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Endocrinology Vol. 143, No. 8 2986-2994
Copyright © 2002 by The Endocrine Society

ARTICLE

Acute Signaling by the LH Receptor Is Independent of Protein Kinase C Activation

Lisa M. Salvador, Evelyn Maizels, Dale Buchanan Hales, Eishichi Miyamoto, Hideyuki Yamamoto and Mary Hunzicker-Dunn

Departments of Cell and Molecular Biology, Northwestern University Medical School (L.M.S., E.M., M.H.-D.), Chicago, Illinois 60611; Department of Physiology and Biophysics, University of Illinois School of Medicine (D.B.H.), Chicago, Illinois 60611; and Department of Pharmacology, Kumamoto University School of Medicine (E.M., H.Y.), 2-2-1 Honjo, Kumamoto 860-0811, Japan

Address all correspondence and requests for reprints to: Dr. Mary Hunzicker-Dunn, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail: . mhd{at}northwestern.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LH receptor activation leads to the phosphorylation/activation of p42/44 MAPK in preovulatory granulosa cells. As the LH receptor can activate both adenylyl cyclase and phospholipase C, we hypothesized that the LH receptor could elicit phosphorylation of p42/44 MAPK through activation of protein kinase A (PKA) and/or protein kinase C (PKC). Preovulatory granulosa cells in serum-free primary cultures were treated with ovulatory concentrations of human chorionic gonadotropin (hCG), an LH receptor agonist, with or without various inhibitors. The PKA inhibitor H89 as well as the myristoylated PKA inhibitor peptide PKI strongly inhibited hCG-stimulated p42/44 MAPK phosphorylation, whereas the PKC inhibitor GF109203X had no effect on p42/44 MAPK phosphorylation. LH receptor-stimulated phosphorylation of cAMP response element-binding protein (CREB), histone H3, and MAPK kinase (MEK) was also strongly inhibited by H89 and not by GF109203X. The extent of PKC activation was assessed in preovulatory granulosa cells using three criteria: translocation of PKC isoforms to the membrane fraction, phosphorylation of a known PKC substrate, and autophosphorylation of PKC {delta} on an activation-related site. By all three criteria PKCs were partially activated before hCG stimulation, and hCG treatment failed to elicit further PKC activation, in vitro or in vivo. Taken together, these results indicate that, under primary culture conditions where physiological levels of signaling proteins are present, hCG signals to activate MEK, p42/44 MAPK, CREB, and histone H3 in a predominately PKA-dependent and PKC-independent manner. Unexpectedly, PKCs were partially activated in the absence of LH receptor activation, and LH receptor activation did not elicit further detectable PKC activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OVULATION OF mature Graafian follicles and subsequent luteinization of granulosa and thecal cells to form the corpus luteum are initiated by the anterior pituitary hormone LH. Granulosa cells from preovulatory-sized follicles and theca interna cells both express surface LH receptors (1, 2). The LH receptor is a hepta-membrane-spanning protein that couples to the stimulatory guanine nucleotide-binding protein Gs and signals to adenylyl cyclase to increase cAMP and activate cAMP-dependent protein kinase (PKA) (3). The LH receptor has also been shown to increase intracellular calcium levels (4, 5). The increase in intracellular calcium probably results predominantly from the ability of the LH receptor to activate phospholipase C (PLC), as demonstrated on transfection of the LH receptor into heterologous cell lines (6, 7, 8). Activation of PLC results in the cleavage of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 goes on to bind to intracellular calcium channels, allowing for increases in intracellular calcium in the cytosol, whereas DAG binds to protein kinase C (PKC), acting as a necessary coactivator for many PKC isoforms (9). In primary rat granulosa cell cultures, the actions of ovulatory concentrations of LH or the LH receptor agonist human chorionic gonadotropin (hCG) can be mimicked by subovulatory concentrations of LH/hCG plus the PKC activator phorbol 12-myristate 13-acetate (PMA) (10, 11). These results suggest that the actions of LH in preovulatory cells are mediated not only by cAMP/PKA, but also by PKC.

PKA is a tetrameric enzyme that consists of dimeric regulatory subunits and two catalytic subunits. There are four isoforms of regulatory subunits: RI{alpha}, RII{alpha}, RIß, and RIIß. These differ in size, phosphorylation potential, and cAMP affinity (12). Rat preovulatory granulosa cells predominately express RII{alpha} and RIIß holoenzymes (13). Upon binding of cAMP to the regulatory subunits, a conformational change occurs that allows for dissociation of the catalytic subunits, which are then free to phosphorylate their substrates.

