Expression of Aryl Hydrocarbon Receptor Repressor in Normal Human Tissues and Inducibility by Polycyclic Aromatic Hydrocarbons in Human Tumor-Derived Cell Lines

Yuki Tsuchiya, Miki Nakajima, Satsuki Itoh, Masashi Iwanari and Tsuyoshi Yokoi1

Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi 13-1, Kanazawa 920-0934, Japan

Received October 23, 2002; accepted December 9, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aryl hydrocarbon receptor repressor (AhRR) has been recently identified as a negative factor that suppresses aryl hydrocarbon receptor (AhR)-mediated transcriptional gene expression. In the present study, the expression level of AhRR in normal human tissues was determined. AhRR mRNA was detected in liver, breast, colon, kidney, lung, bladder, uterus, testis, ovary, and adrenal gland. The expression level in the testis was prominently high. AhRR mRNA was also detected in various human tissue–derived cell lines, HepG2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), LS-180 (colon carcinoma), ACHN (renal carcinoma), A549 (lung carcinoma), HT-1197 (bladder carcinoma), HeLa (cervix of uterus adenocarcinoma), NEC14 (testis embryonal carcinoma), and OMC-3 (ovarian carcinoma). Since the expression level of AhRR mRNA was prominently high in HeLa cells, it is suggested that the high expression level of AhRR might work as a negative factor for the low inducibility of the CYP1 family in HeLa cells. The expression of AhRR mRNA was induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylchoranthrene (3-MC) in HepG2, MCF-7, LS-180, and OMC-3 cells, but not in ACHN, A549, HT-1197, HeLa, and NEC14 cells. The responsiveness was similar to the cell-specific inducibility of the CYP1 family. The inducibility of AhRR mRNA by nitropolycyclic aromatic hydrocarbons (NPAHs) as well as their parent PAHs was compared in HepG2 and OMC-3 cells. The chemical-specific inducibility of AhRR was also similar to that of the CYP1 family determined in our previous study. These results indicated that AhRR is also induced in chemical- and cell-specific manners.

Key Words: aryl hydrocarbon receptor repressor; aryl hydrocarbon receptor; aryl hydrocarbon receptor nuclear translocator; cytochrome P450; induction; TCDD.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl hydrocarbon receptor repressor (AhRR) has been recently identified as a negative factor that suppresses the aryl hydrocarbon receptor (AhR) function (Mimura et al., 1998Go). Polycyclic aromatic hydrocarbons (PAHs) bind to AhR that exists in cytoplasm, and the liganded receptor subsequently translocates to the nucleus, where it switches its partner molecule from Hsp90 to the AhR nuclear translocator (ARNT) (Hankinson, 1995Go; Schmidt and Bradfield, 1996Go; Whitolck, 1999). In the nucleus, the formed AhR/ARNT heterodimer binds to the xenobiotic responsive element (XRE) sequences present in the 5‘-flanking region of target genes such as cytochrome P450 (CYP) 1A1, glutathione S-transferase, quinone reductase, aldehyde dehydrogenase-3, and UDP-glucuronosyltransferase (Asman et al., 1993Go; Emi et al., 1996Go; Favreau and Pickett, 1991Go; Fujisawa-Sehara et al., 1987Go; Rushmore et al., 1990Go). It has been reported that AhRR inhibits the AhR function by competing with AhR for dimerization with ARNT and binding to the XRE (Mimura et al., 1998Go). At present, AhRR gene has been identified in mice (Mimura et al., 1998Go), humans (Watanabe et al., 2001Go), and Atlantic killifish (Fundulus heteroclitus; Karchner et al., 2002Go). In the 5‘-flanking region of the AhRR genes, there are XRE sequences. Therefore, it has also been reported that the expression of AhRR is also induced by PAHs via binding of the AhR/ARNT heterodimer to the XREs in the 5‘-flanking region of the AhRR gene (Baba et al., 2001Go).

