Differential roles of PKC isoforms (PKCs) in GnRH stimulation of MAPK phosphorylation in gonadotrope derived cells.

The role of protein kinase C (PKC) isoforms (PKCs) in GnRH-stimulated MAPK [ERK1/2, JNK1/2 and p38) phosphorylation was examined in gonadotrope derived cells. GnRH induced a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2 and p38MAPK. Gonadotropes express conventional PKCα and PKCßII, novel PKCδ, PKCε and PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein (GFP)-PKCs constructs revealed that GnRH induced rapid translocation of PKCα and PKCßII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs) has revealed differential role for PKCα, PKCßII, PKCδ and PKCε in ERK1/2, JNK1/2 and p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in MAPKs phosphorylation may be explained by persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane. Thus, we have identified the PKCs involved in GnRH stimulated MAPKs phosphorylation in gonadotrope derived cells. Once activated, the MAPKs will mediate the transcription of the gonadotropin subunits and GnRH receptor genes.

Mitogen- and stress-activated protein kinase 1 is required for gonadotropin-releasing hormone-mediated activation of gonadotropin α-subunit expression.

Pituitary gonadotropin hormones are regulated by gonadotropin-releasing hormone (GnRH) via MAPK signaling pathways that stimulate gene transcription of the common α-subunit (Cga) and the hormone-specific ß-subunits of gonadotropin. We have reported previously that GnRH-induced activities at these genes include various histone modifications, but we did not examine histone phosphorylation. This modification adds a negative charge to residues of the histone tails that interact with the negatively charged DNA, is associated with closed chromatin during mitosis, but is increased at certain genes for transcriptional activation. Thus, the functions of this modification are unclear. We initially hypothesized that GnRH might induce phosphorylation of Ser-10 in histone 3 (H3S10p) as part of its regulation of gonadotropin gene expression, possibly involving cross-talk with H3K9 acetylation. We found that GnRH increases the levels of both modifications around the Cga gene transcriptional start site and that JNK inhibition dramatically reduces H3S10p levels. However, this modification had only a minor effect on Cga expression and no effect on H3K9ac. GnRH also increased H3S28p and H3K27ac levels and also those of activated mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 inhibition dramatically reduced H3S28p levels in untreated and GnRH-treated cells and also affected H3K27ac levels. Although not affecting basal Cga expression, MSK1/2 inhibition repressed GnRH activation of Cga expression. Moreover, ChIP analysis revealed that GnRH-activated MSK1 targets the first nucleosome just downstream from the TSS. Given that the elongating RNA polymerase II (RNAPII) stalls at this well positioned nucleosome, GnRH-induced H3S28p, possibly in association with H3K27ac, would facilitate the progression of RNAPII.

Ubiquitin C-terminal hydrolase l1 is expressed in mouse pituitary gonadotropes in vivo and gonadotrope cell lines in vitro.

The ubiquitin-proteasome system (UPS) plays a fundamental role in regulating various biological activities. Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme, belonging to the UPS. To date, it has been reported that UCH-L1 is highly and restrictedly expressed in neural and reproductive tissues and plays significant roles in these organs. Although the expression of UCH-L1 in the anterior pituitary gland has been reported, the detailed localization and the role of UCH-L1 remain obscure. In the present study, we detected UCH-L1 protein exclusively in hormone-producing cells, but not non-hormone producing folliculostellate cells in the anterior pituitary lobe. In addition, the cytoplasmic expression of UCH-L1 varied and was limited to gonadotropes and mammotropes. To investigate the role of UCH-L1 in anterior pituitary cells, we performed a comparative analysis using genetically UCH-L1-deficient gad mice. Significant decreases in the numbers of gonadotropes and mammotropes were observed in gad mice, suggesting a close involvement of UCH-L1 in these cells. Moreover, we also determined the expression of UCH-L1 in cultured gonadotropes. Taken together, this is the first report to definitely demonstrate the presence of UCH-L1 in mouse anterior pituitary gland, and our results might provide a novel insight for better understanding the role of UCH-L1 in the hypothalamic-pituitary-gonadal axis and in the reproduction.

