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Endocrinology Vol. 143, No. 3 877-888
Copyright © 2002 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Requirement for ERK1/2 Activation in the Regulation of Progesterone Production in Human Granulosa-Lutein Cells Is Stimulus Specific

D. A. Dewi, D. R. E. Abayasekara and C. P. D. Wheeler-Jones

Department of Veterinary Basic Sciences, Royal Veterinary College, London, United Kingdom NW1 0TU

Address all correspondence and requests for reprints to: Dr. Caroline P. D. Wheeler-Jones, Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London, United Kingdom NW1 0TU. E-mail: . cwheeler{at}rvc.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was conducted to determine whether the ERK1/2 family of MAPKs can be modulated by physiological regulators of the human corpus luteum, and whether this activation is important for progesterone secretion in human granulosa-lutein (hGL) cells. Human LH (hLH), hCG, and agents that indirectly elevate cAMP [cholera toxin, forskolin, (Bu)2cAMP], time- and dose-dependently activated ERK1/2 in hGL cells. ERK1/2 activation was reduced by preincubation with PKA inhibitors, including myristoylated PKI, suggesting that cAMP mediates ERK1/2 activation. Two structurally distinct inhibitors of MAPK kinase (MEK), PD 98059 and U 0126, abrogated hLH/hCG-induced ERK1/2 activation, but had no effect on hLH-, hCG-, or 22R-hydroxycholesterol-stimulated progesterone secretion. In contrast, both inhibitors blocked cholera toxin-, forskolin-, and (Bu)2cAMP-induced ERK1/2 phosphorylation concomitant with a reduction in progesterone secretion. The known luteotropin, PGE2, promoted MEK- and cAMP-dependent activation of ERK1/2, and inhibitors of either MEK or PKA decreased PGE2-induced progesterone synthesis. Our findings demonstrate that the requirement for ERK1/2 activation as a regulator of progesterone synthesis in hGL cells is stimulus dependent, and that the MEK inhibitor-sensitive step is distal to cAMP generation, but proximal to the conversion of cholesterol to pregnenolone.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE CORPUS LUTEUM (CL) is a transient endocrine organ formed from the ruptured follicle at ovulation. Its main function is to produce the steroid hormone, progesterone, under the influence of LH, a gonadotropin secreted by the anterior pituitary gland. The adenylyl cyclase/cAMP/PKA pathway has been widely accepted as the primary signaling cascade through which LH exerts its steroidogenic action. Thus, upon interaction with its Gs protein-coupled receptor, LH promotes cAMP elevation and increases progesterone production in a PKA-dependent manner (1, 2). The importance of PKA in steroidogenesis is further supported by the absence of steroid synthesis in YI adrenocortical tumor cell mutants lacking PKA activity (3, 4) and by pharmacological blockade of steroidogenesis with PKA inhibitors or the cAMP antagonist Rp diastereoisomer of adenosine 3',5'-cyclic phosphorothioate (RpcAMPS) (5). Once activated, PKA phosphorylates components of steroidogenic pathways, resulting in the synthesis of steroidogenic acute regulatory protein (StAR), an important labile steroidogenic protein. StAR plays a key role in acute progesterone production by facilitating the transport of cholesterol from the outer to inner mitochondrial membrane and hence its conversion to pregnenolone by the P450scc system (reviewed in Ref. 6).

Other pathways in addition to PKA have been implicated in regulating the steroidogenic actions of LH (reviewed in Refs. 7 and 8). Potentially important elements in gonadotropin-mediated signaling in the ovary include members of the MAPK family (ERK1/2) that are activated in response to gonadotropins in both porcine and rat granulosa cells (9, 10). MAPKs are serine-threonine kinases implicated mainly in the transduction of extracellular signals into nuclear signals that regulate gene expression. However, MAPKs are also capable of activating cytoplasmic substrates (e.g. cPLA2{alpha}) and are involved in regulating acute responses, including PG production. The best characterized of the MAPKs are ERK1 and ERK2, whose activation is thought to be critical for mitogenesis and cellular differentiation. MAPKs are activated by phosphorylation on threonine and tyrosine residues mediated by upstream, dual specificity kinases, MAPK kinases (MEKs), and these, in turn, are activated by MEK kinases, including Raf-1 and B-Raf. The molecular mechanisms by which G protein-coupled receptors (GPCRs) regulate ERK1/2 are poorly understood, but several intracellular signaling elements have been shown to modulate ERK1/2 activation in response to GPCR agonists, including PKC, PKA, and PI3K (reviewed in Refs. 11 and 12).

