Regulation of AP1 (Jun/Fos) Factor Expression and Activation in Ovarian Granulosa Cells

AP1 transcription factors control rapid responses of mammalian cells to stimuli that impact proliferation, differentiation, and transformation. To determine which AP1 factors are present in and regulated by hormones in ovarian cells during specific stages of proliferation and differentiation, we used both in vitro and in vivo models, Western blotting, immunohistochemistry, DNA binding assays, and transfections of AP1 promoter-reporter constructs. The expression patterns of Jun and Fos family members in response to hormones (follicle-stimulating hormone (FSH), luteinizing hormone (LH), and cAMP) were distinct. JunB, c-Jun, c-Fos, and Fra2 were rapidly but transiently induced by FSH in immature granulosa cells. JunD and Fra2 were induced by LH and maintained as granulosa cells terminally differentiated into luteal cells. Forskolin and phorbol myristate acetate acted synergistically to enhance transcription of an AP1(−73COL)-luciferase construct. JunD appears to be one mediator of this effect, since JunD was a major component of the AP1-DNA binding complex in granulosa cells, and menin, a selective inhibitor of JunD, blocked transcription of −73COL-luciferase. Thus, FSH and LH via cAMP induce specific AP1 factors, the AP1 expression patterns are distinct, and that of JunD and Fra2 correlates with the transition of proliferating granulosa cells to terminally differentiated, non-dividing luteal cells.

Growth of ovarian follicles involves regulated proliferation and differentiation of granulosa cells (1,2). Exponential growth of granulosa cells in preovulatory follicles is followed by their terminal differentiation and formation of the corpus luteum, a transition initiated by the ovulatory surge of luteinizing hormone (LH). 1 This transition of proliferating granulosa cells to non-dividing luteal cells is associated with specific changes in the expression of cell cycle regulatory molecules that control specific kinase cascades. The cell cycle activators, cyclin D2 and cyclin E, are increased by FSH and steroids in rapidly growing preovulatory follicles but are rapidly turned off by the surge of LH that terminates follicular growth (1,2). Conversely, the cell cycle inhibitors, p27 KIP1 and p21 CIP1 , are induced by the LH surge (1,2). Thus, the gonadotropins impact cell cycle progression at multiple steps depending on the stage of granulosa cell differentiation.
The effects of the gonadotropins on ovarian cell proliferation and differentiation are mediated by changes in intracellular cAMP that activates cAMP-dependent protein kinases (protein kinase A) (3), as well as several other kinases (4,5). Known targets of protein kinase A are the cAMP regulatory element (CRE)-binding protein CREB and the CREB-binding protein CBP (6). CREs are essential for transcriptional activation of aromatase (7) and inhibin ␣ (8), as well as the AP1 factors, c-fos (9) and fra2 (10). AP1 factors, in turn, mediate FSH-regulated expression of inhibin ␤A by binding to a variant CRE in the promoter of the ␤A gene (11). Members of the AP1 transcription factor superfamily are proto-oncogenes that regulate cell proliferation and transformation (12). They have also been associated with differentiation (13,14). These observations suggest that specific AP1 factors or the combination of specific AP1 factors may regulate different sets of functions at specific stages of granulosa cell differentiation. Despite the intense research efforts that have analyzed the function of the Jun/Fos family in many different cell types, relatively few studies have analyzed how hormones regulate the expression of AP1 factors in granulosa cells or how these factors might impact FSH or LH activation of specific genes. Therefore, it becomes imperative to know which AP1 factors are present in granulosa cells and which might be regulated or activated by FSH and LH.
The AP1 transcription factor family is composed of Jun family members (c-Jun, JunB, and JunD) that can form homo-or heterodimers among themselves and bind to AP1 consensus DNA (TGACTCA) (10,12,15). Jun proteins also dimerize with Fos family members (c-Fos, FosB, Fra1, and Fra2) (10,12,16). Thus, the functional activity of AP1 in any given cell is dependent in part on the relative amount of specific Jun/Fos factors present in that cell. Most AP1 transcription factors are present at low levels in cells but are rapidly induced and activated in response to specific stimuli. For example, the c-fos gene has a complex promoter that contains a CRE, a serum response element (SRE), and a Sis-inducible enhancer allowing it to respond to multiple hormones, growth factors, and cytokines (10,15,17). Not surprisingly then, c-fos has been shown to be induced by FSH in granulosa cells (9). The promoter of the junB gene is also complex with Stat3, CRE, CAAT enhancer (CAAT) binding domains, as well as an interleukin-6 regulatory region (10,18). Different promoter regions are utilized in different cell types (18). Although JunB is expressed in ovarian cells (19), the factors regulating its expression have not been analyzed. In contrast, the promoter of c-jun is simpler (10,15), and junD is constitutively expressed in many cell lines (19).
The functional activation of AP1 factors in mammalian cells requires phosphorylation (15). The activated AP1 factors have been shown to regulate gene expression by direct as well as indirect mechanisms (12,16). Direct activation requires that AP1 factors bind an AP1 regulatory domain present in the promoters of target genes and that the AP1 factors be phosphorylated in their activation domain. The AP1 DNA binding domain was originally known as the 12-O-tetradecanoylphorbol-13-acetate response element, since in response to phorbol ester tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate, the AP1 factors are phosphorylated, activated, and induced transcription (15). However, AP1 factors can be phosphorylated and activated in response to numerous other agonists, suggesting that their activation profile is more complex than originally thought. In fact, PMA which activates c-Jun N-terminal kinase does not activate Fos-regulating kinase, Frk (15). Activated AP1 factors can also enhance gene transcription by indirect mechanisms that involve their binding to other transcription factors via protein-protein interactions but do not require their binding to DNA. For example, members of the Jun/Fos family of transcription factors have recently been shown to interact with both Sp1/Sp3 and Smad proteins to enhance transcriptional activity of specific promoters with GCrich and SMAD-binding regulatory elements, respectively (14,20). This may be one attractive way in which FSH or LH regulate the expression of cyclins, p21 cip or p27 kip , as well as sgk. c-Jun has also been shown recently to recruit CBP for enhanced activation of CREB (21). Such a mechanism may be contribute to FSH activation of aromatase. The importance of c-Jun is underscored by the embryonic lethality of mice null for c-jun (22). In contrast, mice null for c-fos are viable but exhibit pleiotropic effects (23).
