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Peroxisome Proliferators Disrupt Retinoic Acid Receptor Alpha Signaling in the Testis¹

^a School of Molecular Biosciences, ^b College of Pharmacy, ^c Center for Reproductive Biology, Washington State University, Pullman, Washington 99164

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Peroxisome proliferators include a diverse group of chemicals, some of which have been demonstrated to be testicular toxicants. However, the mechanism by which peroxisome proliferators, such as phthalates, cause testicular damage is not clear. It is known that retinoic acid receptor alpha (RAR) and its retinoic acid ligand, the acid form of vitamin A, are required for spermatogenesis. It has been demonstrated that the absence of RAR gene or vitamin A in the animal leads to testis degeneration and sterility. Therefore, any compound that disrupts the action of vitamin A in the testis could potentially be damaging to male fertility. The current investigation examined a novel hypothesis that a mechanism of degeneration by peroxisome proliferators in the testis is due, in part, to disruption of the critical RAR signaling pathway. We show that peroxisome proliferators were able to disrupt the retinoic acid-induced nuclear localization of RAR and the retinoic acid-stimulated increase in transcriptional activity of a retinoic acid-responsive reporter gene in Sertoli cells. Concomitantly, peroxisome proliferators increased the nuclear localization of PPAR and the transcriptional activity of a peroxisome proliferator-responsive reporter gene in these cells. These results indicate that peroxisome proliferators can indeed shift the balance of nuclear localization for RAR and PPAR, resulting in deactivation of the critical RAR transcriptional activity in Sertoli cells.

Sertoli cells, spermatogenesis, steroid hormone receptors, testis, toxicology

	INTRODUCTION

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Peroxisome proliferators include a wide variety of compounds that can be categorized as plasticizers, hypolipidemic drugs, nonsteroidal anti-inflammatory agents, industrial solvents, and herbicides [1, 2]. These compounds were named for their ability to cause peroxisome proliferation in the liver and other tissues. Some of these compounds have also been shown to cause hepatocarcinogenesis [1, 2] and damage to the testis [1, 3, 4]. Among the peroxisome proliferators, phthalates are of particular interest in reproductive biology because they are reported to be testicular toxicants, specifically to Sertoli cells [5].

Phthalates have been used as plasticizers since the 1930's to impart flexibility to plastic materials and are found in such commercially available products as plastic food wraps and containers, blood transfusion bags, and children's toys [3]. However, phthalates are not covalently bound to the plastic material and may leach out to contaminate their products, which can then be easily ingested [1]. In fact, phthalates have been found in all human urine samples analyzed [1, 6]. Di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate and is metabolized to mono-(2-ethylhexyl) phthalate (MEHP), the active testicular toxicant [1, 7]. DEHP treatment of rodents leads to testicular degeneration, which includes Sertoli cell vacuolation, shedding of spermatocytes and spermatids, and ultimately decreased sperm production [1].

Several hypolipidemic drugs that have the ability to lower the levels of triglycerides in humans have also been used extensively to demonstrate the effects of peroxisome proliferators on the liver [2, 8]. Of the two hypolipidemic drugs used in this study, clofibrate is an approved drug and is used in the United States, whereas Wyeth-14,643 (Wy-14,643), a more potent peroxisome proliferator, is not approved [2]. Previous studies have reported that clofibrate treatment of dogs and monkeys arrests spermatogenesis [4]. However, the effect of clofibrate and Wy-14,643 on rodent or human spermatogenesis is not yet clear.

Peroxisome proliferators have been shown to act through peroxisome proliferator-activated receptors (PPAR), of which there are at least three subtypes: , ß, and [2, 8–10]. These receptors are members of the steroid/thyroid hormone receptor superfamily, which, upon activation, form heterodimers with retinoid X receptors (RXR) and regulate the transcription of genes that contain peroxisome proliferator-responsive elements (PPREs) [2, 8–10]. Whether phthalates act as ligands for PPAR is not known, but a recent study suggests that Wy-14,653, arachidonic acid synthetic analog (6,8,11,14-eicosatetraynoic acid), leukotriene D4 antagonist (LY-171883), and clofibric acid can bind PPAR and cause conformational changes in vitro [11].

PPAR knockout mice studies demonstrated that PPAR, which has been shown to regulate genes involved in lipid catabolism [2, 8–10], is responsible for the peroxisome proliferation and hepatocarcinogenesis and that PPARß and are not involved in this phenomenon [12–14]. Consistently, the PPAR-null mice showed no evidence of peroxisome proliferation or hepatocarcinogenesis after treatment with Wy-14,643 [13, 14]. The PPAR knockout studies also demonstrated that PPAR is only partially responsible for the testicular damage seen with DEHP treatment and there was a second pathway, a non-PPAR-mediated pathway, responsible for testicular damage that remained in PPAR-null mice treated with DEHP [15]. In the normal testis, PPAR is expressed both in Sertoli cells and germ cells in rats and in germ cells in humans [16].

