ToxSci Advance Access originally published online on August 23, 2006
Toxicological Sciences 2006 94(1):163-174; doi:10.1093/toxsci/kfl090
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In Utero Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Induces Amphiregulin Gene Expression in the Developing Mouse Ureter
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* Department of Pharmacology, University of Toronto, Toronto, Ontario M5S 1A8, Canada; and
Program in Developmental Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada
1To whom correspondence should be addressed at Program in Developmental Biology, Hospital for Sick Children, Room 14-306 TMDT—14th floor East Tower 101 College Street, Toronto, ON M5G 1L7, Canada. Fax: (416) 813-5252. E-mail: pharper{at}sickkids.ca.
Received July 21, 2006; accepted August 17, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure to the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), produces hydronephrosis in developing mice, the etiology of which involves hyperplasia within the ureteric luminal epithelium. Dysregulation of epidermal growth factor receptor (EGFR), EGF, and transforming growth factor-
expression has been implicated as playing a role in TCDD-induced hydronephrosis. In this study, changes in the expression of genes encoding the EGFR and its cognate ligands in response to TCDD were evaluated within the developing ureter. C57BL/6 dams were injected ip with 30 µg/kg TCDD on gestational day (GD) 13 or 16 and fetal tissues removed on GD 17. Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator messenger RNA (mRNA) were expressed in control and treated fetal tissues at GD 14 and 17. Prototypical AHR target genes, Cyp1a1, Cyp1a2, and Cyp1b1 were upregulated in TCDD-exposed fetal tissues, demonstrating AHR transcriptional activity at these developmental stages. Amphiregulin (AREG) and epiregulin, ligands for the EGFR, were induced at the transcriptional level in ureters of fetuses exposed to TCDD for 24 h. AREG mRNA was also induced by TCDD dose- and time-dependently in the mouse hepatoma cell line Hepa-1c1c7 (Hepa-1), mimicking the induction patterns of CYP1A1 mRNA. Other AHR ligands also induced AREG mRNA in Hepa-1 cells. Furthermore, variant Hepa-1 cells (TAOBPrc1 cells) virtually deficient in the AHR failed to display an increase in AREG mRNA in response to TCDD. Taken together, these data suggest that the AHR cross talks with the EGFR signaling pathway by directly inducing the expression of growth factors that are important for EGFR signaling in the developing mouse ureter. Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; aryl hydrocarbon receptor; hydronephrosis; epidermal growth factor receptor; amphiregulin.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure to halogenated aromatic hydrocarbons (HAHs) and structurally related polycyclic aromatic hydrocarbons (PAHs) leads to numerous adverse effects in many species (Birnbaum and Tuomisto, 2000
; Grassman et al., 1998). These compounds exert their toxic effects by binding to the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor. Once activated by ligand, the AHR translocates into the nucleus and heterodimerizes with the aryl hydrocarbon nuclear translocator (ARNT), generating a complex that binds target genes at dioxin responsive elements, initiating gene transcription (Denison et al., 2002; Swanson, 2002). The AHR has best been characterized as playing a vital role in the activation and detoxification of xenobiotics by regulating the expression of the enzymes responsible for their metabolism. The AHR also plays a role in development as evident in models where AHR signaling is disrupted. For example, abolishing the expression of the AHR in mice results in decreased body weight, poor fecundity, and defects in liver, cardiac, and vascular development (Lin et al., 2001; Lund et al., 2003; Peters et al., 1999; Walisser et al., 2004). Development is also affected in mice harboring the wild type AHR when they are exposed to potent AHR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In mice, TCDD produces several teratogenic effects, such as thymic involution, cleft palate, hydronephrosis, and aberrant cardiac, and prostatic bud development (Couture et al., 1990; Ko et al., 2002; Thackaberry et al., 2005). Studies using TCDD-sensitive versus TCDD-resistant strains (Hassoun and Stohs, 1996; Silkworth et al., 1989) and AHR-null mice (Peters et al., 1999) demonstrate that these TCDD-induced teratogenic responses require the AHR.
Hydronephrosis is a sensitive teratogenic response to TCDD in mice (Couture et al., 1990) resulting from either in utero or lactational exposure (Couture-Haws et al., 1991). Hydronephrosis induced by TCDD is characterized by hyperplasia of the developing ureteric luminal epithelium with resultant thickening of the epithelium (Abbott and Birnbaum, 1990a; Abbott et al., 1987). Consequently, necrotic debris is found within the lumen, which narrows and occludes the ureter, restricting the flow of urine. The result is hydroureter and hydronephrosis, the severity of which increases with time.
