Functional and Subcellular Changes in the A-Kinase-Signaling Pathway: Relation to Aromatase and Sgk Expression during the Transition of Granulosa Cells to Luteal Cells

Ignacio J. Gonzalez-Robayna, Tamara N. Alliston, Patricia Buse, Gary L. Firestone and JoAnne S. Richards

Department of Cell Biology (I.J.G.R., T.A., J.S.R.) Baylor College of Medicine Houston, Texas 77030
Department of Molecular and Cell Biology (P.B., G.L.F.) University of California at Berkeley Berkeley, California 94720


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The responsiveness of granulosa cells to FSH (cAMP) changes as these cells switch from the proliferative stage in growing follicles to the terminally differentiated, nonproliferating stage after LH-induced luteinization. To analyze this transition, two well characterized culture systems were used. 1) Granulosa cells isolated from immature rats were cultured in serum-free medium, a system that permits analysis of dynamic, short-term responses to hormones/cAMP. 2) Granulosa cells from preovulatory (PO) follicles that have been exposed in vivo to surge concentrations of hCG (PO/hCG) were cultured in medium containing 1% FBS, a system that permits analyses of cells that have undergo irreversible, long-term changes associated with luteinization. To analyze the biochemical basis for the switch in cAMP responsiveness, the localization of A-kinase pathway components was related to the expression of two cAMP target genes, aromatase (CYP19) and serum-and glucocorticoid-induced kinase (Sgk). Components of the A-kinase pathway were analyzed by Western blotting and indirect immunofluorescence using specific antibodies to the C subunit, RII{alpha}/ß subunits, CREB (cAMP-regulatory element binding protein), phospho-CREB, CBP (CREB binding protein), and Sgk. Cellular levels of C subunit and CREB were similar in all cell types and hormone treatments. CREB and CBP were nuclear; RII{alpha}/ß was restricted to a cytoplasmic basket-like structure. Addition of FSH to immature granulosa cells caused rapid nuclear import of C subunit within 1 h. Nuclear C subunit decreased by 6 h after FSH but could be rapidly reimported to the nucleus by the addition of forskolin at 6, 24, or 48 h. Nuclear C subunit was associated with the rapid but transient increases in phospho-CREB. FSH induced Sgk in a biphasic manner in which the protein was nuclear at 1 h and cytoplasmic at 48 h. Aromatase mRNA was only expressed at 24–48 h after FSH, a pattern that was not altered by phosphodiesterases or phosphatases. In the luteinized (PO/hCG) granulosa cells, immunoreactive C subunit was localized in a punctate pattern in the nucleus as well as to a cytoplasmic basket-like structure, a distribution pattern not altered by forskolin. Aromatase, Sgk, and phospho-CREB were expressed at elevated levels in a non-forskolin-responsive manner. Most notable, both phospho-CREB and Sgk were preferentially localized in a punctate pattern within the cytoplasm and not altered by forskolin. Collectively, these data indicate that when granulosa cells differentiate to luteal cells the subcellular localization (nuclear vs. cytoplasmic) of A-kinase pathway components changes markedly. Thus, either the mechanisms of nuclear import and export or the presence of distinct docking sites (and functions ?) dictate where A-kinase, phospho-CREB and Sgk are localized in granulosa cells compared with the terminally differentiated luteal cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pituitary glycoproteins, FSH and LH, by binding to their cognate receptors stimulate adenylyl cyclase activity and increase the production of the intracellular signal, cAMP. cAMP, in turn, activates cAMP-dependent protein kinase (A-kinase), which leads to the phosphorylation of critical cellular proteins, some of which control the transcription of specific genes (1, 2, 3, 4).

The A-kinase pathway itself is regulated by many factors that control its cellular location and state of activation. For example, the catalytic (C) subunit of A-kinase is inhibited by the regulatory (R) subunit of which there are several isoforms (RI{alpha}/ß, RII {alpha}/ß) encoded by distinct genes (Refs. 3, 4 and references therein). R subunits have recently been shown to bind cellular scaffolding proteins, called A-kinase anchor proteins (AKAPs) (5, 6, 7, 8, 9, 10). Because R binds these scaffolding sites, they are presumed to regulate the site of localization of the holoenzyme and provide convenient, local target sites for the activated enzyme. However, once A-kinase is activated and C is released from the holoenzyme, the C subunit moves rapidly to the nucleus (11) where it phosphorylates transcription factors such as the cAMP-regulatory element binding protein, CREB (12, 13, 14). It has been well documented that phosphorylation of serine 133 CREB (phospho-CREB) is obligatory for transcriptional activation of CREB (15, 16) and its association with CREB binding protein, CBP/p300 (17, 18). This paradigm has now been established in many cells indicating that this pathway is a major route by which A-kinase regulates transcription.

The A-kinase pathway is negatively regulated by numerous inhibitory mechanisms. Phosphodiesterases (PDEs) rapidly catabolize cAMP, thereby rapidly reducing intracellular levels of cAMP and favoring the reestablishment of the holoenzyme (19). Specific inhibitors of the PDE isoforms present in the ovary can sustain elevated intracellular levels of cAMP in granulosa cells vs. ooctyes (19, 20). Protein kinase inhibitor (PKI) exists in multiple isoforms (PKI{alpha}, PKIß, and PKIß subtypes), all of which bind and inactivate the A-kinase C subunit (21, 22, 23, 24). Although the physiological function of PKIs is not yet clear, PKI{alpha} has been shown to enhance the export of C from the nucleus providing one function for its binding and the inactivation of C activity in the nucleus (25, 26, 27). By restricting residence time of the C subunit in the nucleus, PKI{alpha} would regulate the transactivation of A-kinase-regulated genes. Lastly, serine protein phosphatases (PPs) are known to control the phosphorylation state of proteins (28, 29, 30), and calcineurin (PP2B), in particular, is known to be translocated to the nucleus in response to certain signals (31).

In the ovary, FSH and LH stimulate the A-kinase pathway and thereby control the growth and differentiation of the ovarian follicle (1). In response to tonic secretion of FSH, granulosa cells of small follicles produce low levels of cAMP, proliferate, and differentiate. Genes such as serum- and glucocorticoid-induced kinase (Sgk) (32, 33) and serum-induced kinase (Snk) (1), as well as the cell cycle-regulatory molecule, cyclin D2 (34, 35, 36), are induced in these cells in an immediate-early expression pattern. In contrast, other genes exhibit a more delayed response to hormone stimulation and do not peak until 24–48 h after exposure to FSH when granulosa cell function has reached the PO stage. Genes induced at this time include aromatase (Refs. 37, 38 and data herein), inhibin {alpha} (39), LH receptor (4, 40), and the secondary rise of Sgk (Ref. 32 and data herein). In response to the LH surge, granulosa cells generate high levels of intracellular cAMP and rapidly initiate a program of terminal differentiation (luteinization) in which proliferation ceases (4, 35, 36). LH dramatically down-regulates genes associated with follicular function such as aromatase (4, 38) and cyclin D2 (35, 36) and rapidly but transiently induces genes required for ovulation (41, 42, 43, 44, 45, 46, 47, 48, 49, 50): progesterone receptor (PR) (41, 42, 43, 44, 45), prostaglandin synthase-2 (PGS-2) (46, 47, 48, 49) and CAAT enhancer-binding protein (C/EBPß) (47, 50). During the process of luteinization, granulosa cells appear to become refractory to further cAMP stimulation. Genes such as cholesterol side-chain cleavage cytochrome P450 (P450scc) (51, 52) are constitutively expressed at elevated levels. Neither forskolin, which increases cAMP, nor H89, which blocks A-kinase activation, alters expression of P450scc in the luteinized cells (52). Thus, the A-kinase pathway controls the expression of numerous genes in granulosa cells at distinct stages of differentiation and by specific molecular events.

The diverse patterns of gene expression that are regulated by FSH in granulosa cells are dramatically altered by the LH surge-induced transition to luteal cells. Notably, luteal cells exhibit altered/impaired responses to cAMP. Therefore, these studies were designed to focus on the functional and subcellular changes in A-kinase pathway components that might regulate the rapid and reversible responses to cAMP that occur in proliferating granulosa cells compared with the irreversible and cAMP nonresponsive state in terminally differentiated luteal cells. In particular, the localization of the A-kinase C subunit has been related to the phosphorylation state of CREB as well as to the expression of two A-kinase-regulated genes, aromatase and Sgk. Because the latter is also a kinase with presumed functions related to proliferation as well as differentiation (32, 33, 53), the subcellular localization of this protein in granulosa cells and luteal cells was also examined. Inhibitors of the A-kinase pathway were used to determine the extent to which they control the pattern of A-kinase activation, its subcellular localization, and the expression patterns of aromatase and Sgk.

Two well characterized culture systems were used to compare the agonist-induced responses of immature, proliferating granulosa cells to those of terminally differentiated, nondividing luteinized granulosa cells. Specifically, the culture of granulosa cells isolated from preantral follicles of immature rats in serum-free medium permits analysis of dynamic, short-term responses to hormones (cAMP) (32, 38). In contrast, the culture of granulosa cells from PO follicles that have been exposed in vivo to surge concentrations of hCG (PO/hCG) permits analyses of responses in cells that undergo irreversible, long-term changes associated with the process of luteinization (4, 51, 52). Results indicate that granulosa cell differentiation is associated with dynamic changes in the subcellular localization of A-kinase pathway components and provide evidence for the intriguing possibility that A-kinase C subunit, phospho-CREB, and Sgk have distinct nuclear import/export controls, docking sites, and functions (?) in proliferating granulosa cells that differ from those in terminally differentiated luteal cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Content and Localization of the A-Kinase Catalytic (C) Subunit, CREB, and phospho-CREB in Immature Granulosa Cells: Time-Dependent Response to FSH
To characterize the cAMP-induced activation kinetics of the A-kinase pathway in immature granulosa cells, we have analyzed the content and subcellular localization of the A-kinase C subunit, R subunits, CREB, and phospho-CREB, as well as CBP, in granulosa cells after hormone stimulation. Granulosa cells were harvested from ovaries of estradiol-primed, immature rats and cultured in defined medium without serum overnight (0 h) (32, 38). FSH (50 ng/ml) and testosterone (T; 10 ng/ml) or in some experiments forskolin (Fo; 10 µM) were then added for selected time intervals. In response to these hormones/agonist, immature granulosa cells differentiate and acquire functional characteristics similar to those of granulosa cells from PO follicles (4). At each interval, cell extracts were prepared for Western blot analyses or the cells plated on coverslips were fixed in 4% paraformaldehyde and processed for immunocytochemistry (54).

