The Aryl Hydrocarbon Receptor Directly Regulates Expression of the Pot
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《毒物学科学杂志》
Graduate Programs in Molecular Medicine and Molecular Toxicology, The Huck Institutes of the Life Sciences, Department of Veterinary Science and The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, Pennsylvania 16802
ABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause a large number of adverse effects, mediated largely by its binding to the aryl-hydrocarbon receptor (AhR) and subsequent modulation of gene expression. It is thought that AhR mediates these effects through the untimely and disproportionate expression of specific genes. However, the exact mechanism, or the genes involved, through which TCDD leads to these effects is still unknown. This study reports the discovery of a novel target gene, epiregulin, which is regulated by TCDD-activated AhR. Epiregulin is a growth regulator which belongs to the epidermal growth factor (EGF) family. Using real time quantitative PCR (qPCR), it was established that TCDD upregulates epiregulin gene expression. The promoter region of epiregulin has a dioxin responsive element (DRE) 56 nucleotides upstream of the transcription start site, along with three potential Sp1 binding sites. Chromatin immunoprecipitation (ChIP) assays with an anti-AhR antibody showed promoter occupancy upon TCDD treatment. Luciferase reporter assays using a vector harboring the first 125 base pairs of the epiregulin rat promoter revealed an increase in signal on TCDD treatment, which was lost upon mutation of the DRE. Epiregulin and TCDD treatment mediated a dose-dependent increase in primary mouse keratinocyte growth. These results demonstrate that AhR directly increases epiregulin expression, which could play an important role in TCDD mediated tumor promotion observed in rodent models.
Key Words: AhR; epiregulin; transcription; growth factor; TCDD.
INTRODUCTION
The aryl hydrocarbon receptor (AhR) is a member of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) family of transcription factors. AhR exists in the cytoplasm complexed with the chaperone HSP90 together with co-chaperones X-associated protein 2 (also referred to as AIP or ARA9) and p23 (reviewed in Petrulis and Perdew, 2002). The AhR translocates to the nucleus after ligand binding and heterodimerizes with AhR nuclear translocator (ARNT). AhR mediates its effects in a fashion similar to nuclear hormone receptors. The AhR/ARNT heterodimer binds to dioxin response elements (DRE) and modulates transcription of target genes. The consensus core binding site for the AhR-ARNT complex is TNGCGTG. ARNT binds the GTG half-site while AhR binds the other half. The majority of the genes known to be regulated by AhR are involved in xenobiotic metabolism; the well-characterized genes include CYP1A1, CYP1A2, CYP1B1, glutathione S-transferase Ya, and aldehyde dehydrogenase 3A1 (reviewed in Nebert et al., 1993, 2000).
Aside from its role in xenobiotic metabolism, there is an increasing body of evidence implicating AhR in a diverse range of patho-physiological processes involving the immune system, liver, and the cardiovascular system (Heid et al., 2001) as well as in cell cycle control (Elizondo et al., 2000). AhR activation in response to xenobiotic exposure leads to a variety of adverse effects, including tumor promotion. Studies using mice deficient in AhR have provided clues to its role in growth and development. AhR null mice exhibit increased hepatic fibrosis, decreased liver weight, abnormalities of the immune system in the form of a reduced number of peripheral lymphocytes (Fernandez-Salguero et al., 1995; Schmidt et al., 1996), decreased fertility, vascular abnormalities in heart, liver, and uterus, delayed wound healing, impaired skin homeostasis, and an overall slower growth (Fernandez-Salguero et al., 1997). In addition, studies using null mice have also shown that AhR might have a role in the formation of primordial follicles and in regulating the number of antral follicles, thus affecting development of the mouse ovary (Benedict et al., 2000). Also, the incidence of cardiac hypertrophy in AhR null mice suggests a role for AhR in normal cardiovascular development and angiogenesis (Lund et al., 2003). However, it has not been clearly defined how AhR mediates these functions. Differential regulation of genes, other than those involved in xenobiotic metabolism, could possibly account for these AhR mediated effects. Alternate mechanisms have also been suggested in the recent years to explain the different functions of AhR. For example, it has been shown to interact with retinoblastoma (Rb) (Ge and Elferink, 1998) and nuclear factor -B (Koo and Kim, 2003) transcription factors and modulate cell proliferation. Co-expression of AhR and BRG-1 restored Rb-sensitivity to a tumor cell line, C33A, which could otherwise progress through the cell cycle even in the presence of active Rb protein (Strobeck et al., 2000).
A number of exogenous and endogenous ligands are capable of activating the AhR (Denison et al., 2002). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to be the most potent exogenous AhR ligand. While there is evidence suggesting that TCDD exerts almost all its toxic effects through the AhR, the exact mechanisms underlying these effects are not clear. TCDD has been shown to be more effective as a tumor promoter than as a tumor initiator, due to the fact that TCDD is essentially not metabolized (reviewed in Dunson et al., 2000). TCDD has been shown to promote skin tumor formation in hairless mice (Poland et al., 1982) as well as promote liver tumor formation (reviewed in Dragan and Schrenk, 2000). The importance of AhR in tumor promotion is also highlighted by the results obtained in a recent study using transgenic mice expressing a constitutively active AhR where a significant increase in hepatocarcinogenesis was noted compared to wild-type mice (Moennikes et al., 2004). In addition, studies using constitutively active AhR have demonstrated that the receptor can induce tumors in the stomach (Andersson et al., 2002). The fact that benzo[a]pyrene, a known AhR ligand, is unable to exhibit its carcinogenic effects in the skin of AhR null mice, provides further evidence supporting a role for AhR in tumorigenesis (Shimizu et al., 2000).
Epidermal growth factor (EGF) and other members of its family are potent peptide growth factors and are involved in a plethora of physiological and pathological processes through their signaling properties (Harris et al., 2003). Mutations and/or over-expression of EGF-family members cause cells to acquire an oncogenic phenotype. Epiregulin is a relatively newly identified member of the EGF family, isolated from the conditioned medium of NIH/3T3/clone 7 cells (Toyoda et al., 1995). It is secreted as a 46-amino acid single chain polypeptide with contrasting growth regulatory properties, inhibiting the growth of several epithelial cell lines while promoting growth of other cell types, like vascular smooth muscle cells (Koo and Kim, 2003), hepatocytes (Komurasaki et al., 2002; Toyoda et al., 1995), and keratinocytes (Shirakata et al., 2000). Epiregulin is expressed during development as well as later on in the genitourinary tract (Sekiguchi et al., 2002, 2004), gastrointestinal tract, vascular smooth muscle cells, and skin. In the latter two tissues, it has been shown to function in a paracrine as well as autocrine fashion (Takahashi et al., 2003).
