Phoenixin Activates Immortalized GnRH and Kisspeptin Neurons Through the Novel Receptor GPR173.
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.
Departments of Physiology (A.K.T., V.L., D.D.B.), Medicine (D.D.B.), and Obstetrics and Gynaecology (D.D.B.), University of Toronto, and Division of Cellular and Molecular Biology (D.D.B.), Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8.
Alice K
AK
Treen
Departments of Physiology (A.K.T., V.L., D.D.B.), Medicine (D.D.B.), and Obstetrics and Gynaecology (D.D.B.), University of Toronto, and Division of Cellular and Molecular Biology (D.D.B.), Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8.
Vicky
V
Luo
Departments of Physiology (A.K.T., V.L., D.D.B.), Medicine (D.D.B.), and Obstetrics and Gynaecology (D.D.B.), University of Toronto, and Division of Cellular and Molecular Biology (D.D.B.), Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8.
Denise D
DD
Belsham
D000577:Amides / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism*; D007031:Hypothalamus / Q000187:drug effects / Q000378:metabolism; D059648:Kisspeptins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011966:Receptors, LHRH / Q000378:metabolism
D000577:Amides; D017362:Cyclic AMP Response Element-Binding Protein; C578985:GPR173 protein, mouse; C491407:Kiss1 protein, mouse; D059648:Kisspeptins; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone