Follicle-Stimulating Hormone Promotes Histone H3 Phosphorylation on Serine-10
Deborah A. DeManno1,
Joshua E. Cottom,
Michael P. Kline,
Carl A. Peters,
Evelyn T. Maizels and
Mary Hunzicker-Dunn
Departments of Cell and Molecular Biology (D.A.D., J.E.C., C.A.P.,
E.T.M., M.H-D.) Northwestern University Medical School
Chicago, Illinois 60611
Departments of Biochemistry,
Molecular Biology and Cell Biology (M.P.K.) Northwestern
University Evanston, Illinois 60201
 |
ABSTRACT
|
---|
FSH promoted the rapid phosphorylation of the
nuclear protein histone H3 in immature rat ovarian granulosa cells
under experimental conditions that lead to cellular differentiation and
not proliferation. FSH-stimulated histone H3 phosphorylation correlated
with cAMP-dependent protein kinase A (PKA) activation and translocation
of the PKA catalytic subunit to a nuclear-enriched fraction and was
inhibited by the PKA inhibitor H89, and histone H3 phosphorylation was
stimulated in cells treated with agents that raise intracellular
cAMP levels such as forskolin and 8-bromo-cAMP. FSH-stimulated histone
H3 phosphorylation in granulosa cells mapped to ser-10, a site
previously identified as the PKA phosphorylation site in various
mitotically active cells as the mitosis-specific phosphorylation site.
Injection of the FSH analog PMSG to immature rats, which is known to
stimulate granulosa cell proliferation as well as differentiation, also
promoted histone H3 phosphorylation on ser-10 in granulosa cells. These
results establish that FSH-stimulated histone H3 phosphorylation in
granulosa cells is linked not only to granulosa cell mitosis but also
to granulosa cell differentiation and that FSH-stimulated histone H3
phosphorylation on ser-10 in isolated granulosa cells is mediated by
PKA. These results also identify the PKA-dependent histone H3
phosphorylation as an early nuclear protein marker for FSH-stimulated
differentiation of granulosa cells. Based on the recently described
function of histone H3 as a coactivator of transcription, these results
are consistent with the hypothesis that phosphorylated histone H3 may
facilitate PKA-dependent gene transcription in granulosa cells leading
to the preovulatory phenotype.
 |
INTRODUCTION
|
---|
Maturation of ovarian follicles from a preantral to preovulatory
state is dependent upon the pituitary gonadotropin FSH. The actions of
FSH are transduced by guanine nucleotide binding protein-coupled FSH
receptors localized exclusively to follicular granulosa cells (GCs).
GCs proliferate slowly in the absence of FSH; FSH accelerates the rate
of GC proliferation in vivo (1, 2). However, FSH alone is
not sufficient to induce (rat) GC proliferation since FSH does not
promote proliferation in serum-free GC cultures (3, 4, 5, 6, 7, 8). The
proliferative response of isolated (rat) GCs to FSH in serum-free
conditions requires transforming growth factor-ß (TGFß) (5, 6) or
activin (7) and peaks approximately 20 h after addition of FSH and
TGFß or activin (6, 7).
In contrast to GC proliferation, differentiation of GCs to a mature,
preovulatory phenotype does not occur in the absence of FSH (9, 10);
GCs readily differentiate in serum-free cultures with the addition of
FSH (3). In response to FSH, immature GCs acquire specific receptors
for various hormones, including LH, epidermal growth factor (EGF),
insulin-like growth factor 1 (IGF-I), PRL, and additional FSH
receptors; exhibit enhanced synthesis of progesterone and estrogen;
demonstrate morphological characteristics of differentiation, including
changes in cell shape and aggregation; and increase synthesis of
certain cellular proteins including the type II ß regulatory subunit
of cAMP-dependent protein kinase A (PKA) (3, 11, 12, 13) and an 80-kDa PKA-
anchoring protein (14). Immature GCs require 48 h of FSH treatment
in vitro to acquire these hallmarks of the differentiated
phenotype (3).
The intracellular signaling pathways by which FSH modulates
proliferation and differentiation are only partly understood. FSH
promotes an increase in cyclin D2 (mRNA and protein) in GCs in
vivo, leading to GC proliferation (2, 15). Forskolin can mimic the
ability of FSH to elevate cyclin D2 mRNA in serum-free GC cultures,
suggesting that FSH-induced cyclin D2 mRNA expression is mediated by
cAMP (2, 15), although it is not known whether cyclin D2 protein levels
are affected since neither FSH nor forskolin promotes GC proliferation
under serum-free in vitro conditions. Consistent with this
result, however, thymidine uptake by GCs in the presence of activin is
enhanced by 8-bromo-cAMP (7).
There is abundant evidence that a cAMP-mediated signal is sufficient
for FSH-dependent GC differentiation. Activators of adenylyl cyclase,
like forskolin and cholera toxin, and cAMP analogs mimic effects of FSH
in serum-free cultures leading to the differentiated GC phenotype
(3), and the PKA inhibitor H89 blocks FSH-stimulated GC differentiation
(D. W. Carr and M. Hunzicker-Dunn, manuscript in preparation). FSH
via cAMP also stimulates an increase in intracellular Ca2+
in GCs mediated via plasma membrane-localized Ca2+ channels
(16, 17). There is also indirect evidence for the presence of a
tyrosine kinase in the FSH-signaling pathway leading to a
differentiated phenotype that is located downstream of adenylyl
cyclase, based on the ability of the tyrosine kinase inhibitor AG18 to
arrest the FSH- and forskolin-stimulated induction of P450 cholesterol
side-chain cleavage and resulting progesterone secretion (18, 19).
Additional components of the FSH-signaling pathway in immature rat GCs
leading to the differentiated phenotype located downstream of adenylyl
cyclase and PKA are the 42- and 44-kDa mitogen-activated protein
kinases (MAPKs), and the p38 MAPK and downstream MAPK-activated protein
kinases (MAPKAP-kinases), all of which are phosphorylated and activated
upon stimulation by FSH or forskolin in the absence but not in the
presence of the PKA inhibitor H89 (20, 21), as well as the 90 kDa
ribosomal S6 protein kinase (RSK) (20). FSH or forskolin also promote
phosphorylation of the cAMP response element binding protein (CREB)
(22). Thus, there is compelling evidence that FSH actions to promote GC
differentiation are initiated by cAMP via a PKA signal transduction
pathway. The FSH signaling pathway leading to GC differentiation can
also be modulated by growth factors. Both IGF-I and TGFß enhance
actions of FSH leading to GC differentiation, while EGF, GnRH, tumor
necrosis factor-
, and protein kinase C (PKC)-activating phorbol
esters inhibit FSH-stimulated rat GC differentiation (11, 23). However,
the point(s) in the FSH-signaling pathway where most of these growth
factors modulate GC differentiation are largely unknown.
Based on the prominent role of PKA in stimulating GC differentiation,
we sought to identify PKA substrates in intact GCs and to determine
whether phosphorylation of these proteins was modulated by the
PKC-activating phorbol esters or by inhibition of tyrosine kinases. A
serum-free, immature rat GC primary culture model was used to examine
the effects of individual factors on these cells in the absence of the
growth factors present in serum.
 |
RESULTS
|
---|
FSH Promotes Activation of PKA
While there is abundant indirect evidence documenting
FSH-stimulated PKA activation, the time course of PKA activation in
response to FSH is not known. Addition of 50 ng/ml FSH to GCs promoted
a time-dependent activation of PKA measured in soluble GC extracts
(Fig. 1A
, solid bars),
evidenced by an increase in the PKA activity ratio. The resulting
translocation of the PKA catalytic subunit to a nuclear-enriched pellet
fraction was detected by Western blots at 30 min and peaked at 60 min
after FSH addition (Fig. 1B
).

View larger version (40K):
[in this window]
[in a new window]
|
Figure 1. FSH Stimulates Time-Dependent PKA Activation
GCs were treated with 50 ng/ml FSH for indicated times and sonicated in
buffer C, and supernatant extracts were assayed for PKA activity in the
absence and presence of cAMP, as indicated. In panel A, PKI-insensitive
kinase activity was subtracted from Kemptide kinase activities measured
in the presence and absence of cAMP. Results are means ±
SEM of triplicate determinations from individual dishes.