All-trans retinoic acid (tRA), an acid form of vitamin A, is required for spermatogenesis [17–19]. The absence of tRA leads to the loss of germ cells by sloughing of the advanced germ cells from the testis and apoptosis of the remaining primary spermatocytes [17–20]. The only germ cells remaining in the testis are the type A spermatogonia and preleptotene spermatocytes [21–24]. Therefore, disruption of this critical pathway could lead to germ cell loss and male infertility.

The action of tRA is postulated to be mediated through the retinoid receptors in the testis [25]. These receptors are members of the steroid/thyroid hormone receptor superfamily that, upon activation by tRA, regulate the transcription of genes that contain retinoic acid-responsive elements (RAREs) [26]. There are two families of retinoid receptors, the retinoid acid receptors (RARs) and retinoid X receptors (RXR), each with the three subtypes , ß, and [25]. These receptors can be regulated at the level of transcription, translation, nuclear localization, ligand binding, heterodimerization, and DNA binding. RAR is required for spermatogenesis. The RAR knockout mice are sterile and their testes have a morphology similar to the vitamin A-deficient testis [26]. Recent studies have shown that the subcellular localization and transcriptional activity of RAR is regulated by tRA [27].

It has been shown that PPARs may inhibit the transcriptional activity of thyroid hormone receptor (TR) by disrupting TR:RXR heterodimers and instead form PPAR:RXR heterodimers [28]. In a similar manner, activation of PPAR in the testis may disrupt the critical retinoid receptor pathway by competing with RAR for RXRs. With this background, we hypothesize that peroxisome proliferator treatment of testicular cells disrupts the retinoid receptor signaling pathway, which could then lead to testicular degeneration. In this study, the effect of peroxisome proliferators on the retinoid signaling pathway at the level of RAR nuclear trafficking and transcriptional regulation was examined in Sertoli cells, the testicular somatic cell type, clearly shown to be the center of action for phthalates and vitamin A [5, 29, 30]. We also characterized the subcellular localization of RXR and PPAR and the transcriptional regulation of PPAR after peroxisome proliferator treatment.

	MATERIALS AND METHODS

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Tissue Culture

Mouse Sertoli cells (MSC-1) are an immortalized mouse Sertoli cell line from a testicular tumor found in transgenic mice with the transforming region of the SV40 virus fused to the 5' flanking region of human Mullerian inhibiting substance [31]. The promoter for Mullerian inhibiting substance, which is only expressed in the testis and ovary, was used to target the DNA construct to Sertoli cells. This cell line has been shown to express many Sertoli cell genes but not follicle-stimulating hormone receptor [31, 32]. MSC-1 cells were cultured in Dulbecco modified eagle medium (DMEM) (Gibco BRL, Gaithersburg, MD) supplemented with penicillin-streptomycin and 5% fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO₂/95% air (v/v). Cells were grown to approximately 50–75% confluency before being serum starved for 24–48 h in 0.1% FCS in DMEM to reduce endogenous tRA.

Primary Sertoli cells were isolated from the testes of 20-day-old rats by sequential enzymatic digestion [33]. Animal experimentation was approved by the Institutional Animal Care and Use Committee and conducted in accordance with the highest standards of humane animal care as outlined in the National Institute of Health guide for the Care and Use of Laboratory Animals. Decapsulated testis fragments were digested first with 0.25% (w/v) trypsin (Gibco BRL) to remove the interstitial cells and then with 0.7 mg/ml collagenase (Sigma Chemical, St. Louis, MO) and 1 mg/ml hyaluronidase (Sigma Chemical). Sertoli cells were then plated under serum-free conditions on either 24-well Falcon plates or 4-well chamber slides (Nalge Nunc, Naperville, IL). Cells were maintained in a 5% CO₂ atmosphere in Ham F-12 medium (Gibco BRL) without serum at 32°C. After allowing for an initial 2-day seeding, media was changed every day for a maximum of 5 days to reduce endogenous tRA.

Primary Sertoli cells were also plated on coverslips, cultured under serum-free conditions for 4 days, and stained with hematoxylin and eosin. The unique looking nucleus of Sertoli cells allows determination of Sertoli cell number in the preparation. Percentage of Sertoli cells was determined to be 91.6% ± 4.2%. The contaminants were myoid cells (4.8%) and germ cells. Myoid cells contaminating the primary Sertoli cell preparation were detected using anti-human -smooth muscle action monoclonal antibody (1:50; DAKO A/S, Glostrup, Denmark).

