Abstract
Purpose:
Several cellular functions follow circadian rhythms, including the daily clearance of photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells. Retinal phagocytosis peaks 2 hours after light onset in rodents, and proceeds following 2 main steps: binding and internalization. Several proteins are implicated, amongst which 2 surface receptors, alphavbeta5 integrin and MerTK, and their cognate ligands, MFG-E8, and Gas6 and Protein S respectively. Mechanisms regulating the rhythmicity of this activity are still largely unknown, therefore we asked whether expression levels of mRNAs and proteins from the phagocytic machinery vary with time of day.
Methods:
Wild-type and beta5 integrin knockout mice, displaying an arrhythmic phagocytic profile, were compared. Mice were sacrificed at 12 different time-points along the light:dark cycle. The retina (photoreceptors) was carefully separated from the rest of the cup (RPE cells) and frozen in liquid nitrogen. RNAs were extracted and expression levels of tested genes were quantified by qPCR using the ribosomal protein Rplp0 and cyclophilin A as controls. In parallel, corresponding protein levels were analyzed on immunoblots of soluble proteins and lysates of the fellow eye from each animal. In addition, immunohistochemistry experiments were used to characterize where the proteins were localized on eye sections.
Results:
Most of the genes tested show variable expression levels, amidst on different scales. MerTK gene expression is increased after the phagocytosis peak in wild-type animals but not in beta5-/- mice. Its ligand Protein S is expressed in RPE and retinal samples, both expression levels peaking just before phagocytosis. Protein S ligands were abundantly observed in the photoreceptor outer segments layer on eye sections, and their bioavailability in the interphotoreceptor space slightly increased around phagocytic peak time. In contrast, Gas6 mRNA or protein expression levels remain quite stable over time.
Conclusions:
Gas6 and Protein S both stimulate phagocytosis in vitro, and are expressed in the retina. MerTK and Protein S genes showed similar expression profiles. Our data suggest that in vivo, Protein S appears to act as a timely ligand for MerTK during POS phagocytosis, while the role of Gas6 remains to be explored.
Keywords: 701 retinal pigment epithelium •
645 phagocytosis and killing •
458 circadian rhythms