Melanopsin Signaling Pathway in HEK293 Cell Line with Stable Expression of Human Melanopsin: Possible Participation of Phospholipase C beta 4 and Diacylglycerol

Abstract

Melanopsin, a member of the G protein‐coupled receptors family, is involved in non–image‐forming functions including circadian rhythm, sleep regulation and pupil response. In spite of significant research efforts, the signaling cascade involving melanopsin photoactivation remains poorly characterized. Here, we analyzed the effects of photoactivation of melanopsin on phospholipase C (PLC) and diacylglycerol. As an in vitro model, HEK293 cells with stable expression of human melanopsin were used. Although both the PLCβ1 and PLCβ4 subtypes were activated by the cell exposure to blue light, only PLCβ4 appeared to play a significant role in the studied melanopsin signaling pathway. We have demonstrated, for the first time, that cells expressing human melanopsin and enriched with 11‐cis‐retinal exhibited significantly increased diacylglycerol level. To determine the role of phospholipase C and involvement of diacylglycerols, two approaches were employed: inhibition of the G protein and phospholipase C (using the BIM‐46187 and U73122 inhibitors, respectively), and gene silencing using siRNA of PLCβ1 and PLCβ4. While silencing the PLCβ4 gene and using U73122 inhibited the diacylglycerol and calcium ion responses, the FOS gene expression level was only partially reduced. These results may facilitate a better understanding of the role of phospholipase C and diacylglycerols in the melanopsin signaling pathway.