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. 2008 Oct 16;150(2):929–935. doi: 10.1210/en.2008-1032

Follicle-Stimulating Hormone Inhibits Adenosine 5′-Monophosphate-Activated Protein Kinase Activation and Promotes Cell Proliferation of Primary Granulosa Cells in Culture through an Akt-Dependent Pathway

Pradeep P Kayampilly 1, K M J Menon 1
PMCID: PMC2646539  PMID: 18927218

Abstract

FSH, acting through multiple signaling pathways, regulates the proliferation and growth of granulosa cells, which are critical for ovulation. The present study investigated whether AMP-activated protein kinase (AMPK), which controls the energy balance of the cell, plays a role in FSH-mediated increase in granulosa cell proliferation. Cells isolated from immature rat ovaries were grown in serum-free, phenol red free DMEM-F12 and were treated with FSH (50 ng/ml) for 0, 5, and 15 min. Western blot analysis showed a significant reduction in AMPK activation as observed by a reduction of phosphorylation at thr 172 in response to FSH treatment at all time points tested. FSH also reduced AMPK phosphorylation in a dose-dependent manner with maximum inhibition at 100 ng/ml. The chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, 0.5 mm) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK inhibitor (compound C, 20 μm) and FSH reduced p27kip expression significantly compared with control. FSH treatment resulted in an increase in the phosphorylation of AMPK at ser 485/491 and a reduction in thr 172 phosphorylation. Inhibition of Akt phosphorylation using Akt inhibitor VIII reversed the inhibitory effect of FSH on thr 172 phosphorylation of AMPK, whereas ERK inhibitor U0126 had no effect. These results show that FSH, through an Akt-dependent pathway, phosphorylates AMPK at ser 481/495 and inhibits its activation by reducing thr 172 phosphorylation. AMPK activation by 5-amino-imidazole-4-carboxamide-1-β-d-ribofuranoside treatment resulted in a reduction of cell cycle regulatory protein cyclin D2 mRNA expression, whereas FSH increased the expression by 2-fold. These results suggest that FSH promotes granulosa cell proliferation by increasing cyclin D2 mRNA expression and by reducing p27 kip expression by inhibiting AMPK activation through an Akt-dependent pathway.

FSH stimulates granulosa cell proliferation by reducing cell cycle inhibitor p27 kip through AMP kinase inhibition.

In a growing ovarian follicle, the growth and proliferation of different cell types are critical for maintaining normal ovulation. Among these cells, granulosa cells play a crucial role of supporting the development and selection of the follicle that is destined for ovulation. The proliferation and differentiation of granulosa cells thus are important to maintain female fertility. FSH plays a major role in controlling the growth and development of follicles (1).

It has been shown that FSH regulates the proliferation of granulosa cells through multiple signaling pathways (2). For example, it has been reported that FSH, by activating the cAMP-protein kinase A (PKA)-ERK pathway, increases the mRNA expression of cell cycle regulatory protein cyclin D2, which leads to proliferation (3). Furthermore, FSH also stimulates the mammalian target of rapamycin (mTOR) signaling, leading to phosphorylation of S6K and increased cyclin D2 mRNA expression (4,5). Recently the role of AMP-activated protein kinase (AMPK) in cell growth and proliferation has captured attention. It has been reported that mTOR signaling can be regulated by AMPK (6,7,8) and that AMPK activation causes G1/S phase cell cycle arrest in cell lines (9,10,11). AMPK activation is associated with the accumulation of tumor suppressor protein p53 and the cyclin-dependent kinase inhibitors p21 and p27 (9,10,12,13). AMPK has also been found to reduce the stability of mRNAs encoding cell cycle regulators such as cyclin A and B1 (14). Thus, AMPK can serve as a negative regulator of cell growth.

AMPK is a serine/threonine kinase and is highly conserved throughout eukaryotes. It is a heterotrimeric protein consisting of a catalytic α- and regulatory β- and γ-subunits (15,16,17). It is known as the fuel gauge of the cell because it mediates a nutrient signaling pathway that senses cellular energy status (18). AMPK is activated by 5′-AMP in three distinct ways. First, AMP binding causes allosteric activation of AMPK. Second, AMP binding makes AMPK a better substrate for the upstream kinase, LKB, which activates the AMPK by phosphorylation of the α-subunit at a specific threonine residue (thr 172) (19). Third, AMP binding to AMPK inhibits dephosphorylation of thr 172 by protein phosphatases (20). In addition to these activating mechanisms, recently it has been reported that hormones also regulate AMPK activation (21), but the effect of these hormones on AMPK varies considerably (22,23,24,25).

