Activation Of Mertk Stimulates A Survival Signaling Pathway During Rpe Phagocytosis

Abstract

Abstract: : Purpose: Our previous studies demonstrated that the receptor tyrosine kinase Mertk is activated during RPE cell ingestion of photoreceptor outer segments (OS) in cell culture. In the current study, we wanted to identify Mertk–dependent intracellular signaling pathways during RPE phagocytosis and began by examining proteins commonly activated by receptor tyrosine kinases. Methods: Primary RPE cells from wild–type Long Evans (LE) and Mertk–deficient RCS rats were treated with OS for different periods of time and whole cell lysates were collected. The PI3 kinase inhibitor LY294002 was added to LE–RPE cells immediately prior to challenge with OS to test the involvement of PI3 kinase in RPE phagocytosis. Akt and Foxo3 protein expression and phosphorylation were examined by immunoblotting, and Foxo3 was localized by immunostaining. Results: Primary RPE cells from wild–type Long Evans (LE) and Mertk–deficient RCS rats were treated with OS for different periods of time and whole cell lysates were collected. The PI3 kinase inhibitor LY294002 was added to LE–RPE cells immediately prior to challenge with OS to test the involvement of PI3 kinase in RPE phagocytosis. Akt and Foxo3 protein expression and phosphorylation were examined by immunoblotting, and Foxo3 was localized by immunostaining. Conclusions: This study demonstrates that activation of the PI3 kinase–Akt–Foxo3 pathway by OS is Mertk–dependent. It has been reported that Foxo3 functions as a transcription factor to trigger the expression of genes critical for cell death. The fact that, in LE–RPE, Foxo3 is phosphorylated following addition of OS and is translocated out of the nucleus suggests an anti–apoptotic effect. Therefore, OS activation of Mertk stimulates a survival signaling pathway in wild–type rat RPE cells.

Keywords: signal transduction • retinal degenerations: cell biology • cell death/apoptosis