Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function

Abstract

The apical-most epithelial intercellular junction, referred to as the tight junction (TJ), regulates paracellular solute flux in diverse physiological and pathological states. TJ affiliations with the apical filamentous actin(F-actin) cytoskeleton are crucial in regulating TJ function. F-actin organization is influenced by the Rho GTPase family, which also controls TJ function. To explore the role of Rho GTPases in regulating TJ structure and function, we utilized Escherichia coli cytotoxic necrotizing factor-1(CNF-1) as a tool to activate constitutively Rho, Rac and Cdc42 signaling in T84 polarized intestinal epithelial monolayers. The biological effects of the toxin were polarized to the basolateral membrane, and included profound reductions in TJ gate function, accompanied by displacement of the TJ proteins occludin and zonula occludens-1 (ZO-1), and reorganization of junction adhesion molecule-1 (JAM-1) away from the TJ membrane. Immunogold electron microscopy revealed occludin and caveolin-1 internalization in endosomal/caveolar-like structures in CNF-treated cells. Immunofluorescence/confocal microscopy suggested that a pool of internalized occludin went to caveolae, early endosomes and recycling endosomes, but not to late endosomes. This provides a novel mechanism potentially allowing occludin to evade a degradative pathway, perhaps allowing efficient recycling back to the TJ membrane. In contrast to the TJ, the characteristic ring structure of proteins in adherens junctions (AJs) was largely preserved despite CNF-1 treatment. CNF-1 also induced displacement of a TJ-associated pool of phosphorylated myosin light chain (p-MLC), which is normally also linked to the F-actin contractile machinery in epithelial cells. The apical perjunctional F-actin ring itself was maintained even after toxin exposure,but there was a striking effacement of microvillous F-actin and its binding protein, villin, from the same plane. However, basal F-actin stress fibers became prominent and cabled following basolateral CNF-1 treatment, and the focal adhesion protein paxillin was tyrosine phosphorylated. This indicates differences in Rho GTPase-mediated control of distinct F-actin pools in polarized cells. Functionally, CNF-1 profoundly impaired TJ/AJ assembly in calcium switch assays. Re-localization of occludin but not E-cadherin along the lateral membrane during junctional reassembly was severely impaired by the toxin. A balance between activity and quiescence of Rho GTPases appears crucial for both the generation and maintenance of optimal epithelial barrier function. Overactivation of Rho, Rac and Cdc42 with CNF-1 seems to mirror key barrier-function disruptions previously reported for inactivation of RhoA.

Regulation of testicular tight junctions by gonadotrophins in the adult Djungarian hamster in vivo

“…The results from the present study strongly indicate that organisation of TJ protein and not TJ protein expression itself is central to barrier functionality. Thus, following other epithelial tissue systems (Hopkins et al 2003, Morimoto et al 2005, testicular tight junction proteins may be recycled into disorganised intracellular pools after gonadotrophin suppression in this model. Upon gonadotrophin stimulation, it is proposed that tight junction proteins would relocate to the cell membrane, providing a mechanism conducive to a rapid reorganisation of functional tight junctions and re-initiation of spermatogenesis.…”

A Key Claudin Extracellular Loop Domain is Critical for Epithelial Barrier Integrity

“…To determine whether these claudin-1 EL mimetic peptides had the capacity to disrupt TJs, we tested the ability of three different EL peptides to interfere with TJ assembly using a calcium switch protocol. [32][33][34] As shown in Figure 1B, the Cldn-1 53-80 peptide was effective at inhibiting the recovery of T84 cell barrier function, because FD-3 permeability remained high. In contrast, FD-3 permeability of either control cells or cells incubated with either Cldn-1 or Cldn-1 146 -160 was markedly reduced, suggesting that TJs were completely reformed under these conditions.…”

Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo

“…These signaling molecules are attractive candidates for this process, given that TNF has been reported to activate Rho, Rac, and Cdc42 (39) and that these proteins may regulate tight junction protein distribution and epithelial barrier function (40)(41)(42)(43). To investigate the role of Rho family GTPases, we included the Rho kinase inhibitor Y27632 (44) or a Rac1 inhibitor, NSC23766 (45), in the perfusion solution of control and anti-CD3-treated animals.…”