The expression of AhRR in tissues has been determined in mice (Mimura et al., 1998Go) and Atlantic killifish (Karchner et al., 2002Go). Recently, Fujita et al.(2002)Go reported that AhRR mRNA is expressed in the lung, kidney, spleen, and thymus of the human fetus, but not in brain, liver, heart, and muscle. However, the constitutive expression of AhRR in human adult tissues and its inducibility is unknown. In the present study, the constitutive expression levels of AhRR mRNA in normal adult human tissues and in various human tissue–derived cell lines were investigated. To investigate the inducibility of human AhRR gene, nine human tissue–derived cell lines were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene (3-MC). In our previous study (Iwanari et al., 2002Go), we found that nitro-substituted PAHs (NPAHs) can induce the human CYP 1 family in a chemical-specific manner. Therefore, the induction potency of AhRR by various PAH and NPAH compounds was also compared in HepG2 and OMC-3 cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents.
Pyrene (Py), 1-nitropyrene (1-NP), and benz[a]anthracene (B[a]A) were obtained from Tokyo Chemical Industry (Tokyo, Japan). 7-Nitrobenz[a]anthracene (7-NB[a]A) and 6-nitrobenzo[a]pyrene (6-NB[a]P) were obtained from the National Cancer Institute (Bethesda, MD). Benzo[a]pyrene (B[a]P) and 6-aminochrysene (6-AC) were from Sigma Chemical (St. Louis, MO). 6-Nitrochrysene (6-NC) and 2-nitrofluoranthene (2-NF) were from Chemsyn Science Laboratories (Lenexa, KS). Fluoranthene (Flu), 3-nitrofluoranthene (3-NF), and 3-methylchoranthrene (3-MC) were from Wako Pure Chemical Industries (Osaka, Japan). 1-Aminopyrene (1-AP), 1,3-dinitropyrene (1,3-DNP), 1,6-dinitropyrene (1,6-DNP), 1,8-dinitropyrene (1,8-DNP), and chrysene (Chry) were from Aldrich Chemical (Milwaukee, WI). TCDD was obtained from Cambridge Isotope Laboratories (Cambridge, MA). ISOGEN, RNA extraction reagent, and random hexamer were from Nippon Gene (Tokyo, Japan) and Takara (Kyoto, Japan), respectively. Moloney murine leukemia virus-reverse transcriptase (MMLV-RT), Taq polymerase, and [{alpha}-32P]dCTP (> 2500 Ci/mmol) were from Toyobo (Osaka, Japan), Greiner Japan (Tokyo, Japan), and Amersham (Buckinghamshire, UK), respectively. All primers were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). Other chemicals were of the highest grade commercially available.

RNA samples from normal human tissues.
Total RNA samples of human liver, breast, colon, kidney, bladder, uterus, and ovary were obtained from Stratagene (La Jolla, CA). Total RNA samples of human testis and adrenal gland were obtained from Clontech (Palo Alto, CA), and the total RNA sample of human lung was from Cell Applications Inc. (San Diego, CA). The liver sample was obtained at the normal margin to trabecular carcinoma from a single donor, a 45-year-old male. The breast sample was pooled tissues from two female donors, 39- and 49-years-old. The colon sample was pooled tissues from two female donors, 62- and 67-years-old. The kidney, lung, and ovary samples were each a single 56-year-old male, a 40-year-old male, and a 73-year-old female, respectively. The bladder sample was pooled tissues from two female donors, 24- and 42-years-old. The uterus sample was pooled tissues from three female donors, 54-, 68-, and 76-years-old. The testis sample was pooled tissues from 45 Caucasians, ages 19–64. The adrenal gland sample was pooled tissues from 62 male/female Caucasians, ages 15–61. Information concerning smoking or medication of the donors was not available.

