Abstract
Abstract: :
Purpose: To investigate the role of the phosphatidylinositol 3–kinase (PI3K) pathway and the signal mediator AP–1 in monocyte chemotactic protein (MCP–1) and interleukin (IL)–8 gene expression in human retinal pigment epithelium (hRPE) cells. Methods: HRPE cells were stimulated by IL–1ß, TNF–α, and by co–culturing with monocytes in the presence or absence of a series of kinase inhibitors. The induced MCP–1 and IL–8 proteins and mRNA were determined by enzyme–linked immunosorbent assay (ELISA) and RT–PCR respectively. Western blot analysis, kinase assays, and electrophoretic mobility shift assays were used to detect the activation of signaling mediators and transcription factors. Results: Concomitant with the induced chemokine expression, phosphorylation of PI3K and the downstream targets of PI3K, namely Akt, GSK, and FKHR were induced by each of the stimuli. Ly294002, a specific inhibitor of PI3K, resulted in time– and dose–dependent blockade of induced RPE MCP–1 mRNA expression and protein production. The IC50 values for inhibition of MCP–1 secretion induced by IL–1ß, TNF–α, and hRPE–monocyte binding were 16, 12 and 3 µM, respectively. In contrast, Ly294002 did not inhibit RPE IL–8 gene expression induced by any of the stimuli. Ly294002 as well as U0126, SB202190 and SP600125, the selective inhibitors of MEK, p38, and JNK respectively, strongly inhibited induced c–fos expression. Ly294002 did not inhibit induction of MEK, p38, and JNK activity. Ly294002 blockade of PI3K/Akt abolished IL–1ß–induced nuclear translocation of AP–1, but the induction of IΚB degradation was unchanged. Conclusions: The Ly294002–sensitive PI3K/Akt pathway regulates MCP–1, but not IL–8 in hRPE cells by a mechanism independent of MAPK activation and IΚB degradation. IL–1, TNF and monocyte binding to hRPE cells result in PI3K–dependent induction of c–fos. This study was supported by NIH Grants EY–09441, EY007003, and Research to Prevent Blindness–Olga Keith Weiss Award (VME).
Keywords: retinal pigment epithelium • signal transduction • cytokines/chemokines