Disruption of epidermal growth factor receptor (EGFR) signaling has been suggested as a mechanism by which TCDD induces hydronephrosis (Abbott and Birnbaum, 1990a; Abbott et al., 2003a; Bryant et al., 1997, 2001b). During development, EGFR signaling plays a vital role in the regulation of cell proliferation and differentiation (Miettinen et al., 1995). Disrupting the expression of the EGFR or its ligands, EGF, and transforming growth factor- (TGF-), has previously been demonstrated to alter TCDD effects on developing mouse embryonic palates (Abbott and Birnbaum, 1989, 1990b; Abbott et al., 2003a), teeth (Partanen et al., 1998), and prostatic bud (Abbott et al., 2003b). The EGFR, EGF, and TGF- also appear to influence the teratogenic effect of TCDD on the developing kidney but their role in hydronephrosis is not well defined. Mice lacking the EGFR still develop TCDD-induced hydronephrosis demonstrating that EGFR itself is not required for the development of hydronephrosis (Miettinen et al., 2004). In this case, it is possible that other members of the EGFR family, such as ErbB4, which also binds certain EGFR ligands, and ErbB2, which serves as a dimerization partner (Olayioye et al., 2000), could compensate for the lack of EGFR. Furthermore, Bryant et al. (2001b) reported that there is no difference in the incidence of TCDD-induced hydronephrosis between mice lacking both EGF and TGF- compared to wild type mice. But when either EGF or TGF- is absent, the incidence and severity of hydronephrosis increase. These observations suggest that neither EGF nor TGF- is essential for TCDD-induced hydronephrosis but that growth factor availability substantially influences the incidence and severity of hydronephrosis. A better understanding of the mechanistic role of the EGFR signaling pathway in hydronephrosis requires a thorough dissection of the effects of TCDD on EGFR signaling within the developing urinary tract.
In the present study, we have established that the AHR signaling pathway is functional in the developing mouse, notably in the fetal kidney and ureter. We also assessed the expression of genes encoding the EGFR and its cognate ligands in fetal kidneys and ureters in response to a maternal dose of TCDD. EGFR ligands include EGF, TGF-, amphiregulin (AREG), betacellulin (BTC), epiregulin (EREG), heparin binding EGF (HB-EGF), and epigen (EPGN). However, only AREG and EREG were upregulated at the transcriptional level in fetal ureters in response to TCDD. Finally, experiments in a cell-based model demonstrate that the induction of AREG messenger RNA (mRNA) by TCDD is AHR dependent.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Corn oil (Mazola brand), dimethyl sulfoxide (DMSO), Benzo[a]pyrene (BaP), and Dibenzo[a,h]anthracene (DBA) were obtained from Sigma-Aldrich, Oakville, ON. TCDD was obtained from Wellington Laboratories, Guelph ON. Tetrachlorodibenzofuran (TCDF) and 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) were kindly provided by Dr Allan B. Okey of the University of Toronto. For cell culture experiments, all chemicals were dissolved in DMSO. For in vivo experiments, TCDD was dissolved in corn oil.
Animals.
A breeding colony of C57BL/6 mice (originally from Jackson Laboratory, Bar Harbor, ME) was housed at the Lab Animal Services at the Hospital for Sick Children (Toronto, ON). Mice were housed in clear plastic cages with litter and bedding under temperature and light (22°C and 12-h light/dark cycle) controlled conditions and given mouse chow and water ad libitum. Care and treatment of the mice were in compliance with the guidelines of the Canadian Council on Animal Care, and the protocol was approved by the Hospital for Sick Children Animal Care Committee. Female mice were housed overnight with male mice and females checked for vaginal plugs the next morning, which was designated as gestational day 0 (GD 0). On GD 13 or 16, dams were injected ip with a single dose of 30 µg/kg of TCDD or corn oil vehicle alone. Dams were sacrificed 24-h posttreatment by cervical dislocation and fetuses removed and decapitated. Maternal and fetal tissues were dissected with the aid of a Zeiss Stemi DV4 light microscope and stored in RNAlater (Ambion, Austin, TX) at – 80°C until processed.
RNA isolation from animal tissues.