Modulation of gonadotropin-releasing hormone-induced extracellular signal-regulated kinase activation by dual-specificity protein phosphatase 1 in LbetaT2 gonadotropes.

As the regulator of pituitary reproductive hormone synthesis, the hypothalamic neuropeptide GnRH is the central regulator of reproduction. A hallmark of GnRH action is the differential control of gene expression in pituitary gonadotropes through varied pulsatile stimulation. Among other signaling events, GnRH activation of the ERK family of MAPKs plays a significant role in the transcriptional regulation of the luteinizing hormone ß-subunit gene and regulation of cap-dependent translation. We evaluated the ERK response to different GnRH pulse amplitudes in the gonadotrope cell line LßT2. We found that low-amplitude stimulation with GnRH invokes a rapid and transient ERK activation, whereas high-amplitude stimulation invokes a prolonged activation specifically in the cytoplasm fraction of LßT2 cells. Nuclear and cytoplasmic targets of ERK, Ets-like gene 1, and eukaryotic initiation factor 4E, respectively, are similarly activated. Feedback control of ERK activation occurs mainly through the dual-specificity protein phosphatases (DUSPs). DUSP1 is localized to the nucleus in LßT2 cells but DUSP4, another member implicated in GnRH feedback, exists in both the nucleus and cytoplasm. Manipulation of nuclear DUSP activity through overexpression or knockdown of Dusp1 modulates the ERK response to low and high GnRH pulse amplitudes and activation of the Lhb promoter. Dusp1 overexpression abolishes sustained ERK activation and inhibits Lhb promoter activity induced by high amplitude pulses. Conversely, Dusp1 knockdown enhances ERK activation by low-amplitude stimulation and increases stimulation of Lhb promoter activity. We conclude that DUSP1 feedback activity modulates ERK activation and the transcriptional response to GnRH.

Mono-(2-ethylhexyl) phthalate (MEHP) regulates glucocorticoid metabolism through 11beta-hydroxysteroid dehydrogenase 2 in murine gonadotrope cells.

Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite mono-(2-ethylhexyl) phthalate (MEHP) have been classified as toxicants to the reproductive system at the testis level and DEHP may also impair reproductive axis function at the pituitary levels. However, MEHP is 10-fold more potent than DEHP in toxicity and little is known about the toxicological effect of MEHP on pituitary. In this study, we demonstrated that 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), not 11beta-HSD1, is strongly expressed in murine gonadotrope LbetaT2 cells. Interestingly, MEHP inhibited Hsd11b2 mRNA level and 11beta-HSD2 enzyme activity in LbetaT2 cells at as low as 10(-7)M. Corticosterone (CORT) at a concentration of 10(-6)M significantly inhibited LbetaT2 cell proliferation after 2-day culture, and 10(-6)M RU486, an antagonist of glucocorticoid receptor (GR), reversed this inhibition. However, in the presence of 10(-5) or 10(-4)M MEHP, the minimal concentration of CORT to inhibit the proliferation of LbetaT2 cells was lowered to 10(-7)M, and 10(-6)M RU486 was not able to completely reverse the CORT effect. In conclusion, along with the regulation of GR, 11beta-HSD2 may have a key role in glucocorticoid metabolism in LbetaT2 cells. MEHP may participate in the glucocorticoid metabolism in LbetaT2 cells through inhibition of 11beta-HSD2 enzyme activity. Such perturbation may be of pathological significance as MEHP may interfere with the reproductive system at pituitary level through regulation of glucocorticoid metabolism, especially in neonates with higher risk of phthalates exposure.

Induction of mPer1 expression by GnRH in pituitary gonadotrope cells involves EGR-1.