Because FSH is known to regulate granulosa cell proliferation through multiple signaling pathways, in the present study, we examined the possible involvement of AMPK in FSH-mediated proliferation of ovarian granulosa cells. Our results show that FSH inhibits AMPK activation in a dose-dependent manner, and its inhibition leads to reduced p27kip expression and increased cyclin D2 mRNA expression, which are the key molecules involved in granulosa cell proliferation. We also found that the mitogenic stimulus from FSH to inhibit AMPK activation signals through an Akt-dependent pathway.

Materials and Methods

The phenol red-free DME-F12 medium and Trizol reagent were the products of Life Technologies Inc. (Gaithersburg, MD). Ovine FSH (NIDDK-oFSH-20) was purchased from Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA). AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and inhibitor compound C [6-(4-[2-piperidn-1-ylethoxy] phenyle)3-pyridin-4-ylpyrazolo (1,5-a)pyrimidine] were purchased form Sigma (St. Louis, MO). Antibodies for p27kip and AMPK were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against phosphorylated AMPK and Akt were from Cell Signaling Technology Inc. (Beverly, MA). Antimouse and antirabbit IgG horseradish peroxidase conjugates and enhanced chemiluminescence Western blotting detection reagents were from Amersham Pharmacia Biotech (Piscataway, NJ). MAPK kinase (MEK) inhibitor U0126 was obtained from Promega (Madison, WI) and Akt inhibitor VIII was from Calbiochem, (La Jolla, CA). Protein A and G agarose beads were obtained from Upstate Cell Signaling Solutions (Lake Placid, NY). Reagents as well as the primers and probes for the cyclin D2 real-time PCR were from Applied Biosystems (Foster City, CA).

Animals and treatments

Immature female rats (22 d old, Sprague Dawley strain) were purchased from Harlan (Indianapolis, IN) and Charles River Laboratories (Wilmington, MA). They were housed in a temperature-controlled room with proper dark-light cycles under the care of University of Michigan Unit of Laboratory Animal Medicine. All the experimental protocols used in this study were approved by the University Committee on the Use and Care of Animals. The animals were primed with estradiol (1.5 mg/d) for 3 d to stimulate the development of large preantral follicles and were killed 24 h after the last estradiol administration by CO2 asphyxiation, and ovaries were collected. Granulosa cells were harvested and cultured in phenol red free DMEM-F12 medium.

Granulosa cell isolation and culture

Granulosa cells from immature female rats were harvested as described previously (26). Briefly, ovaries were cleared from the surrounding fat and punctured with 25-gauge needles. Cells were collected in phenol red-free DMEM-F12 containing 0.2% BSA, 10 mm HEPES, and 6.8 mm EGTA; incubated for 15 min at 37 C under 95% O2-5% CO2; and centrifuged for 5 min at 250 × g. The pellets were suspended in a solution containing 0.5 m sucrose, 0.2% BSA, and 1.8 mm EGTA in DMEM-F12 and incubated for 5 min at 37 C. After incubation, the suspension was diluted with 3 vol DMEM-F12, centrifuged at 250 × g, and treated sequentially with trypsin (20 μg/ml) for 1 min, 300 μg/ml soybean trypsin inhibitor for 5 min, and deoxyribonuclease I (100 μg/ml) for 5 min at 37 C to remove dead cells. The cells were then rinsed twice with serum-free media, suspended in DMEM-F12, and the cell number determined. Cell viability was examined by trypan blue exclusion method. Cells were cultured in serum-free DME-F12 media supplemented with 20 mm HEPES (pH 7.4), 4 mm glutamine, 100 IU penicillin per milliliter, and 100 μg /ml streptomycin. Before seeding, the culture dishes were coated with 10% fetal calf serum for 2 h at 37 C and washed with DMEM-F12.

Western blot analysis

To study the time- and dose-dependent effects of FSH on AMPK, granulosa cells, after overnight attachment, were treated with FSH (50 ng/ml) for 0, 5, and 15 min or with 0, 25, 50, and 100 ng/ml of FSH for 15 min. In those studies in which AMPK activation or inhibition were tested using pharmacological agents, AMPK was activated by pretreating the cells with AICAR (0.5 mm) for 1 h, and AMPK was inhibited by incubating cells with compound C (20 μm). ERK inhibition was attained by incubating cells with MEK inhibitor U 0126 (30 μm) 30 min before FSH treatment. For Akt inhibition the preincubation period with inhibitor (5 μm, Akt inhibitor VIII) was 30 min. These preincubations were followed by FSH treatment for the time intervals indicated in each experiment. Reactions were stopped by removing the media, and total protein was solubilized using radioimmunoprecipitation assay buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholeate, 50 mm NaF, and 0.1% sodium dodecyl sulfate) or AMPK buffer (15 mm HEPES, 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm sodium pyrophosphate, 2 mm EDTA, 50 mm NAF, 1% Nonidet P-40, 10% glycerol, and protease inhibitors). AMPK was immunoprecipitated using an antibody against total AMPK. Proteins were separated using SDS-PAGE (10%) and transferred to nitrocellulose membrane and probed with antibodies against phosphorylated thr 172 or serine 485/491. Protein loading was normalized by reprobing the same blots with antibody against the total AMPK. Detection of signals was performed with an enhanced chemiluminescence Western blotting detection system.