Based on the roles of AP1 factors in cell proliferation, differentiation, and transformation, we sought to determine which AP1 factors are present in and regulated by hormones in granulosa cells during follicular growth and terminal differentiation to luteal cells. For these studies we have used both in vitro and in vivo models, Western blotting, and DNA binding-electrophoretic mobility shift assays to identify and quantify Jun (c-Jun, JunD, and JunB) and Fos (c-Fos, FosB, Fra1, and Fra2) family members present in granulosa cells. The hormonal activation of AP1 factors in granulosa cells was analyzed by transiently transfecting cells with AP1 promoter-luciferase reporter constructs. Specific cellular signaling cascades were activated by adding forskolin, an agonist that stimulates cAMP, as well as the phorbol ester PMA that is a known activator of AP1 factors in other cell types. Results show that each AP1 factor exhibited a specific expression pattern and response to hormones. Rapid induction of JunB, c-Fos, and Fra2 by FSH corresponds to the pattern of other immediate-early genes in granulosa cells, whereas rapid induction of junD and fra2 by LH in granulosa cells of preovulatory follicles resulted in their prolonged, stable expression during the transition to luteal cells. JunD, in addition to c-Jun and c-Fos, was present as a major Jun factor comprising the AP1-DNA binding complex and controlled transactivation of the AP1-promoter reporter construct. Thus, JunD and Fra2 as well as c-Jun and c-Fos appear to be selectively expressed in terminally differentiated luteal cells indicating the composition of AP1 factors changes during granulosa cell proliferation and differentiation.

EXPERIMENTAL PROCEDURES
Reagents-Media and cell culture reagents and materials were purchased from Life Technologies, Inc., Sigma, Research Organics (Cleveland, OH), Fisher, Corning Glass, and HyClone (Logan, UT). Trypsin, soybean trypsin inhibitor, DNase, phorbol myristate acetate (PMA), dithiothreitol, 17␤-estradiol (E), and propylene glycol were all purchased from Sigma. Ovine FSH (oFSH-16) was a gift of the National Hormone and Pituitary Program (Rockville, MD). Human chorionic gonadotropin (hCG) was from Organon Special Chemicals (West Orange, NJ). Forskolin was from Calbiochem. Antibodies for c-Jun (PC06) and c-Fos (PC05) were obtained from Calbiochem. Antibodies for JunB (SC-73 for Western blots and immunohistochemistry; SC46 for EMSA), JunD (SC74x), FosB (SC-48x), Fra1 (SC605x), and Fra2 (SC-604x) were from Santa Cruz Biotechnology (Santa Cruz, CA). TRIzol reagent (number 15596) was obtained from Life Technologies, Inc. Electrophoresis and molecular biology grade reagents were purchased from Sigma, Bio-Rad, and Roche Molecular Biochemicals. Oligonucleotides were purchased from Genosys (The Woodlands, TX . To obtain granulosa cells for primary cultures, immature rats were injected with estradiol (E, 1.5 mg/ day for 3 days) (7). To analyze the expression of AP1 factors during follicular development in vivo, H rats were treated with E (HE, as above) to stimulate the growth of large preantral follicles. To analyze the response of granulosa cells to acute exposure to FSH, some HE rats were injected intravenously with 5 g of oFSH. Ovaries (for immunohistochemistry) and granulosa cells (for whole cell extracts) were isolated from H and HE rats prior to FSH (0 h) or from HE rats exposed to FSH for 2 and 8 h (HE, FSH 2, 8 h). Other HE rats were injected subcutaneously with 1.0 g of oFSH twice daily for 2 days (HEF, 48 h) to stimulate the growth of preovulatory follicles that express aromatase, LH receptor, and inhibin-␣ (24). HEF rats were subsequently injected intravenously with 10 IU of hCG, an LH-like hormone, to stimulate ovulation and luteinization. Ovaries and granulosa cells were prepared prior to (0 h) and at 2, 4, 8, 12, and 24 h after hCG for immunohistochemistry and WCE, respectively (shown schematically in Fig. 1).
Granulosa Cell Cultures-Granulosa cells were harvested from estradiol-primed intact immature (day 25) rats as described previously (7). Briefly, cells were cultured at a density of 1 ϫ 10 6 cells per 3 ml of serum-free medium (Dulbecco's modified Eagle's medium:Ham's F-12 medium containing penicillin and streptomycin) in multiwell (35-mm) dishes that were serum-coated. Cells were cultured in defined medium overnight (0 h) followed by the addition of FSH (50 ng/ml) and testosterone (T; 10 ng/ml), forskolin (10 M), and other agonists as indicated in the figures legends. FSH/T were used to stimulate the differentiation of granulosa cells to a preovulatory phenotype in which aromatase (7), LH receptor (25), and inhibin (8) are expressed. Forskolin was added to these cells to mimic the LH surge and luteinization. Forskolin alone was used to determine the relative effects of cAMP on specific cell functions (4,7). PMA was used as an agonist for protein kinase C (15,26) (shown schematically in Fig. 1). Hormones, agonists, and antagonists were added as indicated in the figure legends.