To obtain sufficient material for each independent experiment, tissues were pooled from one or more litters or adult mice within the same treatment group to make up one biological replicate. Total RNA was isolated from maternal kidneys and ureters and fetal skin, lungs, heart, liver, bladder, kidneys, and ureters using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Contaminating DNA within RNA samples was digested with DNase I (Amersham Biosciences, Piscataway, NJ).
Cells culture, treatments, and RNA isolation from cells.
The mouse hepatoma cell line, Hepa-1c1c7 (Hepa-1), was kindly provided by Dr David S. Riddick of the University of Toronto, and the TAOBPrc1 cell line was purchased from the American Type Culture Collection (Manassas, VA). Cells were grown as a monolayer in 75-cm2 tissue culture flasks in -minimal essential medium (-MEM) (Wisent, Saint-Jean-Baptiste de Rouville, Quebec, Canada) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and maintained at 37°C in a 5% CO2 incubator. At near confluency, cells were passaged using trypsin digestion and media replaced every 48–72 h. For experiments, cells were seeded at 3 x 105 cells/60-mm tissue culture plate in -MEM + 10% FBS 24 h prior to chemical treatment. Cells were then treated at 
50% confluency with test chemicals in -MEM in the absence of serum. For dose-response studies, Hepa-1 cells were treated with 10–12–10–7M TCDD for 24 h and for time-course studies, Hepa-1 cells were treated with 10–9M TCDD for 3, 6, or 24 h. In experiments testing the effects of other AHR ligands, Hepa-1 cells were treated with 10–9M TCDD, 10–7M PeCDD, 10–6M TCDF, 10–5M BaP, and 3 x 10–5M DBA for 24 h. In experiments using the TAOBPrc1 cell line, Hepa-1 and TAOBPrc1 cells were treated with 10–9M TCDD for 24 h. In all experiments, DMSO served as the vehicle control and was added to the media at a final concentration of 0.1% for all test chemicals except DBA for which the final DMSO concentration used was 0.5%. Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Mississauga, ON, Canada), which included DNA digestion using the RNase-Free DNase Set (Qiagen).
Reverse transcription–PCR.
One microgram of total RNA was reverse-transcribed using Superscript II Reverse Transcriptase (Invitrogen) and target genes amplified by PCR using Platinum Taq DNA Polymerase (Invitrogen) according to the manufacturer's instructions. PCR reactions were performed in a PTC-220 DNA Engine Dyad Cycler or PTC-100 Thermal Cycler (MJ research, Waltham, MA). Oligonucleotides used in PCR amplifications were commercially synthesized (IDT, Coralville, IA). Table 1 lists the sequences of primer pairs along with primer pair-specific PCR conditions and expected product size. PCR parameters included predenaturation of DNA at 94°C for 2 min with subsequent cycling, which included denaturation at 94°C for 20 s, annealing at a primer pair-specific temperature for 20 s, and extension at 72°C for 30 s. A final extension at 72°C for 5 min terminated the PCR reaction. PCR products from in vivo experiments were resolved by electrophoresis on 1.5% agarose gels stained with ethidium bromide and visualized under a UV transilluminator (Diamed, Mississauga, ON, Canada) and digitally recorded using a Gel Documentation System (Diamed). Band intensity was quantified using ImageQuant software (Amersham Biosciences). PCR products from cell culture experiments were radiolabeled during the PCR reaction and quantified as previously described (Li et al., 1998). Briefly, PCR products were radiolabeled during the PCR reaction by incorporation of [-32P]2'-deoxycytidine 5'-triphosphate (2 µCi per reaction) (Perkin Elmer, Boston, MA) in the presence of sufficient unlabeled dCTP. Radiolabeled PCR products were resolved by electrophoresis on a 5% polyacrylamide gel, which was subsequently vacuum dried for 1 h at 80°C and exposed to a Phosphor Screen for 1–2.5 h (Molecular Dynamics, CA). The screen was then scanned using a PhosphorImager (Molecular Dynamics), and band intensity was quantified using ImageQuant software.
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Statistical analyses.
