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Originally published In Press as doi:10.1074/jbc.M402143200 on April 6, 2004

J. Biol. Chem., Vol. 279, Issue 24, 25204-25210, June 11, 2004
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A Constitutively Active Arylhydrocarbon Receptor Induces Growth Inhibition of Jurkat T Cells through Changes in the Expression of Genes Related to Apoptosis and Cell Cycle Arrest*

Tomohiro Ito{ddagger}, Shin-ichi Tsukumo{ddagger}, Norio Suzuki§, Hozumi Motohashi§, Masayuki Yamamoto§, Yoshiaki Fujii-Kuriyama§, Junsei Mimura§, Tien-Min Lin¶, Richard E. Peterson¶, Chiharu Tohyama{ddagger}, and Keiko Nohara{ddagger}||

From the {ddagger}Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba 305-8506, Japan, the §Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba 305-8575, Japan, and the School of Pharmacy, University of Wisconsin, Madison, Wisconsin 53705

Received for publication, February 26, 2004 , and in revised form, March 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress T cell-dependent immune reactions through the activation of the arylhydrocarbon receptor (AhR). Our previous findings suggest that TCDD inhibits the activation and subsequent expansion of T cells following antigen stimulation in mice, leading to a decreased level of T cell-derived cytokines involved in antibody production. In the present study, we investigated the effects of activated AhR on T cells by transiently expressing a constitutively active AhR (CA-AhR) mutant in AhR-null Jurkat T cells. In agreement with our previous findings, CA-AhR markedly inhibited the growth of Jurkat T cells. The inhibited cell growth was found to be concomitant with both an increase in the annexin V-positive apoptotic cells and the accumulation of cells in the G1 phase. The growth inhibition was also shown to be mediated by both xenobiotic response element (XRE)-dependent and -independent mechanisms, because an A78D mutant of the CA-AhR, which lacks the ability of XRE-dependent transcription, partially inhibited the growth of Jurkat T cells. Furthermore, we demonstrated that CA-AhR induces expression changes in genes related to apoptosis and cell cycle arrest. These expression changes were shown to be solely mediated in an XRE-dependent manner, because the A78D mutant of the CA-AhR did not induce them. To summarize, these results suggest that AhR activation causes apoptosis and cell cycle arrest, especially through expression changes in genes related to apoptosis and cell cycle arrest by the XRE-dependent mechanism, leading to the inhibition of T cell growth.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)1 is known to exert a variety of toxicities such as reproductive toxicity, immunotoxicity, teratogenicity, and neurotoxicity (1, 2). Previous studies using arylhydrocarbon receptor (AhR) knock-out mice indicate that most, if not all, of the TCDD-induced toxicity is mediated by the AhR, a basic helix-loop-helix periodicity/ARNT/single-minded (PAS) transcription factor (3, 4). In the absence of a ligand, the AhR is located in the cytoplasm in association with heat shock protein 90, X-associated protein 2, and heat shock protein 90 co-chaperone p23 (5). Once a ligand, such as TCDD, binds to the AhR, the complex is translocated into the nucleus where it forms a heterodimer with an AhR nuclear translocator (ARNT). The AhR/ARNT heterodimer binds to specific DNA sequences termed xenobiotic response elements (XREs), and it enhances the expression of genes such as cytochrome P-450 1A1 (CYP1A1) (4, 6). On the other hand, it has also been shown that the ligand-activated AhR directly interacts with retinoblastoma protein (RB) (7, 8) and NF-{kappa}B (RelA) (9), and their direct interactions modulate the signaling pathways involved in many physiological functions. Although many studies have been conducted, the precise mechanism for individual toxicities of TCDD remains to be clarified.

