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Biology of Reproduction 67, 29-37 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Positive Regulation of Retinoic Acid Receptor Alpha by Protein Kinase C and Mitogen-Activated Protein Kinase in Sertoli Cells

Kirt W. Brauna, My-Nuong Voa, and Kwan Hee Kim1,a

a School of Molecular Biosciences, Center for Reproductive Biology, Washington State University, Pullman, Washington 99164


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoic acid receptor {alpha} (RAR{alpha}) is required for normal testis function. Similar to other steroid hormone receptors, RAR{alpha} appears to undergo an activation process by which it translocates from the cytoplasm to the nucleus where it acts as a transcription factor. In this report, we demonstrate that RAR{alpha} nuclear trafficking in Sertoli cells is positively regulated by phorbol-12-myristate-13-acetate-activated protein kinase C without the requirement of ligand, retinoic acid. Protein kinase C then stimulates the downstream mitogen-activated protein kinase, and the nuclear localization of RAR{alpha} is dependent on activation of both kinases. The increase in RAR{alpha} nuclear translocation is also coupled with enhanced transcriptional activity of RAR{alpha}. This mechanism of RAR{alpha} positive regulation is unique, different from that of its negative regulation, that has previously been shown to be dependent on cAMP-dependent protein kinase A and more importantly, dependent on its ligand. However, the mechanism by which retinoic acid positively influences the nuclear localization of RAR{alpha} is not due to retinoic acid directly increasing protein kinase C or mitogen-activated protein kinase activities. Nonetheless, the positive influence of retinoic acid is also dependent on these two kinases as determined by inhibitor studies. These results suggest two mechanisms for RAR{alpha} activation in Sertoli cells: one involving only the two kinases, the other involving both the ligand and the two kinases. These regulatory mechanisms for RAR{alpha} activation, both positive and negative, may be critical for the proper function of RAR{alpha} in the testis.

gamete biology, mechanisms of hormone action, Sertoli cells, steroid hormone receptors, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The majority of the physiologic effects exerted by retinol (vitamin A), such as cellular proliferation and differentiation, are due to the transcriptional activity of retinoid receptors [1]. The retinoid receptors consist of two families of nuclear receptors: the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), each containing three isotypes, {alpha}, ß, and {gamma} [1]. They act as ligand-dependent transcription factors that regulate the transcription of retinoic acid-responsive genes through retinoic acid response elements (RARE) [1]. In testis, RAR{alpha} is expressed both in Sertoli cells and germ cells [2, 3], and the expression level is decreased in vitamin A-deficient testis, whereas it is increased after retinol replenishment [4]. Moreover, RAR{alpha} knock-out male mice are sterile, illustrating unequivocally, the physiologic importance of RAR{alpha} in the testis [5].

RAR{alpha} belongs to the steroid/thyroid hormone receptor superfamily. Many members of the classical steroid hormone receptor subfamily, including the glucocorticoid, progesterone, and estrogen hormone receptors, undergo an activation process, usually involving the binding of ligand, followed by translocation of the receptor from the cytoplasm to the nucleus [6–8]. More recently, two members of the nonclassical steroid hormone receptor subfamily, the thyroid and vitamin D hormone receptors, tagged with the green fluorescent protein, have been demonstrated to undergo a similar nuclear translocation in the presence of ligand in living cells [9, 10]. Additionally, we have shown that all-trans-retinoic acid (tRA), the ligand for RAR{alpha}, induces the receptor nuclear localization, leading to a dose-dependent increase in its transcriptional activity in mouse Sertoli cells [11].

The transcriptional activity of nuclear receptors is also known to be influenced by second messenger signaling systems, cAMP-dependent protein kinase A (PKA) [12–14] or protein kinase C (PKC) [15–17]. Recently, we found that FSH acting through the PKA pathway inhibited the tRA-induced RAR{alpha} nuclear localization, with a subsequent decrease in its transcriptional activity in Sertoli cells [11]. As for PKC, down-regulation of PKC by a 48-h pretreatment with phorbol-12-myristate-13-acetate (PMA) decreased RAR{alpha} nuclear localization and RARE-dependent transcriptional activation in COS-7 cells [17]. In contrast, the activation of PKC by a short PMA treatment enhanced the tRA-dependent RARE-dependent transcriptional activity of RAR{alpha} approximately 4-fold over tRA alone in 2B4.11 T cells [18]. This transcriptional enhancement was shown not to be due to an increased affinity for the RARE or to the presence of AP-1 transcriptional complex. However, the nuclear localization pattern of RAR{alpha}, when the PKC level was increased, was not determined.

