Diet, microbiome, and the intestinal epithelium: an essential triumvirate?

The intestinal epithelium represents a critical barrier protecting the host against diverse luminal noxious agents, as well as preventing the uncontrolled uptake of bacteria that could activate an immune response in a susceptible host. The epithelial monolayer that constitutes this barrier is regulated by a meshwork of proteins that orchestrate complex biological function such as permeability, transepithelial electrical resistance, and movement of various macromolecules. Because of its key role in maintaining host homeostasis, factors regulating barrier function have attracted sustained attention from the research community. This paper will address the role of bacteria, bacterial-derived metabolism, and the interplay of dietary factors in controlling intestinal barrier function.

Oxymatrine prevents NF-κB nuclear translocation and ameliorates acute intestinal inflammation.

Oxymatrine is a traditional Chinese herbal product that exhibits anti-inflammatory effects in models of heart, brain and liver injury. We investigated the impact of oxymatrine in an acute model of intestinal injury and inflammation. Oxymatrine significantly decreased LPS-induced NF-κB-driven luciferase activity, correlating with diminished induction of Cxcl2, Tnfα and Il6 mRNA expression in rat IEC-6 and murine BMDC. Although oxymatrine decreased LPS-induced p65 nuclear translocation and binding to the Cxcl2 gene promoter, this effect was independent of IκBα degradation/phosphorylation. DSS-induced weight loss and histological damage were ameliorated in oxymatrine-treated C57BL/6-WT-mice. While this effect correlated with reduced colonic Il6 and Il1ß mRNA accumulation, global NF-κB activity as measured in NF-κB(EGFP) mice was unaffected. Our data demonstrate that oxymatrine reduces LPS-induced NF-κB nuclear translocation and activity independently of IκBα status, prevents intestinal inflammation through blockade of inflammatory signaling and ameliorates overall intestinal inflammation in vivo.

Inhibition of caspase-3 by Survivin prevents Wee1 Kinase degradation and promotes cell survival by maintaining phosphorylation of p34Cdc2.

The anti-apoptotic protein Survivin and the cyclin-dependent kinase p34Cdc2 regulate cell cycle progression and apoptosis. p34Cdc2 activation is required for its pro-apoptotic activity and phosphorylation of p34Cdc2 at Tyrosine-15 (Tyr15) maintains p34Cdc2 in an inactive state. In BaF3 IL-3-dependent murine hematopoietic cells, over-expression of wild-type (wt)-Survivin increased Tyrosine phosphorylation of p34Cdc2, while over-expression of dominant-negative (dn) T34A-Survivin decreased Tyr15 phosphorylation. The increased phospho-Tyr15 levels associated with ectopic wt-Survivin directly correlated with enhanced BaF3 cell survival upon growth factor withdrawal, while conversely, low phospho-Tyr15 levels and decreased survival were seen in BaF3 cells expressing ectopic dn-Survivin. Tyrosine-15 phosphorylation of p34Cdc2 is mediated by the Wee1 Kinase, a known target of caspase-3. In BaF3 cells over-expressing wt-Survivin, 2-fold higher levels of Wee1 protein were detected compared to cells expressing vector or dn-Survivin. Treatment of control vector-transduced BaF3 cells with the selective caspase-3 inhibitor Ac-DEVD-CHO increased p34Cdc2-Tyr15 phosphorylation and Wee1 protein levels. In a similar fashion, over-expression of wt-Survivin maintained high levels of phospho-Tyr15-p34Cdc2 and Wee1 protein. Since Survivin requires Hsp90 for stability, we treated cells with the Hsp90 inhibitors AICAR and 17-AAG to further link Survivin to blocking p34Cdc2 activation. Treatment of BaF3 cells expressing ectopic wt-Survivin with AICAR or 17-AAG significantly reduced p34Cdc2-Tyr15 phosphorylation compared to vehicle-treated controls. These results suggest that Survivin protects the p34Cdc2-Tyr15-targeting kinase Wee1 from degradation by blocking caspase-3 activation leading to inhibition of the pro-apoptotic function of p34Cdc2 and enhanced cell survival.

Cell type-specific expression of the IkappaB kinases determines the significance of phosphatidylinositol 3-kinase/Akt signaling to NF-kappa B activation.

Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-kappa B through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-kappa B activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-kappa B DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-kappa B is retained in the cytoplasm by inhibitory proteins, I kappa Bs, which are phosphorylated and targeted for degradation by I kappa B kinases (IKK alpha and IKK beta). Expression and the ratios of IKK alpha and IKK beta, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKK alpha (the IKK kinase activated by Akt) to IKK beta were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKK beta diminished the capacity of the inhibitors to block NF-kappa B DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-kappa B DNA binding in Ikk beta-/- but not Ikk alpha-/- or wild-type cells in which the ratio of IKK alpha to IKK beta is low. Thus, noncoordinate expression of I kappa B kinases plays a role in determining the cell type-specific role of Akt in NF-kappa B activation.