PKC exists as a family of isoforms. The conventional isoforms require Ca2+, phosphaditylserine (PS) and DAG for their activation. The novel isoforms require only PS and DAG, whereas the atypical isoforms require only PS. Upon binding of the regulators, calcium, DAG, and PS or the DAG analog PMA, a conformational change occurs that allows the enzyme to open up, revealing the substrate-binding site and allowing for phosphorylation of substrates (9). Rat preovulatory granulosa cells predominately express PKC {alpha}, ßI, ßII, {delta}, {epsilon}, and {zeta} (14, 15).

Based on the underlying hypothesis that LH actions in mature preovulatory granulosa cells are mediated not only by cAMP and PKA, but also by PKC, in the following studies we sought to determine whether the ability of the LH receptor to activate classic signaling intermediates was dependent on PKC. Based on reports that p42/44 MAPK can be activated downstream of PKC (16), we hypothesized that activation of p42/44 MAPK could serve as a marker in preovulatory granulosa cells for PKC activation downstream of the LH receptor activated by ovulatory concentrations of hCG. We additionally sought direct evidence for PKC activation downstream of the LH receptor in preovulatory granulosa cells. However, the results show that p42/44 MAPK activation is independent of PKC activation, and indeed, that PKCs do not appear to be activated in granulosa cells in response to ovulatory concentrations of LH. Rather, at least PKC {alpha}, ßII, {delta}, and {epsilon} are already partially activated in preovulatory granulosa cells before LH receptor activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The following were purchased: Profasi hCG from Serono Laboratories, Inc. (Randolph, MA); H89, PMA, and GF109203X (GFX) from Alexis Biochemicals (San Diego, CA); myristolated PKA inhibitor (PKI) peptide (amino acid residues 14, 15, 16, 17, 18, 19, 20, 21, 22) from Calbiochem (San Diego, CA); PAGE reagents from Bio-Rad Laboratories, Inc. (Richmond, CA); X-OMAT AR film from Eastman Kodak Co. (Rochester, NY); enhanced chemiluminescence reagents and Hybond C-extra nitrocellulose from Amersham Pharmacia Biotech (Arlington Heights, IL). All culture media were purchased from Life Technologies, Inc. (Lexington, KY), and all other chemicals were purchased from Sigma (St. Louis, MO).

Antibodies
The following antibodies were purchased or donated: phospho-specific p42/44 MAPK polyclonal antibody (T202,Y204; Promega Corp., Madison, WI); p44 MAPK control monoclonal antibody (Zymed Laboratories, Inc., San Francisco, CA); PKC {alpha}, ßII, {delta}, and {epsilon} polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); phospho-specific cAMP response element-binding protein (CREB; S133) and phospho-specific histone H3 (S10; Upstate Biotechnology, Inc., Lake Placid, NY); phospho-specific p38 MAPK (T180, Y182) and phospho-specific p42/44 MAPK kinase (MEK1/2; S217 and S221; New England Biolabs, Inc., Beverly, MA); StAR polyclonal antibody (17); phospho-specific myristolated alanine-rich C kinase substrate (MARCKS) polyclonal antibody (S152 and S156) (18); phospho-specific PKC {delta} polyclonal antibody (T662; a gift from New England Biolabs, Inc.) (15).

Primary granulosa cell cultures
Twenty-six- to 27-d-old Sprague Dawley rats (Sasco, Baltimore, MD), maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and with the Northwestern University institutional animal care and use committee, were injected sc with 10 IU pregnant mare serum gonadotropin (PMSG). Ovaries were harvested 44–48 h post injection; trimmed to remove the bursa, fat, and oviducts; and then incubated for 15–30 min at 37 C in 6 mM EGTA in DMEM/Ham’s F-12 medium. Ovaries were then incubated for 5–20 min in 0.5 M sucrose in DMEM/F-12, and granulosa cells were expressed by subjecting large preovulatory follicles to penetration with a 30-gauge needle. The cells were then plated at a density of 5–7 x 106 in serum-free DMEM/F-12 on plates coated with 0.5 µg/ml fibronectin and cultured as previously described (19, 20). Cells were treated the next day, as indicated in the text.

