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

Abstract

The appropriate control of synthesis and secretion of the gonadotropin hormones LH and FSH by pituitary gonadotropes is essential for the regulation of reproduction. The hypothalamic neuropeptide GnRH is the central regulator of both processes, coordinating secretion with transcription and translation of the gonadotropin hormone subunit genes. The MAPK family of second messengers is strongly induced in gonadotropes upon GnRH stimulation, and multiple pathways activate these kinases. Intracellular reactive oxygen species participate in signaling cascades that target MAPKs, but also participate in signaling events indicative of cell stress. The NADPH oxidase (NOX)/dual oxidase (DUOX) family is a major enzymatic source of intracellular reactive oxygen, and we show that GnRH stimulation of mouse primary pituitary cells and the LβT2 gonadotrope cell line elevates intracellular reactive oxygen via NOX/DUOX activity. Mouse pituitary and LβT2 cells abundantly express NOX/DUOX and cofactor mRNAs. Pharmacological inhibition of NOX/DUOX activity diminishes GnRH-stimulated activation of MAPKs, immediate-early gene expression, and gonadotropin subunit gene expression. Inhibitor studies implicate the calcium-activated DUOX family as a major, but not exclusive, participant in GnRH signaling. Knockdown of DUOX2 in LβT2 cells reduces GnRH-induced Fshb, but not Lhb mRNA levels, suggesting differential sensitivity to DUOX activity. Finally, GnRH pulse-stimulated FSH and LH secretion are suppressed by inhibition of NOX/DUOX activity. These results indicate that reactive oxygen is a potent signaling intermediate produced in response to GnRH stimulation and further suggest that reactive oxygen derived from other sources may influence the gonadotrope response to GnRH stimulation.

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“…DUOX inhibition also decreased GnRH-induced immediate early gene responses, which led to decreased gonadotropin subunit production and secretion. 67 Fshb appears to be preferentially mediated by Duox2. This important link between cellular metabolism and gonadotrope cell function may have important implications on how the reproductive system perceives and manages adverse metabolic conditions.…”

“…[68][69][70][71] NOX/DUOX subunit mRNAs and protein levels were increased by GnRH stimulation in mouse pituitary cells in primary culture and gonadotrope cell lines. 67 Pharmacological inhibition of NADPH oxidase decreased GnRH-induced ROS production, indicating that the NOX/DUOX pathway is activated through GnRH signaling. Interestingly, NOX/DUOX enzymes appear to be necessary for JNK and ERK activation and subsequent regulation of the gonadotropin subunit genes; inhibition of NOX/DUOX or the use of a ROS scavenger decreased GnRH-induced JNK and ERK activation.…”

“…Kim and Lawson demonstrated that in both the gonadotrope-derived LßT2 cell line and in primary pituitary cell cultures, GnRH stimulation increased intracellular reactive oxygen species (ROS) such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide. 67 These free radicals are produced from NADPH oxidase (NOX) and dual oxidase (DUOX) enzyme family, which use NADPH as an electron donor and create ROS as a by-product. 68 ROS signaling and NOX family members have been linked to control of the reproductive axis; however, their specific role(s) in the pituitary gonadotrope had not yet been elucidated.…”

“…This important link between cellular metabolism and gonadotrope cell function may have important implications on how the reproductive system perceives and manages adverse metabolic conditions. 67…”

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“…Reactive oxygen species (ROS) are metabolites of oxygen that are only partially reduced thus carrying reactive potential (Terasaka et al 2017). In mammals, evidence suggests that ROS is involved in the regulation of the pulse interaction of GnRH on the pituitary through the MAP1/3 pathway (Kim and Lawson 2015).…”