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Endocrinology Vol. 142, No. 4 1554-1560
Copyright © 2001 by The Endocrine Society

ARTICLES

Adenosine Triphosphate Activates Mitogen-Activated Protein Kinase in Human Granulosa-Luteal Cells1

Chen-Jei Tai, Sung Keun Kang2, Chii-Ruey Tzeng and Peter C. K. Leung3

Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5; and Taipei Medical College Hospital (C.-R.T.), Taipei, Taiwan

Address all correspondence and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, Room 2H30-4490, Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ATP has been shown to activate the phospholipase C/diacylglycerol/protein kinase C (PKC) pathway. However, little is known about the downstream signaling events. The present study was designed to examine the effect of ATP on activation of the mitogen-activated protein kinase (MAPK) signaling pathway and its physiological role in human granulosa-luteal cells. Western blot analysis, using a monoclonal antibody that detected the phosphorylated forms of extracellular signal-regulated kinase-1 and -2 (p42mapk and p44 mapk, respectively), demonstrated that ATP activated MAPK in a dose- and time-dependent manner. Treatment of the cells with suramin (a P2 purinoceptor antagonist), neomycin (a phospholipase C inhibitor), staurosporin (a PKC inhibitor), or PD98059 (an MAPK/ERK kinase inhibitor) significantly attenuated the ATP-induced activation of MAPK. In contrast, ATP-induced MAPK activation was not significantly affected by pertussis toxin (a Gi inhibitor). To examine the role of Gs protein, the intracellular cAMP level was determined after treatment with ATP or hCG. No significant elevation of intracellular cAMP was noted after ATP treatment. To determine the role of MAPK in steroidogenesis, human granulosa-luteal cells were treated with ATP, hCG, or ATP plus hCG in the presence or absence of PD98059. RIA revealed that ATP alone did not significantly affect the basal progesterone concentration. However, hCG-induced progesterone production was reduced by ATP treatment. PD98059 reversed the inhibitory effect of ATP on hCG-induced progesterone production. To our knowledge, this is the first demonstration of ATP-induced activation of the MAPK signaling pathway in the human ovary. These results support the idea that the MAPK signaling pathway is involved in mediating ATP actions in the human ovary.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXTRACELLULAR ATP is coreleased with neurotransmitter granules from nerve endings by exocytosis (1). After binding to a G protein-coupled P2 purinoceptor, ATP activates phosphoinositides hydrolysis, generating diacylglycerol and inositol 1,4,5-trisphosphate, which stimulate protein kinase C (PKC) and cytosolic calcium mobilization, respectively (2, 3). Thereafter, ATP may participate in various types of physiological responses, including secretion, membrane potential, cell proliferation, platelet aggregation, neurotransmission, cardiac function, and muscle contraction (3, 4). Considering that the ovary is a well innervated organ, it is tempting to speculate that the coreleased ATP from nerve endings may play a role in regulating ovarian function. We reported previously the expression of P2U purinoceptor in human granulosa-luteal cells (hGLCs) (5), further supporting a physiological role of ATP in the human ovary.

Mitogen-activated protein (MAP) kinases (MAPKs) are a group of serine-threonine kinases involved in converting extracellular stimulus into intracellular signals. Extracellular signal-regulated kinases (ERKs), one of the MAPK subfamilies, have been shown to be activated by extracellular agonists such as cytokines, growth factors and neurotransmitters (6, 7). It is believed that two classes of cell surface receptors, G protein-coupled receptor and receptor tyrosine kinases, are associated with the activation of MAPKs (8, 9, 10). When activated, ERK1 and ERK2 (also known as p42mapk and p44 mapk, respectively), phosphorylate a variety of substrates, including transcription factors, which have been implicated in the control of cell proliferation and differentiation (11, 12, 13).

The demonstration of P2U purinoceptor in hGLCs highlights the significance of ATP in regulating ovarian function, but little is known about the signaling events and cellular responses subsequent to the binding of ATP to its receptor in the human ovary. Activation of P2 purinoceptor has been shown to increase MAPK activity (14). However, the role of MAPK in ovarian cells is poorly understood. In the present study the signaling cascade proximal to MAPK activation subsequent to ATP exposure was determined in hGLCs. In addition, the functional role of activated MAPK after ATP treatment was studied.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and materials
ATP, suramin, pertussis toxin (PTX), neomycin, staurosporin, and hCG were obtained from Sigma (St. Louis, MO). PD98059, an MAPK/ERK kinase (MEK) inhibitor, was purchased from New England Biolabs, Inc. (Beverly, MA). DMEM, penicillin-streptomycin, and FBS were obtained from Life Technologies, Inc. (Burlington, Canada). Staurosporin and PD98059 were dissolved in dimethyl sulfoxide as suggested by manufacturers.