This study demonstrates, for the first time, that ligand activated AhR binds to its cognate element in the epiregulin promoter and mutation of this binding element results in loss of AhR-driven reporter activity. AhR increases epiregulin transcription in cultured primary mouse keratinocytes, immortalized hepatocytes, and mouse hepatoma derived cell line. It is also shown that epiregulin is capable of significantly enhancing the proliferation of cultured primary mouse keratinocytes. This could possibly account for one of the mechanisms by which TCDD exerts its tumor promotional effects in rodent skin.
MATERIALS AND METHODS
Cell culture.
Primary mouse keratinocytes were obtained from two-day old neonatal C57BL/6 mice and cultured using a previously described method (Dlugosz et al., 1995). NIH/3T3 cells and Hepa1c1c7 were grown in -minimal essential medium (-MEM) supplemented with 10% FBS (HyClone Laboratories, Logan, UT), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin (Sigma) at 37°C in 5% CO2 atmosphere. Hepatocytes from two-week old C57BL/6N mice were infected with SV40 virus to prepare temperature sensitive SV40 immortalized mouse hepatocytes in our laboratory (Murray et al., in press) using a previously described protocol with modifications (Chou, 1985; Lorenzen et al., 1997). Three cell lines of immortalized hepatocytes were established. Cells from line 2 were used for experiments. These cells were maintained in 4% FBS and 0.1 μM dexamethasone at 34°C.
Real time quantitative PCR (qPCR).
Real time qPCR was performed on the DNA Engine Opticon (MJ Research, Inc.) using DyNAmo Hot Start SYBR Green qPCR kit purchased from MJ Research, Inc. cDNA synthesis was carried out using High Capacity cDNA Archive Kit from Applied Biosystems. The reverse transcription reactions were set up according to the manufacturer's instructions. cDNA synthesized from 50 ng of total RNA was used per qPCR reaction. Epiregulin mRNA was detected using 5'-TGGGTCTTGACGCTGCTTTGTCTA-3' and 5'-AAGCAGTAGCCGTCCATGTCAGAA-3' primers. Epiregulin promoter in ChIP assays was detected using 5'-TTCCTGAGAGGGAGGATGACAT-3' and 5'-CCCACCAAGTCGCTGTGACT-3' primers. Thermal cycling conditions were setup according to the manufacturer's protocol.
Plasmids.
The following plasmids were used for reporter assays: pEpi125 – derived by inserting rat epiregulin promoter region –125/+12 (genomic contig: NW_047424.1) in pGL3-Basic luciferase vector, pmutABC – pEpi125 with all three GT and CT boxes mutated (Sato et al., 2003), pmutARNT – pEpi125 with ARNT half-site of DRE mutated and pmutAhR – pEpi125 with AhR half-site of DRE mutated. pEpi125 and pmutABC were a kind gift of Dr. Kaoru Miyamoto (Fukui Medical University, Japan). pmutARNT and pmutAhR were modified forms of pEpi125, generated by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene). Forward and reverse primer pairs used to mutate the ARNT and AhR half-sites, respectively were (5'-GTAAGTCCTCGCTGGCCTAAGCACC-3' and 5'-GGTGCTTAGGCCAGCGAGGACTTAC-3') and (5'-GTAAGTCCTCGAGTGCCTAAGCACC-3' and 5'-GGTGCTTAGGCACTCGAGGACTTAC-3').
Transient transfections and luciferase reporter assays.
These experiments were carried out using NIH/3T3 cells, obtained from the American Type Culture Collection (ATCC). Transfections were performed using Lipofectamine Plus (Invitrogen). Cells at 70% confluency in 6-well plates were washed with PBS and 1.5 ml Optimem (Life Technologies) containing 5 μl of Lipofectamine, 1 μl of Plus, and 1.5 μg DNA were added per well. The DNA was comprised of 500 ng of a luciferase reporter vector harboring wild-type or mutated versions of the rat epiregulin promoter, 100 ng of pDJM/-gal to control for transfection efficiency and empty expression vector. After 4 h cells were washed with PBS and -MEM containing 10% FBS and antibiotics was added. Cells were allowed to grow overnight and treated with 10 nM TCDD or DMSO the next day for 7 h and lysed with 1X cell lysis buffer (25 mM Tris buffer pH 7.8, 2 mM DTT, 2 mM EDTA, 10% glycerol, and 1% Triton X-100). Luciferase activity from cell lysates was measured using a Turner TD-20e luminometer (Turrner Designs, Sunnyvale, CA) the and values were normalized to -gal values.
Chromatin Immunoprecipitation assay and PCR.
Chromatin Immunoprecipitation (ChIP) assays were performed as previously described (Spencer et al., 2003) with some modifications. Briefly, 80% confluent cells were treated with 10 nM TCDD or DMSO for 100 min and crosslinked for 8 min at room temperature with 0.33% formaldehyde (SIGMA, F-8775). Cells were harvested and lysed with 750 μl/flask lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). Lysates were sonicated with Branson Sonifier 250 using 40% duty cycle and output set at 4 for 8 cycles of 12 pulses each. Protein-DNA complexes were immunoprecipitated from 200 μl lysate using either 6 μl of rabbit polyclonal anti-AhR antibody (Biomol, SA-210), control IgG or no antibody and 50 μl Goat Anti-Rabbit IgG-Agarose resin (SIGMA, A1027). Resin was washed once each with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8 and 150 mM NaCl), 1X RIPA buffer (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 10 mM Tris-HCl pH 8) and 1xMENG buffer (25 mM MOPS, 2 mM EDTA, 0.02% sodium azide, 10% glycerol pH 7.5) in that order. Immunoprecipitated protein-DNA complexes were incubated in 300 μl digesting buffer (50 mM Tris-HCl pH 8, 1 mM EDTA, 100 mM NaCl, 0.5% SDS, 100 μg/ml Proteinase K). DNA was isolated by phenol-chloroform extraction and concentrated using ethanol precipitation. PCR was performed using following primers: Epiregulin (5'-TTCCTGAGAGGGAGGATGACAT-3' and 5'-CCCACCAAGTCGCTGTGACT-3'; located 107 bases upstream and 75 bases downstream relative to TATA box), Cyp1A1 (5'-GCCGAGCATCGCACGCAAACC-3' and 5'-GGATCCACGCGAGACAGCAGG-3'; located 1168 and 784 bases upstream relative to TATA box) and GAPDH (5'-CATGGCCTTCCGTGTTCCTA-3' and 5'-GCGGCACGTCAGATCCA-3'). Ten percent DMSO was added to the reactions.
Keratinocyte proliferation studies.
Equivalent number of keratinocytes were seeded in 12 well plates and cultured in low calcium medium (0.05 mM) containing 8% chelexed FBS for 24 h before treatment. To test the effect of epiregulin or TCDD on keratinocyte growth, cells were cultured in low calcium medium containing epiregulin (R&D Systems, 1068-EP), at a concentration from 1– 20 ng/ml, or TCDD at a concentration from 0.1– 1.0 nM, continuously with medium change after 24 and 72 h. After 24, 72, or 120 h incubation, cell number was measured using a Z1 coulter particle counter (Beckman Counter, Inc., Hialeah, FL).
