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Nadezda Stepicheva, Peng Shang, Imran Ahmed Bhutto, J Samuel Zigler, Stacey L Hose, Debasish Sinha; βA3/A1-crystallin affects circadian rhythm of the retinal pigmented epithelium through regulation of GSK3β expression. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3996.
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© ARVO (1962-2015); The Authors (2016-present)
Recently it has been reported that βA3/A1-crystallin, a protein known to be important for lysosomal acidification and amino acid sensing, is expressed in a circadian pattern, but its possible role in entrainment of circadian rhythmicity in the eye has not been examined. We hypothesize that oscillations in the βA3/A1-crystallin expression might regulate circadian entrainment in the retinal pigmented epithelium (RPE) cells through regulation of glycogen synthase kinase 3 beta (GSK3β).
Wild type (WT) and Cryba1 (gene encoding βA3/A1-crystallin) KO mice were sacrificed at 5 mo old and the RPE+choroid was collected. The samples were submitted to DNA Link USA, Inc., for RNA extraction and RNA-seq. Pathway enrichment analysis was performed using the online Pathway Data Integration Portal; differentially expressed genes were submitted to the KEGG pathway database. For the circadian studies, WT and Cryba1 KO mice (6-8 weeks old) were maintained at LD 12:12 cycle and sacrificed at ZT0, ZT1, ZT2, ZT3, ZT4, ZT6, ZT9, ZT12 (Zeitgeber time; hours after lights on). RPE+choroid was collected and lysed in RIPA buffer. Western blotting was performed with antibodies to GSK3β, p-GSK3β (Ser9), β-catenin and p-β-catenin (Ser33/37).
Pathway enrichment analysis of the genes differentially expressed in the RPE from WT compared to the Cryba1 KO mice revealed enrichment in genes involved in circadian rhythm (p=0.0069) and the β-catenin signaling pathway (p=0.203). We also found that the protein levels of GSK3β oscillate during the day in the RPE from WT mice, with one of the peaks coinciding with the peak of RPE activity. Importantly, in the mice lacking Cryba1, the amplitude of this oscillation is decreased and the peak of GSK3β protein expression occurs 1 hour earlier than in control mice. In addition, knockout of Cryba1 almost eliminated oscillations of p-β-catenin (Ser 33/37), a downstream target of GSK3β, which in WT mice closely followed the expression pattern of GSK3β.
GSK3β is a known regulator of circadian rhythm, but we are the first to demonstrate that βA3/A1-crystallin is an upstream regulator of GSK3β expression, suggesting its role in the entrainment of the circadian rhythmicity in the RPE cells. This work contributes to a fuller understanding of the circadian rhythm entrainment in the RPE cells and introduces a novel member into the circadian network.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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