Extracellular signal-regulated kinase (ERK)–dependent phosphorylation of Y-Box binding protein 1 (YB-1) enhances gene expression in granulosa cells in response to follicle stimulating hormone (FSH)

ABSTRACT
Within the ovarian follicle, immature oocytes are surrounded and supported by granulosa cells (GCs). Stimulation of GCs by FSH leads to their proliferation and differentiation, events that are necessary for fertility. FSH activates multiple signaling pathways to regulate genes necessary for follicular maturation. Herein, we investigated the role of Y-box binding protein-1 (YB-1) within GCs. YB-1 is a nucleic acid binding protein that regulates transcription and translation. Our results show that FSH promotes an increase in the phosphorylation of YB-1 on Ser102 within 15 min that is maintained at significantly increased levels until ~8 h post treatment. FSH-stimulated phosphorylation of YB- 1(Ser102) is prevented by pretreatment of GCs with the PKA-selective inhibitor PKI, the MEK inhibitor PD98059, or the ribosomal S6 kinase-2 (RSK-2) inhibitor BI-D1870. Thus phosphorylation of YB-1 on Ser102 is PKA, ERK, and RSK-2 dependent. However, pretreatment of GCs with the protein phosphatase 1 (PP1) inhibitor tautomycin increased phosphorylation of YB-1(Ser102) in the absence of FSH; FSH did not further increase YB-1(Ser102) phosphorylation. This result suggests that the major effect of RSK-2 is to inhibit PP1 rather than to directly phosphorylate YB-1 on Ser102. YB-1 coimmunoprecipitated with PP1β catalytic subunit and RSK-2. Transduction of GCs with the dephospho-adenoviral(Ad)-YB-1(S102A) mutant prevented the induction by FSH of Egfr, Cyp19a1, Inha, Lhcgr, Cyp11a1, Hsd17b1, and Pappa mRNAs and estradiol-17β production. Collectively, our results reveal that phosphorylation of YB-1 on Ser102 via the ERK/RSK-2 signaling pathway is necessary for FSH-mediated expression of target genes required for maturation of follicles to a preovulatory phenotype.

INTRODUCTION
In the female, fertility requires maturation of the ovarian follicle that contains the oocyte surrounded by granulosa cells (GCs)2 and theca cells. Follicular maturation is a tightly regulated process that is initiated by FSH. FSH regulates at least 4000 target genes within GCs whose expression drives development of the follicle, allowing it to respond to the surge of luteinizing hormone (LH) that promotes ovulation, oocyte maturation, and formation of the corpus luteum that serves to support the developing embryo after fertilization and implantation (reviewed in (1,2)).The mechanism by which FSH signals to regulate gene and protein expression in GCs has been extensively investigated. FSH binds to its G protein-coupled receptor (GPCR) expressed exclusively on GCs to activate adenylyl cyclase, raise intracellular cAMP levels, and activate PKA (3-6). PKA then either directly phosphorylates proteins that regulate transcription or indirectly activates signaling cascades whose targets regulate primarily transcription and translation. Direct PKA targets in GCs include cAMP-response elementbinding protein (CREB) (Ser133) (7), histone H3 (Ser10) (8), and β-catenin (Ser552 and Ser675) (9). Upon phosphorylation, these direct PKA target proteins participate in the regulation of gene expression though interactions with DNA and additional transcription factors.One of the signaling cascades activated by FSH is the MAPK/ERK pathway (8,10), evidenced by the phosphorylation of ERK (Thr202/Tyr204) within 10 min of FSH treatment (11), and phosphorylation of its downstream target RSK-2 (Ser380) (8), shown schematically in Fig 1. FSH also activates the PI3K/protein kinase B (AKT) pathway (12,13). In a PKA–dependent manner, FSH enhances phosphorylation of the pivotal kinase AKT on both the proximal Thr308 and the distal Ser473 sites within 5 min following FSH treatment (12,14), followed by the rapid phosphorylation of downstream AKT targets, tuberin and forkhead box-containing protein O1 (FOXO1) on Thr1462 and Ser256, respectively (12,15,16).However, the mechanisms by which these signaling pathways regulate gene expression are less well understood.

One of the more important pathways activated by FSH is the PI3K/AKT pathway, based on evidence that this pathway is necessary for GC differentiation (9,15,17,18). Yet the only transcriptional factor regulated by this pathway that has been shown to modulate gene expression in GCs is FOXO1 (15,18). Our recent results, however, suggest the PI3K/AKT pathway is likely regulating additional transcriptional modulators, based on the ability of exogenous insulin-like growth factor 1 (IGF1) at concentrations greater than 1 ng/mL to synergize with FSH to enhance gene expression without further increasing the phosphorylation of FOXO1 (Ser256) (19). Strong evidence in cancer cells identified YB-1 as an AKT target (20-23) that, upon phosphorylation on Ser102, acts as a transcription factor to promote expression of genes such as Egfr (21,24-26), a recognized FSH gene target (27).YB-1 was first described as a major component of the inactive mRNA-protein complex (mRNP) (28,29). Further studies demonstrated that YB-1 interacts with both RNA and DNA specifically though its cold shock domain (30,31), and nonspecifically via basic and acidic clusters in its C-terminus (reviewed in (32)). YB-1 functionsin the cytosol to regulate mRNA translation and to stabilize mRNA; in the nucleus, YB-1 can regulate DNA repair, pre-mRNA splicing, and transcription (reviewed in (32)). Transcriptional targets for YB- 1, identified mostly using cancer cell lines, in addition to Egfr, include multidrug resistance gene1(Mdr1) (33), protein tyrosine phosphatase 1B (PTP1B) (34), cyclin A (Ccna) (35), and vascular endothelial growth factor (Vegf) genes (36).Herein, we show that FSH promotes the protracted phosphorylation of YB-1 on Ser102.

