Follicle-stimulating hormone (FSH) is necessary and sufficient to induce maturation of ovarian follicles to a mature, preovulatory phenotype in the intact animal, resulting in the generation of mature eggs and production of estrogen. FSH accomplishes these actions by inducing a complex pattern of gene expression in target granulosa cells that is regulated by input from many different signaling cascades, including those for the extracellular regulated kinases (ERKs), p38 mitogen-activated protein kinases (MAPKs), and phosphatidylinositol-3 kinase (PI3K). The upstream kinase that appears to be responsible for initiating all of the signaling that regulates gene expression in these epithelial cells is protein kinase A (PKA). PKA not only signals to directly phosphorylate transcription factors like cAMP response element binding protein and to promote chromatin remodeling by phosphorylating histone H3, this versatile kinase also enhances the activity of the p38 MAPK, ERK, and PI3K pathways. Additionally, accumulating evidence suggests that activation of a single signaling cascade downstream of PKA is not sufficient to activate target gene expression. Rather, cross-talk between and among signaling cascades is required. We will review the signaling cascades activated by FSH in granulosa cells and how these cascades contribute to the regulation of select target gene expression. KeywordsFollicle-stimulating hormone; Mitogen-activated protein kinase; Female reproduction; Hypoxiainduced factor 1; Histone H3; Protein kinase A Abbreviations AKAP, A kinase anchoring protein; Aromatase, P450 aromatase; CBP, CREB binding protein; ChIP, chromatin immunoprecipitation assay; CREB, cAMP response element binding protein; EGF, epidermal growth factor; Egr-1, early growth response protein-1; ERK, extracellular regulated kinase; Epac, exchange proteins activated by cAMP; FSH, follicle-stimulating hormone; GIOT-1, gonadotropin-inducible ovarian transcription factor-1; GPCR, G-protein-coupled receptor; HIF-1, hypoxia-induced factor-1; IGF, insulin-like growth factor; LH, luteinizing hormone; LRH-1, liver receptor homolog-1; MAP2D, microtubule-associated protein 2D; MAPK, mitogen-activated protein kinase; MEK, mitogen-and extracellular-regulated kinase kinase; MK, MAPK-activated protein kinases; MNK, MAPK-interacting kinase; mTOR, mammalian target of rapamycin; p70S6K, p70 ribosomal S6 kinase; PDE, phosphodiesterase; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PKI, PKA inhibitor peptide; PTP, protein tyrosine phosphatase; R, PKA regulatory subunits; RSK, p90 ribosomal S6 protein kinase; SCC, P450 cholesterol side chain cleavage; SF-1, steroidogenic factor-1; SGK, serum glucocorticoid kinase; Sp1/Sp3, specific

We sought to elucidate the role of AKT in folliclestimulating hormone (FSH)-mediated granulosa cell (GC) differentiation. Our results define a signaling pathway in GCs whereby the inactivating phosphorylation of tuberin downstream of phosphatidylinositol (PI) 3-kinase/AKT activity leads to Rheb (Ras homolog enriched in brain) and subsequent mTOR (mammalian target of rapamycin) activation. mTOR then stimulates translation by phosphorylating p70 S6 kinase and, consequently, the 40 S ribosomal protein S6. Activation of this pathway is required for FSH-mediated induction of several follicular differentiation markers, including luteinizing-hormone receptor (LHR), inhibin-␣, microtubuleassociated protein 2D, and the PKA type II␤ regulatory subunit. FSH also promotes activation of the transcription factor hypoxia-inducible factor-1 (HIF-1). FSHstimulated HIF-1 activity is inhibited by the PI 3-kinase inhibitor LY294002, the Rheb inhibitor FTI-277 (farnesyltransferase inhibitor-277), and the mTOR inhibitor rapamycin. Finally, we find that the FSH-mediated upregulation of reporter activities for LHR, inhibin-␣, and