* Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23113; and Department of Microbiology and Immunology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23113
1 To whom correspondence should be addressed. Fax: (804) 828-0676. E-mail: pnagark{at}hsc.vcu.edu.
Received June 2, 2003; accepted November 17, 2003
ABSTRACT
We have used pathway-specific cDNA arrays coupled with analysis of gene promoter regions to identify novel genes that may mediate the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the thymus. C57BL/6 mice were injected ip with 50 µg/kg TCDD, and 3, 6, or 24 h later, RNA was extracted from the thymus and subjected to microarray analysis. Several members of the TNF and TNFR family were induced following TCDD exposure, including receptor/ligand pairs Ltß-R/LIGHT, OX40/OX40L and TNF-/TNFR1. In addition, Fas and CD30 were also upregulated. Pro-apoptotic bcl-2 gene family members Bax and Hrk, among others, were also induced, as were pro-survival bcl-2 family genes Bcl-x and Bcl-w. Cell-cycle regulator p21Cip1 was also induced. In addition, we analyzed the promoter regions of genes induced by TCDD for the presence of dioxin-responsive elements (DREs). The Fas and LIGHT gene promoters were found to contain DREs as analyzed by Matinspector Web-based search algorithm. Furthermore, binding of the aryl hydrocarbon receptor (AhR) to the DREs present on these genes was confirmed by chromatin immunoprecipitation. Given that several of the genes, including Fas, LIGHT, and CD30 are involved in negative selection of T cells in the thymus, our studies suggest that TCDD-induced upregulation of these genes may enhance negative selection leading to thymic atrophy.
Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin; Fas; LIGHT; apoptosis; negative selection.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is the prototype for a class of halogenated aromatic hydrocarbons that are common environmental contaminants. The toxic effects of TCDD have been well characterized and include hepatotoxicity, teratogenicity, fetotoxicity, chloracne, and immune suppression (Grassman et al., 1998; Poland and Knutson, 1982
). TCDD is also a potent tumor promoter (Davis et al., 2000
; Schwarz et al., 2000
; Walker et al., 2000
). The immunotoxic effects of TCDD are many and varied. TCDD has been shown to suppress both cell-mediated and humoral immune response (Kerkvliet, 2002
). In addition, TCDD has been shown to affect the process of thymocyte maturation. Studies using fetal thymic organ cultures have shown that TCDD induces cell-cycle arrest in double-negative thymocytes and increases the relative number of CD8+ T cells (Lai et al., 2000
). Therefore, TCDD may affect the process of T cell proliferation and differentiation involving positive/negative selection. Recent studies from our laboratory have shown that TCDD-induced atrophy of the thymus is mediated, at least in part, by apoptosis of thymocytes through Fas/FasL interactions (Kamath et al., 1999
; Rhile et al., 1996
). Specifically, Fas-deficient lpr and FasL-defective gld mice were shown to be less sensitive to TCDD-induced thymic atrophy (Kamath et al., 1999
). Moreover, TCDD also triggered apoptosis in antigen-activated T cells involving Fas and FasL (Camacho et al., 2001
, 2002
; Dearstyne and Kerkvliet, 2002
; Kerkvliet, 2002
; Pryputniewicz et al., 1998
). The toxic effects of TCDD are thought to be mediated largely by transcriptional regulation through the aryl hydrocarbon receptor (AhR) (Rowlands and Gustafsson, 1997
). Upon binding of TCDD or a congener, the ligand-AhR complex translocates to the nucleus, where it heterodimerizes with the aryl hydrocarbon nuclear translocator (ARNT). This complex (ligand-AhR-ARNT) then binds to dioxin-responsive elements (DREs) with the consensus sequence 5'-GCGTGNN(A/T)NNN(C/G)-3' located in the regulatory regions of dioxin-responsive genes, where it acts as a transcription factor (Yao and Denison, 1992
). Therefore, TCDD has the potential to directly alter the expression of a large number of genes. For example, TCDD exposure has previously been shown to induce the expression of several genes through the above mechanism, including cytochrome P4501A1/2, cytochrome P4501B1, glutathione S-transferase Ya, aldehyde dehydrogenase 3, and UDP-glucuronosyl transferase 1*06 (Nebert et al., 2000
).
