PERGAMON
The effect of the arylhydrocarbon receptor on the human steroidogenic acute regulatory gene promoter activity
Teruo Sugawara a,*, Eiji Nomura b, Noriaki Sakuragi b, Seiichiro Fujimoto b
a Department of Biochemistry, Hokkaido University School of Medicine, Kita-ku, Kita 15, Nishi 7, Sapporo 060-8638, Japan
b Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, Kita-ku, Kita 15, Nishi 7, Sapporo 060-8638, Japan
Received 31 July 2000; accepted 18 April 2001
Abstract
The steroidogenic acute regulatory (StAR) protein is a rate-limiting factor in steroid hormone production. The StAR protein plays a role in the movement of cholesterol from the outer membrane to the inner membrane, where cholesterol side chain cleavage enzyme exists. Dioxins, which may act as ‘endocrine disruptors’, mimic and antagonize endogenous hormone actions in vivo. Although the mechanism of endocrine disruption is not clear, the actions of dioxins are known to be mediated by binding to the arylhydrocarbon receptor (AhR), and it is known that dioxins act as transcription factors to endocrine-associated gene expression. In the present study, we examined the effect of the AhR on the human StAR gene promoter, and we clarified the action mechanisms of environmental endocrine disruptors. We transfected constructs containing the human StAR gene promoter sequences pGL2 1.3-kb StAR (nt - 1293 to + 39) into mouse Y-1 adrenal tumor cells and measured the promoter activity of the StAR gene. With the addition of ß-napthoflavone (BNF), which is a ligand of AhR, to the culture medium, the activity of the StAR gene promoter increased significantly (P <0.05), and with the addition of 1 µM of BNF, it became maximum (3.1 ± 0.6-fold higher than the control value). When the AhR and ARNT were co-transfected together in Y-1 cells or human adrenocortical carcinoma H295R cells, the promoter activity of the StAR gene significantly (P <0.05) increased, to a level 1.4 ± 0.01-fold higher in Y-1 cells and to a level 1.6 +0.04-fold higher in H295R cells than the control level, when 1 uM of BNF was added. We examined the effect of induction of cAMP with transfection with AhR or ARNT. With the addition of 1 mM 8-Br-cAMP, there were no differences between the StAR gene promoter activities in the group in which AhR and ARNT was introduced and in the group in which they were not introduced. The results suggest that AhR plays a role in the promoter activity of the human StAR gene and that the effect of AhR on StAR gene expression may cause a disturbance to the human endocrine system. @ 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Arylhydrocarbon receptor; Steroidogenic acute regulatory protein; StAR; Ah receptor nuclear translocator; B-napthoflavone
1. Introduction
Steroidogenic acute regulatory (StAR) protein is a rate-limiting factor in steroid hormone production. The StAR protein plays a role in the movement of choles- terol from the outer membrane to the inner membrane, where the cholesterol side chain cleavage enzyme exists [1]. StAR gene expression is present in adrenal cortical tissue, testis and ovary and has been shown to be induced by cAMP signals [2]. The human, mouse and
porcine StAR genes all contain SF-1 binding sites in their promoters [3-6]. SF-1 is an orphan receptor for which a ligand has not yet been identified, and it plays an important role in controlling synthesis of steroid hormones [7-9]. SF-1 also controls basal and cAMP- stimulated expression of the human StAR gene [5].
The arylhydrocarbon receptor (AhR) belongs to a family of transcription factors in which the N-terminal domain has a basic helix-loop-helix (bHLH) protein and a Per-Arnt-Sim (PAS) domain in the middle of the domain [10]. AhR needs a ligand to activate the gene expression like steroid hormone receptor families. AhR binds heat shock protein 90 in the cell cytoplasm [11]. As AhR binds a ligand, it changes its conformation and
* Corresponding author. Tel .: + 81-11-706-5047; fax: + 81-11-727- 6006.
binds other bHLH partners, that is, Ah receptor nu- clear translocator (ARNT) [12-14]. AhR-ARNT protein complexes enter the nucleus and bind to a xenobiotic response element (XRE) to control gene transcriptions [15,16].