vascular endothelial growth factor is dependent upon HIF-1 activity, because a dominant negative form of HIF-1␣ interferes with the up-regulation of these genes. These results show that FSH enhances HIF-1 activity downstream of the PI 3-kinase/AKT/Rheb/mTOR pathway in GCs and that HIF-1 activity is necessary for FSH to induce multiple follicular differentiation markers.The anterior pituitary hormone follicle-stimulating hormone (FSH) 1 provides the key stimulus that promotes proliferation and differentiation of the ovarian follicle to the pre-ovulatory phenotype (1, 2 FSH signaling results in increased transcription of a number of characteristic follicular differentiation markers, including the steroidogenic enzymes side-chain cleavage cytochrome P-450 and aromatase cytochrome P-450 (12), the luteinizing hormone receptor (LHR) (3), inhibin-␣ (13), the signaling intermediates microtubule-associated protein 2D (MAP2D) (14), and the PKA type II␤ regulatory subunit (RII␤) (15). FSH also stimulates antrum formation and angiogenesis in the peripheral follicle thecal cells mediated in part by the vascular endothelial growth factor (VEGF) (16,17). Our investigation into the mechanisms by which two of these FSH targets, MAP2D and RII␤, are up-regulated in response to FSH revealed that their up-regulation at the protein level is blocked by the PI 3-kinase inhibitors LY294002 and wortmannin. FSH-mediated PI 3-kinase activation is also necessary for the expression of the cartilage link protein (8) in GCs and for optimal transferrin secretion and lactate production by Sertoli cells (9). In support of a role for PI 3-kinase/AKT activity in GC differentiation, insulin-like growth factor-1 (IGF-1), which activates the PI 3-kinase/AKT pathway via the IGF-1 receptor (18), synergizes with FSH in GCs to increase the expression of LHR (19), inhibin-␣ (20), and side-chain cleavage cytochrome P-450 and is sufficient for the expres...

Ovarian follicles undergo exponential growth in response to follicle-stimulating hormone (FSH), largely as a result of the proliferation of granulosa cells (GCs). In vitro under serum-free conditions, rat GCs differentiate in response to FSH but do not proliferate unless activin is also present. In the presence of FSH plus activin, GCs exhibit enhanced expression of cyclin D2 as well as inhibin-alpha, aromatase, steroidogenic factor-1 (SF-1), cholesterol side chain (SCC), and epiregulin. In this report we sought to identify the signaling pathways by which FSH and activin promote GC proliferation and differentiation. Our results show that these responses are associated with prolonged Akt phosphorylation relative to time-matched controls and are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) and Smad2/3 signaling, based on the ability of the PI 3-kinase inhibitor LY294002 or infection with adenoviral dominant negative Smad3 (DN-Smad3) mutant to attenuate induction of cyclin D2, inhibin-alpha, aromatase, SCC, SF-1, and epiregulin. The DN-Smad3 mutant also abolished prolonged Akt phosphorylation stimulated by FSH plus activin 24 h post-treatment. Infection with the adenoviral constitutively active forkhead box-containing protein, O subfamily (FOXO)1 mutant suppressed induction of cyclin D2, aromatase, inhibin-alpha, SF-1, and epiregulin. Transient transfections of GCs with constitutively active FOXO1 mutant also suppressed cyclin D2, inhibin-alpha, and epiregulin promoter-reporter activities. Chromatin immunoprecipitation results demonstrate in vivo the association of FOXO1 with the cyclin D2 promoter in untreated GCs and release of FOXO1 from the cyclin D2 promoter upon addition of FSH plus activin. These results suggest that proliferation and differentiation of GCs in response to FSH plus activin requires both removal of FOXO1-dependent repression and positive signaling from Smad2/3.

