scholarly journals Steroidogenic Factor-1 and The Gonadotrope-Specific Element Enhance Basal and Pituitary Adenylate Cyclase-Activating Polypeptide-Stimulated Transcription of the Human Glycoprotein Hormone α-Subunit Gene in Gonadotropes

2003 ◽  
Vol 17 (11) ◽  
pp. 2177-2188 ◽  
Author(s):  
Robert C. Fowkes ◽  
Marion Desclozeaux ◽  
Mayur V. Patel ◽  
Simon J. B. Aylwin ◽  
Peter King ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Promoter Activity ◽  
Kinase Inhibitor ◽  
Mutant Form ◽  
Orphan Nuclear Receptor ◽  
Glycoprotein Hormone ◽  
Steroidogenic Factor 1 ◽  
Specific Element ◽  
Α Subunit ◽  
Pituitary Adenylate Cyclase

Abstract In the anterior pituitary, expression of the common glycoprotein hormone α-subunit (αGSU) is mediated in part by multiple response elements residing in the distal promoter (−435 bp). One such site is the gonadotrope-specific element (GSE), which is bound by the orphan nuclear receptor steroidogenic factor-1 (SF-1) and confers pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated αGSU expression. Here we investigated the functional importance of the GSE and SF-1 phosphorylation in both basal and stimulated αGSU transcription. Mutation of the GSE reduced basal and PACAP-stimulated αGSU promoter activity in the αT3-1 gonadotrope cell line. Overexpression of wild-type SF-1, but not an S203A mutant form of SF-1, enhanced basal and PACAP-stimulated αGSU promoter activity. The effect of PACAP on αGSU promoter activity was inhibited after overexpression of MAPK phosphatase. Helix assembly of the SF-1 ligand-binding domain was stimulated by PACAP in vitro via a MAPK-dependent pathway, as determined using a mammalian two-hybrid assay. PACAP quickly activated MAPK (within 5 min) and also resulted in elevated levels of phospho-cAMP response element-binding protein and phospho-SF-1, as judged by a specific antiphospho-S203 antibody; this effect was blocked by the MAPK kinase inhibitor, UO126. Collectively, these data demonstrate that SF-1 binds to the GSE and activates both basal and PACAP-stimulated αGSU transcription, which is further increased by phosphorylation at Ser203 via MAPK. These data suggest strongly that the induction of αGSU gene expression by peptide hormone signaling is coupled directly to the posttranslational status of SF-1.

scholarly journals Steroidogenic factor-1 enhances basal and forskolin-stimulated transcription of the human glycoprotein hormone alpha-subunit gene in GH3 cells

2003 ◽  
Vol 179 (2) ◽  
pp. R1-R6 ◽  
Author(s):  
RC Fowkes ◽  
JM Burrin
Keyword(s):  
Binding Site ◽  
Promoter Activity ◽  
Phosphorylation Site ◽  
Alpha Subunit ◽  
Gh3 Cells ◽  
Subunit Gene ◽  
Glycoprotein Hormone ◽  
Direct Role ◽  
Steroidogenic Factor 1 ◽  
Specific Element

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.

scholarly journals The Relationship between Steroidogenic Factor 1 and DAX-1 Expression and in Vitro Gonadotropin Secretion in Human Pituitary Adenomas1

2001 ◽  
Vol 86 (6) ◽  
pp. 2476-2483
Author(s):  
S. J. B. Aylwin ◽  
J. P. Welch ◽  
C. L. Davey ◽  
J. F. Geddes ◽  
D. F. Wood ◽  
...  
Keyword(s):  
Pituitary Adenomas ◽  
Messenger Rna ◽  
Gene Promoters ◽  
Orphan Nuclear Receptors ◽  
Glycoprotein Hormone ◽  
Human Pituitary ◽  
Gonadotropin Secretion ◽  
Steroidogenic Factor 1 ◽  
Α Subunit

The orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX-1, are involved in gonadotroph differentiation, and SF-1 has been shown to activate the LH-β and glycoprotein hormone α-subunit (αGSU) gene promoters. Pituitary adenomas from 34 patients [13 somatotroph tumors, 4 prolactinomas, and 17 clinically nonfunctioning pituitary adenomas (NFPAs)] were enzymatically dispersed and cultured in vitro for 48 h. Tissue culture medium was collected and assayed for LH, FSH, and αGSU; messenger RNA was extracted from adherent cells, and expression of SF-1 and DAX-1 messenger RNA was determined by RT-PCR and verified by direct DNA sequencing. The presence of DAX-1 protein in tumor tissue was confirmed by immunocytochemistry. DAX-1 was demonstrated in all NFPAs, 7 of 13 somatotroph tumors and 0 of 4 prolactinomas. SF-1 expression occurred in 8 of 16 NFPAs, 4 of 12 somatotroph tumors, and 1 of 4 prolactinomas. LH secretion in vitro was greater in NFPAs that were SF-1 positive (P < 0.05). Neither FSH secretion nor αGSU secretion in vitro were significantly related to the expression of SF-1 or DAX-1. SF-1-positive somatotroph tumors immunostained positively for LH-β and/or FSH-β and secreted gonadotropins in vitro. SF-1 expression is associated with the in vitro secretion of LH by NFPAs. A proportion of somatotroph tumors also express SF-1 and DAX-1 and secrete gonadotropin hormones in vitro.

scholarly journals Targeted Pituitary Overexpression of Pituitary Adenylate-Cyclase Activating Polypeptide Alters Postnatal Sexual Maturation in Male Mice

Endocrinology ◽  
2012 ◽  
Vol 153 (3) ◽  
pp. 1421-1434 ◽  
Author(s):  
Joseph P. Moore ◽  
Rong Q. Yang ◽  
Stephen J. Winters
Keyword(s):  
Transgenic Mice ◽  
Adenylate Cyclase ◽  
Sexual Maturation ◽  
Gnrh Receptor ◽  
In Vivo Studies ◽  
Mrna Levels ◽  
Gonadotropin Secretion ◽  
Α Subunit ◽  
Pituitary Adenylate Cyclase

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is present in high concentrations within the hypothalamus, suggesting that it may be a hypophysiotropic factor, whereas pituitary expression suggests a paracrine function. PACAP stimulates gonadotropin secretion and enhances GnRH responsiveness. PACAP increases gonadotropin α-subunit (αGSU), lengthens LHβ, but reduces FSHβ mRNA levels in adult pituitary cell cultures in part by increasing follistatin. PACAP stimulates LH secretion in rats; however, acceptance of PACAP as a regulator of reproduction has been limited by a paucity of in vivo studies. We created a transgenic mouse model of pituitary PACAP overexpression using the αGSU subunit promoter. Real-time PCR was used to evaluate PACAP, follistatin, GnRH receptor, and the gonadotropin subunit mRNA in male transgenic and wild-type mice of various ages. Transgenic mice had greater than 1000-fold higher levels of pituitary PACAP mRNA; and immunocytochemistry, Western blot, and ELISA analyses confirmed high peptide levels. FSH, LH, and testosterone levels were significantly suppressed, and the timing of puberty was substantially delayed in PACAP transgenic mice in which gonadotropin subunit and GnRH receptor mRNA levels were reduced and pituitary follistatin expression was increased. Microarray analyses revealed 1229 of 45102 probes were significantly (P < 0.01) different in pituitaries from PACAP transgenic mice, of which 83 genes were at least 2-fold different. Genes involved in small molecule biochemistry, cancer, and reproductive system diseases were the top associated networks. The GnRH signaling pathway was the top canonical pathway affected by pituitary PACAP excess. These experiments provide the first evidence that PACAP affects gonadotropin expression and sexual maturation in vivo.

scholarly journals Extracellular Signal-Regulated Kinase and c-Src, But Not Jun N-Terminal Kinase, Are Involved in Basal and Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone α-Subunit Promoter

Endocrinology ◽  
2003 ◽  
Vol 144 (2) ◽  
pp. 612-622 ◽  
Author(s):  
Dagan Harris ◽  
Dana Chuderland ◽  
David Bonfil ◽  
Sarah Kraus ◽  
Rony Seger ◽  
...  
Keyword(s):  
Fold Increase ◽  
Dominant Negative ◽  
Glycoprotein Hormone ◽  
Mapk Activation ◽  
Specific Element ◽  
Α Subunit ◽  
Mapk Cascades ◽  
Extracellular Signal ◽  
Dominant Negative Form ◽  
Signal Specificity