hGLC culture
hGLCs were collected from patients undergoing in vitro fertilization treatment who ranged in age from 23–43 yr. Forty-nine percent had severe male factor infertility, and the remainder had various female factors or long-standing unexplained infertility. Ovarian stimulation entailed a long luteal phase down-regulation protocol for women under 40 yr or a follicular phase flare protocol for women over 40 yr, as previously described (15). The use of hGLCs was approved by University of British Columbia clinical screening committee for research and other studies involving human subjects. Granulosa cells were separated from red blood cells in follicular aspirates by centrifugation through Ficoll-Paque, washed, and suspended in DMEM containing 100 U penicillin G/ml, 100 µg streptomycin/ml, and 10% FBS as described previously (5). The cells were plated at a density of approximately 150,000 cells in 35-mm culture dishes. Cells were incubated at 37 C under a water-saturated atmosphere of 5% CO2 in air for 3 days.

Treatments
hGLCs were incubated in serum-free medium for 4 h before treatment. To examine the dose-response relationship, hGLCs were treated with increasing concentrations of ATP (100 nM, 1 µM, 10 µM, or 100 µM) for 5 min. For time-course experiments, hGLCs were treated with 10 µM ATP for 1, 5, 10, or 20 min.

To determine the intracellular signaling pathway, hGLCs were treated with suramin (300 µM; an inhibitor of P2 purinergic receptor), PTX (200 ng/ml; a Gi inhibitor), neomycin [10 mM; a phospholipase C (PLC) inhibitor], staurosporin (1 µM; a PKC inhibitor), or PD98059 (50 µM; a MEK inhibitor) in the presence or absence of 10 µM ATP. hGLCs were pretreated with suramin for 15 min, with PTX for 1 h, with neomycin for 15 min, with staurosporin for 15 min, and with PD98059 for 1 h before ATP treatment. The cells were collected 5 min after ATP exposure.

Western blot analysis
The hGLCs were washed with ice-cold PBS and lysed with 100 µl cell lysis buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1.0 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 100 µg/ml aprotinin] at 4 C for 30 min. The cell lysate was centrifuged at 10,000 x g for 5 min, and the supernatant was collected for Western blot analysis. The amount of protein was quantified using a protein assay kit (Bio-Rad Laboratories, Inc., Richmond, CA) following the manufacturer’s protocol. Aliquots (30 µg) were subjected to 10% SDS-PAGE under reducing conditions as previously described (16). The proteins were then electrophoretically transferred from the gels onto nitrocellulose membranes (Amersham Pharmacia Biotech, Oakville, Canada) according to the procedures of Towbin et al. (17). These nitrocellulose membranes were probed with a mouse monoclonal antibody directed against the phosphorylated forms of ERK1 and ERK2 (P-MAPK, p42mapk and p44 mapk, respectively) at 4 C for 16 h. Alternatively, the membranes were probed with a rabbit polyclonal antibody for p42/p44 MAPK, which detected total MAPK (T-MAPK) levels (New England Biolabs, Inc., Beverly, MA). After washing, the membranes were incubated with HRP-conjugated goat-antimouse secondary antibody, and the signal was visualized using ECL system (Amersham Pharmacia Biotech) followed by autoradiography. The autoradiograms were quantified using a laser densitometer (Bio-Rad Laboratories, Inc., model 620, Video Densitometer).

MAPK assay
To measure MAPK activity, a nonradioactive method was used (p44/42 MAP Kinase Assay Kit, New England Biolabs, Inc.). Briefly, active MAPK of cell lysate (200 µg) from hGLCs treated with 10 µM ATP for 5 min was selectively immunoprecipitated with an immobilized monoclonal antibody to phospho-p44/42 MAP kinase. For a positive control, active MAPK (provided by the manufacturer) was added to the control cell extract. The resulting precipitate was incubated with an Elk-1 fusion protein in the presence of ATP, which allowed immunoprecipitated active MAPK to phosphorylate Elk-1. Phosphorylated Elk-1 was detected by Western blot using a phospho-Elk-1 antibody.

