title: Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx.
abstract: Retinitis pigmentosa (RP) is a degenerative retinal disease leading to photoreceptor cell loss. In 2011, our group identified the synthetic progesterone 'Norgestrel' as a potential treatment for RP. Subsequent research showed Norgestrel to work through progesterone receptor membrane component 1 (PGRMC1) activation and upregulation of neuroprotective basic fibroblast growth factor (bFGF). Using trophic factor deprivation of 661W photoreceptor-like cells, we aimed to further elucidate the mechanism leading to Norgestrel-induced neuroprotection. In the present manuscript, we show by flow cytometry and live-cell immunofluorescence that Norgestrel induces an increase in cytosolic calcium in both healthy and stressed 661Ws over 24 h. Specific PGRMC1 inhibition by AG205 (1 μm) showed this rise to be PGRMC1-dependent, primarily utilizing calcium from extracellular sources, for blockade of L-type calcium channels by verapamil (50 μm) prevented a Norgestrel-induced calcium influx in stressed cells. Calcium influx was also shown to be bFGF-dependent, for siRNA knock down of bFGF prevented Norgestrel-PGRMC1 induced changes in cytosolic calcium. Notably, we demonstrate PGRMC1-activation is necessary for Norgestrel-induced bFGF upregulation. We propose that Norgestrel protects through the following pathway: binding to and activating PGRMC1 expressed on the surface of photoreceptor cells, PGRMC1 activation drives bFGF upregulation and subsequent calcium influx. Importantly, raised intracellular calcium is critical to Norgestrel's protective efficacy, for extracellular calcium chelation by EGTA abrogates the protective effects of Norgestrel on stressed 661W cells in vitro.
authors.affiliation: Cell Development and Disease Laboratory, Biochemistry Department, Bioscience Research Institute, University College Cork, Western Road, Cork, Ireland.
authors.forename: Alice C
authors.initials: AC
authors.lastname: Wyse-Jackson
authors.affiliation: Cell Development and Disease Laboratory, Biochemistry Department, Bioscience Research Institute, University College Cork, Western Road, Cork, Ireland.
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Roche
authors.affiliation: Cell Development and Disease Laboratory, Biochemistry Department, Bioscience Research Institute, University College Cork, Western Road, Cork, Ireland.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Ruiz-Lopez
authors.affiliation: Cell Development and Disease Laboratory, Biochemistry Department, Bioscience Research Institute, University College Cork, Western Road, Cork, Ireland.
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Moloney
authors.affiliation: Cell Development and Disease Laboratory, Biochemistry Department, Bioscience Research Institute, University College Cork, Western Road, Cork, Ireland.
authors.forename: Ashleigh M
authors.initials: AM
authors.lastname: Byrne
authors.affiliation: Cell Development and Disease Laboratory, Biochemistry Department, Bioscience Research Institute, University College Cork, Western Road, Cork, Ireland.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Cotter
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D009644:Norgestrel / Q000008:administration & dosage*; D010786:Photoreceptor Cells / Q000187:drug effects* / Q000378:metabolism*; D011374:Progesterone / Q000008:administration & dosage / Q000031:analogs & derivatives*; D011980:Receptors, Progesterone / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects*
chemical_list: D008565:Membrane Proteins; C508868:PGRMC1 protein, mouse; D011980:Receptors, Progesterone; D016222:Fibroblast Growth Factor 2; D009644:Norgestrel; D011374:Progesterone
keywords: 661W; Norgestrel; PGRMC1; neuroprotection; retina