Rev-Erbα and Photoreceptor Outer Segments modulate the Circadian Clock in Retinal Pigment Epithelial Cells

Retinal photoreceptor outer segments (POS) are renewed daily through phagocytosis by the adjacent retinal pigment epithelial (RPE) monolayer. Phagocytosis is mainly driven by the RPE circadian clock but the underlying molecular mechanisms remain elusive. Using ARPE-19 (human RPE cell-line) dispersed and monolayer cell cultures, we investigated the influence of cellular organization on the RPE clock and phagocytosis genes. PCR analysis revealed rhythmic expression of clock and phagocytosis genes in all ARPE-19 cultures. Monolayers had a tendency for higher amplitudes of clock gene oscillations. In all conditions ARNTL, CRY1, PER1-2, REV-ERB alpha, ITGBS, LAMP1 and PROS1 were rhythmically expressed with REV-ERB alpha being among the clock genes whose expression showed most robust rhythms in ARPE-19 cells. Using RPE-choroid explant preparations of the mPer2(Luc) knock-in mice we found that Rev-Erb alpha deficiency induced significantly longer periods and earlier phases of PER2-bioluminescence oscillations. Furthermore, early phagocytosis factors beta(5)-Integrin and FAK and the lysosomal marker LAMP1 protein levels are rhythmic. Finally, POS incubation affects clock and clock-controlled phagocytosis gene expression in RPE monolayers in a time-dependent manner suggesting that POS can reset the RPE clock. These results shed some light on the complex interplay between POS, the RPE clock and clock-controlled phagocytosis machinery which is modulated by Rev-Erb alpha.