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BOR - Papers in Press, published online ahead of print March 28, 2007.
Biol Reprod 2007, 10.1095/biolreprod.106.053918
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BIOLOGY OF REPRODUCTION 77, 147–155 (2007)
DOI: 10.1095/biolreprod.106.053918
© 2007 by the Society for the Study of Reproduction, Inc.

Protein Kinase A-Independent cAMP Stimulation of Progesterone in a Luteal Cell Model Is Tyrosine Kinase Dependent but Phosphatidylinositol-3-Kinase and Mitogen-Activated Protein Kinase Independent1

Elie Needle 5, Kathy Piparo 3 5, Donna Cole 6, Carolyn Worrall 4 5, Ian Whitehead 7, Gwen Mahon 7, and Laura T. Goldsmith 2 5 6

Departments of Biochemistry and Molecular Biology,5 Obstetrics, Gynecology and Women's Health,6 and Microbiology and Molecular Genetics,7 New Jersey Medical School and Graduate School of Biomedical Sciences, University of Medicine & Dentistry of New Jersey, Newark, New Jersey 07103

ABSTRACT

Comprehensive understanding of the cellular mechanisms utilized by luteal cells in response to extracellular hormonal signals resulting in the normal synthesis and secretion of their steroid and peptide products has yet to be achieved. Previous studies have established that cAMP functions as a second messenger in mediating gonadotropin stimulated luteal progesterone secretion. Classically, increased intracellular concentrations of cAMP result in activation of protein kinase A (PKA), which in turn phosphorylates gene regulatory transcription factors. Recent studies demonstrate that non-PKA mediated actions of cAMP exist, yet the mechanisms are not well understood. In addition to gonadotropic hormones, such growth factors as insulin, insulin-like growth factor 1, and epidermal growth factor have been shown to modulate luteal steroid hormone synthesis and steroidogenic enzyme expression as either independent effects or via amplification or modulation of the action of gonadotropic hormones or cAMP. Thus, mechanisms independent of cAMP and also downstream to cAMP that do not involve PKA are likely to be important in steroidogenesis in mammalian cells. The present studies were performed to help define the cellular mediators involved in cAMP-stimulated progesterone expression. Our data demonstrate that, in an in vitro steroidogenic cell model, 1) cAMP-stimulated progesterone occurs in a manner that is independent of PKA, 2) neither phosphatidylinositol-3-kinase nor mitogen-activated protein kinase are involved in PKA-independent cAMP-stimulated progesterone production, 3) tyrosine kinase activity does mediate cAMP-stimulated progesterone production, and 4) cAMP directly activates the Ras protein. These data suggest novel mediators of cAMP-stimulated progesterone production.

corpus luteum, corpus luteum function, female reproductive tract, progesterone, signal transduction

INTRODUCTION

Normal function of the corpus luteum is essential for successful reproduction in most mammalian species. Inadequate function of the mammalian corpus luteum generally precludes establishment and maintenance of pregnancy. Despite the importance of the function of the mammalian corpus luteum, a comprehensive understanding of the cellular mechanisms utilized by luteal cells in response to the extracellular signals responsible for normal synthesis and secretion of their steroid and peptide products has yet to be achieved. The importance of cAMP and its activation of protein kinase A (PKA) in the luteal response to gonadotropic hormones are well established [1, 2]. However, a nascent body of evidence demonstrates that cAMP may regulate hormone dependent signaling without the need for activation of PKA. Also, in addition to the classic gonadotropins, various growth factors such as insulin, insulin-like growth factor 1 (IGF1), and epidermal growth factor (EGF), which generally use other signaling mediators, modulate luteal steroid hormone synthesis and steroidogenic enzyme expression [3–10]. Thus, it is likely that mechanisms independent of cAMP and also downstream to cAMP that do not involve PKA are important in steroidogenesis in mammalian cells. The present studies were performed to help define the cellular mediators involved in cAMP-stimulated progesterone expression. We have observed that, in a cell line of rat steroidogenic cells, increased intracellular cAMP results in significantly increased progesterone production in a manner that is independent of PKA, yet does involve tyrosine kinase activity. However, neither mitogen-activated protein kinase (MAPK) nor phosphatidylinositol-3-kinase (PI3-K) activities appear to be involved, despite stimulation of Ras activity by cAMP.

