Mechanism of IFN-γ-induced Endocytosis of Tight Junction Proteins: Myosin II-dependent Vacuolarization of the Apical Plasma Membrane

Abstract

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.

LIGHT Signals Directly to Intestinal Epithelia to Cause Barrier Dysfunction via Cytoskeletal and Endocytic Mechanisms

“…While a primary barrier defect may be present in IBD kindreds 12-14 , it is also clear that epithelial barrier defects can be induced by inflammatory cytokines [15][16][17][18][19][20][21][22][23][24] . Recent work has demonstrated that barrier dysfunction induced by inflammatory processes can be due to epithelial damage as well as non-apoptotic regulation of tight junction permeability 19,22,[25][26][27][28] . In vitro and in vivo studies have shown that TNF signals directly to intestinal epithelia to regulate barrier function via myosin light chain kinase (MLCK) activation 20, 29-33 .…”

Commensal bacterial internalization by epithelial cells: An alternative portal for gut leakiness

“…Death-dependent TJ disruption or cell death-independent signaling for TJ impairment have been documented in intestinal epithelial cells following exposure to ROS and NO, [27][28][29] as well as IFNg, TNFa, and IL-1b. 21,22,26 We and others demonstrated that bacterial endocytosis by epithelial cells preceded TJ damage under inflammatory stress in a doseand time-dependent manner. 15,29,38,57 Therefore, we suspect that bacterial entry through intermicrovillous lipid rafts may trigger activation of intracellular NLRs by the sensing of endosomally derived bacterial structural components (unrelated to virulence factors).…”

“…19,20 On the other hand, phosphorylation of MLC by Rho-associated kinase (ROCK) leads to the endocytosis of TJ proteins into apical, vacuolar-associated compartments and the impairment of paracellular junctions. [21][22][23] Paracellular permeability is modulated by host-derived factors (e.g., mucosal immune cells, cytokines, enteric neurons) and gut environmental factors (e.g. dietary nutrients, commensals, and pathogens; for more information, see review articles 24,25 ).…”

“…dietary nutrients, commensals, and pathogens; for more information, see review articles 24,25 ). Numerous studies using epithelial cell lines in vitro have shown that the proinflammatory cytokines, e.g., IFNg, TNFa, and IL-1b, cause TJ disruption without affecting cell viability, 21,22,26 whereas free radicals induce cell death-dependent or -independent TJ disruption. [27][28][29] …”

The role of epithelial malfunction in the pathogenesis of enteropathogenic E. coli-induced diarrhea

“…Indeed, IFNg, alone or in synergy with TNFa, is known to induce epithelial barrier dysfunction, independently of its proapoptotic properties. [67][68][69] Furthermore, a recent study has shown the ability of IL-1b to alter epithelial tight junction permeability in an NF-kBdependent manner. 70 Further studies investigated the hypothesis that, during EPEC infection, such proinflammatory mediators may contribute to epithelial barrier damage and diarrhea.…”