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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{alpha}, 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo, 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. 1BGo).



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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. 2Go, 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. 2Go, lanes 2 and 3). Phosphorylation of the 16-kDa protein was also stimulated by 10 µM forskolin (Fig. 2Go, 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. 2Go, lane 7). Phosphorylation of the 16-kDa band in response to FSH was consistently detectable by 30 min, elevated at 60 min (Fig. 3AGo), and undetectable at 3 h (see Fig. 7Go), 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. 3BGo).



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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 4–8), 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.

 


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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. 2Go. Results in panels A and B are representative of five and three experiments, respectively.

 


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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 10–180 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. 4AGo). 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. 4AGo). Phosphorylation of the 16-kDa protein was not stimulated by either EGF at 50 ng/ml (Fig. 4BGo) 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. 5AGo) 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. 5BGo). 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. 5CGo).



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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.

 


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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. 2Go. 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. 6Go (lanes 5–8), 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. 6Go, lanes 1–4). On two-dimensional isoelectric focussing (pH 3–10) 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. 6Go, lanes 9–12), 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).



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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. 7AGo) core histones (H) H2A, H2B, H3, and H4 from the acid-soluble (Fig. 7BGo) histone H1. The 16-kDa protein was insoluble in 5% perchloric acid (Fig. 7AGo), 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. 7AGo). 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. 7BGo). 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. 7BGo). 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. 8AGo, left panel), the 16-kDa phosphoprotein (Fig. 8AGo, right panel) corresponded to histone H3. This conclusion was supported by the migration position of immunoreactive histones (Fig. 8BGo). 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. 9Go, left panel). As seen on the autoradiogram (Fig. 9Go, 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.



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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 4–9; 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 2–4). 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.

 


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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 3–7. 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. 10Go, 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. 10Go, lower panel).



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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. 9Go. 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. 11AGo). 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. 11BGo). 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).



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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 [{gamma}-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. 12Go). 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. 13Go. 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. 13Go, lane 1). PMSG injection promoted the abundant phosphorylation of histone H3 on Ser-10 (Fig. 13Go, 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).



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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.

 


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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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 2Go, 8Go, 10Go). 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 (0.75–3 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 23–25 to promote growth of preantral follicles (72). Cells were plated at a density of 5–7 x 105 cells/ml on fibronectin (2–4 µg/cm2)-coated dishes in serum-free DMEM/Ham’s 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 (8500–9120 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 8–10.5; 20%, pH 3–10) 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. Back

2 A lower concentration of PMA (100 nM) yielded barely detectable phosphorylation of the 16-kDa protein (not shown). Back

Received for publication February 10, 1998. Revision received September 11, 1998. Accepted for publication October 2, 1998.


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