As regards immunotoxicity, TCDD induces thymus atrophy and suppresses both humoral and cellular immunity in an AhR-dependent manner (10, 11). Recent studies using chimeric mice with the AhR in either hemopoietic or stromal tissues showed that TCDD induces thymus atrophy by directly affecting thymocytes (immature T cells) and not dendritic or stromal cells (12, 13). Additionally, it has been suggested that TCDD-induced thymus atrophy can be attributed to the inhibition of G1/S cell cycle progression in thymocytes at the double negative stages of T cell development (12). In terms of cellular immunity, it has been reported that full suppression of the cytotoxic T lymphocyte response by TCDD required AhR expression in both CD4+ and CD8+ T cells; this indicates that T cells are direct targets of TCDD (14). With regard to the suppressive effect of TCDD on humoral immunity, primary effects on both B cells and T cells have been reported by several groups (11, 15). Recently, we showed that TCDD considerably reduces the production of the T cell growth factor IL-2 and CD4+ type 2 helper T cell-derived cytokines prior to the inhibition of antibody suppression in mice immunized with ovalbumin (16–18). Furthermore, TCDD suppressed the increase in the number of T cells in the spleen, following immunization. This suggests that TCDD inhibits the activation of antigen-specific T cells and their subsequent expansion, which probably leads to deteriorated antibody production (16). All these findings strongly suggest that T cells are a vulnerable target of TCDD toxicity, with regard to not only thymus atrophy and inhibition of cellular immunity but also the suppression of humoral immunity. Our recent finding that primary T cells have functional AhR also supports this mechanism (19).

In the present study, to investigate the effects of activated AhR on T cells and their underlying mechanism, we transiently expressed a constitutively active AhR (CA-AhR) mutant in human leukemic Jurkat T cells, because all the T cell lines examined thus far, including Jurkat T cells, do not have functional AhR (20, 21). We used a CA-AhR mutant lacking the minimal PAS B motif, which is constitutively localized in the nucleus and activates AhR-dependent transcription independent of the ligand (22, 23). We also generated an A78D mutant of the CA-AhR, which lacks the ability of XRE-dependent transcription (24), and examined the involvement of XRE-dependent transcription in the effects of CA-AhR on Jurkat T cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—pEB6CAGFP (an Epstein-Barr virus-based expression vector for green fluorescent protein (GFP) driven by a CAG promoter) (25) and pEB6CAGMCS (containing multicloning sites) were kindly provided by Dr. Yoshihiro Miwa (University of Tsukuba, Tsukuba, Japan). A CA-AhR cDNA2 was subcloned into a KpnI-HindIII site of pEB6CAGMCS, and pEB6CAG-CA-AhR-GFP, encoding a CA-AhR fused to GFP, was generated by inserting the SalI-AflII fragment of pEGFP-N3 (Clontech Laboratories, Inc., Palo Alto, CA). To examine whether the effects of CA-AhR on Jurkat T cells are mediated by XRE-dependent transcription, the mutation changing the alanine at position 78 to aspartic acid (A78D) (24) in the CA-AhR was introduced by the use of a QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. A primer with the sequence 5'-GTCAGCTACCTGAGGGACAAGAGCTTCTTTGATG-3' and its complementary equivalent were employed.

Cell Line, Transient Transfection, and Sorting—Jurkat T cells were obtained from the Cell Resource Center for Biomedical Research (Tohoku University, Sendai, Japan) and maintained in RPMI 1640 (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 10 mM HEPES (pH 7.1), 1 mM pyruvate, and 50 µM 2-mercaptoethanol at 37 °C in 5% CO2. Jurkat T cells (2 x 106 cells) were transiently transfected with 6 µg of pEB6CAGFP, pEB6CAG-CA-AhR-GFP, or pEB6CAG-A78D-GFP using DMRIE-C reagent (Invitrogen) according to the manufacturer's instructions. After 2 days, GFP-positive cells from each transfectant were sorted using a FACSVantage SE (BD Biosciences). The efficiency of the sorting was confirmed using a FACSCalibur (BD Biosciences), and 98–99% of the sorted cells were GFP-positive. The sorted cells were cultured at 1 x 105 cells/ml, and then growth rate, apoptosis, and cell cycle distribution were examined as described below. The results obtained in each experiment were confirmed in another independent experiment, and a set of representative results has been shown under "Results."