RIA for intracellular cAMP
hGLCs (2 x 105 cells) were plated onto 35-mm culture dishes and cultured for 4 days. The cells were then incubated in serum-free medium containing 0.1% BSA and 0.5 mM 3-isobutyl-1-methylxanthine (Sigma) for 30 min. To determine ATP- or hCG-induced intracellular cAMP accumulation, hGLCs were treated with ATP (10 µM) or hCG (1 IU/ml) for 20 min. Intracellular cAMP levels were measured using the [3H]cAMP assay system following the protocol provided by manufacturer (Amersham Pharmacia Biotech).

RIA for progesterone
After culture in DMEM with 10% FBS for 3 days, hGLCs were incubated in DMEM for 4 h before treatment for steroidogenesis experiments. To determine the role of MAPK in steroidogenesis, hGLCs were treated with ATP (10 µM), hCG (1 IU/ml), or ATP plus hCG in the presence or absence of PD98059 for 6 h.

Progesterone levels in the culture medium were measured by established RIA (18). Antiprogesterone antibody was provided by Dr. D. T. Armstrong (University of Western Ontario, London, Ontario, Canada). Briefly, samples were incubated with antibody and tracer, with a final concentration of 7000 cpm/ml [1,2,6,7,16,17-3H]progesterone (Amersham Pharmacia Biotech). After incubation for 16–24 h, a charcoal/dextran solution was added to remove unbound progesterone or tracer. Scintillation cocktail (Amersham Pharmacia Biotech) was added to each sample, and the vials were counted with a ß-counter (LKB Wallac, Inc., Turku, Finland). The cells in each dish were harvested for quantifying protein amount using a protein assay kit (Bio-Rad Laboratories, Inc.). Samples were assayed in triplicate, and progesterone concentrations were standardized against total protein contents.

hCG and MAPK in hGLCs
Gonadotropins have been demonstrated to activate MAPK in porcine granulosa cells (19). To examine the effect of hCG on MAPK activation, hGLCs were treated with 1 IU/ml hCG for 1, 5, 10, or 20 min, and cell lysates were collected for Western blot analysis. The effect of MAPK on hCG-stimulated progesterone production was studied by treating cells with 1 IU/ml hCG in the presence or absence of PD98059 for 6 h.

Statistical analysis
MAPK and progesterone levels were expressed as a relative ratio of basal levels. Intracellular cAMP levels were shown as picomoles per 2 x 105 cells. Independent replicates of experiments in this study were performed with cells from different patients. Data were represented as the mean ± SE. Statistical analysis was performed by one-way ANOVA, followed by Tukey’s multiple comparison test. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of ATP on MAPK activation
To demonstrate the ability of ATP in activating MAPK, hGLCs were treated with increasing concentrations (100 nM to 100 µM) of ATP for 5 min. For time-course analysis, the cells were treated with 10 µM ATP for varying time intervals (1–20 min). As shown in Fig. 1Go, ATP activated MAPK in hGLCs in a dose-dependent manner. A significant effect was observed at 1 µM, with a maximum effect noted at 10 µM, and there was no statistical difference between cells treated with 10 and 100 µM ATP. ATP was capable of rapidly inducing MAPK activity. A significant effect was seen within 5 min after treatment, and the activation of MAPK was sustained for at least 15 min (Fig. 2Go).



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  		Figure 1. The dose response of ATP on MAPK activation in hGLCs. hGLCs were treated with increasing concentrations of ATP (0, 100 nM, 1 µM, 10 µM, or 100 µM) for 5 min as described in Materials and Methods. The MAPKs were detected by Western blot analysis. The data are shown as the relative ratio to basal levels. Values are presented as the mean ± SE of three individual experiments. Statistical analysis was performed by one-way ANOVA, followed by Tukey test. Differences were considered significant at P < 0.05 (*).

 


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  		Figure 2. The time course of ATP on MAPK activation in hGLCs. hGLCs were treated with 10 µM ATP for 0, 1, 5, 10, or 20 min as described in Materials and Methods. MAPKs were detected by Western blot analysis. The data are shown as the relative ratio to basal levels. Values are presented as the mean ± SE of three individual experiments. Differences were considered significant at P < 0.05 (*).

 
MAPK activity
In vitro MAP kinase activity was measured using a p44/42 MAP kinase assay kit. As shown in Fig. 3Go, ATP significantly increased MAPK activity.



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  		Figure 3. MAPK activity in hGLCs measured using a MAPK assay kit. hGLCs were treated with 10 µM ATP for 5 min as described in Materials and Methods. Active p42 MAPK was included as a positive control. Values are presented as the mean ± SE of three individual experiments. Differences were considered significant at P < 0.05 (*).