Antibodies against peptides of either the amino terminus (NT) or carboxy terminus (CT) of the A-kinase C subunit (NT-{alpha}C and CT-{alpha}C, respectively) recognize a 41- to 39-kDa immunoreactive band that remained constant in granulosa cells cultured with FSH/T for 0–48 h (Fig. 1AGo). Likewise, the cellular content of CREB (43 kDa) remained unchanged in response to hormone stimulation. An antibody that specifically recognizes activated, phosphorylated (serine 133) CREB showed that immunoreactive phospho-CREB was low in cells cultured overnight in the absence of hormone (0 h) but was markedly increased 6.5 ± 0.3-fold by FSH within 1 h. This increase was transient; levels of phospho-CREB declined to basal levels by 6 h. However, a secondary 2.1 ± 0.3-fold increase in phospho-CREB was observed at 24 h (Fig. 1AGo). Since each antibody recognized only a specific immunopositive band, these same antibodies were used for immunolocalization of these proteins within granulosa cells during hormone stimulation.



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Figure 1. FSH Stimulates Temporal Changes in Content and Cellular Localization of A-Kinase Components in Cultured Granulosa Cells

Granulosa cells were harvested from E-primed immature rats and cultured in defined medium on serum-coated plates. FSH (50 ng/ml) and testosterone (T; 10 ng/ml) were added for 0–48 h. Cell extracts or coverslips were prepared for Western blot analyses (panel A) or immunocytochemistry (panels B and C) as described in Materials and Methods. A, For Western blots, antibodies to phospho-CREB (P-CREB, 1:1000), CREB (1:1000), and A-kinase C subunit (NT-{alpha}C, 1 µg/ml final dilution; and CT-{alpha}C, 1 µg/ml final dilution) were diluted in buffers recommended by the supplier. The experiments have been repeated three times with ECL, quantitated by phosphoimage analysis and the results given as the mean ± SEM in the text. A, In response to FSH/T, phospho-CREB increased 6.5 ± 0.3 fold and 2.1 ± 0.3 at 1 h and 24 h, respectively. Phospho-CREB at 6 and 48 h was not different from 0 h. B, The morphology of cells cultured overnight in defined (serum-free) medium (0 h) or in the presence of FSH/T for 48 h is shown in the upper panels. For immunocytochemistry, antibodies to phospho(P)-CREB, NT-{alpha}C, and CT-{alpha}C were diluted 1:250, 1:1500, and 1:1500, respectively. For each antibody, the same exposure time was used at each time interval. The immunofluorescent experiments have been repeated at least five times with highly reproducible results. C, Antibodies to CREB, CBP, and RII{alpha}/ß subunits were used at a dilution of 1:250, 1:250 and 1:1000, respectively. Antibody specificity was determined by using secondary antibody alone or, in the case of the C-subunit, by neutralizing the CT-{alpha}C antibody with excess peptide. Data for CBP, CREB, and RII{alpha}/ß were obtained at 0 h but are representative of all time intervals. All fluorescent images were taken with a 63x objective.

 
Granulosa cells cultured overnight (0 h) in defined medium on serum-coated glass coverslips exhibit a rounded morphology (Fig. 1BGo). When cultured for 48 h with FSH/T, some cells retain the rounded shape whereas others are more flattened and cobblestone in appearance. Immunocytochemical analyses of A-kinase pathway components within these granulosa cells revealed that distinct changes in the staining and subcellular localization for each protein occur after agonist (FSH/T) stimulation. Immunoreactive phospho-CREB was low in granulosa cells cultured overnight in the absence of hormone (Fig. 1BGo; upper panel, 0 h). However, within 1 h of exposure to FSH/T, essentially all (95 ± 0.5%) granulosa cell nuclei were immunopositive for phospho-CREB (Fig. 1BGo, upper panel). Nuclear staining of phospho-CREB declined markedly by 6 h but was present at low levels at 24 h and 48 h. In a subset (20%) of granulosa cells cultured with FSH/T for 48 h, immunoreactive phospho-CREB was detected in the cytoplasm rather than the nucleus (Fig. 1BGo; upper panel, 48 h). In contrast, immunoreactive CREB, as well as its binding protein, CBP, were present in all cell nuclei at each time interval irrespective of hormone treatment (Fig. 1CGo; 0 h data are presented).

Antibodies to either the N terminus (NT-{alpha}C) or the C terminus (CT-{alpha}C) of the A-kinase C subunit also exhibited specific patterns of immunolocalization (Fig. 1BGo, middle and lower panels, respectively). NT-{alpha}C, preferentially recognized C subunit present in the nucleus with only weak recognition of the kinase complexed to cytoplasm structures (Fig. 1BGo; middle panels, 0 h). Specifically, immunostaining with NT-{alpha}C was low and diffuse in the untreated granulosa cells but increased markedly in nuclei of essentially all cells in response to FSH/T (1 h). This intense nuclear staining of C subunit with NT-{alpha}C was transient and returned to lower levels by 6 h. However, at 24–48 h, C subunit staining in nuclei with NT-{alpha}C appeared to increase slightly in 80% of the cells (Fig. 1BGo, middle panels). This is consistent with the observation that addition of H89, an A-kinase-selective inhibitor, to granulosa cells cultured with FSH/T for 41 h completely blocks the expression of aromatase and reduces expression of Sgk at 48 h (data not shown). In contrast, CT-{alpha}C intensely stained a discrete, small structure [possibly Golgi-related (9)] exterior to the nucleus of untreated granulosa cells (Fig. 1BGo; lower panel, 0 h). CT-{alpha}C staining was undetectable or low in the nucleus and other regions of the cells, respectively (Fig. 1BGo; lower panel, 0 h). After the addition of FSH for 1 h, immunostaining with CT-{alpha}C was more diffuse and spread throughout the cells with punctate staining clearly visible in granulosa cell nuclei. These changes in the distribution of immunoreactive C subunit were rapid but transient and occurred in all cells. At 6 h, C subunit (as detected by CT-{alpha}C) had returned to the basket-like structure in the cytoplasm. At 24 h and 48 h of culture with FSH/T, C subunit remained in the basket-like region of the cytoplasm but was also detected in a punctate pattern in nuclei, especially at 24 h (Fig. 1BGo, middle and lower panels). Thus, although the amount of immunoreactive C that is localized to nuclei at 24–48 h is less than that at 1 h, it appears to be functional and required at this stage of granulosa cell differentiation since inhibitors of A-kinase activity (i.e. H89) block aromatase gene expression.

These results indicate further that each C subunit antibody recognizes a specific epitope within the protein and that in the denatured protein both epitopes are clearly detected by each antibody (i.e. Western blot; Fig. 1AGo) but are selectively detected by the antibodies in tissue sections where proteins have been cross-linked by fixation procedures (i.e. as in immunofluorescent analyses). The N-terminal antigenic site of the C subunit appears to be masked (NT-{alpha}C) when C is bound to R subunits (and other proteins?) tethered to specific cytoplasm structures. The C-terminal antigenic site, however, is exposed when the C subunit is present in the cytoplasmic structures as well as when the protein is imported to the nucleus (CT-{alpha}C). In contrast to the transient, agonist-induced release and transport of the C subunit to the nucleus, immunostaining of the A-kinase RII({alpha}/ß) subunits in untreated (Fig. 1CGo) and hormone-stimulated (not shown) granulosa cells was localized to the basket-like region within the cytoplasm, most likely related to the Golgi (9). Changes in the intensity/pattern of RII {alpha}/ß subcellular distribution were not observed at the time intervals examined.

Effects of PDE Inhibitors on Activation of the A-Kinase Pathway
To determine whether the transient, FSH-induced increase in phospho-CREB between 0 to 6 h was related to changes in intracellular concentrations of cAMP and the residence time of C subunit in the nucleus, two inhibitors of PDEs were added to the cultures to maintain elevated concentrations of intracellular cAMP. Isobutylmethylxanthine (IBMX) is a PDE inhibitor with broad specificity whereas rolipram is a more selective inhibitor of the PDE4 isoform known to be selectively present in granulosa cells (19). As above, granulosa cells were cultured overnight in defined medium. At that time either rolipram (10 µM), IBMX (50 µM-1 mM) or vehicle was added to the cells for 30 min (pretreatment) after which forskolin (10 µM) was added (0 h). Western blots show that phospho-CREB was low in untreated cells as well as in those pretreated with rolipram (Fig. 2AGo). Forskolin alone or forskolin and rolipram stimulated similar increases in phospho-CREB at 1 h (Fig. 2AGo). However, at 6 h, phospho-CREB remained higher (7-fold) in the forskolin- and rolipram-treated cells compared with those exposed to forskolin alone. In addition, the decline in phospho-CREB at 24 and 48 h appeared to be more gradual, and phospho-CREB remained higher in the rolipram-treated cells compared with those stimulated by forskolin alone. Levels of phospho-CREB were also enhanced in granulosa cells cultured in the presence of forskolin and high levels of IBMX (1 mM) (Fig. 2AGo) whereas cells cultured with lower concentrations of IBMX (0.05–0.10 mM) exhibited a pattern of phospho-CREB similar to that of cells cultured with forskolin alone (data not shown).



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Figure 2. Inhibitors of PDE(4 ) Delay but Do Not Prevent the Eventual Decline in Forskolin-Induced Phosphorylation of CREB: Relation to the Expression of Aromatase and Sgk

Granulosa cells were harvested and cultured as described in the legend of Fig. 1Go. To stimulate granulosa cell differentiation, forskolin (10 µM) was added in the absence or presence of the PDE inhibitors, rolipram (10 µM) and IBMX (1 mM) for 0–48 h. A, Cell extracts were prepared in SDS buffer and analyzed by Western blot analysis of Sgk using an affinity-purified antibody (1:2000), or RNA was prepared for RT-PCR analysis of aromatase mRNA using specific primers for aromatase and ribosomal protein L19 (as the internal standard) and conditions (20 cycles) as previously described (see Materials and Methods and Refs. 39 and 74). These experiments have been repeated two times for each inhibitor with similar results. B, Cellular localization of phospho-CREB (P-CREB) and A-kinase C subunit were visualized by immunofluorescent analyses using specific antibodies diluted 1:250. For each antibody, the same exposure time was used for each time interval. Note the nuclear retention of C subunit at 6 h and 24 h and the elevated levels of nuclear phospho-CREB at 6 h compared to those observed in Fig. 1BGo. Also note the cytoplasmic localization of P-CREB in cells at 24–48 h. These experiments have been done at least five times with similar results.