Preovulatory ovarian extracts
Twenty-six- to 27-d-old Sprague Dawley rats (Sasco) were injected sc with 25 IU PMSG 48 h before injection with 25 IU hCG. Ovaries were harvested at 0, 1, or 8 h post hCG injection; cooled to 4 C in an iced 10 mM potassium phosphate buffer, pH 7.0; dissected free of bursa, fat, and oviducts; weighed; and homogenized (5:1 ratio of homogenization buffer/wet weight) in homogenization buffer [10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM ß-glycerol phosphate, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 40 µg/ml phenylmethylsulfonylfluoride, 0.5% Nonidet P-40, and 0.1% deoxycholate] using 15–20 strokes in a ground-glass homogenizer. A supernatant fraction (cytosol) was obtained by centrifugation at 105,000 x g at 4 C for 70 min.

Electrophoresis and Western blotting
Total cell extracts were made by harvesting the cells into 300 µl SDS-PAGE sample buffer (21) and heat denaturation. Protein concentrations were controlled by plating equal number of cells in each dish for each experiment, followed by loading equal volumes onto an SDS-PAGE gel. These cells do not divide under serum-free conditions. The proteins of the cell lysates were separated by SDS-PAGE (22) (10% or 10.5% acrylamide running gel), transferred to Hybond C-extra nitrocellulose overnight at 4 C and stained for protein loading using Ponceau-S staining. The nitrocellulose blots were incubated with primary antibody at 4 C overnight. Antigen-antibody complexes were detected using enhanced chemiluminescence. Quantitation of bands on blots was performed using the Molecular Analyst/PC image analysis software for the densitometer (Bio-Rad Laboratories, Inc., model GS-670). Results were analyzed by t test (P < 0.05) (23).

Cell fractionation
Preovulatory granulosa cells were treated and then collected in 300 µl buffer [10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 50 mM ß-glycerol phosphate, 1 mM sodium orthovanadate, 2 mM dithiothreitol, 100 µg/ml pepstatin, and 10 µg/ml leupeptin] (24). Lysates were made by sonicating the samples for 1 min on ice and centrifuging at 105,000 x g at 4 C for 70 min. The volume of the supernatant or cytosol fraction was measured. The pellets were then resuspended in a volume of lysis buffer plus 0.1% Triton X-100 equal to that of the cytosol fraction. Triton-soluble fractions were obtained by stirring at 4 C for 1 h, followed by centrifugation at 105,000 x g at 4 C for 30 min (25). The Triton-insoluble pellets that remained were resuspended in a volume of lysis buffer equal to that of the cytosol fraction. SDS-PAGE sample buffer was added in a ratio of 1:2 (vol/vol), and samples were heat denatured. Protein concentrations in the cytosol fraction were measured with BSA as standard (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
hCG stimulates the phosphorylation of p42/44 MAPK in a time- and PKA-dependent manner
Treatment of preovulatory granulosa cells with hCG resulted in a transient activation of p42/44 MAPK (Fig. 1Go). p42/44 MAPK phosphorylation was detectable by 5 min, peaked by 10 min, and then slowly decreased to undetectable levels by 2 h. Treatment of preovulatory granulosa cells with the adenylyl cyclase activator, forskolin, which increases intracellular levels of cAMP, also resulted in a robust increase in the phosphorylation of p42/44 MAPK, indicating that the phosphorylation of p42/44 MAPK in preovulatory granulosa cells is cAMP dependent (Fig. 2AGo, lane 5). Pretreatment of cells with 10 µM H89, a PKA inhibitor (27, 28), resulted in strong inhibition of the phosphorylation of p42/44 MAPK stimulated by both hCG (89 ± 11% inhibition) and forskolin (91 ± 9% inhibition; Fig. 2AGo, lanes 4 and 6). To further verify the PKA dependence of hCG-induced p42/44 MAPK phosphorylation, preovulatory granulosa cells were pretreated with 100 µM myristolated PKI. PKI inhibits the catalytic subunit of PKA by binding to the substrate-binding site and, by acting as a pseudosubstrate, prevents PKA substrates from binding, thus preventing their phosphorylation (29). Pretreatment with the myristolated PKI before hCG treatment inhibited hCG-stimulated p42/44 MAPK phosphorylation by 79 ± 15% (n = 3; P < 0.05; Fig. 2BGo, lane 4). These results indicate that the hCG-dependent phosphorylation of p42/44 MAPK is predominately mediated by and dependent on PKA. That hCG-stimulated p42/44 MAPK activation is PKA dependent was also recently reported for a preovulatory granulosa cell line cotransfected with mutated p53 Ha-ras genes and the LH receptor (30).



