ELSEVIER
MCE
Molecular and Cellular Endocrinology
Mono-(2-ethylhexyl) phthalate (MEHP) induces nuclear receptor 4A subfamily in NCI-H295R cells: A possible mechanism of aromatase suppression by MEHP
Mariko Noda, Shuji Ohno, Shizuo Nakajin *
Department of Biochemistry, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan Received 2 October 2006; received in revised form 18 April 2007; accepted 11 May 2007
Abstract
Phthalate esters are widely used as plasticizers for polyvinylchloride and are suspected of functioning as endocrine disrupters. Di-(2-ethylhexyl) phthalate (DEHP), the most important phthalate ester in commercial use, has been reported to act as a rodent reproductive toxicant. In the present study, we investigated the effects of phthalate esters on aromatase (CYP19) activity and on its gene expression in a human adrenocortical carcinoma cell line, NCI-H295R. Mono-(2-ethylhexyl) phthalate (MEHP), a principle metabolite of DEHP, dose-dependently suppressed aromatase activity and its transcription level. Furthermore, MEHP rapidly and transiently induced transcription of the genes which encode nuclear receptor 4A subfamily members (Nur77, Nurr1 and NOR-1), and up-regulated Nur77 promoter activation and Nur77 protein expression in the cells. MEHP- induced Nur77 transcription was inhibited by bisindolylmaleimide I (protein kinase C inhibitor) and wortmannin (phosphoinositide 3-kinase inhibitor). Finally, ectopic expression of Nur77 markedly suppressed forskolin-induced transcriptional activation of promoters I.3 and II of the CYP19 gene. These results suggest that the suppression of aromatase activity and its transcription level by MEHP exposure to NCI-H295R cells was regulated through the rapid and transient expression of Nur77 gene. @ 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: MEHP; Aromatase; NCI-H295R cells; NR4A
1. Introduction
Phthalate esters are widely used as plasticizers for poly- vinylchloride (PVC) plastics (Waring and Harris, 2005). In particular, di-(2-ethylhexyl) phthalate (DEHP) is the one most commonly used in the production of medical devices. DEHP exposure leads to abnormalities in several tissues, mainly of the reproductive tract, in rodents (Latini, 2005; Tickner et al., 2001). Mono-(2-ethylhexyl) phthalate (MEHP), a principle metabolite of DEHP, suppresses aromatase transcript levels in cultured rat granulosa cells (Davis et al., 1994; Lovekamp-Swan and Davis, 2001). However, there have been no reports using human ori- gin cells that investigated the effects of these phthalate esters. Therefore, we decided to investigate the effects of DEHP, dibutyl phthalate (DBP) and their monoesters on aromatase expression in human origin cells because they are the most commonly used phthalate esters and are produced in the greatest amounts.
Aromatase cytochrome P450 (P450arom) is the enzyme that catalyzes the conversion of C19 androgenic steroids to the cor- responding estrogens and is encoded by a single gene (CYP19) in the human genome. A special feature of the CYP19 gene is its multiple exon I (Simpson et al., 1997). Furthermore, at least 10 exon I are selected in a tissue-specific fashion (Fig. 1). For exam- ple, exon PII or exon I.3 is selected in the ovary. After alternative splicing, mRNA with a tissue-specific 5’-untranslated region is formed (Bulun et al., 2004; Sasano and Harada, 1998; Sebastian and Bulun, 2001; Simpson et al., 1997). In this tissue-specific selection of exon I, promoter regions that abut on the 5’-end of each exon I are activated by a distinct signal pathway (Simpson et al., 1997). For example, promoters II and I.3 are activated by intracellular cAMP. On the other hand, promoter I.4 is acti- vated by glucocorticoids. The ovary produces a large amount of estrogen, which is closely involved in female reproductive physiology. Factors influencing this estrogen production that is controlled by aromatase, a rate-limiting enzyme of estrogen, may have a significant impact on human reproductive physio- logical function. We reported previously that aromatase activity and CYP19 gene transcript are up-regulated by forskolin (FSK)
* Corresponding author. Tel .: +81 3 5498 5775; fax: +81 3 5498 5776. E-mail address: nakajin@hoshi.ac.jp (S. Nakajin).
Untranslated exon I
.1
2a
.4
.5
1.7
1f
.2
1.6
1.3
PII
II
III
placenta
placenta
bone
fetal tissue
endothelial cells
brain
placenta
monocyte / macrophage
ovary
adipose stromal cells
ovary
adipose stromal cells
and promoters I.3 and II are activated in NCI-H295R cells, a human adrenocortical carcinoma cell line and as well in ovary (Watanabe and Nakajin, 2004). Therefore, we used this cell as an aromatase expression model in this study.
MEHP suppresses aromatase transcript levels via activa- tion of the nuclear receptor peroxisome proliferator-activated receptor-a (PPARa) and PPARy in cultured rat granulosa cells (Lovekamp-Swan et al., 2003). PPARa is also activated by mono n-butyl phthalate (MBP) (Bility et al., 2004); however, MBP does not suppress estradiol production in cultured rat granulosa cells, although MEHP does (Lovekamp-Swan and Davis, 2003). Accordingly, the suppression mechanism of aro- matase expression by MEHP via these PPARs may also involve other transcription factors which are specifically induced by MEHP.