Chemicals

Cells were treated with mono-(2-ethylhexyl) phthalate (MEHP), 2-(p-chlorophenoxy)-2-methylpropionic acid (clofibrate), 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy-14,643), tRA, or phorbol 12-myristate 13-acetate. MEHP was generously synthesized by Dr. Jan Wahlstrom in the laboratory of Dr. Jeffrey Jones (Department of Chemistry, Washington State University). The phthalate products were purified by high-performance liquid chromatography and the purity determined by nuclear magnetic resonance and mass spectroscopy. Preparations of MEHP were greater than 99% pure. Other chemicals were purchased from Sigma Chemical and dissolved in dimethyl sulfoxide (DMSO). Final concentration of DMSO was 0.1% (v/v), and the chemicals were carried by 0.1% fatty acid-free bovine serum albumin (Boeringer Mannheim, Indianapolis, IN) to the cells.

Immunofluorescence

Cells grown on chamber slides were fixed by immersion in cold methanol (-20°C) for 15 min, washed in PBS, blocked with 10% goat or rabbit serum for 10 min, and then incubated with the anti-RAR, RXR, and PPAR antibodies diluted 1:200 in 10% goat or rabbit serum overnight at 4°C. The cells on the slides were further washed in PBS and treated with biotinylated goat anti-rabbit antibody or rabbit anti-goat antibody (1:300; Vector Laboratories, Burlingame, CA), followed by fluorescein avidin D (1:300; Vector Laboratories). The primary antibodies, the anti-RAR, RXR, PPAR peptide antibodies, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-RAR antibody was raised against a human RAR peptide, consisting of amino acids 443–462. The anti-RXR antibody was against a human RXR peptide, consisting of amino acids 2–21. The anti-PPAR antibody was against a human PPAR peptide, consisting of amino acids 1–98. Anti-RAR and anti-RXR were characterized previously [20, 34, 35]. Anti-PPAR antibody that was used on a Western blot analysis of Sertoli cell and total testicular extracts recognized one single protein of molecular weight 55 kD (data not shown). Three separate immunostaining experiments were performed for each antibody per treatment. For a negative control, the primary antibody was incubated with immunizing peptide (Santa Cruz Biotechnology) overnight at 4°C prior to addition to fixed cells. For additional negative controls, MSC-1 and Sertoli cells were fixed and immunofluorescence conducted but without any primary and secondary antibody. All digital images were obtained using a laser scanning confocal system (MRC 1024; BioRad, Hercules, CA) or Leitz DMRB with epifluorescence (Wetzlar, Germany) and a Magnafire digital camera (Optronics, Goleta, CA).

DNA Constructs

The luciferase reporter constructs used were pRARE-tK-Luc, which contains three RAREs from the RARß promoter [36], and pPPRE-tk-Luc, which contains three PPREs from rat acyl-CoA oxidase promoter [37], both under the control of the Herpes simplex virus thymidine kinase promoter. The mouse PPAR expression vector, pCMXmPPAR, has a PCR fragment cloned into the EcoRV site of the pCMX expression vector, under the influence of cytomegalovirus promoter [38]. The RAR expression vector, pHookRAR, was constructed in two steps, first by excising the 2.8-kb RAR cDNA from the retroviral vector LRARSN [36] and cloning into the EcoR1 and BamHI sites of pBluescript and then subcloning into the HindIII and XbaI sites of the pHook vector (Invitrogen, San Diego, CA) [27]. The pHook expression vector is also under the control of cytomegalovirus promoter. The pHook-LacZ was purchased from Invitrogen and served as a transfection efficiency control.

Transient Transfections and Luciferase Assays

MSC-1 cells were grown in DMEM containing 5% FCS on 24-well plates to 75% confluency. Cells were then serum starved in 0.1% FCS containing media for 24 h and transfected with 42 ng of the pRARE-tk-Luc reporter plasmid [36] or pPPRE-tk-Luc reporter plasmid [37] and 42 ng pHookLacZ per well in 150 µl MEM and 150 µl transfection mix from the lipofectAMINE transfection system (Life Technologies, Grand Island, NY). PPAR expression plasmid (pCMXmPPAR) [38] was added to the transfection mix at 17 ng per well when it was used. Five hours after transfection, the transfection medium was removed and 1 ml of DMEM minus phenol red containing 2% FCS was added. In addition, cells were treated for 24 h with various compounds using 0.1% fatty acid-free BSA as carrier and then were harvested for luciferase and ß-galactosidase assays.

Primary Sertoli cells were plated on 24-well plates in Ham F-12 media without serum. After 3–4 days, Sertoli cells were transfected with a gene construct using calcium phosphate transfection system (Gibco BRL) coupled with hyperosmotic shock, as previously described [39]. Briefly, 0.8 µg of pRARE-tk-Luc reporter plasmid [36] or pPPRE-tk-Luc reporter plasmid [37] and 1.1 µg of pHook-LacZ in 150 µl transfection buffer were added to each well containing 1 ml DMEM minus phenol red media and incubated for 4 h. PPAR expression plasmid (pCMXmPPAR) [38] was added to the transfection mix at 208 ng per well when it was used. After aspirating transfection media, 1 ml of 10% glycerol in Hank buffer was added for 3 min for hyperosmotic shock. Then hyperosmotic shock media was diluted with an additional 1 ml of Hank buffer and washed two times with 1 ml of Hank buffer, and finally fresh DMEM minus phenol red media was added. In addition, cells were treated with various agents.

