Tight junctions on the move: molecular mechanisms for epithelial barrier regulation

Abstract

Increasing evidence suggests that the tight junction is a dynamically regulated structure. Cytoskeletal reorganization, particularly myosin light chain phosphorylation--induced actomyosin contraction, has increasingly been recognized as a mediator of physiological and pathophysiological tight junction regulation. However, our understanding of molecular mechanisms of tight junction modulation remains limited. Recent studies using live cell and live animal imaging techniques allowed us to peek into the molecular details of tight junction regulation. At resting conditions, the tight junction is maintained by dynamic protein-protein interactions, which may provide a platform for rapid tight junction regulation. Following stimulation, distinct forms of tight junction protein reorganization were observed. Tumor necrosis factor (TNF-α) causes a myosin light chain kinase (MLCK)--mediated barrier regulation by inducing occludin removal from the tight junction through caveolar endocytosis. In contrast, MLCK- and CK2-inhibition--caused tight junction regulation is mediated by altered zonula occludens (ZO)-1 protein dynamics and requires ZO-1--mediated protein-protein interaction, potentially through regulating claudin function. Although some of the molecular details are missing, studies summarized above point to modulating protein localization and dynamics that are common mechanisms for tight junction regulation.

Figure 1

Components and dynamics of the tight junction. (A) The tight junction is composed of multiple interacting transmembrane and cytoplasmic proteins that are linked to the actin cytoskeleton. (B) In epithelial monolayers expressing both red fluorescent protein-tagged occludin (red) and EGFP-tagged clauidn-1 (green). Following photobleaching of both fluorescent proteins (shown with arrows), significantly more fluorescent recovery occurred for occludin than claudin-1, making the bleached region appear red. Bar: 5 µm.

Figure 2

Occludin overexpression limits TNF-induced tight junction regulation. (A) Overexpression of EGFP-occludin in intestinal epithelium determined by SDS-PAGE immunoblot. Jejunal epithelial cells were isolated from wild-type and EGFP-occludin transgenic mice and were subjected to immunoblot. (B) EGFP-occludin expression preserves occludin localization at the tight junction following TNF treatment. Jejunal tissues from wild-type and EGFP-occluidn transgenic mice treated with 5 µg TNF for 120 min were sectioned and stained for occludin (top, and green in merge), and F-actin (red in merge). Regions of the tight junction lacking occludin developed in wild-type, but not EGFP-occludin transgenic mice. Bar: 10 µm. (C) Intestinal epithelial EGFP-occludin overexpression limits TNF-induced barrier loss and water secretion in small intestine. In vivo perfusion assays show increased paracellular BSA flux (left) and water secretion (right) in wild-type mice. TNF-induced paracellular BSA flux (left) was attenuated, while TNF-induced water secretion did not occur in EGFP-occludin transgenic mice. (D) Schematic presentation of the pathways for TNF to regulate tight junction in intestinal epithelial cells. TNF activates MLCK in intestinal epithelial cells to phosphorylate MLC and cause actomyosin contraction (left side). These events lead to tight junction reorganization to cause occludin to internalize in a caveolin-1–dependent fashion (right side). The original data are adapted from Ref. .

Figure 3

MLCK inhibition regulates ZO-1 dynamics and tight junction permeability. (A and B) MLCK inhibition by peptide inhibitorPIK decreaseswild-type ZO-1 protein, but not ZO-1 mutant lacking the ABRexchangeatthe tightjunction. ZO-1 knockdown of Caco-2 monolayers expressing either the EGFP-ZO-1 or EGFP-ZO-1 mutant lacking the ABR were treated with 300 µM MLCK inhibitor PIK and then subjected to FRAP experiments. Representative kymographs of ZO-1 protein fluorescent recovery in FRAP experiments are shown in A. Bar: 5 µm. Mobile fractions of the wild-type ZO-1 and ZO-1 mutants lacking the ABR with or without MLCK inhibition were determined on the basis of these experiments (B). (C) The ZO-1 ABR domain overexpression affects the MLCK inhibition–induced TER increase in ZO-1–sufficient, but not ZO-1 knockdown, cells. Treating wild-type Caco-2 monolayers with 300 µM PIK increased TER, which was blocked by ZO-1 ABR expression. In contrast, ZO-1 knockdown cells had minimal TER increases following PIK treatment, which could not be blocked by ZO-1 ABR expression. The original data are adapted from Ref. .

Figure 4

CK2 inhibition alters tight junction protein interaction and barrier function. (A) GST-occludin C-terminal tails (383– 522) immobilizedon glutathione-agarose beads were used tocapture proteins from Caco-2 lysates. Recovered proteins were assessed by SDS-PAGE immunoblot. (B) GST-occludin C-terminal tails (383–522) were used to capture control and ZO-1 knockdown Caco-2 lysates. Recovered proteins were assessed as in A. (C) In the presence of CK2 activity, claudin-2 forms Na⁺ permeable channels and does not form a complex with occludin. (D) When CK2 activity is blocked, occludin is not phosphorylated at S408, which allows it to bind to ZO-1. Occludin, ZO-1, and claudin-2 form a multimolecular complex to disrupt claudin-2 function, which leads to decreased paracelllar ion permeability. The original data are adapted from Ref. .