It has been reported that some transcription factors and nuclear receptors are involved in the regulation of aromatase expression, such as JunB (Ghosh et al., 2005), liver recep- tor homologue-1 and steroidogenic factor-1 (SF-1) (Pezzi et al., 2004), nuclear receptor subfamily 4A (NR4A) (Wu et al., 2005), and PPARy and 9-cis retinoic acid X receptor (RXR) (Rubin et al., 2002). Among these factors, NR4A is a group of orphan nuclear receptors that includes Nur77 (also called NR4A1, NGFI-B, N10, TIS1, Nak-1 or TR3), Nurr1 (NR4A2) and NOR-1 (NR4A3). They are rapidly and transiently induced by a variety of stimuli, including cAMP and growth factors, and have been known conventionally as regulators of apopto- sis in different cells, mainly immune cells. Recently, Nur77 and Nurr1, members of the NR4A subfamily, have been identified as important regulators of the endocrine system that alter the gene expression of steroidogenic enzymes, such as aromatase (CYP19) (Wu et al., 2005), P450aldo (CYP11B2) (Bassett et al., 2004b), 3ß-hydoroxysteroid dehydrogenase (HSD)/45-44 isomerase type2 (HSD3B2) (Bassett et al., 2004a; Martin and Tremblay, 2005), CYP17 (Song et al., 2004), 20x-HSD (Stocco et al., 2000) and 21-hydroxylase (CYP21B) (Wilson et al., 1993b), in several gonadal tissues including the ovary, testis and adrenals. In addition, effects on steroid biosynthesis by various chemicals, such as bisphenol A, through NR4A induction have been reported (Song et al., 2002).
Therefore, we have decided to focus our attention on the NR4A subfamily from among the factors which transcrip- tionally regulate aromatase expression, and to investigate the
possibility that they participate in the effects of phthalate esters (DEHP, DBP and their monoesters) on human aromatase expression.
The following three results were obtained: (i) MEHP sup- presses aromatase expression in NCI-H295R cells as well as rat ovary-derived cells, and this is the first evidence of the sup- pressive effect of MEHP on aromatase using cells of human origin; (ii) MEHP rapidly and transiently induced NR4A gene expression and also activated the Nur77 promoter; (iii) Ectopic expression of Nur77 within the cells inhibited transcription driven by the promoter I.3/II of the CYP19 gene. Based on these results, we propose that Nur77 may act as a novel mediator of aromatase suppression by MEHP.
2. Materials and methods
2.1. Chemicals
Forskolin (FSK), di-(2-ethylhexyl) phthalate (DEHP), dibutyl phthalate (DBP), mono-(2-ethylhexyl) phthalate (MEHP) and monobutyl phthalate (MBP) were purchased from Wako Pure Chemical Industries (Osaka, Japan). 3- Isobutyl-1-methylxanthine (IBMX) was purchased from Sigma-Aldrich Corp. (Milwaukee, WI). These compounds were dissolved in ethanol. H89 was pur- chased from Seikagaku Corporation (Tokyo, Japan). PD98059 and KN-93 were purchased from Cosmo Bio Co., Ltd. (Tokyo, Japan). Bisindolylmaleimide I and wortmannin were purchased from Calbiochem (San Diego, CA). These protein kinase inhibitors were dissolved in dimethyl sulfoxide. The final con- centration of each solvent in the medium was 0.1% (v/v). D-MEM/F-12, D-MEM/F-12 without phenol red and a mixture of penicillin (5000 U/ml) and streptomycin (5000 µg/ml) were purchased from Invitrogen Japan K.K. (Tokyo, Japan). ITS plus was obtained from Nippon Becton Dickinson Com- pany, Ltd. (Tokyo, Japan). Nu-Serum I and fetal calf serum (FCS) were purchased from Cosmo Bio Co., Ltd. and Sanko Junyaku (Tokyo, Japan), respectively.
2.2. Cells
A human adrenocortical carcinoma cell line, NCI-H295R, was purchased from American Type Culture Collection (Manassas, VA). NCI-H295R cells were cultured in D-MEM/F-12 supplemented with 50 U/ml of penicillin, 50 mg/ml of streptomycin, 1% (v/v) of ITS plus and 2.5% (v/v) of Nu-Serum I. The human granulosa-like cell line, KGN (Nishi et al., 2001), was obtained from Riken Cell Bank (Tsukuba, Japan). KGN were cultured in D-MEM/F-12 medium supple- mented with penicillin (50 U/ml), streptomycin (50 µg/ml) and FCS (10%, v/v). Cells were maintained as monolayer cultures in 10cm dishes at 37℃ in an atmosphere of 5% CO2-95% air.
2.3. Plasmids
The expression vector of human full-length Nur77 (GenBank accession No. NM_002135) was purchased from OriGene Technologies, Inc. (Rockville, MD). This is termed Nur77-WT. For the construction of dominant-negative Nur77, we took into account the previous report about a dominant-negative form of mouse Nur77 (N10) (Davis et al., 1993). Site-directed mutagenesis was performed with the GeneTailor Site-Directed Mutagenesis System (Invitrogen). Plasmid Nur77- WT was used as a template, and 5’-gcatggtgaaggaagttgtcTgaacagacagcctga-3’ (sense) and 5’-gacaacttccttcaccatgcccaccgccag-3’ (antisense) (mutated residue is capitalized) were used as primers. The change results in replacement of Arg- 338 with a stop codon. The construct is termed Nur77-DN. A luciferase reporter plasmid that contains the 740 bp promoter I.3/II of CYP19 has been described previously (Watanabe and Nakajin, 2004). The construct is termed PI.3/II-pGL3. The promoter I.3/II of CYP19 gene was also cloned into the high-sensitivity fire- fly luciferase reporter vector pGL4.10 luciferase reporter vector (Promega). The construct is termed PI.3/II-pGL4. The promoter region of the human Nur77 gene
(-2112 to +89 bases of the promoter region of the human TR3 gene; GenBank accession No. HSU17590) was amplified by PCR from human genomic DNA (Promega) and cloned into a pGL4.10 using Xho I/Bgl II sites. The following primer pairs were used: sense, 5’-ccgCTCGAGatatgagtggacctacacagttcaaacg-3’ (Xho I site is capitalized) and antisense 5’-ggaAGATCTccgaagttettctgtgcactcc- ☒ 3’ (Bgl II site is capitalized). A luciferase reporter plasmid harboring 3 x tandem repeats of Nur77 (NGFI-B)-binding response element (NBRE) was constructed as described previously (Maira et al., 1999). All constructs were confirmed by sequencing.