Real-time PCR

Aliquots of total RNA (50 ng) extracted from different experimental groups (control, AICAR, FSH, and AICAR followed by FSH) of granulosa cells were reverse transcribed in a reaction volume of 20 μl using 2.5 μm random hexamer, 500 μm deoxynucleotide triphosphates, 5.5 mm MgCl2, 8 U ribonuclease inhibitor, and 25 U multiscribe reverse transcriptase. The reactions were carried out in a PTC-100 (MJ research, Watertown, MA) thermal controller (25 C for 10 min, 48 C for 30 min, and 95 C for 5 min). The resulting cDNAs were diluted with water. The real-time PCR quantification was then performed using 5 μl of the diluted cDNAs in triplicates and predesigned primers and probes for cyclin D2 (TaqMan Assay on Demand gene expression product Applied Biosystems, Foster City, CA). Reactions were carried out in a final volume of 25 μl using Applied Biosystems 7300 real-time PCR system for 40 cycles (95 C for 15 sec, 60 C for 1 min) after initial incubation for 10 min at 95 C. The fold change in cyclin D2 expression was calculated using the standard curve method with 18S rRNA as the internal control.

Statistical analysis

Statistical analysis was carried out using unpaired t test using GraphPad Prism computer software (version 3.0 cx; GraphPad Inc., San Diego, CA). Each experiment was repeated at least three times with similar results. Each blot is a representative of one experiment and the graphs are mean ± se of three experiments.

Results

FSH inhibits AMPK activation in a time and dose-dependent manner

The effect of FSH on AMPK regulation was studied by examining the expression of AMPK phosphorylated at Thr 172. Phosphorylation of this site is shown to be necessary for the activation of AMPK (19). Treatment with 50 ng of FSH showed a time-dependent reduction in AMPK phosphorylation (0, 5, 10, and 15min) with maximum response seen at 15 min (Fig. 1). FSH also produced a dose-dependent inhibition of AMPK with doses of 0, 25, 50, and 100 ng/ml (Fig. 2). These results indicate that FSH inhibits AMPK activation in a time- and dose-dependent manner.

Figure 1.

Figure 1

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Time course study of FSH treatment on AMPK phosphorylation in granulosa cells. Granulosa cells were isolated from 3-d estradiol primed immature rats as described in Materials and Methods. After overnight attachment, cells were treated with FSH (50 ng/ml) for 0, 5, 15, and 30 min. Cell lysate was prepared using the AMPK lysis buffer and total AMPK was immunoprecipitated. Western blot analysis was performed using antibody against phosphorylated AMPK (thr172; A). The same blot was stripped and reprobed with antibody for total AMPK (B). The lower panel (C) shows the quantitative expression of phosphorylated AMPK normalized for total AMPK. Blots are representative of one experiment, and the graph represents the mean of three experiments. Error bars represent mean ± se. *, Significant differences (P < 0.05) when compared with 0 min. p, Phosphorylated.

Figure 2.

Figure 2

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Dose-response study of FSH-mediated AMPK phosphorylation. Granulosa cells were harvested as described in Materials and Methods. After overnight incubation in serum-free media, cells were treated with 0, 25, 50, and 100 ng/ml FSH for 15 min. Total AMPK was immunoprecipitated from cell lysate. Western blot analysis was carried out for AMPK phosphorylated at thr172 (A), and protein loading was normalized by reprobing the blot with total AMPK antibody (B). C, Densitometric scanning of phosphorylated thr172 normalized for total AMPK. Error bar represents the mean ± se of three experiments. *, Significant difference (P < 0.05) when compared with control. p, Phosphorylated.

AMPK regulation alters p27 kip expression in granulosa cells

Because AMPK regulation is known to influence the cell cycle by activation of cyclin-dependent kinase inhibitors (CDKI), we next examined whether AMPK activation or inhibition by pharmacological agents changes the expression of CDKI protein, p27kip in granulosa cells. p27 kip is an inhibitor of cell cycle progression by blocking the cyclin D/cyclin-dependent kinase complex formation (27). AICAR is a pharmacological activator of AMPK that has been extensively used to activate AMPK in various cell types (28). After entering the cell, AICAR is phosphorylated to AICAR monophosphate (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranotide) and mimics both the allosteric activation of AMPK and promotes the phosphorylation of AMPK without altering the levels of ATP, ADP, or AMP (29). Compound C is a reversible ATP competitive inhibitor of AMPK and is widely used to inhibit AMPK (30). As shown in Fig. 3, treatment with 0.5 mm AICAR for 4 h significantly increased (P < 0.05) the p27 kip protein expression, whereas FSH (75 ng/ml) or compound C (20 μm) treatment significantly (P < 0.05) reduced this expression compared with the control. These results suggest that AMPK activation increases the expression of cell cycle inhibitor p27 kip, whereas AMPK inhibition reduces it.