 
To determine whether the elevated levels of phospho-CREB in cells treated with forskolin and PDE inhibitors were related to functional changes in the granulosa cells, the expression of two genes, Sgk and aromatase, known to be induced in granulosa cells by FSH/T or forskolin was analyzed. Western blot analyses showed that Sgk protein was increased by forskolin alone in a biphasic pattern; a transient peak occurred at 1 h in association with the initial increase in nuclear C subunit and again at 24–48 h (Fig. 2AGo), a pattern confirming our previous observations (32). A similar biphasic pattern of Sgk protein was observed when cells were treated with forskolin in the presence of either rolipram or IBMX (Fig. 2AGo). Semiquantitative RT-PCR analyses showed that aromatase mRNA was low (undetectable) in untreated granulosa cells (0 h). Forskolin alone increased expression of aromatase: mRNA was detectable by 1 h, and then increased 1.5-, 7-, and 10-fold at 6, 24, and 48 h, respectively (Fig. 2AGo). Rolipram did not alter either the magnitude or the temporal pattern of aromatase expression in response to forskolin.

Immunostaining of phospho-CREB and the A-kinase C subunit in granulosa cells of these same cultures showed that forskolin alone (data not shown) stimulated the same pattern as observed in response to FSH/T in Fig. 1Go. In contrast, the PDE inhibitors promoted the retention of the C subunit in the nucleus at 6–48 h (Fig. 2BGo). Although immunoreactive phospho-CREB remained present in granulosa cell nuclei at 6 h, the levels declined markedly at 24 and 48 h, and a subset of cells (20%) exhibited cytoplasmic staining (Fig. 2BGo; 48 h). Thus, despite the longer retention of C subunit in the nucleus and the higher levels of nuclear phospho-CREB at 6 h, the pattern of gene expression was not dramatically altered. In addition, the heterogeneous localization of phospho-CREB in cells at 48 h indicated that some of these cells might be acquiring characteristics of luteal cells.

Effect of Phosphatase Inhibitors on the A-Kinase Pathway
To determine whether the rapid changes in levels of nuclear phospho-CREB were related to phosphatase activity, two inhibitors of serine protein phosphatases were used. Okadaic acid (OA) preferentially inhibits PP1 and PP2A, whereas cyclosporin A (CsA) preferentially inhibits calcineurin (PP2B). OA (10 nM) and CsA (100 nM) were added to granulosa cell cultures 30 min before the addition of forskolin (0 h). OA alone increased the levels of phospho-CREB 2-fold in the untreated cells (Fig. 3Go, 0 h). Cells treated with forskolin and OA also had higher levels of phospho-CREB at 1, 6, and 24 h (1.5-, 3.0- and 3.5-fold, respectively) compared with cells treated with forskolin alone. In these same cells, the level of CREB remained constant (Fig. 3Go). Surprisingly, the expression of Sgk protein was completely abolished by exposing the cells to OA. The FSH/T-induced peaks of Sgk at 1 h and 24 h were completely absent if OA was also present in the cultures (Fig. 3Go). In contrast, CsA had no apparent effect on the levels of phospho-CREB or the expression of Sgk; the relative increases in reponse to forskolin were not changed (Fig. 3Go).



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Figure 3. Inhibitors of Serine PPs Delay but Do Not Prevent the Eventual Decline in Agonist-Induced Phosphorylation of CREB

Granulosa cells were harvested and cultured for 0–24 h in the presence of forskolin alone or after 30 min pretreatment with OA (10 nM) or CsA (100 nM). Western blot analyses were done for CREB, phospho-CREB, and Sgk (as described in Figs. 1Go and 2Go). These experiments have been repeated three times with similar results.

 
Localization of A-Kinase C Subunit and Phosphorylation of CREB Change as Granulosa Cells Differentiate
Because the inhibitors of PDE and phosphatases delayed but did not prevent the eventual decline in levels of nuclear C subunit and phospho-CREB, we sought to determine whether the C subunit targeted to the cytoplasmic structure(s) of granulosa cells cultured for 6, 24, and 48 h with FSH/T (Fig. 1BGo) could be relocalized to the nucleus. In addition, if C subunit was relocalized to the nucleus in response to cAMP, would it phosphorylate CREB? Granulosa cells were cultured in the presence of FSH/T for 0–48 h. Forskolin (10 µM) was added at 0.5, 6, 24, or 48 h after FSH to test for acute responses to further increases in intracellular cAMP. Before and at 45 min after the addition of forskolin, cell extracts were either prepared for Western blotting (Fig. 4Go) or cells plated on coverslips were fixed for immunocytochemical analyses (Fig. 5Go).



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Figure 4. Forskolin-Stimulated Phosphorylation of CREB as Granulosa Cells Differentiate

Granulosa cells were cultured in the presence of FSH and T for 0–48 h as in Fig. 1Go. At 0.5, 6, 24, and 48 h of culture, cells were exposed to the acute addition of forskolin for 45 min. Cell extracts were prepared for Western blot analyses as in Fig. 1Go. In three separate experiments the increases in phospho-CREB stimulated by FSH/T at 0.5 and by forskolin at 6, 24, and 48 h were 9.7 ± 0.3, 6.4 ± 0.3, 5.9 ± 0.5, and 5.0 ± 0.5, respectively.

 


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Figure 5. Forskolin-Stimulated Phosphorylation of CREB and the Subcellular Distribution of C Subunit as Granulosa Cells Differentiate

Granulosa cells were cultured as in Figs. 1Go and 4Go. Immunocytochemistry for P-CREB and C-subunit were done using both NT-{alpha}C and CT-{alpha}C antibodies as described in Fig. 1Go. For each antibody the same exposure time was used for each time interval. Similar observations have been obtained in three separate experiments. Note that forskolin stimulated phosphorylation of CREB at 0, 6, 24, and 48 h is associated with increased nuclear localization of C subunit. Also note that the distribution of phospho-CREB at 48 h is heterogeneous: both nuclear and cytoplasmic staining is observed after forskolin treatment. Also note that NT-{alpha}C recognizes nuclear C subunit whereas CT-{alpha}C recognizes C targeted to cytoplasmic structures (0, 6, 24, and 48 h) and the nucleus (in forskolin-treated cells at 0, 6, 24, and 48 h).

 
FSH stimulated a marked increase (9.7 ± 0.3-fold; n = 3) in phospho-CREB within 0.5 h compared to untreated cells (0 h) (Fig. 4Go). After 6 (not shown), 24, and 48 h of culture with FSH/T alone, the levels of phospho-CREB had declined (Fig. 4Go) as shown previously (Fig. 1AGo). When forskolin was added to the FSH/T-cultured cells at 0.5 h, the high levels of phospho-CREB were not increased further (+Fo 45 min), indicating that FSH had maximally activated adenylyl cyclase within 30 min. However, the addition of forskolin (45 min) to the FSH/T-treated cells did increase in phospho-CREB at 6 h (6.4 ± 0.3-fold; n = 3; not shown), 24 h (5.9 ± 0.5-fold; n = 3), or at 48 h (5.0 ± 0.5-fold; n = 3).

Immunofluorescent analyses showed that low levels of phospho-CREB were present in nuclei of untreated granulosa cells (Fig. 5Go; upper panels, 0 h). Forskolin (or FSH/T; not shown) stimulated marked increases in nuclear phospho-CREB within 45 min. As shown in Fig. 1BGo, this response was transient. After 6 h of culture with FSH/T alone, only a few (<5%) cells exhibited intense levels of phospho-CREB; most cells exhibited low levels of phospho-CREB (Fig. 5Go; upper panels, -Fo). At 24 h of culture with FSH/T alone, essentially all cells showed a low level of nuclear phospho-CREB. As in previous experiments, the staining of phospho-CREB was more heterogeneous at 48 h. Some cells exhibited low levels of phospho-CREB in the nucleus, whereas other (larger?) cells showed phospho-CREB localized to a perinuclear region in the cytoplasm (Fig. 5Go; two panels, 48 h; -Fo). The addition of forskolin at 6 h and 24 h increased immunoreactive phospho-CREB in nuclei of nearly all cells within 45 min (Fig. 5Go; upper panels, +Fo). At 48 h many cells (80 ± 0.6%) exhibited nuclear staining but a subset of cells (~20%) immunoreactive for phospho-CREB remained localized to the cytoplasm (Fig. 5Go; two panels, 48 h; +Fo).

These dynamic changes in the CREB phosphorylation were associated with the activation of A-kinase and its nuclear import/export (Fig. 5Go, middle and lower panels). Specifically, in untreated granulosa cells, very low levels of C-subunit were detected within the nucleus (NT-{alpha}C), whereas intense staining was observed in a small basket-like structure within the cytoplasm (CT-{alpha}C) (Fig. 5Go; -Fo; middle and lower panels, respectively). In response to FSH/T or forskolin, rapid and marked changes in the subcellular distribution of immunoreactive C subunit were observed (Fig. 5Go). Immunostaining to the basket-like structure rapidly decreased as a more diffuse staining pattern of C subunit appeared throughout the cytoplasm (CT-{alpha}C; Fig. 5Go, lower panels; +Fo). Increased immunostaining of C-subunit in the nucleus was also apparent (CT-{alpha}C) and was even more pronounced when the N-terminal antibody was used to detect the kinase (NT-{alpha}C; Fig. 5Go, middle panels; +Fo). These changes were transient and by 6 h the cellular distribution of C subunit was similar to that observed in the untreated cells. Note, however, that the size/intensity of staining to the basket-like structure increased in cells cultured with FSH/T for 24 and 48 h (Fig. 5Go; CT-{alpha}C; -Fo). Cells stimulated with forskolin at 6, 24, and 48 h responded in a fashion similar to that of untreated cells: rapid but transient increases in nuclear C subunit were associated with increased phospho-CREB. The cellular levels of C subunit and CREB remain unchanged as indicated by Western blotting. Therefore, the immunolocalization data indicate that there are agonist-induced and time-dependent changes that control the rapid and transient subcellular trafficking (nuclear import/export) of the C subunit as well as CREB phosphorylation in granulosa cells. Although this culture system provides an ideal model for characterizing the functional changes in granulosa cells as they differentiate in response to FSH/T, the heterogeneous staining pattern observed in cells at 48 h indicated that a more precise model was needed to analyze the cellular localization of A-kinase pathway components and Sgk in luteal cells. Thus, we used another culture model in which granulosa cells luteinize and exhibit altered responses to exogenous hormones and increased intracellular cAMP (4, 51, 52).