Cell lines and cell culture.
The human cell lines HepG2 (hepatocellular carcinoma), A549 (lung carcinoma), HeLa (cervix of uterus adenocarcinoma), OMC-3 (ovarian carcinoma), and NEC14 (testis embryonal carcinoma) were obtained from Riken Gene Bank (Tsukuba, Japan). MCF-7 (breast carcinoma), LS-180 (colon carcinoma), ACHN (renal carcinoma), and HT-1197 (bladder carcinoma) were from American Type Culture Collection (Rockville, MD). Cells were plated on 100-mm diameter dishes. HepG2, A549, and HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) with 10% fetal bovine serum (FBS; Invitrogen, Melbourne, Australia). MCF-7, LS-180, ACHN, and HT-1197 cells were cultured in DMEM with 0.1 mM nonessential amino acid (Invitrogen) and 10% FBS (Invitrogen). NEC14 cells were cultured in RPMI 1640 medium (Nissui Pharmaceutical) with 10% FBS (Invitrogen). OMC-3 cells were cultured in Ham’s F12 medium (Nissui Pharmaceutical) with 10% FBS (Bio Whittaker, Walkersville, MD). These cells were cultured in an atmosphere of 5% CO2/95% air at 37°C.

Treatment of cells and isolation of total RNA.
To determine the concentration-dependent induction of AhRR mRNA, HepG2 cells were treated with 0.1 pM–10 nM TCDD for 24 h or 0.1 nM–10 µM 3-MC for 6 h. To determine the time-dependent induction of AhRR mRNA, HepG2 cells were treated with 10 nM TCDD or 10 µM 3-MC for 0.5, 1, 2, 3, 6, 12, 24, 48, and 72 h. To determine the AhRR inducibility in various human tissue–derived cell lines, cells were treated with 10 nM TCDD or 10 µM 3-MC for 24 h. To determine the chemical-dependent induction of AhRR, HepG2 and OMC-3 cells were treated with 1 µM of pyrenes (Py, 1-NP, and 1-AP), 1 µM of dinitropyrenes (1,3-, 1,6-, and 1,8-DNPs), 10 µM of fluoranthenes (Flu, 2-NF, and 3-NF), 10 µM of chrysenes (Chry, 6-NC, and 6-AC), 5 µM of benzo[a]pyrenes (B[a]P and 6-NB[a]P), 10 µM of benz[a]anthracenes (B[a]A and 7-NB[a]A), 10 nM of TCDD, or 10 µM of 3-MC for 24 h. The concentrations of various chemicals were determined in our previous study (Iwanari et al., 2002Go). It was confirmed that the concentrations of these chemicals did not affect the cell growth or viability. All chemicals were dissolved in DMSO and the final concentration of the solvent in the culture medium was 0.1%. Control cells were treated with 0.1% DMSO. Total RNA was isolated from the cells using ISOGEN according to the protocol supplied by the manufacturer. The RNA concentration and its purity were determined spectrometrically.

Reverse transcriptase-polymerase chain reaction (RT-PCR).
A 1-µl portion of the RT mixture was added to a PCR mixture containing 0.4 µM of each primer, 0.2 mM dNTPs, 1.0 µCi [{alpha}-32P]dCTP, 1.0 U Taq DNA polymerase, 1.5 mM MgCl2, 67 mM Tris–HCl buffer (pH 8.8), 16.6 mM (NH4) 2SO4, 0.45% Triton X-100, and 0.2 mg/ml gelatin in a final volume of 25 µl. PCR reactions were performed with a DNA Thermal Cycler (Takara). RT-PCR analysis of human AhRR was performed as follows: DNA was denatured at 94°C for 3 min and cycled immediately for 30 cycles (cell lines) and 28 cycles (human tissues): denaturing at 94°C for 45 s, annealing at 56°C for 45 s, and extension at 72°C for 1 min. The primers were AhRR-S: 5‘-AGACTCCAGGACCCACAA-3‘ and AhRR-AS: 5‘-CAGCGTCGGACCACACA-3‘. RT-PCR analyses of human CYP1A1, CYP1A2, CYP1B1, and ß-actin were also performed as we described previously (Iwanari et al., 2002Go). A 20-µl portion of the PCR reaction mixture was electrophoresed on a 10% polyacrylamide gel that was subsequently dried. The data were analyzed with a Fujix Bio-Imaging Analyzer BAS 1000 (Fuji Film, Tokyo, Japan). To normalize RNA loading and PCR variations, the signals of targets were corrected with the signals of ß-actin mRNA as the internal standard.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression of AhRR mRNA in Normal Human Tissues and Human Tissue–Derived Cell Lines
The expression levels of AhRR mRNA in normal human tissues, liver, breast, colon, kidney, lung, bladder, uterus, testis, ovary, and adrenal gland were determined. As shown in Figure 1AGo, AhRR mRNA was detected in all tissues. The most intense band was observed with the sample from testis and the intensity was 39-fold as compared with that in liver. In other tissues, AhRR mRNA was expressed at similar levels to that in liver.



