PT  - JOURNAL ARTICLE
AU  - Cong, Xin
AU  - Zhang, Yan
AU  - Li, Jing
AU  - Mei, Mei
AU  - Ding, Chong
AU  - Xiang, Ruo-Lan
AU  - Zhang, Li-Wei
AU  - Wang, Yun
AU  - Wu, Li-Ling
AU  - Yu, Guang-Yan
TI  - Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells
AID  - 10.1242/jcs.165878
DP  - 2015 Jun 15
TA  - Journal of Cell Science
PG  - 2271--2286
VI  - 128
IP  - 12
4099  - http://jcs.biologists.org/content/128/12/2271.short
4100  - http://jcs.biologists.org/content/128/12/2271.full
SO  - J. Cell Sci.2015 Jun 15; 128
AB  - The epithelial cholinergic system plays an important role in water, ion and solute transport. Previous studies have shown that activation of muscarinic acetylcholine receptors (mAChRs) regulates paracellular transport of epithelial cells; however, the underlying mechanism is still largely unknown. Here, we found that mAChR activation by carbachol and cevimeline reduced the transepithelial electrical resistance (TER) and increased the permeability of paracellular tracers in rat salivary epithelial SMG-C6 cells. Carbachol induced downregulation and redistribution of claudin-4, but not occludin or ZO-1 (also known as TJP1). Small hairpin RNA (shRNA)-mediated claudin-4 knockdown suppressed, whereas claudin-4 overexpression retained, the TER response to carbachol. Mechanistically, the mAChR-modulated claudin-4 properties and paracellular permeability were triggered by claudin-4 phosphorylation through ERK1/2 (also known as MAPK3 and MAPK1, respectively). Mutagenesis assay demonstrated that S195, but not S199, S203 or S207, of claudin-4, was the target for carbachol. Subsequently, the phosphorylated claudin-4 interacted with β-arrestin2 and triggered claudin-4 internalization through the clathrin-dependent pathway. The internalized claudin-4 was further degraded by ubiquitylation. Taken together, these findings suggested that claudin-4 is required for mAChR-modulated paracellular permeability of epithelial cells through an ERK1/2, β-arrestin2, clathrin and ubiquitin-dependent signaling pathway.