The mechanisms regulating activation of ERK1/2 by gonadotropins in luteal cells and the functional significance of their activation are unknown. Recent studies, however, have shown that inhibition of ERK1/2 activity can increase StAR protein expression in porcine granulosa cells (13, 14), suggesting that ERK1/2 may be involved in regulating progesterone production in the ovary. Hence, the principal aims of this study were to determine whether physiological regulators of luteal function modulate ERK1/2 in human granulosa-lutein (hGL) cells and to investigate the potential role of ERKs in mediating the steroidogenic action of LH in these cells. Our results show that ERK1/2 can be activated by modulators of luteal cell function and that activation is mediated in part through cAMP generation. The requirement for ERK1/2 activity in the regulation of progesterone production is stimulus dependent, and the MEK inhibitor-sensitive step lies distal to cAMP generation, but proximal to conversion of cholesterol to pregnenolone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Human LH (hLH; NIDDK hLH-B-SIAFP-2) and hCG (CR127) were gifts from A. F. Parlow of the NIDDK’s National Hormone and Pituitary Program (Bethesda, MD). Forskolin, PGE2, (Bu)2cAMP, 22-hydroxycholesterol, cholera toxin (CT), leupeptin, 4-(2-aminoethyl)benzene sulfonyl fluoride, DMEM/F-12 nutrient mix, BSA, FBS, glutamine, penicillin-streptomycin, Rp-isomer of cAMP, and progesterone were purchased from Sigma (Poole, UK). Anti-active ERK1/2 antibody was obtained from Promega Corp. (Southampton, UK), the anti-ERK1/2 antibody was purchased from Santa Cruz Biotechnology, Inc. (Calne, Wiltshire, UK), and the phospho-specific MEK1/2 antibody was obtained from New England Biolabs, Inc. (Hitchin, UK). The goat antirabbit secondary antibody and the bicinchoninic acid assay were obtained from Pierce Chemical Co. (Chester, UK). The progesterone antibody was purchased from the Central Veterinary Laboratory (Reading, UK). [3H]Progesterone, [125I]cAMP-TME, and x-ray Hyperfilm were purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). cAMP antibody and standard, U 0126, PD 98059, and myristoylated PKI (myr PKI) were all obtained from Calbiochem (Nottingham, UK). Polyacrylamide (Protogel) and SDS tank buffer were purchased from National Diagnostics (Hull, UK). X-Ray developer and fixer were purchased from Kodak (London, UK). Other reagents used in these studies were obtained from Sigma (Poole, UK), Fisher (Loughborough, UK), or BDH (Poole, UK) at the equivalent of Analar grade.

Cell isolation and culture
Human granulosa cells were obtained from follicular aspirates of patients undergoing oocyte retrieval for assisted-conception at the London Women’s Clinic, Hallam Medical Center (London, UK), with informed patient consent (in accordance with the Declaration of Helsinki) and with approval of the local ethics committee. Cells were prepared using a 60% Percoll density gradient centrifugation as previously described (15). Briefly, the granulosa cell band (interface between aqueous and Percoll bands) was removed, and cells were washed in sterile PBS. The viable cell number was assessed using trypan blue (0.2%, vol/vol) exclusion. Cells obtained by this method were routinely found to be 80–90% viable and were approximately 90% steroidogenic as assessed by 3ß-hydroxysteroid dehydrogenase cytochemistry. The granulosa cells were cultured at a range of densities depending on the type of experimental procedure. For measurement of progesterone and cAMP, cells were cultured in 96-well plates at 104 cells/well, whereas for immunoblotting studies, cells were seeded in either individual p35 dishes or in six-well plates at 106 cells/well. Cell culture was performed at 37 C in a humidified CO2 incubator (95% O2/5% CO2). Cells were cultured for 3 d in DMEM/F-12 nutrient mix supplemented with 10% (vol/vol) FBS, 2 mM glutamine, and 1 IU penicillin-streptomycin with daily replacement of medium (16). Experiments were therefore performed in hGL cells that had assumed the granulosa-lutein cell phenotype during culture. Two hours before experimental treatments, the FBS in the medium was replaced with 0.1% (wt/vol) BSA.

Preparation of cell lysates
All incubations were carried out in serum-free DMEM supplemented with 0.1% (wt/vol) BSA. After treatment, the medium was aspirated from the dish, and the cells were rinsed with ice-cold PBS (pH 7.5) containing 200 µM Na3VO4. The PBS was immediately aspirated to dryness, and the cells were lysed in a buffer comprising 63.5 mM Tris-HCl (pH 6.8), 10% (vol/vol) glycerol, 2% (wt/vol) SDS, 1 mM Na3VO4, 1 mM 4-(2-aminoethyl)benzene sulfonyl fluoride, and 50 µg/ml leupeptin. The dishes were then incubated on ice for 10 min, and the monolayers were scraped into 1.5-ml microfuge tubes. The protein content of cell lysates was quantified using the bicinchoninic acid method according to the manufacturer’s instructions (Pierce Chemical Co.). Before gel electrophoresis, bromophenol blue and ß-mercaptoethanol were added to the lysates to give final concentrations of 5% (vol/vol) and 0.02% (wt/vol), respectively.

