Steroidogenic factor-1 (SF-1) is a key regulator of endocrine development, and mediates expression of gonadotrophin-specific genes in the pituitary. Basal and hormone stimulated transcription of the human glycoprotein hormone alpha-subunit gene (alphaGSU) in gonadotrophs involves SF-1 and its cognate binding site, the gonadotroph-specific element (GSE). In this study, we demonstrate that SF-1 significantly enhances basal and forskolin-stimulated transcription of the human alphaGSU promoter in GH(3) cells. Mutation of the GSE abolished the SF-1-mediated transactivation of basal alphaGSU promoter activity, and significantly attenuated the forskolin effect by 50%. Mutation of the Ser203 residue in SF-1 to Ala blocked basal transactivation of alphaGSU promoter activity, and halved the forskolin effect. These data collectively reveal a direct role for SF-1 and the GSE in mediating basal and forskolin-stimulated transcription of the human alphaGSU promoter in GH(3) cells. The phosphorylation site at Ser203 appears to be required for these effects.
RC Fowkes and JM Burrin
RC Fowkes, J Burch and JM Burrin
The putative hypophysiotropic factor pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates glycoprotein hormone alpha-subunit (alpha GSU) gene transcription and secretion in the clonal gonadotroph alpha T3-1 cell line. The specific signalling pathways regulating these actions of PACAP have not been clearly defined. We have examined the possibility that mitogen activated protein kinases (MAPKs) may play a role in mediating the effects of PACAP on alpha T3-1 gonadotrophs. Treatment of alpha T3-1 cells with PACAP (100 nM) or epidermal growth factor (EGF, 10 nM) for 5 min significantly stimulated extracellular signal-regulated kinase activity (ERK, a component of the MAPK pathway) as determined by an immunocomplex assay. Pre-treatment of alpha T3-1 cells with the specific MAPK kinase (MEK) inhibitor, U0126, blocked PACAP and EGF-induced activation of ERK. Transcriptional stimulation of a human alpha GSU-luciferase reporter construct by PACAP was unaffected by U0126 treatment. However, pre-treatment with U0126 significantly inhibited PACAP stimulation of [(3)H]-thymidine incorporation in alpha T3-1 cells. Thus our results suggest that PACAP stimulates ERK activation in alpha T3-1 cells, and that the functional effect of this ERK activation is increased DNA synthesis and cell proliferation rather then transcriptional activation of the alpha GSU gene.
RC Fowkes, W Forrest-Owen and CA McArdle
C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, has been found at its highest tissue concentrations in the anterior pituitary, where it is localised in gonadotrophs. Its specific guanylyl cyclase-containing receptor, GC-B, is also expressed on several anterior pituitary cell types, and CNP potently stimulates cGMP accumulation in rat pituitary cell cultures and pituitary cell lines. The mouse gonadotroph-derived alpha T3-1 cell line has been shown to express CNP as well as GC-B (but not GC-A) receptors, suggesting that CNP may well be an autocrine regulator of gonadotrophs. Comparing effects of three natriuretic peptides (atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and CNP) on cGMP accumulation in four pituitary cell lines (alpha T3-1, TtT-GF, AtT-20 and GH(3)) we find that CNP is most potent and effective in alpha T3-1 cells. In these cells, CNP-stimulated cGMP accumulation was found to desensitise during a 30 min exposure to CNP. Pretreatment with CNP for up to 6 h also caused a significant reduction in the ability of CNP to subsequently stimulate cGMP accumulation. This effect was receptor specific, because pretreatment with sodium nitroprusside (an activator of nitric oxide-sensitive guanylyl cyclase), or with ANP or BNP, did not cause desensitisation of CNP-stimulated cGMP accumulation. Protein kinase C activation with phorbol esters also inhibited CNP-stimulated cGMP accumulation and such inhibition was also seen in cells desensitised by pretreatment with CNP. Thus it appears that the endogenous GC-B receptors of alpha T3-1 cells are subject to both homologous and heterologous desensitisation, that the mechanisms underlying these forms of desensitisation are distinct, and that cGMP elevation alone is insufficient to desensitise GC-B receptors.
RC Fowkes, C Chandras, EC Chin, S Okolo, DR Abayasekara and AE Michael
Luteinizing granulosa cells synthesize high concentrations of progesterone, prostaglandin (PG) E(2) and PGF(2 alpha). The objective of this study was to explore the relationship between prostaglandin and progesterone output from human granulosa cells as they undergo functional luteinization in culture. Granulosa cells were partially purified from ovarian follicular aspirates and cultured at a density of 10(5) cells/ml in serum-supplemented DMEM:Ham's F(12) medium for 0, 1 or 2 days. Cells were then switched to serum-free medium for 24 h before measuring hormone concentrations in this spent medium by specific radioimmunoassays. Over the first 3 days in culture, PGF(2 alpha) and PGE(2) production declined progressively by up to 82+/-3% coincident with a 55+/-11% increase in progesterone output. In subsequent experiments, cells were treated for 24 h on the second day of culture with either 0.01 to 10 microM meclofenamic acid or with 10 microM and 100 microM aminoglutethimide. Meclofenamic acid inhibited synthesis of PGF(2 alpha) and PGE(2) by up to 70+/-9% and 64+/-7% respectively without affecting progesterone output. Likewise, 100 microM aminoglutethimide inhibited progesterone production by 62+/-6% without affecting concentrations of either PGF(2 alpha) or PGE(2). We have concluded that the progressive decline in prostaglandin production and the rise in progesterone output from luteinizing human granulosa cells occur independently of each other.