Detection of Apoptosis—For the detection of apoptotic cells by annexin V binding and propidium iodide (PI) staining, we used an Annexin V-biotin apoptosis detection kit (BioVision, Palo Alto, CA) according to the manufacturer's instructions, with minor modifications. At 0, 2, and 4 days after sorting, the cells were incubated with biotin-labeled annexin V for 5 min at room temperature. After washing, the cells were incubated with streptavidin-labeled allophycocyanin (SA-APC, BD Biosciences) for 15 min at room temperature. After washing, PI was added, and the cells were analyzed using a FACSCalibur.

Furthermore, the induction of apoptosis was confirmed by apoptotic morphological changes. The sorted cells were cultured for 2 days and then stained with 4 µM bisbenzimide (Hoechst 33342, ICN Biomedicals Inc., Aurora, OH) for 15 min. The apoptotic cells were examined by the changes in their nuclear morphology, i.e. nuclear fragmentation, under a UV-visible fluorescence microscope. Approximately over 100 cells were counted in four microscopic fields, and the percentage of apoptotic cells was estimated.

Cell Cycle Analysis—At 0, 2, and 4 days after sorting, the cells were stained with PI using a CycleTEST Plus DNA reagent kit (BD Biosciences) according to the manufacturer's instructions, and their DNA content was measured using a FACSCalibur. The percentages of cells in the G1, S, and G2/M phases were analyzed using ModFit software (BD Biosciences).

Affymetrix GeneChip Analysis—Affymetrix GeneChip analysis was performed according to the Affymetrix expression analysis technical manual (Affymetrix, Santa Clara, CA), with some modifications. After sorting, total RNA was isolated using an RNeasy Mini Kit (Qiagen, Chatsworth, CA). Double-stranded cDNA was synthesized from 1 µg of total RNA using SuperScript II reverse transcriptase (Invitrogen) and T7 oligo(dT)24 primer (Affymetrix). The double-stranded cDNA was purified by the phenol/chloroform extraction method, followed by ethanol precipitation. The in vitro transcription reaction was performed using a BioArray high yield RNA transcript-labeling kit (Enzo Diagnostics, Farmingdale, NY). 15 µg of the biotin-labeled cRNA was fragmented and hybridized to a Human Genome U133A array (Affymetrix). The hybridized probe array was washed, stained, and scanned. The data were analyzed using Affymetrix Microarray Suite 5.0 software. A comparison analysis was performed to obtain genes with at least 2-fold changes in Jurkat T cells expressing CA-AhR-GFP as compared with cells expressing GFP alone.

Semiquantitative RT-PCR—To confirm the gene expression changes observed by the Affymetrix GeneChip analysis, semiquantitative RT-PCR was performed on the double strand cDNA prepared for Affymetrix GeneChip analysis. Primers used in the present study were designed using PRIMER3 (frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) and NCBI UniSTS (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unists), based on human sequences published in the NCBI data base. Primer sequences, PCR cycle numbers, and the annealing temperatures of each gene are shown in Table I. Each double-stranded cDNA was amplified in the exponential phase of PCR using TaKaRa Taq (TaKaRa Shuzo Co., Ltd., Tokyo, Japan). The amplification was carried out by an initial incubation at 94 °C for 2 min, followed by 19–30 cycles of 94 °C for 30 s, 55 or 60 °C for 30 s, and 72 °C for 30 s, and a final extension at 72 °C for 7 min. The PCR products were separated in a 1.2% Synergel (Diversified Biotech, Boston, MA) containing 0.5 µg/ml ethidium bromide. The net intensity of the bands was quantified using Kodak EDAS 290. The expression level of each gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase or {beta}-actin.


