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.

As the regulator of pituitary reproductive hormone synthesis, the hypothalamic neuropeptide GnRH is the central regulator of reproduction. A hallmark of GnRH action is the differential control of gene expression in pituitary gonadotropes through varied pulsatile stimulation. Among other signaling events, GnRH activation of the ERK family of MAPKs plays a significant role in the transcriptional regulation of the luteinizing hormone β-subunit gene and regulation of cap-dependent translation. We evaluated the ERK response to different GnRH pulse amplitudes in the gonadotrope cell line LβT2. We found that low-amplitude stimulation with GnRH invokes a rapid and transient ERK activation, whereas high-amplitude stimulation invokes a prolonged activation specifically in the cytoplasm fraction of LβT2 cells. Nuclear and cytoplasmic targets of ERK, Ets-like gene 1, and eukaryotic initiation factor 4E, respectively, are similarly activated. Feedback control of ERK activation occurs mainly through the dual-specificity protein phosphatases (DUSPs). DUSP1 is localized to the nucleus in LβT2 cells but DUSP4, another member implicated in GnRH feedback, exists in both the nucleus and cytoplasm. Manipulation of nuclear DUSP activity through overexpression or knockdown of Dusp1 modulates the ERK response to low and high GnRH pulse amplitudes and activation of the Lhb promoter. Dusp1 overexpression abolishes sustained ERK activation and inhibits Lhb promoter activity induced by high amplitude pulses. Conversely, Dusp1 knockdown enhances ERK activation by low-amplitude stimulation and increases stimulation of Lhb promoter activity. We conclude that DUSP1 feedback activity modulates ERK activation and the transcriptional response to GnRH.

The neuropeptide gonadotropin‐releasing hormone (GnRH) regulates the synthesis of reproductive hormone luteinizing hormone (LH) in pituitary gonadotropes through regulation of both transcription and translation. This study investigated the ability of GnRH to regulate translation of specific mRNAs. GnRH‐treated LβT2 gonadotrope cell extracts were fractionated to separate mRNAs in actively translating polysomes from mRNAs in translationally inefficient ribonucleoprotein complexes (RNPs). Overall, GnRH stimulation redistributed RNA to RNPs, independent of transcriptional events. Quantitative real‐time PCR and Affymetrix GeneChip were used to examine specific mRNAs. Despite the overall shift of RNA to RNPs, individual transcripts were redistributed differently. Lhb and Cga, subunits of LH, redistributed to RNPs, whereas C‐myc and Egr1 redistributed to polysomes, and the receptor for GnRH was not regulated. Microarray analysis confirmed the regulation of Lhb and identified other regulated genes. The results show that GnRH exerts control at the translational level and differentially targets specific mRNAs, directing their movement in translational complexes. The targeted mRNAs include those important for gonadotrope function, indicating that GnRH utilizes translational control as a regulatory mechanism to modulate physiologically relevant gene expression.