Effect of Luteinization on the Content and Cellular Localization of A-Kinase Pathway Components and A-Kinase-Regulated Gene Expression
The LH surge stimulates an acute elevation of intracellular cAMP in granulosa cells of PO follicles. Within 4 h, the granulosa cells of these LH-stimulated follicles cease dividing and differentiate to a stable luteal cell phenotype (4, 35, 36, 51, 52). This reprogramming or terminal differentiation of granulosa cells to luteal cells occurs rapidly, is irreversible, and is characterized by a regulated, genetic switch. Several genes induced by cAMP in granulosa cells are expressed in a constitutive, non-cAMP-inducible manner in luteal cells. Specifically, neither the addition of forskolin, which stimulates cAMP, nor H89, which blocks A-kinase activation by cAMP, alters expression of P450 scc (52), aromatase (37, 55), or as shown herein, sgk. To analyze the content and cellular localization of components of the A-kinase pathway in luteinized granulosa cells, a second culture system (Refs. 51, 52 ; see Materials and Methods) was used. In the second model system, immature female rats were injected subcutaneously with a low dose (0.15 IU) of hCG twice daily for 2 days to stimulate the growth of PO follicles (51, 52). The PO follicles were dissected manually from the ovaries isolated from rats either before or 6 h after an intraperitoneal (IP) injection of an ovulatory dose (10 IU) hCG (PO/hCG). Granulosa cells were harvested from the PO and PO/hCG-treated follicles by needle puncture and cultured in DMEM-F12 containing 1% FBS as previously described (4, 51, 52). Granulosa cells harvested from PO/hCG follicles luteinize, attain a stable luteal phenotype, and thereby permit analysis of responses in cells that undergo irreversible, long-term changes associated with the transition of granulosa cells to luteal cells. In contrast, PO granulosa cells fail to luteinize and require forskolin/cAMP to stimulate the maintenance of a differentiated phenotype and expression of P450scc and aromatase (52) and as shown herein, Sgk.

When A-kinase pathway components were analyzed in this second model, both the A-kinase C subunit and CREB were present and expressed at similar levels in PO granulosa cells and the luteinized PO/hCG granulosa cells cultured for 6 days (0 h) in medium containing 1% FBS (Fig. 6Go). The addition of forskolin did not alter the levels of C subunit or CREB. Immunoreactive phospho-CREB was detected at low levels in the PO cells before the addition of forskolin, likely reflecting a response to some serum factor(s). Forskolin did not stimulate a rapid increase in phospho-CREB between 45–120 min in the PO granulosa cells (Fig. 6Go; 2-h data are shown). However, at 48 h the levels of phospho-CREB were increased 6.1 ± 0.5-fold. At this time (but not at 0–2 h), Sgk protein and aromatase mRNA were also expressed, indicating that forskolin (cAMP) is required in these cells to induce/maintain the differentiated state. In the luteinized PO/hCG granulosa cells, the A-kinase C subunit and CREB were present at levels similar to the PO granulosa cells (Fig. 6Go). However, immunoreactive phospho-CREB was elevated (5.6 ± 0.4 fold) in luteinized cells (0 h) compared with that observed in the PO granulosa cells (0 h) (Fig. 6Go). Furthermore, in the luteinized cells phospho-CREB was not increased by the addition of forskolin at either 2 h or 48 h (Fig. 6Go). The amount of Sgk protein in the luteinized cells was even higher than that induced by forskolin in the PO granulosa cells; no increases in Sgk protein were observed 48 after forskolin in the luteal cells (Fig. 6Go). In a similar manner, aromatase mRNA was constitutively expressed and nonresponsive to forskolin treatment in the luteinized cells (Fig. 6Go).



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Figure 6. Preovulatory (PO) but Not Luteinized (PO/hCG) Granulosa Cells Respond to Forskolin

Granulosa cells were harvested from either PO follicles or from PO follicles exposed to an ovulatory dose of hCG (PO/hCG) in vivo for 6 h. PO and PO/hCG-treated cells were cultured in defined medium containing 1% FBS for 6 days (media changes on day 3 and 6). On day 6 (0 h), PO and PO/hCG cells were exposed to forskolin for 0–48 h. Cell extracts were prepared for Western blots, and RNA was prepared for RT-PCR analysis of aromatase mRNA. PO granulosa cells retained responsiveness to forskolin, which increased phospho-CREB, aromatase, and Sgk at 48 h. PO/hCG cell luteinize spontaneously; forskolin did not alter the elevated expression of aromatase and Sgk or the level of phospho-CREB. These experiments have been repeated at least three times with similar results.

 
When the subcellular distribution of the A-kinase components was examined in PO and luteinized PO/hCG granulosa cells cultured for 6 days in medium containing 1% FBS, several specific features were noted. First, PO granulosa cells appear fibroblastic, whereas PO/hCG cells exhibit a typical luteal morphology (i.e. lipid droplets, prominent nucleus, expanded cytoplasm; Fig. 7Go). In both cell types, CREB and CBP were nuclear proteins (Fig. 7Go), as was observed in the immature granulosa cells (Fig. 1CGo). Likewise, immunoreactive RII{alpha}/ß was localized to a discrete, basket-like region in the cytoplasm of PO and PO/hCG cells (Fig. 7Go). However, the subcellular distribution of phospho-CREB and the C subunit exhibited distinct patterns in the luteinized PO/hCG granulosa cells compared with the nonluteinized PO granulosa cells. Specifically, immunoreactive phospho-CREB was readily detected in the nuclei of PO granulosa cells on day 6 of culture in medium containing 1% FBS (0 h) (Fig. 8AGo). This contrasts with the extremely low level of phospho-CREB observed by Western blotting or immunofluorescent staining in immature granulosa cells cultured in serum-free conditions (Fig. 1BGo, 0 h). The addition of forskolin to the PO granulosa cells increased phospho-CREB at 48 h (~50% of the cells) but not at 2 h (Fig. 8AGo). When the C subunit was analyzed with the N-terminal and C-terminal antibodies, immunoreactive C subunit was detected both in a basket-like region of the cytoplasm (CT-{alpha}C) as well as in nuclei (NT-{alpha}C) of the PO granulosa cells cultured for 6 days in 1% FBS (0 h) (Fig. 8AGo; 2 h). After exposure to forskolin for 2 h, the pattern of immunoreactive C subunit as detected by CT-{alpha}C was more diffuse with punctate staining obvious in the nuclei. At 48 h, C subunit appeared to be retargeted to the cytoplasmic basket, but immunoreactive C subunit clearly remained nuclear (as detected by both NT-{alpha}C and CT-{alpha}C; Fig. 8AGo, 48 h). Immunostaining with NT-{alpha}C preferentially detected C subunit in the nucleus before and after forskolin treatment; marked changes in intensity were not observed (Fig. 8AGo). The apparent absence of aromatase and Sgk at 0 h by RT-PCR and Western blot, respectively (Fig. 6Go), and the low level of Sgk observed by immunofluorescence at 0 h (Fig. 9BGo) occur when nuclear phospho-CREB and C subunit are present, suggesting that other transcription factors (phosphorylation?), as well as nuclear phospho-CREB, are required for the expression of these genes in PO granulosa cells cultured in the presence of 1% serum. Of note, the C-kinase activator phorbol myristate stimulated the phosphorylation of CREB in these PO granulosa cells but did not induce aromatase (data not shown). However, since forskolin increases phospho-CREB (Western blot; Fig. 6Go) and mobilizes A-kinase C subunit between 2–48 h, activation of A-kinase does occur in these PO cells and is associated with an increase in Sgk and aromatase at 48 h.



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Figure 7. Immunolocalization of CREB, CBP, and RII Subunits in PO and PO/hCG Granulosa Cells

Granulosa cells were cultured as in Fig. 6Go. On day 6 (0 h) cells were fixed and processed for morphology and immunocytochemical analyses of CREB, CBP, and RII{alpha}/ß as described in Fig. 1Go. Note the flattened fibroblast-like appearance of the PO granulosa cells and the luteinized appearance (lipid droplets and prominent nucleus) of the PO/hCG cells. Note also that the images of CREB and CBP in the PO/hCG cells were taken with a 40x objective; all others were with a 63x objective.

 


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Figure 8. Immunolocalization of phospho-CREB and the A-Kinase C Subunit Exhibit Distinct Patterns in PO Granulosa Cells Compared with Luteinized (PO/hCG) Cells

Granulosa cells were cultured as in Fig. 6Go. On day 6 (0 h) cells were exposed to forskolin for 2 h and 48 h. The subcellular localization of P-CREB and C-subunit was analyzed in cells before (0 h) and at 2 and 48 h after exposure to forskolin in PO granulosa cells (panel A) and PO/hCG luteinized cells (panel B). A, Note the nuclear presence of phospho-CREB and C subunit (NT-{alpha}C), as well as the distinct cytoplasmic targeting of C subunit to a basket and ring-like structure around the nucleus (CT-{alpha}C) (0 h). Forskolin stimulated marked changes in the distribution of C subunit detected by CT-{alpha}C: at 2 h immunostaining was diffuse in the cytoplasm and nucleus; at 48 h some CT-{alpha}C staining was distinctly punctate in the nucleus whereas most was retargeted to the cytoplasmic basket-like structure. B, Immunostaining of phospho-CREB was distinctly different in the PO/hCG cells where it was observed primarily in punctate regions within the cytoplasm at 0 h, 2 h (not shown), and 48 h after forskolin. C-subunit was nuclear (NT-{alpha}C) and also targeted to a distinct cytoplasmic basket-like structure around the nucleus (CT-{alpha}C) at 0 h. This distribution was not altered by forskolin at 2 h (data not shown) or 48 h. These experiments have been repeated at least eight times with the same results.