Immunoblotting
Proteins within aliquots of whole cell lysates (75 µg/lane) were separated using 10% PAGE and transferred to an Immobilon polyvinylidene difluoride membrane using a semidry Western blotting apparatus (Bio-Rad Laboratories, Inc., Hemel Hempstead, UK). Nonspecific antibody binding was blocked by incubation of membranes in 50 mM Tris, 150 M NaCl, and 0.02% (vol/vol) Tween 20, pH 7.4 (TBST)/10% BSA for 2 h. Membranes were subsequently incubated with the appropriate primary antibody in TBST/10% BSA overnight. After washing (six times, 10 min each time) with TBST, membranes were incubated with goat antirabbit peroxidase-conjugated IgG in TBST/0.2% BSA for 1 h, followed by further washes in TBST (8 times, 10 min each time). Immunoreactive proteins were visualized by ECL according to the manufacturer’s instructions (Amersham Pharmacia Biotech). Where indicated, densitometric analysis of immunoblots was performed using a Gel Doc 1000 system and Molecular Analyst software (Bio-Rad Laboratories, Inc.).

Measurement of progesterone secretion and cAMP formation
Human GL cells in 96-well tissue culture plates were treated as described in the figure legends. Reactions were terminated by the addition of 3 M perchloric acid (final concentration, 0.107 M), followed by freezing at -20 C. Before RIA the medium was neutralized by the addition of 2.16 M K3PO4 to give a final concentration of 0.154 M. Progesterone (17) and cAMP (18) RIAs were carried out on neutralized samples of medium as previously described. The intra- and interassay coefficients of variation for the progesterone RIA were 10% and 10.5%, respectively; those for the cAMP RIA were 13.9% and 14.4%, respectively.

Statistical analyses
Each experiment was performed on cells from two to five individual patients. For the immunoblotting studies, representative immunoblots are shown, and where indicated, a quantitative assessment of phosphorylation was carried out by densitometric analysis of immunoblots from similar experiments. For studies involving measurement of progesterone, each treatment was performed in quadruplicate for cells from each patient. Before pooling the results from at least three different patients, the result from each individual patient was expressed as a percentage of the control value. The combined results from at least three different patients were then expressed as the mean ± SEM. Statistical analyses of the data were performed by ANOVA, followed by Dunnett’s test, or by unpaired t test, as appropriate, using a PRISM software package (GraphPad, Inc., San Diego, CA). P <= 0.05 was accepted as significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gonadotropins activate ERK1/2 in a time- and concentration-dependent manner
Activation of ERK1/2 was assessed by immunoblotting with a phospho-specific, anti-active ERK antibody that detects dually phosphorylated ERK1 and ERK2. Although ERK activation was not measured directly, the use of this antibody is a widely accepted method for indirect assessment of ERK activity. Cells were incubated with either hLH (100 ng/ml) or hCG (100 ng/ml) for up to 3 h. Active ERK1/2 was present under basal conditions, and the phosphorylation of ERK1/2 increased in a time-dependent manner after exposure to gonadotropin. The presence of ERK in whole cell lysates of hGL cells was confirmed by immunoblotting with an antibody that recognizes total ERK1 (p44MAPK) and ERK2 (p42MAPK; Fig. 1BGo). ERK1/2 was maximally stimulated after 5–10 min of incubation with either hLH (Fig. 1Go, A and C) or hCG (Fig. 1DGo). This activation was maintained for up to 30 min, then started to decline after 30–60 min. Both hLH and hCG activated ERK1/2 in a concentration-dependent manner, with maximum stimulation achieved at 100 ng/ml (Fig. 1EGo).



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  		Figure 1. Effects of LH and hCG on activation of ERK1/2 in hGL cells. hGL cells were treated with 100 ng/ml hLH or hCG for the times indicated (A, B, and D). Alternatively, cells were exposed to increasing concentrations of hLH or hCG for 20 min (E). Aliquots of whole cell lysates were subjected to SDS-PAGE and immunoblotting using antibodies recognizing either active, dually phosphorylated ERK1/2 (A, D, and E) or total ERK1/2 (T-ERK; B). Immunoreactive bands were visualized by ECL. C, Densitometric analysis of three individual experiments represented by the blots shown in A and B (mean ± SEM; n = 3). Immunoblots shown in D and E are each representative of results obtained from three individual patients.

 
LH-induced MEK1/2 activation correlates with ERK1/2 activation
We investigated whether hLH also activates MEK and whether the kinetics of activation correlated with those of ERK1/2 activation. Immunoblotting studies using an antibody that recognizes active MEK1/2 showed that MEK is basally active and that its phosphorylation increased within 1 min of exposure to hLH (Fig. 2Go, A and C). Maximal activation occurred between 3 and 10 min and decreased after 1 h of hLH exposure, a time course consistent with that of ERK1/2 phosphorylation (Fig. 1Go). Reprobing the membrane with anti-active ERK1/2 antibody confirmed that ERK1/2 was phosphorylated with similar kinetics as MEK (Fig. 2BGo). Studies over an extended time course showed that both MEK (Fig. 2Go, D and F) and ERK1/2 (Fig. 2Go, E and F) were phosphorylated at early time points and showed a sustained activation at 4 and 24 h compared with time-matched controls.