This phosphorylation is mimicked by forskolin and inhibited by expression of the selective PKA inhibitor PKI, indicating its dependence on PKA activity. Phosphorylation of YB-1 on Ser102 is unaffected by the PI3K inhibitor wortmannin, but is blocked by both the MEK/ERK inhibitor PD98059 and the RSK-2 inhibitor BI-1870, showing dependence on ERK and downstream RSK-2 signaling (Fig 1). Our results surprisingly suggest that the major RSK-2 target is not YB-1; rather RSK-2 promotes inactivation of PP1 to enhance YB-1(Ser102) phosphorylation. FSH leads to an increase of phospho-YB-1 in the nucleus. Using adenoviral YB-1 phosphorylation mutants, we show that phosphorylation of YB-1 on Ser102 is necessary for the expression of multiple gene targets, including Cyp19a1, Lhcgr, Cyp11a1, Egfr, Hsd17b1, Pappa, and Inha. Together these results suggest that the PKA- dependent activation of ERK/RSK-2 pathways in GCs in response to FSH, by promoting phosphorylation of YB-1 on Ser102, contributes to the expression of multiple FSH dependent gene targets that are necessary for follicular maturation. These results also suggest a previously underappreciated role for the ERK signaling pathway in immature GCs.The following were purchased: ovine FSH (oFSH-19) from the National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases (Torrence, CA); recombinant human IGF1 from Atlanta Biologicals; purified mouse EGF, anti–Flag (F3165), and testosterone propionate from Sigma–Aldrich; human fibronectin from BD Biosciences; antiphospho- YB-1 (Ser102; CST 2900), anti-YB-1 (CST 4202),anti-phospho–AKT (Ser473; CST 9271), anti-phospho–ERK (Thr202/Tyr204; CST 9107), anti- phospho–RSK-2 (Ser380; CST 9335), and anti– GST (CST 2622) from Cell Signaling Technologies; anti-SRC homology-2 (SH2) domain-containing tyrosine phosphatase (SHP2) (SC 7384) and Protein A/G PLUS Agarose (SC 2003) from Santa Cruz Biotechnology; wortmannin, PD98059, tautomycin, okadaic acid, forskolin, BI-D1870, 8-chlorophenylthiol (CPT)- cAMP and myristoylated (Myr)PKI from Calbiochem/EMD. 8-CPT-2’-O-methyl (Me)- cAMP was purchased from Life Sciences Institute; TRIzol-RNA Lysis Reagent and Fast SYBR Green Master Mix from Life Technologies; qScript from VWR.