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  		TABLE I
List of primers used for semiquantitative RT-PCR

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CA-AhR Inhibits Growth of Jurkat T Cells—To examine the effect of AhR activation on T cells, we used a CA-AhR mutant lacking the minimal PAS B motif (Fig. 1A). The CA-AhR mutant has been reported to form a heterodimer with ARNT and induce XRE-dependent gene expression in Chinese hamster ovary cells and MCF-7 cells in the absence of a ligand (22, 23). First, we examined CYP1A1 expression to confirm that CA-AhR induces XRE-dependent gene expression in Jurkat T cells. Jurkat T cells, which expressed ARNT but not AhR (data not shown), were transiently transfected with an expression vector for either CA-AhR-GFP or GFP alone. Two days after the transfection, GFP-positive cells were sorted and RT-PCR for CYP1A1 mRNA was performed. As shown in Fig. 1B, CA-AhR-GFP, but not GFP alone, markedly induced CYP1A1 mRNA expression, indicating that the CA-AhR is functional in Jurkat T cells. In addition, the green fluorescence emitted from CA-AhR-GFP was mainly found in the nuclear compartment (data not shown).



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  		FIG. 1.
Structure of a constitutively active mutant of an arylhydrocarbon receptor (CA-AhR) and CYP1A1 induction in Jurkat T cells expressing CA-AhR. A, the structures of wild-type AhR and a CA-AhR fused to GFP (CA-AhR-GFP) are shown. The CA-AhR lacks the minimal PAS B motif. B, the expression vector for either CA-AhR-GFP or GFP alone was transiently transfected into Jurkat T cells using DMRIE-C reagent. Two days after transfection, GFP-positive cells were sorted using a FACSVantage SE. Total RNA was isolated from the cells, and mRNA expression levels of CYP1A1 and glyceraldehyde-3-phosphate dehydrogenase were examined by semiquantitative RT-PCR.

 
To examine the effect of CA-AhR on the growth rate of Jurkat T cells, the sorted cells were cultured for up to 4 days, and the cell numbers were counted at the indicated times. As shown in Fig. 2, the cells expressing GFP alone increased 10-fold, 4 days after sorting. In contrast, the expression of CA-AhR-GFP completely inhibited the increase in cell numbers up to 4 days after sorting, indicating that the activation of AhR greatly inhibits the growth of Jurkat T cells.



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  		FIG. 2.
CA-AhR inhibits growth of Jurkat T cells. The expression vector for either CA-AhR-GFP or GFP alone was transiently transfected into Jurkat T cells using DMRIE-C reagent. After 2 days, GFP-positive cells from each transfected cells were sorted using a FACSVantage SE and then cultured at 1 x 105 cells/ml. The cell numbers at the indicated times were determined by trypan blue exclusion.

 
CA-AhR Induces Apoptosis in Jurkat T Cells—Because CA-AhR was shown to induce growth inhibition, we examined whether the expression of CA-AhR induces apoptosis in Jurkat T cells. Apoptotic cells were detected with annexin V, which monitors the appearance of phosphatidylserine on the cell surface during apoptosis. In addition, PI was used to distinguish between early and late apoptosis, because PI is excluded only by live and early apoptotic cells. As shown in Fig. 3, where annexin V-positive, PI-negative cells (upper left quadrant) represent early apoptotic cells and annexin V, PI-double positive cells (upper right quadrant) represent late apoptotic/necrotic cells, GFP-alone-transfected cells did not show remarkable changes in the ratio of dead cells. On the other hand, CA-AhR-GFP increased the percentage of apoptotic cells, especially 2 and 4 days after sorting. Two days after sorting, the percentage of early apoptotic cells was 3-fold higher in cells expressing CA-AhR-GFP than in those expressing GFP alone. Moreover, 4 days after sorting, CA-AhR-GFP augmented the percentages of early apoptotic cells and late apoptotic/necrotic cells by approximately 8- and 6-fold, respectively, as compared with GFP alone.



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  		FIG. 3.
CA-AhR induces apoptosis in Jurkat T cells. The expression vector for either CA-AhR-GFP or GFP alone was transiently transfected into Jurkat T cells using DMRIE-C reagent. After 2 days, GFP-positive cells were sorted from the transfected cells using a FACSVantage SE and then cultured for the indicated time periods. The cells were incubated with biotin-labeled annexin V, followed by staining with SA-APC and PI, and analyzed using a FACSCalibur. The upper left quadrant (annexin V-positive, PI-negative) represents early apoptotic cells, whereas the upper right quadrant (annexin V, PI-double positive) represents late apoptotic/necrotic cells.