RESULTS
TCDD Treatment Upregulates Epiregulin Transcription
The principal mode of action of AhR is through binding its cognate cis-regulatory element, the DRE, in the promoter region of its target genes. AhR heterodimerizes with ARNT and then recruits coregulators to enhance transcription (reviewed in Swanson et al., 2002). Despite the fact that AhR is implicated in a diverse range of physiological processes, as discussed in the introduction, few genes outside the gamut of xenobiotic metabolism have been reported to be directly regulated by the AhR pathway. In an attempt to identify novel target genes, whose expression is under the regulation of AhR, microarray experiments (unpublished data) were performed using SV-40 immortalized mouse hepatocytes. These experiments revealed epiregulin as one of the target genes whose mRNA levels increased on TCDD treatment. Microarray results were confirmed with real-time qPCR (Fig. 1). Effect of TCDD on epiregulin mRNA levels was also tested in Hepa1c1c7, a mouse hepatoma cell line. Epiregulin was upregulated by 2.6- fold, 90 min after TCDD exposure. As TCDD exerts significant adverse effects on skin, including tumor promotion, upregulation of epiregulin in response to TCDD was assessed in primary keratinocytes. Primary mouse keratinocytes were treated with 10 nM TCDD for 90 min and a change in the level of epiregulin mRNA was compared by real time qPCR. TCDD increased epiregulin mRNA expression by 3.3-fold (Fig. 1) in primary mouse keratinocytes also. Thus, AhR activation resulted in epiregulin upregulation in primary cells as well as immortalized cell lines.
AhR Binds the DRE in the Epiregulin Promoter
In accordance with the currently accepted theory, AhR must bind its response element in the promoter of a gene to directly regulate its expression. A search for binding sites for AhR-ARNT heterodimer by sequence analysis identified a consensus DRE (TCGCGTG) 56 nucleotides upstream of the transcription start site in the epiregulin promoter in mouse and rat genomic DNA. To assess if AhR binds the DRE in the epiregulin promoter sequence, ChIP assays were performed with SV40 virus immortalized mouse hepatocytes treated with TCDD. DNA fragments isolated from ChIP assays were analyzed using PCR (Fig. 2A). CYP1A1 was used as a positive control. GAPDH and immunoprecipitations with no antibody or with control IgG were used as negative controls. CYP1A1 showed a strong signal in the TCDD treated sample, compared to a carrier solvent treated sample, while there was no difference in the case of GAPDH (data not shown). Immunoprecipitations with control IgG or no antibody showed minimal background signals. While the epiregulin signal was more intense in TCDD samples compared to carrier solvent treated samples, the difference was less than that of CYP1A1. This difference could be explained by the fact that epiregulin has only one DRE in its promoter while the CYP1A1 promoter has multiple DREs, and that AhR upregulates CYP1A1 expression by a greater magnitude than epiregulin. These results were confirmed by realtime qPCR (Fig. 2B) performed on DNA isolated from ChIP assays. The relative signal for epiregulin in TCDD treated samples was 1.7-fold greater than carrier solvent treated samples. Combined, these results demonstrate an increase in epiregulin promoter occupancy by AhR in response to TCDD treatment.
AhR Driven Promoter Activity Is Dependent on the DRE
To further confirm the role of the DRE in modulating epiregulin gene expression, a luciferase reporter assay was performed using a construct containing the first 125 bases of the rat epiregulin promoter cloned into pGL3-Basic vector (pEpi125). There is a single nucleotide difference between the first 125 bases of the mouse (genomic contig: NT_039308.3) and the rat (genomic contig: NW_047424.1) epiregulin promoters, which is not a part of the DRE or the Sp1 binding sites. This construct has been previously used by Sekiguchi et al. to study transcriptional regulation of the epiregulin gene in the rat ovary (Sekiguchi et al., 2002). They showed by deletion and mutation analyses that the region encompassing 125 bp upstream of the transcription start site was essential for controlling transcription of the epiregulin gene. According to their results, Sp1/Sp3 proteins bind to one or more of the two CT boxes and one GT box within this 125 bp region and are involved in regulating epiregulin gene expression.
The relative contributions of the AhR and Sp1/Sp3 were studied using plasmids derived from pEpi125 by mutating their binding sites, the DRE and the CT and GT boxes, respectively (Fig. 3A). Luciferase reporter plasmid pEpi125 was transiently transfected in NIH/3T3 cells, while pDJM/-gal vector was cotransfected to control for transfection efficiency. Reporter assays were also carried out with pmutABC plasmid, derived from pEpi125 by mutating both the CT boxes as well as the GT box, pmutARNT plasmid, derived from pEpi125 by mutating the ARNT half-site of DRE and pmutAhR plasmid, derived from pEpi125 by mutating the AhR half-site of DRE (Fig. 3C). There was a two-fold increase (2.3-fold) in luciferase reporter activity observed upon TCDD treatment (Fig. 3B) with the pEpi125 transfections. Surprisingly, a similar increase in luciferase activity (2.2-fold) was observed with the pmutABC transfections compared to vehicle treated control samples. There was no increase in luciferase activity on TCDD treatment when the AhR and ARNT binding half-sites were mutated in pmutARNT and pmutAhR, respectively (1.2-fold in pmutARNT and pmutAhR each). Interestingly, the reporter gene activity in pmutABC transfections was 3.2 times lower compared to pEpi125 transfections, both in TCDD and carrier solvent treated samples. This decrease in reporter activity was not observed when the DRE was mutated (pmutAhR and pmutARNT transfections). These results, along with ChIP assays, demonstrate that the activated AhR binds to the DRE within the epiregulin promoter and is responsible for the observed increase in transcriptional activity.
Effect of Epiregulin and TCDD on Mouse Keratinocyte Proliferation
To characterize the functional role of increased epiregulin expression on cell growth, the effect of epiregulin treatment was determined using primary mouse keratinocytes. It has previously been shown that epiregulin stimulates proliferation of cultured human keratinocytes in a dose-dependent manner and acts as an autocrine growth factor (Shirakata et al., 2000). Recombinant human epiregulin at a concentration of 1 ng/ml resulted in a three-fold increase in cell growth. In the present study, primary mouse keratinocytes showed a dose-dependent increase in cell number when treated with recombinant mouse epiregulin, even in the presence of other growth factors found in fetal bovine serum (Fig. 4A). Epiregulin stimulated a statistically significant proliferation of primary mouse keratinocytes across a dose range of 1–20 ng/ml at 24, 72, and 120 h of treatment, as determined by ANOVA (p < 0.05). Significant increase in proliferation was observed at all three time points with the higher doses (10 and 20 ng/ml), whereas 1 ng/ml epiregulin induced proliferation only at 72 and 120 h.
