PKC activation affects, via ELAVL1/HuR protein, VEGF expression in pericytic/endothelial coculture

Abstract

AbstractPurpose To explore whether, following direct contact, there is a mutual influence between pericytes (PC) and endothelial cells (EC), and to establish whether PKC activation, a condition associated with hyperglycaemia in diabetic retinopathy (DR), can affect, via the mRNA‐binding ELAVL1/HuR protein, VEGF expression.Methods Retinal PC and EC were cultured separately (MONO) or in direct contact (1:1 ratio; coculture; COCU), and exposed or not to PKC activator phorbol esthers (100 nM PMA, 15 min.). PKCβI/βII, HuR, VEGF proteins were analysed by Western blotting; VEGF secretion in cell culture medium was evaluated by ELISA. VEGF mRNA analysis was performed by real‐time qPCR. Immunocytochemistry was performed to evaluate PMA‐induced HuR translocation.Results In MONO, VEGF mRNA/protein basal levels are higher in PC than EC. In COCU, VEGF protein amount is up‐regulated in EC and down‐regulated in PC, respectively. VEGF release is favoured by physical contact between PC and EC and further increased by PMA exposure. PKCβ activation induces HuR translocation from nucleus to cytoplasm, and it increases the protein levels of the kinase itself, HuR and VEGF in PC and EC in both culture conditions.Conclusion VEGF expression varies in function of the cell culture conditions. In all cultures, PKC activation increases PKCβI/βII, HuR and VEGF protein levels, changes which may occur also at very early stages of DR. The COCU model may be useful to study PC/EC interactions in both physiological and pathological conditions.