Activation of the mitogen-activated protein kinase (MAPK) pathway has also been documented to regulate other nuclear receptors, including the glucocorticoid, peroxisome proliferator-activated, and estradiol receptors [19–22]. However, it has not been investigated whether MAPK is involved in modulating RAR{alpha} nuclear localization and transcriptional activity [23, 24]. In this study, we demonstrate that PMA-activated PKC leads to the downstream stimulation of MAPK, which in turn promotes RAR{alpha} nuclear trafficking in Sertoli cells in a ligand-independent manner. This apparent increase in RAR{alpha} nuclear translocation is associated with enhancement of the tRA-induced RARE-dependent transcriptional activity of RAR{alpha}.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture

The MSC-1 cell line was established from transgenic mice carrying a fused gene composed of human müllerian inhibiting substance transcriptional regulatory sequences and the SV40 T antigen gene [25]. This cell line has been shown to display numerous characteristics of primary Sertoli cells, but does not express the receptor for FSH [26]. These cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 5% fetal calf serum (FCS), supplemented with penicillin (105 IU/L) and streptomycin (100 mg/L) at 37°C in a 5% CO2 atmosphere. Cells were grown to approximately 50% confluency before being serum starved for 24–48 h with 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 [27]. Animal experimentation was conducted in accordance with the highest standards of humane animal care as outlined in the National Institutes 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, Gaithersburg, MD) to remove the interstitial cells and then with 0.7 mg/ml collagenase (Sigma, St. Louis, MO) and 1 mg/ml hyaluronidase (Sigma). Sertoli cells were then plated under serum-free conditions either in 24-well Falcon plates or on 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 for a maximum of 5 days with a media change every other day.

Cells were treated with various concentrations of tRA (Sigma), PMA (Sigma), and the RAR{alpha}-specific agonist Am580 (Biomol Research Lab, Plymouth Meeting, PA). Am580 has a higher binding affinity for RAR{alpha} than tRA (6 versus 13 nM), with a significantly higher activation constant (AC50) of 0.36 nM for Am580 versus 2.1 nM for tRA. In addition, various concentrations of the PKC-selective inhibitor calphostin C (Calbiochem, San Diego, CA) and the MAPK kinase (Mek)-selective inhibitor PD98059 (Calbiochem) were added 30 min before treatment with tRA and PMA. Calphostin C is a highly specific inhibitor of PKC that interacts with its regulatory domain by competing at the binding site of diacylglycerol and phorbol esters [28]. The Ki value of calphostin C is 0.05 µM. PD98059 is a selective and cell-permeable inhibitor of Mek and thus prevents the subsequent activation of MAPK. The Ki value of PD98059 is 2.0 µM. Bis[amino[(2-aminophenyl)thio]methylene] (U0126) (Biomol), a second selective Mek inhibitor used for nuclear localization studies, has an IC50 of 72 nM for Mek1 and 58 nM for Mek2. U0126 has been shown to be more potent than PD98059 as a selective inhibitor of the MAPK system but has negligible effects on the kinase activities of PKC, Abl, Raf, Erk, and c-Jun N-terminal kinase [29].

Immunofluorescence and Microscopy

MSC-1 cells and primary rat Sertoli cells were plated on four-well chamber slides (Nalge Nunc) and fixed with methanol for 10 min at -20°C. Cells were blocked with 10% goat serum for 1 h before incubation with the anti-RAR{alpha} antibody at a 1:300 dilution overnight at 4°C. Cells were washed three times with PBS and incubated with a biotinylated secondary antibody at 1:300 dilution for 1 h. Detection of antibody complexes was conducted using fluorescein avidin D (Vector Laboratories, Burlingame, CA). For a negative control, the anti-RAR{alpha} antibody was incubated with immunizing peptide (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C before addition to fixed cells. For additional negative controls, MSC-1 and Sertoli cells were fixed and immunofluorescence was conducted, but without any primary or secondary antibody. All digital images were obtained using a laser scanning confocal system (MRC 1024; BioRad, Hercules, CA) or a Leitz DMRB with epifluorescence and a Magnafire digital camera (Optronics, Goleta, CA).

Protein Extract Preparation

Nuclear extracts were prepared as described previously [11]. Briefly, cells were washed with PBS, pelleted, and resuspended in 200 µl of prechilled hypotonic buffer A (10 mM Hepes, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM dithiothreitol; 0.5 mM PMSF). After the suspension was held for 10 min on ice, 20 µl of 10% IGEPAL detergent (Sigma) was added to the suspension, and cells were lysed by vortexing. After centrifugation at 15 700 x g for 30 sec, the supernatant was removed, and soluble nuclear extracts were prepared by resuspending the pellet in 50 µl of prechilled hypertonic buffer C (20 mM Hepes, pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM dithiothreitol; 1.5 mM PMSF), followed by incubation on ice for 30 min with occasional mixing. After centrifugation at 15 700 x g for 10 min, the supernatant was collected and constituted the nuclear extract.

Western Blotting

Western blot analyses were performed as described previously [11]. In brief, 20 µg of protein was separated by electrophoresis and transferred to Immobilon-P membranes (Millipore Co., Bedford, MA). Membranes were blocked with 5% (w/v) Blotto (Carnation, Los Angeles, CA) and incubated with a polyclonal anti-RAR{alpha} antibody (Santa Cruz Biotechnology) at 1:300 dilution, a polyclonal anti-active MAPK antibody (Promega, Madison, WI) at a 1:1000 dilution, or anti-Erk2 antibody (Santa Cruz Biotechnology) at 1:400 dilution. Membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody at 1:2500, and antibody-antigen complexes were detected by the Enhanced Chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). Densitometric analysis of RAR{alpha} levels was performed using densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The relative levels of RAR{alpha} were normalized to levels of several nuclear proteins revealed by Coomassie blue staining on the same membrane. The amount of nuclear proteins on the Coomassie-stained membrane was quantitated by drawing a box that included several nuclear proteins using ImageQuant software. Quantification and normalization were carried out for nuclear proteins obtained from at least three separate sets of treatment.