View larger version (28K):
[in this window]
[in a new window]
 
FIG. 1. Constitutive expression levels of mRNAs for AhRR in human normal tissues and in human tissue–derived cell lines. (A) Commercially available total RNA samples of liver, breast, colon, kidney, lung, bladder, uterus, testis, ovary, and adrenal gland were used. (B) Total RNA samples were obtained from HepG2, MCF-7, LS-180, ACHN, A549, HT-1197, HeLa, NEC14, and OMC-3 cells after the treatment with 0.1% DMSO for 24 h. RT-PCR analyses of human AhRR were performed as described in Materials and Methods. The expression level of AhRR was corrected with the expression level of ß-actin. It was confirmed that the signals of ß-actin mRNA were similar in all tissues or cell lines. The values are expressed as the ratio of the AhRR/signal of ß-actin relative to that in liver (A) or in HepG2 cells (B). Data are typical of three independent experiments.

 
The constitutive expression levels of AhRR mRNA in various human tissue-derived cell lines were determined (Fig. 1BGo). AhRR mRNA was detected in all cells. However, the expression levels in ACHN, A549, and HT-1197 were low. In contrast, the expression level in HeLa cells was very high and the intensity was 33-fold as compared with that in HepG2.

Concentration-Dependent Induction of AhRR in HepG2 Cells by TCDD and 3-MC
To determine the concentration-dependent induction of AhRR mRNA, HepG2 cells were treated with various concentrations of TCDD for 24 h, and 3-MC for 6 h. To normalize RNA loading and PCR variations, the expression levels were corrected with the expression level of ß-actin mRNA as the internal standard. We confirmed that the chemicals at the indicated concentrations did not significantly influence the ß-actin mRNA levels. As shown in Figure 2Go, AhRR mRNA was induced by TCDD and 3-MC in a concentration-dependent manner. Treatment with 0.1 nM of TCDD induced AhRR mRNA by three-fold, and treatment with 1 nM and 10 nM TCDD induced AhRR mRNA by seven- and eight-fold, respectively. Treatment with 1 µM of 3-MC resulted in the induction of AhRR mRNA by five-fold, and the maximum induction (nine-fold) was observed by 10 µM of 3-MC.



View larger version (15K):
[in this window]
[in a new window]
 
FIG. 2. Concentration-dependent induction of AhRR mRNA in HepG2 cells. HepG2 cells were treated for 24 h with 0.1 pM–10 nM TCDD or 0.1 nM–10 µM 3-MC. AhRR mRNA levels were determined by RT-PCR as described in Materials and Methods. Fold induction is expressed as the ratio of the AhRR/signal of ß-actin relative to that ratio in control (0.1% DMSO). Data represent the mean ± SD of three independent experiments.