MATERIALS AND METHODS

Materials

All cell culture media and characterized fetal bovine serum were purchased from Invitrogen Life Technologies (Carlsbad, CA). 8-bromo-cAMP and lactalbumin hydrolysate were purchased from Sigma-Aldrich (St. Louis, MO). H-89 [N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide, 2HCl (C20H20BrN3O2S.2HCl)], HNMPA(AM)3 [hydroxy-2-naphthalenyl methylphosphonic acid trisacetoxymethyl ester], AG 879 [{alpha}-cyano-(3,5,di-t-butyl-4-hydroxy) thiocinnamide (C18H24N2OS)] and Rp-cAMPS [adenosine-3',5'–cyclic monophosphorothioate, Rp-isomer, triethylamine salt [C10H11N5O5PS . (CH3CH2)3NH] were purchased from Calbiochem (San Diego, CA). LY-294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (C19H17NO3 )] was purchased from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). PD98059 [2'-amino-3'-methoxyflavone (C16H13NO3)] and UO126 [1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene] were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Polyvinylidene difluoride (PVDF) membranes were purchased from Millipore (Bedford, MA). Kaleidoscope Broad Range and Unstained Broad Range protein molecular weight markers and the DC Protein Assay Kit were purchased from Bio-RAD Laboratories (Hercules, CA). Enhanced chemiluminescence reagents were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL) and Cell Signaling Technology, Inc. (Beverly, MA). Protease inhibitor cocktail tablets were purchased from Roche Diagnostics (Indianapolis, IN). Glutathione sepharose 4B beads were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). PepTag Assays for nonradioactive detection of PKA activity were obtained from Promega (Madison, WI). COS1 cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA). Progesterone RIA reagents were purchased from Diagnostic Products Corporation (Los Angeles, CA). Rabbit polyclonal anti-MAPK antibody (catalog no. 9102), rabbit polyclonal anti-phospho p42/p44 MAPK antibody (catalog no. 9101), affinity purified horseradish peroxidase conjugated goat anti-rabbit IgG (catalog no. 7074), agarose immobilized mouse anti-AKT1 monoclonal antibody (catalog no. 9279), rabbit polyclonal anti-AKT1 antibody (catalog no. 9272), and rabbit polyclonal anti-phophoGSK3A/B antibody (catalog no. 9331) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Mouse anti-pan Ras monoclonal antibody (Clone RAS 11) and horseradish peroxidase conjugated goat anti-mouse IgG were purchased from Calbiochem (San Diego, CA). A 1.1-kb fragment of the human glyceraldehyde 3-phosphate dehydrogenase cDNA, consisting of the entire protein coding region, which also recognizes rat glyceraldehyde 3-phosphate dehydrogenase mRNA, was purchased from Clontech (Palo Alto, CA) [11, 12]. The 1.2-kb P450SCC (Cyp11a1) cDNA probe, which includes the 5'untranslated region and the entire protein coding region, was prepared by EcoRI digestion of the pRatSCC plasmid obtained from Dr. Joanne Richards, Houston, Texas [13]. Polyclonal anti-3ßHSD (HSD3B) antibody was obtained from Dr. Van Luu-The, Quebec, Canada [14].

Methods

Cell culture. The rat corpus luteum (RCLP) cell line was derived from a primary culture of rat luteal cells isolated from whole ovaries taken at Day 16 of pregnancy. Timed pregnant Sprague Dawley rats obtained from Taconic (Germantown, NY) were housed in the University of Medicine & Dentistry of New Jersey Medical School's Association for the Assessment and Accreditation of Laboratory Animal Care-accredited animal facility in accordance with the guidelines presented in the National Research Council's Guide for Care and Use of Laboratory Animals published by the Institute for Laboratory Animal Research of the National Academy of Sciences. The protocol was approved by the New Jersey Medical School Institutional Animal Care and Use Committee. Ovaries were removed from Day 16 timed-pregnant Sprague Dawley rats, corpora lutea were enucleated from the ovaries, minced into 1-mm3 pieces, and incubated in a mixture of 0.2% collagenase and 0.05% DNAse in Earles balanced salt solution at 25 mg tissue/ml for 30 min at 37°C in a shaking water bath, followed by trituration for 10 min. Cells were centrifuged at 146 x g for 10 min, resuspended in Medium 199, counted and tested for viability using trypan blue exclusion. Viability was greater than 95%. Cells were maintained as adherent monolayer cultures (designated RCL cells) maintained in Medium 199 (M199) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) (complete M199) at 37°C in a humidified atmosphere of 95% air/5% CO2. These cells have been repeatedly passaged and demonstrated a division time of between 24 to 30 h. The RCLP cell line has been used previously as a model for study of cAMP regulation of the prolactin receptor-associated protein/17ß-hydroxysteroid dehydrogenase-7 gene promoter [15]. To obtain a highly homogeneous population of cells, RCLP cells were cloned by limiting dilution. The parental cell line was plated in 24-well dishes at a density of no more than 1 cell per well. After 2 wk of culture, single colonies were retrieved and expanded. Constitutive and cAMP-stimulated progesterone expression was determined for 8 individual clones, designated RCL1 through RCL8, which exhibited similar morphology and constitutive and cAMP-stimulated progesterone production.

The RCL1 cell line was used for all studies described herein. The morphology of the RCL1 cells is shown in Figure 1. Expression of Cyp11a1 mRNA and HSD3B protein were verified in clone RCL1 cells by Northern blotting and Western blotting analyses respectively, as shown in Figure 2. RCL1 cells were seeded at 2 x 105 cells per well in 12-well tissue culture plates and maintained in complete M199 at 37°C in a humidified atmosphere of 95% air/5% CO2 for 72 h. Media were removed from the wells and the cells were then serum starved in M199 containing antibiotics plus 0.2% lactalbumin hydrolysate. After 48 h, cells were incubated in M199 in the presence or absence of kinase inhibitors (PKA, PI3-K, MEK1 and 2 kinases, or tyrosine kinases) for 3 h, after which medium were removed and replaced with complete M199 plus inhibitors plus 0.25 mM 8-bromo cAMP for 48 h. After 48 h, conditioned medium from each well were collected and saved frozen for subsequent determination of progesterone. Cells were trypsinized from the plate and the number of cells in each well was determined using a Coulter counter. From additional replicate wells for each treatment, cells were trypsinized and cell viability was assessed using trypan blue exclusion methodology.


Figure 01
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  		FIG. 1. Morphology of RCL1 cells. Cells were incubated as described in Materials and Methods, stained with hematoxylin and eosin and photographed under bright field microscopy showing distinct luteal morphology typically observed. Original magnification x200. Bar = 10 microns.