PKC Assay

PKC assays were performed using an ELISA-based PKC assay kit (Calbiochem). The manufacturer's protocol was followed, with minor modification. In brief, approximately 3.0 x 106 MSC-1 cells were collected after specific treatments, and then cells were pelleted, resuspended in buffer A (50 mM Tris-HCl, pH 7.0; 10 mM EGTA; 5 mM EDTA; 10 mM benzamidine; 1.0 mM PMSF; 50 mM ß-mercaptoethanol) and lysed by sonication on ice. Cytosolic fractions were obtained by centrifugation at 100 000 x g for 1 h. To obtain the membrane fraction, the resulting pellet was resuspended in buffer A also containing 0.1% NP-40, 1.0 mM PMSF, and 50 mM ß-mercaptoethanol and centrifuged at 100 000 x g for 1 h. The supernatant was collected as the membrane fraction. Protein concentrations from the cytosolic and membrane fractions were determined using the Bradford method [30]. PKC assays were performed according to protocol using 3 µg of total protein from each fraction. Quantitative analysis of PKC activity was conducted using a Titertek Multiskan MCC/340 microplate reader (Flow Laboratories, McLean, VA) at an OD of 490 nm.

Transient Transfection Assays Using Luciferase and ß-Galactosidase Reporters

Transient transfections and reporter assays were conducted as previously described [11]. MSC-1 cells were grown to approximately 50% confluency in 24-well plates containing 5% FCS in DMEM before being serum starved in 0.1% FCS in DMEM for 24 h. Cells were transfected with 60 ng of the luciferase reporter plasmid pRARE-tk-Luc [31], which contains three RAREs from the RARß promoter, and 30 ng pHook-LacZ (Invitrogen, San Diego, CA) using LipofectAMINE transfection method (Life Technologies, Grand Island, NY). The medium containing the transfection mix was replaced with 1% FCS in DMEM 5 h after transfection. The following day, the medium was again replaced with 3% FCS in DMEM, and the cells were treated with various agents.

Primary Sertoli cells were transfected with a reporter gene construct by the calcium phosphate method coupled with hyperosmotic shock (10% glycerol) as previously described [32]. Briefly, 0.8 µg of pRARE-tk-Luc reporter plasmid and 0.4 µg of pHook-LacZ in 150 µl of transfection buffer (250 mM CaCl2 mixed 1:1 [v/v] with 2x Hepes [28 mM NaCl, 50 mM Hepes, and 1.7 mM Na2HPO4, pH 7.05]) were added to each well of a 24-well plate containing 1 x 106 cells in 1 ml Ham F12 and incubated for 4 h. After the medium was aspirated, 1 ml of 10% glycerol in Hanks buffer was added for hyperosmotic shock and incubated for 3 min. After incubation, an additional 1 ml of Hanks buffer was added, followed by two washes with 1 ml of Hanks buffer, and 1 ml of fresh Ham F12 media was added. The following day, the medium was changed with 1 ml Ham F12 media, and the cells were treated with various agents.

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

Statistical Analysis

Statistical analysis of normalized RAR{alpha} protein levels determined by Western blot analysis and the analysis of normalized RAR{alpha} transcriptional activity consisted of one-way ANOVA followed by pairwise comparison of the means at {alpha} = 0.05 (Tukey-Kramer method, Minitab 10 Xtra; Minitab Inc., State College, PA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of RAR{alpha} Subcellular Localization by PMA and the Involvement of the PKC and MAPK Pathways

To investigate factors that regulate RAR{alpha} nuclear trafficking and thus potentially regulate the transcriptional activity of RAR{alpha} in mouse Sertoli cells, MSC-1 cells were treated with PMA and/or tRA for 30 min. Additionally, MSC-1 cells were pretreated for 30 min with the PKC-selective inhibitor calphostin C or with two Mek-selective inhibitors, PD 98059 and U0126, followed by incubation with PMA and tRA for an additional 30 min. Cells were fixed with methanol, and the subcellular localization of RAR{alpha} was determined using an anti-RAR{alpha} primary antibody and a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibody followed by immunofluorescence confocal microscopy (Fig. 1). Previously, this anti-RAR{alpha} antibody has been shown to recognize both cytoplasmic and nuclear RAR{alpha} in MSC-1 cells and in vivo in testicular cells [2, 3, 11, 33]. In MSC-1 cells, only a single major band was detected on previous Western blot analysis [11]. Treatment with tRA increased the level of RAR{alpha} in the nucleus and near the nuclear envelope (Fig. 1B). Interestingly, PMA treatment alone, without tRA, also promoted the ligand-independent translocation of RAR{alpha} into the nucleus and near the nuclear envelope (Fig. 1C). In addition, the increase of RAR{alpha} associated with the nucleus after treatment with tRA and/or PMA appeared to be dependent on the PKC and MAPK pathways because pretreatment with the PKC- and Mek-specific inhibitors blocked the apparent translocation of RAR{alpha} to the nucleus or near the nuclear envelope (Fig. 1, E–M). For negative controls, cells were incubated with anti-RAR{alpha} antibody that was preadsorbed with immunizing peptide (Fig. 1N) or with no primary or secondary antibodies (Fig. 1, O and P).