Transfections-After culture overnight, granulosa cells were transfected by the calcium phosphate precipitation method (27) using 5 g of vector DNA:Ϫ73COL-luciferase (15,28), ERE-E1b-luciferase (29), 4X-Gal-luciferase and Gal-4-Elk-1 (30), or menin (31) as indicated in the figure legends. Four hours later the cells were washed thoroughly in fresh medium. At that time (0 h) the cells were stimulated with agonists. After 6 h of agonist stimulation, cell lysates were prepared and used to measure luciferase activity according to standard protocols.
Relative light units were normalized to protein (mini-Bradford assay; Bio-Rad) in each sample. All transfections were run in triplicate, and each set of experiments was repeated at least three times. Unless otherwise indicated, values represent the mean Ϯ S.E. of three experiments.
Cell Extracts and Western Blot Analyses-Total cell extracts were prepared from cultured granulosa cells according to a method of Ginty et al. (32) by adding to each well hot (100°C) Tris buffer containing 10% SDS and ␤-mercaptoethanol (4). The cells were rapidly scraped with a rubber policeman. The cell extract was immediately transferred to an Eppendorf tube for 5 min at 100°C and then stored at 4°C. Equal volumes (50 l) of samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). Following SDS-PAGE, proteins were electrophoretically transferred to nylon filter, washed briefly in phosphatebuffered saline, and blotted with either 3% bovine serum albumin 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 ECL reagents according to the specification of the supplier (Pierce). Immunoreactive bands were quantified by image analysis of autoradiograms (ECL) using AlphaImager 2000 (3.3), Alpha Innotech Corp., San Leandro, CA.
Electrophoretic Mobility Shift Assays (EMSA)-Whole cell extracts (WCE) were prepared from hormonally stimulated granulosa cells in culture or from hormonally primed H rats as described previously (29) and in the figure legends. WCE were incubated 20 min on ice with a 32 P-labeled consensus AP1 oligonucleotide probe and then subjected to non-denaturing electrophoresis (0.5ϫ TBE, Tris borate-EDTA) at 150 V. Where indicated, either unlabeled probe or antibodies to specific AP1 transcription factors were added to the reactions 30 min on ice before the addition of labeled DNA.
Immunohistochemical Analyses-Immunohistochemical analyses of AP1 factors in whole ovaries isolated from hormonally primed rats were performed as described previously (24). Ovaries were fixed in 4% paraformaldehyde and were paraffin-embedded. Sections (6 microns) were processed according to routine procedures and then blocked with 10% non-immune goat serum followed by incubation with specific antibodies diluted 1:50 in 10% goat serum overnight at room temperature. After washing in phosphate-buffered saline, biotinylated anti-rabbit antiserum (Vector, Berlingame, CA) was added for 30 min, slides were washed, and streptavidin-conjugated horseradish peroxidase was applied for 30 min. Sections were incubated with DAB substrate (3,3Јdiaminobenzidene) for 2 min, dehydrated without counter-staining, and mounted.
Statistical Analyses-Transfection data were analyzed by one-way analysis of variance or Student's t test. Values were considered significantly different if p Ͻ 0.05.

Models for Analyzing Hormone-induced Changes in Granulosa Cell Expression of AP1 Factors
The AP1 transcription factor superfamily represents an important signal transduction system in mammalian cells. How-ever, relatively little is known about which AP1 factors are expressed or activated in ovarian granulosa cells. Therefore, our initial goals were to determine which members of the AP1 family of transcription factors were present in undifferentiated granulosa cells and which factors might be regulated by hormones in association with proliferation versus differentiation.
Two models were used to analyze hormone-induced changes in AP1 expression in granulosa cells (Fig. 1).
In Vitro Cultures-In the first model, undifferentiated granulosa cells were harvested and cultured in defined medium overnight. At that time the undifferentiated cells were either stimulated acutely (1.5 h) with forskolin to increase cAMP, the phorbol ester PMA (a known activator of AP1 factors), or the combination of forskolin and PMA. To stimulate the differentiation of granulosa cells to a preovulatory phenotype, FSH/T were added to the cells for 48 h. These differentiated cells (FSH/T), which express aromatase and LH receptor, were also stimulated acutely with forskolin, PMA, or the combination to mimic the acute effects of the LH surge. Finally, the differentiated cells were cultured with forskolin for longer periods to mimic the LH-induced process of luteinization in which granulosa cells cease to divide and are terminally differentiated.
Hormonal Treatment of rats in Vivo-In the second model, hypophysectomized immature rats (H) were injected with estradiol (E; HE rats) to stimulate granulosa cell proliferation and the growth of large preantral follicles. Subsequently, FSH (F) was administered to the HE rats (HEF) to examine rapid effects as well as long term effects of the gonadotropins, leading to the development of preovulatory follicles (see "Experimental Procedures" for details). HEF rats were injected with an ovulatory dose of the LH-like hormone, hCG, to stimulate terminal differentiation of granulosa cells. This process of luteinization leads to the formation of corpora lutea.

Differential Induction of Jun/Fos Transcription Factors in Undifferentiated Granulosa Cells
To analyze the response of undifferentiated cells, granulosa cells were harvested from immature rats and cultured with forskolin to stimulate cAMP production for different time intervals (0, 0.33 (20 min), 0.66 (40 min), 1.5, 3, 6, 12, 24, and 48 h). Cell extracts were prepared and analyzed by Western blotting using antibodies specific for individual members of the AP1 transcription factor family as follows: c-Jun, JunB, JunD, c-Fos, FosB, Fra1, and Fra2 (Fig. 2).