2.4. Measurement of aromatase activity
The catalytic activity of aromatase was determined by measuring the amount of3H2O released with the conversion of [1]-3H] androstenedione into estrone as previously described (Watanabe and Nakajin, 2004). Briefly, NCI-H295R cells were seeded at a density of 5.0 x 105 cells/well on 24-well plates. After 48 h of culture, the medium was replaced with the treatment medium (D-MEM/F-12 without phenol red with 1% (v/v) ITS plus, penicillin (50 U/ml) and streptomycin (50 µg/ml)). After incubation for 24 h (serum starvation), the cells were treated with phthalates and/or FSK. The cells were then incubated with 60 nM [1]-3H] androstenedione for an additional 2 h. The medium was treated with chloroform and centrifuged. The aqueous phase was then mixed with 5% charcoal/0.5% dextran and centrifuged. An aliquot of the supernatant was added to 1 ml of scintillation fluid and the radioactivity of 3H2O, which was released by arom- atization, counted. This result was standardized from the protein concentration of the cells determined using a BCA protein assay kit (PIERCE Biotechnology, Rockford, IL).
2.5. RNA isolation and RT-PCR
NCI-H295R cells were seeded at a density of 3.0 x 105 cells/well on 6- or 12-well plates. After 48 h of culture, the cells were then serum-starved for 24 h. On the following day, the cells were treated with treatment medium that contained phthalates and/or FSK. Total RNA was extracted using ISOGEN (Nippongene, Toyama, Japan). The first strand cDNA library was obtained by RT-reaction carried out using Ready-to-Go™M RT-PCR beads (GE Healthcare Bio-Sciences Corp., Piscataway, NJ), and oligo (dT)12-18 as the first strand primer. The negative controls were performed using the same system after heat denaturation of reverse transcriptase according to the manual. RT-PCR of the gene transcripts of aromatase, NR4A and glyceraldehyde-3-phosphate dehy- drogenase (GAPDH) were performed on 2 ul of first-strand cDNA using Taq polymerase and oligonucleotide primers. The PCR primers and conditions took into account the following previous reports, with some modifications being made [aromatase coding region (Richards and Brueggemeier, 2003); aromatase exon PII and GAPDH (Agarwal et al., 1995); Nur77 and Nurr1 (Lu et al., 2004); NOR-1 (Bassett et al., 2004a)]. Human gene specific primers used to amplify aromatase coding region (exons IX-X) PCR were 5’-gaatattggaaggatgcacagact- 3’ (sense) and 5’-gggtaaagatcatttccagcatgt-3’ (antisense), which amplifies a PCR fragment of 293 bp; primers for aromatase exon PII (exons PII-III) were 5’-gcaacaggagctatagat-3’ (sense) and 5’-caggaatctgccgtgggaga- 3’ (antisense), generating a PCR fragment of 305 bp; primers for GAPDH were 5’-cggagtcaacggatttggtegtat-3’ (sense) and 5’-agcettetccatggtggtgaagac- 3’ (antisense), generating a PCR fragment of 307 bp; primers for Nur77 were 5’-tgctcaggcctggtgeta-3’ (sense) and 5’-gcaccaagtcetccagett-3’ (anti- sense), generating a PCR fragment of 355 bp; primers for Nurr1 were 5’-cgaaccctgactatcaaatgagtg-3’ (sense) and 5’-caatgcaggagaaggcagaa-3’ (anti- sense), generating a PCR fragment of 354 bp; primers for NOR-1 were 5’-ccttctcctccaatctgcatg-3’ (sense) and 5’-cttggatatcaaggttcaggctct-3’ (anti- sense), generating a PCR fragment of 424 bp. Each PCR amplification for aromatase coding region, Nur77, Nurr1, NOR-1 and GAPDH was started by an initial denaturing reaction at 95 ℃ for 5 min, followed by 30, 25, 30, 30 and 20 cycles of denaturing (30 s, 95℃) and elongation (30 s, 72℃) reactions, respec- tively. Annealing was performed for 30 s at 64 ℃ for aromatase coding region, 63 ℃ for Nur77, 66 ℃ for Nurr1, 66 ℃ for NOR-1 and 63 ℃ for GAPDH. The PCR cycles for aromatase exon PII were: 94℃ for 5 min; 10 cycles of 94 ℃ for 30 s, 60℃ for 30 s and 72℃ for 30 s; 10 cycles of 94 ℃ for 30 s, 55 ℃ for 30 s and 72 ℃ for 30 s; 7 cycles of 94 ℃ for 30 s, 50 ℃ for 30 s and 72 ℃ for
30 s. The RT-PCR products were electrophoresed on 1.5% agarose gel contain- ing 0.5 mg/ml ethidium bromide. The relative expression levels of each mRNA were determined by measuring the band intensity, and GAPDH was used as an internal control.
