Abstract
Purpose:
Daily phagocytosis of spent photoreceptor outer segment fragments (POS) is a critical task of the retinal pigment epithelium (RPE). The phagocytic capacity of the mammalian RPE is regulated to match diurnal POS shedding resulting in vigorous phagocytosis after light onset and little uptake at other times. Here, we investigated the role of protein inactivation by proteolytic cleavage in terminating the RPE’s phagocytic activity at the end of the daily phagocytic burst.
Methods:
We used immunoblotting and immunofluorescence microscopy to quantify surface expression and cytoskeletal anchorage of phagocytic receptors of murine RPE in situ at different times of the dark-light cycle and of RPE cells in culture challenged with isolated POS with or without pharmacological inhibition or expression silencing of cytosolic proteases. We also measured how these treatments affected phagocytic binding and engulfment by RPE cells in culture. We applied fluorogenic protease substrates followed by fluorescence quantification and confocal microscopy to determine changes in protease activity in relation to POS uptake by RPE cells in culture and in the eye. We used immunoblotting to detect proteolytic cleavage of candidate proteins of the phagocytic machinery of the RPE in response to phagocytic uptake.
Results:
Cytoskeletal anchorage of αvβ5 integrin decreased in the RPE in vivo and in cell culture after phagocytic uptake. This receptor destabilization and the resulting inhibition of POS binding were prevented by protease inhibition or downregulation. In vivo, RPE protease activity followed a precise diurnal rhythm with a peak just after the burst of POS phagocytosis after light onset that was regulated by changing levels of endogenous protease inhibitor proteins. POS uptake was sufficient to increase activity of endogenous proteases in RPE cells in culture. Finally, we detected proteolytic cleavage of integrin-associated proteins but not of surface receptor proteins αvβ5 integrin or MerTK following POS uptake.
Conclusions:
Our data reveal a novel molecular mechanism used by RPE cells in the eye and in culture to limit their phagocytic activity toward POS. In a negative feedback loop, POS uptake increases activity of cytosolic proteases in RPE cells causing cleavage of proteins linking the POS binding receptor αvβ5 integrin to the F-actin cytoskeleton and preventing further POS phagocytosis.
Keywords: 701 retinal pigment epithelium •
662 proteolysis •
493 cytoskeleton