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

Abstract

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-ERBα , ITGB5 , LAMP1 and PROS1 were rhythmically expressed with REV-ERBα 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-Erbα deficiency induced significantly longer periods and earlier phases of PER2-bioluminescence oscillations. Furthermore, early phagocytosis factors β 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-Erbα .

Cysteinyl leukotriene receptor 1 modulates autophagic activity in retinal pigment epithelial cells

“…Basal autophagic flux in RPE cells exhibits a circadian rhythm in vivo 36 . Furthermore, ex vivo cultured ARPE-19 cells express clock and phagocytosis genes in a rhythmic manner 37 . An excellent short summary of the circadian rhythm and clock genes can be found in a review by Cox and Tagahashi 38 .…”

“…Thus, basal autophagy in murine RPE cells is rhythmically regulated, as visualized by peaks of LC3-II levels and the number of autophagosomes over a period of time 36 . Interestingly, ARPE-19 cells exhibit rhythmic expression of clock and phagocytosis genes in vitro 37 . In line with this previous study, rhythmic regulation of autophagic activity in the absence of external stimuli was observed in the present study.…”

“…Milicevic et al reported rhythmic expression of clock and phagocytosis genes lasting < 24 h, which is different from the rhythm of ≥ 48–56 h observed in the present study. This discrepancy may be explained by the serum shock used to synchronize the cell rhythm applied by Milicevic et al 37 . Nevertheless, ARPE-19 cells follow an intrinsic rhythmic cell metabolism that is independent of external stimuli.…”

Phagocytosis by the Retinal Pigment Epithelium: Recognition, Resolution, Recycling

“…Here, at least Gas6 and Protein S are provided to the interphotoreceptor matrix by their synthesis and release from the RPEs themselves (35,37,40). At least some of these bridging molecules were recently shown to be transcribed by the RPE in a circadian fashion (41). Fittingly, experiments challenging RPEs with POS in vitro are often performed by augmenting the culture with exogenous Gas6 and/or Protein S (42) suggesting their requirement and some mechanism(s) of regulation to their expression by light.…”

Association of Expression Levels of Clock Genes and Autophagy-Related Genes under Abnormal Light/Dark Cycle Stimulation in NAFLD Mice

“…Lamp1, a lysosomalassociated membrane protein 1, is involves in intracellular lipid autophagic clearance [8]. Moreover, the rhythmic expression of lamp1 was found in the liver of mice and in human retinal pigment epithelial cells [22,23].…”