 


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Figure 9. Sgk Is Not Only Induced by cAMP but Exhibits Distinct Subcellular Localization in Immature Granulosa Cells Compared with Differentiated, Luteinized Granulosa Cells

A, Granulosa cells were harvested from immature E-primed immature rats and cultured as described in Fig. 1Go. Cells were fixed for immunocytochemical localization of Sgk before and at 2, 12, 24, and 48 h after treatment with FSH. The controls were prepared in the absence of the primary Sgk antibody (no {alpha}-Sgk); nuclear specific staining was verified with the nuclear marker B1C8. B, Granulosa cells were harvested from PO and PO/hCG follicles and cultured as in Fig. 6Go. On day 6 (0 h), cells were treated with forskolin for 0, 2, and 48 h and prepared for immunolocalization of Sgk. Affinity-purified antibody to Sgk was diluted 1:250. Note: To provide fluorescent images in which the details of localization could be visualized for the purpose of printing, PO cells were exposed for 7 sec whereas PO/hCG cells were exposed for only 1 sec. Thus, the intensities shown do not match those observed on the microscope where greater amounts of Sgk were clearly visible in the luteal cells (Fig. 6Go). Note the change in the distribution of Sgk from the nucleus in granulosa cells (immature and PO) to the cytoplasm in luteal cells (PO/hCG). These experiments have been repeated at least four times with identical results.

 
In the luteinized PO/hCG granulosa cells, intense staining of C subunit was observed in the basket-like structure of the cytoplasm (CT-{alpha}C) as well as to discrete punctate regions within the nucleus (NT-{alpha}C) on day 6 of culture in medium containing 1% FBS (0 h) (Fig. 8BGo). The addition of forskolin to these cells had no effect on the cellular distribution of C subunit at either 2 h (data not shown) or 48 h (Fig. 8BGo). As indicated above by Western blot analyses, these luteinized PO/hCG granulosa cells contained elevated amounts of phospho-CREB even in the absence of forskolin. Equally dramatic, but unexpected, was the subcellular distribution of phospho-CREB in these luteinized cells. Notably, immunoreactive phospho-CREB was localized to punctate sites within the cytoplasm in the large luteinized cells (Fig. 8BGo). Only a few cells (~5%) had immunostaining within the nucleus; these cells did not exhibit a luteal cell morphology but rather appeared as nonluteinized granulosa cells (not shown). Thus, phospho-CREB and the A-kinase C subunit appear to be targeted to different regions of the cell depending on the stage of granulosa cell differentiation. In contrast, CREB and CBP remained nuclear and the A-kinase RII{alpha}/ß subunits were localized to a basket-like structure in the cytoplasmic region of the cell (Fig. 7Go).

To determine whether the changes in the subcellular localization of the C subunit and phospho-CREB in luteal cells was specific, we examined time-dependent changes in the cellular localization of another kinase, Sgk, in each culture system. When granulosa cells from estradiol-treated, immature rats were cultured in a defined medium overnight, immunoreactive Sgk protein was low and appeared diffuse (Fig. 9AGo). After stimulation with FSH, immunoreactive Sgk increased at 2 h, a temporal pattern similar to that observed by Western blot (Fig. 4Go) and Northern blot (32). Notably, immunoreactive Sgk was localized to granulosa cell nuclei in a distinct punctate pattern of staining. Immunoreactive Sgk remained nuclear for 6–24 h, after which it was preferentially localized to the cytoplasm (Fig. 9AGo). In PO granulosa cells, immunoreactive Sgk exhibited a low level of distinct punctate staining in nuclei (Fig. 9BGo). By 48 h of culture with forskolin, the intensity of Sgk staining increased. Although this is harder to capture by immunofluorescence than by Western blotting (Fig. 6Go), Sgk remained nuclear in most of the forskolin-treated PO granulosa cells but was also observed in the cytoplasm of some cells (50%) where staining was more intense (Fig. 9BGo; two panels, 48 h). In the luteinized PO+hCG granulosa cells, intense immunostaining of Sgk was preferentially localized in a punctate pattern within the cytoplasm and remained cytoplasmic even after the addition of forskolin for 2 h (data not shown) or 48 h (Fig. 9BGo). Thus, whereas Sgk is nuclear in immature (Fig. 9AGo) and PO granulosa cells (Fig. 9BGo), it exhibits marked cytoplasmic staining as granulosa cells differentiate to luteal cells (Fig. 9Go), a pattern similar to that of phospho-CREB (Fig. 8BGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Ovarian follicular and luteal development is accompanied, at the cellular level, by a transition from proliferating undifferentiated granulosa cells to terminally differentiated luteal cells. This cellular and molecular reprogramming is manifest, in part, in the changing responses of granulosa and luteal cells to hormones and cAMP. Specifically, we demonstrate that this cellular transition from proliferation to differentiation is associated with a loss of hormone (cAMP) responsiveness, and an altered subcellular distribution of A-kinase C subunit, as well as cytoplasmic localization of immunoreactive phospho-CREB. Equally striking is the induction of Sgk by cAMP in immature granulosa cells and the distinct transition of Sgk from the nucleus to the cytoplasm as granulosa cells differentiate, luteinize, and cease dividing.

The activation of the A-kinase pathway in granulosa cells exhibits dynamic short-term changes and critical long-term induced changes that appear irreversible. During the acute response of undifferentiated granulosa cells to FSH (or forskolin), the A-kinase C subunit is rapidly but transiently imported to the nucleus (Fig. 1Go; Ref. 11), a response pattern observed in other cell types and first documented by the studies of Adams et al. (11). The acute phase of FSH action and nuclear import of the A-kinase C subunit are associated with specific changes in downstream targets in the A-kinase pathway, most notable of which is the phosphorylation of CREB (present report and Refs. 54, 56), leading to the transactivation of several ovarian expressed genes that contain functional CRE promoter regions: aromatase (shown herein and Refs. 4, 54), CREM (cAMP regulatory element modulator; Ref. 57), and inhibin {alpha} (39). In addition, we have recently shown that expression of Sgk is induced in a biphasic pattern by A-kinase with the first peak in Sgk mRNA (32) and protein (Ref. 32 and present data) occurring between 1–2 h after FSH/forskolin stimulation and dependent on the transcription factors Sp1/Sp3 (32). The newly expressed Sgk protein is localized to the nucleus, but in contrast to the A-kinase C subunit, remains nuclear from 2–24 h. Increased nuclear C subunit in granulosa cells at 1–2 h after FSH is also associated with the rapid induction of mRNA encoding the cell cycle-regulatory molecule cyclin D2 (34, 35, 36). Thus, the early FSH-stimulated events that are associated with proliferation as well as the onset of differentiation appear to be dependent on the nuclear localization of C subunit and the phosphorylation of nuclear transcription factors, such as CREB (13, 14, 15, 54) and Sp1/Sp3 (32).

After the rapid decline in nuclear levels of C subunit and phospho-CREB at 6 h there is a transient secondary increase that occurs at about 24 h. These secondary changes in C subunit localization and phospho-CREB are associated with maximal induction of aromatase mRNA (Fig. 3Go and Ref. 38) and activity (38) and the secondary increase in Sgk mRNA and protein (present data and Ref. 32). As differentiation proceeds to the PO phenotype, most (80%) granulosa cells retain responsiveness to cAMP, indicating that C subunit rapidly shuttles between the nucleus and cytoplasm. However, as granulosa cells luteinize, their responsiveness is altered. Thus, when immature granulosa cells were cultured with FSH/T for 24 and 48 h and then stimulated by forskolin to reactivate additional bursts in cAMP production, immunoreactive C subunit appears in the nucleus of most cells (80%) but the magnitude of the increase (based on the intensity of nuclear immunostaining) and the number of cells responding were less than observed in response to the initial FSH stimulus, indicating that some of these cells have begun to luteinize. The decrease in the cAMP-induced localization of C subunit to the nucleus as granulosa cells differentiate was associated with a subset of cells in which nuclear phospho-CREB had decreased, indicating that CREB is one key substrate that the A-kinase C subunit regulates during granulosa cell differentiation.

Interestingly, the inhibitors of PDE activity delayed but did not prevent the eventual, progressive decline in nuclear phospho-CREB and C subunit 48 h after FSH/forskolin stimulation. Equally notable, the PDE inhibitors had little or no effect on the downstream targets of A-kinase: neither the magnitude nor the temporal pattern of induction of aromatase mRNA or Sgk protein was markedly altered between 6–24 h. These results indicate that once the program of differentiation is set in motion by the rapid increases in cAMP and nuclear C subunit at 1–2 h, the program cannot be acutely changed by the presence of more cAMP, nuclear C subunit, or phosphoproteins, such as CREB, suggesting that low levels of A-kinase activation and phospho-CREB are sufficient. That A-kinase remains critically important at 24–48 h is emphasized by the fact that the addition of H89 to granulosa cells at either 24 h or 42 h of culture with FSH/T completely abolished (or markedly reduced) the expression of aromatase and Sgk at 48 h (our unpublished data). Thus, A-kinase, in concert with other factors and pathways, appears to control the cellular levels of aromatase mRNA, the expression of Sgk, and the transition to the differentiated phenotype that occurs between 6–48 h after agonist stimulation. The complexity of events and multiplicity of factors associated with differentiation are emphasized by the observation that the phosphatase inhibitor OA completely blocked the early and secondary induction of Sgk expression by forskolin in granulosa cells but did not alter other cell functions, such as the induction of aromatase. Thus, a delicate balance of phosphorylation-dephosphorylation of key regulatory protein(s) (Sp1/Sp3?; Refs. 32, 58, 59) controlling the expression of Sgk may exert potent positive or negative regulatory functions. Equally striking is the distinct transition of Sgk protein from the nucleus in immature, proliferative granulosa cells to punctate sites within the cytoplasm of differentiated, nonproliferative granulosa cells at 48 h of culture. Thus, differentiation proceeds by a progressive, irreversible change in the induced expression of specific genes and the subcellular localization of specific proteins.