Similar cell proliferation studies were performed using TCDD as a stimulant (Fig. 4B). Primary mouse keratinocytes were treated with 0.1 nM, 0.5 nM, and 1 nM TCDD, or vehicle solvent, and cells counted at 24, 72, and 120 h after addition of TCDD. TCDD-induced cell proliferation was statistically significant, as determined by ANOVA (p < 0.05). Individually, all three doses induced statistically significant increase in cell proliferation when compared to vehicle treated cells. However, a statistically significant difference was not observed amongst the different doses, as determined by Tukey's HSD test. When the effects of different doses were compared at individual time-points, an increase in the number of cells was observed at all three time-points with 0.1 nM and 0.5 nM TCDD treatments. Primary mouse keratinocytes treated with 1 nM TCDD showed a slight reduction in cell numbers at the 120 h time point. This could be due to increasing toxicity owing to prolonged TCDD exposure at this latest time point.
DISCUSSION
TCDD causes a wide range of toxic effects including tumor promotion, but the exact mechanisms responsible for these effects are not known. However, most of the efforts investigating genes regulated by AhR have been focused on enzymes involved in xenobiotic metabolism. Though there are indications for involvement of AhR in a number of cellular processes (Carlson and Perdew, 2002), there is no well-described role for the AhR in normal cellular function or tumor promotion that has been delineated at the gene expression level. In this report we have examined the ability of the AhR to regulate expression of the potent mitogen epiregulin.
Skin and liver have been observed to be two of the most common sites of tumor development in TCDD tumor promotion studies. AhR-dependent upregulation of genes involved in growth promotion could be an important mechanism for TCDD mediated carcinogenesis. One of the functional classes of genes that could mediate these effects would be growth factors. The ability of TCDD to increase epiregulin mRNA in cell types known to exhibit TCDD-mediated toxicity, as revealed by the microarrays and real time qPCR experiments in hepatocytes and keratinocytes, support the hypothesis that TCDD exposure could stimulate the growth potential of precancerous cells through the enhanced expression of potent mitogens like epiregulin. Further, results from the ChIP assay and luciferase reporter assay, reveal that an increase in epiregulin mRNA levels is brought about by ligand-activated AhR binding to the DRE in the epiregulin promoter and thus, epiregulin is directly regulated by the AhR in response to TCDD. Earlier studies have demonstrated that two of the three Sp1 binding elements, in the epiregulin promoter, are actively involved in regulating epiregulin mRNA levels. A cooperative interaction between AhR-ARNT and Sp1 in the CYP1A1 gene promoter has been reported previously (Kobayashi et al., 1996). According to their results, binding of either AhR-ARNT or Sp1 to its element in the CYP1A1 promoter facilitated binding of the other factor and increased reporter activity in a synergistic fashion. The DRE and GC/CT boxes are found in close proximity in a number of other AhR regulated gene promoters like UDP-glucuronosyl transferase, aldehyde dehydrogenase-3, and quinine reductase. An interaction between the AhR/ARNT complex and Sp1 has been shown to exist even in the absence of exogenous ligand, for the Cathepsin D gene promoter (Shelly et al., 1998). Since the epiregulin promoter has both the AhR and Sp1 binding elements, it was interesting to determine whether this kind of cooperative interaction existed at the epiregulin promoter. Notably, as revealed in the reporter assays, even when all three Sp1 binding sites were mutated within the epiregulin promoter (pmutABC transfections), the fold-increase in luciferase activity on TCDD induction was similar to that observed in the wild-type promoter, 2.2- and 2.3-fold respectively. However, the overall amount of luciferase signal decreased by approximately two-thirds in pmutABC transfections. This shows that Sp1 is not necessary for AhR-mediated induction of epiregulin gene expression, but enhances the overall transcription rate of the epiregulin promoter. After demonstrating that TCDD-activated AhR increases epiregulin transcription, the next logical step would be to show a corresponding increase in epiregulin secreted in conditioned media. However, attempts to detect epiregulin using Western blot were unsuccessful (data not shown).
Increased epiregulin mRNA expression has been observed in pancreatic cancer tissue samples (Zhu et al., 2000) as well as in prostate and pancreatic cell lines (Torring et al., 2000; Zhu et al., 2000). It has been proposed that over-expression of epiregulin is an important factor in growth regulation of malignant fibrous histiocytoma (Yamamoto et al., 2004), colon cancer cells (Baba et al., 2000), and various gynecological malignancies (Freimann et al., 2004). These data, along with the fact that ligands and receptors of the EGF-family have been implicated in a number of tumors, suggest that epiregulin might be an important factor in mediating TCDD dependent skin tumor promotion in mice. To show that modulation of epiregulin levels has a functionally significant role in primary mouse keratinocytes, cells were treated with increasing concentrations of epiregulin. Epiregulin treatment caused an increase in proliferation of primary mouse keratinocytes as observed at three time points (24, 72, and 120 h), even at a concentration as low as 1 ng/ml. It is worth noting that this increase occurred in the presence of 8% fetal bovine serum. Different concentrations of TCDD also increased cell proliferation, albeit at a lower rate than epiregulin. Statistical analysis revealed this effect of TCDD to be significant, even though apparently modest. Although it is not possible to rule out the contribution of other growth factors, it is possible that this effect is due, at least in part, to an increase in epiregulin level.
Results from these studies also suggest that caution should be taken when extrapolating results across different species. The International Agency for Research on Cancer (IARC) updated its classification of TCDD as a group 1 carcinogen in 1997 from its previous evaluation that classified it as a group 2B (possible) human carcinogen (IARC, 1997). This upgrade has been debated in the literature (Cole et al., 2003; Steenland et al., 2004). However, it is clear from these discussions that not all data obtained from animal studies can be applied directly to humans. The toxic effects of TCDD appear to be less pronounced in humans than in rodents. A number of factors could be responsible for this, including differences in AhR, its cytosolic or nuclear binding partners, its affinity for ligands, endogenous ligands, transcriptional coregulators or the battery of genes whose transcription is modulated by AhR. If a gene involved in a cellular process is regulated differently in humans and mice, it could be an important reason for the possible differences in response to TCDD. We compared the first 125 bases of epiregulin promoter sequence from human, mouse, and rat and found that the DRE is absent in humans (Fig. 5). Thus, TCDD-activated AhR may not regulate expression of epiregulin in humans. However, further studies in human tissue samples or cell lines will be needed to clarify this issue. In conclusion, we have shown for the first time that epiregulin is a direct target gene for the AhR pathway in rodents. Considering the mitogenic potential of epiregulin and the possibility that it might not be regulated through the AhR pathway in humans, these results point to a potential inter-species difference in regulation of a gene that could participate in TCDD-mediated tumor promotion.
ACKNOWLEDGMENTS
We thank Dr. Kaoru Miyamoto at the Fukui Medical University, Japan, for kindly providing pEpi125 and pmutABC plasmids. We also thank Marcia Perdew and Dr. Iain Murray for editorial review of the manuscript. This study was funded by NIH grants ES04869 and ES011834.