Results are graphed in the right panel as the PKA
activity ratio. In panel B, pellet fractions from the same cells were
evaluated for PKA catalytic (C) subunit by Western analysis.
|
|
Characterization of FSH-Stimulated Protein Phosphorylation in Total
GC Extracts
When 32P-labeled GCs were incubated with 50 ng/ml of
FSH for 1 h, a 16- kDa phosphorylated protein was visible in total
cell extracts of FSH-treated but not control cells (Fig. 2
, compare lanes 4 and 5). A prominent
protein band at this molecular mass detected by Coomassie blue
staining was present in both control and FSH-treated cells (Fig. 2
, lanes 2 and 3). Phosphorylation of the 16-kDa protein was also
stimulated by 10 µM forskolin (Fig. 2
, lane 6); however,
the extent of phosphorylation of the 16-kDa band was not increased
further when GCs were incubated with FSH plus forskolin (Fig. 2
, lane
7). Phosphorylation of the 16-kDa band in response to FSH was
consistently detectable by 30 min, elevated at 60 min (Fig. 3A
), and undetectable at 3 h (see
Fig. 7
), consistent with the time course for the translocation of the
PKA catalytic subunit. The 16-kDa protein was maximally phosphorylated
during the 1-h treatment with FSH doses ranging from 1 ng/ml to 1
µg/ml (Fig. 3B
).

View larger version (89K):
[in this window]
[in a new window]
|
Figure 2. FSH and Forskolin Stimulate the Phosphorylation of
a 16-kDa Protein in Immature GCs
32P-labeled GCs were incubated under control (Con)
conditions (lanes 2 and 4) or with 50 ng/ml FSH (lanes 3 and 5), 10
µM forskolin (lane 6, FSK), or FSH and forskolin (lane 7)
for 1 h. Total cell extracts were separated on SDS-PAGE, stained
with Coomassie blue (lanes 2 and 3), and processed for autoradiography
(lanes 48), as described in Materials and Methods.
Molecular weight standards are shown in lane 1 and indicated at the
left in kilodaltons. Results are representative of 5
(for forskolin treatment) or greater than 10 (for FSH) experiments.
|
|

View larger version (49K):
[in this window]
[in a new window]
|
Figure 3. Effect of FSH Dose and Incubation Time on the
Phosphorylation of the 16-kDa Protein
32P-labeled GCs were incubated in the absence or presence
of 50 ng/ml FSH for the indicated times (panel A) or in the absence or
presence of indicated concentrations of FSH for 1 h (panel B).
Total cell extracts were separated by SDS-PAGE. For additional details,
see legend to Fig. 2 . Results in panels A and B are representative of
five and three experiments, respectively.
|
|

View larger version (98K):
[in this window]
[in a new window]
|
Figure 7. Localization of the 16-kDa Phosphoprotein to the
Perchloric Acid-Insoluble Cell Fraction
32P-labeled GCs were incubated under control conditions
(Con) or with 50 ng/ml FSH for 10180 min. Cells were harvested in
protease and phosphatase inhibitor buffer (buffer A), and 5000 x
g pellets were extracted with 5% perchloric acid.
Acid-soluble and -insoluble fractions were separated on SDS-PAGE and
processed for autoradiography. Autoradiograms of perchloric
acid-insoluble phosphoproteins (panel A) and perchloric acid-soluble
phosphoproteins (panel B) are shown. Results are representative of
three experiments.
|
|
When 32P-labeled GCs were incubated with 100 ng/ml of hCG,
an LH analog, there was no phosphorylation of the 16 kDa protein (not
shown), confirming the immature stage of these GCs. Indistinguishable
FSH-stimulated phosphorylation of the 16 kDa protein was also observed
in GCs prepared from rats that had not been primed with estradiol
injections as well as in GCs cultured in the absence of estradiol (not
shown). However, since the estradiol injections promoted an increase
both in the number of GCs per ovary (13, 24) and the apparent
uniformity of the follicles (D. A. DeManno, personal observation),
estradiol was used throughout in this study.
Incubation of GCs with 10 mM 8-bromo-cAMP for 1 h
mimicked the ability of FSH to promote phosphorylation of the 16 kDa
protein (Fig. 4A
). Lower concentrations
of 8-bromo-cAMP, however, were ineffective (not shown). A modest
increase in 32P content in the 16-kDa protein band was also
noted in cells treated with the PKC activator phorbol 12-myristate
13-acetate (PMA2) at 200
nM (Fig. 4A
). Phosphorylation of the 16-kDa protein was not
stimulated by either EGF at 50 ng/ml (Fig. 4B
) or activin at 1 ng/ml
(not shown). FSH-stimulated phosphorylation of the 16-kDa protein was
not affected by preaddition of 200 nM PMA 15 min before
addition of FSH (Fig. 5A
) or by an
equivalent preaddition of 50 ng/ml EGF (not shown) or by coaddition of
1 ng/ml activin (not shown), and was not inhibited by the tyrosine
kinase inhibitor AG18 at 50 µM, added 1 h before
addition of FSH (Fig. 5B
). The 16-kDa protein was also phosphorylated
in response to FSH under Ca2+-free incubation conditions (1
h preincubation in the presence of 11 mM EGTA, a
concentration more than 5-fold greater than the molar concentration of
Ca2+ in the medium) to an extent equivalent to that in
Ca2+-replete medium (Fig. 5C
).

View larger version (47K):
[in this window]
[in a new window]
|
Figure 4. Effect of 8-bromo-cAMP, EGF, and PMA on the
Phosphorylation of the 16-kDa Protein
In panel A, 32P-labeled GCs were incubated in the absence
(lane 1) or presence of 50 ng/ml FSH (lane 2), 10 mM
8-bromo-cAMP (8-Br, lane 3), or 200 nM PMA (lane 4) for
1 h. Cells were sonicated in buffer A, and the resulting pellet
fraction was solubilized in SDS-PAGE sample buffer and subjected to
SDS-PAGE. Results are representative of two separate experiments. In
panel B, 32P-labeled GCs were incubated in the absence
(lane 1) or presence of 50 ng/ml FSH (lane 2) or 50 ng/ml EGF (lane 3).
For rest of details, see above. Results are representative of two
experiments.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Figure 5. Effects of Pretreatment with PMA or the Tyrosine
Kinase Inhibitor AG18 and of EGTA on FSH-stimulated Phosphorylation of
the 16-kDa Protein
In panel A, 32P-labeled GCs were preincubated 15 min in the
absence or presence of 200 nM PMA, as indicated. FSH (50
ng/ml) or vehicle was then added to cells and the incubation was
continued for 1 h. Total cell extracts were separated by SDS-PAGE,
as detailed in the legend to Fig. 2 . Results are representative of two
experiments. In panel B, 32P-labeled GCs were preincubated
1 h in the presence of 50 µM AG18 or vehicle (Veh),
as indicated. FSH (50 ng/ml) or forskolin (10 µM) was
then added to cells and incubation was continued for an additional
hour. Cells were sonicated in buffer A, and extracts were separated
into soluble (S) and pellet (P) fractions, as described in
Materials and Methods. Results are representative of two
experiments. In panel C, 32P-labeled GCs were preincubated
1 h in the absence or presence of 11 mM EGTA. Media or
FSH (50 ng/ml) was then added and the incubation continued for 1
h. Cells were sonicated in buffer B, and the resulting pellet fraction
was solubilized in SDS-PAGE sample buffer and subjected to SDS-PAGE.
Results are representative of three experiments. Equivalent results
were also seen, in one experiment, when cells were sonicated in buffer
A, localizing the 16-kDa phosphoprotein to the pellet fraction (as in
panel B).
|
|
Solubility and Localization of the 16-kDa Phosphoprotein
Studies were performed to determine the cellular location of the
16-kDa phosphoprotein. To this end, we determined whether the 16-kDa
phosphoprotein was localized to the 5000 x g
pellet or supernatant fraction in 32P-labeled control and
FSH-treated GCs harvested in different buffers. As shown in Fig. 6
(lanes 58), the 16-kDa phosphoprotein
was localized in the pellet when GCs were harvested in a hypotonic
buffer (buffer A). When GCs were harvested in a solution containing 0.6
M KCl and 1% Triton-X 100, conditions in which
intermediate filaments are insoluble (25), the 16-kDa phosphoprotein
was soluble (Fig. 6
, lanes 14). On two-dimensional isoelectric
focussing (pH 310) followed by SDS-PAGE of 32P-labeled GC
extracts, the 16-kDa phosphoprotein did not enter the isoelectric
focusing gel (not shown), indicating that the isoelectric point (pI) of
the 16-kDa protein was very basic. The 16-kDa protein was insoluble in
8 M urea, 5% ß-mercaptoethanol, and 2% Nonidet P-40
(Fig. 6
, lanes 912), ruling out its classification as a profilin
(26). These combined characteristics of the 16-kDa phosphoprotein
molecular mass, insolubility in 8 M urea, solubility in
high salt, and apparent basic isoelectric point (pI) are
consistent with the characteristics of histones (27).

View larger version (84K):
[in this window]
[in a new window]
|
Figure 6. Fractionation and Solubility of FSH-Stimulated
Phosphoproteins
Equivalent dishes of control (Con) and FSH-stimulated (50 ng/ml, 1
h) 32P-labeled cells were harvested in one of three
solutions: 0.6 M KCl containing 1% Triton X-100; protease
and phosphatase inhibitor buffer (buffer A; PIB); or 8 M
urea buffer containing 5% 2-mercaptoethanol and 2% Nonidet P-40
(buffer B), as described in Materials and Methods. Equal
volumes of supernatant (S) and pellet (P) fractions were prepared and
separated on SDS-PAGE and autoradiography was performed. Migration
positions of mol wt standards are indicated on the left.
Results are representative of two experiments.
|
|
Extraction of the 16-kDa Phosphoprotein with Histones
After a time course of control and FSH incubations from 10
to 180 min with 32P-labeled cells, cells were harvested (in
buffer A) and 5000 x g pellets were extracted with
perchloric acid to separate the acid-insoluble (Fig. 7A
) core histones (H) H2A, H2B, H3, and
H4 from the acid-soluble (Fig. 7B
) histone H1. The 16-kDa protein was
insoluble in 5% perchloric acid (Fig. 7A
), suggesting that it was a
core histone. In agreement with our time course studies of total cell
extracts, phosphorylation of the 16-kDa protein in the perchloric
acid-insoluble fraction was readily detectable by 30 min of FSH
treatment, peaked at 60 min, and was no longer detectable above
background at 120 min (Fig. 7A
). Although the 16-kDa protein was absent
from the perchloric acid-soluble fraction, we noted that a 40-kDa
protein was phosphorylated in an FSH- and time-dependent manner (Fig. 7B
). The phosphorylation level of the 40-kDa protein was increased
above the control with 30 min of FSH treatment and continued to
increase throughout the 180 min time course (Fig. 7B
). Coomassie blue
staining of this gel demonstrated that unlabeled pure calf thymus
histone H1 comigrated with the 40 kDa protein (not shown). H1 has a
calculated mass of approximately 23 kDa but exhibits anomalous
migration on SDS-PAGE with a higher apparent molecula mass of 40 kDa
(28).
Identification of the 16-kDa Protein as Histone H3
Based on the insolubility of the 16-kDa phosphoprotein in
5% perchloric acid, and consistent with its identification as a core
histone, we compared the migration position of the 16-kDa protein with
that of pure core histones on standard SDS-PAGE. To provide
histone-enriched samples, pellets were prepared from
32P-labeled control and FSH-stimulated GCs extracted with 8
M urea buffer (buffer B). Based on the migration position
of pure core histones, identified by Coomassie blue staining (Fig. 8A
, left panel), the 16-kDa
phosphoprotein (Fig. 8A
, right panel) corresponded to
histone H3. This conclusion was supported by the migration position of
immunoreactive histones (Fig. 8B
). To confirm identity of the 16-kDa
band as histone H3, we also used the well-established Triton-acid-urea
(TAU) PAGE system, which separates histones by shape and mass (due to
differential binding of the Triton X-100 on the basis of
hydrophobicity) (29). The TAU-PAGE system allowed for clear separation
of the core histones in a different order of migration as compared with
SDS-PAGE (Fig. 9
, left panel).
As seen on the autoradiogram (Fig. 9
, right panel) the
16-kDa protein, whose phosphorylation was increased in cells treated
with FSH, comigrated with histone H3. The 40-kDa phosphoprotein, which
migrated at the same position as H1 in the TAU-PAGE system, also
exhibited an increased level of phosphorylation after 1 h of FSH
treatment.

View larger version (62K):
[in this window]
[in a new window]
|
Figure 8. Comparison of the Migration of FSH-Stimulated
Phosphoproteins in GCs to the Migration of Histone Standards on
SDS-PAGE
In panel A, 32P-labeled cells were incubated in the absence
(Con) and presence of FSH (50 ng/ml) for 1 h and then harvested in
8 M urea buffer (buffer B), and extracts were separated
into soluble and pellet fractions. GC pellets were subjected to
SDS-PAGE (lanes 2 and 3), and gel was stained with Coomassie blue
(left panel), and then processed for autoradiography
(right panel). The migration of unlabeled calf thymus
histone standards is shown in lanes 49; mol wt markers are shown in
lane 1. Results are representative of two experiments. Panel B,
32P-labeled GCs were incubated in the presence of FSH (50
ng/ml) for 1 h, harvested in buffer A, sonicated, mixed with
SDS-PAGE sample buffer, and boiled 20 min, and then frozen at -70 C.
After 32P had decayed for 3 half-lives, aliquots (50 µg)
of total cell extracts were subjected to Western blot analyses using
purified monoclonal antihistone antibodies (lanes 24). Lane 1 is an
autoradiograph (AR) of freshly 32P-labeled GCs incubated
with FSH for 1 h. Migration of the 16-kDa protein in lane 1 is
indicated by the open arrow. Individual blots were
aligned by the migration position of the 12-kDa marker. Results are
representative of three separate experiments.
|
|

View larger version (73K):
[in this window]
[in a new window]
|
Figure 9. Comparison of the Migration of FSH-Stimulated
Phosphoproteins in GCs to that of Histone Standards on TAU-PAGE
32P-labeled cells were incubated in the absence (Con) and
presence of FSH (50 ng/ml) for 1 h and harvested in 8
M urea buffer (buffer B), and extracts were separated into
soluble and pellet fractions. GC pellets were subjected to TAU-PAGE
(lanes 1 and 2), and gel was stained with Coomassie blue (left
panel) and then processed for autoradiography (right
panel). Migration of unlabeled calf thymus histone standards is
shown in lanes 37. Results are representative of two experiments. *
Marks the migration position of histone H32 (29 76 ).
|
|
FSH-Stimulated Histone H3 Phosphorylation Is Inhibited by a PKA
Inhibitor
When GCs were pretreated 1 h with 10 µM H89, a
selective PKA inhibitor (30, 31, 32), FSH-stimulated phosphorylation of
histone H3 was inhibited (Fig. 10
, lanes 2 and 3), consistent with the ability of forskolin and
8-bromo-cAMP to mimic the actions of FSH to stimulate histone H3
phosphorylation. Coomassie blue staining of histones is shown for
reference (Fig. 10
, lower panel).

View larger version (75K):
[in this window]
[in a new window]
|
Figure 10. Effect of H89 on the Phosphorylation of Histones
H3 and H1 in GCs Stimulated by FSH
GCs labeled with 32P for 1 h were then incubated in the
presence of 10 µM H89 or vehicle for 1 h, and then in the
absence or presence of FSH (50 ng/ml) for 1 h. Cells were harvested and
subjected to TAU-PAGE as described in the legend to Fig. 9 . The
upper panel is the autoradiograph; lower panel
shows Coomassie blue staining of histone bands to assess protein
loaded. Results are representative of three experiments.
|
|
Phosphoamino Acid Analysis and Phosphopeptide Mapping
Both histones H3 and H1 were phosphorylated in FSH-treated GCs
exclusively on serine residues (Fig. 11A
). When two-dimensional peptide maps
were carried out on tryptic peptides prepared from histone H3
phosphorylated in GCs stimulated 1 h with FSH, two prominent
closely migrating spots were resolved (labeled 1 and 2; Fig. 11B
).
Histone H3 phosphorylated in vitro by addition of the
catalytic subunit of PKA revealed the same two spots as seen in
vivo in FSH-treated cells (spots 1 and 2) plus a third spot
("3"). The two closely migrating tryptic peptides (spots 1 and 2)
correspond to phosphorylation on Ser-10 (33, 34, 35, 36, 37) and derive from
incomplete cleavage by trypsin at Lys-14 (33, 36). The additional
tryptic peptide (spot 3) detected on phosphorylation with PKA in
vitro but not detected in in vivo studies corresponds
to phosphorylation on Thr-118, a site that is reported to be available
in DNA-free but not DNA-bound histone H3 (36).