Animals- Sprague-Dawley, CD-outbred rats (breeders from Charles River Laboratories) were from a colony maintained by our laboratory in a pathogen-free facility at Washington State University. The facility is maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals using protocols approved by the Washington State University Animal Care and Use Committee.GC culture and Western Blotting- Immature female rats were primed with subcutaneous injections of 1.5 mg estradiol-17β (E2) in propylene glycol on days 21-23 to promote growth of preantral follicles. GCs were collected from ovaries on day 24 by puncturing individual follicles using 27-gauge needles (37). Cells were plated on fibronectin-coated plates at a density of~1×106 cells/mL of serum–free media supplemented with 100 U/mL penicillin (P), 100 µg/mL streptomycin (S), and 1 nM E2 or (6). For the experiments measuring secreted estradiol-17β levels, 10 ng/mL androstendione was added instead of E2. GC cultures are reported to be 88.6± 6.4% pure, based on the presence of the FSHR as detected by flow cytometry (38); are free of contaminating theca cells, based on the absence of response to LH (39); and initially contain ~ 0.02% oocytes (40) that are removed with addition of fresh media. These GC cultures faithfully mimic differentiation responses to FSH (reviewed in (3)), but do not mimic proliferation response to FSH (15,41,42) Indicated treatments were added to cells ~20 h following plating, and terminated by aspirating media and washing once with PBS. For western blotting, total cell extracts were collected by scraping cells in SDS sample buffer (43),followed by heat denaturation. Equal protein loading was accomplished by plating equal numbers of cells, collecting at 50 µL/1×106 cells in SDS sample buffer, and loading equal volumes of extract per gel lane. Equal loading was verified by probing for total SHP2 or total AKT, as indicated. Proteins were separated by SDS/PAGE and transferred onto either Hybond C-extra or Protran (Amersham Biosciences) nitrocellulose membrane (8). The membrane was incubated with primary antibody overnight at 4°C, and antigen–antibody complexes were detected using enhanced chemiluminescence (ThermoFisher). Westerns were scanned using an Epson Perfection V500 scanner and Adobe Photoshop CS2 9.0 software with minimal processing and quantified using Quantity One software (BioRad Laboratories). Experimental densitometric values were divided by load control protein values and expressed relative to vehicle values. Results were analyzed using GraphPad Prism and significance was determined using either a one way ANOVA with Tukey’s Multiple Comparison Test or a one tailed Student’s t test.Immunoprecipitation- Briefly, ~107 cells were scraped into 0.5 mL Immunoprecipitation Lysis buffer (14), sonicated twice on ice for 45 s, and clarified by centrifugation at 10, 000 x g for 5 min at 4°C. Following removal of 0.03 mL sample for input, soluble GC extracts were precleared using species matched matrix (ExactaCruz Preclearing Matrix, Santa Cruz Biotechnology) for 60 min at 4°C. Samples were then incubated by rotating at 4°C overnight with the specified antibodies and Protein A/G PLUS Agarose (Santa Cruz Biotechnology; SC 2003) or antibody– agarose conjugate along with a species matched IgG antibody control (Santa Cruz Biotechnology). Following centrifugation at 10,000 x g for 5 min at 4°C, 0.4 mL of the supernatant fraction, representing the unbound protein fraction, was collected in SDS sample buffer. Agarose beads were washed by rotating 5 min at 4°C with 1 mL Immunoprecipitation Wash Buffer (14) 4 times.

Bound proteins were collected in SDS sample buffer and subjected to SDS/PAGE and western blotting. Inputs represent ~2%, and bound sample~98% of total sample.Transfection of GCs and Luciferase Assay- Egfr (-1109 to -16 bp) (44)-, Inha (-2021 to+68 bp) (45)-, and Cyp19a1 (-294 to + 20 bp)(46)- promoter-luciferase constructs were previously described. Cells were plated on fibronectin-coated plates at a density of 1×106 cells/mL in OptiMEM + P/S + E2 with expression constructs (500 ng/ 1×106 cells) and transfected using LipofectAMINE 2000 (Invitrogen) according to manufacturer’s instructions. After 6 h incubation, media was removed and fresh DMEM+P/S +E2 was added. Cells were treated as indicated following ~16 h recovery period then lysed in 0.1 mL Luciferase Assay Buffer (25 mM Hepes pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1mM DTT, 1% Triton X -100). Samples were analyzed for luciferase activity using a Burthold Technologies Lumat LB 9507 luminometer using the Promega Luciferase Assay System according to manufacturer’s instructions.Adenoviral production- Full length YB-1- Flag was purchased from OriGene (Rockville, MD). YB-1–Flag mutants were obtained using QuikChange Lightning Site-Directed Mutagenesis Kit from Agilent Technologies (Santa Clara, CA) according to manufacturer’s instructions. The following primers were used: YB-1(S102D) forward 5′ GTC TCT CCA TCT CCT ACA TCG CGA AGG TAC TTC CTG GG 3′ reverse 5′ CCC AGG AAG TAC CTT CGC GAT GTA GGA GAT GGA GAG AC 3′; YB-1(S102A) forward 5′CCA GGA AGT ACC TTC GCG CTG TAG GAG ATG GAG AGA 3′ reverse 5′ TCT CTC CAT CTC CTA CAG CGC GAA GGT ACT TCC TGG 3′. YB-1-Flag, YB-1 (S102D)–Flag and YB-1(S102A)–Flag were cloned into pShuttleX and then cloned into Adeno –X by Vector Development Laboratory (Baylor College of Medicine, Houston TX). Adenoviral (Ad)-YB- 1(S102D)- Flag was used at (1.4 x 108 pfu/mL) and Ad-YB-1(S102A)– Flag was used at (1 x 109 pfu/mL).Cellular Fractionation- Fractionation was performed using a modified protocol previously described (47). GCs were treated as indicated, washed once with cold PBS, and collected in 0.5 mL Cytoplasmic Lysis buffer (10 mM Hepes pH 7.4, 10 mM KCl, 0.01mM EDTA, 0.1 mM EGTA,2mM DTT, 5 mM Na2VO4, 10 mM NaF, 20 mMsodium β- glycerophosphate, 0.1% NP40, Halt protease inhibitor mixture) and incubated on ice for 20 min. Nuclei were pelleted though centrifugation at 5,000 x g 4°C for 10 min, and the supernatant containing the cytoplasmic fractionGTA 3’; ribosome L19 forward 5’ GTG ACC TGG ATG CGA AGG A 3’ reverse 5’ GCC TTG TCT GCC TTC AGT TTG 3’.Estradiol-17β radioimmunoassay (RIA)- GCs were treated as described above, in the presence on 10 ng/ml androstenedione. Following treatment, cellular media was collected. Estradiol- 17β levels were measured by RIA at the UVA Center for Research in Reproduction Ligand Assay and Analysis Core (48).