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ABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause a large number of adverse effects, mediated largely by its binding to the aryl-hydrocarbon receptor (AhR) and subsequent modulation of gene expression. It is thought that AhR mediates these effects through the untimely and disproportionate expression of specific genes. However, the exact mechanism, or the genes involved, through which TCDD leads to these effects is still unknown. This study reports the discovery of a novel target gene, epiregulin, which is regulated by TCDD-activated AhR. Epiregulin is a growth regulator which belongs to the epidermal growth factor (EGF) family. Using real time quantitative PCR (qPCR), it was established that TCDD upregulates epiregulin gene expression. The promoter region of epiregulin has a dioxin responsive element (DRE) 56 nucleotides upstream of the transcription start site, along with three potential Sp1 binding sites. Chromatin immunoprecipitation (ChIP) assays with an anti-AhR antibody showed promoter occupancy upon TCDD treatment. Luciferase reporter assays using a vector harboring the first 125 base pairs of the epiregulin rat promoter revealed an increase in signal on TCDD treatment, which was lost upon mutation of the DRE. Epiregulin and TCDD treatment mediated a dose-dependent increase in primary mouse keratinocyte growth. These results demonstrate that AhR directly increases epiregulin expression, which could play an important role in TCDD mediated tumor promotion observed in rodent models.
Key Words: AhR; epiregulin; transcription; growth factor; TCDD.
INTRODUCTION
The aryl hydrocarbon receptor (AhR) is a member of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) family of transcription factors. AhR exists in the cytoplasm complexed with the chaperone HSP90 together with co-chaperones X-associated protein 2 (also referred to as AIP or ARA9) and p23 (reviewed in Petrulis and Perdew, 2002). The AhR translocates to the nucleus after ligand binding and heterodimerizes with AhR nuclear translocator (ARNT). AhR mediates its effects in a fashion similar to nuclear hormone receptors. The AhR/ARNT heterodimer binds to dioxin response elements (DRE) and modulates transcription of target genes. The consensus core binding site for the AhR-ARNT complex is TNGCGTG. ARNT binds the GTG half-site while AhR binds the other half. The majority of the genes known to be regulated by AhR are involved in xenobiotic metabolism; the well-characterized genes include CYP1A1, CYP1A2, CYP1B1, glutathione S-transferase Ya, and aldehyde dehydrogenase 3A1 (reviewed in Nebert et al., 1993, 2000).
Aside from its role in xenobiotic metabolism, there is an increasing body of evidence implicating AhR in a diverse range of patho-physiological processes involving the immune system, liver, and the cardiovascular system (Heid et al., 2001) as well as in cell cycle control (Elizondo et al., 2000). AhR activation in response to xenobiotic exposure leads to a variety of adverse effects, including tumor promotion. Studies using mice deficient in AhR have provided clues to its role in growth and development. AhR null mice exhibit increased hepatic fibrosis, decreased liver weight, abnormalities of the immune system in the form of a reduced number of peripheral lymphocytes (Fernandez-Salguero et al., 1995; Schmidt et al., 1996), decreased fertility, vascular abnormalities in heart, liver, and uterus, delayed wound healing, impaired skin homeostasis, and an overall slower growth (Fernandez-Salguero et al., 1997). In addition, studies using null mice have also shown that AhR might have a role in the formation of primordial follicles and in regulating the number of antral follicles, thus affecting development of the mouse ovary (Benedict et al., 2000). Also, the incidence of cardiac hypertrophy in AhR null mice suggests a role for AhR in normal cardiovascular development and angiogenesis (Lund et al., 2003). However, it has not been clearly defined how AhR mediates these functions. Differential regulation of genes, other than those involved in xenobiotic metabolism, could possibly account for these AhR mediated effects. Alternate mechanisms have also been suggested in the recent years to explain the different functions of AhR. For example, it has been shown to interact with retinoblastoma (Rb) (Ge and Elferink, 1998) and nuclear factor -B (Koo and Kim, 2003) transcription factors and modulate cell proliferation. Co-expression of AhR and BRG-1 restored Rb-sensitivity to a tumor cell line, C33A, which could otherwise progress through the cell cycle even in the presence of active Rb protein (Strobeck et al., 2000).
A number of exogenous and endogenous ligands are capable of activating the AhR (Denison et al., 2002). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to be the most potent exogenous AhR ligand. While there is evidence suggesting that TCDD exerts almost all its toxic effects through the AhR, the exact mechanisms underlying these effects are not clear. TCDD has been shown to be more effective as a tumor promoter than as a tumor initiator, due to the fact that TCDD is essentially not metabolized (reviewed in Dunson et al., 2000). TCDD has been shown to promote skin tumor formation in hairless mice (Poland et al., 1982) as well as promote liver tumor formation (reviewed in Dragan and Schrenk, 2000). The importance of AhR in tumor promotion is also highlighted by the results obtained in a recent study using transgenic mice expressing a constitutively active AhR where a significant increase in hepatocarcinogenesis was noted compared to wild-type mice (Moennikes et al., 2004). In addition, studies using constitutively active AhR have demonstrated that the receptor can induce tumors in the stomach (Andersson et al., 2002). The fact that benzo[a]pyrene, a known AhR ligand, is unable to exhibit its carcinogenic effects in the skin of AhR null mice, provides further evidence supporting a role for AhR in tumorigenesis (Shimizu et al., 2000).
Epidermal growth factor (EGF) and other members of its family are potent peptide growth factors and are involved in a plethora of physiological and pathological processes through their signaling properties (Harris et al., 2003). Mutations and/or over-expression of EGF-family members cause cells to acquire an oncogenic phenotype. Epiregulin is a relatively newly identified member of the EGF family, isolated from the conditioned medium of NIH/3T3/clone 7 cells (Toyoda et al., 1995). It is secreted as a 46-amino acid single chain polypeptide with contrasting growth regulatory properties, inhibiting the growth of several epithelial cell lines while promoting growth of other cell types, like vascular smooth muscle cells (Koo and Kim, 2003), hepatocytes (Komurasaki et al., 2002; Toyoda et al., 1995), and keratinocytes (Shirakata et al., 2000). Epiregulin is expressed during development as well as later on in the genitourinary tract (Sekiguchi et al., 2002, 2004), gastrointestinal tract, vascular smooth muscle cells, and skin. In the latter two tissues, it has been shown to function in a paracrine as well as autocrine fashion (Takahashi et al., 2003).
This study demonstrates, for the first time, that ligand activated AhR binds to its cognate element in the epiregulin promoter and mutation of this binding element results in loss of AhR-driven reporter activity. AhR increases epiregulin transcription in cultured primary mouse keratinocytes, immortalized hepatocytes, and mouse hepatoma derived cell line. It is also shown that epiregulin is capable of significantly enhancing the proliferation of cultured primary mouse keratinocytes. This could possibly account for one of the mechanisms by which TCDD exerts its tumor promotional effects in rodent skin.