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  		FIG. 1. Immunofluorescence demonstrating regulation of RAR{alpha} subcellular localization in MSC-1 cells. MSC-1 cells were grown on four-well chamber slides and incubated for 30 min with vehicle alone, 0.1% ethanol (A), 1 µM tRA (B), 10 nM PMA (C), or 1 µM tRA and 10 nM PMA (D). In addition, MSC-1 cells were also pretreated with 0.6 µM calphostin C (PKC-I) (E–G), 75 µM PD98059 (Mek-I) (H–J), or 1 µM U0126 (K–M) for 30 min before incubation with 1 µM tRA (E, H, K), 10 nM PMA (F, I, L), or 10 nM PMA and 1 µM tRA (G, J, M) for an additional 30 min. The MSC-1 cells were fixed, and the subcellular localization of RAR{alpha} was detected using an anti-RAR{alpha} antibody, followed by confocal microscopy. For negative controls, cells were incubated with anti-RAR{alpha} that was preincubated with immunizing peptide (N), or the same immunocytochemical procedures were followed except omitting either the primary (O) or secondary (P) antibodies. Nomarski interference optics were used to visualize the cells treated with vehicle (Q), tRA (R), and PMA (S) to show the cytoplasmic boundaries. These experiments were conducted at least three times. Bar in A = 50 µm

Cells were also visualized by Nomarski interference optics (Fig. 1, Q–S) to demonstrate that tRA- and PMA-treated cell cytoplasm did not collapse around the nucleus, but that these cells still possessed discernable cytoplasm. In this cellular context, the antibody staining showed that RAR{alpha} had clearly moved away from the cytoplasmic membrane boundaries and was located perinuclearly or within the nucleus after tRA or PMA treatments (Fig. 1, B and C). Whether RAR{alpha} is located just inside or outside the nuclear envelope after tRA or PMA treatment requires further investigation with higher-resolution techniques such as immunoelectron microscopy.

To investigate whether RAR{alpha} nuclear trafficking is regulated in a similar manner as in the MSC-1 cell line, we treated primary Sertoli cells isolated from 20-day-old rats with tRA or PMA, plus and minus Mek or PKC inhibitors (Fig. 2). Cells were doubly stained with an anti-RAR{alpha} antibody followed by biotinylated goat anti-rabbit antibody and fluorescein avidin D (green FITC) to locate RAR{alpha} and propidium iodide (red PI) to stain DNA in the nucleus to assess the positions of the nuclei. Previous Western blot analysis using this anti-RAR{alpha} antibody on cell lysate from primary Sertoli cells showed a major band around 55 kDa and on further exposure, two minor bands that were thought to be differentially phosphorylated products [3, 34]. The staining in the vehicle-treated cells was seen both in the cytoplasm and in the nucleus as depicted in Figure 2A or stained primarily in the cytoplasm as in Figure 2C. This variability of subcellular localization may be due to the fact that primary Sertoli cells are known to store retinoids [35], one of which is retinoic acid, which can influence the nuclear localization of RAR{alpha} in the vehicle-treated cells. Some cells are likely more depleted of retinoids than others in culture. In contrast, it was clear that both tRA and PMA increased the nuclear staining of RAR{alpha} compared with that seen for vehicle-treated primary Sertoli cells (Fig. 2, compare E and G with A and C). Then, in the presence of the Mek inhibitor with concomitant treatment of tRA or PMA (Fig. 2, I and K), RAR{alpha} was distributed more in the cytoplasm. The PKC inhibitor treatment also increased the level of cytoplasmic staining, but not to the same extent as the Mek inhibitor treatment did (Fig. 2, M and O).



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  		FIG. 2. Immunofluorescence demonstrating regulation of RAR{alpha} subcellular localization in primary Sertoli cells. Primary Sertoli cells were grown on four-well chamber slides and incubated for 60 min with vehicle (A–D), 1 µM tRA (E, F, I, J, M, N), or 10 nM PMA (G, H, K, L, O, P). In addition, primary Sertoli cells were also pretreated with 75 µM PD98059 (Mek-I) (I–L) or 0.6 µM calphostin C (PKC-I) (M–P) for 30 min before incubation with 1 µM tRA or 10 nM PMA for an additional 60 min. Primary Sertoli cells were methanol fixed and doubly stained with an anti-RAR{alpha} antibody followed by biotinylated goat anti-rabbit antibody and fluorescein avidin D (FITC) to locate RAR{alpha} and with propidium iodide (PI) to stain DNA in the nucleus to assess the positions of the nuclei and the density of plating. Bar = 50 µm

Western Blot Analysis Demonstrates That PMA Increased RAR{alpha} in the Nucleus Without tRA Ligand