Three biological replicates were performed for all studies in which the data were quantified unless otherwise stated. Local average background correction was employed during densitometry using ImageQuant, and the band intensity of each sample for target genes was normalized to the respective ß-actin signal intensity. Fold-induction (over control corn oil or control DMSO values) was calculated for each sample and data were represented as the mean of three replicates ± the standard deviation (SD). The Student's t-test was applied to determine statistical significance (p < 0.05) between treated and control samples.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AHR Transcriptional Activity in Developing Mice
TCDD-induced hydronephrosis is an AHR-mediated teratogenic response, yet limited information is available on the integrity of AHR signaling during development. To determine the transcriptional activity of the AHR in developing mice, mRNA levels of prototypical AHR target genes were evaluated in tissues of fetuses exposed to a maternal dose of 30 µg/kg of TCDD for 24 h. These genes include those encoding the cytochrome P450s 1A1, 1A2, and 1B1 (CYP1a1, CYP1a2, and CYP1b1). Total RNA was isolated from mouse fetal tissues from two developmental stages, GD 14 and 17, and mRNA levels of target genes detected by reverse transcription–PCR (RT-PCR). Tissues examined include lungs, liver, bladder, heart, skin, kidneys, and ureters. Figure 1A to 1E demonstrates the presence of AHR and ARNT transcripts in all fetal tissues examined at GD 14 and GD 17. As expected, CYP1A1 mRNA was not detected in tissues of control fetuses. However, CYP1A1 mRNA levels were clearly upregulated in tissues of fetuses exposed to TCDD in utero for 24 h. CYP1A2 mRNA was also not detected in tissues of control fetuses but was upregulated in TCDD-exposed livers. Substantial basal expression of CYP1B1 mRNA was detected in bladders and kidneys but little or no CYP1B1 was expressed in all other tissues examined from control fetuses. Exposure to TCDD induced CYP1B1 mRNA in all tissues except the heart. The results obtained for both basal and AHR ligand inducible CYP1A1 and CYP1A2 expression are similar to those seen in adult (Dey et al., 1999
; Kimura et al., 1986) and developing (Dey et al., 1989) mice as previously reported. The constitutive expression of CYP1B1 along with its inducibility by AHR ligands has also been reported in adult mice (Ryu and Hodgson, 1999). The findings from this study indicate the presence of a transcritionally active AHR in several fetal tissues in response to TCDD, notably in the fetal kidney and ureter, which are target organs of TCDD-induced teratogenesis.
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Effects of TCDD on Candidate Genes in Developing Mouse Kidney and Ureter
Since TCDD administration was shown to activate the AHR into a functional transcription factor within the developing kidney and ureter, we evaluated the potential for TCDD to alter the expression of genes that may be involved in hydronephrosis. We investigated changes in gene transcription in response to TCDD primarily because the function of the AHR within the nucleus, presumably as a transcription factor, is necessary for TCDD-induced teratogenesis (Bunger et al., 2003
). Genes encoding the EGFR and its ligands EGF and TGF- are reported to be involved in models of AHR-mediated teratogenesis. Therefore, a candidate gene approach was taken and changes in gene expression of the EGFR and its ligands, EGF, TGF-, AREG, BTC, EREG, HB-EGF, and EPGN were assessed by RT-PCR. mRNA levels were evaluated in tissues of fetuses exposed to a maternal dose of 30 µg/kg of TCDD for 24 h at GD 17. As shown in Figure 2A, AREG mRNA was minimally expressed in fetal ureters and kidneys from control animals but was substantially induced in GD 17 fetal ureters after 24 h of in utero exposure to TCDD. EREG mRNA was also minimally expressed in control ureters and kidneys but was induced in ureters of TCDD-exposed fetuses, the degree of induction being similar between AREG and EREG for a given experiment. Notably, fetal kidneys did not demonstrate any changes in the expression of either AREG or EREG. In addition, the expression of other candidate genes such as those encoding the EGFR itself and other EGFR ligands did not differ in the developing kidney or ureter between control and treated groups (data not shown).
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In order to determine the organ specificity of the induction of AREG and EREG by TCDD, the expression of these genes were assessed in several other fetal tissues. As shown in Figure 2B, EREG mRNA was significantly induced by 2.7-fold in bladders (n = 3, p = 0.033) and by 1.9-fold in lungs (n = 3, p = 0.049) from 24-h TCDD-exposed fetuses compared to those of control fetuses. An increase in AREG mRNA was also detected in GD 17 bladders and lungs of 24-h TCDD-exposed fetuses with fold inductions of 1.3 (n = 3, p = 0.014) and 1.3 (n = 3, p = 0.001), respectively, compared to vehicle-exposed fetuses. The expression of AREG and EREG were not significantly altered, however, in fetal liver, heart, and skin after exposure to TCDD (data not shown).