 
Time-Dependent Induction of AhRR and CYP1 Family in HepG2 Cells by TCDD and 3-MC
To determine the time-dependent induction of AhRR mRNA, HepG2 cells were treated with 10 nM of TCDD or 10 µM of 3-MC for each time period, whereas control cells were treated with 0.1% DMSO for each time period. As shown in Figure 3AGo, AhRR mRNA was induced by TCDD by two-fold after 6 h treatment and increased time-dependently to maximum induction after 72 h treatment. The expression levels of the CYP1 family mRNA were also induced by TCDD in a time-dependent manner. The maximal induction levels of CYP1A1, CYP1A2, and CYP1B1 were 24-fold after 48 h treatment, four-fold after 24 h treatment, and 28-fold after 48 h treatment, respectively. Treatment of cells with 10 µM of 3-MC also induced AhRR and CYP 1 family mRNAs in a time-dependent manner (Fig. 3BGo). Interestingly, the response of AhRR toward 3-MC induction was more rapid than those of the CYP1 family. The expression level of AhRR mRNA was induced by eight-fold at 6 h, and remains the induced level at 8- to 11-fold until 72 h. The expression levels of CYP1A1, CYP1A2, and CYP1B1 were induced by 15-, 4- and 15-fold after 24 h treatment, respectively.



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 3. Time-dependent induction of CYP1A1, CYP1A2, CYP1B1, and AhRR mRNAs in HepG2 cells. HepG2 cells were treated with 10 nM of TCDD (A) or 10 µM of 3-MC (B) for 0.5, 1, 2, 3, 6, 12, 24, 48, and 72 h. Each mRNA level was determined by RT-PCR as described in Materials and Methods. Fold induction is expressed as the ratio of the target/signal of ß-actin relative to that in control. Data represent the mean ± SD of three independent experiments.

 
Inducibility of AhRR mRNA in Human Tissue–Derived Cell Lines by TCDD and 3-MC
Nine human tissue–derived cell lines were treated with 10 nM TCDD or 10 µM 3-MC for 24 h. The inducibility of AhRR mRNA was compared with those of the CYP1 family (Fig. 4Go). AhRR mRNA was induced by TCDD or 3-MC in HepG2, MCF-7 and LS-180, and in OMC-3 cells. In these cells, CYP1A1, CYP1A2, and CYP1B1 were also induced. In contrast, in ACHN, A549, HT-1197, and NEC14 cells, AhRR and CYP1 family were not induced by TCDD and 3-MC. Interestingly, HeLa cells with high constitutive expression levels of AhRR failed to show the inducibility of AhRR by TCDD or 3-MC.



View larger version (52K):
[in this window]
[in a new window]
 
FIG. 4. Induction of CYP1A1, CYP1A2, CYP1B1, and AhRR mRNA by TCDD or 3-MC in nine kinds of cell lines. Cells were treated by 10 nM of TCDD (A) or 10 µM of 3-MC (B) for 24 h. RT-PCR was performed as described in Materials and Methods. Fold induction is expressed as the ratio of the target/signal of ß-actin relative to that in control in each cell line. The numbers on column represent fold induction. Data represent the mean of at least two independent experiments. ND, not detectable.

 
Effects of PAHs and NPAHs on AhRR mRNA Levels in HepG2 and OMC-3 Cells
To determine the inducibility of AhRR mRNA, HepG2 and OMC-3 cells were treated with various PAHs and NPAHs for 24 h (Fig. 5Go). In HepG2 cells (Fig. 5AGo), Chry induced AhRR to the same extent as TCDD (10-fold), and 6-NC and 6-AC also induced AhRR mRNA by six- and seven-fold, respectively. B[a]P, 6-NB[a]P, B[a]A, and 7-NB[a]A induced AhRR mRNA by four- to six-fold. 2-NF strongly induced AhRR mRNA by seven-fold, whereas Flu and 3-NF induced AhRR mRNA only by two- to three-fold. The induction efficiency of pyrenes and dinitropyrenes was low (two- to four-fold). As shown in Figure 5BGo, TCDD (10-fold) and 3-MC (eight-fold) showed potent inducibility of AhRR mRNA in the OMC-3 cells. 6-NC and 6-AC induced AhRR by six- and two-fold, respectively, whereas Chry did not affect the expression level of AhRR mRNA. B[a]P and B[a]A induced AhRR mRNA by six-fold, but the nitro-substituted 6-NB[a]P and 7-NB[a]A induced AhRR only by two-fold. The induction efficiency of 2-NF (four-fold) was higher than that of 3-NF and Flu. Pyrenes did not induce AhRR mRNA.