We reported earlier that gonadotropin-releasing hormone (GnRH) activates period1 (mPer1) gene expression in immortalized gonadotropes through protein kinase C and p42/44 mitogen-activated protein kinase pathways. GnRH stimulation also leads to the upregulation of early growth response protein 1 (EGR-1), a critical transcription factor for GnRH-induced luteinizing hormone beta (LHbeta) synthesis. The parallels between the GnRH-LHbeta and the GnRH-mPer1 pathways led us to explore whether EGR-1 is involved in the regulation of mPer1 expression in gonadotropes. Of particular interest was the presence of an EGR-1 binding site in the proximal promoter of the mPer1 gene. Stimulation of LbetaT2 gonadotrope cells with a GnRH agonist caused the rapid induction of Egr-1 mRNA, which was rapidly followed by mPer1 expression. Chromatin immunoprecipitation revealed that the mPer1 promoter can bind EGR-1, while site-directed mutagenesis experiments confirmed the involvement of Egr-1 sequences in maintaining basal and allowing GnRH-stimulated mPer1 transcription. By means of RNA interference experiments, it could also be demonstrated that silencing of Egr-1 expression resulted in markedly lower mPer1 transcript levels. This silencing effect of the Egr-1 siRNA could be rescued by transfecting the cells with an EGR-1 overexpression vector. In summary, these results all point to a role for the EGR-1 protein in transactivating both the LHbeta as well as the mPer1 gene in pituitary gonadotrope cells.

Activins regulate 17beta-hydroxysteroid dehydrogenase type I transcription in murine gonadotrope cells.

Activins are pleiotropic members of the TGFbeta superfamily and were initially characterized based on their abilities to stimulate FSH synthesis and secretion by gonadotrope cells of the anterior pituitary gland. Here, we identified the gene encoding the steroidogenic enzyme, 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD1; Hsd17b1), as an activin-responsive gene in immortalized gonadotrope cells, LbetaT2. 17beta-HSD1 catalyzes the conversion of estrone to the more active 17beta-estradiol, and activin A stimulated an increase in this enzymatic activity in these cells. We demonstrated that activins signaled via the type I receptor, activin receptor-like kinase (ALK4), and the intracellular signaling protein, SMAD2, to regulate Hsd17b1 transcription in immediate-early fashion. Critical cis-elements, including a minimal SMAD-binding element, were mapped to within 100 bp of the start of transcription. Activin/ALK4 signaling also regulated Hsd17b1 transcription in both immortalized and primary cultured murine granulosa cells. The promoter regions mediating basal and activin/ALK4-regulated promoter activity were generally conserved across the different cell types. The data show that activin A rapidly regulates Hsd17b1 transcription in gonadotrope and granulosa cells and may thereby regulate local 17beta-estradiol synthesis.

Protein kinase A serves as a primary pathway in activation of Nur77 expression by gonadotropin-releasing hormone in the LbetaT2 mouse pituitary gonadotroph tumor cell line.