Jun Family Members-When undifferentiated granulosa cells were acutely stimulated with forskolin, different patterns of AP1 protein expression were observed (Fig. 2). Among the Jun family members, immunoreactive c-Jun was present at low levels in granulosa cells cultured overnight in medium alone (0 h). The addition of forskolin stimulated 1-, 5-, 6-, and 8-fold increases in c-Jun at 20, 40, and 90 min and 3 h, respectively. Levels of c-Jun decreased at 6 h and remained at this level at subsequent time intervals. JunB exhibited a similar pattern of response as that of c-Jun, but the magnitude of induction was greater. JunB increased 1-, 3-, 14-, and 16-fold at 20, 40, and 90 min and 3 h, respectively. Levels of JunB protein were decreased at 6 h and remained low. In contrast, JunD was present in granulosa cells at 0 h, and its expression was relatively unaffected by the addition of forskolin.
Fos Family Members-Among the Fos family members, immunoreactive c-Fos increased rapidly in response to forskolin; 2-and 6-fold increases occurred at 20 and 90 min, respectively (Fig. 2). However, this increase was transient; immunoreactive c-Fos returned to the base-line (0 h) level between 3 and 6 h and remained low thereafter. Similarly, FosB and Fra1 exhibited increases in response to forskolin at 20 and 40 min but declined to basal or near undetectable levels, respectively, between 6 and 24 h of culture. Fra2 showed the most dramatic changes in response to forskolin (Fig. 2, bottom panel). Fra2 increased 3-, 3-, and 11-fold at 20, 40, and 90 min, respectively, remained elevated at 3 h, and then declined gradually at 6 -12 h and was low again at 24 -48 h.
Collectively, these data indicate that JunD is constitutively expressed in cultured granulosa cells, whereas other AP1 factors, especially c-Jun, JunB, Fra2, and c-Fos, are rapidly but transiently induced/increased by forskolin/cAMP. Immunocytochemical analyses documented that AP1 factors were localized to the nuclei of granulosa cells (data not shown).

Induction of AP1 Transcription Factors in Undifferentiated and Differentiated Granulosa Cells, Comparison of the Effects of Forskolin and PMA
The transcriptional activation of AP1 factors has been shown in other cells types to be stimulated by the phorbol ester, PMA. Therefore, we next determined if the effects of forskolin (cAMP) on AP1 factor expression were similar to, different from, or synergistic with those of PMA and if the effects were dependent on the stage of granulosa cells differentiation. For these experiments, granulosa cells were cultured overnight in defined medium. At that time (0 h), the undifferentiated cells were treated acutely (for 1.5 h) with either forskolin (10 M), PMA (20 nM), or the combination of forskolin and PMA. Some cells were exposed to forskolin for 24 h. To examine the responses of differentiated granulosa, additional cells were cultured in the presence of FSH/T for 48 h. The differentiated cells were stimulated acutely (for 1.5 h) with agonists to mimic the LH surge or were exposed to forskolin for 24 and 48 h to induce luteinization. Cell extracts were prepared, and the inducibility of two factors, JunB and c-Fos, was analyzed by Western blotting (Fig. 3).
In untreated granulosa cells, immunoreactive JunB increased 44-, 19-, or 52-fold, respectively, by exposure to forskolin, PMA, or the combination for 1.5 h (Fig. 3, upper panel). The induction by forskolin was transient, and JunB protein returned to basal levels by 24 h. JunB was also low in the FSH/T-differentiated cells but could be induced by forskolin, PMA, or the combination 28-, 21-, and 34-fold, respectively. These results indicate that JunB is rapidly but transiently induced by forskolin in control and differentiated granulosa cells. PMA alone or in combination with forskolin also increased JunB. However, since the effects were not additive or synergistic, these two agonists appear to induce JunB by similar or overlapping mechanisms. Likewise, c-Fos was induced equally well by forskolin or PMA in undifferentiated granulosa cells, and the combined treatment was additive (Fig. 3, lower  panel). In the FSH/T-differentiated cells, the response to forskolin appeared less in this experiment than in subsequent experiments (compare results in Fig. 3 and Fig. 4). These differences may be due to the rapid and transient nature of the response. c-Fos was consistently increased by PMA or PMA and forskolin. Other cells were cultured with FSH (50 ng/ml) and testosterone (T; 10 ng/ml) for 48 h to stimulate differentiation. At that time, agonists were added to the differentiated cells as indicated. Immunoreactive JunB and c-Fos were detected by Western blotting as in Fig. 1.

Differential Induction of AP1 Factors in Differentiated Granulosa Cells
To analyze the effects of forskolin and PMA on the expression of other AP1 factors in the differentiated cells and to determine if the tumor promoter, okadaic acid (OA) (33), selectively affected their expression in ovarian cells, additional cultures were studied (Fig. 4). Granulosa cells were either cultured overnight in medium alone (0 h) or with FSH/T (48 h) as controls. Forskolin was added to the differentiated cells, and extracts were prepared 1.5 and 24 h later. In addition, FSH/Ttreated granulosa cells were exposed to PMA for 2 h or OA for 3 h.
Immunoreactive c-Fos was low in the control and FSH/Ttreated cells (Fig. 4, lanes 1 and 2) but increased 5-and 2.5-fold with forskolin at 1.5 and 24, respectively (lanes 3 and 4), 4-fold in response to PMA (lane 5), and 5-fold in response to OA (lane 6). In contrast, levels of FosB and Fra1 decreased from 0 to 48 h (lanes 1 and 2). FosB was increased with OA (lane 6), whereas Fra1 increased with forskolin, 1.5 h (lane 3); otherwise their expression remained low and unchanged. As in previous experiments, Fra2 was low in control cells but increased 8-fold after FSH/T, 48 h. Most dramatic were the 12-and 36-fold increases in Fra2 in response to forskolin (lanes 3 and 4)

Components of the AP1-DNA Binding Complex Change during Granulosa Cells Differentiation
To determine if the AP1 factors present in granulosa cells were capable of binding to AP1 consensus DNA, WCE were prepared from granulosa cells cultured overnight in medium alone (control, undifferentiated cells; 0 h) or in the presence of forskolin, PMA, or the combination for 1.5 h. Additional extracts were prepared from FSH/T-differentiated cells (48 h) followed by forskolin for 24 h, a regimen shown (Fig. 2) to increase selectively levels of immunoreactive JunD and Fra2. WCE were incubated with a labeled AP1 consensus oligonucleotide probe alone, a 100-fold excess of unlabeled oligonucleotide, or in the presence of antibodies specific for c-Jun, JunD, c-Fos, FosB, Fra1, and Fra2. As shown in Fig. 5, two protein-DNA complexes, one major and one minor, were formed when WCE from control ( When WCE of differentiated granulosa cells (FSH/T ϩ forskolin) were analyzed by EMSA, the major protein-DNA complex was markedly increased (Fig. 5, lane 9). Supershift analyses indicated that c-Jun (lane 10), JunD (lane 11), c-Fos (lane 12), and Fra2 (lane 15) were the AP1 components of the complex since each antibody caused a marked shift in the major protein-DNA complex. In contrast, antibodies to FosB (lane 13) and Fra1 (lane 14) did not cause supershifts, consistent with their lower abundance in extracts of differentiated cells (Fig. 3). Although no antibody alone caused a complete supershift of the complex, the addition of two or more antibodies to the reaction mixture caused a more complete supershift of the complex (data not shown). These data indicate that c-Jun, JunD, c-Fos, and Fra2 are more abundant in the differentiated granulosa cells than in the undifferentiated cells.
To analyze the binding activity of JunB, extracts of undifferentiated granulosa cells were prepared at 0 h or after treatment with forskolin, PMA, or forskolin and PMA for 1.5 h. The protein-DNA complexes formed with WCE from control cells were not supershifted with JunB antibody, whereas extracts from the forskolin-treated cells (containing high levels of immunoreactive Jun B; Figs. 1 and 2) were supershifted with the JunB antibody (data not shown) Thus, JunB is an inducible component of the AP1 complex in cells exposed to acute stimulation by forskolin.

Forskolin and PMA Act Synergistically to Induce Expression of the Ϫ73COL-luciferase Transgene
To examine the ability of granulosa cells to transactivate promoters containing AP1 regulatory domains, three promoterluciferase reporter constructs were initially tested. The promoters contained either Ϫ73 base pairs of the human collagenase gene (Ϫ73COL), 4 concatamers of a consensus AP1-binding site (4XAP1), or a single AP1 domain (1XAP1). Since in initial tests each responded in a similar manner to forskolin, PMA, or the combination, we have used the Ϫ73COL-luciferase construct for the studies described herein. When the Ϫ73COLluciferase construct was transfected into control granulosa cells (0 h), forskolin stimulated a 9-fold increase, whereas PMA stimulated only a 3-fold increase. The combination of forskolin and PMA induced a 30-fold increase in luciferase activity, indicating they exert a synergistic response (Fig. 6).
To determine if the effects of forskolin and PMA on the AP1 promoter elements were specific, we tested the response of two other promoter-reporter constructs to forskolin and PMA (Fig.  6). We chose an estrogen receptor response element E1b-luciferase construct since we have previously shown that it responds to forskolin (29), and others have reported activation of estrogen receptor by MAPK (34). When this vector was transfected into granulosa cells, forskolin alone induced a 10-fold increase, PMA a 7-fold increase, and the combination a 12-fold increase. Thus the effects of forskolin and PMA were additive but not synergistic.
Since c-Jun and c-Fos were preferentially increased by PMA alone, we sought to determine if a PMA-specific pathway was operative in granulosa cells. For this, we tested the functional activation of Elk-1, a ternary complex factor that binds serum response factor and enhances transactivation from SREs. Granulosa cells were co-transfected with a 4X-Gal-luciferase reporter construct and an expression vector containing a chimeric gene in which the Gal-4 DNA binding domain was ligated to the activation domain of Elk-1 (Fig. 6, 15). Luciferase activity was low in the absence of agonists, increased 2-fold with forskolin, 31-fold with PMA, and 39-fold with forskolin and PMA (Fig. 6).
Finally, since JunD was present in the highest concentrations in unstimulated granulosa cells, we sought to determine if the activity of the AP1 complex could be altered by menin, a specific inhibitor of JunD. Additional cells were co-transfected with the Ϫ73COL-luciferase reporter construct and a menin expression vector or empty vector (Fig. 6). As shown, menin markedly decreased (75%) the forskolin ϩ PMA-mediated luciferase activity but did not affect the control or forskolinstimulated effects. Thus, JunD, mostly likely in complex with FosB or Fra2 (factors rapidly induced by forskolin and PMA), composes the functional AP1 complex in forskolin-stimulated (undifferentiated) granulosa cells (Fig. 6).

FSH and LH Regulate Transient Versus Stable AP1 Expression in Granulosa Cells in Vivo
To determine if the expression of AP1 factors was regulated by hormones in vivo, hypophysectomized (H) rats were administered estradiol (E), FSH, and LH/hCG to stimulate the growth of antral follicles in which granulosa cells are proliferative, preovulatory follicles in which granulosa cells have acquired a transitional state of differentiation and luteinized follicles in which the granulosa cells have terminally differentiated to non-dividing cells, respectively ( Fig. 1 and "Experimental Procedures").