View larger version (53K):
[in this window]
[in a new window]
|
Figure 11. Phosphoamino Acid Analysis of FSH-Stimulated
Histones and Two-Dimensional Phosphopeptide Mapping of FSH-Stimulated
Histone H3
In panel A, 32P-labeled GCs were treated with 50 ng/ml of
FSH for 1 h, harvested in 8 M urea buffer (buffer B),
and 5000 x g pellets were prepared. Phosphoamino
acid analysis was performed on histones H3 and H1 proteins (see
Materials and Methods), and resultant chromatograms were
exposed for autoradiography. The position of cold, stained phosphoamino
acid standards for each chromatogram is indicated by the dashed
ovals. The areas of radioactivity migrating below P-Tyr are
incompletely hydrolyzed protein. Results are representative of two
experiments. In upper panel, 32P-labeled GCs
were treated 1 h with 50 ng/ml FSH and harvested in protease
phosphatase inhibitor buffer (buffer A), and solubilized 5000 x
g pellets were subjected to SDS-PAGE. For the
lower panel, PKA catalytic subunit (50 U) was mixed with
10 µg histone H3, 50 mM Tris-HCl, pH 7.0, 10
µM [ -32P]ATP, and 10 mM
MgCl2, incubated 15 min at 30 C, and reaction mix was
subjected to SDS-PAGE. After digestion of phosphorylated histone H3
with tosylphenylalanine chloromethyl ketone-trypsin, sample was spotted
at the origin of the thin layer plate and mapped as described in
Materials and Methods, and the resultant chromatograms
were exposed for autoradiography. The phosphorylated tryptic peptides
are designated as 1, 2, and 3. The origin is marked by the .
|
|
FSH Stimulates Histone H3 Phosphorylation on Ser-10 in GCs Both in
Intact Rats and in Vitro
Finally, we compared histone H3 phosphorylation in GCs treated
in vitro with FSH vs. that in GCs obtained after
the subcutaneous injection of estrogen or PMSG to immature rats. FSH or
PMSG injection to immature rats not only promotes GC differentiation
but also accelerates GC proliferation (1, 2, 3, 38). Histone H3
phosphorylation on Ser-10 was detected in GCs using an antibody
specific for histone H3 phosphorylated on Ser-10 (39). The
phospho-specific histone H3 antibody proved to be more a sensitive
detector of H3 phosphorylation compared with H3 phosphorylation in
32P-labeled GCs. Phosphorylation of histone H3 on Ser-10
was detected by the phospho-histone H3 antibody 10 min after the
in vitro addition of FSH to GCs, peaked at 1 h, and was
decreased nearly to basal levels by 4 h, while total histone
protein (seen on Ponceau staining) was unchanged (Fig. 12
). Phosphorylation of CREB, detected
in the same samples using an antibody specific to CREB phosphorylated
on Ser-133, showed an equivalent time dependence to that of histone H3
phosphorylation. Histone H3 phosphorylation in GCs of intact rats after
in vivo hormone treatments is shown in Fig. 13
. By the fourth day of estrogen
treatment to immature rats, GCs cease to proliferate (38), and histone
H3 phosphorylation on Ser-10 was undetectable (Fig. 13
, lane 1). PMSG
injection promoted the abundant phosphorylation of histone H3 on Ser-10
(Fig. 13
, lanes 3 and 4). The in vivo phosphorylation of
CREB in response to PMSG injection showed an equivalent time dependence
to that of histone H3 phosphorylation, consistent with a report by
Mukherjee et al. (22) while CREB protein levels did not
change (22).

View larger version (42K):
[in this window]
[in a new window]
|
Figure 12. FSH Stimulates the in Vitro
Phosphorylation of Histone H3 on Ser-10
GCs were treated with 50 ng/ml FSH or vehicle for indicated times,
total cell extracts were separated by SDS-PAGE, proteins were blotted
onto Hybond, and blots were probed with antibodies specific for
phosphohistone H3 (Ser-10) or phospho-CREB (Ser-133). Ponceau staining
of histones is shown.
|
|

View larger version (60K):
[in this window]
[in a new window]
|
Figure 13. FSH Stimulates the in Vivo
Phosphorylation of Histone H3 on Ser-10
Immature 26-day-old rats were not injected (lane 2), were
injected subcutaneously with estrogen for 3 days, and ovaries obtained
on day 4 (lane 1), or were injected with 25 IU PMSG and ovaries were
obtained after 5 or 24 h. GCs were isolated by the same protocols
used to place cells in culture (14 ), requiring 2 h;
however, after counting cells, GCs were immediately lysed and proteins
were denatured by the addition of SDS-PAGE sample buffer, followed by
boiling for 20 min. Proteins in total cell lysates were separated by
SDS-PAGE and transferred to Hybond, and blots were stained for proteins
with Ponceau, and then probed with phosphohistone H3 antibody, which
detects phospho-Ser-10, and with phospho-CREB antibody, which detects
phospho-Ser-133.
|
|
 |
DISCUSSION
|
---|
These results show that FSH stimulates the phosphorylation of a
16-kDa protein in a hormone-specific and time-dependent manner in
immature GCs. On the basis of several characteristics, including size,
subcellular localization, solubility, immunoreactivity, and migration
patterns on SDS- and TAU-PAGE with histone standards, we conclude that
the 16-kDa protein is the core histone H3.
FSH-stimulated histone H3 phosphorylation in GCs is mimicked by
forskolin and 8-bromo-cAMP and inhibited by the PKA inhibitor H89,
consistent with a PKA-dependent pathway for FSH. However, the well
documented inhibitory effects of phorbol esters and EGF on
FSH-stimulated GC differentiation (11) are mediated either downstream
of histone H3 phosphorylation or by a distinct FSH-stimulated pathway,
based on the inability of EGF (50 ng/ml) or PMA (200 nM),
at concentrations exceeding those that inhibit FSH-stimulated GC
differentiation (10 ng/ml EGF, 10 nM PMA; reviewed in Ref.
11), to modulate FSH-stimulated histone H3 phosphorylation in GCs
coupled with the ability of 200 nM PMA even to modestly
stimulate histone H3 phosphorylation. FSH-stimulated histone H3
phosphorylation is not mediated by extracellular Ca2+ entry
through plasma membrane-localized Ca2+ channels and is not
inhibited by the tyrosine kinase inhibitor Ag18, suggesting that the
Ag18-sensitive step in the induction of P450 cholesterol side-chain
cleavage P450 (18, 19) is also either downstream of H3 phosphorylation
or is in a distinct FSH-stimulated pathway.
Phosphoamino acid and tryptic peptide analyses of H3 phosphorylated in
FSH-stimulated GCs showed serine phosphorylation and mapped as two
closely aligned spots on two-dimensional analysis corresponding to
Ser-10 (33, 34, 35, 36, 37). An identical phosphorylation pattern for Ser-10 was
seen when H3 was phosphorylated in vitro by PKA, as well as
phosphorylation at a second site corresponding to Thr-118, consistent
with previous reports (33, 36, 37, 40). FSH-stimulated phosphorylation
of histone H3 on Ser-10 was further confirmed using an antibody that
detects specifically histone H3 phosphorylated on Ser-10. Therefore,
since Ser-10 on H3 is phosphorylated both in vitro by PKA
and in intact GCs in a PKA-dependent manner in response to FSH, it is
concluded that FSH-stimulated phosphorylation of histone H3 on Ser-10
in GCs is catalyzed by PKA.
We also detected FSH-dependent phosphorylation of histone H1. H1 is
hyperphosphorylated on a number of different sites during the cell
cycle (41) by the 34-kDa cell division cycle and the 33-kDa
cyclin-dependent kinases to regulate progression through the cell cycle
(reviewed in Refs. 42, 43, 44). H1 is additionally phosphorylated in
vitro on Ser-37 by PKA (33, 45) and is phosphorylated in
vivo on Ser-37 in rat liver on stimulation by glucagon (reviewed
in Ref. 46) and in N18 neuroblastoma cells stimulated to differentiate
with forskolin or cAMP analogs (45). Notably, H1 phosphorylation showed
a different time course than that of H3, especially after 2 h when
the phosphorylation of H1 continued to increase while that of H3 was no
longer detectable. FSH-stimulated H1 phosphorylation was blocked in
cells treated with H89, consistent with FSH-stimulated H1
phosphorylation in GCs via PKA.