Both MSC-1 cells and primary Sertoli cells were harvested 16–24 h posttreatment and accumulated luciferase activity was analyzed using a Luciferase Assay System (Promega, Madison, WI) and a luminometer (EG&G Microlumat; Berthold Systems, Aliquippa, PA). The ß-galactosidase activity was determined using Galactosidase Assay System (Promega) and was used to normalize transfection efficiency. Experiments were performed three times in triplicate.

Statistics

Statistical analysis of normalized transcriptional activity consisted of one-way analysis of variance (ANOVA), followed by pairwise comparisons of the means at = 0.05 (Tukey-Kramer test, Minitab 10 Xtra; Minitab Inc., State College, PA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of Peroxisome Proliferators on RAR Subcellular Localization

To investigate whether peroxisome proliferators can influence the pattern of nuclear localization of RAR, immunofluorescence studies were performed with MSC-1 cells that had been treated with tRA, Wy-14,643, MEHP, or clofibrate (Figs. 1 and 2). The tRA was used as a positive control because it was previously shown to increase the amount of RAR in the nucleus of MSC-1 cells [27]. As expected, tRA was able to increase RAR in the nucleus within 15 min compared with vehicle-treated cells (Fig. 1; compare D with A–C). On the other hand, Wy-14,643, an exceptionally potent peroxisome proliferator, inhibited the tRA-induced nuclear localization of RAR (Fig. 1, J–L). Similarly, MEHP also inhibited the tRA-induced nuclear localization of RAR in MSC-1 cells (Fig. 1, P–R).

View larger version (85K): [in this window] [in a new window]   FIG. 1. Subcellular localization of RAR after peroxisome proliferator treatment of MSC-1 cells. MSC-1 cells were treated with vehicle (A), 0.1% BSA (B), or 0.1% BSA and DMSO (C) for 60 min. MSC-1 cells were also treated with 0.5 µM tRA (D–F), 500 µM Wy-14,643 (G–I), 500 µM Wy-14,643 and 0.5 µM tRA (J–L), 500 µM MEHP (M–O), and 500 µM MEHP and 0.5 µM tRA (P–R) for 15, 30, and 60 min. The cells were fixed by methanol and immunofluorescence was performed using an anti-RAR peptide antibody. For negative controls, cells were incubated with anti-RAR that was preabsorbed with immunizing peptide (S), or the same immunofluorescence method was followed except omitting either the primary (T) or secondary (U) antibody. Bar in L = 50 µm

View larger version (137K): [in this window] [in a new window]   FIG. 2. Subcellular localization of RAR after clofibrate treatment of MSC-1 cells. MSC-1 cells were treated with 100 µM clofibrate (A–C), 100 µM clofibrate and 0.5 µM tRA (D–F), 1 mM clofibrate (G–I), or 1 mM clofibrate and 0.5 µM tRA (J–L) for 15, 30, and 60 min. The cells were fixed by methanol and immunofluorescence was performed using an anti-RAR peptide antibody. Bar in L = 50 µm

Interestingly, clofibrate, a weaker peroxisome proliferator, only delayed the tRA-induced nuclear localization at the higher concentration of 1 mM for 60 min (Fig. 2, J–L). At a lower concentration of 100 µM, clofibrate only partially inhibited nuclear localization of RAR (Fig. 2, D–F). In contrast, both Wy-14,643 and MEHP at lower concentrations of 100 and 200 µM still inhibited tRA-induced nuclear localization of RAR (data not shown). In the absence of tRA, Wy-14,643, MEHP, and clofibrate had virtually no effect on the subcellular localization of RAR in MSC-1 cells (Fig. 1, G–I and M–O; Fig. 2, A–C and G–I). In negative controls, no staining was detected (Fig. 1, S–U).

Furthermore, primary rat Sertoli cells were doubly stained with an anti-RAR peptide antibody and propidium iodide or 4',6'-diamidino-2-phenylindole (DAPI) to stain DNA in the nucleus to assess the positions of the nuclei and the density of plating (Fig. 3). Although primary Sertoli cells are known to store retinoids [40], it was clear that extracellularly added tRA further increased the nuclear RAR compared with that seen for vehicle-treated primary Sertoli cells (Fig. 3; compare C with A). In addition, pretreatment with Wy-14,643 or MEHP decreased tRA-induced nuclear localization of RAR in primary Sertoli cells (Fig. 3; compare E or G with C).