These results indicate that changes in growth factor expression by TCDD in the developing mouse, although marginal for the most part, are not limited to target organs of teratogenesis. However, the simultaneous and robust inductions of AREG and EREG by TCDD in developing ureters, which are normally devoid of AREG and EREG mRNA, is unparalleled in any other fetal tissue. These results raise the possibility that these EGFR growth factors are involved in stimulating the hyperplasia within the ureteric lumen that is thought to give rise to TCDD-induced hydronephrosis.
AREG is transcriptionally regulated by the Wilms tumor suppressor (WT1), a critical factor in mouse kidney development (Lee et al., 1999). The WT1(–KTS) isoform, which is a product of the WT1(–KTS) splice variant, is thought to encode the transcriptionally active form of WT1 that is responsible for upregulating AREG. Interestingly, both WT1 and WT1(–KTS) mRNA splice variants are induced in kidneys exposed to the AHR ligand BaP in an AHR-dependent manner with no change in total WT1 mRNA (Falahatpisheh and Ramos, 2003). Considering our findings on the induction of AREG by TCDD, we investigated the effect of TCDD on WT1 and WT1(–KTS) transcript levels in the developing kidney and ureter. As shown in Figure 3, neither WT1 nor WT1(–KTS) were detectable at the transcriptional level in GD 17 ureters of control or TCDD-treated fetuses. Although, detectable levels of both WT1 and WT1(–KTS) mRNA were observed in GD 17 fetal kidneys, there was no apparent change in levels with TCDD exposure. The lack of TCDD-mediated WT1 and WT1(–KTS) mRNA induction within developing ureters or kidneys indicates that these WT1 splice variants are not under the direct transcriptional control of the AHR in our model. Furthermore, an induction of AREG mRNA upon exposure to TCDD in the absence of the WT1(–KTS) splice variant indicates that WT1(–KTS) is not a transcriptional activator of Areg in developing ureters. These findings suggest that the AHR may directly regulate Areg gene expression in the presence of TCDD.
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Effects of TCDD on AREG Gene Expression in the Adult Mouse Ureter
TCDD-induced hydronephrosis is specific to the developing kidney and does not occur in adult mice. Therefore, TCDD-induced changes that lead to hydronephrosis are also thought to be specific to the developing mouse. To determine whether TCDD-mediated induction of AREG is specific to fetal ureters, the effects of TCDD on AREG mRNA levels in adult mice were assessed by RT-PCR. AREG mRNA was not expressed at detectable levels in kidneys of control or treated mice (Fig. 4A). Conversely, AREG mRNA was detectable in control ureters, the level of which was not significantly altered in ureters of 24-h TCDD-treated mice (p = 0.136). To demonstrate AHR functionality, TCDD induced CYP1A1 mRNA in kidneys and ureters, which possess detectable levels of AHR and ARNT mRNA (Fig. 4B). Thus, the induction of AREG by TCDD observed in fetal ureters is specific to the developing mouse.
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TCDD-Mediated Induction of AREG in a Cell Culture Model
TCDD-induced teratogenesis depends on a functional AHR within the nucleus. Therefore, genes that are directly modulated by the AHR within the developing ureter in response to TCDD are likely those genes that are important in hydronephrosis. Accordingly, we tested the AHR dependency of TCDD-mediated induction of AREG mRNA. To this end, we employed a cell culture model, which enabled us to effectively determine the features of AREG induction by TCDD and whether they were characteristic of AHR regulated genes. The mouse hepatoma cell line Hepa-1 was used in our studies in order to retain the AHR machinery specific to the mouse and because this cell line exhibited a significant induction of AREG when treated with TCDD in preliminary experiments.
TCDD Dose-Response of AREG Induction
Hepa-1 cells were treated with increasing concentrations of TCDD (10–12–10–7M) for 24 h and AREG mRNA levels assessed via semiquantitative RT-PCR. As shown in Figure 5A, a TCDD dose-response relationship was observed in that increasing the concentration of TCDD resulted in concomitant increases in AREG mRNA levels. AREG mRNA was significantly increased in cells treated with 10–10–10–7M TCDD compared with cells treated with vehicle alone (p = 0.0289, p = 0.0006, p = 0.0140, and p = 0.0001, respectively). Maximal AREG induction was achieved between 10–10 and 10–9M TCDD with a fold induction of 2 over control. This dose of TCDD, at which maximal AREG induction is elicited, is in accordance with the concentration that elicits maximal CYP1A1 induction (Israel and Whitlock, 1983).