Figure 02
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  		FIG. 2. Cyp11a1 mRNA (A) and HSD3B protein (B) expression in RCL1 cells. Cells were incubated in the absence or presence of 0.25 mM 8 bromo-cAMP for 48 hours. RNAs were isolated and subjected to Northern blot analysis and protein extracts were prepared and subjected to Western blot analysis as described in Materials and Methods.

Radioimmunoassay. Progesterone concentrations in each spent medium sample were determined in duplicate by radioimmunoassay using commercially available reagents (Coat-a-Count, Diagnostic Products Corporation, Los Angeles, CA). The sensitivity of the assay was 28–35 pg/ml, the intraassay coefficient of variation was 6.7% (n = 10 observations) and the interassay coefficient of variation (n = 10 assays) was 5.7%.

RNA isolation and Northern blot analysis. Isolation of RNA and Northern blot analysis were performed as previously described [16]. Total RNA was isolated from cells using the acid guanidinium thiocyanate-phenol-chloroform extraction procedure [17]. Cells were lysed in solution D (4M guanidium thiocyanate, 0.1 MB-mercaptoethanol, 0.5% sodium N-laurylsarcosine, 25-mM sodium citrate, pH 7.0), 0.1 volume 2M sodium acetate pH 4.0, 1 volume water-saturated phenol, and 0.2 volume chloroform:isoamyl alcohol (25:1) were added. After centrifugation, the aqueous phase was removed and RNA was precipitated with the addition of 2 volumes of ethanol. RNA pellets were resuspended in a smaller volume of solution D and precipitated a second time by the addition of 2 volumes ethanol. Following centrifugation, RNA pellets were resuspended in RNase-free dH2O and optical density was assessed at 260 and 280 nm.

Total RNA was denatured in the presence of formaldehyde and formamide and fractionated electrophoretically on 1.2% agarose-formaldehyde gels. Equal amounts of RNA (20 µg), as determined by absorbance at 260 nm, were loaded and equivalence was verified by ethidium bromide stained 18 S and 28 S ribosomal RNA bands. RNA was then transferred to nylon membranes using downward neutral capillary transfer in 20x SSC overnight [18]. RNAs were fixed by UV-cross-linking and membranes were probed with 32P-labeled cDNA probes. Membranes were prehybridized in buffer containing 50% formamide, 10% dextran sulfate, 1% SDS, 1 M NaCl, 0.2 mg/ml denatured salmon sperm DNA and 5x Denhardts solution at 42°C for 2 h in a glass bottle in a minihybridization oven (Hybaid, Labnet, Woodbridge, NJ). cDNA probes were labeled by random priming with {alpha}-32PdCTP (Perkin Elmer, Boston, MA) using the Random Primers DNA labeling system (Invitrogen Life Technologies, Rockville, MD). Radiolabeled probes were separated from free nucleotides using Chroma Spin + TE-10 columns (Clontech, Palo Alto, CA). Incorporated radioactivity was counted in a scintillation counter. Heat-denatured radiolabeled probe was added to the buffer and the membranes were incubated for an additional 18 h at 42°C. Membranes were then washed successively twice in 2x SSC at 42°C for 5 min, twice in 2x SSC/0.1% SDS at 60°C for 30 min, and twice in 0.1x SSC/0.1% SDS at room temperature for 30 min. After washing, membranes were subjected to autoradiography.

Western blot analysis. Western blot analyses were performed as we have described previously [16, 19]. Polyclonal anti HSD3B antibody, previously demonstrated to detect rat HSD3B protein, was used at a 1:3000 dilution [20].

Total protein extracts were prepared by lysing cells in boiling 1x SDS-PAGE sample loading buffer (0.125 M Tris-HCl pH 6.8, 20% glycerol, 2% SDS). DNA was sheared by passing through a 26-gauge needle. Protein concentrations of the extracts were measured by a modified Bradford method using the Bio-Rad DC reagent and BSA as a standard. Equal protein amounts of extracts were heat denatured with 2% ß-mercaptoethanol and loaded onto denaturing acrylamide gels. Following separation by SDS-PAGE, proteins were transferred to PVDF membranes and visualized by Ponceau-S staining to confirm equal loading of lanes. Membranes were then blocked in blotting buffer [5% Carnation nonfat milk in TBS/T (10 mM Tris, 150 mM NaCl, pH 8.0; 0.1% Tween-20)] for 30 min and incubated for 2 h or overnight with primary antibody diluted in blotting buffer. Blots were washed three times for 10 min each with TBS/T and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody diluted in blotting buffer. After washing three times for 10 min each with TBS/T, bound antibody was visualized using enhanced chemiluminescence and autoradiography.