2.6. Western blot analysis
NCI-H295R cells were seeded at a density of 3.0 × 105 cells/dish on 10-cm dishes. After 48 h of culture, the cells were then serum-starved for 24 h. After serum starvation, the cells were treated with 100 µM MEHP for the indicated time periods. Nuclear extracts of these cells were obtained by the method of Maira et al. (1999) with slight modifications. The cells were washed twice with cold phosphate-buffered saline (PBS), and then the pellet was rapidly frozen at -80℃. The frozen pellet was resuspended and homogenized in 50 ul of buffer A (20 mM potassium phosphate buffer (KPB, pH 7.4), 0.25 M sucrose, 10% glycerol, 0.5 mM DTT, 1 mM PMSF, 1 µg/ml pepstatin A) and then cen- trifuged (1000 x g, 7 min at 4℃). The supernatant was aspirated off, and the pellet resuspended in 20 ul buffer A +0.5% Nonidet P-40 and then sonicated moderately. The amount of protein was quantitated by the BCA protein assay mentioned above. Equal amounts of proteins and High-Range Rainbow Molec- ular Weight Markers (GE Healthcare) were separated electrophoretically and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Japan, Tokyo). For immunoblotting, membranes were blocked with 1% non-fat dried milk for 1 h and then incubated with anti-Nur77 (at 1:200 dilution; sc-5569; Santa Cruz Biotechnology Inc., Santa Cruz, CA) overnight at 4℃. The mem- branes were washed and incubated with anti-rabbit IgG conjugated to peroxidase (at 1:1000 dilution; PI-1000; Vector Laboratories Inc., Burlingame, CA) for 3 h. Immunoreaction was visualized by Immunostaining HRP-1000 (Konica Minolta, Tokyo).
2.7. Transfection and luciferase assay
NCI-H295R cells were seeded at a density of 5.0 x 105 cells/well on 24-well plates. After 24 h of culture, the cells were transfected using FuGENE 6 transfec- tion reagent (Roche Diagnostics KK, Tokyo) according to the manufacturer’s instructions with the following vectors: (1) 250 ng of PI.3/II-pGL3, (2) 50 ng of PI.3/II-pGL4, (3) 100 ng of phRL-TK sea pansy luciferase internal control vector (Promega), (4) 1 ng of hRluc-CMV sea pansy luciferase internal control vector (Promega), (5) 10-250 ng of the Nur77-WT, (6) 100 ng of the Nur77- DN and (7) 50 ng of the Nur77-pGL4. Vectors (1-6) were used to measure the CYP19 promoter I.3/II activity, and vectors (4 and 7) were used to mea- sure the Nur77 promoter activity. After the 48 h transfection, the medium was replaced with treatment medium containing the chemical compounds and incu- bated. Luciferase activity was measured using Dual-Luciferase reporter assay (Promega) and Sirius luminometer (Berthold Detection Systems, Pforzheim, Germany).
2.8. ELISA of intracellular cAMP
NCI-H295R cells were seeded at a density of 1.0 x 105 cells/well on 96-well plates. The concentration of cAMP in the cells that were treated with FSK plus 300 µM IBMX, or MEHP plus IBMX for 2 h was measured using a cAMP Bio- trak EIA System (GE Healthcare) according to the manufacturer’s instructions. This assay involves a non-acetylation method and the data were standardized from the protein concentration determined using a BCA protein assay kit (Pierce Biotechnology Inc.).
2.9. Statistical analysis
Statistically significant differences between the experimental groups were determined by one-way analysis of variance (ANOVA) followed by the Dun- nett test or Tukey test for multiple comparisons. Statistical analysis was performed using the software program Prism 4 for Windows (GraphPad Soft- ware, San Diego, CA). P-values below 0.05 were considered to be signi- ficant.
(A)
80
Aromatase Activity (pmol/mg protein/2h)
T
T
T
(B)
100
T
Aromatase Activity (pmol/mg protein/2h)
T
60
T
T
75
T
T
T
40
50
20
25
0
0
0
1
10
30
100
300
0
1
10
30
100
300
DEHP(LM)
DBP (uM)
(C)
100
Aromatase Activity (pmol/mg protein/2h)
(D)
125
**
**
Aromatase Activity (pmol/mg protein/2h)
75
**
100
T
T
T
**
75
T
50
**
50
25
25
0
0
1
10
30
100
300
0
0
1
10
30
100
300
MEHP(u.M)
MBP (uM)
3. Results
3.1. MEHP suppresses aromatase activity in NCI-H295R cells
To investigate whether MEHP has any effects on human ori- gin cells, we first examined the effects of phthalate esters on aromatase activity in NCI-H295R cells. As shown in Fig. 2, MEHP was the only phthalate ester that significantly suppressed 25 MM FSK-induced aromatase activity in a dose-dependent manner. Although the data are not presented, FSK at a con- centration of 25 p.M induced aromatase activity by 20.2-fold compared to the basal level (vehicle), as described previously (Watanabe and Nakajin, 2004), and the FSK-induced aromatase activity was decreased by 41.3% and 53.0% by exposure to 100 and 300 µM MEHP, respectively (Fig. 2C). Other phthalate esters, such as DEHP, DBP and MBP had no effect on aro- matase activity (Fig. 2A, B and D). Meanwhile, cytotoxicity, which was estimated by the leakage of lactate dehydrogenase (LDH) activity, associated with the indicated concentrations of these phthalate esters was not observed compared with the basal level (data not shown). These results suggest that only MEHP, among the phthalate esters tested, suppressed FSK-induced aro- matase activity without showing any cytotoxicity in these cells of human origin.