Dioxins are very toxic, and death can result from the exposure of animals to dioxins. Dioxins mimic and antagonize endogenous hormone actions in vivo and they may act as ‘endocrine disruptors’ [17]. Although the mechanisms of the actions of endocrine disruptors are not clear, it is known dioxins mediated by binding to AhR and act as transcription factors to endocrine- associated gene expression. AhR regulates the gene to act by direct DNA binding through protein-protein interactions [18-20]. AhR modulates cathepsin D gene transcription by interfering with the DNA-binding abil- ity of an estrogen receptor and Sp1 complex [21]. AhR interacts with COUP-TF [22] and Sp1 [23] to control the expression. In this study, we investigated whether AhR influences the StAR promoter activity and StAR gene expression, and we analyzed the mechanism of endocrine disruption of steroid hormone production.
2. Materials and methods
2.1. Plasmid constructs
The 1.3-kb HindIII fragment of the StAR gene (nt - 1293 to + 39) was cloned into the pGL2 plasmid vector (Promega Corp, Madison, WI), which contains firefly luciferase as a reporter gene, as previously de- scribed [24]. Various deletion constructs were prepared by the polymerase chain reaction (PCR), as previously described [5]. The galactosidase expression vector (pCH110, Amersham Pharmacia Biotech) was used for normalization of luciferase data. A human AhR expres- sion plasmid, pSporthAhr2, and a human ARNT ex- pression plasmid, pSportARNT, were kindly provided by Dr Christopher A. Bradfield of the University of Wisconsin.
2.2. Cell culture
Mouse Y-1 adrenal tumor cells were obtained from the RIKEN Cell BANK (Tsukuba, Japan). Human adrenocortical carcinoma H295R cells were a gift from Dr Mitsuhiro Okamoto, Osaka University Medical School (Osaka, Japan). The cells were grown in 35-mm plastic dishes. The Y-1 cells were cultured with Dulbec- co’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 50 µg/ml of gentamycin. H295R cells were grown in DMEM/F12 containing 2% ULTROSER G (BioSepra, Cergy-Pon- toise, France) and 1% ITS Premix (Becton Dickinson and Co, Franklin Lakes, NJ).
2.3. Transfection and luciferase assays
Y-1 cells were cultured at 40-60% confluence in a medium with serum. One-tenth ml of serum-free medium contained pGL2 plasmids, pSporthAhr2, pSportARNT and pCH110 with 3 ul of Fugene 6 (Roche Molecular Biochemicals, Mannheim, Germany) per 1 µg DNA. H295R cells were washed with serum- free medium before adding 0.5 ml of serum-free medium containing plasmids with 4 ul of lipofectamine and PLUS Reagent (Life Technologies, Inc/BRL, Washington, DC) per 1 µg DNA. After 3 h of incuba- tion, the medium was replaced with 2 ml of medium containing 2% ULTROSER G and 1% ITS Premix. Some dishes were treated with various amounts of B-napthoflavone (BNF) (0.001-100 µM) or 8-Br-cAMP (1 mM) during the final 24 h of culture. Cells were harvested 48 h after transfection, and extracts were prepared in lysis buffer (Promega). One aliquot of 100 ul of 400 ul (total extract volume) was used for the luciferase assay (Luciferase Assay System, Promega), and 150 ul was used for the ß-galactosidase assay (ß-Galactosidase Enzyme Assay System, Promega). The ‘blank’ luciferase value was measured in extracts of untransfected cells. The luciferase assay results were normalized to ß-galactosidase activity to compensate for variations in transfection efficiency. Triplicate cul- tures were used in each treatment group, and each experiment was repeated three or four times.
2.4. Northern blot
Northern blotting and hybridization were carried out as previously described [2]. Total RNA was isolated from cultured mouse adrenal tumor Y-1 cells. Y-1 cells were subcultured every 3 days to maintain the cells. The Y-1 cells were cultured for one day after being subcul- tured and were then treated with various amounts of BNF in the culture medium for 24 h. Detailed protocols for the preparation, culture, and isolation of total RNA from Y-1 cells were described previously [2]. Northern blots were probed with mouse StAR cDNA. Each blot was stripped and hybridized with a probe specific for mouse ß-actin cDNA. The relative abundance of StAR mRNA signals was quantified with Desk Scan II (Ver- sion 2.1, Hewlett Pakard) using NIH Image 1.55 f (Ohlendorf Research, Inc, Ottawa, IL), normalized against levels of ß-actin mRNA, and expressed as a percentage of the control value.