We examined the phosphorylation and acetylation of histone H3 in ovarian granulosa cells stimulated to differentiate by follicle-stimulating hormone (FSH). We found that protein kinase A (PKA) mediates H3 phosphorylation on serine 10, based on inhibition exclusively by PKA inhibitors. FSH-stimulated H3 phosphorylation in granulosa cells is not downstream of mitogenactivated protein kinase/extracellular signal-regulated kinase, ribosomal S6 kinase-2, mitogen-and stressactivated protein kinase-1, p38 MAPK, phosphatidylinositol-3 kinase, or protein kinase C. Transcriptional activation-associated H3 phosphorylation on serine 10 and acetylation of lysine 14 leads to activation of serum glucocorticoid kinase, inhibin ␣, and c-fos genes. We propose that phosphorylation of histone H3 on serine 10 by PKA in coordination with acetylation of H3 on lysine 14 results in reorganization of the promoters of select FSH responsive genes into a more accessible configuration for activation. The unique role of PKA as the physiological histone H3 kinase is consistent with the central role of PKA in initiating granulosa cell differentiation.Maturation of ovarian follicles to a preovulatory stage requires follicle-stimulating hormone (FSH) 1 production by the pituitary gland. The FSH receptor is a member of the G protein-coupled seven-transmembrane receptor family and is coupled to adenylyl cyclase (1). It is expressed exclusively on ovarian granulosa cells in female mammals (2). Most of the actions of FSH are mediated by cAMP formation and activation of protein kinase A (PKA), based on the ability of cell-permeable cAMP analogs to mimic the known differentiation responses to FSH in granulosa cells (2) and on the ability of the PKA inhibitors H89 2 (3) and KT5720 2 to inhibit granulosa cell differentiation. The downstream consequences of FSH are well established and include, for example, the induction of receptors for luteinizing hormone (LH) and prolactin, induction of enzymes associated with the increased steroidogenic capacity of granulosa cells including P450 aromatase and cholesterol side chain cleavage, induction of proteins associated with PKA signaling including RII␤ (2, 4) and AKAP80 (5), and expression of the hormone inhibin (6). However, gene and/or protein induction for these responses to FSH is generally delayed by at least 24 h (2-4, 7). The more immediate responses to FSH, which lead to the induction of immediate early genes such as c-fos and serum glucocorticoid kinase (SGK) (3,8), are less well understood. Further elucidation of the FSH signaling pathways that lead to the induction of immediate early genes would be useful to understand how FSH initiates granulosa cell differentiation. We have previously shown that FSH (via PKA) promotes activation of the p42/p44 mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway (9). FSH also activates the p38 MAPK pathway (10) and downstream phosphorylation of the heat shock protein (HSP) 27, leading to granulosa cell rounding (10). We 3 and ot...

In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca 2؉ channels are already partially activated in vehicletreated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicletreated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca 2؉ channel blocker, and chelation of extracellular Ca 2؉ . These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase.The cytoplasmic p42/p44 mitogen-activated protein kinase (MAPK) 1 /extracellular signal-regulated kinases (ERKs) comprise a critical convergence point in the signaling pathways initiated by a variety of receptor agonists that promote cellular differentiation or proliferation. For the classic receptor tyrosine kinase-initiated pathway, growth factors like epidermal growth factor (EGF) induce the autophosphorylation of their receptors and create specific binding sites for Src homology 2-containing proteins such as Grb2 (1). Grb2 complexed to Sos associates with the receptor tyrosine kinase, and Sos stimulates GDP release from Ras, leading to Ras activation. Active Ras then binds to Raf-1, leading to its activation, and Raf-1 in turn catalyzes the serine phosphorylation and activation of the MAPK/ERK kinase MEK. MEK then catalyzes the phosphorylation of ERK on regulatory Thr and Tyr residues, resulting in ERK activation.Guanine nucleotide-binding protein-coupled receptors (GPCRs) are also well known activators of ERK; however, there are a variety of pathways by which GPCRs promote ERK activation. Often, GPCRs such as those activated by lysophosphatidic acid or angiotensin II promote the transactivation of a receptor tyrosine kinase as evidenced by its increased tyrosine phosphorylation (2). Receptor tyrosine kinase transactivation directs the tyrosine phosphorylation of adaptor proteins such as Shc, recruitment of the Grb2-Sos complex, and subsequent Ras activation. It is less clear how GPCRs promote the tyrosine phosphorylation of the receptor tyrosine kinase, although Src activation downstream of the G␤␥ has been implicated in some cells (3, 4). For those GPCRs whose activated G␣ subunits promote...