Western Blots-Each AP1 factor exhibited its own specific pattern of expression in granulosa cells of growing, ovulating, and luteinizing follicles (Fig. 7). Immunoreactive JunB was negligible in granulosa cells of H and HE rats. However, JunB increased dramatically (13-fold) within 2 h of exposure to FSH. Three distinct immunoreactive bands of JunB indicate that they are either phosphorylated forms or degraded products of JunB. The latter may be more likely since JunB was no longer detected at 8 h. JunB was also induced rapidly by hCG in granulosa cells of preovulatory follicles; JunB was highest at 8 h (24-fold increase) but was non-detectable at 24 h. Multiple bands were also observed in the 8-h sample, indicating that JunB is rapidly synthesized and processed (phosphorylated or degraded).
Fra2, like JunB, was negligible in granulosa cells of H and HE rats but was increased rapidly and transiently by acute exposure to FSH (Fig. 7). However, the expression of Fra2 in granulosa cells of preovulatory follicles was distinct from that of JunB. Fra2 was rapidly induced by hCG, but the elevated levels were then sustained in luteinizing granulosa cells (HEF-hCG, 4 -12 h) and in corpora lutea (HEF-hCG, 24 h; corpus luteum). In this manner, Fra2 is similar to JunD.
Immunoreactive JunD was absent in immature granulosa cells of H and HE rats but was increased rapidly (2-fold) by 2 h following acute injection of FSH. The increase was transient FIG. 5. Granulosa cell AP1 factors bind an AP1 consensus DNA. WCE were prepared from undifferentiated granulosa cells cultured overnight in defined medium (0 h) or from differentiated granulosa cells that had been cultured in the presence of FSH/T for 48 h followed by forskolin for 24 h to stimulate luteinization. Extracts (5 g of protein) were incubated with labeled AP1 oligonucleotide with or without unlabeled competitor DNA or were preincubated with antibody prior to the addition of labeled probe. Protein-DNA complexes were resolved by PAGE in 0.5ϫ TBE. Complex I contained AP1 factors, whereas the minor complex II appeared to be nonspecific. Supershifted complexes are depicted by the brackets. Note that in the differentiated cell extracts, the increased binding activity appeared to be composed of JunD and Fra2, as well as c-Jun and c-Fos. since levels of JunD returned to control levels at 8 h. JunD also increased rapidly in granulosa cells of HEF rats following administration of hCG. In contrast to the transient response of immature HE granulosa cells to FSH, the response of differentiated HEF granulosa cells to hCG was sustained from 4 to 24 h. The 11-fold increase in JunD at 8 h was maintained in the ovaries of HEF-hCG, 24-h rats that are composed mostly of corpora lutea.
The JunD inhibitor, menin, exhibited a different pattern of expression (Fig. 7). Levels of menin were low in granulosa cells of H, HE, and HEF rats but were increased in luteinized ovaries of HEF-hCG,24 h rats, and in corpora lutea isolated from pregnant rats in early pregnancy (day 7 of gestation) but low in later pregnancy (day 22). Thus, menin is also regulated and may modify the functional activity of JunD at specific stages of granulosa cells differentiation.
EMSAs-Changes in the relative binding of AP1 factors to a consensus AP1 oligonucleotide exhibited patterns similar to those observed by Western blotting. AP1 factor binding activity was low in WCE of granulosa cells from H and HE rats (Fig.  8A). AP1 binding increased rapidly (11-fold) but transiently in response to acute exposure to FSH at 2 h. Low levels of AP1 in granulosa cells of preovulatory follicles (HEF; lane 5) were increased by acute stimulation with hCG and were then maintained at levels between 11-and 30-fold above that observed in the H control (Fig. 8A, lane 1).
Collectively, these results show that the hormonal regulation of AP1 factor expression in granulosa cells of growing follicles in vivo is similar to that in cultured granulosa cells (Fig. 10). Western blots and EMSAs confirm that JunB is rapidly but transiently induced by FSH (and forskolin) in undifferentiated as well as by LH in differentiated cells. FosB and Fra1 are present in undifferentiated cells but not in luteal cells. In contrast, Fra2 as well as c-Fos and c-Jun are rapidly but transiently induced in undifferentiated cells but are expressed at elevated levels in luteinizing granulosa cells and luteal cells. JunD is expressed in granulosa cells of growing follicles and is elevated in luteinized cells. In vivo, JunD is also regulated by FSH and LH. Thus, JunD and Fra2 as well as c-Jun and c-Fos are likely different sets of AP1-regulated genes during the transition of proliferating granulosa cells to terminally differentiated luteal cells.

Immunohistochemical Localization of AP1 Factors in Ovarian Cells
Immunohistochemical data confirm the hormonal regulation and nuclear localization of JunD and Fra2 in granulosa cells and further demonstrate regulation of these factors in theca cells (Fig. 9). JunD was present but low in ovaries of HEF rats. JunD increased in theca cells of preovulatory follicles in response to hCG at 2 h. By 4 h immunoreactive JunD was detected in granulosa cells and remained elevated and in nuclei of granulosa cells during luteinization. Immunoreactive Fra2 (and JunB, data not shown) was negligible in granulosa cells and theca cells of preovulatory follicles. Immunoreactive Fra2 and JunB (not shown) was increased first in theca cells at 2 and 4 h after hCG and then appeared in granulosa cells at 4 h after which it reached maximal levels between 8 and 12 h. Immunoreactive Fra2, unlike JunB (not shown), was present in nuclei of corpora lutea of immature rats 24 h after hCG as well as of pregnant rats on day 7 of gestation. Thus, JunB, JunD, and Fra2 are low in preovulatory follicles but are increased rapidly in response to hCG, appearing first in the theca cells and then in the granulosa cells. JunD and Fra2, but not JunB, remain persistent in luteal cell nuclear suggesting that they are selectively associated with terminal differentiation of the granulosa cells.

