Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca2+ signaling in human retinal pigment epithelial cells

Rina Ikarashi,Takashi Ebisawa,Eri Morioka,Masayuki Ikeda,Kouhei Takeuchi,Alsawaf Ahmad,Masaaki Ikeda,Takashi Sugiyama,Yuzuki Kanda,Honami Akechi

doi:10.1038/srep44175

Abstract

Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca2+-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca2+ signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks.

Highlights

Retinal pigmental epithelium (RPE) cells[19,20]
Live hRPE-yellow cameleon (YC) cells were stimulated with various receptor agonists and the responses were screened by Ca2+ imaging
The magnitude of the Ca2+ responses was analyzed as a function of circadian time (CT), which is defined by the average Bmal1-luciferase rhythms in a culture dish

Summary

Introduction

Retinal pigmental epithelium (RPE) cells[19,20]. Within various peripheral (i.e., non-SCN) circadian clocks, the importance of the clock in the eye should be emphasized as its exceptional role for the photic input (i.e., resetting) system to the central SCN clock. Of the many molecular oscillators, the clock gene Bmal[1] may play a pivotal role in the retina, because a conditional knockout of Bmal[1] in the retina using CHX10-Cre resulted in a loss of circadian rhythm of inner retinal electrical activity in response to light[21]. CHX10-Cre might not knockout Bmal[1] in RPE cells, because CHX10 is a transcriptional factor localized to the inner nuclear layer, in bipolar cells[22,23]. It is still unknown how clock gene oscillations in RPE cells[19,20] contribute to physiological rhythm generations in the eye. Because we observed consistent cytosolic Ca2+ mobilizations via M3 muscarinic acetylcholine receptors in hRPE-YC cells, the effect of a muscarinic agonist (carbamylcholine, carbachol) on phase responsiveness in Bmal1-luciferase rhythms was analyzed in detail

Methods

Results

Conclusion