Phoenixin Activates Immortalized GnRH and Kisspeptin Neurons Through the Novel Receptor GPR173

Abstract

Reproductive function is coordinated by kisspeptin (Kiss) and GnRH neurons. Phoenixin-20 amide (PNX) is a recently described peptide found to increase GnRH-stimulated LH secretion in the pituitary. However, the effects of PNX in the hypothalamus, the putative signaling pathways, and PNX receptor have yet to be identified. The mHypoA-GnRH/GFP and mHypoA-Kiss/GFP-3 cell lines represent populations of GnRH and Kiss neurons, respectively. PNX increased GnRH and GnRH receptor (GnRH-R) mRNA expression, as well as GnRH secretion, in the mHypoA-GnRH/GFP cell model. In the mHypoA-Kiss/GFP-3 cell line, PNX increased Kiss1 mRNA expression. CCAAT/enhancer-binding protein (C/EBP)-β, octamer transcription factor-1 (Oct-1), and cAMP response element binding protein (CREB) binding sites are localized to the 5' flanking regions of the GnRH, GnRH-R, and Kiss1 genes. PNX decreased C/EBP-β mRNA expression in both cell models and increased Oct-1 mRNA expression in the mHypoA-GnRH/GFP neurons. PNX increased CREB phosphorylation in both cell models and phospho-ERK1/2 in the mHypoA-GnRH/GFP cell model, whereas inhibiting the cAMP/protein kinase A pathway prevented PNX induction of GnRH and Kiss1 mRNA expression. Importantly, we determined that the G protein-coupled receptor, GPR173, was strongly expressed in both GnRH and kisspeptin cell models and small interfering RNA knockdown of GPR173 prevented the PNX-mediated up-regulation of GnRH, GnRH-R, and Kiss1 mRNA expression and the down-regulation of C/EBP-β mRNA expression. PNX also increased GPR173 mRNA expression in the mHypoA-GnRH/GFP cells. Taken together, these studies are the first to implicate that PNX acts through GPR173 to activate the cAMP/protein kinase A pathway through CREB, and potentially C/EBP-β and/or Oct-1 to increase GnRH, GnRH-R, and Kiss1 gene expression, ultimately having a stimulatory effect on reproductive function.

Contingent role of phoenixin and nesfatin‐1 on secretions of the male reproductive hormones

“…A lately discovered novel endogenous neuropeptide, phoenixin (PNX), has been isolated and identified from multiple locations, such as the hypothalamus, in the brain (Yosten et al, ). PNX, which is post‐translationally processed from a relatively smaller integral membrane protein 20 (SMIM20), has been described to have regulatory implications on the reproductive system (Yosten et al, ) via G‐protein‐coupled receptor (GPCR; Treen, Luo, & Belsham, ). The first report about PNX indicated that the peptide is chiefly produced by hypothalamus and pituitary gland in the brain and a number of peripheral tissues (Yosten et al, ).…”

Nesfatin-1: functions and physiology of a novel regulatory peptide

“…Furthermore, the recently discovered neuropeptide phoenixin, which is colocalized with NUCB2/nesfatin-1 in a number of hypothalamic nuclei (ARC, PVN, ventromedial and lateral hypothalamus) has been suggested as a potential mediator for nesfatin-1's action with regard to reproduction. Phoenixin increases GnRH, GnRH receptor and Kiss1 gene expression (Yosten et al 2013, Treen et al 2016 and potentiates GnRH-stimulated LH release in vitro (Yosten et al 2013). These findings were substantiated by an in vivo study demonstrating that i.c.v.…”

Phoenixin: a novel brain-gut-skin peptide with multiple bioactivity

“…Moreover, in vivo siRNA knockdown of phoenixin resulted in a delayed appearance of oestrus and a reduced expression of GnRH receptors in the pituitary [8] . More recently, the orphan G-protein coupled receptor GPR173 has been proposed as the primary receptor candidate mediating the reproductive effect of phoenixin [6,7] . Reproduction is controlled by the hypothalamic-pituitary-gonadal axis which responds to cues from energy stores.…”

“…Viewed in this context, a clear and present need for information relative to novel GPCRs and their endogenous ligands is obvious. Phoenixin is a recently discovered peptide ligand that is reported to target the GPR173 [6,7] . Similar to many peptide ligands, phoenixin is distributed broadly in the central and peripheral nervous systems with a particular concentration in the hypothalamus, spinal cord and myenteric plexus, establishing an anatomical as well a functional connection between the gut and brain.…”

“…By utilizing the bioinformatics algorithm from information provided by the Human Genome Project, several previously unrecognized, secreted, highly conserved neuropeptides have been identified, one of which being neuronostatin with orphan receptor GPR107 as its cognate receptor [3][4][5] . Below is a brief account of the discovery of a second peptide ligand, phoenixin, which may target an orphan G-protein coupled receptor, GPR173 [6,7] .…”