REPRODUCTION-DEVELOPMENT
Follicle-Stimulating Hormone Inhibits Adenosine
5ⴕ-Monophosphate-Activated Protein Kinase Activation
and Promotes Cell Proliferation of Primary Granulosa
Cells in Culture through an Akt-Dependent Pathway
Pradeep P. Kayampilly and K. M. J. Menon
Departments of Obstetrics and Gynecology and Biological Chemistry, University of Michigan Medical School, Ann
Arbor, Michigan 48109
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-␤-D-ribofuranoside,
0.5 mM) increased the cell cycle inhibitor p27 kip expression significantly, whereas the AMPK
inhibitor (compound C, 20 ␮M) 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-␤-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. (Endocrinology 150: 929 –935, 2009)
I
n 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 – 8) and that AMPK activation causes G1/S phase cell
cycle arrest in cell lines (9 –11). AMPK activation is associated
with the accumulation of tumor suppressor protein p53 and the
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2009 by The Endocrine Society
doi: 10.1210/en.2008-1032 Received July 10, 2008. Accepted October 9, 2008.
First Published Online October 16, 2008
Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide-1-␤-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.
Endocrinology, February 2009, 150(2):929 –935
endo.endojournals.org
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Kayampilly and Menon
FSH-Mediated AMPK Regulation in Granulosa Cells
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 ␣- and regulatory ␤- and ␥-subunits (15–17). 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 ␣-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 (22–25).
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-␤-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
Endocrinology, February 2009, 150(2):929 –935
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 ␮g/ml) for 1 min, 300 ␮g/ml
soybean trypsin inhibitor for 5 min, and deoxyribonuclease I (100 ␮g/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 ␮g /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 ␮M). ERK inhibition was attained
by incubating cells with MEK inhibitor U 0126 (30 ␮M) 30 min before
FSH treatment. For Akt inhibition the preincubation period with inhibitor (5 ␮M, 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 ␮l using
2.5 ␮M random hexamer, 500 ␮M 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 ␮l 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 ␮l 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
Endocrinology, February 2009, 150(2):929 –935
endo.endojournals.org
at 95 C. The fold change in cyclin D2 expression was calculated using the
standard curve method with 18S rRNA as the internal control.
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.
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
pAMPK (thr 172)
A
Total AMPK
B
1.5
C
*
1
*
*
0.5
0
5
10
15
pAMPK expression normalized for total AMPK
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.
pAMPK expression normalized for total AMPK
A
pAMPK (thr 172)
B
Total AMPK
Statistical analysis
931
1.25
C
1
0.75
*
0.5
*
0.25
0
0
25
50
100
FSH (ng/ml)
FIG. 2. 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.
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-4carboxamide-1-␤-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 ␮M) 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.
FSH (50 ng/ml) treatment (min)
FIG. 1. 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.
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 ␣-subunit. Phosphorylation of these residues is implicated in the reduction of thr 172 phosphorylation,
Kayampilly and Menon
FSH-Mediated AMPK Regulation in Granulosa Cells
p27kip
A
B
*a
pAMPK (thr 172)
A
pAMPK (ser485/491)
B
Total AMPK
C
1.5
*a
2
1.5
*b
1
*b
Compound C
FSH
Control
0
AICAR+FSH
0.5
AICAR
Densitometric scanning of p27kip expression
2.5
Endocrinology, February 2009, 150(2):929 –935
pAMPK (thr 172) and pAMPK (Ser 485/491) expression
normalized for total AMPK
932
FIG. 3. 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 ␮M), 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.
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 FSHmediated 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.
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
D
thr 172
Ser 485/491
*
1
0.5
*
*
*
0
Control
FSH
AKT in
+ FSH
AKT in
FIG. 4. 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 serumfree, phenol red-free DME-F12 media. One set of cultures was preincubated with
Akt inhibitor (Akt inhibitor VIII 5 ␮M) 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.
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.
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.
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 sur-
Endocrinology, February 2009, 150(2):929 –935
endo.endojournals.org
pAMPK (thr 172)
3
C
2
1
ERK in
AKT in
ERK in+ FSH
AKT in + FSH
FSH
0
FIG. 5. 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 ␮M) 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.
rounded 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
(32–34). 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 – 40). In the present study, we show that FSH is able to inhibit
Cyclin D2 mRNA normalized for 18S rRNA
B
Control
pAMPK expression normalized for total AMPK
250
A
Total AMPK
933
*a
200
*b
150
100
50
0
Control
FSH
AICAR
+FSH
AICAR
FIG. 6. 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.
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 ␣-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).
934
Kayampilly and Menon
FSH-Mediated AMPK Regulation in Granulosa Cells
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 ␣-subunit through an Aktdependent 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 FSHmediated 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.
Address all correspondence and requests for reprints to: Dr. K. M. J.
Menon, 6428 Medical Science I, 1150 West Medical Center Drive,
Endocrinology, February 2009, 150(2):929 –935
University of Michigan Medical School, Ann Arbor, Michigan 48109.
E-mail: [email protected].
This work was supported by National Institutes of Health Grant HD
38424.
Disclosure Statement: The authors have nothing to disclose.
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