Abstract
The insulin-like growth factor 1 (IGF-1) stimulates expression and secretion of vascular endothelial growth factor-A (VEGF-A), the main actor in ocular neovascularization, by RPE cells. Activity of IGF-1 is regulated by interaction between its receptor and Caveolin-1 (Cav-1), the main component of caveolae. The aim of this study was to investigate whether modulation of Cav-1 expression affects synthesis and secretion of VEGF-A. ARPE-19 cells were transfected with small interfering RNA for Cav-1 (si-Cav-1) and with control siRNA (si-CTR) and stimulated with IGF-1. We found that down-regulation of Cav-1 did not affect activation of IGF-1R but regulated in an opposite manner the phosphorylation of Akt and Erk1/2. Moreover, we found that IGF-1 increased mRNA levels of VEGF-A in both si-CTR and in si-Cav-1 ARPE-19 cells and that Cav-1 silencing significantly reduced basal and IGF-1-stimulated VEGF-A release. Then we investigated the response of the microvascular endothelial cell line HMEC-1 to secretory products of ARPE-19 cells by evaluating wound healing closure, finding that conditioned media from si-Cav-1-ARPE-19 cells reduced endothelial cell migration rate. These data demonstrate that Cav-1 regulates secretion of VEGF-A, and that the depletion of Cav-1 reduces IGF-1 induced VEGF-A secretion in ARPE-19 cells and the migratory potential of their secretory products.
Highlights
Caveolin-1 (Cav-1) is the principal protein of caveolae, specialized lipid rafts which regulate many cellular functions, including endocytosis, intracellular trafficking and signaling [1]
Since we previously demonstrated that IGF-1R localizes in caveolae and that insulin-like growth factor 1 (IGF-1) signaling may be regulated by Cav-1 [13,14], the aim of this study was to investigate whether Cav-1 is involved in mediating IGF-1-induced vascular endothelial growth factor-A (VEGF-A) expression and secretion in the retinal pigment epithelium (RPE) cell line ARPE-19
All the results obtained in si-CTR ARPE-19 cells are comparable with those obtained in untransfected cells (CTR, CTR + IGF-1)
Summary
Caveolin-1 (Cav-1) is the principal protein of caveolae, specialized lipid rafts which regulate many cellular functions, including endocytosis, intracellular trafficking and signaling [1]. RPE cells play multiple roles in the retina: they produce several growth factors, are part of the outer blood-retinal barrier (BRB), perform transcytosis and phagocytosis, and digest the photoreceptor outer segments [2,3,4]. Cav−/− mice develop a normal RPE [5], caveolin-1 seems to play an important role in RPE function. It affects ion transport activities and is required for phagolysosomal digestion of photoreceptors [5,6]. RPE cells are a main source of vascular endothelial growth factor-A (VEGF-A), which plays a critical role in maintaining the homeostasis of the retinal and choroidal vasculature [3]. Caveolin-1 siRNA inhibition reduced retinal neovascularization in a murine model of oxygen-induced retinopathy [7] but worsened choroidal and retinal neovascularization in Cav-1-deficient mice [8]
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