View larger version (91K): [in this window] [in a new window]   FIG. 3. Subcellular localization of RAR after peroxisome proliferator treatment of primary rat Sertoli cells. Primary rat Sertoli cells were treated for 60 min with vehicle (A, B), 1 µM tRA (C, D), 500 µM Wy-14,643 and 1 µM tRA (E, F), and 500 µM MEHP and 1 µM tRA (G, H). The cells were fixed by methanol and doubly stained with an anti-RAR peptide antibody followed by biotinylated goat anti-rabbit antibody and fluorescein avidin D (green) and propidium iodide (red) or DAPI (blue) to stain DNA in the nucleus. Digital images were obtained to display fluorescein avidin D stain (A, C, E, G), propidium iodide stain (B, D, F), or DAPI (H). Bar in H = 50 µm

Effect of Peroxisome Proliferators on PPAR and RXR Subcellular Localization

To determine the effects of MEHP on the subcellular localization of PPAR, MSC-1 cells and primary rat Sertoli cells were treated with Wy-14,643 or MEHP in the absence or presence of tRA. In the presence of Wy-14,643 and MEHP, PPAR was more abundant in the nucleus in both MSC-1 cells and in rat primary Sertoli cells (Fig. 4, A, C, and E; Fig. 4, B, C, and E). Treatment of tRA in addition to Wy-14,643 and MEHP further distributed the receptor more into the nucleus in both cell types (Fig. 4, A, D, and F; Fig. 4, B, D, and F).

View larger version (132K): [in this window] [in a new window]   FIG. 4. Subcellular localization of PPAR after peroxisome proliferator treatment of MSC-1 cells and primary rat Sertoli cells. MSC-1 cells (A) and primary rat Sertoli cells (B) were treated for 60 min with vehicle (A), 1 µM tRA (B), 500 µM Wy-14,643 (C), 500 µM Wy-14,643 and 1 µM tRA (D), 500 µM MEHP (E), 500 µM MEHP and 1 µM tRA (F). The cells were fixed by methanol and immunofluorescence was performed using an anti-PPAR peptide antibody. Bar = 50 µm

In addition, the subcellular localization of RXR, a potential heterodimer partner of RAR and PPAR, was examined by immunofluorescence in MSC-1 and primary Sertoli cells after tRA, Wy-14,643, or MEHP treatment. Unlike the subcellular distribution of RAR and PPAR, which changed with treatments, albeit in an opposite manner for each receptor, RXR was present predominantly in the nucleus regardless of treatment both in MSC-1 (data not shown) and primary Sertoli cells (Fig. 5).

View larger version (142K): [in this window] [in a new window]   FIG. 5. Subcellular localization of RXR after peroxisome proliferator treatment of primary rat Sertoli cells. Primary rat Sertoli cells were treated for 60 min with vehicle (A), 1 µM tRA (B), 500 µM Wy-14,643 (C), 500 µM MEHP (E), 500 µM Wy-14,643 and 1 µM tRA (D), 500 µM MEHP and 1 µM tRA (F). The cells were fixed by methanol and immunofluorescence was performed using an anti-RXR peptide antibody. Bar in F = 50 µm

Effect of Peroxisome Proliferators on the Transcriptional Activity of RARE-Containing Reporter Gene Construct

The transcriptional activity of RAR has been shown to directly relate to the amount of RAR present in the nucleus of MSC-1 cells [27, 39]. To analyze whether disruption of tRA-induced RAR nuclear localization after peroxisome proliferator treatment was linked to changes in retinoid-regulated transcription, MSC-1 cells and primary rat Sertoli cells were transiently transfected with a RARE-containing luciferase reporter gene and then treated with peroxisome proliferators (Figs. 6 and 7). As expected from previous results [27], tRA treatment increased the transcriptional activity of the reporter construct 3- to 4-fold compared with the transcriptional activity found in vehicle treated cells. In addition, cotransfection of RAR expression vector only slightly increased the luciferase activity (data not shown), indicating that the amount of endogenous RAR is sufficient to activate luciferase from the RARE-containing luciferase reporter gene. However, Wy-14,643 and MEHP, which altered the tRA-induced nuclear localization of RAR, decreased the tRA-stimulated increase in transcription in a dose-dependent manner (Fig. 6, A and B).

View larger version (27K): [in this window] [in a new window]   FIG. 6. Effect of peroxisome proliferators on a RARE-luciferase reporter gene in MSC-1 cells. MSC-1 cells were cotransfected with a RARE-luciferase reporter and ß-galactosidase constructs to assess the effect of peroxisome proliferators on the transcription of tRA-regulated RARE-containing genes. After transfection, MSC-1 cells were treated for 16–24 h and then assayed for luciferase and ß-galactosidase activities. A) Cells were treated with vehicle (V), 1 µM tRA, and varying concentrations of Wy-14,643 (100, 200, 500 µM). B) Cells were treated with vehicle (V), 1 µM tRA, and varying concentrations of MEHP (100, 200, 500 µM). C) Cells were treated with vehicle, 1 µM tRA, 500 µM Wy-14,643, and 1 mM clofibrate. After normalization of transfection efficiency to ß-galactosidase activity, the relative fold luciferase activities are presented on the y axis. Data are represented as the mean ± SD (n 3). Means bearing different value labels are significantly different from each other (P 0.05)