 
P2 purinergic receptor and ATP-induced MAPK activation
P2U purinergic receptor has been demonstrated in hGLCs (5, 20). To investigate the involvement of P2 purinoceptor in ATP-induced MAPK activation, hGLCs were pretreated with 300 µM suramin, a P2 purinoceptor antagonist (21), for 15 min before the administration of ATP. As demonstrated in Fig. 4Go, ATP activated MAPK to about 230% of the basal (control) level. The cotreatment with suramin and ATP significantly reduced MAPK activity by 85% compared with ATP treatment alone.



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  		Figure 4. The effect of suramin, a P2 purinoceptor inhibitor, on ATP-induced MAPK activation in hGLCs. hGLCs were treated with 10 µM ATP in the presence or absence of suramin (300 µM) as described in Materials and Methods. MAPKs were detected by Western blot analysis. The data are shown as the relative ratio to basal levels. Values are presented as the mean ± SE of three individual experiments. Differences were considered significant at P < 0.05. a, P < 0.05 vs. control; b, P < 0.05 vs. ATP.

 
PTX and ATP-induced MAPK activation
A PTX-insensitive G protein, G{alpha}q/11, is known to be expressed in hGLCs (22, 23). To identify the subclass of G protein involved in the ATP-induced activation of MAPK, hGLCs were pretreated with PTX for 1 h before exposure to ATP. Pretreatment of PTX did not alter ATP-induced MAPK activity, indicating that ATP acts through a PTX-insensitive G protein-coupled. PTX had no effect on ATP-induced MAPK activation up to 500 ng/ml (data not shown).

PLC and ATP-induced MAPK activation
Neomycin, an aminoglycoside antibiotic, has been demonstrated to inhibit PLC (24). In this study hGLCs were pretreated with 10 mM neomycin for 15 min before stimulation of ATP. As shown in Fig. 5AGo, treatment of hGLCs with neomycin significantly inhibited the ATP-induced activation of MAPK. The combined treatment with neomycin and ATP significantly attenuated MAPK activity by 90% compared with ATP treatment alone.



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  		Figure 5. A, The effect of neomycin, a PLC inhibitor, on ATP-induced MAPK activation in hGLCs. hGLCs were treated with 10 µM ATP in the presence or absence of neomycin (10 mM). B, The effect of staurosporin, a PKC inhibitor (PKCI), on ATP-induced MAPK activation in hGLCs. hGLCs were treated with 10 µM ATP in the presence or absence of staurosporin (1 µM). MAPKs were detected by Western blot analysis. The data are shown as the relative ratio to basal levels. Values are presented as the mean ± SE of three individual experiments. Differences were considered significant at P < 0.05. a, P < 0.05 vs. control; b, P < 0.05 vs. ATP.

 
PKC and ATP-induced MAPK activation
Staurosporin, a potent inhibitor of PKC (25), significantly attenuated the ATP-induced activation of MAPK (Fig. 5BGo). Concomitant treatment with the PKC inhibitor and ATP attenuated MAPK activation by 70% compared with the level stimulated by ATP alone.

MEK- and ATP-induced MAPK activation
In the MAPK activation cascade, MEK is the immediate activator of MAPK. MEK is also known as MAPK kinase (7). MEK inhibitor, PD98059, significantly decreased the ATP-induced activation of MAPK in hGLCs (Fig. 6Go). Simultaneous treatment with PD98059 and ATP reduced MAPK activity to about 50% of the level stimulated by ATP alone (data not shown).



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  		Figure 6. The effect of PD98059, a MEK inhibitor (MEKI), on ATP-induced MAPK activation in hGLCs. hGLCs were treated with 10 µM ATP in the presence or absence of PD98059 (50 µM) as described in Materials and Methods. The activated MAPK were detected by Western blot analysis. The data are shown as the relative ratio to basal levels. Values are presented as the mean ± SE of three individual experiments. Differences were considered significant at P < 0.05. a, P < 0.05 vs. control; b, P < 0.05 vs. ATP.

 
Effect of ATP on intracellular cAMP accumulation
ATP has been demonstrated to increase intracellular cAMP production by activating adenylyl cyclase in several cell systems (26, 27). To examine the effect of ATP on intracellular cAMP production, hGLCs were treated with 10 µM ATP for 20 min, whereas 1 IU/ml hCG was used as a positive control. hCG markedly increased intracellular cAMP level. In contrast, ATP was not able to increase intracellular cAMP accumulation in hGLCs compared with control group (data not shown). This result indicates that the P2U purinoceptor expressed in hGLCs is not coupled to adenylyl cyclase.