View larger version (22K):
[in this window]
[in a new window]
 
FIG. 5. Effects of various PAHs and NPAHs on AhRR mRNA expression in HepG2 and OMC-3 cells. HepG2 (A) and OMC-3 (B) cells were treated with 1 µM of pyrenes (Py, 1-NP, and 1-AP), 1 µM of dinitropyrenes (1,3-, 1,6-, and 1,8-DNPs), 10 µM of fluoranthenes (Flu, 2-NF, and 3-NF), 10 µM of chrysenes (Chry, 6-NC, and 6-AC), 5 µM of benzo[a]pyrenes (B[a]P and 6-NB[a]P), 10 µM of benz[a]anthracenes (B[a]A and 7-NB[a]A), 10 nM of TCDD, or 10 µM of 3-MC for 24 h. RT-PCR was performed as described in Materials and Methods. Fold induction is expressed as the ratio of the AhRR/signal of ß-actin relative to that in control. Data represent the mean ± SD of three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AhRR is a member of the superfamily bHLH/PAS transcriptional factors and has recently been found to work as a repressor of AhR function. The AhRR gene has been identified in mice (Mimura et al., 1998Go), humans (Watanabe et al., 2001Go), and Atlantic killifish (Karchner et al., 2002Go). The mouse AhRR acts as a negative regulator of the AhR function by competing with AhR for ARNT and by forming AhRR/ARNT complex that bind to XREs. The expression of AhRR in tissues has been determined in the mouse (Mimura et al., 1998Go) and Atlantic killifish (Karchner et al., 2002Go). In 3-MC treated mice, AhRR mRNA was detected in lung, heart, liver, kidney, and intestine, but not in thymus, whereas in untreated mice the expression of AhRR mRNA could not be detected in any of these tissues (Mimura et al., 1998Go). In the untreated Atlantic killifish, the expression of AhRR mRNA was detected in gill, ovary, and gut, but not in brain, kidney, spleen, liver, and heart. TCDD treatment of the Atlantic killifish induced the AhRR expression in all tissues. However, the constitutive expression of AhRR in human adult tissues and its inducibility is unknown. Recently, Fujita et al.(2002)Go reported that AhRR mRNA was expressed in lung, kidney, spleen, and thymus, but not in brain, liver, heart, and muscle in the human fetus. In the present study, we first demonstrated that human AhRR is constitutively expressed in normal human tissues, liver, breast, colon, kidney, lung, bladder, uterus, testis, ovary, and adrenal grand. The expression level of AhRR in testis was particularly high. Taking these results into consideration, it is suggested that the expression of AhRR is possibly regulated developmentally in human liver. Furthermore, there might be species differences in the constitutive expression of AhRR in various tissues. Although information concerning smoking or medication of the donors was not available, we should note the possibility that the constitutive expression of AhRR in human tissues might be affected by smoking or exposure to environmental pollutants.