 
Furthermore, the induction of apoptosis was confirmed by nuclear morphological changes. Each group of sorted cells was cultured for 2 days and stained with Hoechst 33342 dye. Fragmented nuclei were observed in a number of cells expressing CA-AhR-GFP (Fig. 4A, arrowheads), and apoptotic cells reached about 30%; however, the apoptotic cells only reached 10% in cells expressing GFP alone (Fig. 4B).



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  		FIG. 4.
CA-AhR induces apoptotic morphological changes in Jurkat T cells. A, the expression vector for either CA-AhR-GFP or GFP alone was transiently transfected into Jurkat T cells using DMRIE-C reagent. After 2 days, GFP-positive cells were sorted from the transfected cells using a FACSVantage SE. The sorted cells were cultured for 2 days and then stained with 4 µM Hoechst 33342 for 15 min. The apoptotic cells were examined by the changes in their nuclear morphology, i.e. with fragmentation under a phase-contrast and a UV-visible fluorescence microscope. The arrowheads indicate the fragmented nuclei of the apoptotic cells. B, approximately over 100 cells were counted in four microscopic fields under a UV-visible fluorescence microscope, and the percentage of the apoptotic cells was estimated.

 
CA-AhR Induces the Accumulation of Cells in the G1 Phase—We also investigated the possibility that CA-AhR induces cell cycle arrest. As shown in Fig. 5, in non-transfected Jurkat T cells, 48% of the cells were in the G1 phase, 37% were in the S phase, and 15% were in the G2/M phase. Immediately after sorting (0 day), no difference was observed in the DNA profile among non-transfected cells, cells expressing GFP alone, and those expressing CA-AhR-GFP. Two days after sorting, the percentage of cells expressing CA-AhR-GFP rose to 61% in the G1 phase and correspondingly decreased to 24% in the S phase, whereas no change in cell cycle distribution was observed in cells expressing GFP alone. Four days after sorting, the percentages in the individual phases were not significantly changed from those obtained 2 days after sorting. These results suggest that CA-AhR affects cell cycle progression, especially in the G1 phase.



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  		FIG. 5.
CA-AhR increases the percentage of cells in the G1 phase. The expression vector for either CA-AhR-GFP or GFP alone was transiently transfected into Jurkat T cells using DMRIE-C reagent. After 2 days, GFP-positive cells were sorted from the transfected cells using a FACSVantage SE and then cultured for the indicated time periods. The cells were stained with PI using a CycleTEST Plus DNA reagent kit, and DNA content was measured using a FACSCalibur. The percentages of cells in the G1, S, and G2/M phases were analyzed using ModFit software.

 
The Inhibition of Growth by CA-AhR Is Mediated by Both XRE-dependent and -independent Mechanisms—To elucidate whether the inhibitory effect of CA-AhR on the growth of Jurkat T cells is mediated by an XRE-dependent or -independent mechanism, we generated an A78D mutant of the CA-AhR. The A78D mutant of wild-type AhR is translocated into the nucleus in the presence of TCDD and forms a heterodimer with ARNT. However, it lacks the potential for XRE-driven gene expression due to impaired XRE binding (24). To confirm that the disruption of transcription by A78D mutation is available for the CA-AhR, we examined the effects of A78D mutation introduced into the CA-AhR on localization and gene expression. When either CA-AhR-GFP or A78D mutant of CA-AhR-GFP (A78D-GFP) was transiently expressed in Jurkat T cells, a FACS analysis showed the same GFP expression patterns between both types of transfected cells, before and after sorting (Fig. 6A). Furthermore, the mRNA expression of CA-AhR-GFP or A78D-GFP was detected at the same level in both transfected cells by semiquantitative RT-PCR (Fig. 6B). The green fluorescence emitted by the GFP was mainly observed in the nuclear compartment in cells expressing A78D-GFP as well as CA-AhR-GFP (data not shown). However, as shown in Fig. 6B, only CA-AhR-GFP, but not A78D-GFP, induced CYP1A1 mRNA expression. These observations show that A78D-GFP is constitutively localized in the nucleus in the absence of TCDD, but it cannot induce gene expression by binding to the XRE.



