Acute regulation of translation initiation by gonadotropin-releasing hormone in the gonadotrope cell line LbetaT2.
The hypothalamic neuropeptide hormone GnRH is the central regulator of reproductive function. GnRH stimulates the synthesis and release of the gonadotropins LH and FSH by the gonadotropes of the anterior pituitary through activation of the G-protein-coupled GnRH receptor. In this study, we investigated the role of translational control of hormone synthesis by the GnRH receptor in the novel gonadotrope cell line LbetaT2. Using immunohistochemical and RIA studies with this model, we show that acute GnRH-induced synthesis and secretion of LH are dependent upon new protein synthesis but not new mRNA synthesis. We examined the response to GnRH and found that activation of cap-dependent translation occurs within 4 h. LHbeta promoter activity was also examined, and we found no increases in LHbeta promoter activity after 6 h of GnRH stimulation. Additionally, we show that increased phosphorylation of translation initiation proteins, 4E-binding protein 1, eukaryotic initiation factor 4E, and eukaryotic initiation factor 4G, occur in a dose- and time-dependent manner in response to GnRH stimulation. Quantitative luminescent image analysis of Western blots shows that 10 nm GnRH is sufficient to cause a maximal increase in factor phosphorylation, and maximal responses occur within 30 min of stimulation. Further, we demonstrate that the MAPK kinase inhibitor, PD 98059, abolishes the GnRH-mediated stimulation of a cap-dependent translation reporter. More specifically, we demonstrate that PD 98059 abolishes the GnRH-mediated stimulation of a downstream target of the ERK pathway, MAPK-interacting kinase. Based on these findings, we conclude that acute GnRH stimulation of LbetaT2 cells increases translation initiation through ERK signaling. This may contribute to the acute increases in LHbeta subunit production.
Nguyen
Kathryn A
KA
Department of Reproductive Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0674, USA.
Santos
Sharon J
SJ
Kreidel
Marit K
MK
Diaz
Alejandro L
AL
Rey
Rodolfo
R
Lawson
Mark A
MA
D000818:Animals; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism; D005419:Flavonoids / Q000494:pharmacology; D005640:Follicle Stimulating Hormone / Q000096:biosynthesis*; D007987:Gonadotropin-Releasing Hormone / Q000502:physiology*; D007986:Luteinizing Hormone / Q000096:biosynthesis*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011966:Receptors, LHRH / Q000378:metabolism*
D004791:Enzyme Inhibitors; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D005419:Flavonoids; D012333:RNA, Messenger; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one