View larger version (20K): [in this window] [in a new window]   FIG. 7. Effect of peroxisome proliferators on a RARE-luciferase reporter gene in primary rat Sertoli cells. Primary rat Sertoli cells were transfected with RARE-luciferase reporter and ß-galactosidase constructs and the effect of peroxisome proliferators on the transcription of the RARE-luciferase gene was determined. Sertoli cells were treated for 16–24 h with (A) vehicle, 1 µM tRA, and varying concentrations of Wy-14,643 (100, 200, 500 µM) and (B) vehicle, 1 µM tRA, and varying concentrations of MEHP (100, 200, 500 µM). Data are represented as the mean ± SD (n 3). Means bearing different value labels are significantly different from each other (P 0.05)

On the other hand, clofibrate at 1 mM concentration was unable to inhibit the tRA-induced transcription (Fig. 6C). This was expected because RAR was translocated into the nucleus by tRA after 60 min even in the presence of clofibrate (Fig. 2L) and the RARE-mediated transcription as determined by luciferase activity is measured after 16–24 h.

In addition, both Wy-14,643 and MEHP decreased the tRA-induced transcriptional activity in primary rat Sertoli cells (Fig. 7, A and B). Interestingly, Wy-14,643, a potent peroxisome proliferator, also lowered the transcription levels in primary Sertoli cells at higher concentrations. This is consistent with peroxisome proliferators possibly countering the effects of endogenous retinoids in primary Sertoli cells. Sertoli cells are known to store retinoids [40]. Considered together, these results clearly suggest that peroxisome proliferators disrupt the retinoid signaling pathway also in primary Sertoli cells. They decrease the nuclear localization of RAR, which is then directly coupled to decreased transcriptional activity of RAR.

Effect of Peroxisome Proliferators on the Transcriptional Activity of PPRE-Containing Reporter Gene Construct

To determine whether peroxisome proliferators indeed increased the PPRE-containing reporter gene transcriptional activity, MSC-1 cells were transiently transfected with a PPRE-containing luciferase reporter gene construct, with and without a PPAR gene construct, and then treated with increasing concentrations of MEHP (Fig. 8A). We found that cotransfection of PPAR and the PPRE-containing luciferase reporter gene construct increased luciferase reporter activity about 30-fold even in the absence of MEHP (Fig. 8A). This is consistent with some unknown factor in the media stimulating the transcriptional activity of the PPRE-containing reporter gene. However, MEHP and Wy-14,643 additionally increased PPRE-luciferase reporter activity significantly, about 2- to 3-fold, above the vehicle-only level with PPAR expression vector cotransfection (Fig. 8, A–C). Both MEHP and Wy-14,643 stimulated the transcriptional activity at a concentration as low as 10 µM in these cells (data not shown). The tRA also synergistically stimulated PPRE-luciferase reporter activity above the MEHP level, suggesting that the RXR partner of PPAR may be an active partner (also called a permissive partner) requiring the tRA ligand (Fig. 8C).

View larger version (29K): [in this window] [in a new window]   FIG. 8. Effect of peroxisome proliferators on a PPRE-luciferase reporter gene in MSC-1 cells. MSC-1 cells were transfected with PPRE-luciferase reporter and ß-galactosidase constructs, treated for 16–24 h, and then assayed for luciferase and ß-galactosidase activities. A) Cells were treated with vehicle after PPAR expression vector cotransfection (V), no PPAR expression vector transfection (No PPAR V), and were treated with varying concentrations of MEHP (50–500 µM) after PPAR expression vector cotransfection. B) Cells were treated with vehicle and varying concentrations of Wy-14,643 (50–500 µM) after PPAR cotransfection. C) Cells were treated with vehicle, 100 µM MEHP, 100 µM Wy-14,643, and/or 1 µM tRA after PPAR cotransfection. Data are represented as the mean ± SD (n 3). Means bearing different value labels are significantly different from each other (P 0.05)

In addition, the PPRE-mediated transcription was examined in primary rat Sertoli cells after cotransfection of the PPRE-containing luciferase gene and the PPAR expression construct (Fig. 9). Both MEHP at 10 and 50 µM and Wy-14,643 at 50 and 100 µM increased the PPRE-mediated transcriptional activity 5- to 9-fold in primary rat Sertoli cells compared with that in vehicle-treated cells. Furthermore, tRA alone stimulated PPRE-luciferase reporter activity by 2.5-fold, as shown similarly in MSC-1 cells, and tRA significantly increased the reporter activity above the MEHP level at 10 µM or Wy-14,643 levels at 100 µM in primary Sertoli cells. These results clearly suggest that peroxisome proliferators regulate the transcriptional activity of PPAR in primary Sertoli cells in a similar manner as in MSC-1 cells and this is also directly coupled to increased nuclear localization of PPAR.