RESULTS
FSH signals to stimulate phosphorylation of YB-1(Ser102) through a PKA-dependent pathway- We initially determined whether YB-1 was expressed in GCs and whether its expression at the protein level was regulated by FSH. Results (Fig. 2A and 2B) show that YB-1 total protein, which migrates on SDS-PAGE at 50 kDa, is readily detected in GCs and its expression is not acutely regulated by FSH. We then determined whether YB-1 was phosphorylated on Ser102 in response to FSH. FSH promoted a relatively slow increase in YB-1(Ser102) phosphorylation first detected 15 min after treatment (Fig. 2A). YB- 1(Ser102) phosphorylation peaked 1 h following FSH treatment; this peak level of phosphorylation was maintained until about 8 h and then declined to undetectable levels by 24 h (Fig. 2B). (The identities of the faster and slower migrating bands in phospho-YB-1 blots are not known.) Increased phosphorylation of YB-1(Ser102) was mimicked by the adenylyl cyclase activator forskolin (Fig. 3A). Phosphorylation of AKT(Ser473) serves as a positive control. YB-1(Ser102) phosphorylation also increased in response to 8-CPT–cAMP (Fig. 3B), a cell permeable cAMP analog that activates both exchange proteins activated by cAMP and PKA (49). But 8-CPT-2’-O-Me-cAMP, a cAMPanalog that does not activate PKA (50), failed to increase YB-1(Ser102) phosphorylation (Fig. 3B). Treatment of GCs with Ad–PKA inhibitor (PKI), a pseudosubstrate for the catalytic subunit of PKA (51), led to a loss of FSH-dependent YB-1(Ser102) phosphorylation [82% ± 17%] (Fig. 3C). Similarly, treatment with Myr–PKI peptide resulted in a loss of YB-1 phosphorylation mediated by FSH (data not shown). Phosphorylation of AKT(Ser473) has been shown to be PKA dependent in GCs (14) and serves as a positive control. Taken together, these dataindicate that FSH stimulation results in phosphorylation of YB-1 on Ser102 in a PKA- dependent manner in GCs.Phosphorylation of YB-1(Ser102) is dependent on the ERK pathway and not PI3K/AKT pathway.

The phosphorylation of YB-1(Ser102), investigated in various cancer cell lines, is most commonly mediated by AKT (20-23), although there is evidence that this site can be phosphorylated by RSK-2 downstream of ERK (26,52). Because FSH in a PKA-dependent manner activates both the PI3K/AKT (14) and ERK signaling pathways (8,10,11,53) in GCs, we investigated both pathways as possible regulators of YB-1 phosphorylation. Pretreatment of GCs with the PI3K inhibitor wortmannin (54), did not decrease FSH stimulated YB-1(Ser102) phosphorylation, while AKT(Ser473) phosphorylation was inhibited significantly [74.2% ± 13.0%] (Fig. 4A). However,pretreatment with the MEK inhibitor PD98059(54) promoted a near complete inhibition of YB- 1(Ser102) phosphorylation [94.2% ± 3.6%] in the presence of FSH (Fig. 4B), similar to the inhibition of ERK(Thr202/Tyr204) phosphorylation [98.1% ±5.9%] that is used as the positive control. Equivalent results were obtained with a different MEK inhibitor U0126 (54) (data not shown). Consistent with these results, treatment of GCs with EGF, a known activator of the MEK/ERK pathway, promoted increased levels of YB- 1(Ser102) phosphorylation as well as RSK-2(Ser380) phosphorylation (Fig. 4C). When GCs were treated with the PI3K/AKT pathway activator IGF1 or FSH, only cells treated with FSH showed enhanced YB-1(Ser102) phosphorylation (Fig. 4C). Unlike many other cell models, IGF1 does not activate the ERK pathway in GCs (55). Additionally, GCs treated with the RSK-2 inhibitor BI-D1870 (56) exhibited a marked inhibition [89.4% ± 5.3%] of YB-1(Ser102) phosphorylation in the presence of FSH (Fig. 5). CREB(Ser133) phosphorylation serves as a negative control. Taken together, these results indicate that FSH-stimulated phosphorylation of YB-1(Ser102) is mediated by RSK-2 downstream of ERK.Phosphorylation of YB-1(Ser102) is dependent on protein phosphatase 1 (PP1) inhibition- Enhanced phosphorylation of YB- 1(Ser102) is relatively slow, compared to thephosphorylation of CREB which is detected within 1 min (11) or of ERK which is detected within 10 min (8,11) of FSH treatment. Moreover, YB- 1(Ser102) phosphorylation is detected up to 8-12 h (Fig. 2B) while phosphorylations of both ERK(11) and RSK-2 (8) are undetectable by 2 h post treatment with FSH. We therefore sought to determine if the inhibition of a Ser/Thr phosphatase contributed to the phosphorylation of YB-1(Ser102).Pretreatment of GCs with the protein phosphatase 2 (PP2) inhibitor okadaic acid (at 200 nM (57,58)) resulted in an increase in YB- 1(Ser102) phosphorylation in both vehicle- and FSH-treated GCs (Fig. 6A).