Determination of PKA activity. Tests were performed to verify the efficacy of the PKA inhibitor/antagonists, and verify the specificity (i.e., lack of effect on PKA activity) of other inhibitors. Confluent cells were serum starved for 48 h in M199 plus antibiotics supplemented with 0.2% lactalbumin hydrolysate. Medium was removed and replaced with M199 containing the selective PKA inhibitor H-89 [21] (or 0.1% DMSO as vehicle control) or the PKA antagonist Rp-cAMPS [22–24] for 3 h. Medium was then removed and replaced with complete M199 containing H-89 or Rp-cAMPS plus 0.25 mM 8 bromo-cAMP and incubated at 37°C for 15 min. Medium was removed from each well and cells were washed with Earles balanced salt solution (EBSS) on ice. Homogenization buffer [25 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 10 mM ß-mercaptoethanol, 1 mM PMSF, and protease inhibitor tablet (1 tablet/10 ml) (Roche Diagnostics, Indianapolis, IN)] was added to each well. Cells in each well were scraped into the buffer, the mixture was transferred into a microcentrifuge tube and homogenized with a dounce homogenizer and centrifuged at 14 000 x g for 15 min at 4°C. Supernatants were removed, and assessed for PKA activity using the PepTag Assay for nonradioactive detection of PKA activity (Promega, Madison, WI) according to the manufacturer's protocol as described in detail previously [25–27]. Briefly, the reaction mixture (25 µl) consisted of 2 µg substrate peptide (Kemptide peptide L-R-R-A-S-L-G, fluorescently labeled), peptide protection solution, and sample extract or homogenization buffer in 1x PKA reaction buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM ATP). Reactions were performed in duplicate, with activator (1 µM cAMP) added to the reaction mix. In positive control reactions, 2 ng PKA catalytic subunit was substituted for sample extract. After incubation at 30°C for 30 min, the reaction was stopped by boiling for 10 min. Phosphorylated substrate was separated from nonphosphorylated substrate by electrophoresis on 0.8% agarose slab gels in 50 mM Tris-HCl, pH 8.0 at 100 V for 15 min. Gels were photographed under ultraviolet illumination, digital images were captured, and the intensities of the peptide bands were determined using Gel Pro Analyzer (version 4.5, Media Cybernetics Inc., Silver Spring, MD). Relative PKA activity is computed as the ratio of phosphorylated to total (nonphosphorylated plus phosphorylated) substrate.

Determination of MAPK activation. Phosphorylated and nonphosphorylated MAPK3 and MAPK1 were assessed as follows: Confluent cells were serum starved for 48 h in M199 plus antibiotics supplemented with 0.2% lactalbumin hydrolysate. Medium was removed and replaced with M199 containing MEK inhibitors PD 98059 (10 µM) [28] or UO 126 (10 µM) [29] (or 0.1% DMSO as vehicle control) for 1 h, after which medium was removed and replaced with complete M199 containing PD 98059 or UO 126 plus 0.25 mM 8 bromo cAMP and incubated at 37°C for 15 min. Medium was removed from each well and cells were washed with EBSS on ice. Cells were lysed on ice by adding buffer [50 mM Tris pH 7.4, 100mM NaCl, 1% NP-40, 10% glycerol, 2mM MgCl2, 1 mM eCG, and protease inhibitor cocktail tablet (1 tablet per 10 ml)] to each well. Cells in each well were scraped into the buffer, the lysates were pulled through a 22-gauge needle to shear genomic DNA and centrifuged at 14 000 x g for 15 min. Protein concentrations in the supernatants were determined using the modified Bradford method and BSA as standard as previously described [16, 19]. Supernatants were mixed with SDS-PAGE buffer with reducing agent, boiled 5 min, separated on 10% acrylamide gels, and electrophoretically transferred to PVDF membranes. Membranes were blocked (5% nonfat milk in TBST [10 mM Tris, 150 mM NaCl, pH 8.0; 0.1% Tween-20]) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies (rabbit anti-phosphoMAPK3/1 or antiMAPK3/1 antibodies used at 1:1000 dilutions in blocking buffer). Blots were washed three times for 15 min each with TBS/T and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (HRP-linked goat anti-rabbit IgG used at a 1:2000 dilution) diluted in blocking buffer. After washing three times for 15 min each with TBST, bound antibody was visualized using enhanced chemiluminescence and autoradiography. The intensity of the bands obtained upon autoradiography was determined by scanning the films and assessing the digitized images using Gel-Pro analyzer (version 4.5, Media Cybernetics Inc, Silver Spring, MD).

Determination of AKT1 kinase activity. Determination of AKT1 kinase activity was performed as described previously [30, 31]. Confluent cells were serum starved for 48 h in M199 plus antibiotics supplemented with 0.2% lactalbumin hydrolysate. Medium was removed and replaced with M199 containing the specific inhibitor of phosphatidylinositol-3-kinase LY-294002 (10 uM) [32] (or 0.1% DMSO as vehicle control) for 1 h. Medium was then removed and replaced with complete M199 containing LY-294002 (or vehicle) plus 0.25 mM 8 bromo-cAMP and incubated at 37°C for 15 min. Medium was removed from each well and cells were washed with EBSS on ice. Cells were lysed on ice in homogenization buffer (20 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1µg/ml leupeptin]) and total protein concentrations in the lysates were measured using the modified Bradford method and BSA as standard as described previously [16, 19]. AKT1 protein was immunopreciptated using an immobilized AKT1 mouse monoclonal antibody. The resulting immunoprecipitate was incubated with GSK3A/B (an effector of AKT1) for 30 min at 30°C in the presence of ATP and kinase buffer (25 mM Tris [pH 7.5], 5 mM ß-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2). AKT1 protein levels and phosphorylation of GSK3A/B were assessed by Western blotting. Proteins were separated on 12.5% acrylamide gels and transferred to PVDF membranes. Membranes were then blocked (5% BSA in TBS/T [10 mM Tris, 150 mM NaCl, pH 8.0; 0.1% Tween-20]) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies (rabbit polyclonal anti-AKT1 antibody, and rabbit polyclonal phospho-GSK3A/B antibody were used at 1:1000 dilutions in blocking buffer). Blots were washed three times for 15 min each with TBST and incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (HRP-linked goat anti-rabbit IgG) diluted in blocking buffer. After washing three times for 15 min each with TBST, bound antibody was visualized using enhanced chemiluminescence and autoradiography. The intensity of the bands obtained upon autoradiography was determined by scanning the films and assessing the digitized images using Gel-Pro analyzer (version 4.5, Media Cybernetics Inc., Silver Spring, MD).

