PI3–Kinase and HIF–1 independent stimulation of VEGF and IGFBP–3 Secretion in the Retinal Pigment Epithelial Cell Line ARPE–19

Abstract

Abstract: : Purpose: To examine the effects of PI3–kinase inhibition (LY294002 and wortmannin), mTOR inhibitor (rapamycin), and the inhibitor of hypoxia inducible factor 1 (HIF–1) stability (cyclosporin A), on insulin like growth factor 1 (IGF–1) and CoCl₂ stimulated HIF–1α expression. The effects of inhibiting these effectors on vascular endothelial growth factor (VEGF) and IGF binding protein 3 (IGFBP–3) secretion by the human retinal pigment epithelial (RPE) cell line ARPE–19 was also determined. Methods: Confluent cells were pretreated with inhibitors followed by stimulation with IGF–1 or CoCl₂. Whole cell lysates were assayed by immunoblot for HIF–1α or phosphorylated Akt expression. Conditioned medium was subjected to TCA precipitation and assayed by immunoblot for VEGF and ligand blot for IGFBP–3 accumulation. Cells grown on coverslips were similarly treated and probed with antibodies to phospho Akt and visualized using epifluorescence microscopy. HIF–1 luciferase reporter transfected cells were assayed for luciferase activity. Results: Pharmacological inhibition of PI3–kinase, mTOR, or HIF–1 led to a complete abrogation of IGF–1 stimulated HIF–1α stability and HIF–1 activity, accompanied by a partial decrease in IGF–1 stimulated VEGF and IGFBP–3 secretion. In contrast, HIF–1 inhibition abolished CoCl₂ stimulated HIF–1 activity and secretion of VEGF and IGFBP–3. Conclusions: Whereas the PI3–K/Akt pathway regulates IGF–1 stimulated HIF–1α protein and HIF–1 expression, this cytokine can stimulated VEGF and IGFBP–3 secretion in a PI3–K/Akt and HIF–1 independent manner as well. This contrasts with the HIF–1 dependent nature of CoCl₂ stimulated VEGF and IGFBP–3 secretion.

Keywords: retinal pigment epithelium • signal transduction: pharmacology/physiology • choroid: neovascularization