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J. Cai, A. Afzal, M. B. Grant, J. Kielczewski, L. Jacobson, M. E. Boulton; -Secretase-Dependent Cleavage and Translocation of CXCR4 in Vascular Endothelial Cells Is Regulated by Pigment Epithelium-Derived Factor (PEDF) and Insulin-Like Growth Factor-I (IGF-I). Invest. Ophthalmol. Vis. Sci. 2008;49(13):2651.
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The interaction of stromal-derived factor (SDF-1) with its cognate membrane bound receptor CXCR4 activates angiogenic signalling pathways in vascular endothelial cells. Intracellular signalling occurs through both activation of PI3 kinase and the translocation of CXCR4 to the nucleus. The aim of this study was to characterize the regulatory mechanisms involved in nuclear translocation of CXCR4 in microvascular endothelial cells.
Primary microvascular endothelial cells were isolated and treated with SDF-1, PEDF, IGF-I or combinations thereof at 100ng/ml in the presence or the absence of a γ-secretase inhibitor. In vitro angiogenesis assays were used to analyze the effects of these factors on microvascular endothelial cell migration, proliferation and tubular formation. Cleavage and intracellular localization of CXCR4 were determined by subcellular fraction followed by Western Blotting analysis at two time points (2 hr and 24 hr) using antibodies against the extracellular and intracellular domains of CXCR4.
SDF-1 and IGF-I were able to promote microvascular endothelial cell migration, proliferation and tubule formation while PEDF was inhibitory. Western Blotting analysis revealed full length 43kDa CXCR4 in the cell membrane of untreated cells. However, following 24hr exposure to SDF-1 the 43kDa CXCR4 was cleaved resulting in the appearance of a 35kDa membrane bound C-terminal fragment. This was associated with a decrease in N-terminal staining for CXCR4. There was no change in CXCR4 within the membrane fraction at 2 hr treatment with SDF-1. PEDF alone or PEDF combined with SDF-1 significantly increased levels of the cleaved 35kDa membrane bound CXCR4 C-terminal fragment at both 2 and 24 hr and this effect was blocked in the presence of a γ-secretase inhibitor or IGF-I. Increased cleavage of CXCR4 was associated with a significant decrease in the nuclear localization of CXCR4.
γ-secretase-mediated cleavage of membrane bound CXCR4 and elimination of the N-terminal domain in vascular endothelial cells prevents translocation of full length CXCR4 to the nucleus and acts as a negative regulator of in vitro angiogenesis. IGF-I appears to be able to prevent CXCR4 cleavage and to promote angiogenesis.
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