MATERIALS AND METHODS
Cell culture.
Primary mouse keratinocytes were obtained from two-day old neonatal C57BL/6 mice and cultured using a previously described method (Dlugosz et al., 1995). NIH/3T3 cells and Hepa1c1c7 were grown in -minimal essential medium (-MEM) supplemented with 10% FBS (HyClone Laboratories, Logan, UT), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin (Sigma) at 37°C in 5% CO2 atmosphere. Hepatocytes from two-week old C57BL/6N mice were infected with SV40 virus to prepare temperature sensitive SV40 immortalized mouse hepatocytes in our laboratory (Murray et al., in press) using a previously described protocol with modifications (Chou, 1985; Lorenzen et al., 1997). Three cell lines of immortalized hepatocytes were established. Cells from line 2 were used for experiments. These cells were maintained in 4% FBS and 0.1 μM dexamethasone at 34°C.
Real time quantitative PCR (qPCR).
Real time qPCR was performed on the DNA Engine Opticon (MJ Research, Inc.) using DyNAmo Hot Start SYBR Green qPCR kit purchased from MJ Research, Inc. cDNA synthesis was carried out using High Capacity cDNA Archive Kit from Applied Biosystems. The reverse transcription reactions were set up according to the manufacturer's instructions. cDNA synthesized from 50 ng of total RNA was used per qPCR reaction. Epiregulin mRNA was detected using 5'-TGGGTCTTGACGCTGCTTTGTCTA-3' and 5'-AAGCAGTAGCCGTCCATGTCAGAA-3' primers. Epiregulin promoter in ChIP assays was detected using 5'-TTCCTGAGAGGGAGGATGACAT-3' and 5'-CCCACCAAGTCGCTGTGACT-3' primers. Thermal cycling conditions were setup according to the manufacturer's protocol.
Plasmids.
The following plasmids were used for reporter assays: pEpi125 – derived by inserting rat epiregulin promoter region –125/+12 (genomic contig: NW_047424.1) in pGL3-Basic luciferase vector, pmutABC – pEpi125 with all three GT and CT boxes mutated (Sato et al., 2003), pmutARNT – pEpi125 with ARNT half-site of DRE mutated and pmutAhR – pEpi125 with AhR half-site of DRE mutated. pEpi125 and pmutABC were a kind gift of Dr. Kaoru Miyamoto (Fukui Medical University, Japan). pmutARNT and pmutAhR were modified forms of pEpi125, generated by site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene). Forward and reverse primer pairs used to mutate the ARNT and AhR half-sites, respectively were (5'-GTAAGTCCTCGCTGGCCTAAGCACC-3' and 5'-GGTGCTTAGGCCAGCGAGGACTTAC-3') and (5'-GTAAGTCCTCGAGTGCCTAAGCACC-3' and 5'-GGTGCTTAGGCACTCGAGGACTTAC-3').
Transient transfections and luciferase reporter assays.
These experiments were carried out using NIH/3T3 cells, obtained from the American Type Culture Collection (ATCC). Transfections were performed using Lipofectamine Plus (Invitrogen). Cells at 70% confluency in 6-well plates were washed with PBS and 1.5 ml Optimem (Life Technologies) containing 5 μl of Lipofectamine, 1 μl of Plus, and 1.5 μg DNA were added per well. The DNA was comprised of 500 ng of a luciferase reporter vector harboring wild-type or mutated versions of the rat epiregulin promoter, 100 ng of pDJM/-gal to control for transfection efficiency and empty expression vector. After 4 h cells were washed with PBS and -MEM containing 10% FBS and antibiotics was added. Cells were allowed to grow overnight and treated with 10 nM TCDD or DMSO the next day for 7 h and lysed with 1X cell lysis buffer (25 mM Tris buffer pH 7.8, 2 mM DTT, 2 mM EDTA, 10% glycerol, and 1% Triton X-100). Luciferase activity from cell lysates was measured using a Turner TD-20e luminometer (Turrner Designs, Sunnyvale, CA) the and values were normalized to -gal values.
Chromatin Immunoprecipitation assay and PCR.
Chromatin Immunoprecipitation (ChIP) assays were performed as previously described (Spencer et al., 2003) with some modifications. Briefly, 80% confluent cells were treated with 10 nM TCDD or DMSO for 100 min and crosslinked for 8 min at room temperature with 0.33% formaldehyde (SIGMA, F-8775). Cells were harvested and lysed with 750 μl/flask lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). Lysates were sonicated with Branson Sonifier 250 using 40% duty cycle and output set at 4 for 8 cycles of 12 pulses each. Protein-DNA complexes were immunoprecipitated from 200 μl lysate using either 6 μl of rabbit polyclonal anti-AhR antibody (Biomol, SA-210), control IgG or no antibody and 50 μl Goat Anti-Rabbit IgG-Agarose resin (SIGMA, A1027). Resin was washed once each with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8 and 150 mM NaCl), 1X RIPA buffer (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 0.5 mM EGTA, 140 mM NaCl, 10 mM Tris-HCl pH 8) and 1xMENG buffer (25 mM MOPS, 2 mM EDTA, 0.02% sodium azide, 10% glycerol pH 7.5) in that order. Immunoprecipitated protein-DNA complexes were incubated in 300 μl digesting buffer (50 mM Tris-HCl pH 8, 1 mM EDTA, 100 mM NaCl, 0.5% SDS, 100 μg/ml Proteinase K). DNA was isolated by phenol-chloroform extraction and concentrated using ethanol precipitation. PCR was performed using following primers: Epiregulin (5'-TTCCTGAGAGGGAGGATGACAT-3' and 5'-CCCACCAAGTCGCTGTGACT-3'; located 107 bases upstream and 75 bases downstream relative to TATA box), Cyp1A1 (5'-GCCGAGCATCGCACGCAAACC-3' and 5'-GGATCCACGCGAGACAGCAGG-3'; located 1168 and 784 bases upstream relative to TATA box) and GAPDH (5'-CATGGCCTTCCGTGTTCCTA-3' and 5'-GCGGCACGTCAGATCCA-3'). Ten percent DMSO was added to the reactions.
Keratinocyte proliferation studies.
Equivalent number of keratinocytes were seeded in 12 well plates and cultured in low calcium medium (0.05 mM) containing 8% chelexed FBS for 24 h before treatment. To test the effect of epiregulin or TCDD on keratinocyte growth, cells were cultured in low calcium medium containing epiregulin (R&D Systems, 1068-EP), at a concentration from 1– 20 ng/ml, or TCDD at a concentration from 0.1– 1.0 nM, continuously with medium change after 24 and 72 h. After 24, 72, or 120 h incubation, cell number was measured using a Z1 coulter particle counter (Beckman Counter, Inc., Hialeah, FL).