Despite intense study, the exact mechanisms of TCDD-induced immunotoxicity remain elusive. Given the varied effects of TCDD exposure on the immune system, and the number of genes whose expression may be altered by TCDD, broad scale approaches are necessary to more fully describe the mechanisms of TCDD-induced immunotoxicity. Recently, we employed low-density microarray technology to investigate the altered expression of genes involved in apoptosis in a number of organs, including the thymus. These studies showed that TCDD upregulated the expression of many genes involved in apoptosis (Zeytun et al., 2002). In the current study, we expanded upon these earlier results by using higher density microarrays, coupled with analysis of the promoter regions of genes differentially expressed in the thymus following exposure to TCDD. This approach enabled us to more completely describe the effects of TCDD on expression of genes related to apoptosis in the thymus and, using analysis of promoter regions, to identify genes induced following activation of the aryl hydrocarbon receptor.
MATERIALS AND METHODS
Mice and TCDD treatment.
Female C57BL/6 mice (5 to 7 weeks old) were purchased from NIH and housed in the VCU animal care facility. Mice were housed in standard polyethylene cages with wood shavings as bedding and given water and rodent chow ad libitum. Mice were housed in rooms maintained at 74 ± 2°F on a 12-h light/dark cycle. TCDD was a gift from Dr. Kun Chae of the National Institute of Environmental Health Sciences (Research Triangle Park, NC). Briefly, TCDD was dissolved in acetone, diluted in corn oil, and the acetone was removed by evaporation. The mice were injected ip with 50 µg/kg TCDD in 100 µl of corn oil. Control mice were injected with vehicle alone.
RNA extraction.
Mice were killed 3, 6, or 24 h following the injection of TCDD or vehicle, and their thymi removed. The pooled thymi from groups of two mice were placed in cold PBS and reduced to a single-cell suspension with a stomacher. Cells were washed twice with PBS. Total RNA was extracted from the cells with Trizol according to the manufacturer's protocol as described. The RNA was further purified using the RNeasy Extraction Kit (Qiagen, Valencia, CA). The concentration of each RNA sample was determined spectrophotometrically, and the integrity of each sample was verified by visualizing an aliquot on a 0.8% agarose gel.
Microarray hybridization and detection.
Nylon expression arrays spotted with cDNA fragments of 96 genes involved in various apoptotic processes were purchased from SuperArray Inc. (Bethesda, MD) (Table 3). Biotin-16-dUTP labeled probes were synthesized using 5 µg of total RNA as template, and using the probe labeling primer mix provided by SuperArray. All steps in the hybridization and detection of the arrays were carried out according to the manufacturer's protocols. Following the addition of a chemiluminescent substrate (CDP-star), arrays were exposed to X-ray film.
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RT-PCR analysis of gene expression.
To confirm the results generated by microarray analysis, the expression of several genes was also analyzed by RT-PCR. RNA was extracted as described previously from an independent replication of the original experiment. cDNA was synthesized using the Sensiscript RT Kit (Qiagen, Valencia, CA). RT-PCR reactions were prepared using Epicentre's PCR premix F (Madison, WI) and Platinum Taq Polymerase (Invitrogen, Carlsbad, CA). Primer sequences and annealing temperatures are listed in Table 1. All reactions were cycled 35x, using the following parameters; 10 s at 95°C, 10 s at the appropriate annealing temperature, and 30 s at 72°C, with a final incubation at 72°C for 2 min PCR products were resolved using 1.2% agarose gels. The amplicon sizes of PCR products are also shown in Table 1.
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Chromatin immunoprecipitation (ChIP) assay.
The ChIP assay was performed using the ChIP Assay Kit (Upstate Biotechnology, Waltham, MA) following the manufacturer's protocol. Briefly, thymocytes (1 x 106) were isolated from vehicle or TCDD-treated mice, and protein/DNA complexes were cross-linked by 10-min incubation in 1% formaldehyde at room temperature with gentle agitation. Cells were then washed twice in ice-cold PBS containing protease inhibitors. Cells were pelleted and lysed by 10-min incubation on ice in 200 µl SDS lysis buffer. DNA in the cell lysate was then sheared into 200- to 1000-bp lengths by sonication. Cellular debris was pelleted by centrifugation for 10 min at 13,000 rpm at 4°C. The supernatant was diluted 10-fold in 1800 µl ChIP dilution buffer with the addition of protease inhibitors. Samples were cleared by incubation with 80 µl Salmon sperm DNA/Protein A agarose slurry for 30 min at 4°C with agitation. The agarose was pelleted, and the supernatant was collected. A 1:10 dilution goat polyclonal anti-mouse AhR antibody (Novus Biologicals, Littleton, CO) was added to each sample and incubated overnight at 4°C with rotation. An isotype control antibody was also included. The antibody/AhR/DNA complexes were collected by incubation with 60 µl Salmon sperm DNA/Protein A agarose slurry for 1 h at 4°C with rotation. Following incubation, the agarose was pelleted, and the supernatant was removed. Protein-DNA complexes were then eluted from the immunoprecipitating antibody by incubation with 250 µl elution buffer for 15 min at room temperature with rotation. Twenty µl 5 M NaCl was added to the elutant (500 µl), and the sample was incubated at 65°C for 4 h to reverse protein/DNA cross-linking. DNA was recovered by phenol/chloroform extraction and ethanol precipitation. Pelleted DNA was resuspended in 40 µl distilled H2O. Primers used in ChIP PCR are detailed in Table 2. PCR analysis was performed essentially as described above.