To quantify the increase of RAR{alpha} protein associated with the nucleus and its apparent regulation by PKC and MAPK, nuclear extracts were obtained as described under Materials and Methods, and 20 µg protein from the nuclear fraction was subjected to SDS-PAGE and analyzed by Western blot technique. Similar to the results observed previously [11], anti-RAR{alpha} antibody revealed a single major band of the correct molecular weight for RAR{alpha} in all lanes in MSC-1 cells (Fig. 3, A and B). To demonstrate equal loading of nuclear proteins, the same membranes were stained with Coomassie blue dye (Fig. 3, C and D). The amount of RAR{alpha} in each lane was quantitated using ImageQuant software, and then this value was normalized to the value obtained from quantitating several nuclear proteins from the same lane. The results from at least three independent experiments were combined (Fig. 3, E and F). There was a significant increase of RAR{alpha} protein in the nuclear fraction of MSC-1 cells after treatment with PMA alone or with tRA plus PMA, approximately a 2.5-fold increase of RAR{alpha} in the nuclear fraction, compared with untreated control cells. In contrast, Western blot analysis of nuclear extracts from MSC-1 cells pretreated with increasing concentrations of calphostin C (PKC-I) (Fig. 3, A and E), and PD98059 (Mek-I) (Fig. 3, B and F), revealed a dose-dependent decrease of RAR{alpha} in the nuclear fraction in the presence of PMA alone, or in the presence of tRA plus PMA. Similar to data presented previously [11], the level of cytoplasmic contamination, as measured by antibody to actin in the nuclear fractions, was determined to be minimal but consistent across all samples (data not shown). Therefore, the changes in RAR{alpha} levels in the nuclear fraction cannot be attributed to differential cytoplasmic contamination or cytoskeletal protein contamination, but are due to changes in amount of RAR{alpha} in the nucleus.



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  		FIG. 3. PMA-induced RAR{alpha} nuclear localization is dependent on PKC and MAPK activity. MSC-1 cells were incubated with vehicle alone as a control, 1 µM tRA alone, 10 nM PMA alone, or 1 µM tRA plus 10 nM PMA for 30 min. In addition, MSC-1 cells were pretreated with three different concentrations of calphostin C (PKC-I) (A, C, E) or PD98059 (Mek-I) (B, D, F) for 30 min before incubation with 10 nM PMA alone or 1 µM tRA and 10 nM PMA. The nuclear extracts were collected, and RAR{alpha} protein was detected by Western blot analysis using an anti-RAR{alpha} antibody (A and B). The loading controls were obtained by staining the same membrane with Coomassie blue dye (C and D). The levels of RAR{alpha} protein in the nuclear fraction were determined by densitometric analysis from three independent experiments. The results are plotted as relative levels of RAR{alpha} protein in the nuclear fraction of treated cells versus untreated control cells (mean ± SD) (E and F). Levels of RAR{alpha} were normalized to levels of several nuclear proteins on the Coomassie blue-stained gels as determined by densitometric and ImageQuant analyses. Asterisks denote a significant difference from the control level (P <= 0.05)

Enhancement of Retinoid-Induced RARE-Dependent RAR{alpha} Transcriptional Activity by PMA Acting Through the PKC and MAPK Pathways

To investigate whether the increase of RAR{alpha} in the nuclear fraction by PMA acting through PKC and MAPK pathways subsequently increases RARE-dependent transcriptional activity, MSC-1 cells were transiently cotransfected with luciferase reporter (pRARE-tk-Luc) and ß-galactosidase (pHook-LacZ) plasmids and treated with various agents, and the relative luciferase activity was determined. As shown previously [11], MSC-1 cells treated with increasing concentrations of tRA had a dose-dependent increase in luciferase activity compared with untreated cells, reaching a maximum induction of approximately 5-fold over untreated control cells (Fig. 4A). When cells were treated with PMA alone, there was no significant increase in RARE-dependent transcriptional activity. This was expected since ligand is required for the receptor to be transcriptionally active.



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  		FIG. 4. Regulation of RAR{alpha} transcriptional transactivation by tRA and Am580 and PMA-induced PKC and MAPK activity in MSC-1 cells. After transfection, MSC-1 cells were treated with vehicle as a control, treated with four concentrations of tRA, 10-8 M PMA alone, or treated with four concentrations of PMA in the presence of 10-6 M tRA for 24 h (A). In addition, cells were treated with five concentrations of Am580, 10-6 M tRA, or three concentrations of PMA in the presence of 10-7 M Am580 for 24 h (C). For inhibitor studies, MSC-1 cells were treated with 10-6 M tRA, 10-6 M tRA plus 10-8 M PMA, or pretreated with three concentrations of calphostin C (PKC-I) or PD98059 (Mek-I) for 30 min before incubation with 10-6 M tRA and 10-8 M PMA for 24 h (B). In addition, cells were treated with 10-7 M Am580 instead of 10-6 M tRA (D). Data are presented as relative luciferase activity compared with control, after normalization to ß-galactosidase activity, and are the average of three independent assays, each conducted in triplicate (mean ± SD). Means bearing different value labels are significantly different (P <= 0.05)

More importantly, when cells were treated concomitantly with PMA and tRA, both 10 and 100 nM PMA enhanced tRA-dependent transcription significantly over the levels seen for 1 µM tRA treatment alone, reaching a maximum of nearly 9-fold over untreated control cells (Fig. 4A). To determine whether the enhancement of tRA-induced RARE-dependent transcriptional activity by PMA involved the activation of the PKC and MAPK pathways, MSC-1 cells were pretreated with increasing concentrations of calphostin C and PD98059 for 30 min before treatment with tRA and PMA. There was a dose-dependent decrease in luciferase activity with increasing concentrations of the PKC- and MAPK-specific inhibitors (Fig. 4B).