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Time-Course of TCDD Induction of AREG
For experiments assessing the time-course of AREG induction by TCDD, Hepa-1 cells were treated with 10–9M TCDD for 3, 6, or 24 h. As shown in Figure 6, the expression of AREG mRNA was significantly increased in Hepa-1 cells within 3 h after TCDD treatment compared to cells treated with vehicle alone. TCDD maximally induced AREG by 6 h (
2-fold), the levels of which remained elevated 24 h after treatment. We have also observed AREG induction by TCDD as early as 1.5 h (data not shown). Our data show that the time-course of AREG induction by TCDD is similar to that of CYP1A1 (Israel and Whitlock, 1983).
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Induction of AREG by Various AHR Ligands
To further characterize the extent to which AREG expression is modulated by an active AHR, we investigated whether other known AHR ligands also induced AREG mRNA levels. The prototypical AHR responsive gene, Cyp1a1, is induced not only by TCDD but by other HAHs (Zacharewski et al., 1989
) and PAHs (Merchant et al., 1992; Shimada et al., 2002) in an AHR-dependent fashion. We therefore tested the ability of several of these compounds to induce AREG. As shown in Figure 7A, the HAHs PeCDD and TCDF increased AREG mRNA levels in Hepa-1 cells significantly (2-fold) compared to cells treated with vehicle alone. The extent of AREG induction by PeCDD and TCDF was similar to that of TCDD. As expected, however, higher doses of PeCDD (10–7M) and TCDF (10–6M) were required in order to achieve the same response as 10–9M TCDD, which is consistent with their relative affinities for the AHR. Conversely, cells treated with the PAHs BaP (10–5M) and DBA (3 x 10–5M) showed an enhanced induction of AREG mRNA levels compared to cells treated with HAHs (Fig. 7B). This phenomenon can be explained by the differential metabolism of PAHs in comparison to HAHs. HAHs are not readily metabolized, and therefore, act primarily through the AHR. PAHs, on the other hand, are readily metabolized by the cytochrome P450s they induce producing active metabolites that can amplify effects initiated by their parent compound through alternative pathways. For example, BaP metabolites can enhance EGFR signaling through the generation of hydrogen peroxide, a reactive oxygen species, resulting in an increase in cell number (Burdick et al., 2003). The findings from these experiments indicate that AREG is induced by TCDD in much the same way as CYP1A1, suggesting that they both share a similar mechanism of induction, i.e., direct transcriptional regulation by the AHR.
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Effect of Reduced AHR Levels on AREG Induction
To determine the extent by which TCDD induction of AREG is AHR dependent, we investigated the consequence of the absence of the AHR on this induction. To this end, we employed the Hepa-1 variant cell line TAOBPrc1, which is virtually deficient in the AHR and has correspondingly reduced TCDD-induced CYP1A1 mRNA and activity levels in comparison to Hepa-1 wild type cells (Israel and Whitlock, 1983
; Miller et al., 1983). Our data confirm that TAOBPrc1 cells possess a significant reduction in AHR mRNA levels compared to wild type cells, while the level of ARNT mRNA is similar to wild type cells (Fig. 8A). In keeping with this result, CYP1A1 mRNA is minimally induced by TCDD in TAOBPrc1 cells compared to wild type cells (data not shown). Consistent with previous results, AREG mRNA was significantly induced in wild type cells by 2-fold after treatment with 10–9M TCDD (Fig. 8B). However, AREG mRNA levels were not significantly altered by TCDD in TAOBPrc1 cells compared to variant cells treated with vehicle alone. Similarly, PAH induction of AREG is AHR-dependent as evidenced by the lack of induction in TAOBPrc1 cells that are exposed to PAHs (data not shown), suggesting that BaP and DBA metabolites enhance the induction of AREG through pathways that are AHR-dependent. This finding demonstrates that the presence of the AHR is required for TCDD-mediated induction of AREG at the transcriptional level.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our studies demonstrate that the AHR is present and functional in the developing kidney and ureter, which are target organs of AHR-mediated teratogenesis. Furthermore, genes encoding the EGFR growth factors AREG and EREG were shown to be strongly upregulated in the developing ureter in response to TCDD, the induction of AREG being AHR dependent.