RESULTS
TCDD Treatment Upregulates Epiregulin Transcription
The principal mode of action of AhR is through binding its cognate cis-regulatory element, the DRE, in the promoter region of its target genes. AhR heterodimerizes with ARNT and then recruits coregulators to enhance transcription (reviewed in Swanson et al., 2002). Despite the fact that AhR is implicated in a diverse range of physiological processes, as discussed in the introduction, few genes outside the gamut of xenobiotic metabolism have been reported to be directly regulated by the AhR pathway. In an attempt to identify novel target genes, whose expression is under the regulation of AhR, microarray experiments (unpublished data) were performed using SV-40 immortalized mouse hepatocytes. These experiments revealed epiregulin as one of the target genes whose mRNA levels increased on TCDD treatment. Microarray results were confirmed with real-time qPCR (Fig. 1). Effect of TCDD on epiregulin mRNA levels was also tested in Hepa1c1c7, a mouse hepatoma cell line. Epiregulin was upregulated by 2.6- fold, 90 min after TCDD exposure. As TCDD exerts significant adverse effects on skin, including tumor promotion, upregulation of epiregulin in response to TCDD was assessed in primary keratinocytes. Primary mouse keratinocytes were treated with 10 nM TCDD for 90 min and a change in the level of epiregulin mRNA was compared by real time qPCR. TCDD increased epiregulin mRNA expression by 3.3-fold (Fig. 1) in primary mouse keratinocytes also. Thus, AhR activation resulted in epiregulin upregulation in primary cells as well as immortalized cell lines.
AhR Binds the DRE in the Epiregulin Promoter
In accordance with the currently accepted theory, AhR must bind its response element in the promoter of a gene to directly regulate its expression. A search for binding sites for AhR-ARNT heterodimer by sequence analysis identified a consensus DRE (TCGCGTG) 56 nucleotides upstream of the transcription start site in the epiregulin promoter in mouse and rat genomic DNA. To assess if AhR binds the DRE in the epiregulin promoter sequence, ChIP assays were performed with SV40 virus immortalized mouse hepatocytes treated with TCDD. DNA fragments isolated from ChIP assays were analyzed using PCR (Fig. 2A). CYP1A1 was used as a positive control. GAPDH and immunoprecipitations with no antibody or with control IgG were used as negative controls. CYP1A1 showed a strong signal in the TCDD treated sample, compared to a carrier solvent treated sample, while there was no difference in the case of GAPDH (data not shown). Immunoprecipitations with control IgG or no antibody showed minimal background signals. While the epiregulin signal was more intense in TCDD samples compared to carrier solvent treated samples, the difference was less than that of CYP1A1. This difference could be explained by the fact that epiregulin has only one DRE in its promoter while the CYP1A1 promoter has multiple DREs, and that AhR upregulates CYP1A1 expression by a greater magnitude than epiregulin. These results were confirmed by realtime qPCR (Fig. 2B) performed on DNA isolated from ChIP assays. The relative signal for epiregulin in TCDD treated samples was 1.7-fold greater than carrier solvent treated samples. Combined, these results demonstrate an increase in epiregulin promoter occupancy by AhR in response to TCDD treatment.
AhR Driven Promoter Activity Is Dependent on the DRE
To further confirm the role of the DRE in modulating epiregulin gene expression, a luciferase reporter assay was performed using a construct containing the first 125 bases of the rat epiregulin promoter cloned into pGL3-Basic vector (pEpi125). There is a single nucleotide difference between the first 125 bases of the mouse (genomic contig: NT_039308.3) and the rat (genomic contig: NW_047424.1) epiregulin promoters, which is not a part of the DRE or the Sp1 binding sites. This construct has been previously used by Sekiguchi et al. to study transcriptional regulation of the epiregulin gene in the rat ovary (Sekiguchi et al., 2002). They showed by deletion and mutation analyses that the region encompassing 125 bp upstream of the transcription start site was essential for controlling transcription of the epiregulin gene. According to their results, Sp1/Sp3 proteins bind to one or more of the two CT boxes and one GT box within this 125 bp region and are involved in regulating epiregulin gene expression.
The relative contributions of the AhR and Sp1/Sp3 were studied using plasmids derived from pEpi125 by mutating their binding sites, the DRE and the CT and GT boxes, respectively (Fig. 3A). Luciferase reporter plasmid pEpi125 was transiently transfected in NIH/3T3 cells, while pDJM/-gal vector was cotransfected to control for transfection efficiency. Reporter assays were also carried out with pmutABC plasmid, derived from pEpi125 by mutating both the CT boxes as well as the GT box, pmutARNT plasmid, derived from pEpi125 by mutating the ARNT half-site of DRE and pmutAhR plasmid, derived from pEpi125 by mutating the AhR half-site of DRE (Fig. 3C). There was a two-fold increase (2.3-fold) in luciferase reporter activity observed upon TCDD treatment (Fig. 3B) with the pEpi125 transfections. Surprisingly, a similar increase in luciferase activity (2.2-fold) was observed with the pmutABC transfections compared to vehicle treated control samples. There was no increase in luciferase activity on TCDD treatment when the AhR and ARNT binding half-sites were mutated in pmutARNT and pmutAhR, respectively (1.2-fold in pmutARNT and pmutAhR each). Interestingly, the reporter gene activity in pmutABC transfections was 3.2 times lower compared to pEpi125 transfections, both in TCDD and carrier solvent treated samples. This decrease in reporter activity was not observed when the DRE was mutated (pmutAhR and pmutARNT transfections). These results, along with ChIP assays, demonstrate that the activated AhR binds to the DRE within the epiregulin promoter and is responsible for the observed increase in transcriptional activity.
Effect of Epiregulin and TCDD on Mouse Keratinocyte Proliferation
To characterize the functional role of increased epiregulin expression on cell growth, the effect of epiregulin treatment was determined using primary mouse keratinocytes. It has previously been shown that epiregulin stimulates proliferation of cultured human keratinocytes in a dose-dependent manner and acts as an autocrine growth factor (Shirakata et al., 2000). Recombinant human epiregulin at a concentration of 1 ng/ml resulted in a three-fold increase in cell growth. In the present study, primary mouse keratinocytes showed a dose-dependent increase in cell number when treated with recombinant mouse epiregulin, even in the presence of other growth factors found in fetal bovine serum (Fig. 4A). Epiregulin stimulated a statistically significant proliferation of primary mouse keratinocytes across a dose range of 1–20 ng/ml at 24, 72, and 120 h of treatment, as determined by ANOVA (p < 0.05). Significant increase in proliferation was observed at all three time points with the higher doses (10 and 20 ng/ml), whereas 1 ng/ml epiregulin induced proliferation only at 72 and 120 h.
