アクアポリン-2発現における細胞間ばらつきを明らかにするGFPレポーターシステム

Vasopressin regulates transcription of the aquaporin-2 gene (Aqp2) in collecting duct principal cells. To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. In the absence of dDAVP, a vasopressin analog, these cells exhibited low or undetectable GFP and Aqp2 expression in all cells. dDAVP stimulation (1nM dDAVP for 48hrs) markedly increased both GFP and Aqp2 expression together with reversal upon dDAVP removal. These observations demonstrate that GFP faithfully tracks Aqp2 expression. Interestingly, fewer than 50% of cells express GFP and Aqp2 after dDAVP or forskolin, indicating significant variability even though they were clonally-derived. We flow-sorted the GFP^- cells (Aqp2^-) and GFP⁺ cells (Aqp2⁺), regrew them, and restimulated them separately with dDAVP. Cells originating from GFP^- cells gave rise to both GFP^- cells and GFP⁺ cells, and GFP⁺ cells similarly regenerated both GFP^- and GFP⁺ populations in the same proportion. Flow cytometry analysis of the DNA content showed variability in cell cycle phases, with most GFP⁺ cells in G0/G1, and more GFP^- cells in G2/S. RNA-seq analysis of the GFP^- and GFP⁺ cells revealed increased abundance of cell-cycle related transcripts in the GFP^- cells. We conclude that: 1) heterogeneity in Aqp2 expression is related to cell cycle state; and 2) the newly generated reporter cell line will likely serve as a useful tool to study Aqp2 transcriptional regulation.