Nur77 belongs to a subfamily of nuclear receptors that includes two other members, Nor-1 and Nurr1. It plays an important role in a number of biological processes, including regulation of signaling functions in the hypothalamo-pituitary-adrenal axis, regulation of thymocyte apoptosis, regulation of steroidogenesis and regulation of tumor cell proliferation and apoptosis. In previous studies, using DNA microarray analysis of the effects of the gonadotropin-releasing hormone (GnRH) on the mouse pituitary gonadotroph cell line LbetaT2, we identified Nur77 as one of the highly regulated immediate early genes involved in this response, with >40-fold upregulation after 1 h of treatment of the cells with the GnRH agonist [D-Ala6GnRH (GnRHA)]. GnRH is a hypothalamic decapeptide that stimulates the secretion and expression of gonadotropins (follicle stimulating hormone, FSH and luteinizing hormone releasing hormone, LH) from anterior pituitary through activation of high affinity receptors present on cell membrane of pituitary gonadotropes. In addition to pituitary, the presence of GnRH high affinity receptors has been reported in various cancers and cancer cell lines. In addition, GnRH and its analogs are clinically used in the treatment of prostate cancer. To elucidate the molecular mechanism involved in regulation of Nur77 by GnRH, we first confirmed upregulation of Nur77 in response to GnRH analog (GnRHA) in LbetaT2 cells. Nur77 mRNA was upregulated within 30 min of GnRHA treatment and returned to nearly basal level after 24 h of treatment. Nur77 protein expression was upregulated after 2 h of treatment and remained steady even after 12 h of treatment. The expression of Nur77 mRNA was induced by GnRHA in a dose-dependent manner. Induction of Nur77 expression was stimulated on treatment of cells with forskolin and 8-Br-cAMP, whereas H-89, a specific inhibitor of PKA pathway significantly inhibited GnRHA-induced Nur77 expression. Treatment of cells with both H-89 and EGTA completely blocked the GnRHA-induced expression of Nur77, indicating that both calcium and cAMP/PKA play an important role in regulation of Nur77 expression by GnRHA. Analysis of the protein kinase C (PKC) signaling pathway using specific inhibitors for PKC, Erk1/2, p38 and JNK demonstrated that these pathways are not involved in GnRHA-induced Nur77 expression. Based on our results, we conclude that activation of protein kinase A is the major mechanism regulating the expression of Nur77 by GnRH which may serve as a down-stream signaling gene to mediate the antitumor effects of GnRH.

The involvement of phosphatidylinositol 3-kinase in gonadotropin-releasing hormone-induced gonadotropin alpha- and FSHbeta-subunit genes expression in clonal gonadotroph LbetaT2 cells.

Akt/protein kinase B (Akt/PKB), which is activated by phosphatidylinositol-3 kinase (PI3-kinase), plays an important role in cell survival and cell proliferation. Using the well differentiated, clonal gonadotroph cell line, LbetaT2, we examined (1) whether Akt/PKB was activated by gonadotropin-releasing hormone (GnRH); (2) the contribution of PI3-kinase-Akt/PKB pathway in each of gonadotropin subunit gene expression; (3) crosstalk between extracellular signal-regulated kinase (ERK) and Akt/PKB pathways. Insulin-like growth factor-1 (IGF-1) was used as Akt/PKBs classic activator. Western blot analyses using antibodies specific for the phosphorylated forms of ERK and Akt/PKB demonstrated that both were rapidly phosphorylated following treatment with GnRH and IGF-1. Akt/PKB activation by GnRH and IGF-1 was completely eliminated in the presence of the PI3-kinase inhibitor, LY 294002, but not in the presence of an Akt/PKB inhibitor. Interestingly, the total amount of Akt/PKB protein was dramatically increased in the presence of LY 294002. Phosphorylation of ERK was significantly increased in the presence of LY 294002 alone, and was further increased when GnRH was used in combination with LY 294002. In experiments using a luciferase reporter construct containing the serum response element (SRE), a known target of the ERK pathway, LY 294002 but not the Akt/PKB inhibitor increased SRE-luciferase activity. GnRH-induced SRE-luciferase activity was significantly increased by LY 294002. GnRH stimulation resulted in gonadotropin LHbeta, FSHbeta, and alpha-subunit promoter activation, while IGF-1 failed to stimulate any of them. GnRH-induced gonadotropin promoter activities were not modulated in the presence of an Akt/PKB inhibitor, but treatment with LY 294002 or Wortmannin resulted in a significant increase in alpha- and FSHbeta-subunit promoter activation, both with and without GnRH. LY 294002, but not the Akt/PKB inhibitor, significantly inhibited cell proliferation. These results suggest that GnRH-induced gonadotropin gene expression is not regulated through the Akt/PKB pathway; however, PI3-kinase may be involved in the negative regulation of alpha- and FSHbeta-subunit gene expression as well as cell proliferation.