Effect of ATP-evoked MAPK activation on hCG-induced progesterone production
To determine the role of MAPK in ovarian steroidogenesis, hGLCs were treated with ATP (10 µM), hCG (1 IU/ml), or ATP plus hCG in the presence or absence of PD98059. As shown in Fig. 7Go, 10 µM ATP had no effect on the basal level of progesterone production, whereas hCG increased progesterone production to 250% of the control level in hGLCs. Cotreatment of hGLCs with ATP and hCG significantly inhibited progesterone production to 50% of the level induced by hCG alone. Further, the presence of MEK inhibitor (PD98059) reversed the inhibitory effect of ATP on hCG-induced progesterone production.



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  		Figure 7. The effect of MAPK on progesterone production in hGLCs. hGLCs were treated with ATP (10 µM), hCG (1 IU/ml), or ATP plus hCG in the presence or absence of PD98059 for 6 h as described in Materials and Methods. Samples were assayed in triplicate, and progesterone concentrations were standardized against total protein content. Values are presented as the mean ± SE of three individual experiments. Differences were considered significant at P < 0.05. a, P < 0.05 vs. control; b, P < 0.05 vs. ATP plus hCG.

 
hCG activates MAPK in hGLCs
As shown in Fig. 8AGo, hCG was capable of activating MAPK in hGLCs in a time-dependent manner. Phosphorylated MAPK increased significantly in 1 min compared with the control and reached a maximum response after treatment with 1 IU/ml hCG for 5 min. The activity of MAPK maintained for at least 20 min. To investigate the role of hCG-stimulated MAPK in steroidogenesis, hGLCs were treated with hCG in the presence or absence of MEK inhibitor, PD98059. RIA demonstrated that there was no significant effect of MEK inhibitor on hCG-induced progesterone production (Fig. 8BGo).



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  		Figure 8. A, The effect of hCG on MAPK activation in hGLCs. hGLCs were treated with 1 IU/ml hCG for various times (1–20 min) as described in Materials and Methods. The activated MAPK were detected by Western blot analysis. B, The effect of PD98059, a MEK inhibitor (MEKI), on hCG-induced progesterone production in hGLCs. Samples were assayed in triplicate, and progesterone concentrations were standardized against total protein content. Values are presented as the mean ± SE of three individual experiments. Differences were considered significant at P < 0.05. a, P < 0.05 vs. control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The MAPKs have been implicated in the regulation of cell growth and differentiation (28). MAPKs are classified into three subfamilies: I) ERKs, including ERK1 and ERK2; II) stress-activated protein kinases, also called c-Jun N-terminus kinases; and III) p38 kinase (9). MEKs, also known as MAPK kinases, activate MAPKs by dual phosphorylation on threonine and tyrosine residues of a TEY (Thr-Glu-Tyr) motif (29). The first MAPKs to be cloned are MAPK/ERK1 and -2, which are phosphorylated and activated by MEKs (30, 31). MAPKs have been identified in several steroidogenic cells (10, 32), but little is known about their role(s) in steroidogenesis. Gonadotropins and growth factors have been shown to activate MAPK in granulosa cells (19, 33). Recently, Kang et al. reported that MAPKs mediate the inhibitory effect of GnRH in progesterone production in hGLCs (34), indicating a role for MAPKs in steroidogenesis. In the present study the phospho-specific MAPK antibody, which detected phosphorylated Thr202 and Tyr204 on ERK1/2, was used to measure activated MAPKs by exogenous ATP in hGLCs. The concentration of ATP in adrenergic granules of sympathetic nerves and in acetylcholine-containing granules of parasympathetic nerves can be as high as 150 mM (35). Our results demonstrated that 10 µM ATP was able to activate MAPKs, and furthermore, MAPKs mediated the antigonadotropic action of ATP in steroidogenesis in hGLCs.

The P2U purinoceptor has been identified in hGLCs (5). Regarding the receptor-coupled G protein, P2U purinoceptors may be coupled to PTX-sensitive or insensitive G proteins (36, 37). It was reported previously that P2U purinoceptors are coupled to PTX-insensitive G protein in hGLCs using microspectrofluorometry (20). In the present study ATP-induced phosphorylation of MAPK was not affected by 200 ng/ml PTX, indicating the involvement of a PTX-insensitive G protein such as G{alpha}q/11 (22, 23). P2 purinoceptors have been reported to couple to adenylyl cyclase in several systems (26, 27, 38). In this study ATP failed to increase intracellular cAMP accumulation, indicating that the P2U purinoceptor expressed in hGLCs is not coupled to adenylyl cyclase.