Phosphorylation of histone H3 has been well documented in mitotically
active cells and is coordinated with condensation of metaphase
chromosomes during mitosis (34, 39, 42, 47, 48). In Chinese hamster
ovary cells, histone H3 is phosphorylated only during mitosis and is
not phosphorylated at other phases of the cell cycle (47, 48, 49). Based on
evidence that histone H3 is phosphorylated on Ser-10 both in
vivo in cells synchronized in metaphase and in vitro in
metaphase chromosomes, phospho-Ser-10 has been tagged the
mitosis-specific phosphorylation site on histone H3 and is considered a
marker for mitosis (33, 39, 40). During mitosis,
50% of the total
histone H3 protein is phosphorylated (48).
The administration of FSH or PMSG to immature rats promotes both GC
differentiation as well as proliferation (1, 2), the latter documented
by increased [3H]thymidine incorporation (1, 38, 50).
Using the phospho-histone H3 antibody, which specifically recognizes
Ser-10 phosphorylation, we detected strong H3 phosphorylation in GCs of
rats injected with PMSG at times consistent with the accelerated
proliferative response of these cells to FSH (T. K. Woodruff,
personal communication) (38). However, FSH in the absence of TGFß,
activin, or other growth factors does not promote chromosome
condensation (M. Hunzicker-Dunn and J. Cottom, personal observation) or
entry of GCs in primary culture into the cell cycle (6, 7, 51, 52, 53).
Nevertheless, we detected transient histone H3 phosphorylation on
Ser-10 in response to FSH. Activin, which promotes GC proliferation
in vitro (7), did not stimulate H3 phosphorylation. In
contrast to the well documented hyperphosphorylation of histones H3 and
H1 during mitosis in a variety of cells, only a small percentage of
histones H3 and H1 appeared to be phosphorylated in isolated GCs
stimulated with FSH (see Figs. 2
, 8
, 10
). Similarly, a small fraction
of histone H3 protein was phosphorylated in quiescent C3H 101/2
cells in response to phorbol esters (54) concomitantly with induction
of early response genes c-fos and c-Jun leading to cellular
differentiation (54, 55). Consistent with evidence that relatively low
concentrations of 8-bromo-cAMP (<0.5 mM) enhance
activin-stimulated thymidine incorporation in GCs (7) while higher
concentrations (0.753 mM) inhibit thymidine uptake but
stimulate GC differentiation in a dose-dependent manner (7, 56),
histone H3 phosphorylation in GCs required 10 mM
8-bromo-cAMP, compatible with induction of differentiation. Histone H3
phosphorylation on Ser-10 in GCs in primary culture therefore occurs in
response to ligands that promote differentiation and not in response to
ligands that promote entry into the cell cycle. Use of the in
vitro cellular model, which supports FSH-stimulated GC
differentiation but not proliferation, therefore allows us to conclude
that histone H3 phosphorylation on Ser-10 in GCs in response to FSH is
linked not only to mitosis but also to GC differentiation.
Histones are highly conserved proteins that function to interact with
DNA and package the chromosomes (57). Each nucleosome consists of
160 bp of DNA wound in two turns around two molecules each of the
four core histones: a tetramer of (H3/H4)2 flanked by two
dimers of (H2A/H2B) (57). Between adjacent nucleosomes is a variable
length of linker DNA. H1 binds to the outer surface of the DNA that
encircles the core histones as well as to the stretches of linker DNA
that connect nucleosomes (58). Of the core histones (H2A, H2B, H3, H4),
histone H3 is believed to play an important role in modulating the
transcriptional activity of specific genes. Histone H3 in an
underacetylated and likely underphosphorylated state interacts, through
its amino terminus, with a family of recently identified proteins to
repress transcription (59, 60, 61). Modifications of H3 that neutralize its
positive charge, such as acetylation or phosphorylation, are known to
alter the conformation of the nucleosome, weakening the association of
H3 with DNA (62, 63). Unfolding of the nucleosome and associated
transcriptional activation have been linked to the altered conformation
of histone H3 (64, 65) resulting from its acetylation (66, 67).
However, the in vitro phosphorylation of histone H3 by PKA
in the N-terminal region has also been shown to change the conformation
of the C-terminal end of the protein, evidenced by reduced binding of
an H3 C-terminal peptide-specific antibody (68). Direct
evidence that histone H3 can participate in regulation of
transcription of specific genes in quiescent cells comes from a
recent report that shows that H3 in a growth factor-dependent manner
can function as a coactivator of the CTF/NF-1 and SP1 families of
transcription factors, binding to specific sites in the transactivation
domain of these transcription factors (63, 69, 70). We hypothesize that
the general transcriptional activation induced by FSH in GCs, leading
to activation of such genes as RIIß, LH receptor, P450 side chain
cleavage, and aromatase, requires histone H3 phosphorylation on Ser-10
to relax nucleosomal structure, thereby promoting access of
transcription factor complexes, such as CREB and CREB-binding proteins,
to DNA promoter regions. It is also possible that H3 in its
phosphorylation-dependent altered conformation can itself function as a
coactivator of cAMP-responsive genes.
In summary, these results establish that FSH via PKA promotes the
phosphorylation of the core histone H3 on Ser-10 and identify
PKA-dependent histone H3 phosphorylation as an early nuclear marker for
FSH-stimulated differentiation of GCs to a preovulatory phenotype.
 |
MATERIALS AND METHODS
|
---|
Materials
Ovine FSH (oFSH-16) was obtained from NIDDK-NIH, and
purified recombinant human activin A was obtained from Dr. Teresa
Woodruff. Monoclonal antihistone and polyclonal anti-H3 antibodies were
generously provided by Drs. Marc Monestier (71) and Sylviane Muller
(68), respectively. The following were purchased: urea and pure calf
thymus histones from Boehringer Mannheim (Indianapolis, IN);
4-(2-aminoethyl)-benzensulfonylfluoride-HCl (AEBSF) from Calbiochem (La
Jolla, CA); PAGE reagents from Bio-Rad (Richmond, CA); AG18 from Alexis
Biochemicals (San Diego, CA); recombinant human EGF from
Collaborative Research, Inc. (Waltham, MA,); X-Omat AR film and
phosphocellulose chromatogram sheets without fluorescent indicator from
Kodak (Eastman Kodak, Rochester, NY); enhanced chemiluminescence (ECL)
reagents from Amersham (Arlington Heights, IL); antiphosphohistone H3
(Ser-10 specific) and antiphospho-CREB (Ser-133 specific) antibodies
from Upstate Biotechnology (Lake Placid, NY); anti-PKA catalytic
subunit antibody from Transduction Laboratory (Lexington, KY). All
culture media (GIBCO/BRL) and PBS were prepared by the Cancer Center of
Northwestern University Medical School. All other chemicals were from
Sigma Chemical Co. (St. Louis, MO).
Primary GC Cultures
GCs were isolated from ovaries of 26-day-old
Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA),
maintained in accordance with the NIH Guide for the Care and Use of
Laboratory Animals, and primed with subcutaneous injections of 1.5 mg
estradiol-17ß in 0.1 ml propylene glycol per day on days 2325 to
promote growth of preantral follicles (72). Cells were plated at a
density of 57 x 105 cells/ml on fibronectin (24
µg/cm2)-coated dishes in serum-free DMEM/Hams F-12
(DMEM/F-12, 1:1) with 10 nM estradiol-17ß and cultured as
previously described (14). Concentrated stocks of test additions were
diluted with indicated vehicles (media for FSH, EGF, PMA, 8-bromo-cAMP;
50% ethanol for forskolin; 50% dimethylsulfoxide for H89; 100%
ethanol for AG18) and then added to culture dishes at 200-fold dilution
to achieve the final concentration indicated in the text. Control
refers to media addition; vehicle refers to specific vehicle other than
media.
[32P]Orthophosphate Labeling of
Cells
Approximately 20 h after plating, culture media were
removed and cells were incubated for 1 h in phosphate-free
DMEM/F-12; 0.5 mCi [32P]orthophosphate (85009120
Ci/mmol; DuPont NEN, Boston, MA) per 60-mm dish was added for a second
hour. Indicated additions were then made to dishes containing equal
numbers of cells at doses and times specified for each experiment. To
terminate treatments, medium was aspirated, and cells were rinsed once
with PBS and harvested in either a protease and phosphatase
inhibitor-enriched hypotonic buffer (buffer A) containing 10
mM potassium phosphate (pH 7.0), 1 mM EDTA, 5
mM EGTA, 10 mM MgCl2, 2
mM dithiothreitol, 1 mM
Na3VO4, 80 mM ß-glycerophosphate,
100 µg/ml pepstatin-A, 21 µM leupeptin, and 1
mM AEBSF (73), or 8 M urea buffer (buffer B)
containing 5% ß-mercaptoethanol and 2% Nonidet P-40 (26). Cells
were then sonicated on ice 1 min and centrifuged at 5000 x
g at 4C for 10 min to prepare supernatant and pellet
fractions. Pellets were resuspended in the appropriate gel sample
buffer or acid extracted as described below. Alternatively, total cell
extracts were prepared when GCs were harvested in 2-fold diluted
SDS-PAGE sample buffer and boiled 20 min. Final volumes of supernatant
and pellet fractions in sample buffer were equal for each
experiment.
