R.K. Rao

c-Jun NH2-terminal kinase-2 mediates osmotic stress-induced tight junction disruption in the intestinal epithelium

Gastrointestinal epithelium faces osmotic stress, both at physiological and pathophysiological conditions. JNK activation is an immediate cellular response to osmotic stress. We investigated the effect of osmotic stress on intestinal epithelial barrier function and delineated the role of JNK2 in osmotic stress-induced tight junction (TJ) regulation in Caco-2 cell monolayers and ileum of Jnk(-/-) and Jnk2(-/-) mice. The role of JNK activation in osmotic stress-induced TJ disruption was evaluated using JNK-specific inhibitor and antisense oligonucleotides. Furthermore, the effect of cold restraint stress in vivo on TJ integrity was determined in rats. Osmotic stress disrupted TJs and barrier function in Caco-2 cell monolayers without affecting cell viability. Osmotic stress activated JNK1 and JNK2 and the inhibition of JNK by SP600125 attenuated osmotic stress-induced TJ disruption. TJ disruption and barrier dysfunction by osmotic stress was associated with JNK-dependent remodeling of actin cytoskeleton. Knockdown of JNK2 accelerated TJ assembly and attenuated osmotic stress-induced TJ disruption in Caco-2 cell monolayers. In mouse ileum in vitro, osmotic stress increased paracellular permeability, which was attenuated by SP600125. Osmotic stress disrupted actin cytoskeleton and TJs and increased paracellular permeability in the ileum of wild-type and JNK1(-/-) mice, but not in JNK2(-/-) mouse ileum. Cold restraint stress activated JNK in rat ileum and caused JNK-dependent remodeling of actin cytoskeleton and redistribution of occludin and zona occluden-1 from the intercellular junctions. These results reveal the role of JNK2 in the mechanism of osmotic stress-induced TJ disruption in the intestinal epithelium.

Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers

Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl]pyrazolo[3-4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-Src(Y527F) enhanced cell migration, whereas the expression of dominant negative c-Src(K296R/Y528F) attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130(CAS) in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism.

Deletion of TLR-4 attenuates fetal alcohol exposure-induced gene expression and social interaction deficits

Fetal alcohol spectrum disorders (FASD) are associated with social interaction behavior and gastrointestinal (GI) abnormalities. These abnormal behaviors and GI abnormalities overlap with autism spectrum disorder (ASD). We investigated the effect of fetal alcohol exposure (FAE) on social interaction deficits (hallmark of autism) in mice. Evidence indicates that exogenous lipopolysaccharide (LPS) administration during gestation induces autism-like behavior in the offspring. LPS regulates the expression of genes underlying differentiation, immune function, myelination, and synaptogenesis in fetal brain by the LPS receptor, TLR-4-dependent mechanism. In this study, we evaluated the role of TLR-4 in FAE-induced social behavior deficit. WT and TLR4-/- pregnant mice were fed Lieber-DeCarli liquid diet with or without ethanol. The control group was pair-fed with an isocaloric diet. Social behavior was tested in the adult offspring at postnatal day 60. Frontal cortex mRNA expression of autistic candidate genes (Ube3a, Gabrb3, Mecp2) and inflammatory cytokine genes (IL-1β, IL-6, TNF-α) were measured by RT-qPCR. Adult male offspring of ethanol-fed WT dams showed low birth weight compared to offspring of pair-fed WT dams. However, their body weights at adulthood were greater compared to the body weights of offspring of pair-fed WT dams. There were no body weight differences in offspring of TLR4-/- dams. Social interaction deficit was observed only in male offspring of ethanol-fed WT dams, but it was not observed in both male and female offspring of ethanol-fed TLR4-/- dams. Expressions of autism candidate genes, Gabrb3 and Ube3a, were elevated, while that of the Mecp2 gene was suppressed in the frontal cortex of male, but not female, offspring of ethanol-fed WT mice. The expressions of inflammatory cytokine genes, IL-1β, IL-6, and TNF-α, were also significantly increased in the frontal cortex of male, but not female, offspring of ethanol-fed dams. The changes in the expression of autistic and cytokine genes were unaffected in the offspring of ethanol-fed TLR4-/- dams. These data also indicate that TLR4 mediates FAE-induced changes in social interactions and gene expression in brain, suggesting that ethanol-induced LPS absorption from the maternal gut may be involved in gene expression changes in the fetal brain.

Lactobacillus Casei Prevents Osmotic Stress-Induced Disruption of Tight Junctions (TJ) and Adherens Junctions (AJ) in CACO-2 Cell Monolayers by a PKC-Dependent Mechanism, but Independent of EGF Receptor Tyrosine Kinase Activity

right ears of mice (N=5) were injected with SbS and the left ears were injected with vehicle as control. The first treatment was administered at 1 hour after 5x10^6 pfu Ad-VEGF-A164 was injected into both ears. The second SbS/vehicle injections were administered 24 hours later. An inhibitory effect of SbS was clearly evident by day 7 with reduced new vessel formation in the right ear compared to the left. The effect of SbS remained evident until the end of the experiment on day 21. 4) DSS (4% for 5 days) was administered to mice to induce colitis. Daily gavage of Sb significantly attenuated weight-loss (N=5,p<0.01). DSS treated mice had significantly increased blood vessel density and volume compared to healthy mice. Sb treatment significantly reduced the neo-vascularization associated with inflammation in the colon. CONCLUSIONS: Sb inhibits VEGFR signaling and reduces neo-vascularization In Vitro and in two separate In Vivo models. Our findings indicate that the probiotic yeast S boulardii can modulate angiogenesis in intestinal injury and repair, which provides a novel mechanism for its beneficial effects.