The dynamics and factors controlling the localization and activation of the A-kinase pathway and its target genes is even more dramatic in fully luteinized PO/hCG granulosa cells. Although the PO and luteinized PO/hCG granulosa cells contained the same cellular levels of the A-kinase C subunit (as determined by Western blotting) as untreated immature granulosa cells, the subcellular distribution of the C-subunit in the luteinized PO/hCG cells was distinct, the movement of the C subunit in response to forskolin was impaired, and genes regulated by cAMP in granulosa cells no longer responded to acute stimulation by forskolin. Specifically, in the luteinized PO/hCG granulosa cells, immunoreactive C was localized to highly punctate regions within the nucleus and the cytoplasm (NT-{alpha}C) as well as to the basket-like cytoplasm structure (CT-{alpha}C). This distribution pattern was not altered by exposing these luteinized cells to forskolin for 2 h or 48 h, indicating that factors other than, or in addition to, cAMP might also be regulating the distribution of C subunit in these cells. Since nuclear localization of C subunit was also observed in PO granulosa cells, factors in 1% serum may stimulate low levels of cAMP, allowing C subunit to shuttle between the cytoplasm and nucleus. However, in the PO cells (unlike the luteinized cells), the addition of forskolin did increase nuclear uptake of C subunit, indicating that these cells retained responsiveness to cAMP. This contrasts with the absence of nuclear immunostaining of C subunit within undifferentiated, immature granulosa cells (cultured in serum-free medium) in which activation of adenylyl cyclase is dependent on addition of hormone/agonist and obligatory for nuclear localization of C subunit. Thus, the immature granulosa cells and the luteal cells represent two extremes with the PO granulosa cells exhibiting an intermediate phenotype.

The localization of Sgk further indicates that immature cells differ markedly from luteal cells. Sgk, which is mostly nuclear in immature and PO granulosa cells, was localized almost exclusively to cytoplasmic sites in the luteinized PO/hCG granulosa cells, even after treatment with forskolin. In addition, Sgk, which is induced by cAMP in immature, proliferative granulosa cells, was constitutively expressed at elevated levels in the nonproliferating, terminally differentiated PO/hCG cells even in the absence of forskolin. In a similar manner, Sgk was observed in mammary epithelial tumor cells to be nuclear in S and G2/M phases of the cell cycle but was in the cytoplasm of cells during the G1 transition or in cells arrested in G1 as a consequence of glucocorticoid-induced differentiation (33, 53). Collectively, these results add Sgk to the list of kinases that are not only regulated by A-kinase but exhibit changes in their subcellular localization during cell differentiation.

The mechanisms controlling nuclear-cytoplasmic shuttling (nuclear import/export) and the cellular localization (by specific docking sites?) of the A-kinase C subunit, as well as Sgk, at each stage of granulosa cell differentiation appear complex. Nuclear transport of molecules is controlled by many factors including nuclear localization signals (NLS), nuclear export signals (NES), ATP and specific GTPases, phosphorylation, and in some cases, chaperones (60, 61, 62, 63, 64, 65, 66, 67). The A-kinase C subunit does not have a specific NLS or NES (25, 26, 27). Rather, studies by Taylor and colleagues (25) have shown that simple diffusion accounts for import and export in the cell types studied (25) whereas PKI{alpha} and PKIß can facilitate nuclear export of the A-kinase C subunit, an effect explained by the ability of PKI to bind C and to the presence of a NES within the PKI{alpha} and PKIß peptide (26, 27). The cellular localization of A-kinase C subunit is also determined by specific docking proteins, such as the R subunit/AKAP complexes (5, 6, 7, 8, 9, 10) as well as I{kappa}B (67). Since the subcellular distribution pattern of the A-kinase C subunit differs in granulosa cells and luteal cells and does not strictly mimic that of RII{alpha} and since the response to cAMP appears impaired in luteinized PO/hCG cells, we hypothesized that either the antigenic sites of C subunit are masked when it binds to R subunit/AKAP complexes or the C subunit binds different tethering sites as granulosa cell differentiation proceeds. Using two different antibodies, we have determined that the N-terminal antigenic sites on C subunit (recognized by NT-{alpha}C antibody) appear masked when C is complexed with the R subunit/AKAP complex. However, it remains possible that there are specific AKAPs present in luteal cells that alter the nuclear import/export behavior of the C subunit. Alternatively, some C subunit may be bound by I{kappa}B (or other docking proteins). If associated with I{kappa}B or some other protein, the C subunit would not released by increased intracellular cAMP (67), providing one explanation for the apparent lack of C-subunit redistribution by forskolin/cAMP in the luteal cells. Interestingly, neither cAMP not C subunit have been shown to increase the phosphorylation of luteal cell proteins, adding further evidence for the apparent diminished effect of A-kinase in these terminally differentiated cells (68, 69). In contrast to the A-kinase C subunit, Sgk has putative NLS and NES sequences in its structure. Therefore, changes in the subcellular trafficking of Sgk from the nucleus in granulosa cells and to a cytoplasmic site in luteal cells may involve modifications (by phosphorylation or protein binding?) of either the NLS or NES to enhance or restrict nuclear import in relation to cell cycle progression (53) or other cell functions.

The amount and subcellular distribution of phospho-CREB were also dramatically altered in the luteinized granulosa cells. First, the cellular levels of phospho-CREB (Western blot analysis) were elevated in luteinized granulosa cells in the absence or presence of forskolin. This is consistent with our previous observations that the addition of the A-kinase inhibitor, H89, to these cells does not alter expression of P450scc (52) or aromatase (37, 55). Furthermore, immunoreactive phospho-CREB was exclusively localized to a cytoplasmic region of the luteal cells. The cytoplasmic localization of phospho-CREB is difficult to explain. In luteal cells, immunodetection of nuclear phospho-CREB may be masked by the binding of specific nuclear proteins (coregulators) to this transcription factor. Alternatively, since immunoreactive CREB is nuclear in luteal cells, it is possible that a set of nuclear kinases are selectively active in luteal cells (but not granulosa cells) and phosphorylate CREB not only at Ser133 (as detected by the Western blot) but also at other amino acid residues to target it for nuclear export. Kinases other than A-kinase that phosphorylate CREB include CaM kinase II and CaM kinase IV, protein kinase C, RSK-2, and ERK-2 (70, 71, 72, 73, 74). Some of these have been shown to alter the nuclear movement of specific proteins, but the identity of the kinase(s) acting in luteal cells that might alter the localization of phospho-CREB remains to be determined.

In summary, the transition of proliferating granulosa cells to terminally differentiated luteal cells is characterized by a dramatic reprogramming of cellular and molecular events. The data provided herein illustrate two diverse patterns in the activation of the A-kinase pathway and the distribution of A-kinase components within the cells. Immature cells respond in a rapid and robust manner to FSH/forskolin/cAMP: C subunit is mobilized to the nucleus and phosphorylates CREB. This response is transient, reversible, and associated with maximal induction of aromatase and Sgk at 24–48 h. In luteal cells forskolin/cAMP do not alter the cellular distribution of C subunit, phosphorylation of CREB, or expression of aromatase and Sgk, which are constitutively elevated. Quite unexpectedly, phospho-CREB resides in a cytoplasmic region, not in the nucleus, of luteal cells. The dramatic switch in the subcellular localization of phospho-CREB, combined with the lack of mobilization of A-kinase C subunit in luteal cells, provides biochemical and cellular evidence to help account for the shift from cAMP-regulated gene expression in granulosa cells to cAMP-independent regulation in luteal cells. Rivaling the changes in A-kinase and phospho-CREB is the dramatic egress of Sgk from the nucleus in granulosa cells to cytoplasmic sites in luteal cells. Although the mechanisms that control the subcellular trafficking of C subunit and Sgk, as well as the cytoplasmic localization of phospho-CREB, are not yet entirely clear, these data provide the novel observation that the cellular distribution patterns of specific kinases are differentiation dependent and may be functionally related to cell cycle progression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
Media and cell culture reagents and materials were purchased from Life Technologies, Inc. (Gaithersburg, MD), Sigma Chemical Co. (St. Louis, MO), Research Organics (Cleveland, OH), Fisher Scientific (Fairlawn, NJ) Corning, Inc. (Corning, NY), and HyClone Laboratories, Inc. (Logan UT). Trypsin, soybean trypsin inhibitor, DNAse, phorbol myristate, CsA, 17ß estradiol (E), propylene glycol, and mineral oil were all purchased from Sigma Chemical Co. Forskolin and OA were from Calbiochem (La Jolla, CA). Ovine FSH (oFSH-16) and LH (oLH-23) were gifts of the National Hormone and Pituitary Program (Rockville, MD). The A-kinase inhibitor H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamine, was from Seikagaku Corp. (Rockville, MD). Human chorionic gonadotropin (hCG) was from Organon (West Orange, NJ). Antibodies to CREB (catalog no. 9192) and phospho-CREB (catalog no. 9191L) were obtained from New England Biolabs, Inc. (Beverly, MA); A-kinase C subunit N-terminal (catalog no. 06–386) and RII subunits (catalog no. 06–411) were from Upstate Biotechnology, Inc. (Lake Placid, NY); and A-kinase C subunit C-terminal (catalog no. sc-903 with competing peptide) and CBP (catalog no. sc-584) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody to Sgk was generated in rabbits and affinity purified in the laboratory of Dr. Gary L. Firestone.

Electrophoresis and molecular biology grade reagents were purchased from Sigma Chemical Co., Bio-Rad Laboratories, Inc. (Richmond, CA), and Roche Molecular Biochemicals (Indianapolis, IN). Oligonucleotides were purchased from Genosys (The Woodlands, TX). All reverse transcriptase-PCR reagents were from Promega Corp. (Madison, WI) except for deoxyribonucleotides (dNTPs; Roche Molecular Biochemicals). {alpha}-32P[dCTP] was from ICN Radiochemicals (Costa Mesa, CA). Hyperfilm was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL).

Animals
Intact immature (day 25 of age) Holtzman Sprague Dawley female rats (Harlan, Indianapolis, IN) were housed under a 16-h light, 8-h dark schedule in the Center for Comparative Medicine at Baylor College of Medicine and provided food and water ad libitum. Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, as approved by the Animal Care and Use Committee at Baylor College of Medicine (Houston, TX).

Granulosa Cell Cultures
For these studies we have used two well characterized cell culture models in which the effects of cAMP on gene expression have been clearly defined. The first system has been used to characterize the immediate-early and delayed effects of FSH on granulosa cell differentiation (32, 38). Immature rats were primed with estradiol (E; 1.5 mg/0.2 ml propylene glycol once daily for 3 days) as previously described (32, 38). After treatment (day 27), granulosa cells were harvested and cultured at a density of 1 x 106 cells per 3 ml serum-free medium (DMEM-F12 containing penicillin and streptomycin) in multiwell (35-mm) dishes that were serum coated. In the presence of FSH/T or forskolin, these cells differentiate to a PO phenotype in which aromatase (38), LH receptor (40), and Sgk (32) are expressed.