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TCDD-Induced Alterations in the Expression of TNF Family Genes
The effect of exposure to 50 µg/kg body weight of TCDD on the expression of 96 genes involved in apoptosis at 3, 6, and 24 h was assessed in the thymus of C57BL/6 mice by microarray analysis. The 96 genes involved in various apoptotic pathways included in the current investigation are shown in Table 3. The expression of several members of the tumor necrosis factor receptor (TNFR) family was altered by a factor of 1.5 or more at 1 or more time points (see Table 4), including TNFR1, Fas, CD30, OX40 (TNFSF4), and lymphotoxin beta receptor (LTßR). TNFR1 was upregulated at all time points, though the degree of increase diminished with time. OX40 was downregulated at 3 and 6 h and upregulated at 24 h postexposure. Expression of LTßR was upregulated at 6 and 24 h postexposure to TCDD. Fas and CD30 were both increased at early time points.
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TCDD-Induced Alterations in the Expression of Bcl-2 Family Genes
Previous studies have shown that pro-apoptotic members of the Bcl-2 gene family such as Bax and Hrk, are dioxin responsive (Matikainen et al., 2001; Park and Lee, 2002
). In the current study, when expression level of Bcl-2 family members was investigated, many pro-apoptotic members of the Bcl-2 gene family were found to be upregulated following TCDD exposure (Table 5). Bax was upregulated at 6 h, bik at 3 and 6 h, bid and Hrk at all 3 time points tested, and mcl-1 at only 24 h. Expression of Bok, also pro-apoptotic, was decreased at 3 h. Among pro-survival bcl-2 family genes, increased expression of Bcl-x was seen at 6 and 24 h, and Bcl-w at 3 h.
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DISCUSSION
In the current study, we observed altered expression of genes involved in apoptosis in the thymus resulting from in vivo exposure to TCDD. We noted that TCDD exposure dysregulates the expression of many pro-apoptotic genes, supporting our earlier findings that TCDD may induce toxicity in the thymus by triggering apoptosis (Kamath et al., 1997). In addition, we demonstrate that TNF family member Fas and TNFR family member LIGHT (tnfsf14) are direct targets of TCDD due to the presence of functional dioxin-responsive elements (DREs) in their promoter regions.
The apoptotic program is initiated either extrinsically, via the death-receptor mediated pathway, or intrinsically via the mitochondrial or endoplasmic reticulum pathway. Initiation of the death-receptor pathway involves ligation of TNF family member containing a cytoplasmic death domain by its cognate ligand (e.g., Fas and FasL). This leads to receptor clustering and the formation of the death-inducing signaling complex (DISC). The DISC includes adaptor molecules such as TRADD, which recruits FADD, which in turn recruits procaspase 8. The execution phase of the apoptotic program is initiated by autocatalytic cleavage of procaspase 8 in association with the DISC, which then processes effector caspases (Locksley et al., 2001). Members of the bcl-2 gene family mediate the intrinsic apoptotic pathways. These gene family members are defined by bcl-2 homology (BH) domains and are divided into pro- and anti-apoptotic groups. Pro-apoptotic members such as Bax and Bad, also known as BH 1-3 proteins, act to mediate the release of components of organelles such as cytochrome c from the mitochondria and Ca2+ from the endoplasmic reticulum (Newmeyer and Ferguson-Miller, 2003
; Nutt et al., 2002a
,b
). Release of these molecules leads to the formation of the apoptosome complex, which includes Apaf-1 and procaspase 9; procaspase 9 is then autocatalytically cleaved, which initiates the caspase cascade through the cleavage of effector caspases. Bcl-2 family BH 1-4 proteins, such as Bcl-2 and Bcl-w, antagonize the pro-apoptotic activities of Bax and Bad, although the precise mechanisms of this antagonism are unknown (Newmeyer and Ferguson-Miller, 2003
). In addition, initiation of the extrinsic/death receptor pathway can also lead to activation of the intrinsic pathway via cross-talk mediated by BH-3-only bcl-2 family members such as Bid.