Although, the AHR and its heterodimerization partner ARNT have been localized in fetal tissues at both the protein and transcriptional levels (Abbott and Probst, 1995; Abbott et al., 1995; Jain et al., 1998; Peters and Wiley, 1995), the transcription of AHR target genes in response to ligand is not well characterized. Our study confirms the presence of the AHR and ARNT at the transcriptional level in several fetal tissues at two developmental stages that are relevant to TCDD-induced hydronephrosis (Figs. 1A–1E). Furthermore, our results demonstrate a transcriptionally active AHR in these tissues, notably in the kidney and ureter, as demonstrated by the induction of CYP1A1, CYP1A2, and CYP1B1 mRNA in response to TCDD administration. Therefore, the AHR is present and functional in the developing kidney and ureter during a time when developing mice are susceptible to TCDD-induced hydronephrosis.
Disruption of EGFR signaling may be a mechanism by which TCDD induces teratogenesis. The EGFR is a receptor tyrosine kinase that binds growth factors with subsequent effects on cellular proliferation and apoptosis. EGFR signaling is vital for normal development as EGFR null mice die during midgestation or perinatally with general growth retardation and a multitude of growth abnormalities (Miettinen et al., 1995; Threadgill et al., 1995). Specifically, EGFR signaling plays an important role in renal development as EGFR null mice display dilated urinary collecting ducts, implicating the EGFR in branching tubulogenesis of the collecting duct system. Furthermore, EGFR ligands account for a large fraction of branching morphogens secreted by the embryonic kidney in culture (Sakurai et al., 1997b), most of which induce tubulogenesis and branching morphogenesis of ureteric bud and collecting duct cells and kidney organ cultures (Barros et al., 1995; Sakurai et al., 1997a,b). These growth factors act through the EGFR to induce branching tubulogenesis as inhibiting EGFR tyrosine kinase activity inhibits cell process formation, an early step in branching tubulogenesis (Sakurai et al., 1997a,b).
Our focus on genes encoding the EGFR and its ligands is supported by reports indicating a role for EGFR signaling in TCDD-induced teratogenicity of the palate (Abbott and Birnbaum, 1989, 1990b; Abbott et al., 2003a), teeth (Partanen et al., 1998), prostatic bud (Abbott et al., 2003b), and kidney (Abbott and Birnbaum, 1990a; Abbott et al., 2003a; Bryant et al., 1997, 2001a,b; Miettinen et al., 2004). In addition, the AHR is reported to regulate EGFR ligands at the transcriptional level, notably the induction of TGF- in human mammary epithelial cells (MCF-10A cells), which translates into a biological effect of enhanced apoptosis through EGFR signaling (Davis et al., 2000, 2001, 2003). In our study, among the genes encoding the EGFR ligands, only Areg and Ereg were altered in their expression specifically in the developing ureter in response to TCDD. AREG and EREG mRNA were substantially induced in ureters (Fig. 2A), with AREG following a similar temporal pattern to that of the Cyp1a and Cyp1b1 genes (data not shown), suggesting a similar mechanism of upregulation. AREG is a regulator of cell proliferation and migration, and the principal physiological role of AREG is in branching morphogenesis of developing organs. In mice, AREG is vital for the normal development of the lung and mammary gland (Luetteke et al., 1999; Schuger et al., 1996) and is an inducer of ureteric bud branching (Lee et al., 1999). Thus, AREG is a developmentally important factor, the dysregulation of which may be detrimental to the developing kidney and ureter. This is consistent with our hypothesis that TCDD dysregulates the expression of developmentally important genes involved in the regulation of cell proliferation and apoptosis within the mouse urinary tract.
Other EGFR ligands, such as EGF and TGF-, were not affected by TCDD at the transcriptional level. The finding that TGF- mRNA was not induced by TCDD suggests that the response to TCDD seen in MCF-10A cells is species and/or tissue specific. These results also substantiate the finding that hydronephrosis induced by TCDD is comparable between the EGF + TGF- double knockout and wild type mice (Bryant et al., 2001b). Together, the data leave open the possibility of a role for EGFR ligands other than EGF and TGF- in the development of hydronephrosis. However, when either EGF or TGF- is absent, the incidence and severity of hydronephrosis increase with the urinary tract being more sensitive to TCDD in the absence of EGF than in the absence of TGF- (Abbott et al., 2003a). Therefore, the availability of EGF and TGF- can influence the outcome of hydronephrosis, but neither is required, suggesting the involvement of other key growth factors. We hypothesize that EGFR ligands whose expression is altered by TCDD, such as AREG and EREG, play a role in TCDD-induced hydronephrosis in balance with the basal expression of EGF and TGF-.