Addition of a GnRH agonist (GnRH-A) to αT3-1 cells stimulates different MAPK cascades: ERK, Jun N-terminal kinase (JNK), and p38. Activation of JNK, ERK, and p38 shows a unique fold activation ratio of 25:12:2, which might encode signal specificity. ERK is translocated to the nucleus within 20 min with a peak at 120 min of GnRH-A stimulation. We used the human α-subunit promoter linked to chloramphenicol acetyl transferase (αCAT) to examine the role of ERK, JNK, and c-Src, which is implicated in MAPK activation, in basal and GnRH-stimulated αCAT. Addition of GnRH-A resulted in a 3-fold increase in αCAT, whereas the Ca2+ ionophore ionomycin and the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect. Addition of GnRH-A and TPA, but not GnRH-A and ionomycin, produced a synergistic response, whereas removal of Ca2+, but not down-regulation of TPA-sensitive PKCs, abolished GnRH-A-stimulated αCAT. Thus, regulation of α-promoter activity by GnRH is Ca2+ dependent and is further augmented by PKC. Cotransfection of αCAT and constitutively active or dominant negative plasmids of ERK and JNK cascade members, or the use of the ERK inhibitor PD98059, revealed that ERK, but not JNK, is involved in basal and GnRH-A-stimulated αCAT. Because c-Src participates in MAPK activation by GnRH, we also studied its role. Cotransfection of αCAT and the dominant negative form of c-Src or incubation with the c-Src inhibitor PP1 reduced GnRH-A-stimulated αCAT. The 5′-deletion analysis revealed that the −846/−420 region participated in basal α-transcription. In addition, the −346/−156 region containing the pituitary glycoprotein hormone basal element, α-basal elements, glycoprotein-specific element, and upstream response element is involved in basal and GnRH-A-stimulated αCAT. ERK contribution to GnRH maps to −346/−280 containing the pituitary glycoprotein hormone basal element and α-basal elements 1/2. Surprisingly, although c-Src is involved in GnRH-A-stimulated ERK, its involvement is mapped to another region (−280/−180) containing the glycoprotein-specific element. Thus, ERK and c-Src but not JNK are involved in basal and GnRH-A-stimulated-αCAT, whereas c-Src contribution is independent of ERK activation.

PPARγ co-activator-1α co-activates steroidogenic factor 1 to stimulate the synthesis of luteinizing hormone and aldosterone

2010 ◽  
Vol 432 (3) ◽  
pp. 473-486 ◽  
Author(s):  
Liuluan Zhu ◽  
Yaojun Ke ◽  
Di Shao ◽  
Ying Cui ◽  
Aijun Qiao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Luteinizing Hormone ◽  
Promoter Region ◽  
Glutathione Transferase ◽  
Peroxisome Proliferator ◽  
Orphan Nuclear Receptor ◽  
Steroidogenic Factor 1 ◽  
Specific Element

The orphan nuclear receptor SF-1 (steroidogenic factor 1) is highly expressed in the pituitary, gonad and adrenal glands and plays key roles at all levels of the hypothalamic–pituitary–steroidogenic tissue axis. In the present study, we show that PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator 1α] interacts with and co-activates SF-1 to induce LHβ (luteinizing hormone β) and αGSU (α-glycoprotein subunit) gene expression, subsequently leading to the increased secretion of LH in pituitary gonadotrope-derived αT3-1 cells. PGC-1α co-activation of LHβ expression requires an SF-1-binding element [GSE (gonadotrope-specific element)] mapped to the promoter region of LHβ. Mammalian two-hybrid and co-immunoprecipitation assays, as well as GST (glutathione transferase) pull-down experiments demonstrated that PGC-1α interacts with SF-1 in vivo and in vitro. Additionally, PGC-1α stimulates the expression of Cyp11b2 (aldosterone synthase gene), Cyp11b1 (steroid 11β-hydroxylase gene) and P450scc (cholesterol side-chain cleavage enzyme), and the synthesis of aldosterone in adrenal-cortex-derived Y-1 cells. Chromatin immunoprecipitation assays confirmed that endogenous PGC-1α co-localizes with SF-1 in the LHβ and Cyp11b2 promoter region. Knockdown of endogenous SF-1 by siRNA (small interfering RNA) abolished the PGC-1α induction of LHβ and Cyp11b2 gene expression in αT3-1 and Y-1 cells respectively. Finally, we demonstrated that PGC-1α induces SF-1 gene expression in both αT3-1 and Y-1 cells. Taken together, our findings reveal the potential role of PGC-1α and suggest that it may play important roles in steroidogenesis, gonad development and sex differentiation through SF-1.

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