3.2. MEHP down-regulates transcription of CYP19 gene via its promoter I.3/II in NCI-H295R cells
To determine if the down-regulation of aromatase in NCI- H295R cells was regulated at the stage of gene transcription, we
performed RT-PCR on first-strand cDNAs from the cells after treatment with 25 µM FSK and 100 µM MEHP. As shown in Fig. 3B, MEHP down-regulated in a dose-dependent manner both of the FSK-induced CYP19 gene transcripts, one of which corresponds to the coding region (exons IX and X) and the other which includes exon PII. This result suggests that MEHP down- regulates FSK-induced aromatase expression at the level of the CYP19 gene expression in NCI-H295R cells, and that promoter II of the CYP19 gene may be involved.
Promoter II abuts the 5’-end of exon PII and this promoter includes the sequence of exon I.3. Promoter I.3 abuts exon I.3 (Fig. 3A). To clarify the possibility that MEHP down- regulates CYP19 gene expression via promoter II, we transfected the firefly luciferase reporter constructs that harbor promoter I.3/II sequences. As shown in Fig. 3C, MEHP down-regulated the FSK-induced transcriptional activation of promoter I.3/II by 61.8% at 300 µM, and the down-regulation was dose- dependent. Furthermore, MEHP down-regulated even in the case of non-inducible promoter activity (without FSK activa- tion) in a dose-dependent manner (Fig. 3D). This result suggests that MEHP-suppressed CYP19 gene expression is involved in the down-regulation of transcriptional activation of promoter I.3/II.
3.3. MEHP induces orphan nuclear receptor NR4A genes in NCI-H295R cells
To investigate whether the action of MEHP on aromatase suppression is mediated through NR4A, the effects of MEHP on NR4A expression at designated time points were analyzed. As shown in Fig. 4A, the NR4A subfamily genes Nur77,
(A)
promoter II
coding region
exon 1.6
exon 1.3
exon PII
exon II
CRE
CRE
aro
aro
TATA
CLS
SF-1
S1
TATA
+
+
4
-717
+1
+23
(C)
Relative Luciferase Activity
6
(B)
KGN
FSK (25 uM) + MEHP (u.M)
4.
10
**
coding
0
1
30
100
300
**
region
2
**
exon PII
0
GAPDH
MEHP (uM)
0
1
10
30
100
300
0
FSK (25 µM)
1.5
+
+
+
+
+
+
+
T
CYP19 mRNA (CYP19/ GAPDH)
coding region exon PII
Reporter vector
PI.3/II-pGL3
pGL3
T
T
WW
T
T
1.0-
T
*
T
*
(D)
15
T
T *
**
Relative Luciferase Activity
**
T
T
T
0.5-
10-
*
5-
0.0
**
MEHP (μM)
**
0
1
10
30
100
300
0
FSK (25 μM)
+ + + + + +
MEHP (LM)
0
1
10
100
100
Reporter vector
PI.3/II-pGL4
pGL4
Nurr1 and NOR-1 were rapidly and transiently expressed by 100 µM MEHP exposure. The Nur77 gene induction reached a maximum at 30 min, while the Nurr1 and NOR-1 genes both reached a maximum at 1 h. Furthermore, the Nur77 expres- sion level is higher than the other NR4A subfamily members in this cell because the PCR cycles after RT reaction are 25 for detection of Nur77 gene expression and 30 for Nurr1 and NOR-1. We further determined whether MEHP induces Nur77 protein levels by Western blotting. After a 3 or 6 h exposure to MEHP, Nur77 protein levels were markedly up-regulated (Fig. 4B).
To gain a better understanding of MEHP-induced Nur77 expression, we tested the effects of phthalate ester exposure on Nur77 gene expression. As shown in Fig. 4C, 100 µM MEHP- induced Nur77 gene expression at 1 h. Furthermore, DEHP, DBP and MBP, which had no effects on aromatase activity at 100 p.M
(Fig. 2A, B and D), did not induce the Nur77 gene. Addition- ally, we tested the effects of MEHP on Nur77 gene expression at different concentrations. As shown in Fig. 4D, MEHP induced Nur77 gene expression in a dose-dependent manner.
These results suggest the possibility that MEHP suppresses aromatase activity and transcription through activation of Nur77 expression, and that MEHP induces Nur77 gene expression in a time- and dose-dependent manner.
3.4. MEHP activates Nur77 gene promoter in NCI-H295R cells
To clarify the possibility that MEHP up-regulates Nur77 gene expression via its promoter, we transfected the Nur77-pGL4. The Nur77 promoter contains some potential cis-acting ele- ments, such as activator protein-1 (AP-1), estrogen responsive
(A)
100 µM MEHP exposure time (h)
2
0
0.5
1
2
3
Nur77
NR4A mRNA (NR4A/ GAPDH)
Nur77
(B)
100 μΜ ΜΕΗΡ
Nurr1
exposure time (h)
1
0
3
6
NOR-1
Nurr1
25kDa
GAPDH
0
NOR-1
0
1
2
3
MEHP exposure time (h)
(C)
100 μΜ
(D)
MEΗΡ (μ.Μ)
EtOH
DEHP DBP MEHP MBP
0
1
10
100
Nur77
Nur77
GAPDH
GAPDH
6
**
6-
*
Nur77 mRNA (Nur77/ GAPDH)
5.
Nur77 mRNA (Nur77/ GAPDH)
5-
4-
4-
3-
3-
2-
2.
1-
1.
0
MEHP (μM)
0
control
DEHP
DBP
MEHP
MBP
0
1
10
100
100 μΜ
element (ERE) and cAMP responsive element (CRE) [Uemura et al., 1995 and Fig. 5A]. As shown in Fig. 5B, the Nur77 pro- moter was activated 3.2-fold by exposure to 100 µM MEHP for 1 h, and 6.1-fold by treatment with 25 µM FSK for 4h. Fur- thermore, the activation level of its promoter by MEHP had returned to the basal level at 4 h after treatment. This result sug- gests that MEHP rapidly and transiently up-regulates activation of the Nur77 promoter.