Phosphorylation of RSK-2(Ser380 ) is indicative of RSK-2 activity (59) and provides insight into the regulation of YB- 1(Ser102) phosphorylation in the presence of okadaic acid. RSK-2(Ser380) phosphorylation was enhanced both in the absence and presence of FSH in response to okadaic acid and appears to account for the increase in YB-1(Ser102) phosphorylation. Fold activation by FSH relative to EtOH vehicle in the absence and presence of okadaic acid for pYB- 1(Ser102) was 6.2 and 16.0, and for pRSK-2(Ser380) was 7.2 and 21.2, respectively. These results suggest that okadaic acid is inhibiting a phosphatase that acts upstream of YB-1 rather than at the level of YB-1.Pretreatment of GCs with the PP1 inhibitor tautomycin (at 1 µM (57,58)) resulted in an increase in YB-1(Ser102) phosphorylation in vehicle-treated cells to levels equivalent to those seen in FSH-treated cells in the absence of tautomycin, while FSH did not further enhance YB-1(Ser102) phosphorylation (Fig. 6B). Tautomycin pretreatment did not enhance FSH- stimulated RSK-2(Ser380) phosphorylation. Fold activation by FSH relative to EtOH vehicle in the absence and presence of tautomycin for pYB- 1(Ser102) was 3.4 and 4.6, and for pRSK-2(Ser380) was 3.7 and 3.2, respectively. These results suggest that PP1 promotes the dephosphorylation of YB-1 on Ser102 in the absence of FSH.If PP1 maintains the dephosphorylation of YB-1(Ser102) in the absence of FSH, then a PP1 catalytic subunit should co-immunoprecipitate with YB-1. As total YB-1 antibody is not immunoprecipitating, we transiently transfected GCs with a full length Flag-tagged YB-1 plasmid, allowed ~16 h for cells to recover, treated GCswith vehicle or FSH, then immunoprecipitated Flag-tagged YB-1 with anti-Flag antibody. Results (Fig. 6C) show that anti-Flag antibody selectively pulls down comparable levels of the catalytic subunit PP1β (PP1cβ) in both vehicle- and FSH- treated samples. Additionally, when the same experiment was performed and the presence of RSK-2 was investigated, results show that RSK-2 also interacts (directly or indirectly) with Flag- tagged YB-1 in a FSH-independent manner (Fig. 6C). These results suggest that both PP1cβ and RSK-2 are in a complex with YB-1 and that these actions are independent of FSH.

Taken together, these results suggest (i) that PP2 inhibits YB-1 phosphorylation on Ser102 indirectly through regulation of RSK-2 itself or on a site upstream of RSK-2, and (ii) that PP2 activity is not regulated by FSH. Additionally, these results suggest (iii) that PP1 maintains YB- 1(Ser102) in a dephosphorylated state in the absence of FSH, and (iv) that FSH signals to inhibit the activity of PP1, preventing the dephosphorylation of YB-1 at Ser102. The inability of FSH to significantly enhance YB-1(Ser102) phosphorylation in the presence of tautomycin suggests that the major effect of PKA via ERK/RSK-2 is to promote inactivation of PP1.YB-1 phosphorylated on Ser102 is readily detected in the nuclear fraction- As the phosphorylation of YB-1 on Ser102 in breast cancer cells is most often linked to its function as a transcriptional activator (21,23,24,26,33) (and as reviewed in (60)), we asked if phospho-YB-1 was preferentially localized to the nuclear fraction of GCs.To address this question, GCs were transfected with Flag-tagged YB-1 plasmids that express wildtype (WT) YB-1, the phospho- YB- 1(S102D) mimic, and dephospho-YB-1(S102A) mutant. Following a 16 h recovery period, GCs were treated with vehicle or FSH and then fractionated into cytosolic and nuclear fractions; GAPDH and histone H3 serve as positive controls for the purity of the cytoplasmic and nuclear fractions, respectively. (Cytoplasmic and nuclear fractions represent 9% and 50% of total sample, respectively). All of the Flag-tagged constructs were readily detected in the cytoplasmic fraction (Fig. 7A), consistent with the presence of both phosphorylated YB-1(Ser102) and non- phosphorylated YB-1(Ser102) in this fraction (asshown below). However, the phospho-YB- 1(S102D) mimic was readily detected in the nuclear fraction at higher levels compared to both YB-1-WT and the dephospho-YB-1(S102A) mutant.Phosphorylated YB-1(Ser102) was also readily detected using the phospho-YB-1(Ser102) antibody in both the nuclear and cytosolic fractions of non-transfected GCs (Fig. 7B, left panel).