Since RARE-dependent transcription can be induced by not only RAR{alpha}, but also by RARß or RAR{gamma}, it was important to determine if the observed increase in RARE-dependent transcription was due to the activity of RAR{alpha} specifically. Therefore, to investigate whether the enhancement of RARE-dependent transcriptional activity by PMA involved the activation of RAR{alpha}, and not RARß or RAR{gamma}, the RAR{alpha}-specific agonist Am580 was used instead of tRA as a ligand. Although there was a dose-dependent stimulation of luciferase reporter activity with increasing concentrations of Am580, Am580 did not stimulate RARE-dependent transcriptional activity equal to tRA (Fig. 4C). This increase in the transcriptional activation by tRA over Am580 is likely due to the additional activation of RARß and RAR{gamma} by tRA. Interestingly, maximal stimulation of RAR{alpha} transcriptional activity was found to occur at 10-7 M with Am580 and not at 10-6 M as with tRA. This is consistent with the report by Delescluse et al. [36] that demonstrated that Am580 has a higher binding affinity for RAR{alpha}, but more importantly, the activation constant (AC50) for Am580 is an order of magnitude lower than that for tRA. Similar to the enhancement of luciferase activity observed with tRA, 100 nM Am580 and 10 nM PMA together increased RAR{alpha} transcriptional activity, higher than the level achieved with the 100 nM Am580 treatment alone (Fig. 4C). It was also determined that the inhibition of PKC and MAPK led to a dose-dependent decrease in RAR{alpha} transcriptional activity after treatment with Am580 and PMA, suggesting that the activation of the PKC and MAPK pathways may play an important role in the Am580 and PMA-induced transcriptional regulation of RAR{alpha} (Fig. 4D).

A Similar Trend Is Observed in Primary Sertoli Cells for RARE-Dependent Transcriptional Activity

To investigate whether the increase of RAR{alpha} in the nuclear fraction by PMA in primary Sertoli cells also led to increases in RARE-dependent transcriptional activity, Sertoli cells were transiently transfected and treated with various agents, and the relative luciferase activity was determined. Primary Sertoli cells treated with tRA or tRA + PMA both demonstrated a significant increase in luciferase activity compared with untreated control cells (Fig. 5). To determine whether the tRA-induced RARE-dependent transcriptional activity by PMA involved the activation of the PKC and MAPK pathways, Sertoli cell were pretreated with increasing concentrations of a PKC inhibitor, calphostin C, and a Mek-selective inhibitor, PD98059, for 30 min before treatment with tRA and PMA. The results demonstrate a decrease in luciferase activity in the presence of the PKC- and MAPK-specific inhibitors similar to that seen for MSC-1 cells (Fig. 5).



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  		FIG. 5. Regulation of RAR{alpha} transcriptional transactivation by tRA and PMA-induced PKC and MAPK activity in Sertoli cells. After transfection, primary Sertoli cells were treated with vehicle as a control, treated with 10-6 M tRA, or treated with 10-6 M tRA plus 10-8 M PMA for 24 h. In addition, Sertoli cells were pretreated with three concentrations of calphostin C (PKC-I) or two concentrations of PD98059 (Mek-I) for 30 min before incubation with 10-6 M tRA plus 10-8 M PMA for 24 h. Data are presented as relative luciferase activity compared with control, after normalization to ß-galactosidase activity, and are the average of three independent assays, each conducted in triplicate (mean ± SD). Means bearing different value labels are significantly different (P <= 0.05)

PKC and MAPK Are Important Regulators for tRA-Induced RAR{alpha} Transcriptional Activity

Figure 1 demonstrates that PKC and MAPK are important regulators of tRA-induced nuclear localization of RAR{alpha} (Fig. 1, E, H, and K). To determine whether the endogenous PKC and MAPK pathways are involved in the ligand-dependent RAR{alpha} transcriptional activity, without additional PMA stimulation, MSC-1 cells were pretreated with calphostin C and PD98059 for 30 min before treatment with only tRA or Am580. Interestingly, inhibition of the PKC and MAPK pathways significantly reduced the tRA-, and Am580-stimulated RARE-dependent transcriptional activity (Fig. 6, A and B). These results demonstrate that PKC and MAPK are important regulators for tRA-induced RAR{alpha} transcriptional activity.



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  		FIG. 6. Ligand-dependent RAR{alpha} transcriptional activity is also dependent on PKC and MAPK. After transfection, MSC-1 cells were treated with vehicle as a control, treated with 10-6 M tRA, or pretreated with three concentrations of calphostin C (PKC-I) or PD98059 (Mek-I) for 30 min before incubation with 10-6 M tRA for 24 h (A). In addition, 10-6 M tRA was replaced with 10-7 M Am580 (B). Data are presented as relative luciferase activity as compared with control, after normalization to ß-galactosidase activity, and are the average of three independent assays, each conducted in triplicate (mean ± SD). Means bearing different value labels are significantly different (P <= 0.05)