Histone Extraction
Histones were acid extracted from pellet fractions of
sonicated cells by modifications of published protocols (45, 46).
Briefly, each pellet was twice extracted with cold 5% perchloric acid,
resulting in a supernatant that contained histone H1 and the high
mobility group proteins (40). The core histones (H2A, H2B, H3, and H4)
remained in the pellet (40). The pellet was resuspended in water, and
proteins in both fractions were precipitated by adding cold 100%
trichloroacetic acid to a final concentration of 20%. Samples were
incubated on ice (15 min) and then centrifuged at 15,000 x
g. Precipitates were washed once with cold acid acetone and
twice with cold acetone. Residual acetone was removed by speed vacuum
centrifugation. Pellets were solubilized in 2-fold diluted SDS-PAGE
sample buffer, boiled 20 min, and placed at -70 C.
Electrophoresis
Proteins in samples denatured in SDS-PAGE sample buffer were
separated by SDS-PAGE using a 5% acrylamide stacking gel and 12.5%
acrylamide resolving gel at 50 mA/gel (74). Western blots were
performed as previously described (75), except that detection of
antigen-antibody complexes was by ECL on Hybond nitrocellulose
membranes. For TAU-PAGE, pellets prepared from GCs harvested in 8
M urea buffer (buffer B) were solubilized at room
temperature in an isoelectric focusing sample buffer containing 8
M urea, 2.5% Triton X-100, Pharmalyte (80%, pH 810.5;
20%, pH 310) and 0.01% Pyronin Y. Samples were run on a 12%
acrylamide, 0.1% bis-acrylamide gel containing 0.4% Triton X-100, 0.9
M acetic acid, and 2.5 M urea with a running
buffer of 0.9 M acetic acid and 0.1% Triton X-100 (29, 76). Before sample loading, the gel was preelectrophoresed at 130 V for
2 h, the last 30 min in the presence of 1 mM
thioglycolic acid in the upper running buffer. Sample electrophoresis
was from the anode to the cathode at 130 V for 3 h. Both SDS-PAGE
and TAU-PAGE gels were processed for autoradiography as previously
described (74).
PKA Assays
Cells were treated as indicated, rinsed, and sonicated at 4 C in
buffer (buffer C) containing 20 mM HEPES, pH 7.4, 20
mM NaCl, 5 mM EDTA, 2 mM
dithiothreitol, 1 mM EGTA, 5 µg/ml pepstatin, 5 µg/ml
aprotinin, 10 µg/ml leupeptin, 50 µg/ml soybean trypsin inhibitor,
10 mM benzamidine (20), 10 mM
MgCl2, and 0.2% Triton X-100, and centrifuged 1 min at
20,000 x g. Pellets were mixed with SDS-PAGE sample
buffer and boiled 20 min for PKA catalytic subunit western. Kemptide
kinase activity was measured in triplicate in supernatant fractions
containing 30 µg protein in the absence and presence of 0.5
µM cAMP and in the absence and presence of 2
µM PKA inhibitor peptide (PKI) (20). PKI-insensitive
kinase activity was subtracted, so that results represent
PKI-inhibitable PKA activity.
Phosphoamino Acid Analysis
Proteins in GC samples were separated by SDS-PAGE and
electroblotted onto Immobilon-P, stained, and exposed for
autoradiography (77). Using the autoradiogram as a guide, the
phosphorylated protein bands of interest were excised from Immobilon-P
and hydrolyzed in 6 N HCl at 110 C for 1 h (78).
Recovery of radioactivity from Immobilon-P was monitored by Cerenkov
counting of the membrane before and after hydrolysis. The hydrolysate
was dried in vacuo, washed with water, and dried several
times. The sample was then redissolved in 3 µl of running buffer,
acetic acid-pyridine-water at 50:5:945, vol/vol/vol, pH 3.5 (79).
Samples and phosphoamino acid standards (2 mg/ml) were electrophoresed
on phosophocellulose TLC plates in one dimension (FBE-3000, Pharmacia,
Piscataway, NJ) at 750 V for 75 min. Plates were dried, stained with
0.2% ninhydrin, dried, and exposed for autoradiography.
Peptide Mapping
The Coomassie blue-stained protein band corresponding to histone
H3 was excised from the dried SDS-polyacrylamide gel, cut into small
pieces, and eluted. Eluted histone H3 was precipitated with
trichloroacetic acid, oxidized with performic acid, and digested twice
with 10 µg tosylphenylalanine chloromethyl ketone-trypsin
(Worthington Biochemical Corp., Freehold, NJ) as described by Boyle
et al. (80). Digested protein was desalted on a 10 ml G-25
Sephadex column and subjected to phosphopeptide mapping by
electrophoresis on a 20 x 20 cm TLC plate (EM Science, Gibbstown,
NJ) using an HTLE-7000 apparatus (C.B.S. Scientific Co., Del
Mar, CA). Electrophoresis in the first dimension was in pH 4.7
electrophoresis buffer (butanol-acetic acid-H20-pyridine,
50:25:900:25) (81) at 600 V for 50 min. Plates were dried and ascending
chromatography was performed in a solution of butanol-acetic
acid-H2O-pyridine (75:150:600:500) (81). TLC plates were
then subjected to autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We gratefully acknowledge the gifts of purified monoclonal
antihistone antibodies from Dr. Marc Monestier, Temple University
School of Medicine (Philadelphia, PA), and of antihistone H3 polyclonal
antibody from Dr. Sylviane Muller, Centre National De La Recherche
Scientifique, Institut de Biologie Moléculaire & Cellulaire
(Strasbourg, France).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Mary Hunzicker-Dunn, Department of Cell and Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, Illinois 60611. E-mail:
mhd{at}nwu.edu
This work was funded by NIH Grants PO1 HD-21921 (to M.H.D.), NRSA F32
HD-07244 (to D.A.D.), and by the P30 Center for Reproductive Science,
Northwestern University (HD-28048). Preliminary results were presented
at the 25th and 26th Annual Meetings of the Society for the Study of
Reproduction, Raleigh, NC, 1992 and Fort Collins, CO, 1993,
respectively.
1 Present address: Fertility Pregnancy and Neurodiagnostics, Research
and Development, Abbott Laboratories, One Abbott Park Road,
Abbott Park, Illinois 60064-3500. 
2 A lower concentration of PMA (100
nM) yielded barely detectable phosphorylation of the 16-kDa
protein (not shown). 
Received for publication February 10, 1998.
Revision received September 11, 1998.
Accepted for publication October 2, 1998.
 |
REFERENCES
|
---|
-
Hirshfield AN 1991 Development of follicles in the
mammalian ovary. Int Rev Cytol 124:43101[Medline]
-
Robker RL Richards JS 1998 Hormone-induced
proliferation and differentiation of granulosa cells: a coordinated
balance of the cell cycle regulators cyclin D2 and p27Kip1.
Mol Endocrinol 12:924940[Abstract/Free Full Text]
-
Hsueh AJW, Adashi EY, Jones PBC, Welsh Jr TH 1984 Hormonal regulation of the differentiation of cultured ovarian
granulosa cells. Endocr Rev 5:76110[Medline]
-
Orly J, Sato G, Erickson GF 1980 Serum suppresses
the expression of hormonally induced functions in cultured granulosa
cells. Cell 20:817827[Medline]
-
Bendell JJ, Dorrington JH 1990 Epidermal growth
factor influences growth and differentiation of rat granulosa
cells. Endocrinology 127:533540[Abstract]
-
Dorrington J, Chuma AV, Bendell JJ 1988 Transforming growth factor B and follicle-stimulating hormone promote
rat granulosa cell proliferation. Endocrinology 123:353359[Abstract]
-
Miro F, Hillier SG 1996 Modulation of granulosa
cell deoxyribonucleic acid synthesis and differentiation by activin.