View larger version (29K): [in this window] [in a new window]   FIG. 9. Effect of peroxisome proliferators on a PPRE-luciferase reporter gene in primary rat Sertoli cells. Primary rat Sertoli cells were transfected with a PPRE-luciferase reporter, PPAR, and ß-galactosidase constructs and the effect of peroxisome proliferators on the transcription of the PPRE-luciferase gene determined. Sertoli cells were treated for 16–24 h with vehicle (V), 1 µM tRA (tRA), MEHP (10, 50, 100 µM), Wy-14,643 (10, 50, 100 µM), MEHP and tRA or Wy-14,643 and tRA. Data are represented as the mean ± SD (n 3). Means bearing different value labels are significantly different from each other (P 0.05)

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The purpose of this study was to determine whether administration of phthalates or other peroxisome proliferators to Sertoli cells disrupted the RAR signaling pathway. Because RAR signaling is essential for testis function, we reasoned that any chemical that antagonized RAR transcriptional activity could be detrimental to testis function. We found that peroxisome proliferator treatment of MSC-1 cells and primary rat Sertoli cells decreased the tRA-induced nuclear localization of RAR and the RARE-driven luciferase activity, which is indicative of transcriptional activity of RAR. Wy-14,643, a potent peroxisome proliferator, and MEHP, a weaker peroxisome proliferator, which is a hydrolyzed product of DEHP [1], were able to inhibit the tRA-induced nuclear localization of RAR and decrease the tRA-stimulated transcriptional activity of RAR. In contrast, clofibrate, a reportedly stronger peroxisome proliferator in liver cells but a weak testicular toxicant, only slightly perturbed the nuclear localization of RAR for a short time and had no long-term effect on transcriptional activity of RAR in the presence of tRA.

Equally important to point out is that the inhibitory action of Wy-14,643 and MEHP on the nuclear localization of RAR was dominant over the stimulatory effect of tRA in localizing RAR to the nucleus. That is, if either Wy-14,643 or MEHP was present, there was a loss of RAR from the nucleus, even if tRA was present. This loss of RAR from the nucleus was subsequently reflected as a decreased tRA-regulated transcriptional activity of RAR. This is consistent with a previous finding in lung fibroblast cells, in which peroxisome proliferators decreased the expression of elastin, a retinoid-regulated gene [41]. Together, these results suggest that some peroxisome proliferators may not only disrupt the retinoid receptor pathway for Sertoli cells but also Sertoli cells may not be the only cell type in which peroxisome proliferators exhibit this mode of regulation.

At the same time that peroxisome proliferators disrupted RAR transcriptional activity in Sertoli cell cultures, PPAR and its PPRE-mediated transcription was activated. Because a regulatory mechanism that is able to explain the testicular damage by phthalates in vivo should include PPAR as a key player [15], the mechanism that we describe, which involves the activation of PPAR by peroxisome proliferators, certainly fits the requirement. Additionally, our results support concomitant down-regulation of the critical RAR activity in Sertoli cells by peroxisome proliferators. Our results also imply that any cell that expresses PPAR and RAR can be potentially regulated by peroxisome proliferators in a similar manner. In the testis, the germ cells may exhibit a similar mode of regulation of the RAR pathway by peroxisome proliferators as observed in Sertoli cells because both PPAR and RAR are expressed in germ cells [16, 34, 35].

A possible biochemical mechanism for disruption of the retinoid receptor pathway is competition between RAR and PPAR for the same heterodimerization partner, one of the RXRs. This was suggested in a previous investigation where analogous competition between PPAR and thyroid hormone receptors for RXR was described [28]. In the present study, we show that at least one of the RXRs, RXR, is present in the nucleus of MSC-1 cells and primary rat Sertoli cells regardless of the amount of tRA and peroxisome proliferators present in Sertoli cells. In this condition, RAR, which localizes to the nucleus in the presence of tRA, could form a heterodimer with RXR in the nucleus. In contrast, when Wy-14,643 or MEHP is added, PPAR predominantly distributes into the nucleus, whereas RAR partitions into the cytoplasmic compartment, leaving PPAR to form a heterodimer with RXR in the nucleus. Our findings certainly suggest there is a potential for partner exchange of RXR in Sertoli cells following peroxisome proliferator treatment. Then RAR or PPAR without its RXR partner may distribute preferentially into the cytoplasm, whereas the receptor interacting with RXR can form a stable complex and be retained within the nucleus. This is consistent with a current model of nuclear localization of steroid/nuclear hormone receptors, which proposes that these receptors shuttle between the cytoplasmic and nuclear compartments but remain in the nucleus when they interact with a heterodimeric partner and multiple transcriptional complex proteins including coactivators and corepressors [42, 43].

