Role of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 1 in Oxidative Burst Response to Toll-Like Receptor 5 Signaling in Large Intestinal Epithelial Cells

The NADPH oxidase 1 (Nox1) is a gp91^phox homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O ₂^-) of 160 nmol/mg protein/h and expressed the Nox1, p22^phox, p67^phox, and Rac1 mRNAs, but not the gp91 ^phox, Nox4, p47^phox, p40^phox, and Rac2 mRNAs. They also expressed novel homologues of p47^phox and p67 ^phox (p41^nox and p51^nox, respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22 ^phox, p51^nox, and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O₂^- (<2 nmol/mg protein/h). Cotransfection of p41^nox and p51 ^nox cDNAs in T84 cells enhanced PMA-stimulated O₂ ^- release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O₂^- release in association with the induction of Nox1 protein. The enhanced O₂^- production by cotransfection of p41^nox and p51^nox vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-β-activated kinase 1 and TGF-β-activated kinase 1-binding protein 1. A potent inhibitor for NF-κB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O₂^- production and induction of Nox1 protein. These results suggest that p41^nox and p51 ^nox are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.