Determination of Ras activity. Ras activity was measured using the well-established glutathione-S-transferase (GST) fusion protein "pull down" assay, described in detail previously [33, 34]. This method is based upon the marked affinity of the activated, GTP-bound form of Ras for specific binding domains of effector proteins, such as RAF1. The binding domain sequences are used as activation probes after precoupling to glutathionine-agarose beads. In contrast, GDP-bound Ras does not avidly bind to Ras effector molecules. The GST fusion proteins containing the Ras binding domain of RAF1 (amino acids 51–131) are used to precipitate in cell lysates the activated, GTP-bound Ras form, which is then subsequently detected by Western analysis using a specific anti-Ras antibody.

RCL1 cells, which had been plated at 2 x 105 cells per well and maintained for 72 h, were serum starved for 48 h in M199 containing 0.2% lactalbumin hydrolysate. Cells were incubated for 15 min in 10% serum without and with 0.25 mM 8 bromo-camp. COS1 cells, which were transiently transfected with HRAS-L61 constitutively active mutant HRAS subcloned into PAX 142 using calcium phosphate method, were used as a positive control. COS1 cells transiently transfected by similar methodology using PAX 142 empty vector were also used as controls. RCL1 and COS1 cells were lysed on ice in GST-FISH buffer (50 mM Tris pH 7.4, 100 mM NaCl, 1% NP-40, 10% glycerol, 2 mM MgCl2, 1 mM PMSF plus protease inhibitor cocktail tablet). Lysates were pulled through a 22-gauge needle to shear genomic DNA and centrifuged at 14 000 x g for 15 min. Supernatants were retained for determination of total protein, Ras activity, and total Ras protein levels.

Preparation of GST-RAF1-Ras binding domain fusion protein was performed as follows: Bacterial BL21 cells expressing the RAF1-Ras binding domain were cultured at 37°C overnight in LB Broth + 50µg/mL ampicillin. The culture was diluted 1:10 into fresh medium and grown for 1 h at 37°C. RAF1-Ras-binding domain protein expression was induced by adding 0.1 mM isopropylthio-ß-galactoside (IPTG). After 2 h of additional growth, cells were pelleted and lysed in bacterial lysis buffer (20% sucrose, 10% glycerol, 50 mM Tris pH 8.0, 0.2 mM Na2S2O5, 2 mM MgCl2, 2 mM DTT, 1 mM PMSF + protease inhibitor cocktail tablet). Cells were sonicated and debris was removed by centrifugation at 14 000 x g for 10 min. The supernatants were incubated with 1.0 ml glutathione sepharose 4B for 1 h at 4°C. The beads were washed three times in bacterial cell lysis buffer and resuspended in 1.0 ml GST-FISH buffer.

Aliquots of the cell lysates (2 mg of RCL1 lysate protein and 200 µg COS1 cell lysate) were incubated with rotation for 1 h at 4°C with 50 µg GST-RAF1-Ras binding domain fusion protein precoupled to glutathione sepharose beads. After centrifugation at 82 x g for 5 min at 4°C, the supernatant was removed and the pellet was washed three times in 1 ml GST-FISH buffer. The pellets were resuspended in SDS-PAGE buffer with reducing agent, boiled for 5 min, separated on 12.5% acrylamide gels, and electrophoretically transferred to PVDF membranes. After blocking (5% nonfat milk in TBST [10 mM Tris, 150 mM NaCl, pH 8.0; 0.1% Tween-20]) for 1 h at room temperature, membranes were incubated overnight at 4°C with anti pan-Ras monoclonal antibody (Clone RAS 11) used at 1:1000 dilution in blocking buffer, followed by incubation for 30 min with horseradish peroxidase coupled goat anti-mouse IgG (1:10 000 in blocking buffer).

Total Ras protein levels in cell lysates were also assessed by Western analyses. Lysates (50 µg RCL1 and 20 µg COS1) were electrophoresed on 12.5% acrylamide gels, transferred to PVDF membranes, and blotted with anti pan-Ras antibody using the identical procedures as for assessment of GTP-bound Ras. After washing four times for 20 min each with TBST, bound antibody was visualized using enhanced chemiluminescence and autoradiography. The intensity of the bands obtained upon autoradiography was determined by scanning the films and assessing the digitized images using Gel-Pro analyzer version 4.5 (Media Cybernetics Inc, Silver Spring, MD). Verifications of the linear relationships between densitometric units and Ras activity and Ras levels were performed. Ras activity is computed as active (GTP-bound) divided by total Ras levels for each cell lysate preparation.

Statistical analysis. Data were assessed to determine if they were normally distributed and of equal variance using the Shapiro-Wilk test. Normally distributed data were compared parametrically using two-tailed t-tests. Data which were not normally distributed were evaluated by nonparametric analysis using Kruskal-Wallis rank-sum testing. All comparisons were performed using JMP statistical software (SAS Institute, Inc., Cary, NC) written for the Macintosh (Apple Computers, Cupertino, CA). P values of less than 0.05 were considered significant.

