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S. C. Finnemann, Y. Chang, M. Anand, M. Sircar; Integrin Receptor-Tyrosine Kinase Signaling Cascades That Synchronize Daily RPE Phagocytosis of Photoreceptor Outer Segments. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2521.
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© ARVO (1962-2015); The Authors (2016-present)
Every morning, each RPE cell in the retina engulfs and digests numerous outer segment fragments (OS) shed in a circadian rhythm by adjacent photoreceptor neurons. Daily synchronized OS clearance by RPE cells is critical for vision. We have previously shown that OS recognition by apical αvß5 integrin-focal adhesion kinase (FAK) complexes of the RPE activates FAK and Mer tyrosine kinase (MerTK). Our ongoing studies aim to identify missing elements of the signaling cascade(s) that trigger OS phagocytosis by RPE cells.
We used immunoprecipitation, comparative immunoblotting, and confocal microscopy to quantify signaling protein expression, localization, and activity in intact retinal tissue obtained from wild-type and mutant mice and rats. We used the same techniques to analyze experimental phagocytosis assays, in which we incubated isolated OS with unpassaged, primary wild-type or mutant RPE or with RPE cell lines whose signaling protein expression we manipulated by transient transfection or adenovirus infection.
Rhythmic phosphorylation of Src family kinases (SFK) precisely coincided with daily RPE phagocytosis in wild-type mouse retina but was lacking from ß5 integrin knockout mouse retina. Overexpression of a dominant-negative mutant of c-Src strongly reduced RPE recognition and engulfment of OS. In contrast, dominant-negative forms of other SFK’s had no effect on OS uptake. C-Src localized to the apical, phagocytic surface of primary wild-type but not αvß5-integrin deficient RPE in culture. Wild-type and MerTK-deficient RPE cells activated FAK and c-Src in a detergent-resistant complex with αvß5 in response to incubation with isolated OS. However, c-Src did not dissociate from the αvß5 complex during engulfment like FAK did. When stimulated with OS in the presence of SFK inhibitors or dominant-negative c-Src, RPE cells ceased MerTK activation and OS engulfment but retained FAK activation. Finally, overexpression of wild-type or constitutively active c-Src was sufficient to re-store OS engulfment by RPE cells expressing dominant-negative FAK.
Our results show that c-Src links OS-αvß5-FAK signaling to MerTK activation. FAK activity is not sufficient to stimulate MerTK phosphorylation or phagocytosis but requires c-Src activation. Furthermore, increasing c-Src is sufficient to stimulate phagocytosis irrespective of FAK activity. These experiments identify an essential role for c-Src in signaling downstream of αvß5 integrin-FAK and upstream of MerTK that is required for RPE phagocytosis.
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