2.5. RT-PCR
Total RNA was isolated from the human liver, ovary, testis and adrenal tissues. Complementary DNA synthesis was carried out at 37 ℃ for 60 min using 150 pmol of oligo dT as a primer, 1 µg total RNA and 200
units of SUPERSCRIPT II Rnase H (Life Technolo- gies, Inc/BRL, Washington, DC). Reverse Transcrip- tase in a 20-ul reaction mixture contained 50 mM Tris-HCI (pH 8.3), 75 mM KCI, 3 mM MgCl2, 20 mM dithiothreitol and 0.5 mM each of dATP, dCTP, dGTP, and dTTP. Next, we designed the oligonucletide primers for amplification of the PAS domain of AhR: sense, 5’-GTCGACCGGTGCAGAAAACAGTAA- AGCCA-3’; antisense, 5’-GTCGACTTGTTCCTTC- CTCATCTGTTAGTGGTCTC-3’. The PCR reaction volume (50 ul) contained 10 mM Tris-HCI (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs and 10 pmol each of the primers. The reaction was subjected to 35 cycles of denaturing at 94 ℃ for 45 s, annealing at 55 ℃ for 45 s, and extension at 72 ℃ for 1 min.
2.6. Data analysis
Values are presented as mean ± S.E. Significance be- tween experimental values was determined by Student’s unpaired t-test. P<0.05 was taken as the level of significance.
3. Results
3.1. Northern blotting analysis of StAR expression
BNF is an AhR agonist that acts as a ligand for a receptor. Northern blotting analysis was performed to examine the effect of AhR on StAR mRNA gene expression with various amounts of BNF in the culture medium. The mouse StAR cDNA probe hybridized mRNA from Y-1 cells and produced mainly two sig- nals, a 3.4-kb band and a 1.6-kb band. The 1.6-kb StAR mRNA band was predominant when BNF was not added to the medium. The 1.6-kb signal increased in proportion to the dosage of BNF (0.02-20 uM) added to the medium. When increasing amounts of BNF were added to the medium, the 3.4-kb StAR signals also increased (Fig. 1(A)). The StAR quantity of both 3.4 and 1.6-kb StAR signals were maximum when 20 µM of BNF was added to the culture medium (Fig. 1(B)). To investigate the effect of BNF on the StAR mRNA, Y-1 cells were cultured in the presence of 20 µM of BNF for 72 h. Both 3.4 and 1.6-kb StAR mRNA signals were increased with ßNF incubation in the culture medium for 48 h, and both signals were de- creased after 48-72 h of culture (Fig. 1(C)).
3.2. Promoter activity of StAR was increased by BNF
To determine whether StAR promoter activity in- creased in the presence of BNF, we prepared a 1.3-kb StAR promoter fragment fused to the pGL2 luciferase reporter gene. The plasmids were transfected into Y-1
cells. The promoter activity increased with increases in the BNF concentration, the maximum level being 1 uM of BNF. The maximum promoter activities of StAR were inhibited when more than 1 µM of BNF was added to the culture medium (Fig. 2(A)). At a BNF concentration in the medium of about 1 µM, the antag- onistic effect of ßNF on the StAR promoter was inves- tigated. Various amounts of BNF (0-4 uM) were added to the culture medium, and luciferase activity was as- sayed. When 1 µM of BNF was added to the medium, StAR promoter activities became maximum (3.1 ± 0.6- fold higher than the control value). BNF at concentra- tions of more than 1 uM still inhibited the StAR promoter activity (Fig. 2(B)).
3.3. Expression of AhR on steroid hormone-producing cells
To examine the effect of BNF on the StAR promoter activity via AhR, we performed RT-PCR to examine the expression of AhR on steroid hormone-producing cells. RT-PCR products were found to be present in the liver, testis, ovary and adrenal gland, which produce steroid hormones (Fig. 3).
3.4. AhR increased StAR promoter activity with ARNT
To determine the effect of AhR on the StAR pro- moter activity, overexpressed AhR and ARNT expres- sion plasmids were transfected in Y-1 cells. In the case of overexpression with AhR or ARNT only, the pro- moter activity of StAR decreased, and ßNF-stimulated activity also decreased with AhR over-expression. In the case of co-transfection with AhR and ARNT over- expression, basal and ßNF-stimulated promoter activi- ties of StAR increased. The promoter activity of the StAR gene significantly (P <0.05) increased, to a level 1.4 ±0.01-fold higher than that of the control, when 1 uM of BNF was added (Fig. 4(A)). To examine the human StAR promoter function in human cells, overex- pression of AhR and ARNT expression plasmids were transfected into human adrenocortical H295 R cells. The promoter activity of the human StAR gene (P < 0.01) increased significantly, to a level 1.6 ± 0.04-fold higher than that of the control, when 1 µM of BNF was added in the culture media (Fig. 4(B)).