EREG has recently been identified as being directly regulated by the AHR through a DRE located 56 base pairs (bp) upstream of the transcriptional start site (Patel et al., 2006). However, Areg has not yet been reported as a direct AHR target gene. Our results indicate that AREG induction by TCDD is dose- and time-dependent with similar induction patterns as that of CYP1A1 in Hepa-1 cells (Figs. 4 and 5). The induction of AREG mRNA by TCDD reaches a plateau at the higher dose range indicative of a saturation event due to a receptor-mediated process. The rapid accumulation of AREG mRNA in response to TCDD is an indication that AREG upregulation is an early and primary cellular response to TCDD much like that of CYP1A1. AREG mRNA was also induced by other AHR ligands (Fig.7 A), a characteristic attributed to AHR target genes. Furthermore, our studies show that cells virtually devoid of the AHR do not display an increase in AREG mRNA when treated with TCDD (Fig.8 B). Therefore, TCDD induction of AREG absolutely requires the AHR and follows similar induction patterns to that of CYP1A1, strongly suggesting that Areg is under the direct transcriptional control of the AHR. Whether or not the increase in transcription of AREG by TCDD occurs as a result of AHR binding to DRE sequences within the promoter region has yet to be established. We have, however, identified 7 core DRE sequences within the first 5 kilobases upstream of the transcriptional start site of Areg, two of which, at positions – 1234 and – 3502 nt upstream of the transcriptional start site are good candidates for binding of the AHR-ARNT heterodimer.
Interestingly, treatment of cells with PAHs resulted in an enhanced induction of AREG mRNA compared to cells treated with HAHs (Fig.7 B), which was found to also require the AHR. Unlike HAHs, PAHs are readily metabolized into active metabolites. We hypothesize that active PAH metabolites are responsible for activating alternative signaling pathways in an AHR-dependent manner in conjunction with the direct induction of AREG by the parent compound. This is consistent with the recent report showing that AREG transcription is induced by tobacco smoke, which contains PAHs, through a cAMP signaling pathway that is dependent on the AHR (Du et al., 2005). The consequence of inducing AREG levels was an enhancement of DNA synthesis, which is thought to contribute to the increase in cell proliferation that leads to smoking-induced carcinogenesis. In fact, tobacco smoke-induced AREG levels stimulate lung cell proliferation (Lemjabbar et al., 2003). This is reminiscent of TCDD-induced hydronephrosis where an increase in cell proliferation found in the developing ureteric epithelium is associated with elevated levels of AREG mRNA. Together, these findings support the notion that disrupting AREG expression affects molecular and cellular processes leading to detrimental effects in target organs.
Differences in HAH and PAH molecular signaling downstream of AHR activation also explains the different effects that TCDD and BaP produce in the developing kidney. In kidney organ culture, BaP inhibits nephrogenesis and branching morphogenesis (Falahatpisheh and Ramos, 2003), whereas TCDD produces hyperplasia of the developing ureteric luminal epithelium in vivo and in cultured ureters (Abbott and Birnbaum, 1990a). Both events are AHR dependent, yet the morphological outcomes are remarkably different, most likely due to signaling of BaP metabolites through pathways distinct from that of TCDD. In addition, the AHR-dependence of TCDD- and BaP-induced effects on the developing kidney, although dissimilar, indicate an important physiological role for the AHR in regulating kidney development.
The role of EGFR signaling in the pathogenesis of TCDD-induced hydronephrosis is supported by other investigators as well as our findings. Here, we report increased levels of the EGFR ligands AREG and EREG at the transcriptional level in response to TCDD in the developing ureter where, it is thought, the initial pathology leading to hydronephrosis occurs. Our data indicate that TCDD-mediated AREG induction is AHR dependent and suggest that it is under the direct transcriptional control of the AHR. Our hypothesis is that exposure to TCDD results in AHR-mediated transcription of genes encoding AREG and EREG, which bind the EGFR, enhancing downstream signaling. The biological consequence is an increase in cell proliferation seen within the developing ureteric luminal epithelium. A change in gene expression does not necessitate a biological effect, and therefore, investigating changes in a biological marker, such as cell proliferation or apoptosis, is warranted. Nonetheless, the findings from the present study provide a molecular basis by which the EGFR pathway may contribute to hydronephrosis and other dioxin-mediated toxicities.
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ACKNOWLEDGMENTS |
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Supported by a grant from the Canadian Institutes of Health Research to P.A.H.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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