Figure 3.

Figure 3

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Effect of AMPK activation and AMPK inhibition on p27 kip protein expression. Twenty-two-day-old rats were given estradiol injections sc for 3 d. Granulosa cells were isolated and after overnight attachment were treated with AMPK activator (AICAR, 0.5 mm), AMPK inhibitor (compound C, 20 μm), or FSH (75 ng/ml) for 4 h. Simultaneously one set of cultures preincubated with AICAR for an hour was also treated with FSH for 4 h. Reaction was stopped by removing the media and cell lysate was prepared using radioimmunoprecipitation assay buffer. p27 kip was immunoprecipitated from equal amounts of total protein and separated on 10% SDS-PAGE and transferred to nitrocellulose for Western blot analysis. A, Expression of p27 kip. B, Densitometric scanning of p27 kip expression. Blot is representative of one experiment, and the graph represents the mean of three experiments. Error bars represent mean ± se. *, Significant difference (P < 0.05) when compared with control and different letters represent significant difference (P < 0.05) between them. p, Phosphorylated.

FSH-mediated inhibition of AMP kinase is mediated through AKT-dependent pathway and is independent of ERK activation

In light of these results, we sought to find out the signaling mechanism by which FSH regulates AMPK activation. Recently it has been shown that insulin, through an Akt-dependent pathway, increases the phosphorylation of AMPK at two serine residues (485/491) at the α-subunit. Phosphorylation of these residues is implicated in the reduction of thr 172 phosphorylation, which inhibits the AMPK activity. FSH (75 ng/ml) treatment for 15 min significantly (P < 0.05) reduced the phosphorylation of AMPK at thr 172 when compared with control. Reprobing the blot with antibody against phosphorylated AMPK at serine 485/491 showed that FSH increases the phosphorylation at these residues, whereas the thr 172 phosphorylation was reduced (Fig. 4). Prior treatment of cells with Akt inhibitor abolished the FSH-mediated phosphorylation at serine 485/491 and also removed its inhibition on AMPK phosphorylation at thr 172 (Fig. 4). Collectively these observations suggest that FSH inhibits AMPK activation by increasing the phosphorylation of serine 485/491 residues and Akt inhibition abolishes this response.

Figure 4.

Figure 4

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Effect of Akt inhibition on FSH-mediated phosphorylation of serine 485/491 and thr172 residues of AMPK. Granulosa cells from 3-d estradiol-primed immature rats were harvested. Cells were allowed to attach overnight in serum-free, phenol red-free DME-F12 media. One set of cultures was preincubated with Akt inhibitor (Akt inhibitor VIII 5 μm) for 30 min and then incubated with FSH (75 ng/ml) for 15 min. Another set was treated with FSH (75 ng/ml) alone and the third with Akt inhibitor alone. Total AMPK was immunoprecipitated from the cell lysate and Western blot analysis for phosphorylated AMPK (thr172) was performed (A). B, Expression of AMPK phosphorylated at serine 485/491 in the same blot. C, Total AMPK expression. D, Densitometric scanning of phosphorylated thr172 and serine 485/491 normalized to total AMPK. Blots are representative of one experiment, and the graph represents the mean of three experiments. Error bars represents mean ± se. Black bars represent AMPK phosphorylation at thr172 and checked bars represent that of serine 485/491. *, Significant differences (P < 0.05) between phosphorylated thr172 and phosphorylated serine 485/491. Akt in, Akt inhibitor; p, phosphorylated.

Previous studies established that FSH stimulates both the phosphatidylinositol 3 kinase/Akt pathway as well as ERK pathway in cultured granulosa cells. Furthermore, we have shown that FSH acts through a PKA-ERK-dependent pathway to increase cyclin D2 expression. We therefore examined whether FSH-mediated regulation of AMPK occurs through a phosphatidylinositol 3 kinase/Akt pathway or PKA/ERK signaling pathway. To test this, cells were treated with Akt or ERK inhibitors followed by stimulation with FSH (75 ng/ml) for 15 min. The results (Fig. 5) showed that preventing ERK activation elicited no effect on FSH-mediated inhibition of AMPK phosphorylation at thr 172, whereas Akt inhibition abolished this response. These data therefore demonstrate that FSH-mediated inhibition of AMPK is independent of ERK activation but involves the AKT pathway.