Recent work from our group and others has supported the involvement of Fas/FasL interactions in TCDD-induced thymic atrophy and peripheral T cell dysfunction, inasmuch as Fas-deficient lpr and FasL-defective gld mice were shown to be less sensitive to TCDD-induced immunotoxicity (Dearstyne and Kerkvliet, 2002; Kamath et al., 1999
). We have further demonstrated that FasL is upregulated in the thymus following exposure to TCDD, although the FasL promoter was not found to contain a DRE (Kamath et al., 1999
). The current study demonstrates for the first time that TCDD upregulates Fas expression and that the Fas promoter contains a functional DRE. The upregulation of Fas and FasL may have serious consequences on T cell differentiation in the thymus. For example, it has been demonstrated that Fas/FasL interactions are involved in the process of negative selection in the thymus (Castro et al., 1996
). Specifically, blockade of Fas/FasL reduced the percentage of apoptotic FashiTCRintCD4+CD8+ thymocytes, which is the predominant stage in thymocyte maturation when negative selection occurs. Coupled with our data demonstrating the induction of Fas by TCDD, these findings support a model in which TCDD may act, in part, by increasing the sensitivity of maturing thymocytes to the apoptotic signals induced by negative selection through increased expression of Fas.
It is likely that TCDD induces immunotoxicity in the thymus via multiple mechanisms. For example, there is a significant body of literature indicating that exposure to TCDD or congeners causes cell cycle arrest, as shown in murine hepatocytes, murine fetal thymic organ cultures, macaque endocervical primary cell cultures, 3T3 fibroblast cells, and MCF-7 cells (Lai et al., 2000; Munzel et al., 1996
; Puga et al., 2000
; Vaziri and Faller, 1997
; Wang et al., 1998
). Further, it has been demonstrated that the cyclin-dependent kinase inhibitor, p21 (WAF1), which acts as a G1/S checkpoint regulator, is dioxin responsive (Enan et al., 1998
; Koliopanos et al., 2002
). The data presented in the current study show that p21 is significantly upregulated in the thymus following TCDD exposure. Therefore, TCDD may also act by inducing cell-cycle arrest in thymocytes, which could lead to apoptosis. Other pro-apoptotic genes known to be dioxin responsive include bcl-2 family members Bax and Hrk (Matikainen et al., 2001
; Park and Lee, 2002
). Both of these genes were shown to be upregulated in our study and, therefore, may contribute to the apoptosis of thymocytes induced by TCDD. In addition, TCDD may induce signals other than those involved in apoptosis. For example, stimulation of both Fas and TNFR1 can trigger NF-
B signaling pathways mediated by the adaptor molecule TRAF2, which also binds to the adaptor molecule TRADD. As shown in this study, Fas, TNFR1, and TRAF2 are induced in the thymus following TCDD exposure, which suggests that TCDD may induce signaling pathways mediated by TRAF2, such as NF-
B or JNK (Liu et al., 1996
; Natoli et al., 1997
; Pensati et al., 1997
; Reinhard et al., 1997
).
The role of TNF family member LIGHT in T cell development has been the subject of much recent study. LIGHT is expressed on T cells and interacts with herpes virus entry mediator (HVEM) and LTßR on stromal cells (Mauri et al., 1998; Zhai et al., 1998
). Recent work has shown that LIGHT plays a pivotal role in the facilitation of negative selection as a costimulatory molecule (Tamada et al., 2000
, 2002
). Studies using LIGHT Tg mice demonstrated that overexpression of LIGHT leads to a decrease in thymus weight and cellularity (Shaikh et al., 2001
; Wang et al., 2001
). Also, LIGHT Tg mice show a marked reduction in the numbers of DP thymocytes, and lesser reductions in the numbers of CD4+ and CD8+ thymocytes (Shaikh et al., 2001
). These reductions were accompanied by an increase in the numbers of apoptotic thymocytes, suggesting that the reduction in thymic cellularity was due to increased apoptosis of thymocytes, rather than increased emigration from the thymus. In addition, using the H-Y TCR Tg model, it was demonstrated that LIGHT is central in the elimination of autoreactive thymocytes via negative selection, because the deletion of autoreactive thymocytes could be partially rescued by blocking the interaction of LIGHT by treatment with LTßR-Ig and HVEM-Ig, both soluble receptors for LIGHT (Wang et al., 2001
). It should be noted that, in the current study, we used thymocytes for gene expression analysis. Because thymocytes contain a mixture of T cells, epithelial cells, and stromal cells, the results do not necessarily suggest that the nature of changes seen in gene expression are restricted to T cells. However, our data showing the transcriptional upregulation of LIGHT, which is expressed on T cells, and increased induction of LTßR, which is expressed on thymic stromal cells, suggests that exposure to TCDD may induce apoptosis in T cells, in part, through enhanced negative selection. Our study also notes that TCDD upregulates expression of CD30, another TNFR family member that has also been implicated in negative selection. Specifically, CD30 KO mice exhibit reduced efficiency of negative selection, while negative selection is enhanced in CD30 Tg mice (Amakawa et al., 1996
