Cyclic Adenosine 3′,5′Monophosphate/Protein Kinase A and Mitogen-Activated Protein Kinase 3/1 Pathways Are Involved in Adenylate Cyclase-Activating Polypeptide 1-Induced Common Alpha-Glycoprotein Subunit Gene (Cga) Expression in Mouse Pituitary Gonadotroph LbetaT2 Cells

Abstract

Adenylate cyclase-activating polypeptide 1 (ADCYAP1) binds both Gs-and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathways in pituitary gonadotrophs. In this study, we investigated the cAMP and MAPK3/1 signaling pathways induced by ADCYAP1 stimulation and examined the effects of ADCYAP1 on the expression of gonadotropin subunit genes using a clonal gonadotroph cell line, LbetaT2. ADCYAP1 increased intracellular cAMP accumulation up to 19-fold in LbetaT2 cells. Common alpha-glycoprotein subunit gene (Cga) promoter activity was strongly activated by both ADCYAP1 and the cyclic-AMP analog, 8-(4-chlorophenylthio) adenosine 3 0 ,5 0 -cyclic monophosphate (CPT-cAMP). Both had little effect on luteinizing hormone beta (Lhb) and follicle-stimulating hormone beta (Fshb) promoter activities. Cga promoter activity was significantly increased by transfection with constitutively active cAMP-dependent protein kinase (PKA). Activities of the Lhb and Fshb promoters were only modestly increased. Both ADCYAP1 and CPT-cAMP induced MAPK3/1 activation in LbetaT2 cells. The MEK inhibitor, U0126, and the PKA inhibitors, H89 and cAMP-dependent protein kinase peptide inhibitor (PKI), completely inhibited MAPK3/1 activation by either ADCYAP1 or CPT-cAMP. Using luciferase reporter constructs containing cis-elements, the cAMP response element (Cre) promoter was stimulated about 4-fold by ADCYAP1. ADCYAP1-induced Cre promoter activity was completely inhibited by H89, but not by U0126. ADCYAP1 also increased the activity of the serum response element (Sre) promoter, a target for MAPK3/1, and treatment of the cells with U0126 completely inhibited ADCYAP1-induced Sre promoter activity. ADCYAP1-increased Cga promoter activity was inhibited partially by both H89 and U0126. Although combining the inhibitors showed an additive inhibition effect, it did not result in complete inhibition. These results suggest that in LbetaT2 cells, ADCYAP1 mainly increases Cga through activation of PKA and MAPK3/1, as well as through an additional unknown pathway.cyclic adenosine monophosphate, hypothalamic hormones, kinases, neuroendocrinology, pituitary hormones

Effects of metformin administration on plasma gonadotropin levels in women with infertility, with an in vitro study of the direct effects on the pituitary gonadotrophs

“…This line expresses the a-, LHb, and FSHbsubunits as well as GnRH receptors [19]. These cells synthesizes and releases LH and FSH in response to GnRH stimulation or other gonadotropin-releasing peptides, such as Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) [20]. The LbT2 line has facilitated the study of pituitary function as it is nearly impossible to isolate individual gonadotrophs from the pituitary.…”

Pulse Frequency-Dependent Gonadotropin Gene Expression by Adenylate Cyclase-Activating Polypeptide 1 in Perifused Mouse Pituitary Gonadotroph LbetaT2 Cells1

“…A physiological concentration of ADCYAP1 increases LH and FSH as well as free common alpha-glycoprotein subunit (CGA) release in normal gonadotrophs [8,[10][11][12]. The stimulatory effects of ADCYAP1 on gonadotropin production also are evident in single gonadotroph cell models, such as aT3-1 cells or LbT2 cells [13,14].…”

Effects of estradiol and progesterone on prolactin transcriptional activity in somatolactotrophic cells

“…TRH is secreted from the hypothalamus and transported via the hypothalamic-hypophysial portal circulation to the pituitary gland, where it stimulates inositol phospholipid metabo-luciferase cDNA in pGL3, as previously described [16]. GH3 cells were transiently transfected by electroporation [18] with 2.0 µg/dish of reporter construct and 0.1 µg of pRL-TK (Promega), and plated on 35-mm tissue culture dishes. To measure the activity of promoter regions containing a CRE and SRE, cells were transfected with either pCre-Luc (1.0 µg DNA) or pSre-Luc (1.0 µg DNA), which have a CRE enhancer (x 4) or five tandem SRE repeats, respectively, upstream of the TATA boxes of the firefly luciferase gene.…”