Figure 5.

Figure 5

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Effect of ERK and Akt inhibition on FSH-stimulated AMPK phosphorylation. Three-day estradiol primed immature rats were used for the experiments. Cells were harvested as described in Materials and Methods. After overnight attachment, one group of cultures was incubated with Akt inhibitor (Akt inhibitor VIII, 5 μm) for 30 min and the second group with ERK inhibitor U0126 for 30 min, and the third group served as control. After the treatments, one set of cultures from both the control and inhibitor-treated groups was stimulated with FSH (75 ng/ml) for 15 min, and the other received vehicle. AMPK protein was immunoprecipitated and Western blot was performed. Upper panel (A) shows the expression of phosphorylated AMPK at thr172. Middle panel (B) shows the total AMPK expression. Phospho-AMPK expression normalized for total AMPK in three separate experiments is depicted in C. *, Significant differences (P < 0.05) when compared with control and same letters represent no significant difference between them. Akt in, Akt inhibitor; ERK in, ERK inhibitor; p, phosphorylated.

AMP kinase activation inhibits cyclin D2 expression in granulosa cells

Because cyclin D2 is a marker for granulosa cell proliferation (26,31) and AMPK activation causes cell cycle arrest, we tested whether activation of AMPK leads to a reduction in cyclin D2 mRNA expression. After overnight attachment, granulosa cells were incubated with AICAR (0.5 mm) or AICAR pretreatment followed by FSH treatment for 2 h. Real-time PCR analysis showed a 2-fold increase in cyclin D2 mRNA expression in response to FSH, whereas AMPK activation by AICAR significantly reduced this stimulation (Fig. 6). This observation demonstrates that AMPK activation results in the inhibition of cyclin D2 mRNA expression.

Figure 6.

Figure 6

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Effect of AMPK activation on cyclin D2 mRNA expression in granulosa cells. Granulosa cells from 3-d estradiol primed immature rats were cultured in serum-free, phenol red-free medium overnight followed by incubation with AMPK activator (AICAR) for 1 h. After treatment, one set of cultures from both the control and activator-treated groups was treated with FSH (75 ng/ml) for 2 h, and the other set received vehicle. Total RNA from the cells was reverse transcribed, and the resulting cDNAs were subjected to real-time PCR using predesigned primers and probes for rat cyclin D2 as described in Materials and Methods. The graph represents the change in cyclin D2 mRNA expression normalized for18S rRNA. Error bars represents the mean ± se of three experiments. *, Significant difference (P < 0.05) when compared with control and different letters represent significant differences (P < 0.05) between them.

Discussion

Ovarian follicular growth and ovulation are dependent on the growth and proliferation of granulosa cells. The primordial follicle in the mammalian ovary consists of a single ovum surrounded by a layer of granulosa cells. The period of follicular growth is characterized by mitotic activity of granulosa cells and the transformation of surrounding stroma into layers of thecal cells. FSH plays an important role in this process along with insulin and other paracrine factors. These mitogenic and growth regulatory signaling pathways do not appear to be independent but are interconnected and often cross talk with each other (1). Our laboratory has been examining the different mitogenic signaling pathways in granulosa cell proliferation in response to FSH under normal conditions and the disruptive effects of androgens on this process. Our studies have shown that FSH regulates granulosa cell proliferation using multiple signaling pathways and the disruption of FSH signaling by androgens adversely affects granulosa cell proliferation (3,26).

We have reported that FSH regulates mTOR signaling and cyclin D2 expression in granulosa cells and this regulation is dependent on the ERK-mediated inhibition of tuberin phosphorylation (5). Tuberin is a negative regulator of mTOR signaling (32,33,34). Recently it has been reported that tuberin can also be regulated by AMPK (35). AMPK is a regulator of energy balance in the cell, and once activated, it shuts down all energy consuming processes to maintain proper energy balance (36,37). In addition to a low-energy status, AMPK is also known to be activated by a number of hormones, including insulin and leptin (38,39,40). In the present study, we show that FSH is able to inhibit AMPK activation and that AMPK inhibition results in decreased p27 kip and increased cyclin D2 mRNA expression. Furthermore, our results show that FSH inhibits AMPK phosphorylation at thr 172 residue in a time- and dose-dependent manner and reduces AMPK activation (Figs. 1 and 2). Phosphorylation of this residue within the activation loop of the kinase domain on the α-subunit has been shown to be necessary for AMPK activation (19,41).