PMA but Not tRA Leads to Increased PKC Activity

It is well known that PMA stimulates the activity of PKC, which translocates from the cytoplasm to the plasma membrane, where it can phosphorylate specific substrates [37]. As for tRA, it has been shown to increase the expression of PKC{alpha} [38–40] in the long term, which could then potentially lead to increases in PKC activity. Thus, to assess whether tRA or PMA stimulates PKC directly in the short term or more indirectly in the long term in MSC-1 cells, cells were treated with tRA, PMA, or tRA and PMA together for 30 min or 24 h or pretreated with calphostin C for 30 min before treatments. The cytosolic and membrane fractions were obtained and used with an ELISA-based PKC assay kit. PMA treatment of MSC-1 cells led to a 6-fold increase in the PKC activity in the membrane fraction after 30 min compared with that in untreated cells (data not shown). Pretreatment with calphostin C abrogated this PKC activity, demonstrating that calphostin C is indeed an effective inhibitor of PKC activity. In contrast, there was neither an increase in PKC activity in the membrane fraction with a tRA treatment alone, nor an additional increase in PKC activity with a combined treatment of PMA and tRA for 30 min. For the 24-h treatment, there was no significant increase in PKC activity in the membrane fraction with any of the treatments. In addition, PKC activity in the cytoplasmic fraction did not change significantly with any of the treatments.

Western Blot Analysis Demonstrates Activation of MAPK by PKC in MSC-1 Cells

The activation of PKC is known to stimulate the MAPK pathway, which is usually independent of Ras, but dependent on activation of Raf [23, 24]. Then, Raf phosphorylates and activates Mek, which in turn activates MAPK (Erk1 and Erk 2) via phosphorylation at two specific sites, Thr183 and Tyr185, located within the catalytic core [41, 42]. To investigate whether PMA or tRA activates the MAPK pathway in MSC-1 cells, cells were treated with PMA or tRA, and total cell lysates were collected. Western blot analysis was then performed using a specific anti-active MAPK antibody that recognizes only the dually phosphorylated forms of Erk1 and Erk2. There was a considerable increase of phosphorylated Erk1 and Erk2 protein in MSC-1 cells after PMA treatment compared with that in untreated cells, whereas there was no increase in the phosphorylated Erk1 and Erk2 with tRA treatment (Fig. 7A). Western blot analysis was also performed using an anti-Erk2 antibody that recognizes both Erk1 and Erk 2 to demonstrate that the amounts of Erk1 and Erk2 do not vary with various treatments (Fig. 7, C and D). These results demonstrate that PMA, but not tRA, leads to the activation of MAPK in these cells. To determine more specifically whether PMA stimulation of PKC leads to the downstream phosphorylation and activation of Erk1 and Erk2, MSC-1 cells were pretreated with increasing concentrations of calphostin C followed by a 30-min treatment with PMA. Western blot analysis performed using the specific anti-active MAPK antibody demonstrated a calphostin C dose-dependent decrease in the amounts of phosphorylated Erk1 and Erk2 (Fig. 7B, compare lane 2 with lanes 3–5). These results demonstrate that the activation of MAPK by PMA is at least partially mediated by the PKC pathway.



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  		FIG. 7. PMA-induced activation of MAPK is dependent on PKC. MSC-1 cells were treated with vehicle as a control, with two concentrations of PMA and two concentrations of tRA for 30 min (A and C). In addition, cells were pretreated with three concentrations of calphostin C (PKC-I) for 30 min before incubation with 10-8 M PMA (B and D). The cellular extracts were collected, and phosphorylated Erk 1 and Erk 2 proteins were detected by Western blot analysis using an anti-active MAPK antibody (A and B), and total Erk1 and Erk2 proteins were detected by Western blot analysis using an anti-Erk2 antibody (C and D). These experiments were conducted at least three times


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There is now a growing list of nuclear hormone receptors that require some type of activation mechanism, primarily the participation of ligand, that promotes receptor translocation from the cytoplasm to the nucleus, where they act as ligand-dependent transcription factors [6–10, 43]. In this report, we provide a novel finding that PMA-induced PKC and MAPK can positively regulate the nuclear localization of RAR{alpha} in absence of its ligand in Sertoli cells. This independence from ligand is unique. It is distinct from what we found previously with cAMP-dependent PKA, which negatively regulates RAR{alpha} nuclear localization, but this regulation of RAR{alpha} occurs in the presence of ligand [11].

Previously, we found that tRA can positively regulate RAR{alpha} nuclear localization [11]. We demonstrate here that this tRA-induced positive regulation of RAR{alpha} was not due to tRA directly increasing new PKC activity or stimulating MAPK. Nevertheless, the tRA-mediated RAR{alpha} nuclear localization was still dependent on PKC and MAPK, since inhibitors of PKC and MAPK decreased the tRA-induced nuclear localization of RAR{alpha}. Thus, these results suggest that there are at least two possible mechanisms for positive regulation of RAR{alpha} nuclear translocation: 1) a ligand-independent mechanism involving PKC/MAPK and 2) a ligand-dependent mechanism using tRA and PKC/MAPK. The increased level of RAR{alpha} in the nuclear fraction of Sertoli cells after PMA treatment was physiologically coupled with a concomitant enhancement in the tRA-dependent RAR{alpha} transcriptional activity. This increase in transcriptional activity was significant beyond the level observed for tRA treatment alone. Nonetheless, it should be noted that the PMA treatment alone had no effect on the RARE-mediated transcriptional activity of RAR{alpha} and that tRA was still required for activation of RARE-mediated transcription. Thus, the PMA-induced PKC and MAPK activities were only able to enhance the ligand-dependent RAR{alpha} transcriptional activity in Sertoli cells. Enhancement of the ligand-dependent RAR{alpha} transcriptional activity has been reported in other cell lines. Yang et al. [18] have shown that an activation of PKC by PMA leads to the superinduction of tRA-responsive gene transcription and that PKC{alpha} is the primary PKC isoform responsible for this superinduction in T cells. Similarly, activation of MAPK has been shown to enhance the transcriptional activity of the estrogen receptor and the glucocorticoid receptor to a level more than that stimulated by their respective ligands [19, 20]. Moreover, the estrogen receptor-stimulated transcription of the transforming growth factor ß3 in HeLa and osteosarcoma MG63 cells has been shown to be blocked by PKC and MAPK inhibitors [44].