The expression level of AhRR is high in testis but not in the testis embryonal carcinoma cell line NEC14. In addition, the expression levels of AhRR is high in uterus adenocarcinoma cell line HeLa but not in the uterus. Thus, there was no apparent relationship between human tissues and cell lines concerning the AhRR expression level. In order to determine the inducibility of human AhRR, various human tissue– derived cell lines were treated with TCDD or 3-MC. AhRR in HepG2 cells was induced by TCDD or 3-MC in concentration- and time-dependent manners. These responses of AhRR were similar to those of the CYP1 family demonstrated in our previous study (Iwanari et al., 2002Go). These results support the notion that AhRR is induced via binding of the AhR/ARNT heterodimer to the XREs in the 5‘-flanking region of the AhRR gene. The differences in the induction levels of AhRR and CYP1A1 might be partly due to differences in the binding affinity of the AhR/ARNT heterodimer to XREs, since it has been reported that its binding affinity to XREs on the AhRR gene was lower than that to the XRE on the CYP1A1 gene (Baba et al., 2001Go). The liganded AhR and ARNT heterodimer activates the expression of the AhRR gene, and the expressed AhRR, in turn, inhibits the function of AhR. Accordingly, it has been suggested that AhR and AhRR constitute a regulatory loop of xenobiotic signal transduction (Mimura et al., 1998Go). However, these phenomena could not be demonstrated, since the inductions of AhRR were parallel with those of the CYP1 family. It has been reported that proteins corresponding to the AhRR/ARNT heterodimer in human fibroblasts bind to XRE (Gradin et al., 1999Go, 2002Go). It was considered that the expression of AhRR resulted in no inducibility of CYP1B1 mRNA in human fibroblasts. In contrast to human fibroblasts, the binding of the proteins corresponding to the AhRR/ARNT heterodimer to XRE in HepG2 cells was not observed (Gradin et al., 1999Go). Therefore, it was suggested that the expression level of AhRR protein in HepG2 cells might not be high enough, although the AhRR mRNA was induced by TCDD or 3-MC.

In our previous study (Iwanari et al., 2002Go), we demonstrated that the CYP1 family is inducible in HepG2, MCF-7, LS-180, and OMC-3 cells, but not in ACHN, A549, HT-1197, HeLa, and NEC14 cells. These results were confirmed in the present study (Fig. 4Go). The inducibility of AhRR in various cells was similar to those of the CYP1 family. We first demonstrated that in HeLa cells, the constitutive expression level of AhRR is remarkably high. In HeLa cells, the CYP1 family was also not induced. It has been reported that the high constitutive expression of AhRR would repress the induction of CYP1A1 in normal human skin fibroblasts (Gradin et al., 1999Go). Therefore, it is suggested that the high expression level of AhRR might work as a negative factor in the low induction of the CYP1 family in HeLa cells. In other noninducible cell lines, ACHN, A549, HT-1197, and NEC14 cells, the expression levels of AhRR mRNA were not so high. Another downregulation mechanism might be involved in the noninducibility of the CYP1 family in these cells.

In our previous study (Iwanari et al., 2002Go), we also found that human CYP1A1, CYP1A2, and CYP1B1 were induced by NPAHs as well as their parent PAHs in chemical- and CYP isoform-specific manners. In the present study, the inducibility of AhRR by NPAHs and PAHs was compared with those of the CYP1 family in HepG2 and OMC-3 cells. The induction profile of AhRR by PAHs and NPAHs was similar to those of the CYP1 family (Iwanari et al., 2002Go), although the extent of induction was different between the AhRR and CYP1 isoforms.

In conclusion, we demonstrated that AhRR is constitutively expressed in almost human tissues, and that AhRR mRNA was induced in a ligand concentration- and treatment time-dependent, and chemical- and cell-specific manner. Further characterization of the AhRR function in humans may contribute to understand the mechanisms responsible for differences in response to PAH exposure among tissues or cell types.


    ACKNOWLEDGMENTS
 
This work was supported in part by the Environmental Health Research Grant from Ministry of Health and Welfare of Japan. We thank Brent Bell for reviewing the manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +81-76-234-4407. E-mail: tyokoi{at}kenroku.kanazawa-u.ac.jp. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asman, D. C., Takimoto, K., Pitot, H. C., Dunn, T. J., and Lindahl, R. (1993). Organization and characterization of the rat class 3 aldehyde dehydrogenase gene. J. Biol. Chem. 268, 12530–12536.[Abstract/Free Full Text]

Baba, T., Mimura, J., Gradin, K., Kuroiwa, A., Watanabe, T., Matsuda, Y., Inazawa, J., Sogawa, K., and Fujii-Kuriyama, Y. (2001). Structure and expression of the Ah receptor repressor gene. J. Biol. Chem. 276, 33101–33110.[Abstract/Free Full Text]

