The functional effects of cytoplasmic actins on epithelial junctions are examined by using isoform-specific siRNAs and cell-permeable inhibitory peptides. Unique roles of cytoplasmic actin isoforms in regulating structure and remodeling of adherens and tight junctions are revealed.

The actin cytoskeleton is a critical regulator of intestinal mucosal barrier permeability, and the integrity of epithelial adherens junctions (AJ) and tight junctions (TJ). Non muscle myosin II (NM II) is a key cytoskeletal motor that controls actin filament architecture and dynamics. While NM II has been implicated in the regulation of epithelial junctions in vitro, little is known about its roles in the intestinal mucosa in vivo. In this study, we generated a mouse model with an intestinal epithelial-specific knockout of NM IIA heavy chain (NM IIA cKO) and examined the structure and function of normal gut barrier, and the development of experimental colitis in these animals. Unchallenged NM IIA cKO mice showed increased intestinal permeability and altered expression/localization of several AJ/TJ proteins. They did not develop spontaneous colitis, but demonstrated signs of a low-scale mucosal inflammation manifested by prolapses, lymphoid aggregates, increased cytokine expression, and neutrophil infiltration in the gut. NM IIA cKO animals were characterized by a more severe disruption of the gut barrier and exaggerated mucosal injury during experimentally-induced colitis. Our study provides the first evidence that NM IIA plays important roles in establishing normal intestinal barrier, and protection from mucosal inflammation in vivo.

The nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARc) has been identified as an important therapeutic target in murine models of colorectal cancer (CRC). To examine whether PPARc inhibition has therapeutic effects in late-stage CRC, the effects of PPARc inhibitors on CRC cell survival were examined in CRC cell lines and a murine CRC model. Low doses (0.1-1 lM) of PPARc inhibitors (T0070907, GW9662 and BADGE) did not affect cell survival, while higher doses (10-100 lM) of all 3 PPARc inhibitors caused caspase-dependent apoptosis in HT-29, Caco-2 and LoVo CRC cell lines. Apoptosis was preceded by altered cell morphology, and this alteration was not prevented by caspase inhibition. PPARc inhibitors also caused dual G and M cell cycle arrest, which was not required for apoptosis or for morphologic alterations. Furthermore, PPARc inhibitors triggered loss of the microtubule network. Notably, unlike other standard antimicrotubule agents, PPARc inhibitors caused microtubule loss by regulating tubulin post-transcriptionally rather than by altering microtubule polymerization or dynamics. Proteasome inhibition by epoxomicin was unable to prevent tubulin loss. siRNA-mediated reduction of PPARc and PPARd proteins did not replicate the effects of PPARc inhibitors or interfere with the inhibitors' effects on apoptosis, cell cycle or tubulin. PPARc inhibitors also reduced CRC cell migration and invasion in assays in vitro and reduced both the number and size of metastases in a HT-29/ SCID xenograft metastatic model of CRC. These results suggest that PPARc inhibitors are a novel potential antimicrotubule therapy for CRC that acts by directly reducing microtubule precursors. ' 2006 Wiley-Liss, Inc.

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins.

Background: Vesicle trafficking proteins play important roles in regulating cell survival. Results: Inhibition of a key membrane fusion protein, ␣SNAP, induced epithelial cell apoptosis, decreased Bcl-2 level, and impaired ER-Golgi communications. Conclusion: ␣SNAP promotes cell survival by controlling ER/Golgi-dependent expression of Bcl-2. Significance: Uncovering mechanisms that link vesicle trafficking and apoptosis is critical for understanding the regulation of cell fate.