RESULTS

Progesterone was expressed by RCL1 cells and expression was stimulated by incubation of the cells with 8 bromo-cAMP. In a total of 24 experiments, mean progesterone concentrations in media from cells incubated without added cAMP were 2.1 ± 0.2 (± SEM) pg/105 cells/24 h, significantly lower than mean progesterone levels in media from cells incubated with 0.25 mM 8 bromo-cAMP, 70.3 ± 12.9 pg/105 cells/24 h (P < 0.0001). For comparison, mean progesterone concentrations in media from the primary cultured luteal cells isolated from whole ovaries taken at Day 16 of pregnancy were 1.47 ± 0.3 ng/105 cells/24 h (± SEM, n = 5 experiments, each conducted in quadruplicate) and mean progesterone levels in media from primary cultured luteal cells which were incubated with 0.25 mM 8 bromo-cAMP were 12.9 ± 1.4 ng/105 cells/24 h.

Inhibition of PKA in RCL1 cells failed to decrease cAMP-stimulated progesterone levels, as shown in Figure 3. In these experiments, progesterone levels in media from cells incubated with 0.25 mM 8 bromo-cAMP were significantly higher than cells incubated with vehicle alone (P = 0.004). Progesterone concentrations in media from cells incubated with 0.25 mM 8 bromo-cAMP in combination with the PKA inhibitor H89 (10 µM) were not significantly different from cells incubated with 8 bromo-cAMP alone (P = 0.51). In addition, use of an additional PKA antagonist, Rp-cAMPS, showed similar findings. Progesterone concentrations in media from cells incubated with 0.25 mM 8 bromo-cAMP in combination with Rp-cAMPS at 10 µM or 20 µM were not significantly different from cells incubated with 8 bromo cAMP alone (P = 0.82 for both doses).


Figure 03
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  		FIG. 3. cAMP stimulation of progesterone in RCL1 cells is PKA independent. Replicate wells of cells were incubated in the presence (or absence) of PKA inhibitors H89 (10µM) or Rp-cAMPs (at doses indicated) with 0.25 mM 8 bromo-cAMP as described in Materials and Methods. Medium progesterone levels in each well were determined by RIA and normalized to cell number. Each bar shows the mean (± SEM) medium progesterone levels in three independent experiments, each conducted in quadruplicate. aP < 0.05 compared to without 8br-cAMP controls . Bars with the same letter are not significantly different.

In contrast to the lack of effect of PKA inhibition upon cAMP-stimulated progesterone expression, inhibition of tyrosine kinase significantly decreased cAMP-stimulated progesterone levels, as shown in Figure 4. In these experiments, progesterone concentrations in media from cells incubated with vehicle alone were significantly lower than progesterone levels in media from cells incubated with 0.25 mM 8 bromo-cAMP. Progesterone concentrations in media from cells incubated with 0.25 mM 8 bromo-cAMP in combination with the tyrosine kinase inhibitor HNMPA (10 µM) were significantly decreased from cells incubated with 8 bromo-cAMP alone. Progesterone concentrations in media from cells incubated with 0.25 mM 8 bromo-cAMP in combination with the tyrosine kinase inhibitor AG 879 (4 µM) were also significantly decreased from cells incubated with 8 bromo-cAMP alone.


Figure 04
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  		FIG. 4. Inhibition of tyrosine kinase inhibits cAMP stimulation of progesterone. Replicate wells of cells were incubated in the presence (or absence) of tyrosine kinase inhibitors HNMPA (10 µM) or AG879 (4 µM) with 0.25 mM 8 bromo-cAMP as described in Materials and Methods. Medium progesterone levels in each well were determined by RIA and normalized to cell number. Each bar shows the mean (± SEM) medium progesterone levels in four independent experiments, each conducted in quadruplicate. Bars with different letters are significantly different (P < 0.05).

Incubation of the cells with 0.25 mM 8 bromo-cAMP significantly increased Ras activity, whereas levels of total Ras p21 protein were not affected, as shown in Figure 5. Ras activity data from four individual experiments are shown in Table 1. As indicated, incubation with 0.25 mM 8 bromo-cAMP caused a significant increase above that of control values.


Figure 05
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  		FIG. 5. cAMP stimulates RAS activity in rat luteal cells. Films developed after exposure to blots from representative experiments in which RAS activity (RAS-GTP) (upper panel) and total RAS protein levels (lower panel) were assessed in cells incubated in the absence (lane 2) and presence of 0.25 mM 8 bromo-cAMP (lane 3) as described in Materials and Methods. Lane 1 shows data from cells incubated in serum free media.


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  		TABLE 1. RAS activity in cAMP stimulated cells.

Inhibition of MEK activity did not affect cAMP-stimulated progesterone levels, as shown in Figure 6B. In these experiments, progesterone concentrations in media from cells incubated with vehicle alone were significantly lower than progesterone levels in media from cells incubated with 0.25 mM 8 bromo-cAMP (P = 0.002). Progesterone concentrations in media from cells incubated with 0.25 mM 8 bromo-cAMP in combination with the MEK inhibitor UO 126 (10 M) were not different from cells incubated with 8 bromo-cAMP alone (P = 0.14). Progesterone concentrations in media from cells incubated with 0.25 mM 8 bromo-cAMP in combination with the MEK inhibitor PD 98059 (10 µM) were higher, but not significantly different from cells incubated with 8 bromo-cAMP alone (P = 0.08). Figure 6A shows a representative blot demonstrating the inhibition of MAPK3 and MAPK1 phosphorylation that occurred in cells incubated with these inhibitors. UO 126 reduced phosphorylated MAPK3/1 levels to 17% ± 4% (mean ± SEM, n = 3 experiments) of levels in cells incubated with 8 bromo-cAMP in the absence of inhibitor. PD 98059 reduced phosphorylated MAPK3/1 levels to 63% ± 7% (mean ± SEM, n = 3 experiments) of levels in cells incubated with 8 bromo-cAMP in the absence of inhibitor. Levels of MAPK3 or MAPK1 protein were not affected.