The tRA activated PPRE-regulated luciferase activity in MSC-1 and primary Sertoli cells, suggesting that the RXR partner of PPAR requires its ligand. Furthermore, tRA synergistically increased the activity of the PPRE-regulated luciferase stimulated by Wy-14,643 and MEHP in MSC-1 cells. These are consistent with previous reports that demonstrated that the RXR partner of PPAR is often an active partner, requiring 9-cis RA, the specific ligand for RXR, which can be isomerized from tRA [8, 44]. It is in contrast with the RXR partner of RAR, which is usually a silent partner [45] not requiring binding of 9-cis RA to RXR for transcriptional activation. This ligand requirement of the RXR partner of PPAR is intriguing because it means less tRA is available for binding to RAR when PPAR is activated, further preventing RAR from acting as a ligand-dependent transcription factor in the presence of peroxisome proliferators.

This RAR/PPAR receptor competition mechanism does not necessarily exclude alternative mechanisms to account for the adverse action of phthalates on the testis. Indeed, the PPAR knockout studies have demonstrated that toxic lesions in testis by DEHP can act through both PPAR-dependent and PPAR-independent pathways [15]. The PPAR-independent pathways may involve another PPAR receptor, such as PPAR, because its transcriptional activity has been shown to be activated by MEHP [46], or no receptor at all. For example, several laboratories have recently reported that in utero exposure to some phthalates such as dibutyl phthalate (DBP) and DEHP have produced an antiandrogenic action in the developing animal [47–50]. This antiandrogenic action appears not to involve the irreversible binding of phthalates to the androgen receptor, but in the case of DBP, it may suppress androgen production in fetal Leydig cells [49, 50, 51]. Androgenic insufficiency is then postulated to alter estrogen levels, resulting in a shift in the balance of action between androgens and estrogens on the testis [52]. In light of recent acceptance that estrogen plays a key role in Sertoli cell function and development of germ cells in the testis [7, 53–56], the alteration in the balance of androgen and estrogen action could potentially be another important mechanism by which peroxisome proliferators can impair testis function.

In comparison, another interesting finding, i.e., that both mechanisms of peroxisome proliferator action, the antiandrogenic mechanism in Leydig cells and the receptor competition mechanism in Sertoli cells, involve a common key player, PPAR, should also be noted. Recently, it has been demonstrated that the antiandrogenic effect of peroxisome proliferators is mediated by PPAR in Leydig cells [57]. Thus, the receptor competition paradigm may also apply to Leydig cells. Peroxisome proliferator activation of PPAR could inhibit the activity of other nuclear hormone receptors, namely, any receptors that require a RXR as a heterodimeric partner. It remains to be investigated whether any nuclear hormone receptor signaling pathway is inhibited by peroxisome proliferators via PPAR in Leydig cells.

In summary, we report a novel biochemical mechanism that peroxisome proliferators can disrupt the retinoid receptor pathway in Sertoli cells. Specifically, treatment with peroxisome proliferators inhibited the nuclear localization of RAR, which was followed by a decrease in the tRA-induced transcriptional activity of RAR. In contrast, Wy-14,643 and MEHP increased the nuclear localization and transcriptional activity of PPAR. RXR was present in the nucleus regardless of treatment. Considered together, these results suggest that there may be a change in the heterodimer partner for the RXR in the nucleus following peroxisome proliferator treatment and the receptor, RAR, without the RXR partner may partition into the cytoplasm. Because a working RAR signaling pathway is essential to testis function, the receptor competition mechanism between RAR and PPAR for the common RXR partner could easily explain the in vivo adverse effect of phthalates on the testis. Further investigation is necessary to determine which heterodimer partners physically interact in the nucleus or in the cytoplasm under particular treatment conditions. Elucidation of the biochemical mechanism by which peroxisome proliferators act in rats is important because only then can we see if the key components of the mechanism exist in the human system to determine risk potentials for humans.

	ACKNOWLEDGMENTS

 
We would like to thank Dr. Vincent Giguere (Children's Hospital, Montreal, Canada) for the reporter plasmid pRARE-luciferase gene, Dr. Bruce Spiegelman (Dana Farber Cancer Institute, Boston, MA) for the PPRE-luciferase gene, and Dr. Ron Evans (Salk Institute, San Diego, CA) for the mouse PPAR expression vector. We also thank Dr. Michael Griswold (Washington State University, Pullman, WA) for providing the MSC-1 cells.

	FOOTNOTES

 
¹ This publication was made possible by grant ES09978 from the National Institute of Environmental Health Sciences.

² Correspondence: Kwan Hee Kim, School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234. FAX: 509 335 1907; khkim{at}wsu.edu

³ These authors contributed equally to this work

Received: 22 August 2002.

First decision: 16 September 2002.

Accepted: 17 October 2002.

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