Abstract
Purpose: :
We have recently reported that PEDF inhibits angiogenesis via regulated intramembrane proteolysis of VEGFR–1 (Cai et al, J. Biol. Chem. 2006). Since VEGFR–1 cleavage is dependent on γ–secretase and presenilin–1 (PS1) is an essential component in the enzymatic activity of the γ–secretase complex we wished to examine how PEDF can regulate PS1 expression and cellular localisation.
Methods: :
Retinal microvascular endothelial cells were cultured to confluence and treated with VEGF, PEDF or VEGF plus PEDF for up to 48 hours. PS1 expression was determined by RT–PCR and Western blotting. Immunohistochemistry and confocal microscopy was used to investigate subcellular changes in the localisation of PS1 following growth factor treatment.
Results: :
RT–PCR demonstrated that neither VEGF, PEDF nor VEGF plus PEDF induced a change in PS1 mRNA relative to control. However, immunolabelling of PS1 demonstrated that PEDF induced a time–dependent translocation of PS1 from the perinuclear region to the plasma membrane. This was confirmed by Western blot analysis which showed that PEDF exposure resulted in a significant increase in the membrane fractions of both full–length PS1 and the C–terminal fragment (CTF) of PS1 at 30 minutes post PEDF treatment and that this increased up to 48 hours. VEGF treatment alone did not affect PS1 while VEGF plus PEDF had the same effect on PS1 translocation as PEDF alone. The translocation of PS1 into the membrane coincided with cleavage of VEGFR–1 and the appearance of an 80 kDa C–terminal domain of VEGFR–1 in the cytosol fraction.
Conclusions: :
There is a direct correlation between an increase in level of the PS1 CTF in the membrane, the cleavage of VEGFR–1 and the kinetics of the appearance of an 80kDa C–terminal fragments of VEGFR–1. This suggests that PEDF may initiate γ–secretase–dependent cleavage of VEGFR1 by interacting with presenilin–1.
Keywords: growth factors/growth factor receptors • neovascularization • vascular cells