Cell confluence regulates claudin-2 expression: possible role for ZO-1 and Rac

Abstract

Claudin-2 (Cldn-2) is a channel-forming tight junction (TJ) protein in the proximal tubules that mediates paracellular Na transport and has also emerged as a regulator of proliferation and migration. Expression of Cldn-2 is altered by numerous stimuli, but the underlying mechanisms remain incompletely understood. Here we show that Cldn-2 protein and mRNA expression were low in subconfluent tubular cells and increased during junction maturation. Cldn-1 or occludin did not exhibit similar confluence-dependence. Conversely, disruption of TJs by Ca removal or silencing of zonula occludens-1 (ZO-1) or ZO-2 induced a large drop in Cldn-2 abundance. Immunofluorescent staining revealed a more uneven Cldn-2 staining in nascent, Cldn-1-positive TJs. Subconfluence and ZO-1 silencing augmented Cldn-2 degradation and reduced Cldn-2 promoter activity, suggesting that insertion into the TJs slows Cldn-2 turnover. Indeed, blocking endocytosis or lysosomal degradation increased Cldn-2 abundance. Cell confluence increased expression of the junctional adapters ZO-1 and -2, and the small GTPase Rac, and elevated Rac activity and p21-activated kinase (Pak) phosphorylation, suggesting that they might mediate confluence-dependent Cldn-2 regulation. Indeed, Rac silencing or Pak inhibition strongly reduced Cldn-2 protein abundance, which was likely the combined effect on turnover, as these interventions reduced Cldn-2 promoter activity and augmented Cldn-2 degradation. Taken together, our data suggest that TJ integrity and maturity, ZO-1 expression/TJ localization, and Rac/Pak control Cldn-2 degradation and synthesis. A feedback mechanism connecting Cldn-2 expression with junction remodeling, e.g., during wound healing, epithelial-mesenchymal transition, or tumor metastasis formation, may have important downstream effects on permeability, proliferation, and migration.

A Bile Duct‐on‐a‐Chip With Organ‐Level Functions

“…Previous studies have suggested that epithelial cell confluence is associated with morphology, polarity, and barrier function . Although changes in transepithelial electronic resistance have been reported to be minimal after cells reach confluence, we found that permeability decreased significantly when cells progressed from confluent to compact, with associated changes in height, suggesting changes in tight junction molecules .…”

“…Previous studies have suggested that epithelial cell confluence is associated with morphology, polarity, and barrier function . Although changes in transepithelial electronic resistance have been reported to be minimal after cells reach confluence, we found that permeability decreased significantly when cells progressed from confluent to compact, with associated changes in height, suggesting changes in tight junction molecules . These results demonstrate that compactness is required for an optimal barrier function, and suggest that confluence may be an intermediate rather than ultimate goal in the repair of an injured epithelial layer.…”

In vivo and ex vivo assessment of bladder hyper-permeability and using molecular targeted magnetic resonance imaging to detect claudin-2 in a mouse model for interstitial cystitis

“…We found in this study that claudin-2 was increased in the URO-MCP-1 LPS-induced model for interstitial cystitis. In addition, IF images of the mouse bladder urothelial, obtained in this study, supports the notion that claudin-2 plays a role in the tight-junction network [ 31 , 32 ].…”

Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells

“…The following antibodies against tight junction molecules were from Thermo/Invitrogen and have been validated earlier (19,69,90): claudin-2: monoclonal mouse antibody (catalog no. 325600) (used for Western blotting) and polyclonal rabbit antibody (catalog no.…”