3.5. cAMP did not respond to AhR or ARNT
We investigated whether the effects of AhR and ARNT on the StAR promoter activity are associated with cAMP signal transduction. AhR and ARNT ex- pression plasmids were co-transfected into Y-1 cells, and the promoter activity of StAR was assayed. The stimulatory effect of cAMP on the promoter activity of
StAR was not changed in Y-1 cells co-transfected with AhR and ARNT when compared with mock trans- fected cells (Fig. 5).
3.6. Deletion analysis of the StAR promoter
Human 1.3-kb StAR promoters, as well as various deletion constructs, were transfected into mouse Y-1 cells. The 1.3-kb StAR promoter contains two Sp1 sites and three SF-1 binding sites (Fig. 6(A)). One or more of these binding sites was removed in the dele-
tion constructs. pGL2 - 885 to + 39 does not contain the most distal SF-1 binding site (-926 to - 918). pGL2 - 235 to + 39, which was constructed from the - 235 to + 39 fragment, contains two SF-1 binding sites (- 105 to - 96 and - 43 to - 36) and an Spl consensus-binding site (- 157 to - 151). pGL2 - 150 to +39 does not contain an Sp1 binding site. pGL2 - 85 to + 39 does not contain the two Spl sites and contains only one SF-1 site. These deletion mutant constructs ablated the response to ßNF in Y-1 cells (Fig. 6(B)).
StAR
kb
+ 3.4
+ 1.6
StAR mRNA expression (% of control)
250
☐ 1. 6kbStAR
3. 4kbStAR
200
B-actin
150
100-
50
BNF
0
0.02
0.2
2
20
μ.Μ
0
(A)
BNF (M)
0
0.02
0.2
2
20
(B)
(kb)
StAR
3.4
- 1.6
B-actin
incubation
0
12
24
48
72
time
(C) (hours)
A
Promoter activity (% of pGL21.3kbStAR)
400
350
300
250
200
150
100
50
0
0. 001
0.01
0.1
BNF ( 4 M)
1
10
100
B
Promoter activity (% of pGL21.3kbStAR)
400
350
300
250
200
150
100
50
0
0
0. 25
0.5
1
2
4
BNF ( MM)
4. Discussion
The StAR protein is a rate-limiting factor that con- trols steroid hormone production. StAR gene expres- sion is controlled by SF-1, which interacts with other factors, C/EBPB [25,26] or Sp1, in the control of gene expression. Dioxins have an effect on the steroid hor- mone action, although the mechanism of its effect is not well known [27,28]. AhR regulates a variety of biological responses to environmentally ubiquitous polycyclic aromatic hydrocarbons, dioxins, metylcon- srane, benzperene and ßNF, but the natural endoge- nous ligand of AhR is not known [29]. BNF is also a well-characterized AhR agonist [30,31]. ßNF has a dif- ferent affinity to XRE than does dioxin, and the rela- tive potencies and efficiencies of these two agonists
have been shown to be essentially identical [32]. An Ah receptor agonist modulates P450 gene expression and related activities, and this can result in tissue-specific changes in hormone levels. The adrenal 21-hydroxylase, testicular 17-hydroxylase and 17,20-lyase activities were decreased in rats treated with dioxins [33,34].
The results of Northern blot analysis showed that the StAR gene expression increased with the addition of BNF. Mouse StAR signals include a 1.6-kb signal and a 3.4-kb signal. The 1.6-kb signal was greatly increased relative to the 3.4-kb signal with the addition of BNF to the culture medium. Both signals were decreased in the presence of BNF from 48 to 72 h. However, the func- tional difference between the 3.4 and 1.6-kb transcripts during this period of incubation is not known.
