Abstract
Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gαs knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gαs knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gαs. In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs.
Highlights
GnRH3 is a hypothalamic neuropeptide that plays a central role in the regulation of mammalian reproductive functions
Pharmacological Inhibition of Secretion Selectively Suppresses Gonadotropin Gene Regulation—Our recent identification of inhibin ␣ and growth differentiation factor 9 (GDF9) as autocrine regulators of FSH gene expression [1, 37] prompted us to study the effects of inhibition of secretion on gonadotropin subunit gene expression
Inhibition of secretion led to a dramatic decline in both basal and gonadotropin-releasing hormone (GnRH)-induced Fshb mRNA expression (Fig. 1A) without affecting induction of immediate early genes Egr1, Jun, and Atf3 by GnRH (Fig. 1B)
Summary
GnRH, gonadotropin-releasing hormone; CGA, common ␣-glycoprotein subunit; CTX, cholera toxin; GDF, growth differentiation factor; iTRAQ, isobaric tags for relative and absolute quantitation; LH, luteinizing hormone; NERP, neuroendocrine regulatory peptide; qPCR, quantitative real time PCR; VGF, VGF nerve growth factor-inducible; SCG, secretogranin; IGFBP, insulin-like growth factor-binding protein; SEMA, semaphorin; STC1, stanniocalcin 1; BMP, bone morphogenetic protein; ANOVA, analysis of variance. The LT2 cell line, which was isolated from pituitary tumors that developed in these mice, has characteristics of late developmental stage gonadotropes, expressing mRNA for the GnRH receptor, the CGA, and the specific  subunits of LH and FSH [16, 17]. GnRH is the main regulator of gonadotropin subunit gene expression, intrinsic pituitary factors may tune gonadotrope responsiveness to GnRH and contribute to the differential regulation of gonadotropin subunits. These include activin, pituitary adenylate cyclase-activating polypeptide, BMPs, inhibin, and growth differentiation factor 9 (GDF9). We identified several novel secreted candidate regulatory factors and identified a VGF nerve growth factorinducible (VGF)-derived peptide as a GnRH-stimulated autocrine factor that inhibits Fshb gene expression
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