested to lead to the thyroid disruption. While there are studies demonstrating endocrine disrupting effects of persistent environmental pollutants (AhR activators) on TR-mediated signaling, the opposite, i.e. the effects of thyroid hormones on AhR signaling were neglected. In the current paper we investigated whether T3 affects basal and TCDD-inducible expression of AhR-target gene, CYP1A1. We used two in vitro models, human hepatocellular carcinoma (HepG2) and seven primary cultures of human hepatocytes. We measured the expression of CYP1A1 mRNA and protein, and also the catalytic activity of CYP1A1.
Table 1 Health and demographic data on human hepatocytes donors. Culture abbreviation
Gender
Age
Smoking
Alcohol use
LH37 LH38 LH42 LH50 LH51 LH59 Hep220770
male male female female female female female
65 58 60 55 58 42 35
unknown unknown unknown positive negative negative negative
unknown unknown unknown positive negative negative negative
were maintained at 37 °C and 5% CO2 in a humidified incubator. Hepatocytes were incubated with the tested compounds, inducers and/ or vehicle (DMSO; 0.1% v/v) for 24 h (RNA) and 48 h (protein, catalytic activity). 2.4. Quantitative reverse transcriptase polymerase chain reaction (qRTPCR) Total RNA was isolated using TRI Reagent® (Molecular Research Center, USA). cDNA was synthesized from 1000 ng of total RNA using M-MuLV Reverse Transcriptase (M0253, New England Biolabs) at 42 °C for 60 min in the presence of random hexamers (3801, Clontech). qRTPCR was carried out on Light Cycler 480 apparatus (Roche Diagnostic Corporation, Prague, Czech Republic). The levels of CYP1A1/1A2/ GAPDH mRNAs were determined using primers and Universal Probes Library (UPL; Roche Diagnostic Corporation, Prague, Czech Republic) technology described earlier (Vrzal et al., 2015). The primers and UPL probe for Spot14 were as follows, forward: Spot14, 5-CATGCACCTCACCGAGAA-3, reverse: 5-TGTCTTCTATCATGTGAAGGGATCT-3, UPL 79. The following program was used for monitoring the expression of all genes: an activation step at 95 °C for 10 min was followed by 45 cycles of PCR (denaturation at 95 °C for 10 s; annealing with elongation at 60 °C for 30 s). The measurements were performed in duplicates. Gene expression was normalized per glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene. Data were processed by delta-delta method (Pfaffl, 2001). Results are expressed as fold induction over DMSO-treated cells.
2. Materials and methods 2.1. Compounds and reagents Dimethylsulfoxide (DMSO; purity ≥ 99.9%), 3,3′,5-Triiodo-L-thyronine sodium salt (T3; purity ≥ 95%), charcoal-stripped fetal serum (FBS) were purchased from Sigma-Aldrich (Prague, Czech Republic). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was from Ultra Scientific (Cincinnati, Ohio, USA). Oligonucleotide primers used in RT-PCR reactions were synthesized by Generi Biotech (Hradec Kralove, Czech Republic). LightCycler 480 Probes Master was from Roche Diagnostic Corporation (Intes Bohemia, Czech Republic). All other chemicals were of the highest quality commercially available.