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  		Figure 1. Time course of hCG-stimulated p42/44 MAPK phosphorylation. Granulosa cells from preovulatory follicles were treated for the indicated times with 1 IU/ml hCG. Cell lysates were collected in SDS-PAGE sample buffer and subjected to SDS-PAGE on a 10.5% acrylamide gel as described in Materials and Methods. The proteins were transferred to nitrocellulose, and a Western blot was performed using an antibody to phosphorylated p42/44 MAPK. Protein loading is reflected by Ponceau-S staining of the histones. Results are representative of three separate experiments. Fold stimulation by hCG could not be determined because p42/p44 MAPK phosphorylation at time zero was below the level of detection.

 


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  		Figure 2. Effect of hCG and forskolin on p42/44 MAPK activation in preovulatory granulosa cells. A, Granulosa cells obtained from preovulatory follicles were pretreated for 1 h with 10 µM H89 and then were treated with vehicle (Veh), 1 IU/ml hCG, or 50 µM forskolin (FOR) for 10 min. For other conditions see Fig. 1Go. Results are representative of two separate experiments. The mean percent inhibition ± range by H89 of hCG- and forskolin-stimulated p42/p44 MAPK activation was 89 ± 11 and 91 ± 9, respectively. B, Cells were pretreated for 45 min with either vehicle or 100 µM myristolated PKI and then treated with either vehicle or hCG for 10 min. Results are representative of three separate experiments.

 
hCG-stimulated phosphorylation of MEK is dependent on PKA
Preovulatory granulosa cells treated for 10 min with hCG also demonstrated increased phosphorylation of the kinase upstream of p42/44 MAPK, MEK (Fig. 3Go). Moreover, after pretreatment with 10 µM H89, hCG-stimulated MEK phosphorylation was inhibited by 64%. This indicates that the majority of hCG-stimulated MEK phosphorylation is also PKA dependent.



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  		Figure 3. Effect of H89 on hCG-stimulated phosphorylation of MEK. Granulosa cells were pretreated for 1 h with 10 µM H89 and then treated with either vehicle or 1 IU/ml hCG for 10 min. Cell lysates were subjected to SDS-PAGE and Western blotting analysis. Results are representative of two separate experiments.

 
hCG-stimulated phosphorylation of p42/44 MAPK and MEK is independent of PKC
In the following experiments we sought to determine whether hCG-stimulated p42/44 MAPK activation was also dependent on PKC activation. Although pretreatment of preovulatory granulosa cells with 10 µM H89 reduced hCG-stimulated p42/44 MAPK phosphorylation by 66 ± 6% (n = 4; P < 0.05; Fig. 4AGo, lane 5), consistent with results seen in Fig. 2Go, pretreatment of the preovulatory granulosa cells with 5 µM GFX, a PKC inhibitor (31), had no effect on hCG-stimulated phosphorylation of p42/44 MAPK (0% inhibition; n = 4; Fig. 4AGo, lane 6). To ensure the efficacy of the GFX employed, granulosa cells were pretreated with vehicle or 5 µM GFX and then treated with the PKC activator, PMA (Fig. 4BGo). PMA stimulated the phosphorylation of p42/44 MAPK, and this response was inhibited 82 ± 7% (n = 4; P < 0.05) by GFX. Preovulatory granulosa cells were also treated with a 10-fold higher concentration of hCG (10 IU/ml) to determine whether larger quantities of hCG were required to activate PKC and thus p42/44 MAPK. However, hCG-stimulated p42/44 MAPK phosphorylation remained insensitive to inhibition by GFX (0% inhibition; n = 2) when cells were treated with 10 IU/ml hCG (Fig. 4CGo). We also investigated the upstream kinase, MEK. Consistent with results in Fig. 3Go, hCG stimulated MEK phosphorylation (Fig. 4DGo), and the MEK inhibitor PD98059 completely inhibited hCG-stimulated p42/44 MAPK phosphorylation (100% inhibition; n = 2), but did not acutely modulate the expression of StAR (Fig. 4EGo). hCG-stimulated MEK phosphorylation was also unaffected by GFX (0% inhibition; n = 3; Fig. 4DGo). Thus, although p42/44 MAPK in granulosa cells can be strongly activated by PKC, these results demonstrate that phosphorylation of MEK and p42/44 MAPK in response to hCG treatment is not dependent on PKC, and that PKC is not involved in the MAPK pathway activated by hCG.