DISCUSSION
The FSH-, LH-, and forskolin-induced changes in the expression and activation of AP1 factors in granulosa cells in vivo and FIG. 6. Forskolin and PMA act synergistically to transactivate the ؊73COL-luciferase reporter construct but not other promoters. Granulosa cells were cultured overnight in defined medium (0 h) at which time the cells were transfected with specific promoterreporter constructs as follows: Ϫ73COLluciferase vector, ERE-E1b-luciferase vector, Gal4(4X)-luciferase in combination with expression vectors for Gal4-ELK and the Ϫ73COL-luciferase vector, and either a menin expression plasmid or empty vector. Following 6 h of transfection, the cells were washed and stimulated with forskolin (Fo), PMA, or the combination (C). Data represent the mean Ϯ S.E. of three separate experiments; LSU, light specific units.
in vitro indicate that the AP1 signaling pathways are important downstream targets of cAMP in granulosa cells. Since the FSH-induced AP1 complex in proliferating granulosa cells is distinct (in composition and temporal pattern) from that of the LH-induced complex in terminally differentiated, non-dividing luteal cells, these changes likely impact specific AP1 target genes during this transitional period (Fig. 10).
In proliferating (undifferentiated) granulosa cells of small follicles, induction by FSH (forskolin) of c-Jun, JunB, c-Fos, FosB, Fra1, and Fra2 is rapid but transient; the most dramatic increases occurred for JunB (16-fold) and Fra2 (11-fold). The increases in these AP1 factors relate temporally to the expression of other immediate-early genes such as sgk (24) and egr-1 (12,35). JunB, c-Jun, c-Fos, and FosB were also induced in granulosa cells by the tumor promoters, PMA and okadaic acid. That the hormone (as well as cAMP-, PMA-, and okadaic acidinduced)-induced increases in AP1 factors are transient in granulosa cells during the normal progression of follicular growth may be important for preventing granulosa cell transformation. In numerous transformed cell lines, several AP1 factors (c-Jun, JunB, Fra1, and Fra2) are expressed at high levels; hence their designation as proto-oncogenes. Of potential relevance to these studies, JunB is expressed at high levels in ovarian cancer cell lines (36). Moreover, the increased levels of JunB in the transformed cells have been associated with sustained activation of MAPK by the Ras/Raf pathway (37). The Ras/Raf MAPK pathways may also mediate the effects of FSH

FIG. 7. FSH and LH regulate the expression of AP1 factors in granulosa cells of growing follicles and during luteinization.
Immature hypophysectomized (H) rats were treated with estradiol (E; HE), FSH (F; HEF) and hCG HEF-hCG to stimulate follicular growth and luteinization (see Fig. 1 and "Experimental Procedures"). WCE were prepared from granulosa cells isolated from the ovaries of H and HE rats and from HE rats 2 and 8 h after an intravenous injection of FSH or 48 h after twice daily injections subcutaneously in FSH. WCE were also prepared from granulosa cells 2, 4, 8, 12, and 24 h after injection of hCG (subcutaneously). Western blots document the presence and hormonal regulation of JunB, Fra2, and JunD in the extracts of granulosa cells from H rats. Menin was also regulated. For Fra2 and JunD, 20 g of WCE protein were loaded in each lane; for JunB 35 g of protein was needed and for menin 75 g were used.
FIG. 8. AP1 binding activity is regulated by hormones in granulosa cells in vivo. Immature hypophysectomized (H) rats were treated with hormones to stimulate follicular growth and luteinization as above (Figs. 1 and 7). EMSAs were run using 2.5 g of WCE protein and a labeled AP1 oligonucleotide as the probe (A). The AP1 factors that were present in WCE of HEF-hCG, 8 and 24 h granulosa cells (B) were identified by supershift assays. Antibodies to the AP1 factors were added 0.5 h prior to the addition to the labeled probe. CL, corpus luteum. on AP1 factor expression and activation in granulosa cells. Recent evidence shows that FSH/cAMP can impact the ERK, p38 MAPK, and phosphatidylinositol 3-kinase pathways (5). Importantly, the effects of FSH/cAMP on these pathways are independent of protein kinase A. Likely, cAMP activates a newly identified group of cAMP-regulated proteins, cAMP-guanine nucleotide exchange factors, that activate Ras-like molecules (38,39). Thus, FSH may modify AP1 factor expression and activity by diverse signaling pathways. Although junB exhibits high sequence homology to c-jun, and like c-jun has no introns, the promoter of junB is more complex than that of c-jun (10). As shown herein, the hormonal induction of JunB in granulosa cells is far more dramatic and transient than that of c-Jun. What factors contribute to JunB induction in the ovary, or its expression in Sertoli cells in the testis (45), remain to be determined. Based on regulatory regions in the junB promoter, these factors could be CREB or related family members (18), Smads (40,41), C/EBP␤ (18), Stat factors (18), or a combination of these all of which are expressed and regulated by hormones in ovarian granulosa cells.
In contrast to JunB and c-Jun, JunD was not regulated markedly by either forskolin or PMA in undifferentiated granulosa cells in culture. Based on the lack of regulation in cultured cells, it was surprising to observe that hormones regulated JunD expression in vivo. Notably, FSH increased JunD transiently in granulosa cells of small follicles, whereas LH increased JunD in granulosa cells of preovulatory follicles, a response that persisted as the cells luteinized. The junD pro- moter contains several potential regulatory elements including a CRE capable of binding CREB, a GC-rich region with potential binding sites for Sp1/Egr-1, an AP1 site, and a CAAT site to which NF-Y binds (10). The complexity of the junD promoter, as well as the presence of multiple hormones and growth factors present in vivo, may explain the more complex pattern of JunD expression in vivo compared with in vitro.