The relationship between FSH-mediated AMPK inhibition and activation of cell cycle progression was then shown by the observed decrease in p27 kip expression. Rattan et al. (12) have shown that chemical activation of AMPK inhibits proliferation in various cell lines by increasing CDKIs like p27 kip. p27 kip inhibits cyclin D/cyclin-dependent kinase complex formation and phosphorylation of retinoblastoma protein that leads to cell cycle arrest (27). Therefore, p27 kip was selected as a target molecule to examine the AMPK-mediated inhibition of proliferation. The involvement of AMPK on p27 kip was further demonstrated with the use of chemical activators and inhibitors of AMPK. AMPK activation was achieved by treating the cells with the chemical activator, AICAR. AICAR treatment increased p27 kip expression, whereas compound C as well as FSH inhibited p27 kip expression (Fig. 3). Exposure of cells to AICAR before FSH treatment reversed the FSH-mediated inhibition of p27 kip. The relationship between AMPK activation and cell cycle inhibition was then shown by a reduction in cyclin D2 mRNA expression, a well-established cell proliferation marker (3,31). FSH treatment increased cyclin D2 mRNA expression approximately 2-fold, whereas AMPK activation using AICAR before FSH treatment significantly reduced this stimulation. It is obvious from these results that once activated, AMPK can inhibit FSH-mediated cell proliferation by reducing cyclin D2 mRNA expression and by increasing the expression of the CDKI p27 kip.

We also examined the intermediary molecules involved in FSH-mediated AMPK regulation. Previous studies have shown that insulin inhibits AMPK activation by phosphorylating two serine residues (serine 485/491) at the α-subunit through an Akt-dependent pathway. Phosphorylation of these residues, in turn, reduces the phosphorylation at thr 172, which is necessary for AMPK activation (39,40). Consistent with these reports, in the present studies, FSH treatments increased the phosphorylation of these residues whereas reducing the phosphorylation at the thr 172 (Fig. 4). We have previously shown that FSH treatment for 15 min can significantly increase Akt phosphorylation in granulosa cells (5). We show here that inhibition of Akt activation using specific Akt inhibitor (Akt inhibitor VIII) reversed FSH-mediated reduction of thr 172 phosphorylation of AMPK (Figs. 4 and 5). Taken together, these results establish that FSH-mediated inhibition of AMPK activity is coupled to the phosphorylation of AMPK at serine 485/491, and this occurs through Akt phosphorylation.

FSH has been shown to increase cell proliferation by stimulating the ERK pathway (3). ERK activation is also involved in FSH-mediated mTOR activation in granulosa cells (5). These observations make ERK a key molecule in FSH-mediated granulosa cell mitogenesis. Examination of the signaling pathways in response to FSH shows that the MEK inhibitor U0126, which is known to block ERK activation, did not affect the FSH mediated inhibition of thr 172 phosphorylation of AMPK (Fig. 5). This finding rules out the role of ERK in FSH-mediated AMPK regulation and substantiate that AMPK inhibition occurs through an Akt-dependent mechanism.

Taken together, the present study shows that AMPK, which so far has been known as a controller of energy balance in cells, can also be regulated by FSH in granulosa cells. Based on this study as well as our previous reports, it is reasonable to conclude that FSH increases granulosa cell mitogenesis not only by increasing the expression of cyclin D2 through an ERK-dependent pathway but also by regulating AMPK through an Akt-dependent pathway to reduce the cell cycle inhibitor protein, p27 kip. In light of these findings, we suggest that in anovulatory conditions often associated with metabolic disorders such as obesity, anorexia, and type 2 diabetes, the dysregulation of appropriate follicle maturation might be mediated through signaling pathways regulated by AMPK.

Acknowledgments

We express our appreciation to Helle Peggel, Dr. Palaniappan Murugesan, Dr. Bindu Menon, and Dr. Thippeswamy Gulappa for critical reading of the manuscript.

Footnotes

This work was supported by National Institutes of Health Grant HD 38424.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 16, 2008

Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside; AMPK, AMP-activated protein kinase; CDKI, cyclin-dependent kinase inhibitor; MEK, MAPK kinase; mTOR, mammalian target of rapamycin; PKA, protein kinase A; thr 172, threonine residue.