Endocrinology 137:464468[Abstract]
-
Peluso JJ, Delidow BC, Lynch J, White BA 1991 Follicle-stimulating hormone and insulin regulation of 17-B estradiol
secretion and granulosa cell proliferation within immature rat ovaries
maintained in perifusion culture. Endocrinology 128:191196[Abstract]
-
Aittomaki K, Lucena JLD, Pakarinen P, Sistonen P,
Tapanainen J, Gromoll J, Sankila E-M, Lehvasiaho H, Engel AR, Nieschlag
E, Huhtaniemi L, de la Chapelle A 1995 Mutation in the
follicle-stimulating hormone receptor gene causes hereditary
hypergonadotropin ovarian failure. Cell 82:959968[Medline]
-
Kumar TR, Wang Y, Lu N, Matzuk MM 1997 Follicle
stimulating hormone is required for ovarian follicle maturation but not
male fertility. Nat Genet 15:201204[Medline]
-
Hsueh AJW, Bicsak TA, Jia X-C, Dahl KD, Fauser BCJM,
Galway AB, Czekala N, Pavlou SN, Papkoff H, Keene J, Boime I 1989 Granulosa cells as hormone targets: the role of biologically
active follicle-stimulating hormone in reproduction. Recent Prog Horm
Res 45:209273[Medline]
-
Amsterdam A, Rotmensch S 1987 Structure-function
relationships during granulosa cell differentiation. Endocr Rev 8:309337[Medline]
-
Richards JS 1980 Maturation of ovarian follicles:
actions and interactions of pituitary and ovarian hormones on
follicular cell differentiation. Physiol Rev 60:5189[Free Full Text]
-
Carr DW, DeManno DA, Atwood A, Hunzicker-Dunn M,
Scott JD 1993 Follicle-stimulating hormone regulation of A-kinase
anchoring proteins in granulosa cells. J Biol Chem 268:2072920732[Abstract/Free Full Text]
-
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 384:470474[CrossRef][Medline]
-
Flores JA, Veldhuis JD, Leong DA 1990 Follicle-stimulating hormone evokes an increase in intracellular free
calcium ion concentrations in single ovarian (granulosa) cells.
Endocrinology 127:31723179[Abstract]
-
Flores JA, Leong DA, Veldhuis JD 1992 Is the calcium
signal induced by follicle-stimulating hormone in swine granulosa cells
mediated by adenosine cyclic 3',5'-monophosphate-dependent protein
kinase? Endocrinology 130:18621866[Abstract]
-
Gomberg-Malool S, Ziv R, Reem Y, Posner I,
Levitski A, Orly J 1993 Tyrphostins inhibit follicle-stimulating
hormone-mediated functions in cultured rat ovarian granulosa cells.
Endocrinology 132:362370[Abstract]
-
Orly J, Rei Z, Greenberg NM, Richards JS 1994 Tyrosine kinase inhibitor AG18 arrests follicle-stimulating
hormone-induced granulosa cell differentiation: use of reverse
transcriptase-polymerase chain reaction assay for multiple messenger
ribonucleic acids. Endocrinology 134:23362346[Abstract]
-
Das S, Maizels ET, DeManno D, St.Clair E, Adam SA,
Hunzicker-Dunn M 1996 A stimulatory role of cyclic adenosine
3',5'-monophosphate in follicle-stimulating hormone-activated
mitogen-activated protein kinase signaling pathway in rat ovarian
granulosa cells. Endocrinology 137:967974[Abstract]
-
Maizels ET, Cottom J, Jones JR, Hunzicker-Dunn M 1998 Follicle stimulating hormone (FSH) activates the p38
mitogen-activated kinase pathway, inducing small heat shock protein
phosphorylation and cell rounding in immature rat ovarian granulosa
cells. Endocrinology 139:33533356[Abstract/Free Full Text]
-
Mukherjee A, Park-Sarge OK, Mayo KE 1996 Gonadotropins induce rapid phosphorylation of the 3',5'-cyclic
adenosine monophosphate response element binding protein in ovarian
granulosa cells. Endocrinology 137:32343245[Abstract]
-
Adashi EY, Resnick CE, DErcole J, Svoboda ME, Van
Wyk JJ 1985 Insulin-like growth factors as intraovarian regulators of
granulosa cell growth and function. Endocr Rev 6:400420[Abstract]
-
Bley MA, Simon JC, Saragueta PE, Baranao JL 1991 Hormonal regulation of rat granulosa cell deoxyribonucleic acid
synthesis: effects of estrogens. Biol Reprod 44:880888[Abstract]
-
Zackroff RV, Goldmann RD 1979 In vitro assembly of
intermediate filaments from baby hamster kidney (BHK-21) cells. Proc
Natl Acad Sci USA 76:62266230[Abstract]
-
Bub B, Temm-Grove C, Henning S, Jockusch BM 1992 Distribution of profilin in fibroblasts correlates with the presence of
highly dynamic actin filaments. Cell Motil Cytoskeleton 22:5161[Medline]
-
Wu RS, Panasz HT, Hatch CL, Bonner WM 1986 Histones
and their modifications. Crit Rev Biochem 20:201263[Medline]
-
Hnilica LS, Grimes SR, Chiu J-F 1978 Electrophoretic
fractionation of histones utilizing starch gels and and sodium dodecyl
sulfate-urea gels. Methods Cell Biol 17:211222[Medline]
-
Zweidler A 1978 Resolution of histones by
polyacrylamide gel electrophoresis in presence of ionic detergents.
Methods Cell Biol 17:223233[Medline]
-
Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayaashi
K, Inoue T, Naito K, Toshioka T, Hidaka H 1990 Inhibition of
forskolin-induced neurite outgrowth and protein phosphorylation by a
newly synthesized selective inhibitor of cyclic AMP-dependent protein
kinase, N-2[-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide
(H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:52675272[Abstract/Free Full Text]
-
Hidaka H, Kobayashi R 1992 Pharmacology of protein
kinase inhibitors. Annu Rev Pharmacol Toxicol 32:377397[CrossRef][Medline]
-
Morris JK, Richards JS 1995 Luteinizing hormone
induces prostaglandin endoperoxide synthase-2 and luteinization
in vitro by A-kinase and C-kinase pathways. Endocrinology 136:15491558[Abstract]
-
Taylor SS 1982 The in vitro
phosphorylation of chromatin by the catalytic subunit of cAMP-dependent
protein kinase. J Biol Chem 257:60566063[Abstract/Free Full Text]
-
Ajiro K, Nishimoto T 1985 Specific site of histone
H3 phosphorylation related to the maintenance of premature chromosome
condensation. J Biol Chem 260:15379 15381[Abstract/Free Full Text]
-
Ajiro K, Yoda K, Utsumi K, Nishikawa Y 1996 Alteration of cell cycle-dependent histone phosphorylations by okadaic
acid. J Biol Chem 271:1319713201[Abstract/Free Full Text]
-
Shibata K, Inagaki M, Ajiro K 1990 Mitosis-specific
histone H3 phosphorylation in vitro in nucleosome
structures. Eur J Biochem 192:8793[Abstract]
-
Shibata K, Ajiro K 1993 Cell cycle-dependent
suppressive effect of histone H1 on mitosis-specific H3
phosphorylation. J Biol Chem 268:1843118434[Abstract/Free Full Text]
-
Rao MC, Midgley Jr JS, Richards JS 1978 Hormonal
regulation of ovarian cellular proliferation. Cell 14:7176[Medline]
-
Hendzel MJ, Wei Y, Mancini MA, VanHooser A, Ranalli
T, Brinkley BR, Bazett-Jones DP, Allis CD 1997 Mitosis-specific
phosphorylation of histone H3 initiates primarily within
pericentromeric heterochromatin during G2 and spreads in an ordered
fashion coincident with mitotic chromosome condensation. Chromosoma 106:348360[CrossRef][Medline]
-
Paulson JR, Taylor SS 1982 Phosphorylation of
histones 1 and 3 and nonhistone high mobility group 14 by an endogenous
kinase in HeLa metaphase chromosomes. J Biol Chem 257:60646072[Abstract/Free Full Text]
-
Langan TA, Zeilig C, Leichtling B 1981 Characterization of multiple-site phosphorylation of H1 histone in
proliferating cells. Cold Spring Harbor Conf Cell Proliferation 8:10391052
-
Bradbury EM 1992 Reversible histone modifications
and the chromosome cell cycle. Bioessays 14:915[Medline]
-
Sherr CJ 1993 Mammalian G1 cyclins. Cell 73:1059
1065[Medline]
-
Pines J 1993 Cyclins and cyclin-dependent kinases:
take your partners. Trends Biochem Sci 18:195197[CrossRef][Medline]
-
Ajiro K, Shibata K, Nishikawa Y 1990 Subtype-specific cyclic AMP-dependent histone H1 phosphorylation at the
differentiation of mouse neuroblastoma cells. J Biol Chem 265:64946500[Abstract/Free Full Text]
-
Langan TA 1978 Methods for the assessment of
site-specific histone phosphorylation. Methods Cell Biol 19:127143[Medline]
-
Gurley LR, Valdez JG, Buchanan JS 1995 Characterization of the mitotic specific phosphorylation of histone H1.