; Chiarle et al., 1999
). Taken together, these findings suggest several mechanisms through which exposure to TCDD may enhance negative selection: (1) Upregulation of LIGHT on thymocytes and LTßR on thymic stroma may increase the avidity of the interaction between TCR and MHC/self-peptide complexes and, thereby, enhance negative selection. (2) Increased expression of Fas, as shown in the current study, or FasL, as shown previously (Kamath et al., 1999
), may enhance negative selection. (3) Increased expression of CD30 may facilitate negative selection. (4) A combination of these mechanisms may together favor negative selection of T cells in the thymus. It is possible that the ability of TCDD exposure to enhance negative selection through multiple mechanisms explains our earlier finding that, while Fas-deficient lpr and FasL-defective gld mice were resistant to thymic atrophy induced by low doses of TCDD, they were susceptible to high doses (Kamath et al., 1999
). Together, the current study demonstrates that some members of the death-receptor gene family, such as Fas and LIGHT, express DRE in their promoters and that TCDD can directly induce their expression. In contrast, other members do not express DREs and may be induced through alternative pathways involving other transcription factors. Because several of such molecules are involved in T cell negative selection, the current study suggests that TCDD may alter the T cell repertoire by enhancing negative selection of T cells in the thymus.
Direct induction of gene transcription by ligand-bound AhR cannot explain most of the changes in gene expression reported here. Several recent studies have described mechanisms other than the classical AhR/DRE pathway through which TCDD can alter gene expression. For example, TCDD exposure has been shown to induce MAPKs independent of the AhR in mouse embryonic fibroblasts (Tan et al., 2002). Another study demonstrated that the Ah receptor is able to directly interact with NF-
B (Tian et al., 1999
, 2002
), and thereby suppress NF-
B-mediated transcription. This result may explain the expression kinetics of several of the genes described in this study, including CD30 and TNF-
. The promoter regions of these genes contain potential NF-
B binding sites, and all have similar kinetics, with initial high levels of induction following TCDD exposure and minimal induction at later time points. In addition, many of the changes in gene expression following TCDD exposure are likely due to secondary effects of TCDD. For example, TCDD exposure results in the production of reactive oxygen species, as TCDD is metabolized by Cyp1A1, resulting in DNA adduct formation and DNA damage (Alsharif et al., 1994
; Bagchi and Stohs, 1993
). One of the primary genes involved in protection against DNA damage is the tumor suppressor p53. In this study, we show that p53 is induced only after 24 h exposure, suggesting that p53 induction may be a secondary effect of TCDD exposure.
Finally, the data presented here support previous results demonstrating importance of a complete DRE binding site for AhR-ARNT-ligand/DNA interaction. Previous studies have shown that, in addition to the absolute requirement for the GTGCG motif, interaction of the ligand-bound AhR with DNA is also dramatically reduced following ablation of the A/T motif at position 8 and the C/G motif at position 12 of the canonical DRE (Yao and Denison, 1992). Our results support these studies inasmuch as only those genes (p21, Fas, and LIGHT), which harbor a complete DRE, were found to be dioxin responsive by ChIP analysis, while the DREs in the CD30 and Mcl-1 promoters were found to be nonfunctional, as they lack bases proximal to the DRE core also involved in AhR binding. In addition, in this study in which we compared human and murine promoter regions, only those genes known to be dioxin responsive, including p21, Bax, and Hrk, as well as genes we have identified as dioxin responsive such as, Fas and LIGHT, were found to contain complete DREs in the promoters of each gene in both human and mouse. This suggests that interspecies comparisons of promoter regions for the presence of complete DREs may provide a powerful tool to identify novel dioxin-responsive genes.
ACKNOWLEDGMENTS
The authors acknowledge funding from NIH grants R01ES09098, R01DA016545, R21DA014885, R01AI053703, and R01HL058641. M.T.F. is supported by NIEHS fellowship F32ES011732.
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