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  		FIG. 6.
CA-AhR inhibits the growth of Jurkat T cells by the XRE-dependent and -independent mechanisms. A, the expression vector for either CA-AhR-GFP, A78D-GFP, or GFP alone was transiently transfected into Jurkat T cells using DMRIE-C reagent. Two days after the transfection, GFP-positive cells were sorted using a FACSVantage SE. The GFP expression of the cells was analyzed before and after sorting, using a FACSCalibur. B, total RNA was isolated from the cells and mRNA expression levels of CYP1A1, CA-AhR-GFP, and glyceraldehyde-3-phosphate dehydrogenase were examined by semiquantitative RT-PCR. C, the sorted cells were cultured at 1 x 105 cells/ml, and the cell numbers at the indicated times were determined by trypan blue exclusion.

 
Using these AhR mutants, the XRE dependence of the inhibitory effect of activated AhR was examined. As shown in Fig. 6C, CA-AhR markedly inhibited the increase in cell number (in agreement with the data shown in Fig. 2). On the other hand, the A78D mutant only partially inhibited the increase. This result indicates that both XRE-dependent and -independent mechanisms are involved in the CA-AhR-induced growth inhibition.

CA-AhR Induces Expression Changes of Genes Related to Apoptosis and Cell Cycle Arrest by an XRE-dependent Mechanism—Because CA-AhR induced apoptosis and the accumulation in the G1 phase in Jurkat T cells, we examined whether CA-AhR changes the expression of genes related to apoptosis and cell cycle arrest and whether the regulations of these genes are mediated by XRE-dependent transcription. Two days after transfection, total RNA was isolated from the GFP-positive cells, and the gene expression was analyzed using an Affymetrix GeneChip. Genes related to apoptosis and cell cycle arrest were selected from the genes that showed at least a 2-fold change in gene expression in the cells expressing CA-AhR-GFP, as compared with cells expressing GFP alone, and their expression changes were confirmed by semiquantitative RT-PCR. Furthermore, we determined the relative -fold induction of each of the confirmed genes in cells expressing CA-AhR-GFP and in cells expressing A78D-GFP by a comparison with the cells expressing GFP alone (Fig. 7 and Table II). We found that CA-AhR up-regulates genes related to apoptosis (caspase 8, c-Jun, and Fas) (26, 27) and cell cycle arrest (cyclin G2, growth arrest and DNA-damage-inducible, alpha (GADD45A), p21waf1, cell division autoantigen-1 (CDA1), and IL-9 receptor) (28–32). CA-AhR also up-regulated the genes involved in both apoptosis and cell cycle arrest (dual specificity phosphatase 6, GADD34, and TGF-{beta} receptor II) (33–36). On the other hand, c-Myc, which plays an important role in the G1/S transition (37), was down-regulated in cells expressing CA-AhR-GFP. Furthermore, our results clarified that all the changes in these genes were dependent on transcription through the XRE, because only CA-AhR, but not A78D, induced expression changes of these genes (Fig. 7 and Table II).



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  		FIG. 7.
CA-AhR induces expression changes of genes related to apoptosis and cell cycle arrest by an XRE-dependent mechanism. The expression vector for either CA-AhR-GFP, A78D-GFP, or GFP alone was transiently transfected into Jurkat T cells using DMRIE-C reagent. Two days after the transfection, GFP-positive cells were sorted using a FACSVantage SE. Total RNA was isolated from the cells, and gene expression was analyzed using an Affymetrix GeneChip. Genes related to apoptosis and cell cycle arrest were collected from the genes that showed at least a 2-fold change in gene expression in the cells expressing CA-AhR-GFP, as compared with the cells expressing GFP alone, and the expression changes of these genes were confirmed by semiquantitative RT-PCR.

