Knock Out of CGN and CGNL1 in MDCK Cells Affects Claudin-2 but Has a Minor Impact on Tight Junction Barrier Function

Cells. 2023 Aug 5;12(15):2004. doi: 10.3390/cells12152004.

Abstract

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.

Keywords: cingulin; claudin-2; paracingulin; permeability; sodium.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Claudin-2* / genetics
  • Claudin-2* / metabolism
  • Claudins / genetics
  • Claudins / metabolism
  • Dogs
  • Madin Darby Canine Kidney Cells
  • Tight Junctions* / metabolism

Substances

  • Claudin-2
  • Claudins

Grants and funding

This research was funded by the Swiss National Science Foundation (Grants n. 31003A_135730, 31003A_152899, 31003A_172809, and 310030_200681 to S. C.) and by the State of Geneva.