SQSTM1/p62 regulates the expression of junctional proteins through epithelial-mesenchymal transition factors.

The epithelial to mesenchymal transition (EMT) is an essential process during development and during tumor progression. Here, we observed the accumulation of the selective autophagy receptor and signaling adaptor sequestosome-1 (SQSTM1/p62) during growth factor-induced EMT in immortalized and tumor-derived epithelial cell lines. Modulation of the p62 level regulated the expression of junctional proteins. This effect was dependent on the ubiquitin-associated domain of p62, which stabilized the TGFß/Smad signaling co-activator Smad4 and the EMT transcription factor Twist. This study highlights a novel function of p62 in a major epithelial phenotypic alteration.

Impairment of swimming motility by antidiarrheic Lactobacillus acidophilus strain LB retards internalization of Salmonella enterica serovar Typhimurium within human enterocyte-like cells.

We report that both culture and the cell-free culture supernatant (CFCS) of Lactobacillus acidophilus strain LB (Lactéol Boucard) have the ability (i) to delay the appearance of Salmonella enterica serovar Typhimurium strain SL1344-induced mobilization of F-actin and, subsequently, (ii) to retard cell entry by S. Typhimurium SL1344. Time-lapse imaging and Western immunoblotting showed that S. Typhimurium SL1344 swimming motility, as represented by cell tracks of various types, was rapidly but temporarily blocked without affecting the expression of FliC flagellar propeller protein. We show that the product(s) secreted by L. acidophilus LB that supports the inhibitory activity is heat stable and of low molecular weight. The product(s) caused rapid depolarization of the S. Typhimurium SL1344 cytoplasmic membrane without affecting bacterial viability. We identified inhibition of swimming motility as a newly discovered mechanism by which the secreted product(s) of L. acidophilus strain LB retards the internalization of the diarrhea-associated pathogen S. enterica serovar Typhimurium within cultured human enterocyte-like cells.

Secreted autotransporter toxin (Sat) triggers autophagy in epithelial cells that relies on cell detachment.

The secreted autotransporter toxin, Sat, which belongs to the subfamily of serine protease autotransporters of Enterobacteriaceae, acts as a virulence factor in extraintestinal and intestinal pathogenic strains of Escherichia coli. We observed that HeLa cells exposed to the cell-free culture supernatant of recombinant strain AAEC185p(Sat-IH11128) producing the Sat toxin (CFCS(Sat) ), displayed dramatic disorganization of the F-actin cytoskeleton before loosening cell-to-cell junctions and detachment. Examination of the effect of Sat on GFP-microtubule-associated protein light chain 3 (LC3) HeLa cells revealed that CFCS(Sat) -induced autophagy follows CFCS(Sat) -induced F-actin cytoskeleton rearrangement. The induced autophagy shows an acceleration of the autophagy flux soon after Sat treatment, followed later by a blockade of the flux leading to the accumulation of large GFP-LC3-positive vacuoles in the cell cytoplasm. CFCS(Sat) did not induce cell detachment in autophagy-deficient mouse embryonic fibroblasts in contrast with wild-type mouse embryonic fibroblasts. The CFCS(Sat) -induced large GFP-LC3 dots do not display the characteristics of autophagolysosomes including expression of cathepsin D and Lamp-1 and 2 proteins, and Lysotracker Red- and DQ-BSA-positive labelling. We provide evidences that CFCS(Sat) -induced autophagy is not a cell response intended to get rid of the intracellular toxin. By a pharmacological blockers approach, we found that the blockade of Erk1/2 and p38 MAPKs, but not JNK, inhibited the CFCS(Sat) -induced autophagy and cell detachment whereas phosphatidylinositol-3 kinase blockers inhibiting canonical autophagy were inactive. When attached CFCS(Sat) -treated cells start to detach they showed caspase-independent cell death and rearrangements of the focal adhesion-associated vinculin and paxillin. Collectively, our results support that Sat triggers autophagy in epithelial cells that relies on its cell-detachment effect.

A protein kinase A-dependent mechanism by which rotavirus affects the distribution and mRNA level of the functional tight junction-associated protein, occludin, in human differentiated intestinal Caco-2 cells.

We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.

Zipper-like internalization of Dr-positive Escherichia coli by epithelial cells is preceded by an adhesin-induced mobilization of raft-associated molecules in the initial step of adhesion.

We undertook a study of the mechanism by which Dr-positive bacteria invade epithelial cells. Our findings show that Dr-positive bacteria enter via a zipper-like mechanism that is independent of the Dr-induced mobilization of F-actin and of the signaling molecules that control Dr-induced F-actin rearrangements. We also observed that Dr-positive IH11128 bacteria entered cells that were positive for the caveola marker VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) to the same extent as those that were not (parental Caco-2 cells). Using fluorescence labeling and confocal laser scanning microscopy, we provide evidence that during the adhesion step, the alpha5beta1 integrin, which plays a pivotal role in Afa/Dr diffusely adhering Escherichia coli bacterial entry, is mobilized around adhering Dr-positive bacteria. We show that the receptor for Afa/Dr adhesins, glycosylphosphatidylinositol-anchored CD55; the raft marker, ganglioside GM1; and VIP21/caveolin are all recruited around adhering Dr-positive bacteria. We also observed that extracting membrane cholesterol with methyl-beta-cyclodextrin (MBCD) did not affect the recruitment of CD55, GM1, or beta1 integrin to adhering Dr-positive bacteria. In contrast, extracting or changing membrane-bound cholesterol by means of drugs that modify lipid rafts (MBCD, filipin III, or mevalonate plus lovastatin plus MBCD) inhibited the entry of Dr-positive IH11128 both into cells that expressed VIP21/caveolin (HeLa and Caco-2/Cav-1 cells) and into those that did not (parental Caco-2 cells). Finally, restoring cholesterol within the cell membrane of MBCD-treated cells restored Dr-positive IH11128 internalization.