Similar cell proliferation studies were performed using TCDD as a stimulant (Fig. 4B). Primary mouse keratinocytes were treated with 0.1 nM, 0.5 nM, and 1 nM TCDD, or vehicle solvent, and cells counted at 24, 72, and 120 h after addition of TCDD. TCDD-induced cell proliferation was statistically significant, as determined by ANOVA (p < 0.05). Individually, all three doses induced statistically significant increase in cell proliferation when compared to vehicle treated cells. However, a statistically significant difference was not observed amongst the different doses, as determined by Tukey's HSD test. When the effects of different doses were compared at individual time-points, an increase in the number of cells was observed at all three time-points with 0.1 nM and 0.5 nM TCDD treatments. Primary mouse keratinocytes treated with 1 nM TCDD showed a slight reduction in cell numbers at the 120 h time point. This could be due to increasing toxicity owing to prolonged TCDD exposure at this latest time point.
DISCUSSION
TCDD causes a wide range of toxic effects including tumor promotion, but the exact mechanisms responsible for these effects are not known. However, most of the efforts investigating genes regulated by AhR have been focused on enzymes involved in xenobiotic metabolism. Though there are indications for involvement of AhR in a number of cellular processes (Carlson and Perdew, 2002), there is no well-described role for the AhR in normal cellular function or tumor promotion that has been delineated at the gene expression level. In this report we have examined the ability of the AhR to regulate expression of the potent mitogen epiregulin.
Skin and liver have been observed to be two of the most common sites of tumor development in TCDD tumor promotion studies. AhR-dependent upregulation of genes involved in growth promotion could be an important mechanism for TCDD mediated carcinogenesis. One of the functional classes of genes that could mediate these effects would be growth factors. The ability of TCDD to increase epiregulin mRNA in cell types known to exhibit TCDD-mediated toxicity, as revealed by the microarrays and real time qPCR experiments in hepatocytes and keratinocytes, support the hypothesis that TCDD exposure could stimulate the growth potential of precancerous cells through the enhanced expression of potent mitogens like epiregulin. Further, results from the ChIP assay and luciferase reporter assay, reveal that an increase in epiregulin mRNA levels is brought about by ligand-activated AhR binding to the DRE in the epiregulin promoter and thus, epiregulin is directly regulated by the AhR in response to TCDD. Earlier studies have demonstrated that two of the three Sp1 binding elements, in the epiregulin promoter, are actively involved in regulating epiregulin mRNA levels. A cooperative interaction between AhR-ARNT and Sp1 in the CYP1A1 gene promoter has been reported previously (Kobayashi et al., 1996). According to their results, binding of either AhR-ARNT or Sp1 to its element in the CYP1A1 promoter facilitated binding of the other factor and increased reporter activity in a synergistic fashion. The DRE and GC/CT boxes are found in close proximity in a number of other AhR regulated gene promoters like UDP-glucuronosyl transferase, aldehyde dehydrogenase-3, and quinine reductase. An interaction between the AhR/ARNT complex and Sp1 has been shown to exist even in the absence of exogenous ligand, for the Cathepsin D gene promoter (Shelly et al., 1998). Since the epiregulin promoter has both the AhR and Sp1 binding elements, it was interesting to determine whether this kind of cooperative interaction existed at the epiregulin promoter. Notably, as revealed in the reporter assays, even when all three Sp1 binding sites were mutated within the epiregulin promoter (pmutABC transfections), the fold-increase in luciferase activity on TCDD induction was similar to that observed in the wild-type promoter, 2.2- and 2.3-fold respectively. However, the overall amount of luciferase signal decreased by approximately two-thirds in pmutABC transfections. This shows that Sp1 is not necessary for AhR-mediated induction of epiregulin gene expression, but enhances the overall transcription rate of the epiregulin promoter. After demonstrating that TCDD-activated AhR increases epiregulin transcription, the next logical step would be to show a corresponding increase in epiregulin secreted in conditioned media. However, attempts to detect epiregulin using Western blot were unsuccessful (data not shown).
Increased epiregulin mRNA expression has been observed in pancreatic cancer tissue samples (Zhu et al., 2000) as well as in prostate and pancreatic cell lines (Torring et al., 2000; Zhu et al., 2000). It has been proposed that over-expression of epiregulin is an important factor in growth regulation of malignant fibrous histiocytoma (Yamamoto et al., 2004), colon cancer cells (Baba et al., 2000), and various gynecological malignancies (Freimann et al., 2004). These data, along with the fact that ligands and receptors of the EGF-family have been implicated in a number of tumors, suggest that epiregulin might be an important factor in mediating TCDD dependent skin tumor promotion in mice. To show that modulation of epiregulin levels has a functionally significant role in primary mouse keratinocytes, cells were treated with increasing concentrations of epiregulin. Epiregulin treatment caused an increase in proliferation of primary mouse keratinocytes as observed at three time points (24, 72, and 120 h), even at a concentration as low as 1 ng/ml. It is worth noting that this increase occurred in the presence of 8% fetal bovine serum. Different concentrations of TCDD also increased cell proliferation, albeit at a lower rate than epiregulin. Statistical analysis revealed this effect of TCDD to be significant, even though apparently modest. Although it is not possible to rule out the contribution of other growth factors, it is possible that this effect is due, at least in part, to an increase in epiregulin level.
Results from these studies also suggest that caution should be taken when extrapolating results across different species. The International Agency for Research on Cancer (IARC) updated its classification of TCDD as a group 1 carcinogen in 1997 from its previous evaluation that classified it as a group 2B (possible) human carcinogen (IARC, 1997). This upgrade has been debated in the literature (Cole et al., 2003; Steenland et al., 2004). However, it is clear from these discussions that not all data obtained from animal studies can be applied directly to humans. The toxic effects of TCDD appear to be less pronounced in humans than in rodents. A number of factors could be responsible for this, including differences in AhR, its cytosolic or nuclear binding partners, its affinity for ligands, endogenous ligands, transcriptional coregulators or the battery of genes whose transcription is modulated by AhR. If a gene involved in a cellular process is regulated differently in humans and mice, it could be an important reason for the possible differences in response to TCDD. We compared the first 125 bases of epiregulin promoter sequence from human, mouse, and rat and found that the DRE is absent in humans (Fig. 5). Thus, TCDD-activated AhR may not regulate expression of epiregulin in humans. However, further studies in human tissue samples or cell lines will be needed to clarify this issue. In conclusion, we have shown for the first time that epiregulin is a direct target gene for the AhR pathway in rodents. Considering the mitogenic potential of epiregulin and the possibility that it might not be regulated through the AhR pathway in humans, these results point to a potential inter-species difference in regulation of a gene that could participate in TCDD-mediated tumor promotion.
ACKNOWLEDGMENTS
We thank Dr. Kaoru Miyamoto at the Fukui Medical University, Japan, for kindly providing pEpi125 and pmutABC plasmids. We also thank Marcia Perdew and Dr. Iain Murray for editorial review of the manuscript. This study was funded by NIH grants ES04869 and ES011834.
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