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  		Figure 4. Effect of GFX on hCG-stimulated p42/44 MAPK and MEK activation. A, Preovulatory granulosa cells were pretreated for 1 h with 10 µM H89 or for 30 min with 5 µM GFX and then treated for 10 min with either vehicle or 1 IU/ml hCG. For details, see Fig. 1Go. Results are representative of four separate experiments. B, Cells were pretreated for 30 min with 5 µM GFX and then treated for 10 min with either vehicle or 200 nM PMA. Results are representative of four separate experiments. C, Cells were pretreated for 30 min with either vehicle or 5 µM GFX, then treated for 10 min with either 1 or 10 IU/ml hCG. Results are representative of two separate experiments. D, Cells were pretreated for 30 min with vehicle or 5 µM GFX and then treated with vehicle or 1 IU/ml hCG for 10 min. Results are representative of three separate experiments. E, Cells were pretreated with vehicle or 5 µM GFX for 30 min, with 10 µM H89 for 60 min, or with 50 µM PD98059 for 90 min, and then treated for 10 min with 1 IU/ml hCG. Results are representative of three separate experiments. The blot was probed for active p42/44 MAPK and StAR protein. Protein loading is reflected by Ponceau-S staining or by an antibody that detects primarily p44 MAPK.

 
PKC is not involved in hCG-stimulated phosphorylation of CREB, p38 MAPK, or histone H3
We next determined whether the phosphorylation of other signaling intermediates in preovulatory cells known or predicted to be downstream of the LH receptor, such as CREB, histone H3, and p38 MAPK, was induced by hCG via PKA- and/or PKC-dependent pathways. As shown in Fig. 5Go, hCG stimulated the phosphorylation of CREB, p38 MAPK, and histone H3. hCG-stimulated phosphorylation of both CREB and histone H3 was strongly inhibited by H89 [74 ± 17% (n = 2) and 86% inhibition, respectively; Fig. 5Go, A and B, compare lanes 1 and 2 vs. lanes 3 and 5], indicating that the pathways leading to these hCG-stimulated responses are also largely dependent on PKA. Pretreatment of cells with the PKC inhibitor GFX did not affect the ability of hCG to stimulate histone H3 or CREB phosphorylation (14 ± 14% and 0% inhibition, respectively; n = 2; Fig. 5Go, A and B; note the reduced protein load in lane 6 and especially in lane 7 in Fig. 5AGo). These results indicate that hCG-stimulated PKC activation is not responsible for the hCG-dependent phosphorylation of CREB or histone H3. However, the hCG-stimulated phosphorylation of p38 MAPK was not affected by either H89 or GFX (15.2 ± 2.5% and 0% inhibition, respectively; n = 2; Fig. 5AGo), indicating that neither PKA nor PKC is necessary for hCG to stimulate p38 MAPK phosphorylation. Taken together, these results indicate that PKC is not involved in the activation/phosphorylation of a number of signaling intermediates that participate in hCG-induced differentiation. Based on these results, we next wanted to test directly whether hCG activates PKC in preovulatory granulosa cells.



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  		Figure 5. Effect of H89 and GFX on hCG-stimulated phosphorylation of CREB, p38 MAPK, histone H3, and MARCKS. Granulosa cells were pretreated for 1 h with 10 µM H89 or for 30 min with 5 µM GFX and then treated for 10 min with vehicle, 1 IU/ml hCG, or 200 nM PMA. For details, see Fig. 1Go. Protein loading is reflected by reactivity to PKC {delta} antibody in A and by Ponceau-S staining in B. Results in A and B are representative of two separate experiments.

 
hCG does not induce the translocation of PKC isoforms to the Triton-soluble fraction
The classic method for measuring the activation of PKC is to examine its translocation from the cytosol to a Triton-soluble membrane fraction (9). Conventional and novel PKC isoforms require the lipid DAG for their activation. DAG is found in the plasma membrane of cells, and for PKC to be activated, PKC must move to the membrane and bind DAG to cause the conformational change required to allow for substrate binding and subsequent phosphorylation (9). We therefore determined whether hCG induced the translocation of DAG-regulated PKCs present in preovulatory granulosa cells from the soluble fraction to the Triton-soluble fraction that contains the plasma membrane. The cells were treated for 2 and 10 min to ensure that a rapid translocation event was not missed (32). Figure 6Go and Table 1Go show that upon treatment with hCG for either 2 or 10 min, no translocation of PKC {alpha}, ßII, {delta}, or {epsilon} from cytosol to the Triton-soluble fraction was observed. However, the positive control PMA was able to promote the translocation of these isoforms from the cytosol to the Triton-soluble fractions at both 2 and 10 min posttreatment, as evidenced by reduced PKCs in the cytosol fraction after PMA treatment. The decreased total protein observed for the 10-min PMA treatment, especially for PKC {alpha} (see Fig. 6Go), is indicative of the ability of phorbol esters to down-regulate PKC protein (33). These results indicate that hCG does not promote activation of PKC {alpha}, ßII, {delta}, or {epsilon}.



