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N. Moningka, A. Afzal, J. Cai, K. Chang, L. Shaw, M. B. Grant, M. E. Boulton; Cleavage and Intracellular Translocation of the Stromal Derived Factor -1 (sdf-1) Receptor, Cxcr4, Regulates Endothelial Cell Function. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1724.
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SDF-1, a potent angiogenic factor, and its receptor CXCR-4 play a critical role in hematopoietic stem cell (HSC) recruitment to ischemia regions of the retina and choroid. The aim of this study was to investigate the cleavage and intracellular translocation of CXCR4 in response to a variety of factors known to modulate HSC function and angiogenesis.
Primary human microvascular endothelial cells were prepared and treated with SDF-1, IGF-1, PEDF (100ng/ml) or vehicle alone for periods up to 24 hours. Intracellular localization and cleavage of CXCR4 was determined by a) subcellular fractionation and Western blotting using antibodies against the extracellular and C-terminal domains of CXCR4, and b) immunolocalization using confocal microscopy. γ-secretase, a membrane-bound enzyme complex associated with the cleavage and translocation of other receptors (Cai et al, J Biol Chem, 2006) was assessed for its activity using ELISA and the effect of a γ-secretase inhibitor (12.5 nM) on CXCR4 translocation and signaling was examined. In vitro angiogenesis was assessed using the Matrigel tubule-forming assay.
Western blot analysis demonstrated both cleavage of CXCR4 within the transmembrane domain and intracellular translocation of the receptor to the nucleus. Immunoprecipitation and western blotting showed co localization of IGF-1 receptor with CXCR-4. Cleavage and translocation were modulated by PEDF, SDF-1 and IGF-1. Confocal microscopy demonstrated nuclear immunolocalization of CXCR4 within 4 hours following test agent treatment. Cleavage and translocation were dependent on γ-secretase activation as well as its membrane translocation and this could be blocked with a γ-secretase inhibitor. PEDF was the most efficient factor at promoting the translocation of γ-secretase to the cell membrane. Blocking CXCR4 translocation reduced tube formation.
Our data indicate that CXCR4 cleavage and intracellular translocation is regulated by its ligand SDF-1 as well as IGF-1 and PEDF. CXCR-4 cleavage and intracellular translocation may be important in SDF-1-induced angiogenesis and serve as a novel pharmacological target.
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