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S. M. J. Farrell, G. Groeger, D. Wallace, C. J. O'Brien, T. G. Cotter; Fibroblast Growth Factor Causes Cyclooxygenase to Produce Reactive Oxygen Species Which Modulate Survival Pathways in Retinal Cell Lines. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5975.
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© ARVO (1962-2015); The Authors (2016-present)
Basic Fibroblast Growth Factor (bFGF) causes phosphorylation of AKT and β-GSK and thereby activates pro-survival pathways in retinal cells. Reactive Oxygen Species (ROS), traditionally thought of as toxic by-products of metabolism are increasingly recognised as important cell messengers. ROS deactivate phosphatases allowing enzymes remain in the active phosphorylated form. We have previously shown that bFGF produces an increase in ROS and that scavenging of these ROS prevents activation of the AKT and β-GSK pathways. This indicates that ROS play a direct role in activating these pathways. Here we identify the molecular source of ROS in response to treatment with bFGF in retinal cell lines.
The 661W mouse photoreceptor derived cell lines and RGC-5 rat retinal ganglion cell derived cell lines were used.For flow cytometry, the chemiluminescent probe dihydrorhodamine was used to measure ROS levels. bFGF was added to cells to achieve a concentration of 50ng/ml. Inhibitors of known sources of ROS were added 1 hour prior to bFGF administration.siRNA knockdown of cyclooxygenase 1 (COX1) and cyclooxygenase 2 (COX2), was confirmed by western blot.To assess the effect of bFGF on expression of COX, cells were treated with bFGF 50ng/ml. Western blot was performed to analyse COX levels.
Flow cytometry demonstrated that the production of ROS in response to bFGF is completely inhibited by pre-treatment with Diclofenac, a non-selective COX inhibitor. Inhibition of NADPH oxidase, the electron transport chain, lipoxygenase and cytochrome P-450 showed no effect on ROS production in response to bFGF.Knockdown of COX 1 and COX2 was achieved 72 hours post transfection with the appropriate siRNA. Cells transfected with siRNA to either COX 1 or COX2 for 72hrs showed attenuation in the increase of ROS in response to bFGF. Cells transfected with siRNA to both COX1 and COX2 showed no increase in levels of intracellular ROS in response to bFGF.Western Blotting demonstrated marked upregulation of COX2 in response to bFGF while levels of COX1 remained unchanged.
bFGF causes upregulation of COX2 in retinal cell lines. Both COX1 and COX2 are responsible for an increase in ROS in response to bFGF. While it is known that COX produces ROS, these were thought to be unwanted by products of prostaglandin production. We show that Reactive Oxygen Species produced by Cyclooxygenase in response to bFGF are capable of modulating survival pathways in Retinal Cell Lines.
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