β-Secretase-dependent Shedding Of The Ectodomain Of VEGFR1 Is A Prerequisite For Transmembrane Cleavage Of VEGFR1 By -secretase In Endothelial Cells

Abstract

Purpose: : Previously, we have shown that the antiangiogenic activity of pigment epithelium-derived growth factor (PEDF) is associated with γ-secretase-catalysed transmembrane cleavage of VEGFR1 (Cai et al., J Biol Chem 2006). The aim of this study was to determine if shedding of the ectodomain of VEGFR1 is requisite prior to the γ-secretase-dependent cleavage of VEGFR1.

Methods: : Retinal microvascular endothelial cell migration, proliferation and tubule formation were assessed following exposure to VEGF, PEDF and VEGF+PEDF in the presence or absence of a β-secretase inhibitor. To better understand to role of β-secretase cleavage of VEGFR1, porcine aortic endothelial cells which lack VEGFR1 were infected with pEGFP-N1 vector containing wild type VEGFR1 (R1-WT) cDNA or a mutant VEGFR1 (R1-V767A) cDNA which lacks the γ-secretase cleavage site. The infected cells were exposed to VEGF and/or PEDF in the presence or the absence of γ-secretase or β-secretase inhibitors. Some cells were also treated with the proteasomal inhibitor Lactacystin to prevent degradation of VEGFR1 fragments. Total lysates or cellular fractions were analyzed by Western blot with antibodies against the C-terminal of VEGFR1 or GFP. Supernatants were probed with antibodies against the N-terminal of VEGFR1 or native soluble VEGFR1.

Results: : PEDF-inhibition of VEGF-induced endothelial cell migration, proliferation and tubule formation was prevented by β-secretase inhibition. VEGF+PEDF in combination resulted in a 100-kDa and a ~110-kDa fragment for R1-WT and R1-V767A, respectively, in addition to the full-length 200 kDa VEGFR1. The amount of these fragments was increased by proteasome inhibition. β-secretase inhibitor prevented the formation of fragments in both R1-WT and R1-V767A transfected cells. γ-secretase inhibitor abolished the 100-kDa fragment in R1-WT cells. Based on the sizes of the fragments; β-secretase releases a 90 kDa ectodomain of VEGFR1 and leaves a 110 kDA transmembrane protein; γ-secretase inhibitor results in a 100 kDa cytosolic fragment indicating that a 10 kDa transmembrane fragment is not released. Neither γ-secretase nor β-secretase inhibitors had an effect on soluble VEGFR1 levels.

Conclusions: : PEDF induces a sequential cleavage of VEGFR1 by β-secretase and γ-secretase which are both required to negatively regulate angiogenesis.

Keywords: growth factors/growth factor receptors • vascular endothelial growth factor • proteolysis