AhR is widely expressed; high levels have been found to be expressed in the placenta, lung, liver, pancreas and heart, and lower levels have been found in the skeletal muscle, brain and kidney [35]. RT-PCR analy- sis showed that AhR expressions are present in steroid- producing organs, adrenal, testis and ovary. AhR binds BNF, thereby changing its conformation, and it also binds another transcription factor, ARNT, and then binds XRE [12]. The StAR promoter activity increased with increases in the amount of BNF added to the culture medium. After reaching a peak, the activity subsequently decreased with increases in the amount of BNF added to the medium. The promoter activity curve was not linear. The curves of estrogenic activities of environmental chemicals and phytoestrogens are also not linear, because the environmental chemicals and phytoestrogens bind to estrogen receptors, this being a complicated phenomenon caused by a number of fac- tors, such as differential effects on the transactivation functionality of the receptor, the particular coactivators recruited, and the cell and target gene promoter context [36]. The effect of a high concentration of BNF on the
kb
3.0-
2.0-
1.5-
1.0-
+ AhR
0.5-
Marker
AhR cDNA
Adrenal
Testis
Ovary
Liver
A
Promoter activity (% of pGL21.3kbStAR)
600
Basal
+
500
₿ NF
400
T
T
300
200
* *
100
0
pGL21.3kbStAR
+
+
+
+
AhR
+
+
ARNT
+
+
B
Promoter activity (% of pGL21.3kbStAR)
**
600
Basal
T
500
₿ NF
400
T
300
T
T
200
100
T
T
T
0
pGL21.3kbStAR
+
+
+
+
AhR
+
+
ARNT
+
+
StAR promoter activity is down-regulated, and it is difficult to estimate the effect in the case of exposure to low concentrations of chemical agents. Northern blot data did not show a biphasic effect. This may be due to the effect of BNF on gene transcription and/or StAR message stability.
AhR is heterodimeric, and it binds DNA and regu- lates the gene [37]. The promoter activity was inhibited by transfection with ARNT or AhR alone, and the
promoter activities of StAR were increased in the case of co-transfection with ARNT and AhR. Co-transfec- tion may change the expression the endogenous AhR
Basal
Promoter activity (% of pGL21.3kbStAR)
CAMP
250
T
200
T
T
150
100
T
T
T
50
0
pGL21.3kbStAR
+
+
+
+
AhR
+
+
ARNT
+
+
Plasmid
pGL2 1.3kbStAR
A
Sp1
SF-1
Sp1
SF-1
SF-1
-1293
SS
+39
(-926 to -918)
(-1159 to -1153)
(-157 to -151)
(-105 to -96)
(-43 to -36)
B
pGL24-85/+39
4
pGL24-150/+39
1
pGL24-235/+39
4
pGL24-885/+39
pGL21. 3kbStAR
0
0.5
1
1.5
2
2.5
3
3.5
4
Fold induction by BNF (fold of basal promoter activity)
and ARNT with over-expressed AhR or ARNT alone or AhR and ARNT together. AhR and ARNT com- pete with other transcription factors to control gene activity [22]. AhR and ARNT bind together, and the AhR-ARNT protein complex enters the nucleus and binds XRE.
StAR gene expression is controlled by SF-1, which interacts with co-activators, including steroid receptor coactivator-1 (SRC-1) [38] and other transcription factors, CREB-binding protein (CBP) [39], C/EBP [26] and Sp1 [40]. Sp1 and AhR physically interact and cooperate in the regulation of gene transcription [23]. It has been reported that AhR binds RIP140, which is a co-activator of ER, and it may compete with steroid receptors for co-activators [41]. Although we did not find consensus XREs in the human StAR promoter sequences, the results of deletion analysis of the human StAR promoter demonstrated that the re- gion between - 1293 and - 885 is important for re- sponse to ßNF stimulation. AhR effect may associate with these factors and other factor including co-acti- vator or co-repressor and function on the human StAR promoter activity.
Although the StAR gene is partly regulated by AhR, when exogenous cAMP was added, it was found that the PKA is not involved in AhR signal transduction. It has been reported that AhR is associ- ated with PKA and is phosphorylated [42]. AhR phosphorylation plays an important role in the ability of an active AhR-ARNT complex to associate with cis-acting regulatory elements. Our results showed that the increase in promoter activity by AhR signal is not related to PKA.
In this study, it was found that AhR affects the promoter activity of the StAR gene. The StAR gene may be regulated by AhR, and AhR may control steroid hormone production via StAR gene expres- sion. The injury mechanism of dioxins, which is a ligand of AhR, is not well known. Further investiga- tion is needed to elucidate the disruption mechanism of steroid hormone production.
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