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  		Figure 6. Effects of hCG and PMA on cellular localization of PKC isoforms. Granulosa cells were treated for 2 or 10 min with vehicle, 1 IU/ml hCG, or 200 nM PMA. The cells were lysed in a protease inhibitor buffer and subjected to extraction with 0.1% Triton X-100 as described in Materials and Methods. The cytosol (Cyt), Triton-soluble (TS), and Triton-insoluble (TI) fractions were then collected, and the fractions were subjected to SDS-PAGE and subsequent Western blotting with antibodies to PKC {alpha}, ßII, {delta}, and {epsilon} isoforms. Equal cytosol protein (50 µg) from each cell treatment was added to gel lanes. Volumes for TS and TI were equalized to that of Cyt during the preparation of cell extracts, and volumes of TS and TI extracts equal to those of Cyt were added to gel lanes. Results are representative of four separate experiments.

 

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  		Table 1. Percentage of total PKC isoform in cells

 
hCG does not induce the phosphorylation of the PKC substrate MARCKS or of PKC {delta} in vitro or in vivo
These data indicated that PKC does not appear to be activated or even involved in the initial signaling pathways activated downstream of the LH receptor leading to hCG-induced differentiation in preovulatory granulosa cells. To further examine whether hCG promotes PKC activation, we tested the ability of hCG to induce the phosphorylation of the universal PKC substrate, MARCKS (34, 35), both in vivo and in vitro. MARCKS was found to be heavily phosphorylated in untreated preovulatory granulosa cells, as detected by Western blotting using a phospho-specific MARCKS antibody that detects the PKC-catalyzed phosphorylation of Ser152 and Ser156 (Fig. 5BGo). hCG did not further stimulate the phosphorylation of MARCKS in vitro in preovulatory granulosa cells, whereas PMA further increased the phosphorylation of MARCKS in these cells by 60% (Fig. 5BGo). The phosphorylation of MARCKS was reduced approximately 40% by GFX in the untreated granulosa cell (Fig. 5BGo, lane 4), suggesting that PKCs are at least partially activated in preovulatory granulosa cells before stimulation with hCG. The partial inhibition of MARCKS phosphorylation in vehicle-treated cells (Fig. 5BGo, lane 4) also suggests that only a portion of these phosphates is readily turning over (i.e. subject to dephosphorylation and phosphorylation during the total treatment time of 40 min) and therefore susceptible to inhibition by GFX in the presence of active PKC. PMA-stimulated phosphorylation of MARCKS was also inhibited about 60% by GFX (Fig. 5BGo, lane 8), indicating that PKC is responsible for the phosphorylation of MARCKS induced by PMA treatment. We also evaluated the in vivo phosphorylation of MARCKS to determine whether results obtained with in vitro granulosa cell cultures were an artifact of the culture model. In vivo, MARCKS was already phosphorylated in preovulatory follicle-enriched ovarian extracts (Fig. 7AGo, lane 1), consistent with the in vitro granulosa cell results in Fig. 5BGo. hCG injection to PMSG-primed rats did not further stimulate MARCKS phosphorylation in these ovarian extracts obtained at 1 or 8 h post-hCG injection (Fig. 7AGo); rather, hCG appeared to reduce MARCKS phosphorylation. hCG treatment of PMSG-primed rats also did not enhance the phosphorylation of PKC {delta} in vivo, as detected by Western blotting of ovarian extracts using a phospho-specific PKC {delta} antibody (Fig. 7AGo) raised against a phosphorylation site (Thr662) that is specific for PKC {delta} activation (25). Interestingly, a high basal level of phosphorylated PKC {delta} was already present at 0 h post-hCG injection in ovaries of PMSG-primed rats (Fig. 7AGo). Similarly, a high basal level of phospho-PKC {delta} was detected in vitro in preovulatory granulosa cells, and PKC {delta} phosphorylation was not increased by hCG treatment, but was increased approximately 2-fold by PMA treatment of these cells (Fig. 7BGo). hCG injection to PMSG-primed rats did, however, stimulate the in vivo phosphorylation of ovarian MEK and p42/44 MAPK (Fig. 7AGo). Taken together, these results indicate that hCG does not appear to stimulate the activation of PKC in preovulatory granulosa cells or in ovaries of PMSG-primed rats. Rather, PKC {alpha}, ßII, {delta}, and {epsilon} are already partially activated in preovulatory granulosa cells before LH receptor activation.