Evidence in the literature suggests that phosphorylation of nuclear receptors may perform a key role in modulating the receptor activity, both to increase or to decrease their transcriptional activation [15, 19–22, 45]. It is postulated that phosphorylation of nuclear receptors modulates the interaction of these receptors with associated proteins such as coactivators or corepressors. In human RAR{alpha} (hRAR{alpha}), there are six potential PKC phosphorylation motifs, two in the ligand-binding E domain and four in the C, D, and F domains [17, 46]. However, when bacterially expressed hRAR{alpha} is phosphorylated by PKC at Ser157, which maps to the D domain, in the region involved in dimerization and DNA binding, this decreases the heterodimerization between hRAR{alpha} and RXR{alpha}, and decreases the transcriptional activity of hRAR{alpha} [46]. Thus, the phosphorylation site for positive regulation of RAR{alpha} has not been identified for either transcriptional activation or nuclear localization. Further investigation is necessary to determine whether PKC or MAPK directly phosphorylates RAR{alpha} protein in vivo in Sertoli cells, and if this occurs, whether it changes the pattern of nuclear localization for the receptor.

A number of reports indicate that primary Sertoli cells respond to PMA by either increasing [47, 48] or decreasing [49, 50] specific gene expression. Some of the resulting effects are shown to be antagonistic to the FSH effect [47, 49], similar to what we observed for the negative regulation of RAR{alpha} nuclear localization and transcriptional activity [11]. In addition, it has been shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) antagonizes the FSH-stimulated cAMP production in a MSC-1 cell line stably transfected with FSH receptor cDNA, similar to the effects observed on PKC activation in rat seminiferous tubules in vitro [51]. Our results demonstrating the negative regulation of RAR{alpha} by PKA even in the presence of ligand [11] and positive regulation of RAR{alpha} by PKC/MAPK in the absence of ligand suggest that RAR{alpha} activity in Sertoli cell may be tightly regulated, not only by ligand, but also by intracellular protein kinases. This type of positive and negative regulation may allow RAR{alpha} to be active in a cyclic manner in the testis, perhaps related to the spermatogenic cycle-specific activity in Sertoli cells. The in vivo role of PKA, PKC, and MAPK interactions converging on RAR{alpha} in the testis remains to be delineated.

The synergism between the retinoid receptor and PKC signaling also has a significant implication with regard to cellular differentiation and to the antitumorigenic effects of retinoids. Both tRA and PKC appear to be required for differentiation of B16 melanoma cells [43], growth inhibition of human breast carcinoma cells [46], and a decrease in the malignant phenotype of human pancreatic carcinoma cells [45]. Furthermore, RAR{alpha} has been demonstrated to be a mediator of the synergy between retinoids and PKC, since PKC{alpha} expression increases the antiproliferative actions of Am580, which selectively activates RAR{alpha}, in human breast carcinoma cells [47].

In summary, for the first time, we report that activation of PKC and MAPK in Sertoli cells results in the ligand-independent translocation of RAR{alpha} from the cytoplasm to the nucleus, leading to the enhancement of the tRA-dependent transcriptional activity of RAR{alpha}. In addition, we found that the positive influence of tRA on receptor nuclear translocation was not due to its direct induction of new PKC activity or activation of MAPK in MSC-1 cells. These results support other previous reports that demonstrated that tRA and PKC work cooperatively to promote cellular differentiation and inhibition of growth in cancer cells [44, 45, 47]. However, further investigation is necessary to determine the detailed molecular mechanisms by which tRA, PKC, and MAPK directly influence RAR{alpha} to promote its nuclear translocation and stimulate its transcriptional activity in Sertoli cells.


   ACKNOWLEDGMENTS
 
We wish to thank Drs. Norah McCabe and Steven Sylvester (Washington State University) for their critical reading of this manuscript. We also thank Dr. Vincent Giguere (Children's Hospital, Montreal, Canada) for the reporter plasmid pRARE-tk-Luc. We are grateful to Dr. Chris Davitt from the Electron Microscopy Center at Washington State University for her assistance in acquiring the confocal micrographs and Asa Oudes for acquiring Nomarski micrographs. We thank Dr. Martin Morgan at Washington State University for his help with statistical analyses.


   FOOTNOTES
 
First decision: 3 January 2002.

1 Correspondence. FAX: 509 335 1907; khkim{at}mail.wsu.edu Back

Accepted: January 28, 2002.

Received: December 1, 2001.


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 DISCUSSION
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