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N. Lara, S.E. Aparicio, S.S. Sawant, C.J. Barnstable, J. Tombran–Tink; Autocrine Signaling Of PEDF In RPE Cells: Regulation Of Akt, Map Kinase, And Stat Pathways . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4902.
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There is increasing evidence to support a role for PEDF in neuroprotection and angiogenesis in the eye. The RPE cells, which secrete PEDF, also play an important role in both biological processes and have been shown to respond to PEDF by differentiating in culture. The purpose of this study was to examine the autocrine signals of PEDF on RPE cells
ARPE–19 cells were grown to confluency in cultures then treated with 100ng/ml PEDF at 0 – 6 hours in serum free medium (SFM). Cytoplasmic proteins were extracted from the cells, loaded onto 10% SDS gels and transblotted onto PVDF membranes. Changes in phosphorylation of signal molecules were determined by western blot analysis using specific antibodies against the phosphorylated and non–phosphorylated derivatives of Akt, Erk1/2, and STAT–3 proteins. Degradation of IkappaB was determined using anti–IkappaB. The blots were scanned and statistical analysis of changes was quantitatively evaluated using ImageJ software (NIH). Band intensity was calculated and percentage was obtained using the control as reference.
PEDF induced activation of Akt by 20% in A–RPE19 cells at 5 min and decreased the levels of IkappaB by 2 hours by almost 50%. There was significant phosphorylation of Erk1/2 at 5 min by 50%, with a maximum increase by 150% at 60 min of treatment. PEDF also increased the phosphorylation of STAT–3 by 15% after 1 hr of treatment.
Our data show that PEDF has strong autocrine effects on RPE cells that lead to changes in phosphorylation of members of multiple signaling pathways. The neuroprotective and antiangiogenic actions of PEDF in the retina may be mediated in part by its action on RPE cells.
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