References

  1. Hunzicker-Dunn M, Maizels ET 2006 FSH signaling pathways in immature granulosa cells that regulate target gene expression: branching out from protein kinase A. Cell Signal 18:1351–1359 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Richards JS, Russell DL, Ochsner S, Hsieh M, Doyle KH, Falender AE, Lo YK, Sharma SC 2002 Novel signaling pathways that control ovarian follicular development, ovulation, and luteinization. Recent Prog Horm Res 57:195–220 [DOI] [PubMed] [Google Scholar]
  3. Kayampilly PP, Menon KMJ 2004 Inhibition of extracellular signal-regulated protein kinase-2 phosphorylation by dihydrotestosterone reduces follicle-stimulating hormone-mediated cyclin D2 messenger ribonucleic acid expression in rat granulosa cells. Endocrinology 145:1786–1793 [DOI] [PubMed] [Google Scholar]
  4. Alam H, Maizels ET, Park Y, Ghaey S, Feiger ZJ, Chandel NS, Hunzicker-Dunn M 2004 Follicle-stimulating hormone activation of hypoxia-inducible factor-1 by the phosphatidylinositol 3-kinase/AKT/Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) pathway is necessary for induction of select protein markers of follicular differentiation. J Biol Chem 279:19431–19440 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kayampilly PP, Menon KMJ 2007 Follicle-stimulating hormone increases tuberin phosphorylation and mammalian target of rapamycin signaling through an extracellular signal-regulated kinase-dependent pathway in rat granulosa cells. Endocrinology 148:3950–3957 [DOI] [PubMed] [Google Scholar]
  6. Inoki K, Zhu T, Guan KL 2003 TSC2 mediates cellular energy response to control cell growth and survival. Cell 115:577–590 [DOI] [PubMed] [Google Scholar]
  7. Bolster DR, Crozier SJ, Kimball SR, Jefferson LS 2002 AMP-activated protein kinase suppresses protein synthesis in rat skeletal muscle through down-regulated mammalian target of rapamycin (mTOR) signaling. J Biol Chem 277:23977–23980 [DOI] [PubMed] [Google Scholar]
  8. Kimura N, Tokunaga C, Dalal S, Richardson C, Yoshino K, Hara K, Kemp BE, Witters LA, Mimura O, Yonezawa K 2003 A possible linkage between AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells 8:65–79 [DOI] [PubMed] [Google Scholar]
  9. Imamura K, Ogura T, Kishimoto A, Kaminishi M, Esumi H 2001 Cell cycle regulation via p53 phosphorylation by a 5′-AMP activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, in a human hepatocellular carcinoma cell line. Biochem Biophys Res Commun 287:562–567 [DOI] [PubMed] [Google Scholar]
  10. Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum MJ, Thompson CB 2005 AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell 18:283–293 [DOI] [PubMed] [Google Scholar]
  11. Igata M, Motoshima H, Tsuruzoe K, Kojima K, Matsumura T, Kondo T, Taguchi T, Nakamaru K, Yano M, Kukidome D, Matsumoto K, Toyonaga T, Asano T, Nishikawa T, Araki E 2005 Adenosine monophosphate-activated protein kinase suppresses vascular smooth muscle cell proliferation through the inhibition of cell cycle progression. Circ Res 97:837–844 [DOI] [PubMed] [Google Scholar]
  12. Rattan R, Giri S, Singh AK, Singh I 2005 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase. J Biol Chem 280:39582–39593 [DOI] [PubMed] [Google Scholar]
  13. Xiang X, Saha AK, Wen R, Ruderman NB, Luo Z 2004 AMP-activated protein kinase activators can inhibit the growth of prostate cancer cells by multiple mechanisms. Biochem Biophys Res Commun 321:161–167 [DOI] [PubMed] [Google Scholar]
  14. Wang W, Fan J, Yang X, Furer-Galban S, Lopez de Silanes I, von Kobbe C, Guo J, Georas SN, Foufelle F, Hardie DG, Carling D, Gorospe M 2002 AMP-activated kinase regulates cytoplasmic HuR. Mol Cell Biol 22:3425–3436 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hardie DG 2004 The AMP-activated protein kinase pathway—new players upstream and downstream. J Cell Sci 117:5479–5487 [DOI] [PubMed] [Google Scholar]
  16. Carling D 2004 The AMP-activated protein kinase cascade—a unifying system for energy control. Trends Biochem Sci 29:18–24 [DOI] [PubMed] [Google Scholar]
  17. Kemp BE, Stapleton D, Campbell DJ, Chen ZP, Murthy S, Walter M, Gupta A, Adams JJ, Katsis F, van Denderen B, Jennings IG, Iseli T, Michell BJ, Witters LA 2003 AMP-activated protein kinase, super metabolic regulator. Biochem Soc Trans 31:162–168 [DOI] [PubMed] [Google Scholar]
  18. Hardie DG, Carling D 1997 The AMP-activated protein kinase—fuel gauge of the mammalian cell? Eur J Biochem 246:259–273 [DOI] [PubMed] [Google Scholar]
