Modulation of Gonadotropin-Releasing Hormone-Induced Extracellular Signal-Regulated Kinase Activation by Dual-Specificity Protein Phosphatase 1 in LβT2 Gonadotropes

Abstract

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.

GnRH Regulates Gonadotropin Gene Expression Through NADPH/Dual Oxidase-Derived Reactive Oxygen Species

“…The mechanisms of ROS action are not well defined, but primary targets of H 2 O 2 are protein tyrosine and dual specificity phosphatases bearing active site cysteine residues that are inactivated by oxidation (69). It is possible that ROS directly participate in regulation of activity through modulation of tyrosine and dual specificity kinases, which control overall MAP kinase and tyrosine kinase activity and are known participants in MAP kinase regulation by GnRH (35,70). The impact of ROS on expression of Atf3, c-Fos, Jun, and Egr1 mRNA, and on promoter activation and mRNA level of both Fshb and Lhb suggests that ROS participates in the major regulatory mechanisms engaged by GnRH in gonadotropes The participation of ROS in the GnRH signaling network is of general significance.…”

“…The cells were plated a density of 2.75 ϫ 10 5 cells/cm 2 and incubated for 24 hours before transfection. Reporter genes were cotransfected with a pGL3-basic reporter plasmid containing the herpesvirus thymidine kinase promoter and a substitution of the luciferase coding sequence with ␤-galactosidase, pGL3-TK-Gal, to serve as an internal control (34,35). Transfected cells were preincubated with 5 M DPI or 10 mM N-acetyl cysteine (NAC) for 30 minutes and then incubated with 10 nM GnRH (Sigma-Aldrich, St Louis, MO) for 5 hours.…”

“…Although most activated MAPK1/3 is cytosolic in L␤T2 cells, acute GnRH treatment induces transient activation and translocation of phosphorylated MAPK1/3 into the nucleus (35) and this is a major signaling component mediating the regulation of transcription in response to GnRH. GnRH activates both cytosolic translation factors (32, 35, 41, 44 -46) and nuclear transcription factors (47) via MAPK1/3 activation.…”

Growth Differentiation Factor 9 (GDF9) Forms an Incoherent Feed-forward Loop Modulating Follicle-stimulating Hormone β-Subunit (FSHβ) Gene Expression

“…Kanasaki et al initially observed that slow GnRH pulses triggered a more rapid and sustained phosphorylation of ERK1/2 than fast pulses (8). Subsequently, the feedback activity of MKP1, which inactivates MAPK via dephosphorylation, was found to modulate GnRH-induced ERK activation and gonadotropin response to GnRH (34,35). Finally, MKP1 expression was shown to be predominant under high GnRH frequency compared with low GnRH frequency (36).…”

Translational control of gene expression in the gonadotrope

“…DUSP1 is constitutively expressed in LβT2 cells, and mRNA and protein levels are increased in response to GNRH stimulation. DUSP1 protein modulates ERK activation in LβT2 cells and affects Lhb promoter activity (Nguyen et al, 2010). The Dusp1 mRNA contains a 3’ UTR ARE which binds HuR, causing improved stabilization and translation (Kuwano et al, 2008).…”