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S.S. Sawant, S. Aparicio, A. Tink, C.J. Barnstable, J. Tombran–Tink; PEDF Is Present In Large Amounts In The Nucleus Of RPE Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4903.
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PEDF is a secreted protein with several important functions including protection of photoreceptors and inhibition of neovascularization in the eye. The purpose of this study is to show that PEDF is also present in the nucleus in significant amounts.
ARPE–19 monolayers were placed in serum free media (SFM) for 12hr prior to treatment with 2–20 ug/ml brefeldin A, 100 ng/ml PEDF, PEDF + Brefeldin A, or SFM alone. Cells were harvested 0–48hr later and used to extract cytoplasmic and nuclear proteins which were then fractionated on 10% SDS PAGE, transblotted onto PVDF membranes which were then processed using anti–PEDF (1:5000) and chemiluminescent reagents. Immunocytochemical studies were done on 4% paraformaldehye fixed RPE cells exposed to anti–PEDF, anti–vimentin and DAPI.
There was strong PEDF signal at 50 kDa in both cytoplasmic and nuclear preparations of RPE cells. At 0hr and 24hr in SFM there were 68% and 60% more PEDF, respectively, in the nucleus compared to the cytoplasm. After 24 and 48 hrs treatment with PEDF there were 49%, and 27% more PEDF, respectively in the cytoplasm and 13%, and 1% more in the nucleus compared to controls. There was intense immunolabeling of PEDF in the nucleus with somewhat weaker signals in the cytoplasm in SF cultures. Nuclear PEDF labeling was confirmed by Dapi colocalization and cytoplasmic labeling by vimentin colocalization. In brefeldin A treated cultures anti–PEDF labeled only the nucleus. Treatment with exogenous PEDF increased PEDF immunolabeling in the nucleus.
Our studies show that PEDF is present in large amounts in the nucleus and that the levels are under very dynamic control. We have previously shown that PEDF contains a nuclear localization signal and propose that it may use this to shuttle into the nucleus. Although specific nuclear structures are labeled with the PEDF antibody, it is not clear whether it can act as a transcription factor or serves to modulate the activity of other proteins in the nucleus.
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