2.5. Western blotting Total protein extracts for each sample were prepared from 1 well of a six-well plate dish. Cells were washed twice with ice-cold phosphate buffered saline (PBS) and scraped into 1 ml of PBS. The suspension was centrifuged (5000 × rpm at 4 °C for 2 min), and the pellet was resuspended in 150 μL of ice-cold lysis buffer (150 mM NaCl; 10 mM Tris pH 7.2; 0.1% (w/v) SDS; anti-protease cocktail, 1% (v/v) Triton X100; anti-phosphatase cocktail, 1% (v/v) sodium deoxycholate; 5 mM ethylenediaminetetraacetic acid [EDTA]). The mixture was vortexed and incubated for 10 min on ice and then centrifuged (13,000 × rpm at 4 °C for 13 min). Supernatant was collected and the protein content was determined with the Bradford reagent. For CYP1A1 protein detection in HepG2 cells, SDS–PAGE gels (8%) were run on a BioRad apparatus according to the general procedure followed by the protein transfer onto the polyvinylidene fluoride (PVDF) membrane. The membrane was saturated with 5% non-fat dried milk for 2 h at room temperature. Blots were probed with primary antibodies against CYP1A1 (goat polyclonal; sc-9828, G-18), CYP1A2 (mouse monoclonal; sc-53614, 3B8C1) and actin (goat polyclonal; sc-1616, 1–19), all purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Chemiluminescence detection was performed using horseradish peroxidase-conjugated secondary antibodies and Western blotting Luminol kit (both from Santa Cruz Biotechnology). Detection of CYP1A1, CYP1A2 and actin in human hepatocytes was performed with Sally Sue Simple Western System according to the
2.2. Cell cultures Human Caucasian hepatocellular carcinoma cells HepG2 (Public Health England, ECACC No. 85011430) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of fetal calf serum, 100 U/mL streptomycin, 100 μg/mL penicillin, 4 mM L-glutamine, 1% non-essential amino acids, and 1 mM sodium pyruvate. Cells were maintained at 37 °C and 5% CO2 in a humidified incubator. HepG2-derived AZ-AhR cell line was incubated under the same condition as parental HepG2. All treatments took place in medium supplemented with charcoal-stripped fetal serum (CS-FBS). 2.3. Human hepatocytes Human liver tissue was obtained from two sources: (i) from multiorgan donors or (ii) long-term human hepatocytes in monolayer (Biopredic International, Rennes, France) (Table 1). Tissue acquisition protocol was in accordance with the requirements issued by local ethical commission in the Czech Republic. Cells were plated into collagen-coated dishes in a hormonally and chemically defined medium consisting of a mixture of William’s E and Ham’s F-12 [1:1 (v/v)] and 78
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We found that neither basal nor TCDD-inducible mRNA level of CYP1A1 was significantly affected by T3 (Fig. 1D). Further, we monitored the effect of T3 on CYP1A1 protein and catalytic activity. The level of CYP1A1 protein (Fig. 1E) or CYP1A1-mediated catalytic activity (Fig. 1F) was not affected by the presence of T3 alone or in combination with TCDD.
manufacturer's instructions (ProteinSimple™). In brief, samples were diluted to adjust protein concentration to 1 μg/μl in 5 μl with sample buffer and further diluted by adding 1.25 μl of the 5× master mix (Protein Simple) to a final concentration of 1× sample buffer, 1× fluorescent molecular weight markers, and 40 mM dithiothreitol (DTT) and then heated at 95 °C for 5 min. The samples, blocking reagent, wash buffer, primary antibodies, secondary antibodies, and chemiluminescent substrate were dispensed into designated wells in the manufacturer provided microplate. After plate loading, the separation electrophoresis and immunedetection steps took place in the capillary system and were fully automated. Simple Western analysis was carried out at room temperature, and instrument default settings were used. The data were analyzed with inbuilt Compass software (ProteinSimple).
3.2. The effect of activated TR on the signaling of AhR in primary cultures of human hepatocytes We further decided to investigate the effect of activated TRs on AhRmediated induction of CYP1A1/2 mRNA, protein and catalytic activity in primary cultures of human hepatocytes. In most cultures, T3 increased basal CYP1A1 mRNA only weakly (Fig. 2A), an exception being the culture LH51. The inducible CYP1A1 mRNA was affected in culture-specific way. In three cultures (LH37, LH38, Hep220770) there was an upregulation with 2 nM T3 while for other cultures there was no effect (LH42, LH51) or decrease in mRNA (LH50, LH59) (Fig. 2B). The basal CYP1A2 mRNA level was increased with T3 to higher extent than CYP1A1 but no reproducible trend was observed (Fig. 2C). The inducible CYP1A2 mRNA was decreased (cultures LH37, LH50, LH51), unaffected (cultures LH42, LH59) or increased (LH38, Hep220770) in the presence of T3 (Fig. 2D). After this step, we monitored the CYP1A proteins level. In cultures, where both mRNA and protein were determined, the protein level displayed similar trend like mRNA, as it can be seen on representative virtual western blot from culture LH50 and quantification for these data (Fig. 2E and F). Moreover, in cultures where CYP1A catalytic activity was measured (Fig. 2G), the level of activity followed the level of protein. However, we observed approx. 50% increase in TCDD-inducible CYP1A enzymatic activity in the presence of T3 in 3 cultures (LH51, LH59, LH64).
2.6. CYP1A enzymatic activity assay 7-Ethoxyresorufin-O-deethylase (EROD) activity was determined as described elsewhere (Vrzal et al., 2013). 2.7. Gene reporter assay Stably transfected cell line derived from human hepatoma HepG2 cells was used for assessment of AhR transcriptional activity (Novotna et al., 2011). Cells were incubated for 24 h with increased concentrations of T3 and/or vehicle (DMSO; 0.1% v/v), in the presence (“Antagonist settings”) or absence (“Agonist settings”) of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD; 5 nM). After the treatment, cells were lysed and luciferase activity was measured with Infinite M200 (TECAN, Austria). 2.8. Statistical analysis Data were analyzed by using GraphPad Prism Version 6.05 (GraphPad Software, San Diego, CA, USA). The differences in luciferase assay, mRNA, protein expression and catalytic aktivity following incubation with the compounds investigated compared with the respective vehicle controls were tested by using ANOVA with Dunnett’s post hoc test; P ≤ 0.05 was considered significant. p < 0.05 was considered statistically significant.
3.3. Functional verification of activated thyroid hormone receptor (TR) It is known, that TR is functional in HepG2 cells as it was demonstrated in some studies (Prieur et al., 2005) and our recently established stably transfected HepG2-derived TR-dependent cell line (Illes et al., 2015). Nevertheless, the inducibility of TR-target gene, Spot14 was verified. The mRNA level of Spot14 was induced in average 1.5-times with 2 nM of T3 only, the higher or lower concentrations were without the effect (Fig. 3A). Due to the atypical induction profile in HepG2 cells, we verified the Spot14 induction in human hepatocytes as well. In each hepatocyte culture, Spot14 was induced concentrationdependently with culture-specific magnitude (Fig. 3B). This demonstrates functional thyroid hormone receptor.
3. Results 3.1. The effect of 3,3′,5-triiodo-L-thyronine on AhR signaling in HepG2 cells At the beginning, we used stably transfected AZ-AhR cell line (Novotna et al., 2011) to investigate the effect of T3 on AhR-mediated luciferase activity. Triiodothyronine did not have any effect on luciferase activity, while positive control dioxin (TCDD) induced the activity in average 1500-fold over negative control, DMSO (Fig. 1A), providing the evidence of the functionality of the system. In the presence of TCDD, the luciferase activity was stimulated concentration-dependently by T3 and the effect was significant for 2 highest concentrations (Fig. 1B). However, since additive or synergistic effects are sometimes better to study with non-saturating concentrations, we tried to verify if the effect would be different for lower concentrations of TCDD. Therefore, we calculated the EC10, EC25 and EC50 from doseresponse curve for TCDD on AZ-AhR cell line. From calculated 95% confidence interval we chose 0.3, 0.6 and 1.2 nM concentrations of TCDD representing EC10, EC25 and EC50, respectively. We found that there is synergistic effect between T3 and TCDD no matter of TCDD concentration (Fig. 1C). Triiodothyronine-stimulated increase reached in average 22% above TCDD alone in its maximum. This is not different from saturating concentration 5 nM of TCDD for AhR (Fig. 1B). Therefore, we further used this concentration only. Due to the synergistic effect of T3 on TCDD-inducible AhR-dependent luciferase activity, we decided to investigate the expression level of AhR-target gene, CYP1A1.
4. Discussion In the current work, we investigated if T3-activated thyroid hormone receptor can affect the activity of AhR and its target gene CYP1A1 expression in human hepatocellular carcinoma HepG2 cells or in primary cultures of human hepatocytes. In HepG2 cells, there was no effect of T3 on basal or TCDD-inducible level of CYP1A1 at mRNA, protein or activity levels. However, we recorded significant stimulation of TCDD-inducible AhR-mediated luciferase activity in stably transfected HepG2 cells. The monitoring of AhR activity via CYP1A1/2 induction in human hepatocytes revealed irreproducible effect of T3, which differed among cultures. In some cultures there was stimulation of TCDD-inducible CYP1A1/2 mRNA, in others no effect or mild inhibition. This was more or less followed at the protein and the activity level of CYP1A enzymes. Our data from human hepatocytes provide the evidence that even with increasing induction of Spot14 and anticipated increasing activation of TRs, the effects on the AhR signaling, monitored as CYP1A1/2 expression, are unlikely to be directly connected with TRs. While we always observed induction of Spot14, the effects especially on TCDD79
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Fig. 1. Cells were treated with 3,3′, 5-Triiodo-L-thyronine sodium salt (T3; 0.2–20 nM) in the absence or presence of TCDD (5 nM) and/or DMSO for 24 h (A–D) or 48 h (E and F). Thereafter, gene reporter assay (A–C), qRT-PCR (D), western blotting (E) or enzymatic activity determination (F) were performed as described in Materials and methods section. The data are the mean ± SD from 3 to 4 independent experiments and are expressed as fold activation/induction over DMSO-treated cells (A, B, D, F) or as % of positive control (TCDD) (B) or as relative luminescence units (RLU) (C). *—value is significantly different from untreated cells (DMSO; p < 0.05) (A, D, F) or TCDD-treated cells (B and C).