3.5. Involvement of protein kinase (PK) C and
phosphoinositide 3-kinase (PI3K) signaling pathways in MEHP-mediated Nur77 induction
We first examined whether MEHP-induced Nur77 expression is mediated through induction of the intracellular cAMP level. As shown in Fig. 6A, the intracellular cAMP level was strongly increased by treatment with 25 p.M FSK for 2h, while exposure to 100 or 300 µM MEHP for the same time had no effect on the level. This result suggests that MEHP induces Nur77 expression through intracellular signaling pathways other than the cAMP- PKA pathway.
Next, to elucidate the signaling pathways which are involved in Nur77 induction by MEHP, we treated the cells with various inhibitors of diverse signaling pathways in combination with 100 µM MEHP. The doses of protein kinase inhibitors used to sufficiently inhibit each signaling pathway were obtained from the corresponding reports as follows: The inhibitor of calcium-calmodulin-kinase II (CaMKII), PKC and mitogen- activated protein kinase kinase (MAPKK) (Doi et al., 2001); PKA (Song et al., 2002); PI3K (Poh and Pervaiz, 2005). As shown in Fig. 6B, MEHP-induced Nur77 gene expression was significantly suppressed to the basal level by the PKC inhibitor bisindolylmaleimide I (GF109203X; 10 M) and the PI3K inhibitor wortmannin (200 nM). Although the MAPKK inhibitor PD98059 (20 µM) did not significantly suppress the induction, it did reach approximately half. Meanwhile, the CaMKII inhibitor KN-93 (5 MM) and the PKA inhibitor H89 (10 µM) had no significant effects. The result that H89 had no effect on MEHP- induced Nur77 expression corresponds to the finding that MEHP had no effect on the intracellular cAMP level (Fig. 6A). Taken together, these results indicate that MEHP induces Nur77 gene expression via PKC and PI3K signaling pathways.
(A)
Nur77
ERE
AP-
TATA
CRE
AP-
AP-
AP-
4
-2112
+1
+89
=transcription start)
(B)
Relative Luciferase Activity
12.5
*
**
10.0-
T
7.5-
**
5.0-
2.5
0.0
FSK (h)
-
0.5
1
4
-
-
4
| MEHP (h) | - | - | - | 0.5 | 1 | 4 - | |
| Reporter vector | Nur77-pGL4 | pGL4 | |||||
Fig. 5. MEHP activates Nur77 gene promoter in NCI-H295R cells. Diagram showing the structure and some potential cis-acting elements [activator protein-1 (AP-1), CRE, estrogen responsive element (ERE), TATA] of the human Nur77 gene promoter (A). NCI-H295R cells were transfected with -2112 to +89 bases of the Nur77 promoter luciferase reporter plasmid (Nur77-pGL4) or pGL4 empty vector, and hRluc-CMV plasmid was used as an internal control. The transfected cells were subsequently treated with 25 p.M FSK or 100 p.M MEHP for the indicated time periods (B). Each column represents the mean with S.E.M. (n=3). Significant differences were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. An asterisk denotes a significant difference compared with the vehicle control; * P<0.05, ** P<0.01.
3.6. Ectopic Nur77 expression represses transcription from promoter I.3/II of CYP19 gene
To investigate whether the induction of Nur77 expression represses aromatase expression in NCI-H295R cells, we cotrans-
fected the cells with Nur77-WT and PI.3/II-pGL3. As shown in Fig. 7A, promoter I.3/II transcription was activated approx- imately 5.5-fold by 25 MM FSK. However, Nur77 ectopic expression impaired FSK-induced transcriptional activation of the promoter I.3/II in a dose-dependent manner. This activa-
(B)
ΜΕΗΡ (100 μΜ)
control
-
PD
KN
H89
BIM
WM
(A)
Nur77
350
**
Intracellular cAMP Level (pmol/ mg protein)
300
GAPDH
250
2
200
150
Nur77 mRNA (Nur77/ GAPDH)
100
50
1
*
*
T
0
EtOH
FSK (25 uM)
MEHP (100 μΜ)
MEHP (300 μΜ)
0
control
PD
KN
H89
BIM
WM
-
MEHΡ (100 μΜ)
(B)
Relative Luciferase Activity
Relative Luciferase Activity
20
(A)
7
**
T
6
5
10-
4
**
3
*
*
2
0
1
**
1
2
3
4
5
6
0
Nur77-WT (ng/well)
10
100
100
-
-
-
FSK (25 µM)
-
+
+
+
+
+
ΜΕΗΡ (100 μΜ)
Nur77-DN (100ng/well)
+
-
-
-
-
+
+
-
-
-
-
-
Nur77-WT
FSK (25uM)
-
-
-
-
+
tion was decreased 50.2% and 76.0% by the transfection of 10 and 100 ng Nur77-WT, respectively. Under the same experimen- tal conditions, decreasing the level of transcriptional activity by 100 µM MEHP was similar to 100 ng Nur77 transfection. Next, we attempted to clarify if MEHP repression of FSK- induced aromatase expression is mediated by Nur77. Cells were cotransfected with PI.3/II-pGL4 and Nur77-WT or Nur77- DN. As shown in Fig. 7B, ectopic expression of Nur77-WT impaired FSK-induced transcriptional activation of the pro- moter I.3/II in a dose-dependent manner (lanes 2-4). (Because luciferase reporter vector was replaced by PI.3/II-pGL4, the con- trol experiment was repeated). However, FSK-induced promoter I.3/II transactivation was not repressed when Nur77-DN was expressed. (Fig. 7B, lane 5). Transfection of equal amounts of both Nur77-WT and -DN impaired slightly, but not significantly, this promoter activity (Fig. 7B, lane 6). In addition, we confirmed that the Nur77-DN vector abolished the activation of 3x NBRE luciferase reporter construct compared with Nur77-WT vector (data not shown). These results suggest that MEHP-suppressed aromatase expression involves induction of Nur77 expression.