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  		Figure 2. LH activates MEK and ERK1/2 in a time- and dose-dependent manner. hGL cells were treated with hLH for up to 1 h (A and B) or for up to 24 h (C and D). Cell lysates were initially analyzed by immunoblotting with a phospho-MEK antibody (A and D). Blots were then stripped and reprobed with an anti-active ERK1/2 antibody (B and E). Immunoreactive bands corresponding to active, phosphorylated MEK and ERK1/2 are indicated. C and F, Densitometric analysis of two individual experiments from each series (mean ± SD).

 
Effects of gonadotropins, CT, and forskolin on cAMP generation
It is generally accepted that interaction of LH with its receptor leads to activation of the adenylate cyclase-cAMP-PKA pathway. To confirm that steroidogenic agents elevate cAMP in hGL cells, cells were exposed to various concentrations of hLH for times ranging between 30 min and 48 h. cAMP levels increased in a time- and concentration-dependent manner upon exposure to hLH (Fig. 3Go, A and C). A significant increase in cAMP production was achieved at 100 ng/ml hLH, exceeded basal levels after 30 min, and continued to increase up to 4 h (mean ± SEM, 1498.3 ± 410.4% of basal; n = 3). After 10 h of incubation with hLH, cAMP had declined to basal levels. hLH did not cause a significant elevation in cAMP before 30 min, whereas forskolin promoted a rapid increase in cAMP that was evident as early as 5 min after exposure (Fig. 3BGo). Another gonadotropin, hCG (100 ng/ml), also elevated cAMP production, and this was mimicked by agents that elevate cAMP in a receptor-independent manner, including forskolin (10-5 M) and CT (50 µg/ml; Fig. 3CGo).



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  		Figure 3. Effects of steroidogenic agents on cAMP generation. hGL cells in 96-well tissue culture trays were exposed to hLH (100 ng/ml) or forskolin (10-5 M) for various times (A and B), to increasing concentrations of hLH for 4 h (C, left panel), or to forskolin (10-5 M), hCG (100 ng/ml), or CT (50 µg/ml) for 4 h (C, right panel). Reactions were terminated by the addition of 3 M perchloric acid, and cAMP levels were quantified by RIA as described in Materials and Methods. Data in each panel are derived from experiments on cells from three individual patients. For the time-course experiments results are expressed as a mean percentage of the control value at time zero ± SEM (n = 3). Other data are given as a mean percentage of the basal value ± SEM (n = 3). Basal cAMP levels ranged between 20–50 pmol/105 cells. *, P < 0.05; **, P < 0.01 (vs. basal).

 
cAMP-elevating agents activate ERK1/2
To evaluate the role of the adenylate cyclase-cAMP-PKA pathway in mediating ERK1/2 activation, we initially examined the effects of cAMP-elevating agents on ERK1/2 activation. Forskolin and (Bu)2cAMP both activated ERK1/2 in a concentration-dependent manner, with maximum activation achieved with 10-5 M and 5 mM, respectively (data not shown). At these concentrations, both forskolin and (Bu)2cAMP mimicked the actions of gonadotropins, in that both reagents caused a time-dependent activation of ERK1/2 (Fig. 4Go). Activation of both ERK1 and ERK2 was detected after 5 min, was maintained for up to 30 min, and declined toward basal after 1 h.



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  		Figure 4. Forskolin and (Bu)2cAMP activate ERK1/2. hGL cells in 35-mm dishes were treated with either forskolin (A) or (Bu)2cAMP (C) for the times indicated. The resulting blots were probed with an anti-active ERK1/2 antibody. Immunoreactive bands corresponding to active ERK1/2 are indicated. Data in each panel are representative of two similar experiments. B and D, Combined densitometric analysis of two individual experiments with each cAMP-elevating agent (mean ± SD).

 
Effects of PKA inhibitors on ERK1/2 activation
Parallel studies were carried out to further investigate the role of cAMP in mediating ERK1/2 activation. In this group of experiments the effects of the PKA inhibitor, myr PKI (19), on ERK1/2 activation were examined. At a concentration (100 µM) that completely abrogated hLH- or forskolin-induced progesterone production (Table 1Go), myr PKI had no effect on basal ERK1/2 activation (Fig. 5CGo), but partially reduced hLH-induced ERK1/2 activation (Fig. 5Go, A and C) and completely inhibited ERK1/2 activation by forskolin (Fig. 5Go, B and C). However, neither hLH- nor forskolin-induced ERK1/2 activation was affected by the cAMP antagonist RpcAMPS (results not shown).