activation with delayed dissociation of N-CoR and SMRT, which in turn may more easily associate with CYP1A1 promoter (Nguyen et al., 1999). In addition, at least one TRα splicing variant, c-erbA α-2, inhibits of T3 action in hormone-independent fashion (Koenig et al., 1989). Another thing that may also contribute to heterogenous effects in hepatocytes is the fact that TRs induce hypoxia-inducible factor 1 alpha (HIF-1α), among many other genes (Otto and Fandrey, 2008). As it is known that this protein associates with HIF-1β (ARNT), it is likely that the response in some hepatocyte culture may be affected by tiny shift in equilibrium between these two proteins. Consequently, CYP1A1/2 expression is affected. Concerning the results, we obtained in HepG2 cells, we may ask the
inducible CYP1A1/2 expression level was inconsistent among cultures with all 3 possible outputs, i.e. potentiation, inhibition, no effect. Thus, we can expect that CYP1A1/2 expression and activity can be modulated by donor-specific manner and among many, genetic polymorphisms of components of TR signaling pathway may play a significant part. This is likely the most plausible explanation when looking at the data. Moreover, in contrast to HepG2 cells, which should be uniform in their TRs expression ratios, each hepatocyte donor may have different level of expression of these isoforms and consequently the results may be affected this way. Thus, diverse results could have been obtained due to the fact that some TR-regulated genes display differential response to T3 with TRα and TRβ, where higher levels of T3 are needed for optimal induction with TRβ (Lin et al., 2013). This may lead to delayed TRβ 80
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Fig. 2. Primary cultures of human hepatocytes were treated with 3,3′,5-Triiodo-L-thyronine sodium salt (T3; 0.2–20 nM) in the absence or presence of TCDD (5 nM) and/or DMSO for 24 h (A–D) or 48 h (E–G). Thereafter, qRT-PCR (A–D), capillary electrophoresis with immunedetection (E and F) or enzymatic activity determination (G) were performed as described in Materials and methods section. Results are expressed as fold induction over DMSO-treated cells ± SD. A representative virtual western blot from culture LH50 is shown (E) together with quantification data from the same culture (F).
SMRT (Chan and Privalsky, 2006), which may lead to higher, i.e. repressive manner on CYP1A1 promoter in HepG2 cells. This may explain why we observed significantly potentiated TCDD-inducible AhR-dependent luciferase activity (Fig. 1B and C) in our stably transfected AZ-AhR cell line with DREs only (Novotna et al., 2011)
question why there was no effect in contrast to hepatocytes, where actually all possibilities (i.e. increase, decrease, no effect) were observed. One thing that might contribute to the explanation is the fact that many hepatocellular carcinomas have mutated TRs (Lin et al., 1999). Some of these mutants have decreased binding to N-CoR or
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Fig. 3. HepG2 cells (A) or primary cultures of human hepatocytes (B) were treated with 3,3′,5-Triiodo-L-thyronine sodium salt (T3; 0.2–20 nM) for 24 h. Thereafter, qRT-PCR was performed as described in Materials and methods section. Results are expressed as fold induction over DMSO-treated cells ± SD. *—value is significantly different from untreated cells (DMSO; p < 0.05).
but no effect on CYP1A1 mRNA level (Fig. 1D), the expression of which comes from the full promoter. In conclusion, there are inconsistent and rather modulatory effects of activated TRs on basal or TCDD-inducible CYP1A1/2 expression or activity in human hepatocytes. The resulting output is likely a mix of several factors, like the level of expression of each isoform of TRs, polymorphisms of components in TR signaling cascade or the shift in the release/recruitment of sharing transcription factors between AhR and TR transcription machinery. Conflict of interest Authors declare no conflict of interest Acknowledgement This work was supported by a grant from the Czech Science Foundation P303/12/G163. References Chan, I.H., Privalsky, M.L., 2006. Thyroid hormone receptors mutated in liver cancer function as distorted antimorphs. Oncogene 25, 3576–3588. Cheng, S.Y., 2000. Multiple mechanisms for regulation of the transcriptional activity of
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