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  		Figure 7. Effect of hCG treatment of isolated granulosa cells and PMSG-treated rats on the phosphorylation of MARCKS, PKC {delta}, MEK, and/or histone H3. A, Twenty-nine-day-old PMSG-primed rats were injected with hCG (25 IU) at time zero and the ovaries were harvested at the times indicated. Ovarian extracts were made, and lysates were analyzed by Western blotting as indicated. Results are representative of two separate experiments. B, Granulosa cells from preovulatory follicles were treated for 10 min with vehicle, 1 IU/ml hCG, or 200 nM PMA. For details, see Figs. 1Go and 6Go.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results show that LH receptor activation in preovulatory granulosa cells promotes the phosphorylation of MEK, p42/44 MAPK, CREB, and histone H3 by a pathway dependent on cAMP and largely inhibited by the PKA inhibitor H89. We also demonstrate that PKA activity is necessary for hCG-stimulated p42/44 MAPK phosphorylation, as treatment of primary cultures of preovulatory granulosa cells with a myristolated PKA inhibitor peptide, PKI, before hCG stimulation strongly inhibits hCG-induced phosphorylation of p42/44 MAPK. That CREB and histone H3 phosphorylations are dependent on PKA is not surprising, because both proteins are in vitro PKA substrates (20, 36, 37, 38), and both have been shown to be phosphorylated in a PKA-dependent manner downstream of FSH in immature granulosa cells (20, 36, 38, 39, 40, 41). That LH receptor activation also promotes MEK phosphorylation in mature granulosa cells suggests that the mechanism by which cAMP/PKA feeds into the p42/44 MAPK activation cascade involves Raf-1, A-Raf, or B-Raf, each of which is expressed in granulosa cells (42), as the upstream activator of MEK, and probably Ras or Rap, the upstream activators of Raf (43, 44). However, the site(s) of entry by cAMP/PKA into this cascade is not currently known and is under investigation. Our evidence for PKA involvement in p42/44 MAPK activation in preovulatory granulosa cells, however, does not support results in PC12 cells showing cAMP-dependent, PKA-independent activation of Rap via a cAMP-regulated guanine nucleotide exchange factor (45, 46). We also detected hCG-stimulated phosphorylation of p38 MAPK in preovulatory granulosa cells. However, in contrast to the phosphorylation of MEK, p42/44 MAPK, CREB, and histone H3 in granulosa cells treated with hCG, as reported herein, and p38 MAPK phosphorylation observed in cells treated with ß-adrenergic receptor agonists or TSH (47, 48), p38 MAPK phosphorylation in hCG-treated preovulatory granulosa cells was not inhibited by the PKA inhibitor H89.

There is compelling evidence that implicates PKCs in LH receptor signaling in mature granulosa cells. For example, the requirement for ovulatory concentrations of LH or hCG to induce prostaglandin synthase-2 (11) and luteinization-associated progesterone production (10, 11) can be met by subovulatory concentrations of hCG only when PMA or GnRH, which activate PKCs, is also included in the cell treatment. Consistent with a presumed role for activation of PKC downstream of the LH receptor, constitutively active human LH receptors promote clear activation of PLC (6, 7, 8). Moreover, when the murine LH receptor is transfected into L cells at a sufficiently high receptor number, receptor activation leads to increased intracellular levels of calcium, consistent with activation of PLC (5). The apparent requirement of high LH receptor numbers to engage the pathway to PLC in preovulatory granulosa cells is a realistic possibility based on the high numbers of LH receptors present in these cells (49, 50). hCG has also been shown to increase IP3 production in mature granulosa cells (51). However, it is not clear whether this increase in IP3 is due to phosphatidylinositol 4,5-bisphosphate cleavage, creating DAG as a by-product, or whether hCG can stimulate de novo synthesis of IP3. One report suggests that hCG induces de novo synthesis of phosphoinositides including IP3, indicating that the IP3 production induced by hCG is not due to hCG activation of PLC and can be distinguished from an increase in DAG production (52, 53).