In the second model system, immature female rats were injected subcutaneously with a low dose (0.15 IU) of hCG twice daily for 2 days to stimulate the growth of PO follicles (51, 52). The PO follicles were dissected manually from the ovaries either before or 6 h after an ip injection of an ovulatory dose (10 IU) hCG (PO/hCG). Granulosa cells were harvested from the PO and PO/hCG-treated follicles by needle puncture, collected by centrifugation, and cultured in 35-mm multiwell dishes in DMEM-F12 containing 1% FBS as previously described (4, 51, 52). Granulosa cells harvested from PO/hCG follicles luteinize and attain a stable luteal phenotype, whereas PO granulosa cells fail to luteinize and require forskolin/cAMP to stimulate the maintenance of a differentiated phenotype.

The culture of immature granulosa cells permits analysis of dynamic, short-term responses to hormone (cAMP), whereas the PO/hCG granulosa cells permit analysis of responses in cells that undergo irreversible, long-term changes associated with the transition of granulosa cells to luteal cells. In each culture system, hormones, agonists (FSH, forskolin), and inhibitors (IBMX, rolipram, OA, CsA) were added at the doses and times indicated in the figure legends. All experiments were repeated at least three times.

RNA Isolation, Northern Blots, and RT-PCR Assays
Cytoplasmic RNA was isolated from cultured cells with a buffer containing 1% NP-40 (32, 38). Each RNA sample was pooled from three replicate wells. The RNA was purified by sequential phenol, phenol-chloroform, and chloroform extraction, followed by ethanol precipitation. The RNA was resuspended in 0.1% diethylpyrocarbonate-treated water and its concentration determined by absorbance at 260 nm.

RT-PCR reactions were performed as previously described (39, 75) using specific primer pairs for rat aromatase (forward, 5'-TGCACAGGCTCGAGTATTTCC-3' and reverse 5'-ATTTCCACAATGGGGCTGTCC-3') (74) and the ribosomal protein L19 (39). The amplified cDNA products were resolved by acrylamide gel electrophoresis, and radioactivity/PCR product band was quantified on a Betascope 603 Blot Analyzer (Betagen Corp., Mt. View, CA). Data are presented as the ratio of radioactivity in the aromatase and L19 bands.

Cell Extracts and Western Blot Analyses
Total cell extracts were prepared according to a method described by Ginty et al. (70) by adding to each well hot (100 C) Tris-buffer containing 10% SDS and ß-mercaptoethanol. The cells were rapidly scraped with a rubber policeman, and the extract transferred to an Eppendorf tube at 100 C for 5 min. Extracts were stored at 4 C until analyzed by SDS-PAGE. After SDS-PAGE, samples were electrophretically transferred to nylon filter, washed briefly in PBS, and blotted with either 3% BSA, or 5% Carnation milk at room temperature for 1 h. Antibodies were added in the same blocking solutions at the dilutions indicated in the figure legends. Immunoreactive proteins were visualized with either [I125]Protein A or with enhanced chemiluminescence (ECL) according to the specification of the supplier (Pierce Chemical Co., Rockford, IL). Immunoreactive bands were quantified by counting the radioactive bands ([I125]Protein A) or by quantitative image analysis of autoradiograms (ECL) using AlphaImager 2000 (3.3) from Alpha Innotech Corporation (San Leandro, CA).

Immunocytochemistry
Granulosa cells in each model system were also cultured (as above) on glass coverslips for various times in the presence or absence of FSH and T or forskolin. Cells were fixed in fresh 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS for 30 min at room temperature, blocked with PBS containing 10 mM glycine, and then washed three times with PBS as described previously (54). The fixed cells were either stored at 4 C or the cells were immediately permeabilized with 0.5% NP-40 in PBS for 10 min and then blocked with 4% BSA in PBS for 1 h at room temperature. The cells were incubated at 4 C for 18 h with specific antibodies diluted in buffers according to the specifications of the companies from which they were obtained. After several PBS washes, cells were incubated with fluorescein-labeled goat antirabbit IgG (1:20, Pierce Chemical Co.) in 4% BSA in PBS for 1 h at room temperature. phospho-CREB, CREB, A-kinase catalytic subunit (C subunit), CBP, and RII{alpha}/ß were visualized on a Axiophot microscope (Carl Zeiss, Thornwood, NY).


    ACKNOWLEDGMENTS
 
The authors wish to thank Dr. David Ginty (Johns Hopkins University, Baltimore, MD) for providing the initial phospho-CREB antibody and protocols for both the Western blotting and the indirect immunocytochemistry of phospho-CREB. We also thank Robert Steiner for suggestions and some reagents used at the initiation of these studies.


    FOOTNOTES
 
Address requests for reprints to: JoAnne S. Richards, Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.

This work was supported, in part, by NIH Grants HD-16272 (J.S.R.) and CA-71514 (G.L.F.).

