Abstract
The synthetic progesterone Norgestrel has been shown to have proven neuroprotective efficacy in two distinct models of retinitis pigmentosa: the rd10/rd10 (B6.CXBI-Pde6b(rd10)/J) mouse model and the Balb/c light-damage model. However, the cellular mechanism underlying this neuroprotection is still largely unknown. Therefore, this study aimed to examine the downstream signalling pathways associated with Norgestrel both in vitro and ex vivo. In this work, we identify the potential of Norgestrel to rescue stressed 661W photoreceptor-like cells and ex vivo retinal explants from cell death over 24 h. Norgestel is thought to work through an upregulation of neuroprotective basic fibroblast growth factor (bFGF). Analysis of 661W cells in vitro by real-time polymerase chain reaction (rt-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting revealed an upregulation of bFGF in response to Norgestrel over 6 h. Specific siRNA knockdown of bFGF abrogated the protective properties of Norgestrel on damaged photoreceptors, thus highlighting the crucial importance of bFGF in Norgestrel-mediated protection. Furthermore, Norgestrel initiated a bFGF-dependent inactivation of glycogen synthase kinase 3β (GSK3β) through phosphorylation at serine 9. The effects of Norgestrel on GSK3β were dependent on protein kinase A (PKA) pathway activation. Specific inhibition of both the PKA and GSK3β pathways prevented Norgestrel-mediated neuroprotection of stressed photoreceptor cells in vitro. Involvement of the PKA pathway following Norgestrel treatment was also confirmed ex vivo. Therefore, these results indicate that the protective efficacy of Norgestrel is, at least in part, due to the bFGF-mediated activation of the PKA pathway, with subsequent inactivation of GSK3β.