Figure 06
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  		FIG. 6. Inhibition of MAP kinase does not inhibit cAMP-stimulated progesterone. Replicate wells of cells were incubated in the presence (or absence) of inhibitors of MAP Kinase UO126 (10 µM) or PD 98059 (10 µM) with 0.25 mM 8 bromo cAMP as described in Materials and Methods. Medium progesterone levels in each well were determined by RIA and normalized to cell number. In B, each bar shows the mean (± SEM) medium progesterone levels in four independent experiments, each conducted in quadruplicate. A) Films developed after exposure to blots from representative experiments assessing phosphorylated MAPK3 and MAPK1 levels (upper panel) and total MAPK3 and MAPK1 levels (lower panel) in cells incubated as described for B. aP < 0.05 compared with those without 8br-cAMP controls. Bars with the same letter are not significantly different.

Inhibition of PI3-K activity had no effect on cAMP-stimulated progesterone expression, as shown in Figure 7B. In these experiments, progesterone concentrations in media from cells incubated with vehicle alone (control) were significantly lower than progesterone levels in media from cells incubated with 0.25 mM 8 bromo-cAMP (P = 0.004). Progesterone concentrations in media from cells incubated with 0.25 mM 8 bromo-cAMP in combination with the PI3-K inhibitor LY 294002 (10 µM) were not different from cells incubated with 8 bromo-cAMP alone (P = 0.24). In these experiments, LY 294002 reduced phosphorylated GSK3A/B levels to 3% ± 0.2% (mean ± SEM, n = 3 experiments) of levels in cells incubated with 8 bromo-cAMP in the absence of inhibitor, as shown in Figure 7A. Protein levels of GSK3A/B and AKT1 were not affected.


Figure 07
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  		FIG. 7. Inhibition of PI-3 kinase does not inhibit cAMP-stimulated progesterone. Replicate wells of cells were incubated in the presence (or absence) of PI-3 kinase inhibitor LY-294002 (10 µM) with 0.25 mM 8 bromo-cAMP as described in Materials and Methods. Medium progesterone levels in each well were determined by RIA and normalized to cell number. In B, each bar shows the mean (± SEM) medium progesterone levels in three independent experiments, each conducted in quadruplicate. A) Films developed after exposure to blots from representative experiments assessing phosphorylated GSK3A/B (upper panel), total GSK3A/B (middle panel), and total AKT1 (lower panel) in cells incubated as described for B. aP < 0.05 compared with those without 8br-cAMP controls. Bars with the same letter are not significantly different.

Since inhibition of PKA had no effect upon cAMP-stimulated progesterone, we performed studies to verify the effectiveness of the PKA inhibitors. PKA activity, as shown in Figure 8, was significantly stimulated in cells incubated with 0.25 mM 8 bromo-cAMP, above that of control cells incubated in the absence of cAMP. PKA activity in cells incubated with 0.25 mM 8 br-cAMP was effectively reduced by both H89 and Rp-cAMPS to activity virtually equivalent to that in cells incubated without cAMP. To determine whether the pronounced inhibition of cAMP-stimulated progesterone seen in cAMP-stimulated cells incubated with tyrosine kinase inhibitors was due to an effect of these inhibitors upon PKA activity, we determined PKA activity in cAMP-stimulated cells incubated with HNMPA. Despite the pronounced inhibition of HNMPA upon cAMP-stimulated progesterone expression, no effect of HNMPA at 5 or 25 µM upon PKA activity was seen, as shown in Figure 8.


Figure 08
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  		FIG. 8. Relative PKA activity. Replicate wells of cells were incubated in the presence (or absence) of PKA inhibitors (H-89 or Rp-cAMPS) or tyrosine kinase inhibitor HNMPA (at doses indicated) with 0.25 mM 8 bromo-cAMP, and PKA activity was determined as described in Materials and Methods. In each experiment, values computed for the control cells incubated without 8 bromo-cAMP were set at 1, and values computed for the treated cells are expressed as relative to the control. Each bar shows the mean (± SEM) of PKA activity in three independent experiments, each conducted in duplicate. aP < 0.05 compared with those without 8br-cAMP controls.

DISCUSSION

Numerous studies have clearly established that cAMP functions as a second messenger to regulate steroidogenesis [35, 36]. In the classical model, intracellular cAMP activates the cAMP-dependent protein kinase, PKA. The current studies provide evidence that in this cell line, the response to increased cAMP involves other downstream mediators in addition to PKA. Although non-PKA mediated actions of cAMP have been previously demonstrated, many have been shown to involve either MAPK or PI3-K mediators [37, 38]. Our current studies show that neither MAPK nor PI3-K activities appear to be involved in the PKA-independent cAMP-stimulated progesterone process. The data also demonstrate the involvement of tyrosine kinase activity, consistent with data from a previous study that suggest that Cyp11a1 gene expression becomes PKA independent following luteinization of rat granulosa cells, yet is dependent upon tyrosine kinase activity [39]. Our studies also show stimulation of Ras activity by cAMP, suggesting the involvement of this mediator.

Various cAMP effects that are PKA independent have been described previously. cAMP has been shown to regulate gene transcription, cellular proliferation, and cytokine signaling through PKA-independent pathways [40–43]. Although many of these effects of cAMP have been shown to be mediated by MAPK [38], MAPK-independent effects have also been shown [44]. No role for MAPK in cAMP-stimulated progesterone was demonstrated in our studies. In certain previous studies, activation of MAPK has been shown to be related to inhibition of steroidogenesis [45]. Thus, a role for MAPK may not be in accord with the cAMP stimulation of steroidogenesis observed in our studies.

A recently discovered example of non-PKA-mediated effects of cAMP is the cAMP activation of guanine nucleotide exchange factors, which activate other noncyclase associated GTP-binding proteins such as Ras and RAP1. Epac (exchange protein directly activated by cAMP), also known as cAMP guanine nucleotide exchange factor (cAMP-GEF) and which is widely expressed in various cell types, including ovarian granulosa cells [36], functions to activate RAP1, a small G protein involved in cell proliferation and cell differentiation in several cell types [46]. The cyclic nucleotide exchange factor CNrasGEF activates Ras in response to elevations of intracellular cAMP [47, 48]. Currently, the lack of existence of CNrasGEF inhibitors or other tools have precluded studies to determine if this exchange factor plays a role in cAMP-stimulated progesterone.

Our data demonstrate that, in our model, cAMP significantly stimulates Ras activity. We previously demonstrated that levels of the KRAS protein in the rat ovary vary with physiological function of the corpus luteum in a pattern that mimics circulating progesterone concentrations [49]. In addition, stable transfection of nonsteroidogenic rat ovarian and adrenocortical cells with viral KRAS has been shown to induce HSD3B activity and steroidogenesis and similar transfection of ovarian granulosa cells resulted in constitutive steroidogenesis [50, 51]. Ras activation of the ovine CYP11A1 promoter has been demonstrated [52]. These data suggest that ras gene products play a role in steroidogenesis. In addition, cAMP activation of Ras can occur by both PKA-dependent and PKA-independent mechanisms [53, 54]. In rat thyroid cells, thyroid-stimulating hormone stimulates a cAMP-mediated pathway that requires Ras activity [54]. This may be cell type selective. Recent studies indicate localization of Ras to different cellular compartments may be responsible for these differences. Early data demonstrated Ras localization to the plasma membrane; whereas more recent data clearly demonstrate Ras activation and signaling also occur on various intracellular membranes, including Golgi [55]. This may explain the demonstration of cAMP inhibition of Ras activity in certain cell types.

In addition to gonadotropic hormones, growth factors such as insulin, IGF1, and EGF have been shown to modulate luteal steroid hormone synthesis and steroidogenic enzyme expression as either independent effects or via amplification or modulation of the action of gonadotropic hormones or cAMP [3–10, 56]. Amplification of cAMP-stimulated progesterone by IGF1 in rat luteal cells has been previously demonstrated [56]. The receptors for these growth factors are transmembrane tyrosine kinases which dimerize in response to ligand binding and autophosphorylate multiple tyrosines in the cytoplasmic domain. Various reports have directly implicated tyrosine kinase pathways in ovarian steroidogenesis. Addition of specific tyrosine kinase inhibitors to cultured rat granulosa cells results in inhibition of LH-stimulated progesterone production and Cyp11a1 mRNA expression [57–59]. Other studies have shown synergistic effects of insulin or IGF1 on gonadotropin-stimulated progesterone production and expression of Cyp11a1 and Hsd3b mRNAs by rat and human granulosa cells [3, 4, 8]. IGF1 stimulates in vitro luteinization and expression of the STAR protein, which stimulates delivery of cholesterol to the mitochondrial inner membrane, in porcine granulosa cells [10].

Our data demonstrate that receptor tyrosine kinase inhibitors HNMPA and AG 879 inhibit cAMP-stimulated progesterone in our in vitro model. The receptor tyrosine kinase inhibitor HNMPA was originally designed to inhibit insulin receptor tyrosine kinase activity. However, it has since been shown to be a more general tyrosine kinase inhibitor [60, 61]. Similarly, AG 879, originally reported to be a selective nerve growth factor receptor tyrosine kinase inhibitor, has also been recently used to effectively inhibit tyrosine kinase activity of the ERBB2 (also known as HER2/Neu) receptor, a member of the EGF family of receptor tyrosine kinases [62, 63].

In summary, we have demonstrated that, in an in vitro model, cAMP-stimulated progesterone does not involve PKA activity, nor does MAPK or PI3-K appear to be involved. Stimulation of Ras activity by cAMP in these cells suggests the involvement of this mediator. A comprehensive understanding of the physiological regulation of luteal function, including the mechanisms involved in luteal development and regression, requires identification of the signaling molecules that subserve gonadotropin action and the actions of other factors that modulate gonadotropin action.

FOOTNOTES

3Current address: 401 Cherryville Rd., Pittstown, NJ 08867. Back

4Current address: 580 E. Market St., Marietta, PA 17547. Back

1Supported by National Science Foundation grant IBN 9600915. Back

Correspondence: 2Laura T. Goldsmith, New Jersey Medical School of UMDNJ, 185 South Orange Ave., Newark, NJ 07103. FAX: 973 972 4574; e-mail: goldsmit{at}umdnj.edu

Received: 30 May 2006.

First decision: 15 June 2006.

Accepted: 14 March 2007.

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