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  		Table 1. Effects of PKA inhibitors on progesterone production

 


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  		Figure 5. PKA inhibitors modulate LH and forskolin-induced ERK1/2 activation. hGL cells were preexposed to myr PKI (100 µM) or vehicle alone for 30 min. Cells were subsequently challenged with either hLH (A) or forskolin (B) for a further 10 min in the continued presence of myr PKI. Immunoreactive bands corresponding to active ERK1/2 are indicated. Results are representative of three experiments. C, Densitometric analysis of three individual experiments (mean ± SEM; n = 3). *, P < 0.05 vs. LH alone; **, P < 0.01 vs. forskolin alone.

 
Effects of MEK inhibitors on agonist-stimulated ERK1/2 activation and progesterone secretion
The robust activation of ERK1/2 by hLH and hCG led us to further investigate the potential role of this activation in gonadotropin-mediated progesterone production. These studies employed two structurally distinct noncompetitive MEK inhibitors, U 0126 (20) and PD 98059 (21). Experiments in which the effects of these inhibitors on hLH- and forskolin-induced cAMP elevation were examined showed that neither PD 98059 nor U 0126 modified hLH- or forskolin-induced cAMP generation (Fig. 6Go). hLH (100 ng/ml) stimulated progesterone production by hGL cells in a time- and concentration-dependent manner (Fig. 7Go, A and B). Progesterone accumulation was significantly elevated over basal levels after 4 h of exposure to 100 ng/ml hLH (mean ± SEM, 187.7 ± 7.5% of basal; n = 3) and continued to increase up to 48 h. Similarly, hCG (100 ng/ml) and cAMP-elevating agents [forskolin (10-5 M), (Bu)2cAMP (5 mM), and CT (50 µg/ml)] significantly increased progesterone accumulation after 4 h (Fig. 7CGo). As shown in Fig. 8Go, preincubation with either of the MEK inhibitors caused a concentration-dependent reduction of hLH- and hCG-induced ERK1/2 activation, with U 0126 having the most potent effect. Both inhibitors also blocked ERK1/2 activation in response to forskolin (Fig. 8Go, C and D), CT, or (Bu)2cAMP (data not shown). Acutely (4 h), inhibition of MEK did not modify either basal (data not shown) or hLH/hCG-stimulated progesterone production (Fig. 9AGo). Similar results were also obtained when submaximal doses of hLH were used (Fig. 10Go). Accumulation of progesterone over a chronic 24-h exposure to gonadotropins was similarly unaffected by pharmacological blockade of MEK (results not shown). In marked contrast to the results obtained with hLH and hCG, preexposure to either PD 98059 or U 0126 decreased progesterone accumulation over 4 h in response to CT, forskolin, or (Bu)2 cAMP (Fig. 9BGo). Similar effects on progesterone secretion caused by a 24-h exposure to cAMP-elevating agents were also observed (results not shown). Progesterone secretion in response to 22R-hydroxycholesterol (ROHC) was not affected by exposure to either PD 98059 or U 0126 (Fig. 9AGo).



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  		Figure 6. Inhibition of MEK does not affect LH- or forskolin-induced cAMP formation. hGL cells were preincubated with vehicle, PD 98059 (50 µM), or U 0126 (10 µM) for 30 min and subsequently exposed to hLH (100 ng/ml) or forskolin (10-5 M) for 4 h in the continued presence or absence of inhibitor. cAMP formation was quantified by RIA as described in Materials and Methods. Results are given as the mean ± SEM. Basal cAMP generation ranged between 15 and 25 pmol/105 cells, and stimulated cAMP generation ranged between 115 and 530 pmol/105 cells. Data are from experiments performed in cells from three individual patients.

 


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  		Figure 7. Effects of steroidogenic agents on progesterone secretion. hGL cells were incubated with hLH for the times indicated (A), with increasing concentrations of hLH (B), or with forskolin (10-5 M), hCG (100 ng/ml), or CT (50 µg/ml) for 4 h (C). Progesterone in the spent medium was quantified by RIA as described in Materials and Methods. For the time-course experiments, results are expressed as a mean percentage of the control value at time zero ± SEM (n = 3). Other data are given as a mean percentage of the basal value ± SEM (n = 3). Basal progesterone accumulation at time zero ranged between 175 and 410 pmol/105 cells, and that over 4 h ranged between 430 and 1585 pmol/105 cells. A: *, P < 0.05; **, P < 0.01 (relative to basal at the matched time point). B and C: **, P < 0.01 vs. basal.

 


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  		Figure 8. Effects of MEK inhibitors on ERK1/2 activation. hGL cells were treated with the indicated concentration of PD 98059 or U 0126 for 30 min and then exposed to hLH (100 ng/ml; A and B) or forskolin (fsk; 10-5 M; C and D) for a further 10 min in the continued presence of inhibitor. Whole cell lysates were analyzed by immunoblotting with a phospho-specific ERK1/2 antibody. Immunoreactive bands corresponding to active ERK1/2 are indicated. B and D, Combined densitometric analysis of phosphorylation in three (B; mean ± SEM) and two (D; mean ± SD) individual experiments. *, P < 0.05 vs. untreated control; {dagger}, P < 0.05 vs. agonist alone.