However, FSH did not promote a change in total levels of YB-1 within the nuclear or cytoplasmic fractions (Fig. 7B, right panel), suggesting either that the phosphorylation of YB-1 on Ser102 occurs within the nuclear fraction on/or that a relatively small fraction of total YB-1 phosphorylated in the cytoplasmic fraction translocates into the nuclear fraction.While the majority (~90%) of total and phosphorylated YB-1 in non-transfected GCs is localized to the cytoplasmic fraction, the presence of phosphorylated YB-1(Ser102) in the nuclear fraction suggests a potential role for this phosphoprotein in the regulation of gene expression.Phospho-YB-1(Ser102) is necessary for expression of select FSH target genes- Based on the increased levels of the phospho-YB-1(S102D) mutant detected in the nuclear fraction compared to WT and dephospho-YB-1(S102A) mutant, we hypothesized that YB-1 could participate in the transcriptional regulation of FSH target genes. While ERK signaling is critical to LH-induced ovulation and luteinization in preovulatory GCs (61), the importance of the ERK signaling pathway to FSH-stimulated differentiation of GCs is less well understood. We therefore initially sought to identify FSH target genes that were regulated by the ERK signaling pathway. Pretreatment of GCs with the MEK inhibitor U0126 resulted in inhibition of FSH-induced Egfr [85.7% ± 6.2%], Inha [82.5% ± 3.2%], Cyp19a1[96.1% ± 2.65%], and Ereg [67.7% ± 4.4%]mRNA levels measured at 48 h post FSH treatment, while Star mRNA levels were not affected (Fig. 8). Treatment with the MEK inhibitor PD98059 showed significant inhibition of FSH-induced expression of the immediate early gene Egr1 [86.4% ± 3.2%] 1 h post FSH treatment (Fig. 8).We next transduced GCs with adenoviral phospho- and dephospho-mutants of YB-1 todetermine if select ERK–regulated gene targets were activated by the phosphorylation of YB-1 on Ser102. We initially focused on the three ERK- regulated target genes that showed the greatest inhibition by the MEK inhibitor at the 48 h time point; Star expression was included as a negative control. Transduction of GCs with Ad-YB- 1(S102D) phospho-mutant resulted in a statistically significant FSH-independent increase in Egfr and Cyp19a1 mRNAs compared to Ad- Empty (E), that was not significantly enhanced further by FSH (Fig. 9A). In contrast, Inha mRNA levels were not different between Ad-E and Ad- YB-1(S102D) treatments.

The ability of the Ad- YB-1(S102D) phospho-mutant to enhance Egfr and Cyp19a1 mRNA levels in the absence of FSH suggests that phosphorylated YB-1(Ser102) is a major contributor to the induction of these genes.Transduction of GCs with the dephospho- Ad-YB-1(S102A) mutant prevented the induction by FSH of Egfr [82.0% ± 9.5%] and Cyp19a1 [88.6% ± 5.5%] mRNAs (Fig. 9B), consistent with results in Fig. 9A. FSH-stimulated induction of Lhcgr [98.6 ± 0.75%], Inha [90.9% ± 2.9%],Cyp11a1 [85.3 ± 3.2%], Hsd17b1 [94.5 ± 3.5%],and Pappa [92.7 ± 6.3%] mRNAs were also abrogated by the dephospho-Ad-YB-1(S102A) mutant. In contrast, FSH-stimulated Star mRNA expression was not reduced by the dephospho-Ad- YB-1(S102A) mutant. The unexpected ability of the dephospho-YB-1(S102A) mutant to induce expression of Egfr in the absence of FSH may be a consequence of YB-1 overexpression, such that the phosphorylation requirement for YB-1 to promote expression of this gene was negated (62). Taken together, we interpret these results to suggest that YB-1 phosphorylated on Ser102 is necessary for the induction of Lhcgr, Cyp19a1, Cyp11a1, Inha, Egfr, Hsd17b1, and Pappa mRNA by FSH, but does not participate in the induction of Star mRNA by FSH.GCs transfected with the indicated promoter-luciferase constructs, followed by transduction with Ad-YB-1(S102A) mutant, promoted a significant (p < 0.05) inhibition of Egfr [64.9% ± 13.2%], Cyp19a1 [59.9% ±10.0%], and Inha promoter-luciferase [72.5% ±5.0%] activities (Fig. 10). These promoter- luciferase results suggest that phosphorylated YB-1 is acting primarily at the level of the gene promoters and not at the level of mRNA stability.The lack of complete inhibition of FSH-stimulated signal by the dephospho-YB-1(S102A) mutant of Cyp19a1 and Inha promoter-luciferase constructs, as opposed to the ~90% inhibition of mRNA expression shown in Fig. 9, either reflects the nature of the promoter-luciferase constructs that may be lacking important regulatory elements, and/or provides indirect evidence that phospho- YB-1 is not only regulating gene transcription but also mRNA stability.Finally, estradiol-17β levels were measured in culture media of GCs that were transduced with Ad-E versus Ad-YB-1(S102A) and treated with vehicle or FSH for 48 h. Consistent with the ability of the dephospho-Ad- YB-1(S102A) mutant to inhibit the induction by FSH of Cyp19a1, Ad-YB-1(S102A) blocked the ability of FSH to enhance estradiol-17β biosynthesis in the presence of exogenous androstenedione [95.3 ± 2.9%] (Fig. 11).Collectively, these results indicate that YB-1(Ser102) phosphorylation is necessary for FSH-mediated induction of Egfr, Cyp19a1, Inha, Lhcgr, Cyp11a1, Hsd17b1, and Pappa genes that characterize the preovulatory GC as well as for estradiol-17β biosynthesis.