  19. Hawley SA, Davison M, Woods A, Davies SP, Beri RK, Carling D, Hardie DG 1996 Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 271:27879–27887 [DOI] [PubMed] [Google Scholar]
  20. Davies SP, Helps NR, Cohen PT, Hardie DG 1995 5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2Cα and native bovine protein phosphatase-2AC. FEBS Lett 377:421–425 [DOI] [PubMed] [Google Scholar]
  21. Kahn BB, Alquier T, Carling D, Hardie DG 2005 AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1:15–25 [DOI] [PubMed] [Google Scholar]
  22. Steinberg GR, Rush JW, Dyck DJ 2003 AMPK expression and phosphorylation are increased in rodent muscle after chronic leptin treatment. Am J Physiol Endocrinol Metab 284:E648–E654 [DOI] [PubMed] [Google Scholar]
  23. Palanivel R, Sweeney G 2005 Regulation of fatty acid uptake and metabolism in L6 skeletal muscle cells by resistin. FEBS Lett 579:5049–5054 [DOI] [PubMed] [Google Scholar]
  24. Kola B, Hubina E, Tucci SA, Kirkham TC, Garcia EA, Mitchell SE, Williams LM, Hawley SA, Hardie DG, Grossman AB, Korbonits M 2005 Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase. J Biol Chem 280:25196–25201 [DOI] [PubMed] [Google Scholar]
  25. Andersson U, Filipsson K, Abbott CR, Woods A, Smith K, Bloom SR, Carling D, Small CJ 2004 AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 279:12005–12008 [DOI] [PubMed] [Google Scholar]
  26. Pradeep PK, Li X, Peegel H, Menon KMJ 2002 Dihydrotestosterone inhibits granulosa cell proliferation by decreasing the cyclin D2 mRNA expression and cell cycle arrest at G1 phase. Endocrinology 143:2930–2935 [DOI] [PubMed] [Google Scholar]
  27. Weinberg RA 1995 The retinoblastoma protein and cell cycle control. Cell 81:323–330 [DOI] [PubMed] [Google Scholar]
  28. Sullivan JE, Brocklehurst KJ, Marley AE, Carey F, Carling D, Beri RK 1994 Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase. FEBS Lett 353:33–36 [DOI] [PubMed] [Google Scholar]
  29. Corton JM, Gillespie JG, Hawley SA, Hardie DG 1995 5-Aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells? Eur J Biochem 229:558–565 [DOI] [PubMed] [Google Scholar]
  30. Zhou G, Myers R, Li Y, Chen Y, Shen X, Fenyk-Melody J, Wu M, Ventre J, Doebber T, Fujii N, Musi N, Hirshman MF, Goodyear LJ, Moller DE 2001 Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 108:1167–1174 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sicinski P, Donaher JL, Geng Y, Parker SB, Gardner H, Park MY, Robker RL, Richards JS, McGinnis LK, Biggers JD, Eppig JJ, Bronson RT, Elledge SJ, Weinberg RA 1996 Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature 384:470–474 [DOI] [PubMed] [Google Scholar]
  32. Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC, Blenis J 2002 Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc Natl Acad Sci USA 99:13571–13576 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC 2002 Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell 10:151–162 [DOI] [PubMed] [Google Scholar]
  34. Inoki K, Li Y, Zhu T, Wu J, Guan KL 2002 TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4:648–657 [DOI] [PubMed] [Google Scholar]
  35. Inoki K, Ouyang H, Zhu T, Lindvall C, Wang Y, Zhang X, Yang Q, Bennett C, Harada Y, Stankunas K, Wang CY, He X, MacDougald OA, You M, Williams BO, Guan KL 2006 TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126:955–968 [DOI] [PubMed] [Google Scholar]
  36. Munday MR, Campbell DG, Carling D, Hardie DG 1988 Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase. Eur J Biochem 175:331–338 [DOI] [PubMed] [Google Scholar]
  37. Hardie DG 2007 AMP-activated protein kinase as a drug target. Annu Rev Pharmacol Toxicol 47:185–210 [DOI] [PubMed] [Google Scholar]
  38. Minokoshi Y, Kim YB, Peroni OD, Fryer LG, Muller C, Carling D, Kahn BB 2002 Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415:339–343 [DOI] [PubMed] [Google Scholar]
  39. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR 2003 Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem 278:39422–39427 [DOI] [PubMed] [Google Scholar]
  40. Horman S, Vertommen D, Heath R, Neumann D, Mouton V, Woods A, Schlattner U, Wallimann T, Carling D, Hue L, Rider MH 2006 Insulin antagonizes ischemia-induced Thr172 phosphorylation of AMP-activated protein kinase α-subunits in heart via hierarchical phosphorylation of Ser485/491. J Biol Chem 281:5335–5340 [DOI] [PubMed] [Google Scholar]
  41. Stein SC, Woods A, Jones NA, Davison MD, Carling D 2000 The regulation of AMP-activated protein kinase by phosphorylation. Biochem J 345(Pt 3):437–443 [PMC free article] [PubMed] [Google Scholar]

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