 


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  		Figure 9. Effects of MEK inhibitors on progesterone production. hGL cells were incubated with either PD 98059 (50 µM) or U 0126 (20 µM) for 30 min. Cells were then challenged with hLH (100 ng/ml), hCG (100 ng/ml), CT (50 µg/ml), forskolin (10-5 M), (Bu)2cAMP (5 mM), or ROHC (5 µM) for a further 4 h in the continued presence of inhibitor. Progesterone accumulation in the medium was quantified by RIA. Results are expressed as the mean percentage of stimulation with agonist alone ± SEM (n = 3). Basal accumulation over 4 h ranged between 55 and 500 pmol/10 5 cells, and stimulated accumulation ranged between 150 and 1500 pmol/105 cells. *, P < 0.05; **, P < 0.01 (relative to stimulation with agonist only).

 


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  		Figure 10. Effects of blocking MEK on progesterone secretion by varying concentrations of hLH. hGL cells were pretreated (30 min) with PD 98059 (50 µM), U 0126 (20 µM), or vehicle alone and then exposed to increasing concentrations of hLH in the absence or presence of inhibitor for 4 h. Progesterone in the medium was quantified by RIA. Data are representative of three separate experiments with similar results and are expressed as the mean percentage of the basal value ± SEM (n = 4). Basal accumulation (4 h) was between 155 and 630 pmol/105 cells. *, P < 0.05; **, P < 0.01 relative to basal.

 
Inhibiting MEK blocks PGE2-stimulated progesterone production
To determine whether the MEK independence of hLH/hCG-stimulated progesterone production is characteristic of all physiological luteal cell stimulants, we assessed the effects of PD 98059 and U 0126 on PGE2-mediated signaling and progesterone secretion. A 4-h exposure to PGE2 (10 µM) elevated cAMP by approximately 15-fold (mean ± SEM, 1498.3 ± 410.4% of basal; n = 3), and increased progesterone release by 1.5-fold (mean ± SEM, 154.1± 15.8% of basal; n = 3). PGE2 also promoted an early, time-dependent activation of ERK1/2 (Fig. 11AGo), which was attenuated by pretreatment with PD 98059, U 0126, or myr PKI (Fig. 11Go, B and C). Inhibition of either MEK or PKA reduced PGE2-stimulated progesterone production (Fig. 11DGo).



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  		Figure 11. Effects of MEK and PKA inhibitors on PGE2-stimulated ERK1/2 activation and progesterone production. hGL cells were exposed to PGE2 (10 µM) for the times indicated (A). Alternatively, cells were treated with PD 98059, U 0126, or myr PKI for 30 min and then challenged with PGE2 (10 µM) for a further 10 min (B and C) or for 4 h (D). Immunoreactive bands corresponding to active ERK1/2 are indicated (A and B). C, Combined densitometric analysis of phosphorylation in three individual experiments (mean ± SEM). Progesterone was quantified by RIA (D), and results are expressed as the mean percentage of stimulation with PGE2 ± SEM (n = 3). Progesterone accumulation ranged between 150 and 455 basally and between 275 and 955 pmol/105 cells in the presence of PGE2. *, P < 0.05; **, P < 0.01 (relative to stimulation with PGE2 alone).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LH is the principal physiological regulator of the primate CL and plays a critical role in the process of luteinization. It is a member of the glycoprotein hormone family and shares structural similarities with other members of the group, including FSH, CG, and TSH (22). LH and CG are similar in structure and bind to the same GPCR in the CL (23). The cell culture system used in our studies was selected as a readily available functional approximation to the luteinized granulosa cells of the CL. The fact that the granulosa cells employed in this and similar studies have been exposed to pharmacological doses of gonadotropins before isolation and culture might be expected to influence the in vitro output of steroid hormones. However, the contribution of this previous exposure to gonadotropins would be minimal after the 3-d culture period employed. The model of cultured hGL cells is a widely used in vitro system that is known to mirror many of the biochemical changes occurring when granulosa cells undergo functional luteinization in vivo. Hence, the biochemical changes reported herein are likely to be an accurate reflection of intracellular signaling events that regulate progesterone secretion by cells of the early human CL in vivo.

In the present studies, hLH and hCG rapidly increased ERK1/2 activation in hGL cells, a finding in agreement with earlier work in porcine granulosa cells (9) and a rat granulosa cell line (14). FSH also activates ERK in rat and porcine granulosa cells (9, 10). In contrast, Zhu et al. (24) found no consistency of ERK stimulation by FSH in porcine granulosa cells, a difference that may reflect the use of granulosa cells derived from follicles at different stages of follicular development. GnRH has also been shown to activate ERK in both hGL cells and an ovarian carcinoma cell line (25, 26).

