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

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Melanopsin Signaling Pathway in HEK293 Cell Line with Stable Expression of Human Melanopsin: Possible Participation of Phospholipase C beta 4 and Diacylglycerol
Photochemistry and Photobiology ( IF 2.6 ) Pub Date : 2021-05-12 , DOI: 10.1111/php.13453
Olga I Krzysztynska-Kuleta ₁ , Magdalena M Olchawa ₁ , Tadeusz J Sarna ₁

Affiliation

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 PLC_β1 and PLC_β4 subtypes were activated by the cell exposure to blue light, only PLC_β4 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 PLC_β1 and PLC_β4. While silencing the PLC_β4 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.

中文翻译：

HEK293 细胞系中黑视蛋白信号通路与人黑视蛋白的稳定表达：磷脂酶 C β 4 和二酰基甘油的可能参与

黑视蛋白是 G 蛋白偶联受体家族的成员，参与非成像功能，包括昼夜节律、睡眠调节和瞳孔反应。尽管进行了大量的研究工作，但涉及黑视蛋白光激活的信号级联仍然缺乏特征。在这里，我们分析了黑视蛋白光活化对磷脂酶 C (PLC) 和二酰基甘油的影响。作为体外模型，使用稳定表达人黑视蛋白的HEK293细胞。虽然 PLC _β1和 PLC _β4亚型都被细胞暴露于蓝光激活，但只有 PLC _β4似乎在所研究的黑视蛋白信号通路中起重要作用。我们首次证明，表达人黑视蛋白并富含 11-顺-视黄醛的细胞表现出显着增加的甘油二酯水平。为了确定磷脂酶 C 的作用和甘油二酯的参与，采用了两种方法：抑制 G 蛋白和磷脂酶 C（分别使用 BIM-46187 和 U73122 抑制剂），以及使用 PLC _β1和 PLC _β4的 siRNA 进行基因沉默。在使 PLC _β4静音时基因和使用U73122抑制甘油二酯和钙离子反应，FOS基因表达水平仅部分降低。这些结果可能有助于更好地理解磷脂酶 C 和二酰基甘油在黑视蛋白信号通路中的作用。

更新日期：2021-05-12

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