Department of Molecular and Cellular Biology, Faculty of Sciences, University of Geneva, 1205 Geneva, Switzerland.
Department of Cellular and Metabolic Physiology, Faculty of Medicine, University of Geneva, 1205 Geneva, Switzerland.
Cells. 2023 Aug 5;12(15):2004. doi: 10.3390/cells12152004.
Cingulin (CGN) and paracingulin (CGNL1) are cytoplasmic proteins of tight junctions (TJs), where they play a role in tethering ZO-1 to the actomyosin and microtubule cytoskeletons. The role of CGN and CGNL1 in the barrier function of epithelia is not completely understood. Here, we analyzed the effect of the knock out (KO) of either CGN or CGNL1 or both on the paracellular permeability of monolayers of kidney epithelial (MDCK) cells. KO cells displayed a modest but significant increase in the transepithelial resistance (TER) of monolayers both in the steady state and during junction assembly by the calcium switch, whereas the permeability of the monolayers to 3 kDa dextran was not affected. The permeability to sodium was slightly but significantly decreased in KO cells. This phenotype correlated with slightly increased mRNA levels of claudin-2, slightly decreased protein levels of claudin-2, and reduced junctional accumulation of claudin-2, which was rescued by CGN or CGNL1 but not by ZO-1 overexpression. These results confirm previous observations indicating that CGN and CGNL1 are dispensable for the barrier function of epithelia and suggest that the increase in the TER in clonal lines of MDCK cells KO for CGN, CGNL1, or both is due to reduced protein expression and junctional accumulation of the sodium pore-forming claudin, claudin-2.
连接蛋白（CGN）和副连接蛋白（CGNL1）是紧密连接（TJ）的细胞质蛋白，在那里它们在将 ZO-1 固定到肌动球蛋白和微管细胞骨架上发挥作用。CGN 和 CGNL1 在上皮屏障功能中的作用尚未完全了解。在这里，我们分析了敲除（KO）CGN 或 CGNL1 或两者对肾上皮（MDCK）细胞单层的旁通透性的影响。KO 细胞显示出单层跨上皮电阻（TER）的适度但显着增加，无论是在稳态下还是在钙开关期间进行连接组装时，而对 3 kDa 葡聚糖的单层通透性不受影响。KO 细胞中钠的通透性略有但显着降低。这种表型与 claudin-2 的 mRNA 水平略有增加、claudin-2 的蛋白水平略有降低以及 claudin-2 的连接积累减少相关，这可以通过 CGN 或 CGNL1 挽救，但不能通过 ZO-1 过表达挽救。这些结果证实了先前的观察结果，表明 CGN 和 CGNL1 对于上皮的屏障功能不是必需的，并表明在 KO 细胞克隆系中 TER 的增加是由于钠孔形成 claudin-claudin-2 的蛋白表达和连接积累减少所致。