Despite the ability of the LH receptor to activate PLC under experimental conditions and the ability of PKC activators to synergize with subovulatory hCG concentrations to elicit responses of ovulatory concentrations of hCG, our results clearly show that at least five of the acute responses to LH receptor activation are not dependent on the activation of PKC in preovulatory granulosa cells. Moreover, we have been unable to demonstrate that hCG promotes PKC activation in preovulatory granulosa cells. This conclusion is based both on the inability of hCG to promote translocation of PKC {alpha}, ßII, {delta}, or {epsilon} from a cytosolic to a membrane-enriched fraction and on the inability to detect increased phosphorylation of the universal PKC substrate MARCKS in response to LH receptor activation. We also did not detect increased phosphorylation of PKC {delta} when probing for an activation-sensitive phosphorylation site. Indeed, both MARCKS and PKC {delta} were already phosphorylated in preovulatory granulosa cells and in PMSG-primed ovaries enriched in preovulatory follicles; if anything, LH receptor activation results in reduced phosphorylation of these PKC substrates. Consistent with this result, the PKC translocation data show that for all of the DAG-sensitive PKC isoforms present in granulosa cells, a substantial fraction is already distributed to the Triton-soluble fraction, consistent with activation of a fraction of the cellular PKCs in the absence of hCG treatment. That mild constitutive PKC activation is detected both in vitro in granulosa cell cultures and in the intact animal in ovarian extracts indicates that the results are not an artifact of the granulosa cell culture model. We recently determined that both phospho-PKC {delta} and phospho-MARCKS are also readily detectable in granulosa cell extracts obtained from estrogen-primed immature rats that have not been exposed to PMSG (unpublished). Also, approximately 50% of PKC {epsilon} is distributed to the Triton-insoluble fraction in vehicle-treated cells of these estrogen-primed rats. Therefore, it is unlikely that PMSG priming of immature rats artificially preactivated PKC pathways. That PMSG-primed immature rats are able to ovulate and carry a pregnancy to term suggests that follicular development in this model is typical of the mature cycling animal (54). Perhaps ovulatory concentrations of LH receptor agonists or subovulatory LH receptor agonists plus PMA, rather than activating PKCs, lead to the inactivation at least of the PMA-sensitive PKC isoforms that phosphorylate MARCKS. Our in vivo results support this hypothesis, based on the ability of hCG to promote reduced MARCKS phosphorylation at 1 and 8 h post-hCG injection to PMSG-primed rats. The functions regulated by the partially activated PKCs in preovulatory granulosa cells before receiving the surge of LH that induces follicle ovulation and luteinization are not known.

Our results therefore allow us to conclude that the phosphorylation of MEK, p42/44 MAPK, CREB, histone H3, and p38 MAPK in response to LH receptor activation are not dependent on PKC activation. Moreover, our results show that activation of PKCs, as detected by the classic translocation assay or by increased MARCKS or PKC {delta} phosphorylation, is not a consequence of LH receptor activation. Future studies are now required to determine how LH receptor activation promotes the apparent inactivation of the PKC(s) that phosphorylates MARCKS as well as the function of partially activated PKC {alpha}, ßII, {delta}, and {epsilon} in granulosa cells before initiation of the ovulatory response.


   Acknowledgments
 


   Footnotes
 
This work was supported by NIH Grants P01-HD-21921 (to M.H.D.) and R01-HD/DK-38060 (to M.H.D.) and by U.S. Army USAMRMC Grant DAMD17-00-1-0386 (to L.M.S.). Preliminary results were presented at the 82nd Annual Meeting of The Endocrine Society, Toronto, Canada, 2000.

Abbreviations: CREB, cAMP response element-binding protein; DAG, diacylglycerol; GFX, GF109203X; hCG, human chorionic gonadotropin; IP3, inositol 1,4,5-triphosphate; MARCKS, myristolated alanine-rich C kinase substrate; MEK, MAPK kinase; PKA, protein kinase A; PKC, protein kinase C; PKI, protein kinase A inhibitor; PLC, phospholipase C; PMA, phorbol 12-myristate 13-acetate; PMSG, pregnant mare serum gonadotropin; PS, phosphaditylserine.

Received January 9, 2002.

Accepted for publication April 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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