Acute Regulation of Translation Initiation by Gonadotropin-Releasing Hormone in the Gonadotrope Cell Line LβT2

Abstract

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.

Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation

“…With initiation of feeding the extent of phosphorylation of eIF4G in the eIF4E immunoprecipitate increased tenfold and remained elevated throughout the feeding period. This observation is in contrast to studies where eIF4G phosphorylation was not sustained in the gonadotroph cell line LbT2 in the presence of gonadotropin-releasing hormone (36). The phosphorylation of eIF4G in the eIF4E immunoprecipitate returned to values observed prior to initiation of meal feeding following the meal.…”

“…Furthermore, the extent of phosphorylation remains elevated over the course of the meal. This is in contrast to studies involving gonadaltropin-releasing hormone where the extent of phosphorylation of eIF4G was only transiently elevated during stimulation (36). The mechanism by which meal feeding-induced eIF4G phosphorylation stimulates eIF4G⅐eIF4E assembly remains unknown; however, increased phosphorylation of eIF4G correlates with conditions known to stimulate protein synthesis (8,44).…”

“…Increased phosphorylation of eIF4G on Ser 1108 is associated with enhanced formation of active eIF4G⅐eIF4E complex in cells in culture (36,44), leading to an increased rate of protein synthesis. Likewise, elevated phosphorylation of eIF4G correlates with mRNA translation in skeletal muscle during perfusion of hindlimb with buffer containing leucine (8) or following oral administration of a single bolus of leucine (10).…”

Rapamycin blunts nutrient stimulation of eIF4G, but not PKCε phosphorylation, in skeletal muscle

“…Numerous reports have linked phosphorylation of eIF4G with corresponding changes in protein synthesis. Increased phosphorylation of eIF4G on Ser 1108 is associated with enhanced assembly of eIF4E⅐eIF4G complex following exposure of cells in culture to serum (34) or gonadotrophin-releasing hormone (26). In vivo, IGF-I-induced stimulation of phosphorylation of eIF4G correlates with acceleration of protein synthesis in muscle (19,46).…”

Activation of a GPCR leads to eIF4G phosphorylation at the 5′ cap and to IRES-dependent translation

“…Inducible phosphorylation of eIF4G on Ser1108 has been correlated with enhanced translation efficacy, and rapamycin-sensitivity has been observed in response to anabolic factors such as insulin-like growth factor 1 or insulin, acting through a tyrosine kinase receptor. Also, the GPCR for gonadotropin-releasing hormone mediates eIF4G phosphorylation in LbT2 cell lysates (Nguyen et al 2004). But to our knowledge, rapamycin-sensitive eIF4G phosphorylation occurring at the 5 0 cap, in response to a ligand-bound GPCR, has never been reported before.…”