LH-induced ERK activation closely correlated with the phosphorylation of its upstream activator, MEK1/2. Moreover, rapid activation was followed by a subsequent decline in activity that did not return to basal levels, but persisted for at least 24 h after exposure to gonadotropin, indicating sustained activation of MEK/ERK. The temporal organization of MAPK activation has been reported to play an important role in determining specific physiological outcomes depending on the cell type. In PC12 cells, for example, sustained activation of ERK1/2 leads to cellular differentiation, whereas transient activation leads to cell proliferation (27). In contrast, prolonged activation of ERK in fibroblasts also leads to cell proliferation (28). In the present studies, the early peak of MEK/ERK phosphorylation followed by a prolonged activation is consistent with a role for ERKs in the regulation of specific biological responses during luteinization, such as steroid production, differentiation, or PG synthesis.

We also investigated the mechanism by which gonadotropins promote ERK activation in hGL cells. Given the importance of the adenylate cyclase-cAMP-PKA pathway in LH signaling, we initially examined whether agents that directly increase intracellular cAMP activate ERK1/2. These agents activated ERK1/2 in a MEK-dependent manner and with kinetics similar to those observed in gonadotropin-stimulated cells. Similarly, the Gs protein activator, CT, also elicited MEK-dependent ERK1/2 activation in hGL cells. ERK activation in response to cAMP-elevating agents has previously been reported in immature rat granulosa cells (10), porcine granulosa cells (9), and YI adrenal cells (29, 30), whereas cAMP inhibits ERK activation in MA-10 tumor Leydig cells (31). Indeed, it is well documented that cAMP can exert various effects on ERK1/2 activation depending on the cell type. Thus, cAMP has been reported to activate ERK1/2 in some cell types (32, 33), but to negatively regulate ERK1/2 activation in others (31, 34, 35). This difference is thought to reflect the differential expression of Raf-1 vs. B-Raf (36).

Parallel studies using PKA inhibitors provided further support for the involvement of the PKA pathway in mediating gonadotropin-induced ERK1/2 activation in hGL cells. Myr PKI is a synthetic peptide derived from the endogenous PKA inhibitor that contains a pseudosubstrate region between residues 14 and 22 and hence binds to and inactivates the catalytic subunit of PKA (19). The partial inhibition of hLH-induced ERK1/2 activation by myr PKI implicates both PKA-dependent and -independent pathways in this response, whereas the complete abolition of forskolin-stimulated ERK activation by this inhibitor indicates an absolute requirement for cAMP elevation. The rapid elevation in cAMP observed in forskolin-stimulated cells coincided with ERK activation, further suggesting that cAMP is an important upstream regulator of ERK in response to forskolin. In contrast, cAMP was not significantly elevated until 30 min after exposure to gonadotropins, suggesting that very early changes in cAMP are likely to be minimal and that hLH-induced ERK activation is therefore less dependent upon cAMP elevation than is forskolin-induced ERK. A role for cAMP is further supported by the finding that neither hLH- nor forskolin-stimulated cAMP generation was affected by blocking MEK, suggesting that cAMP lies upstream of ERK1/2. These findings are in agreement with recent work in immature rat granulosa cells showing cAMP-dependent activation of ERK1/2 by FSH (10). In contrast, TSH activates ERK1/2 through cAMP-independent pathways in astrocytes (37) and human thyroid follicles (38). The partial dependence of ERK activation on cAMP in hGL cells suggests that other pathways may be recruited by gonadotropins to activate ERK1/2 (11). Similarly, the involvement of cAMP-binding proteins other than PKA in mediating ERK1/2 activation by gonadotropins cannot be ruled out (39).

We also examined the potential functional role of ERK1/2 in the ovary. Evidence drawn from studies in granulosa cells of nonprimate species supports a role for ERKs in growth factor-induced mitogenesis (40, 41, 42, 43). However, as hGL cells do not proliferate over the time course of our studies, ERK activation is unlikely to regulate the proliferative state of these cells. ERKs may therefore be important for regulating other processes in luteal cells, including hypertrophy, steroidogenesis, or secretion of auto/paracrine factors. We employed pharmacological inhibitors of MEK, PD 98059 and U0126, to determine the significance of MEK/ERK1/2 activation for progesterone production. Our results demonstrated that under conditions where ERK1/2 activation was abolished, blocking MEK did not modify hLH- or hCG-stimulated progesterone production, indicating that ERK1/2 is not required for gonadotropin-stimulated progesterone synthesis. In marked contrast, inhibition of MEK caused a decrease in CT-, forskolin-, and (Bu)2cAMP-stimulated progesterone production, suggesting that ERK1/2 plays a role in regulating receptor-independent, but not gonadotropin-dependent, progesterone generation. It should be noted, however, that hLH and hCG are physiological regulators of the CL, whereas forskolin, CT, and (Bu)2cAMP are pharmacological agents that elevate intracellular cAMP. Differences in the absolute levels of cAMP generated in response to gonadotropins or receptor-independent agents cannot explain this phenomenon, as LH and forskolin induce equivalent increases in cAMP. Differential cellular localization of pools of cAMP (44) or ERK (36) in response to gonadotropins vs. cAMP-elevating agents may explain these results.