4. Discussion
Phthalate esters are ubiquitous environmental contaminants since they are widely used as plasticizers for PVC plastics that are used in medical devices and food container manufacturing. Furthermore, these chemicals have been shown to elute at a constant rate from plastic products into the environment (Latini, 2005). Among these chemicals, DEHP is the most commonly used plasticizer, so it is estimated that the exposure rate from the environment is high (Latini, 2005). DEHP produces a spectrum of toxic effects in rats of all ages in multiple organs includ- ing the testis (such as testicular atrophy and degeneration) and the ovary (such as delayed ovulation and polycystic ovaries)
(Tickner et al., 2001). Thus, DEHP may act as a reproductive toxicant in male and female rodents. DEHP is rapidly converted to its monoester, MEHP, in the liver, the kidney, the lung, the pancreas and the blood plasma (Tickner et al., 2001). MEHP suppresses aromatase transcript levels and estradiol production in cultured rat granulosa cells (Lovekamp-Swan and Davis, 2001; Lovekamp-Swan et al., 2003). Aromatase is expressed in gonadal tissues and other tissues, and this expression is regu- lated by its tissue-specific promoters (Simpson et al., 1997). The change in aromatase activity leads to a change in the concen- tration of estrogen which is required for homeostatic regulation. Therefore, it is important to investigate the effects of DEHP and its principle metabolite MEHP on aromatase activity and its transcription.
In this study, we investigated the effects of phthalate esters on aromatase activity using human derived NCI-H295R cells which express aromatase. First we investigated the effects of DEHP, DBP and their monoesters, which are commonly used phthalate esters, on aromatase expression in NCI-H295R cells. MEHP was the only phthalate ester that suppressed aromatase activity with- out showing cytotoxicity. In addition, MEHP down-regulated both of the CYP19 gene transcripts, one which corresponds to the coding region and the other which includes exon PII, and down-regulated transcriptional activation of promoter I.3/II of CYP19 gene.
In cultured rat granulosa cells, MEHP suppresses aromatase transcript levels via activation of the nuclear receptors PPARa and PPARy (Lovekamp-Swan et al., 2003). PPAR& is also acti- vated by MBP (Bility et al., 2004). However, MBP does not suppress estradiol production in the same cultured rat granu- losa cells (Lovekamp-Swan and Davis, 2003), and had no effect on aromatase activity even in our study. Therefore, we thought the possibility that the suppression mechanism of aromatase expression by MEHP via these PPARs may also involve other
transcription factors which are specifically induced by MEHP. So, we decided to focus on the orphan nuclear receptor NR4A subfamily as the candidate mediators of aromatase suppres- sion by MEHP. The NR4A subfamily genes are known to be expressed in several tissues, including the adrenals (Davis and Lau, 1994), the brain (Maheux et al., 2005), the ovary (Richards, 1994) and the testis (Song et al., 2001). Intriguingly, bisphenol A (BPA), one of the well-known endocrine disrupting chemi- cals, induces Nur77 gene expression and subsequently alters the steroidogenesis in K28, a mouse Leydig tumor cell line (Song et al., 2002). In this study, we determined that all NR4A subfamily members are expressed in NCI-H295R cells, and we are the first to report that they are rapidly and transiently induced by MEHP. The time until reaching maximum induction of Nur77 was shorter than for the other members of the NR4A subfamily, and the expression level of Nur77 was expected to be much higher than the other members. Therefore, we focused on Nur77 in this study. Nur77 expression was specifically and dose-dependently induced by MEHP among the phthalate esters tested. Nur77 protein expression was also induced by short-term exposure to MEHP. Interestingly, DEHP had no effect on aromatase activity and Nur77 expression in this study, though it has been shown to act as a reproductive toxicant in a rodent (Tickner et al., 2001). DEHP is rapidly metabolized to its monoester MEHP in vivo, so it is thought that MEHP may be partially involved in the repro- ductive adverse effect of DEHP. In addition, MEHP rapidly and transiently activated the Nur77 promoter in this study. There are some potential cis-acting elements on the Nur77 promoter, such as AP-1, CRE and ERE, so it is conceivable that the associa- tion of MEHP with those sites or a non-canonical site causes the activation of its promoter. It is interesting that the maximal induction level of the Nur77 promoter by MEHP was about half of that by FSK. To the best of our knowledge, there has only been one report, other than the present paper, indicating that the well-known endocrine disrupting chemical BPA induces NR4A subfamily expression, especially Nur77, and the induction of Nur77 by BPA was lower than the case of luteinizing hormone (LH) in the testicular Leydig cell line K28 (Song et al., 2002). It was clearly demonstrated in this study that activation of the Nur77 promoter by MEHP was also lower than in the cases of FSK, a cAMP stimulator, as well as LH.
Next, to explore the induction mechanism of Nur77 by MEHP, we first examined the possibility that cAMP, a well-known intracellular second messenger, participates in the induction. Intracellular cAMP accumulation was strongly increased by FSK, but MEHP exposure by a concentration that induced Nur77 expression had no effect on its accumulation. Furthermore, to elucidate the signaling pathways which are involved in Nur77 induction by MEHP, we also treated the cells with various inhibitors of diverse signaling pathways. MEHP- induced Nur77 gene expression was significantly suppressed to the basal level by the PKC inhibitor bisindolylmaleimide I and the PI3K inhibitor wortmannin, clarifying that Nur77 induction by MEHP is mediated via PKC and PI3K signaling pathways. However, the PKA inhibitor H89 had no effect on Nur77 induc- tion by MEHP, confirming again that Nur77 induction by MEHP was not mediated through the cAMP-PKA pathway. Nur77
induction by BPA was shown to be mediated through cAMP- PKA and MAPK pathways in mouse Leydig K28 cells (Song et al., 2002), findings which are different from the present results.