Emi, Y., Ikushiro, S., and Iyanagi, T. (1996). Xenobiotic responsive element-mediated transcriptional activation in the UDP-glucuronosyltransferase family 1 gene complex. J. Biol. Chem. 271, 3952–3958.[Abstract/Free Full Text]

Favreau, L. V., and Pickett, C. B. (1991). Transcriptional regulation of the rat NAD(P)H: quinone reductase gene. Identification of regulatory elements controlling basal level expression and inducible expression by planar aromatic compounds and phenolic antioxidants. J. Biol. Chem. 266, 4556–4561.[Abstract/Free Full Text]

Fujisawa-Sehara, A., Sogawa, K., Yamane, M., and Fujii-Kuriyama, Y. (1987). Characterization of xenobiotic responsive elements upstream from the drug-metabolizing cytochrome P-450c gene: A similarity to glucocorticoid regulatory elements. Nucleic Acids Res. 15, 4179–4191.[Abstract]

Fujita, H., Kosakai, R., Yoshihashi, H., Ogata, T., Tomita, M., Hasegawa, T., Takahashi, T., Matsuo, N., and Kosaki, K. (2002). Characterization of the aryl hydrocarbon receptor repressor gene and association of its Pro185Ala polymorphism with micropenis. Teratology 65, 10–18.[CrossRef][ISI][Medline]

Gradin K., Mimura, J., Poellinger, L., and Fujii-Kuriyama, Y. (2002). Nonresponsiveness of normal human fibroblasts to dioxin is mediated by the aryl hydrocarbon receptor repressor. 14th International Symposium on Microsomal and Drug Oxidations Abstracts, p. 170.

Gradin, K., Toftgård, R., Poellinger, L., and Berghard, A. (1999). Repression of dioxin signal transduction in fibroblasts. J. Biol. Chem. 274, 13511–13518.[Abstract/Free Full Text]

Hankinson, O. (1995). The aryl hydrocarbon receptor complex. Annu. Rev. Pharmacol. Toxicol. 35, 307–340.[CrossRef][ISI][Medline]

Iwanari, M., Nakajima, M., Kizu, R., Hayakawa, K., and Yokoi, T. (2002). Induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs by nitropolycyclic aromatic hydrocarbons in various human tissue-derived cells: Chemical-, cytochrome P450 isoform-, and cell-specific differences. Arch. Toxicol. 76, 287–298.[CrossRef][ISI][Medline]

Karchner, S. I., Franks, D. G., Powell, W. H., and Hahn, M. E. (2002). Regulatory interactions among three members of the vertebrate aryl hydrocacrbon receptor family: AHR repressor, AHR1, and AHR2. J. Biol. Chem. 277, 6949–6959.[Abstract/Free Full Text]

Mimura, J., Ema, M., Sogawa, K., and Fujii-Kuriyama, Y. (1998). Identification of a novel mechanism of regulation of Ah (dioxin) receptor function. Genes. Dev. 13, 20–25.[ISI]

Rushmore, T., King, R. G., Paulson, K. E., and Pickett, C. B. (1990). Regulation of glutathione S-transferase Ya subunit gene expression: Identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds. Proc. Natl. Acad. Sci. USA 87, 3826–3830.[Abstract]

Schmidt, J. V., and Bradfield, C. A. (1996). Ah receptor signaling pathways. Annu. Rev. Cell. Biol. 12, 55–89.[CrossRef][ISI][Medline]

Watanabe, T., Imoto, I., Kosugi, Y., Fukuda, Y., Mimura, J., Fujii, Y., Isaka, K., Takayama, M., Sato, A., and Inazawa, J. (2001). Human arylhydrocaron receptor repressor (AhRR) gene: Genomic structure and analysis of polymorphism in endometriosis. J. Hum. Genet. 46, 342–346.[CrossRef][ISI][Medline]

Whitlock, J. P., Jr. (1999). Induction of cytochrome P4501A1. Annu. Rev. Pharmacol. Toxicol. 39, 103–125.[CrossRef][ISI][Medline]