Previous reports have suggested a possible link between ERK1/2 activation and steroidogenesis. In Y1-adrenal cells, forskolin-induced pregnenolone synthesis is decreased by exposure to MEK inhibitors (30). However, it is still unclear whether this effect is physiologically relevant, as ACTH, an agonist known to stimulate adrenal steroidogenesis via cAMP, apparently does not use the adenylate cyclase-cAMP-PKA pathway to activate ERK1/2 in Y1 cells (45). In contrast, PD 98059 potentiates FSH-mediated induction of StAR mRNA in porcine granulosa cells (13) and rat granulosa cell lines (14) and enhances steroid synthesis (14). Similarly, it has been reported that PD 98059 can restore LH-induced progesterone production inhibited by platelet-derived growth factor in porcine thecal cells (46), and the inhibition of progesterone production by GnRH in hGL cells can also be reversed by inhibition of MEK (26). Therefore, it appears that ERK can play differential roles in progesterone production depending on the cell type/cell stage as well as on the stimulus employed.

Having demonstrated that ERK1/2 is active and is necessary for receptor-independent, but not gonadotropin-dependent, progesterone production, we asked whether MEK-independent progesterone production was a specific characteristic of agonists using the LH/CG receptor or was also observed with other steroidogenic agents whose interaction with their receptors raises cAMP levels. PGE2 stimulates progesterone synthesis in the CL (47), and its interaction with the EP2 receptor raises cAMP levels in hGL cells (48). This prostanoid caused an early and sustained activation of ERK1/2 in hGL cells, which was reduced by blocking PKA, suggesting a dependence of ERK1/2 activation on cAMP elevation. Inhibition of MEK abrogated PGE2-driven progesterone secretion, indicating that ERK1/2 activation is necessary for PGE2-stimulated progesterone secretion. These findings hence suggest that PGE2 exerts its steroidogenic action through a mechanism distinct from that employed by hLH and hCG, although all of these agents elevate cAMP in hGL cells.

Finally, to further define the MEK inhibitor-sensitive step, the effects of MEK inhibitors on ROHC-stimulated progesterone production were examined. ROHC is a hydrophilic cholesterol that is membrane permeant and serves as a substrate for P450scc, the enzyme that converts cholesterol to pregnenolone (49). ROHC-stimulated progesterone secretion was not affected by blocking MEK, indicating that MEK inhibitors do not directly affect the activities of steroidogenic enzymes, that ERK1/2 activation is not required for steroidogenic enzyme activity, and that the MEK inhibitor-sensitive step is proximal to the conversion of cholesterol to pregnenolone.

Although ERK1/2 activation clearly plays a role in PGE2-stimulated progesterone secretion, it is not required for steroidogenesis mediated by gonadotropins, and the physiological significance of gonadotropin-induced ERK1/2 activation remains to be determined. ERK1/2 activation does not appear to be required for cell rounding in response to gonadotropins (Dewi, D. A., D. R. E. Abayasekara, and C. P. D. Wheeler-Jones, unpublished observations). However, gonadotropin-induced ERK1/2 activation may play a role in the maintenance of human luteal cell function, as enhanced ERK1/2 activation was reported in climacteric women whose regressing CLs exhibited persistence of luteal cell characteristics (50). ERK1/2 may also be involved in the process of luteinization of hGL cells, including the secretion of autocrine factors such as PGE2 and PGF2{alpha}.

In summary, we have shown for the first time that the requirement for ERK1/2 activity in the regulation of progesterone production in human granulosa-lutein cells is stimulus dependent and that the adenylate cyclase-cAMP-PKA pathway mediates activation of the ERK1/2 cascade. We have also demonstrated that the site of action of ERK1/2 in the regulation of progesterone production is distal to cAMP generation, but proximal to conversion of cholesterol to pregnenolone. We are currently examining whether the stimulus-dependent role of ERK1/2 in steroidogenesis reflects differential translocation of discrete pools of ERK1/2 to distinct subcellular locations. The present studies together with previous reports from a number of other laboratories (9, 10, 25, 26, 40, 41, 42) strongly support a central role for ERK1/2 in ovarian cell signaling.


   Acknowledgments
 
We are grateful to Mr. Stanley Okolo and colleagues at the London Women’s Clinic, Hallam Medical Center (United Kingdom), for the provision of follicular aspirates.


   Footnotes
 
Abbreviations: CL, Corpus luteum; CT, cholera toxin; GPCR, G protein-coupled receptor; hGL, human granulosa-lutein; hLH, human LH; MEK, MAPK kinase; myr PKI, myristoylated PKI; ROHC, 22R-hydroxycholesterol; Rp-cAMPS, Rp diastereoisomer of adenosine 3',5'-cyclic phosphorothioate; StAR, steroidogenic acute regulatory protein; TBST, 50 mM Tris, 150 M NaCl, and 0.02% (vol/vol) Tween 20, pH 7.4.

Received June 26, 2001.

Accepted for publication November 6, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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