RESUMO
The neuropeptide gonadotropin-releasing hormone stimulates synthesis and secretion of the glycoprotein gonadotropic hormones and activates the unfolded protein response, which causes a transient reduction of endoplasmic reticulum-associated mRNA translation. Hormone-treated cell extracts were fractionated to resolve mRNA in active polyribosomes from mRNA in inactive complexes. Quantitative real-time PCR and expression array analysis were used to determine hormone-induced redistribution of mRNAs between fractions and individual mRNAs were found to be redistributed differentially. Among the affected mRNAs relevant to gonadotropin synthesis, the luteinizing hormone subunit genes Lhb and Cga were enriched in the ribonucleoprotein pool. The MAP kinase phosphatase Dusp1 was enriched in the polyribosome pool. Enrichment of Dusp1 mRNA in the polyribosome pool was independent of the unfolded protein response, sensitive to ERK inhibition, and dependent on the 3'untranslated region. The results show that GnRH exerts translational control to modulate physiologically relevant gene expression through two distinct signaling pathways.
Assuntos
Hormônio Liberador de Gonadotropina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Polirribossomos/metabolismo , Ribonucleoproteínas/metabolismo , eIF-2 Quinase/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Proteínas ELAV/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Proteína Quinase C/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sirolimo/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
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 ß-subunit gene and regulation of cap-dependent translation. We evaluated the ERK response to different GnRH pulse amplitudes in the gonadotrope cell line LßT2. 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 LßT2 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 LßT2 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.