DISCUSSION
Our results show that YB-1 is phosphorylated on Ser102 in response to FSH. However, in contrast to our original premise, YB- 1 in GCs is not an AKT target. Rather, our results reveal that YB-1 is phosphorylated on Ser102 in response to FSH in a PKA- and ERK/RSK-2- dependent manner, consistent with previous reports that YB-1(Ser102) is a RSK-2 target (26,52). Unexpectedly, our results suggest that the major effect of RSK-2 on YB-1(Ser102) phosphorylation is to inactivate PP1 rather than to promote the direct phosphorylation of YB-1, as discussed below. YB-1 phosphorylated on Ser102 is readily detected to the nuclear fraction and is required for the ability of FSH to induce key target genes required for follicular maturation. Together, our results also reveal a previously unidentified role for YB-1 and an unappreciated role for the ERK/RSK-2 signaling pathway in FSH-dependent GC maturation.YB-1 is a multifunctional DNA and RNA binding protein and consequently participates in most DNA- and RNA-dependent cellular functions(30,31). In the cytoplasm, YB-1 is one of two primary proteins (along with poly-A binding protein) that comprise the mRNP complex (as reviewed in (32)). Hence, YB-1 is associated with both free nontranslated and polysomal mRNA, and is a major regulator of translation (63-65). YB-1 also functions as a transcriptional factor to both repress and activate transcription by binding directly to gene promoters or in complex with other transcriptional factors (as reviewed in (22,32)). YB-1 is generally viewed as an “oncogenic” transcription factor, based on its overexpression in breast and lung cancer cells (21,66) and on its gene targets, that include Egfr, Her2, Pi3kca, and Mdr1 (as reviewed in (32)). While YB-1 can be phosphorylated on a number of Ser and Tyr residues, phosphorylation of Ser102 within the cold shock domain is most commonly associated with the regulation of translation and transcription (as reviewed in (32)).

Phosphorylation of YB-1 on Ser102 promotes its nuclear localization in ovarian and breast cancer cells (20,22,26) and is required for Egfr expression in breast cancer cells (21,23,24,26).Our results show that YB-1 is also phosphorylated on Ser102 in ovarian GCs, and that a phospho-YB-1(S102D) mutant is readily detected in the nuclear fraction. Unexpectedly, our results also reveal that YB-1 phosphorylated on Ser102 is necessary for the expression of select FSH gene targets that define a mature, preovulatory GC, such as Lhcgr, Cyp11a1, Egfr, Hsd17b1, Pappa, Cyp19a1, and Inha.Lhcgr encodes the LH receptor that is required for ovulation, cumulus cell expansion, and the resumption of meiosis (67); Cyp11a1 encodes cytochrome P450 side chain cleavage, the rate-limiting enzyme for progesterone biosynthesis (18,68); the EGFR contributes to LH-stimulated expansion of cumulus granulosa cells, resumption of meiosis, and ovulation (27,69,70); Cyp19a1 encodes aromatase, the rate limiting enzyme in estrogen biosynthesis (71); Inha encodes for the inhibin hormone subunit inhibin-α that inhibits FSH expression by the anterior pituitary (72); Hsd17b1 encodes the enzyme that converts androstenedione and estrone to testosterone and estradiol-17β, respectively, and is required for normal ovulation and corpus luteum formation (73); and Pappa encodes a secreted metalloproteinase that cleaves IGF bindingproteins and contributes to estrogen and progesterone biosynthesis and to ovulation (74).FSH-stimulated gene expression in GCs is complex and requires activation of a number of transcriptional factors coupled with inhibition of transcriptional repressors. These events are mediated by multiple signaling pathways activated by FSH and PKA (as reviewed in (2)). CREB, SF- 1(Nr5a1)/LRH-1(Nr5a2), and GATA-4/6 enhance expression of both Cyp19a1 and Inha (46,75-77), and FOXO1 (15) and T-cell factor 3 (9) repress expression of both genes. Many transcriptional proteins that regulate expression of the Lhcgr (9,78,79) and Cyp11a1 (76,80,81) have been elucidated, although the mechanisms by which FSH enhances Egfr, and Pappa expression have not been reported.