J Biol Chem 270:2765327660[Abstract/Free Full Text]
-
Gurley LR, DAnna JA, Barham SS, Deaven LL, Tobey
RA 1978 Histone phosphorylation and chromatin structure during mitosis
in Chinese hamster cells. Eur J Biochem 84:115[Medline]
-
Gurley LR, Walters RA, Tobey RA 1975 Sequential
phosphorylation of histone subfractions in the Chinese hamster cell
cycle. J Biol Chem 250:39363944[Abstract]
-
Woodruff TK, Lyon RJ, Hansen SE, Rice GC, Mather JP 1990 Inhibin and activin locally regulate rat ovarian folliculogenesis.
Endocrinology 127:31963205[Abstract]
-
Anderson E, Lee GY 1993 The participation of growth
factors in simulating the quiescent, proliferative, and differentiative
stages of rat granulosa cells grown in a serum-free medium. Tissue Cell 25:4972[CrossRef][Medline]
-
Bendell JJ, Dorrington J 1988 Rat
thecal/interstitial cells secrete a transforming growth factor-B-like
factor that promotes growth and differentiation of rat granulosa cells.
Endocrinology 123:941948[Abstract]
-
Adashi EY, Resnick CE, Svoboda ME, Van Wyk JJ 1985 Somatomedin-C synergizes with follicle-stimulating hormone in the
acquisition of progestin biosynthetic capacity by cultured rat
granulosa cells. Endocrinology 116:21352142[Abstract]
-
Mahadevan LC, Willis AC, Barratt MJ 1991 Rapid
histone H3 phosphorylation in response to growth factors, phorbol
esters, okadaic acid, and protein synthesis inhibitors. Cell 65:775783[Medline]
-
Barratt MJ, Hazzalin CA, Cano E, Mahedevan LC 1994 Mitogen-stimulated phosphorylation of histone H3 is targeted to a small
hyperacetylation-sensitive fraction. Proc Natl Acad Sci USA 91:47814785[Abstract]
-
Knecht M, Amsterdam A, Catt K 1981 The regulatory
role of cyclic AMP in hormone-induced granulosa cell differentiation.
J Biol Chem 256:1062810633[Free Full Text]
-
Wolffe AP 1992 New insights into chromatin function
in transcriptional control. FASEB J 6:33543361[Abstract/Free Full Text]
-
Wolffe AP 1994 Nucleosome positioning and
modification: chromatin structures that potentiate transcription.
Trends Biochem Sci 19:240244[CrossRef][Medline]
-
Bortvin A, Winston F 1996 Evidence that Spt6p
controls chromatin structure by a direct interaction with histones.
Science 272:14731476[Abstract]
-
Palaparti A, Baratz A, Stifani S 1997 The
groucho/transducin-like enhancer of split transcriptional repressors
interact with the genetically defined amino-terminal silencing domain
of histone H3. J Biol Chem 272:2660426610[Abstract/Free Full Text]
-
Wan JS, Mann RK, Grunstein M 1995 Yeast histone H3
and H4 N termini function through different GAL1 regulatory elements to
repress and activate transcription. Proc Natl Acad Sci USA 92:56645668[Abstract]
-
Wu C 1997 Chromatin remodeling and the control of
gene expression. J Biol Chem 272:2817128174[Free Full Text]
-
Wolffe AP, Pruss D 1996 Chromatin: hanging on to
histones. Curr Biol 6:234237[Medline]
-
Chen TA, Allfrey VG 1987 Rapid and reversible
changes in nucleosome structure accompany the activation, repression,
and superinduction of murine fibroblast protooncogenes c-fos and
c-myc. Proc Natl Acad Sci USA 84:52525256[Abstract]
-
Walia H, Chen HY, Sun J-M, Holth LT, Davie JR 1998 Histone acetylation is required to maintain the unfolded nucleosome
structure associated with transcribing DNA. J Biol Chem 23:1451614522[CrossRef]
-
Kuo MH, Zhou J, Jambeck P, Churchill ME, Allis CD 1998 Histone acetyltransferase activity of yeast Gcn5p is required for
the activation of target genes in vivo. Genes Dev 12:627639[Abstract/Free Full Text]
-
Struhl K 1998 Histone acetylation and
transcriptional regulatory mechanisms. Genes Dev 12:599606[Free Full Text]
-
Muller S, Mazen A, Martinage A, Van Regenmortel MHV 1984 Use of histone antibodies for studying chromatin topography and
the phosphorylation of chromatin subunits. EMBO J 3:24312436[Abstract]
-
Alevizopoulos A, Dusserre Y, Tsai-Pflugfelder M, von
der Weid T, Wahli W, Mermod N 1995 A proline-rich TGF-beta-responsive
transcriptional activator interacts with histone H3. Genes Dev 9:30513066[Abstract]
-
Alevizopoulos A, Mermod N 1996 Antagonistic
regulation of a proline-rich transcription factor by transforming
growth factor beta and tumor necrosis factor alpha. J Biol Chem 271:2967229681[Abstract/Free Full Text]
-
Monestier M, Fasy TM, Bohm L 1989 Monoclonal
anti-histone H1 autoantibodies from MRL lpr/lpr mice. Mol Immunol 266:749758[CrossRef]
-
Ratoosh SL, Lifka J, Hedin L, Jahnsen T, Richards JS 1987 Hormonal regulation of the synthesis and mRNA content of the
regulatory subunit of cyclic AMP-dependent protein kinase type II in
cultured rat ovarian granulosa cells. J Biol Chem 262:73067313[Abstract/Free Full Text]
-
Meier KE, Licciardi KA, Haystead TAJ, Krebs EG 1991 Activation of messenger-independent protein kinases in wild-type and
phorbol ester-resistant EL4 thymoma cells. J Biol Chem 266:19141920[Abstract/Free Full Text]
-
Hunzicker-Dunn M 1981 Selective activation of rabbit
ovarian protein kinase isozymes in rabbit ovarian follicles and corpora
lutea. J Biol Chem 256:1218512193[Abstract/Free Full Text]
-
Hunzicker-Dunn M, Cutler Jr RE, Maizels ET, DeManno
DA, Lamm MLG, Erlichman J, Sanwal BD, LaBarbera AR 1991 Isozymes
of cAMP-dependent protein kinase present in the rat corpus luteum.
J Biol Chem 266:71667175[Abstract/Free Full Text]
-
Hardison R, Chalkley R 1978 Polyacrylamide gel
electrophoretic fractionation of histones. Methods Cell Biol 17:235251[Medline]
-
Ji H, Baldwin GS, Burgess AW, Mortiz RL, Ward LD,
Simpson RJ 1993 Epidermal growth factor induces serine phosphorylation
of stathmin in a human colen carcinoma cell line (LIM 1215). J
Biol Chem 268:1339613405[Abstract/Free Full Text]
-
Kamps MP, Sefton BM 1989 Acid and base hydrolysis of
phosphoproteins bound to immobilon facilitates analysis of phosphoamino
acids in gel-fractionated proteins. Anal Biochem 176:2227[Medline]
-
Hunter T, Sefton BB 1980 Transforming gene product
of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci USA 77:13111315[Abstract]
-
Boyle WJ, van der Geer P, Hunter T 1991 Phosphopeptide mapping and phosphoamino acid analysis by
two-dimensional separation on thin-layer cellulose plates. Methods
Enzymol 202:110148
-
Ajiro K, Borun TW, Shulman SD, McFadden GM, Cohen LH 1981 Comparison of the structure of human histones 1A and 1B and their
intramolecular phosphorylation sites during the Hela S-3 cell cycle.
Biochem J 20:14541464