 


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  		TABLE II
Relative-fold induction of CA-AhR-regulated genes in the XRE-dependent and -independent fashion Gene expression changes by CA-AhR were examined by Affymetrix GeneChip analysis, and genes related to apoptosis and cell cycle arrest with at least two-fold changes in CA-AhR-GFP-transfected cells, as compared with GFP-alone transfected cells, were selected. Relative expression levels of the selected genes in cells expressing either CA-AhR-GFP or A78D-GFP were determined by semiquantitative RT-PCR.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, CD4+ T cell line Jurkat T cells were transiently expressed with a CA-AhR, a model of ligand-activated AhR. We demonstrated that CA-AhR remarkably inhibits the growth of Jurkat T cells. We also clarified that CA-AhR induces both apoptosis and accumulation in the G1 phase, which strongly suggests that these effects induce growth inhibition in Jurkat T cells. Furthermore, we showed that CA-AhR-induced growth inhibition is mediated by both XRE-dependent and -independent mechanisms, using an A78D mutant of the CA-AhR. With regard to the XRE-dependent mechanism, our results demonstrate that CA-AhR induces expression changes in genes related to apoptosis and cell cycle arrest. On the other hand, the XRE-independent growth inhibition, which is caused by the A78D mutant of the CA-AhR, may result from the interaction between CA-AhR and its target molecules. For instance, it has been reported that RB specifically interacts with both an LXCXE motif in PAS B of the AhR and the C-terminal region of the AhR and that their direct interaction inhibits E2F-driven gene expression, leading to G1 arrest (7, 8, 38). Although the CA-AhR protein lacks a PAS B region containing an LXCXE motif, it possesses the ability to specifically interact with RB through the C-terminal region. The semiquantitative RT-PCR data in our present and previous study (19) suggest that the CA-AhR was expressed in Jurkat T cells at a much higher level than the native AhR in mouse thymocytes and splenocytes. Therefore, we also cannot rule out the possibility that the XRE-independent growth inhibition was due to artifactual effects, such as stress response, of overexpression of the AhR protein in the nucleus. Further studies will be needed to elucidate the mechanism of XRE-independent growth inhibition.

The gene expression analysis suggests the involvement of several signaling pathways in the growth suppression of Jurkat T cells. The increase in Fas and caspase 8 transcripts by CA-AhR suggests that the Fas signaling pathway is involved in the CA-AhR-induced apoptosis. In agreement with our present data, previous studies using mice having a deficiency in the Fas signaling pathway have shown that TCDD decreases the cell number of anti-CD3-activated T cells through a Fas signaling pathway (39, 40). The study by Zeytun et al. (40) reported that Fas ligand was up-regulated in the spleen cells of mice exposed to TCDD. However, CA-AhR did not increase the expression level of Fas ligand in Jurkat T cells. This discrepancy between our study and that of Zeytun et al. suggests that TCDD-induced up-regulation of Fas ligand is due to the effect on non-T cells in spleen cells. As another apoptosis-related gene, we found that CA-AhR increases TGF-{beta} receptor II transcript. The TGF-{beta} signaling pathway has been reported to induce apoptosis and cell cycle arrest in T cells (36). Although CA-AhR induces no changes in the expression level of the TGF-{beta} family (TGF-{beta}1, {beta}2, and {beta}3) in Jurkat T cells (data not shown), the up-regulation of TGF-{beta} receptor II by AhR activation in T cells may cause an increase in their susceptibility to TGF-{beta}. CA-AhR also regulated a number of genes related to cell cycle arrest. We found that CA-AhR induces expression changes of genes involved not only in G1 arrest (cyclin G2 and c-Myc) (28, 37) but also in G2 arrest (GADD45A) (29), in both G1 and G2 arrest (p21waf1) (30), and in multiphase cell cycle arrest (CDA1) (31). Through a cell cycle analysis, we showed that CA-AhR induced the accumulation in the G1 phase by culturing for 2 days after sorting. However, further accumulation in the G1 phase was not found by culturing for 4 days after sorting. These results may suggest that CA-AhR causes alteration of the G1/S transition at an early stage and then inhibits cell cycle progression at various stages of the cell cycle.