Received for publication December 15, 1998. Revision received May 7, 1999. Accepted for publication May 20, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Walsh DA, Perkins JP, Krebs EG 1968 An adenosine 3',5'-monophosphate-dependent protein kinase from rabbit skeletal muscle. J Biol Chem 243:3763–3768[Abstract/Free Full Text]
  2. Taylor SS 1989 cAMP-dependent protein kinase. J Biol Chem 264:8443–8446[Free Full Text]
  3. McKnight GS, Cadd GG, Clegg AD, Correll LA 1988 Expression of wild-type and mutant subunits of the cAMP-dependent protein kinase. In: Cold Spring Harbor Symposia on Quantitative Biology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, vol 3:111–119
  4. Richards JS 1994 Hormonal control of gene expression in the ovary. Endocr Rev 15:725–751[Medline]
  5. Pawson T, Scott JD 1997 Signaling through scaffold, anchoring and adapter proteins. Science 278:2075–2080[Abstract/Free Full Text]
  6. Scott JD, McCartney S 1994 Localization of A-kinase through anchoring proteins. Mol Endocrinol 10:5–113
  7. Dong F, Feldmesser M, Casadevall A, Rubin CS 1998 Molecular characterization of a cDNA that encodes six isoforms of a novel murine A-kinase anchor protein. J Biol Chem 273:6533–6541[Abstract/Free Full Text]
  8. Chen Q, Lin R-Y, Rubin CS 1997 Organelle-specific targeting of protein kinase AII (PKAII). J Biol Chem 272:15247–15257[Abstract/Free Full Text]
  9. Rios RM, Celati C, Lohmann SM, Bornens M, Keryer G 1992 Identification of a high affinity binding protein for the regulatory subunit of RIIß of cAMP-dependent protein kinase in Golgi enriched membranes of human lymphoblasts. EMBO J 11:1723–1731[Abstract]
  10. Hunzicker-Dunn M, Scott JD, Carr DW 1998 Regulation of expression of A-kinase anchoring proteins in rat granulosa cells. Biol Reprod 58:1496–1502[Abstract]
  11. Adams SR, Harootunian AT, Buechler YJ, Taylor SS, Tsien RY 1991 Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349:694–697[CrossRef][Medline]
  12. Deutsch PJ, Jameson JL, Habener JF 1987 Cyclic AMP responsiveness of human gonadotropin-{alpha} gene transcription is directed by a repeated 18-base pair enhancer. J Biol Chem 262:12169–12174[Abstract/Free Full Text]
  13. Gonzalez GA, Montminy MR 1989 Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell 59:675–680[Medline]
  14. Habener JF, Miller CP, Vallejo M 1995 cAMP-Dependent regulation of gene transcription by cAMP response element-binding protein and cAMP response-element modulator. Vitam Horm 51:1–57[Medline]
  15. Ginty DD, Kornhauser JM Thompson MA, Bading H, Mayo KE, Takahashi JS, Greenberg ME 1993 Regulation of CREB phosphorylation in the suprachiasmatic nucleus by light and a circadian clock. Science 260:238–241[Medline]
  16. Chrivia JC, Kwok, RPS, Lamb N, Haglwara M, Montminy MR, Goodman, RH 1993 Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365:855–859[CrossRef][Medline]
  17. Kwok RPS, Lundblad JR, Chrivia JC, Richards JP, Bachinger HP, Brennan RG, Roberts SGE, Green MR, Goodman RH 1994 Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370:223–226[CrossRef][Medline]
  18. Arias J, Alberts AS, Brindle P, Claret FX, Smeal T, Karin M, Feramisco J, Montminy M 1994 Activation of cAMP and mitogen responsive genes relies on a common nuclear factor. Nature 370:226–229[CrossRef][Medline]
  19. Conti M, Nemoz G, Sette C, Vicini E 1995 Recent progress in understanding the hormonal regulation of phosphodiesterases. Endocr Rev 16:370–389[Medline]
  20. Wiersma A, Hirsch B, Tsafriri A, Hanssen RGJM, Van de Kant M, Kloosterboer HJ, Conti M, Hsueh AJW 1998 Phosphodiesterase 3 inhibitors suppress oocyte maturation and consequent pregnancy without affecting ovulation and cyclicity in rodents. J Clin Invest 102:532–537[Abstract/Free Full Text]
  21. Van Patten SM, Howard P, Walsh DA, Maurer RA 1992 The {alpha}- and ß-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression. Mol Endocrinol 6:2114–2122[Abstract]
  22. Kumar P, Van Patten SM, Walsh DA 1997 Multiplicity of the ß form of the cAMP-dependent protein kinase inhibitor protein generated by post-translational modification and alternate translational initiation. J Biol Chem 272:20011–20020[Abstract/Free Full Text]
  23. Olsen SR, Uhler MD 1991 Inhibition of protein kinase-A by overexpression of the cloned human protein kinase inhibitor. Mol Endocrinol 5:1246–1256[Abstract]
  24. Beale EG, Dedman JR, Means AR 1977 Isolation and characterization of a protein from rat testis which inhibits cyclic AMP-dependent protein kinase and phosphodiesterase. J Biol Chem 252:6322–6327[Medline]
  25. Wen W, Meinkoth JL, Tsien RY, Taylor SS 1995 Identification of a signal for rapid export of proteins from the nucleus. Cell 82:463–473[Medline]
  26. Fantozzi DA, Harootunian AT, Wen W, Taylor SS, Feramisco JR, Tsien RY, Meinkoth JL 1994 Thermostable inhibitor of cAMP-dependent protein kinase enhances the rate of export of the kinase catalytic subunit from the nucleus. J Biol Chem 269:2676–2686[Abstract/Free Full Text]
  27. Wen W, Harootunian AT, Adams SR, Feramisco J, Tsien RY, Meinkoth JL, Taylor SS 1994 Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal. J Biol Chem 269:32214–32220[Abstract/Free Full Text]
  28. Hunter T 1995 Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80:225–236[Medline]
  29. Hagiwara M, Alberts A, Brindle P, Meinkoth J, Feramisco J, Deng T, Karin M, Shenolikar S, Montminy M 1992 Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB. Cell 70:105–113[Medline]
  30. Wadzinski BE, Wheat WH, Jaspers S, Peruski Jr L, Lickteig RL, Johnson GL, Klemm DJ 1993 Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 13:2822–2834[Abstract]
  31. Shibasaki F, Price ER, Milan D, McKeon F 1996 Role of kinases and the phosphatase calcineurin in the nuclear shuttling of transcription factor Nf-AT4. Nature 382:370–373[CrossRef][Medline]
  32. Alliston TN, Maiyar AC, Buse P, Firestone GL, Richards JS 1997 Follicle stimulating hormone-regulated expression of serum/glucocorticoid-inducible kinase in rat ovarian granulosa cells: a functional role for the Sp1 family in promoter activity. Mol Endocrinol 11:1934–1949[Abstract/Free Full Text]
  33. Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL 1993 Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum. Mol Cell Biol 13:2031–2040[Abstract]
  34. 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 a cAMP-responsive gene involved in gonadal cell proliferation and oncogenesis. Nature 384:470–474[CrossRef][Medline]
  35. 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 12:924–940[Abstract/Free Full Text]
  36. 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. Biol Reprod 59:476–482[Free Full Text]
  37. Hickey GJ, Krasnow JS, Beattie WG, Richards JS l 990 Aromatase cytochrome P450 in rat ovarian granulosa cells prior to, after luteinization: cyclic AMP-dependent, independent regulation. Cloning, sequencing of rat aromatase cDNA, 5' genomic DNA. Mol Endocrinol 4:3–12
  38. Fitzpatrick SL, Richards JS 1991 Regulation of cytochrome P450 aromatase mRNA and activity by steroids and gonadotropins in rat granulosa cells. Endocrinology 129:1452–1462[Abstract]
  39. Pei L, Dodson R, Schoderbek WE, Maurer RA, Mayo KE 1991 Regulation of the {alpha} inhibin gene by cyclic adenosine 3',5'-monophosphate after transfections into rat granulosa cells. Mol Endocrinol 5:521–534[Abstract]
  40. Segaloff DL, Wang H, Richards JS 1990 Hormonal regulation of LH/CG receptor mRNA in rat ovarian cells during follicular development and luteinization. Mol Endocrinol 4:1856–1865[Abstract]
  41. Natraj U, Richards JS 1993 Hormonal regulation, localization and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. Endocrinology 133:761–769[Abstract]
  42. Park O-K, Mayo K 1991 Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge. Mol Endocrinol 5:967–978[Abstract]
  43. Richards JS, Russell DL, Robker RL, Dajee M, Alliston TN 1998 Molecular mechanisms of ovulation and luteinization. Mol Cell Endocrinol 145:47–54[CrossRef][Medline]
  44. Clemens JW, Robker RL, Kraus WL, Katzenellenbogen BS, Richards JS 1998 Hormone induction of progesterone receptor (PR) messenger ribonucleic acid and activation of PR promoter regions in ovarian granulosa cells: evidence for a role of cyclic adenosine 3',5'-monophosphate but not estradiol. Mol Endocrinol 12:1201–1214[Abstract/Free Full Text]
  45. Lydon JP, DeMayo F, funk CR, Mani SK, Hughes AR, Montgomery CA, Shyamala G, Conneely OM, O’Malley BW 1995 Mice lacking progesterone receptor exhibit reproductive abnormalities. Genes Dev 9:2266–2278[Abstract]
  46. Wong WYL, Richards JS 1991 Evidence for two antigenically distinct molecular weight variants of prostaglandin H synthase in the rat ovary. Mol Endocrinol 5:1269–1279[Abstract]
  47. Sirois J Richards JS 1993 Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. J Biol Chem 268:21931–21938[Abstract/Free Full Text]
  48. Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD, Covington MB, Contel NR, Eng VM, Collins RJ, Czerniak PM, Gorry SA, Trzaskos JM 1995 Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II. Nature 378:406–409[CrossRef][Medline]
  49. Morham SG, Langenback R, Loftin CD, Tiano HF, Vouloumanos N, Jennette JC, Mahler JF, Kluckman KD, Ledford A, Lee Ca, Smithies O 1995 Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell 83:473–482[Medline]
  50. Sterneck E, Tassarollo L, Johnson PF 1997 An essential role for C/EBPb in female reproduction. Genes Dev 11:2153–2162[Abstract/Free Full Text]
  51. Richards JS, Hedin L, Caston L 1986 Differentiation of rat ovarian theca cells: evidence for functional luteinization. Endocrinology 118:1660–1668[Abstract]
  52. Oonk RB, Beattie WG, Richards JS 1989 Cyclic AMP-dependent and -independent regulation of cholesterol side-chain cleavage cytochrome P450 (P450scc) in rat ovarian granulosa cells and corpora lutea: cDNA and deduced amino acid sequence of rat P450scc. J Biol Chem 264:21934–21942[Abstract/Free Full Text]
  53. Buse P, Tran SH, Luther E, Phu PT, Aponte GW Firestone GL 1999 Cell cycle and hormonal control of nuclear-cytoplasmic localization of the serum and glucocorticoid inducible protein kinase, Sgk, in mammary tumor cells: a novel convergence point of anti-proliferative and proliferative cell signaling pathways. J Biol Chem 274:7253–7263[Abstract/Free Full Text]
  54. Carlone D, Richards JS 1997 Functional interactions, phosphorylation and levels of CREB and SF-1 mediate hormone-regulated and constitutive expression of aromatase in gonadal cells. Mol Endocrinol 11:292–304[Abstract/Free Full Text]
  55. Krasnow JS, Hickey GJ, Richards JS 1990 Regulation of aromatase mRNA and estradiol biosynthesis in rat ovarian granulosa cells and luteal cells by prolactin. Mol Endocrinol 4:13–21[Abstract]
  56. Mukherjee A, Park-Sarge O-K, Mayo KE 1996 Gonadotropins induce rapid phosphorylation of the 3',5'-cyclic adenosine monophosphate response element binding protein in ovarian granulosa cells. Endocrinology 137:3234–3245[Abstract]
  57. Mukherjee A, Urban J, Sassone-Corsi P, Mayo KE 1998 Gondadotropins regulate inducible cyclic adenosine 3',5'-monophosphate early repressor in the rat ovary: implications for inhibin {alpha} gene expression. Mol Endocrinol 12:785–800[Abstract/Free Full Text]
  58. Rohlff C, Ahmad S, Borellini F, Lei J, Glazer RI 1997 Modulation of transcription factor Sp1 by cAMP-dependent protein kinase. J Biol Chem 272:21137–21141[Abstract/Free Full Text]
  59. Pal S, Claffey KP, Cohen HT, Mukhopadhyay D 1998 Activation of Sp1-mediated vascular permeability factor/vascular endothelial growth factor transcription requires specific interaction with protein kinase C{zeta}. J Biol Chem 273:26277–26280[Abstract/Free Full Text]
  60. Jans DA, Hubner S 1996 Regulation of protein transport to the nucleus: central role of phosphorylation. Physiol Rev 76:651–685[Abstract/Free Full Text]
  61. Moore MS 1998 Ran and nuclear transport. J Biol Chem 273:22857–22860[Free Full Text]
  62. Heist EK, Srinivasan M, Schulman H 1998 Phosphorylation at the nuclear localization signal of Ca2+/calmodulin-dependent protein kinase II blocks its nuclear targeting. J Biol Chem 273:19763–19771[Abstract/Free Full Text]
  63. Whiteside ST, Goodbourn S 1993 Signal transduction and nuclear targeting: regulation of transcription factor activity by subcellular localization. J Cell Sci 104:949–955[Free Full Text]
  64. Topham MK, Bunting M, Zimmerman GA, McIntyre TM, Blackshear PJ, Prescott SM 1998 Protein kinase C regulates the nuclear localization of diacylglycerol kinase-{zeta}. Nature 394:697–700[CrossRef][Medline]
  65. Beals CR, Sheridan CM, Turck CW, Gardner P, Crabtree GR 1997 Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science 275:1930–1933[Abstract/Free Full Text]
  66. Shibaski F, Kondo E, Akagi T, Mckeon F 1997 Suppression of signaling through transcription factor NF-AT by interactions between calcineurin and Bcl-2. Nature 386:728–731[CrossRef][Medline]
  67. Zhong H, Voll RE, Ghosh S 1998 Phosphorylation of NF-{kappa}B p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1:661–671[Medline]
  68. Richards JS, Sehgal N, Tash JS 1983 Changes in content and cAMP-dependent phosphorylation of specific proteins in granulosa cells of preantral and preovulatory ovarian follicles and in corpora lutea. J Biol Chem 258:5227–5232[Abstract/Free Full Text]
  69. Richards JS, Kirchick HJ 1984 Changes in the content and phosphorylation of cytosol proteins in luteinizing ovarian follicles and corpora lutea. Biol Reprod 30:737–751[Abstract]
  70. Ginty DD, Bonni A, Greenberg ME 1994 Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell 77:713–725[Medline]
  71. Dash PK, Karl KA, Colicos MA, Prywes R, Kandel ER 1991 cAMP response element-binding protein is activated by Ca2+/calmodulin as well as cAMP-dependent protein kinase. Proc Natl Acad Sci USA 88:5061–5065[Abstract]
  72. DeGroot RP, den Hertog J, Vandenheeds JR, Goris J, Sassone-Corsi P 1993 Multiple and cooperative phosphorylation events regulate CREM activator function. EMBO J 12:3903–3911[Abstract]
  73. Xing J, Ginty DD, Greenberg ME 1996 Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 273:959–963[Abstract]
  74. Deisseroth K, Heist EK, Tsien RW 1998 Translocation of calmodulin to the nucleus supports CREB phosphorylation in hippocampal neurons. Nature 392:198–202[CrossRef][Medline]
  75. 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 134:2336–2346[Abstract]