However, a role for YB-1, and indeed for the ERK signaling pathway in the transcriptional activation of FSH-target genes is novel.FSH dependent phosphorylation of YB-1 on Ser102 is mimicked by treatment of GCs with EGF. Although ERK phosphorylation is FSH- dependent, we previously reported that the canonical pathway upstream of ERK is constitutively active, yet necessary for ERK phosphorylation in immature GCs (8,11). Consistent with this premise, FSH-stimulated ERK phosphorylation is blocked by the EGFR antagonist AG1478 (11). We expect that, in a similar manner, FSH-stimulated ERK/RSK-2 dependent YB-1 phosphorylation on Ser102 is dependent on an active upstream EGFR/MEK pathway that would be blocked by inhibition of upstream signaling proteins, such as the EGFR, RAS and RAF.While YB-1 phosphorylated on Ser102 is readily detected in the nuclear fraction of GCs, the great majority of both phosphorylated YB- 1(Ser102) and total YB-1 is present in the cytoplasmic fraction (see Fig. 7, where the cytoplasmic and nuclear fractions represent 9% and 50% of the total fraction). Cytoplasmic YB-1 has been linked with both translated polysomal mRNPs and free untranslated mRNPs (reviewed in (32)). YB-1 is recognized to stabilize both long- and short-lived mRNAs at high YB-1/mRNA ratios effectively by burying mRNAs within YB-1 protein complexes (reviewed in (32)). In NIH3T3 cells, AKT-dependent phosphorylation of YB-1 on Ser102 stimulated Cap-dependent translation (64).It is thus likely that cytosolic YB-1 is playing a major role in both stabilizing GC mRNA and, upon phosphorylation, enhancing translation.FSH-stimulated phosphorylation of YB-1 on Ser102 in GCs is mediated by PKA, ERK, and its downstream kinase RSK-2. However, to our surprise, our results suggest that the major effect of RSK-2 is to inhibit the phosphatase activity of PP1. This conclusion is based largely on the inability of FSH to increase phospho-YB-1(Ser102) signal over that seen in vehicle-treated cells in the presence of the PP1 inhibitor tautomycin (see Fig. 6B). Moreover, the RSK-2 inhibitor BI-D1870 abrogates YB-1(Ser102) phosphorylation in vehicle- and FSH-treated cells, even though PKA remains active (evidenced by CREB phosphorylation on Ser133; see Fig.5). Additionally the PP1β catalytic subunit, RSK-2, and YB-1 co- immunoprecipitate in a complex. The presence of phosphorylated YB-1(Ser102) in GCs pretreated with tautomycin and treated with vehicle (i.e., in the absence of FSH) further suggests that while RSK-2 may also directly phosphorylate YB-1 in the presence of FSH, RSK-2 is not the kinase that phosphorylates YB-1 on Ser102 in vehicle-treated cells. We hypothesize that Ser102 may be phosphorylated by the constitutively active kinase casein kinase 2, as the phosphorylation motif on YB-1 (KYLRS102VGD) partially aligns with that of casein kinase 2 (S*E/DXE/D) (82).

We do not know the mechanism by which RSK-2 inactivates PP1 activity. The catalytic subunits of PP1 can associate with a large number of regulatory subunits (up to 200 have been identified), many of which inhibit the catalytic activity of PP1 (83). Unfortunately, we have not been able to identify the PP1 regulatory subunit inhibited by RSK-2. We hypothesize, however, that RSK-2-dependent phosphorylation of the PP1 regulatory subunit in GCs promotes its degradation, allowing for the prolonged phosphorylation of YB-1 that persists up to 8 h post FSH treatment, despite the shorter-lived phosphorylation of RSK-2 that is undetectable within 2 h post FSH treatment (8).The prolonged phosphorylation of YB-1 is not observed with other transcriptional targets in GCs, such as CREB and histone H3, whose phosphorylation has returned to the levels of untreated cells by 4 h post FSH treatment (8). We do not know the functional significance of thissustained phosphorylation of YB-1. It is unlikely to be required for transcriptional regulation. The prolonged YB-1 phosphorylation is more likely to participate either in mRNA stabilization and/or translation.While Ser102 is the most studied phosphorylation site on YB-1, multiple kinases have been shown to phosphorylate YB-1. ERK and glycogen synthase kinase 3β are reported to phosphorylate YB-1 on Thr38 and/or Ser41 and/or Ser45 (36). Indeed, mass–spectrometric analysis has identified additional phosphorylations on YB- 1, including Tyr162, Ser165 and/or Ser167, Ser174 and/or Ser176, and Ser313 and/or Ser314 (84,85). Therefore, we cannot rule out the possibility that FSH promotes phosphorylation of YB-1 on additional sites by other kinases.

In conclusion, we have shown that FSH promotes YB-1(Ser102) phosphorylation through a PKA- and ERK/RSK-2-dependent signaling pathway. The major effect of RSK-2 appears to be to inhibit the Ser/Thr phosphatase PP1 rather than to directly phosphorylate YB-1 on Ser102. YB-1 phosphorylated on Ser102 is necessary for the expression of FSH-dependent gene targets that BI-D1870 are essential for GC maturation. Taken together, our results not only establish a functional role for YB- 1 as a transcriptional activator of FSH target genes in the GCs, but also reveal an underappreciated role for ERK signaling pathway in the activation of gene expression by FSH in GCs.