With regard to how CA-AhR regulates the transcription of these genes, a search for the human genome sequences of the NCBI demonstrated that the 5'-flanking regions of GADD34, IL-9 receptor, CDA1, and c-Jun genes contain the core consensus sequence of XRE (5'-TNGCGTG-3' or 5'-CACGCNA-3'), suggesting that these genes are directly regulated by activated AhR through the XRE, whereas other genes seem to be regulated by indirect mechanisms. The expression of Fas, GADD45A, p21waf1, and caspase 8 are known to be up-regulated by the activation of p53 (41, 42). Recently, it has been reported that GADD34 induces phosphorylation of p53 and enhances p21waf1 expression (35). Likewise, CA-AhR may up-regulate genes such as Fas, GADD45A, and caspase 8 through induction of GADD34 and following p53 activation. In addition, it has been reported that the induction of CYP1A1 causes DNA damage (43), probably leading to the activation of p53. Therefore, this p53-dependent pathway may be involved in CA-AhR-induced apoptosis and cell cycle arrest.

Previous studies have reported that TCDD induces apoptosis in AhR-null T cell clones, including Jurkat T cells, in an AhR-independent manner (20, 44). However, Jurkat T cells used in this study were not susceptible to apoptosis even in the presence of 10 nM TCDD (data not shown). Although the reason for the discrepancy is unclear, our results well indicate that activated AhR is essential for the inhibition of T cell growth by TCDD, in agreement with the findings that AhR expression is indispensable for TCDD-induced immunosuppression in vivo (10, 11).

In summary, we demonstrated that CA-AhR induces the growth inhibition of Jurkat T cells, with an increase in apoptosis and the accumulation of the cells in the G1 phase. Furthermore, we showed that both XRE-dependent and -independent mechanisms are involved in CA-AhR-induced growth inhibition and that CA-AhR regulates the expression of several genes related to apoptosis and cell cycle arrest in an XRE-dependent manner. Further studies will aim to identify target gene(s) and protein(s) mainly responsible for the inhibition of T cell growth by the XRE-dependent and -independent mechanisms. CD4+ helper T cells play an important role in both humoral and cellular immunity, where TCDD inhibited the increase in the number of CD4+ T cells, following immunization (16, 45). The present data may provide a mechanism for the suppression of both humoral and cellular immunity.


   FOOTNOTES
 
* This work was supported by grants from the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (to C. T., K. N., T. I., and S. T.) and the Ministry of the Environment. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: National Institute for Environmental Studies, Tsukuba 305-8506, Japan. Tel.: 81-29-850-2500; Fax: 81-29-850-2574; E-mail: keikon{at}nies.go.jp.

1 The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, arylhydrocarbon receptor; ARNT, arylhydrocarbon receptor nuclear translocator; XRE, xenobiotic response element; CA-AhR, constitutively active arylhydrocarbon receptor; PAS, periodicity/ARNT/single-minded; RB, retinoblastoma protein; IL-2, interleukin-2; GFP, green fluorescent protein; PI, propidium iodide; RT, reverse transcriptase; CYP1A1, cytochrome P-450 1A1; FACS, fluorescence-activate cell sorting; TGF, transforming growth factor; CDA1, cell division autoantigen-1; GADD45A, growth arrest and DNA-damage-inducible, alpha. Back

2 Y. Fujii-Kuriyama and J. Mimura, unpublished data. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Drs. Yoshihiro Miwa and Junko Tanaka (University of Tsukuba) for providing pEB6 expression vectors and technical advice on plasmid construction, and Kazuhiro Shiizaki (National Institute for Environmental Studies) for technical advice on point mutation in the AhR. We also thank Michiyo Matsumoto for their excellent technical and Kyoko Nakazawa for secretarial assistance.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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