Abstract
Purpose :
To determine the expression of water channel protein aquaporins (Aqps) in the lens epithelial cells of connexin 50 (Cx50, encoded by Gja8) knockout mice with microphthalmia and MEK1(E) transgenic mice with macrophthalmia. To investigate the mechanism about how Cx50 and MEK1 mediated signal transduction pathways regulate water channels and water transport to affect the lens size and homeostasis.
Methods :
Single-cell RNA-sequencing (scRNA-seq) analysis was performed to determine the expression levels of genes including aquaporins in lens epithelial cells of Gja8 knockout (Cx50KO) and MEK1(E) transgenic mice. Aqp1 and Aqp5, two major aquaporins expressed in lens epithelial cells, were further characterized for their protein levels and distributions in vivo and in vitro by using western blot analysis and immunostaining.
Results :
Altered Aqp1 and/or Aqp5 protein expressions were observed in the epithelial cells of Cx50KO and MEK1(E) lenses. The scRNA-seq data revealed up-regulated Aqp1 in Cx50KO lens epithelial cells while both Aqp5 and Cx46 remained unchanged. Western blot analysis demonstrated Aqp1 protein level was 2.5-fold increased in the Cx50KO lens epithelial cell homogenates compared to wild-type control samples and was 3-fold increased in cultured Cx50KO lens epithelial cells compared to the wild-type control. Immunostaining further confirmed increased Aqp1 expression in cultured lens epithelial cells. Aqp5 protein levels were insignificantly changed in the Cx50KO lens epithelial cells. In contrast, the Aqp5 level was significantly increased in lens epithelial cells of the MEK1(E) mice while Aqp1 remained unchanged.
Conclusions :
Upregulated Aqp1 in Cx50KO lens epithelial cells indicates that both Cx50 and Aqp1 likely play important roles in regulating lens water homeostasis. Increased Aqp1 may result from compensatory mechanism for water transport due to a loss of Cx50. Increased Aqp5 in MEK1(E) lens epithelium likely contributes to enlarged lenses leading to macrophthalmia. These data indicate new regulatory mechanisms of Aqp1 and Aqp5 in water transport of lens epithelial cells in Cx50KO and MEK1(E) lenses.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.