The molecular mechanisms by which hormones, forskolin (cAMP), and PMA regulate expression of Fos family members in proliferating (undifferentiated) granulosa cells are equally diverse (10,12,17,37). For example, although the gene structure and promoter regulatory regions of the c-fos and fosB genes are highly conserved, their regulation in granulosa cells by cAMP and the tumor promoters differs markedly. In the undifferentiated granulosa cells, c-Fos increased more in response to forskolin (and PMA) than did FosB. In contrast, both were increased by okadaic acid. The cAMP response elements (CREs) and AP1 elements within the c-fos promoter are likely targets of cAMP induction in granulosa cells (15,17). In contrast, the SRE of the c-fos promoter is likely to be the target of the PMA response, especially since PMA, but not forskolin/ cAMP, was a potent stimulator of transactivation of the Elk-Gal and Gal-luciferase reporter system in granulosa cells. These two different promoter sites and the preferential activation of protein kinase A by FSH and ERK by PMA appear to contribute to the additive effects of forskolin and PMA on induction of c-Fos in the undifferentiated cells. The mechanisms by which Fra2 is induced clearly involves cAMP and protein kinase A since H89 completely blocked induction of Fra2 by FSH or forskolin (data not shown) confirming the strong induction of Fra2 by cAMP in other cell types (10).
The LH/hCG (forskolin)-induced expression patterns of the AP1 factors in differentiated granulosa cells of preovulatory follicles differed markedly from that in undifferentiated cells. Despite the similarities of the c-fos and fosB promoters, expression of FosB but not c-Fos was turned off in response to hCG. In marked contrast, induction of c-Jun and c-Fos, as well as JunD and Fra2, was sustained and stable as granulosa cells terminally differentiated to luteal cells. This change from transient to stable expression of JunD and Fra2 suggests that they exert specific functions during granulosa cell proliferation and differentiation. That JunD is a functional component of the AP1-DNA binding complex in differentiated granulosa cells suggests that it is an important regulator of specific AP1responsive genes. In other systems, JunD can act as an inhibitor or an activator of transcription, dependent on the composition of the heterodimeric complex, the promoter element, and the cell type. For example, c-Fos/c-Jun or c-Fos/ JunB, but not c-Fos/JunD, activates the Ϫ73COL-luciferase construct in vascular endothelial cells (42). In contrast, a Fra2-JunD complex was more effective than c-Fos/c-Jun in activating the promoter of the oncostatin gene in ROS 17/2.8 osteosarcoma cells (43). Thus, based on the composition of AP1 factors in differentiating granulosa cells, JunD/Fra2 heterodimers may activate one set of AP1-responsive genes, whereas c-Fos-c-Jun complexes regulate other AP1-responsive genes. Furthermore, other factors such as menin, a specific JunD inhibitor, can alter the activity of JunD. In support of this, co-expression of menin blocked forskolin-induced transcription of Ϫ73COL-luciferase, suggesting that JunD is a functional component of the AP1 complex in granulosa cells. In terminally differentiating granulosa cells, JunD may play a critical role in terminating cell proliferation. Specifically, JunD has been shown to be low in certain ovarian cancer cell lines, and overexpression of JunD in these cells can suppress cell growth in a cell line-specific manner. JunD and Fra2 have recently been shown to increase during osteoblast differentiation (43). Thus, the increase in JunD and Fra2 in granulosa cells of preovulatory follicles exposed to an LH surge may control the transcription of specific genes that regulate the exit of granulosa cells from the cell cycle, thereby terminating granulosa cell proliferation (1,2). Of note, JunD is low in the testis (19), perhaps because the number of non-proliferative, JunDpositive Sertoli cells (data not shown) is low compared with the number of proliferating germ cells.
There are likely to be many genes regulated in granulosa cells by members of the Jun/Fos family of transcription factors. First, AP1 factors appear to control their own expression (10,15). Other genes in which AP1 factor regulation has been determined include inhibin ␤A (11), the GnRH receptor (26), TIMP-1 (44,45), and as already mentioned p21 cip (1,2,14). Each of these genes is hormonally regulated in granulosa cells, but only the promoter of the inhibin ␤A gene has been specifically examined in granulosa cells. The inhibin ␤A promoter contains a variant CRE that binds AP1-like factors and is inducible by forskolin and PMA as well as by overexpression of JunB and FosB proteins in granulosa cells (11). These synergistic effects of forskolin and PMA on the inhibin ␤A promoter mimic that observed herein for activation of the AP1 site within the human collagenase gene. Synergism between cAMP and PMA has also been observed recently to control induction of progesterone receptor mRNA in granulosa cells (46). Based on the results described herein, the synergy in granulosa cells could involve the selective induction by PMA of c-Jun and c-Fos and by cAMP of Fra2, JunB, and JunD. In addition to the induction of AP1 factors, forskolin and PMA activate several kinase cascades (protein kinase A, p38 MAPK, protein kinase C, and ERKs) that would lead to the phosphorylation and activation of specific AP1 factors and their co-activators. For example, c-Jun but not JunB is a target of N-terminal c-Jun kinase; c-Fos is activated by Frk (15). Finally, AP1 factors may regulate the expression of ovarian genes by indirect mechanisms that involve protein-protein interactions with other transcription factors. Of note, c-Jun has been shown to interact with the cell cycle regulator Rb (retino blastoma protein) (47). c-Jun also interacts with CBP to activate CREB (21), with Sp1/Sp3 to activate p21 cip1 gene (14), and with Smads (20) to regulate specific chimeric reporter genes. Thus, hormonal regulation of AP1 factors in the ovary has far reaching effects on many cellular signaling cascades that regulate proliferation and differentiation. The studies herein provide the specific observation that JunD and Fra2, as well as c-Jun and c-Fos, may be critical for regulating specific sets of AP1-responsive genes that control the terminal differentiation of granulosa cells, their exit from the cell cycle, and the prevention of granulosa cell transformation associated with elevated levels of AP1.