Purchase this article with an account.
Nemanja Milićević, Ivanka de Bruin, Esmeé Wils, Jacoline ten Brink, Anneloor ten Asbroek, Marie-Paule Felder-Schmittbuhl, Arthur A Bergen; Common and distinct circadian expression of clock and phagocytosis genes in ARPE-19 monolayers and dissociated cell cultures. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4029.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal photoreceptor outer segments (POS) undergo daily phagocytosis by a monolayer of retinal pigment epithelial (RPE) cells. This process occurs as a daily peak and is driven, in part, by circadian clocks. These clocks are molecular pacemakers comprised of transcriptional-translational feedback loops with a period of roughly 24h. However, little is known about the role of monolayer morphology on the molecular machinery regulating phagocytosis. We hypothesize that RPE monolayers and dissociated cell cultures show distinct circadian clock and phagocytosis gene expression profiles.
The ARPE-19 dissociated cell cultures and monolayers grown on matrigel-coated surfaces were either synchronized using the serum-shock (SS) procedure (incubation with 50% bovine-serum for 2h) or received no intervention (control groups). The cells were harvested at 3h intervals up to 54h (n=3 per intervention per time-point), followed by RNA extraction, cDNA synthesis and sqRT-PCR analysis. The periodicity of the relative gene expression levels was assessed with nonlinear least squares fitting to cosine wave function y=y0+c cos[2π (t-Φ)/τ] with τ representing the endogenous period (h), Φ phase (h), and c amplitude.
Rhythmic expression of the clock gene Bmal1 and phagocytosis genes Itgb5 and Pros1 were observed in all conditions (see statistics in table). In all conditions, except for the ARPE19 control monolayer, the clock genes Per1, Per2, and Rev-Erbα also showed significant rhythmic expression. The clock gene Cry1 showed rhythmic expression in three out of four conditions (not in dissociated control cells). Interestingly, Ptk2 was distinctly rhythmic in monolayers, whereas Mfge8 and Cry2 rhythmicity is observed in SS dissociated cell cultures. No rhythmic expression was found for the clock gene Clock, and the phagocytosis gene Gas6.
Our results reveal common and distinct oscillating clock and phagocytosis gene expression profiles in the RPE monolayers and dissociated cell cultures. Further research is required to probe the functional significance of these commonalities and differences.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Results of non-linear regression fitting to cosine function are shown in the table.
This PDF is available to Subscribers Only