It is possible that rapid and transient induction of Nur77 by MEHP may repress aromatase expression in NCI-H295R cells. To confirm this, we cotransfected the full-length Nur77 expression vector and a luciferase reporter construct harboring promoters I.3 and II of CYP 19 gene into the cells. Ectopic expres- sion of Nur77 impaired FSK-induced transcriptional activation of the promoter I.3/II in a dose-dependent manner. Decreas- ing the level of transcriptional activity with 100 p.M MEHP was similar to 100 ng Nur77 transfection. Furthermore, expo- sure of MEHP under the ectopic expression of Nur77 tended to attenuate the transcriptional activity of this promoter more than independent exposure of MEHP (data not shown). More- over, to obtain additional evidence that MEHP-induced Nur77 expression might lead to aromatase suppression, the promoter I.3/II reporter construct was cotransfected into the cells with the Nur77-WT or Nur77-DN expression vector. Transfection of Nur77-WT impaired promoter I.3/II activity in a dose-dependent manner, whereas no impairment was observed with Nur77- DN. However, exactly what factors are directly involved in the aromatase suppression by Nur77 is not clear at present. It is inter- esting to note that there has been a report about the involvement of Nur77 in the regulation of another transcription factor expres- sion, which controls aromatase expression. There is a recent report indicating that Nur77 induces gonadotropin-inducible ovarian transcription factor-1 (GIOT-1) gene expression in tes- ticular Leydig cell lines and that GIOT-1 acts as a novel corepressor of the SF-1 (Song et al., 2006). Therefore, even in the case of NCI-H295R cells, there is a possibility that Nur77 induces GIOT-1 and GIOT-1 suppresses aromatase expression via repression of SF-1. We hope to examine this hypothesis in the near future.
Recently, there was a report that Nur77 and Nurr1, which are rapidly and transiently induced by LH (Cortinez et al., 2005), both impair FSK-induced transcriptional activation of the CYP19 gene promoter II in KGN, a human granulosa-like tumor cell line (Wu et al., 2005). Moreover, they revealed in that report that FSK induces CYP19 gene expression as well. However, the contradiction between the induction of aromatase by FSK and the suppression of aromatase by FSK-induced Nur77 and Nurr1 remains to be clarified. Meanwhile, Park et al. reported that atypical PKC{ mediates the LH action on the induction of Nur77 in rat granulosa cells of preovulatory follicles (Park et al., 2003). Differences in the signaling pathways (e.g. cAMP-PKA or Ca2+-PKC pathway) for Nur77 induction may lead to the difference in the physiological role induced by Nur77, includ- ing transcriptional regulation of its target gene. Promoter II is involved in aromatase expression in NCI-H295R cells as well as in the ovary, thus, NR4A subfamily members may regulate CYP19 gene expression in this cell as well. In addition, aro- matase suppression by MEHP may be caused by Nur77 (and Nurr1 as well) which is directly induced by MEHP.
In this study, we observed aromatase suppression with expo- sure to a relatively high dose of MEHP. The maximum serum level of MEHP in humans within the general population is about
two orders of magnitude below our effective dose (Kato et al., 2004). However, both MEHP and MBP were found in >75% of subjects within the general population tested (Blount et al., 2000). Moreover, the maximum level of MEHP within human breast milk samples reached about 1400 µg/1, which is two orders of magnitude above the maximum serum level of MEHP in humans (Main et al., 2006). Therefore, MEHP levels should be measured not only in serum, but also in other tissues as well.
In gonadal tissues and adrenals, Nur77 expression is induced by various tropic hormones, such as follicle stimulating hor- mone, LH and adrenocorticotropic hormone, and this induction is mediated via activation of the cAMP-PKA pathway (Bassett et al., 2004b; Havelock et al., 2005; Song et al., 2001). In this study, MEHP-induced Nur77 expression in a human adreno- cortical carcinoma cell line, although the detailed regulatory mechanism of this induction remains to be elucidated. BPA and proto-oncogene c-jun concertedly transactivate Nur77 gene pro- moter in K28 cells (Song et al., 2002), thus both MEHP and c-jun may also concertedly have an effect on Nur77 expression. The underlying mechanism of NR4A-mediated transcriptional repression remains to be elucidated. The NR4A subfamily binds as a monomer to NBRE or Nur response element (NurRE) on the target gene promoter to transactivate these genes (Maira et al., 1999; Wilson et al., 1993a). It has also been demonstrated that it binds as a homodimer to NurRE on that promoter (Maira et al., 1999), and that Nur77 and Nurr1 (but not NOR-1) bind as a heterodimer with RXR to direct repeats separated by five nucleotides (Zetterstrom et al., 1996). On the other hand, Nur77 and Nurr1 both impair transcriptional activation of promoter II, and it is not mediated via the interaction of NBRE (Wu et al., 2005), however, the attenuated mechanism has not yet been elucidated. Elucidation of the mechanism of suppression of NR4A target gene expression in the future will hopefully provide valuable information on the detailed suppression mechanism of aromatase by MEHP.
Acknowledgements
This work was supported in part by a Grand-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Kiyoshi Kawana and Rurika Miyake for their technical assistance.
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