After binding to the G protein-coupled receptor, ATP has been reported to activate PLC (36, 39), resulting in the production of inositol trisphosphate and diacylglycerol, which, in turn, activate PKC. PLC-ß and PLC-{gamma} isoforms have been identified in hGLCs (22). Neomycin has been demonstrated to inhibit all three isoforms of PLCs (24). In the present study 10 mM neomycin significantly reduced the level of phosphorylated form of MAPKs, indicating the role of PLC in ATP-induced MAPK activation. PKC has been shown to exert its effects in the ovary (40, 41, 42, 43). In this study ATP-induced MAPK activation was significantly attenuated in hGLCs pretreated with staurosporin, a potent PKC inhibitor (25), indicating the involvement of PKC in the MAPK activation cascade. MEK is an immediate activator of MAPK. Our data demonstrated that the MEK inhibitor, PD98059, significantly decreased ATP-induced activation of MAPK. Taken together, this study delineated the ATP signaling pathway in hGLCs from PTX-insensitive G protein-coupled receptor, PLC, and PKC, with a MEK to MAPK activation. In addition, the observation that staurosporin at a relatively high dose (1 µM) only partially attenuated ATP-induced MAPK activity leads us to speculate that other mechanisms may be involved in the activation of MAPK in response to exogenous ATP.

ATP has been demonstrated to induce the production of steroid hormones in steroidogenic cells (44, 45). In the ovary, 100 µM ATP, ADP, and AMP have been shown to regulate basal levels of progesterone and estrogen in hGLCs, indicating the effects of ATP metabolites on steroidogenesis. However, UTP has no effect on the basal progesterone level in hGLCs, implying that the stimulatory effects of purine nucleotides on progesterone production are not through P2U purinoceptors, but via A2 adenosine receptors (46). As shown in the present study, a lower concentration of ATP (10 µM) had no effect on the basal level of progesterone production in hGLCs. However, cotreatment of hGLCs with ATP significantly inhibited the progesterone production induced by hCG, indicating an antigonadotropic action of ATP in hGLCs. Furthermore, pretreatment of hGLCs with MEK inhibitor reversed the inhibitory effect of ATP on hCG- induced progesterone production.

The precise mechanism by which MAPKs affect ovarian steroid hormone is not clear. Several steroidogenic enzymes, such as steroidogenic acute regulatory protein, cytochrome P450 cholesterol side-chain cleavage enzyme, and 3ß- hydroxysteroid dehydrogenase, have been demonstrated in the human ovary (47, 48). Considering the nuclear translocation of activated MAPKs (7, 11, 12, 13), it can be postulated that MAPKs are involved in steroidogenesis through altering the production of steroidogenic enzymes.

Oliver et al. reported that PD98059 (100 µM) induced apoptosis in luteinized granulosa cells cultured in serum-free medium (49). In our observations, hGLCs were viable and had no morphological change after treatment with 50 µM PD98059 in DMEM supplemented with 5% FBS for 24 h or in serum-free conditions for 6 h.

LH has been demonstrated to increase MAPK activity in porcine granulosa cells (19). In the present study hCG activated both ERK1 and ERK2 in a time-dependent manner. However, hCG-induced MAPK did not alter hCG-stimulated progesterone production. Taken together, these observations support the idea that a diverse array of ligands, including hormones, neurotransmitters, and growth factors, are able to activate MAPK, and cells may contain several MAPK signaling cascades, potentially regulated independently (50).

To our knowledge, this is the first demonstration of ATP-induced activation of a MAPK signaling pathway in the human ovary. It is proposed that through a PTX-insensitive G protein and without affecting intracellular cAMP production, ATP activates MAPK subsequent to PLC and PKC activation in hGLCs. These findings support a role for the MAPK signaling pathway in mediating the ATP modulation of steroidogenesis in the human ovary.


   Acknowledgments
 
We thank Dr. Margo Fluker and the Genesis Fertility Center (Vancouver, Canada) for the provision of human granulosa-luteal cells.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada. Back

2 Recipient of a studentship award from the British Columbia Research Institute for Children’s and Women’s Health. Back

3 Recipient of a career investigator award from the British Columbia Research Institute for Children’s and Women’s Health. Back

Received August 4, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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