Previous studies addressing the role of NOD1 in H. pylori infection of the gastric mucosa clearly showed that this innate immune receptor is an important part of the mucosal host defense program. These studies, however, did not consider the possibility that NOD1 activation may also play a role during established infection that addresses H. pylori induction of Cdx2 and IM, a feature of chronic H. pylori infection that is considered a precursor to gastric cancer. In our initial studies, we found that H. pylori infection of both cancer cell line cells and nonneoplastic cell line led to prompt and robust expression of Cdx2. In addition, we showed that such induction was greatly enhanced by H. pylori expression of the cagPAI virulence factor possibly because CagA induces activation of NF-B. This in vitro Cdx2 induction system allowed us to probe the effect of NOD1 signaling on H. pylori Cdx2 induction and, subsequently, to conduct studies that defined the mechanism of such effects. Our basic observation was that downregulation of NOD1 signaling led to increased H. pyloriinduced Cdx2 expression, and upregulation had the opposite effect. These in vitro studies thus clearly established that NOD1 signaling has a regulatory function with respect to H. pylori induction of Cdx2. In further studies focused on the nature of the NOD1 signaling mechanism responsible for downregulating Cdx2 induction, we showed initially that increased Cdx2 expression in gastric cancer cells lines by H. pylori infection depends on NF-B activation. This finding is consistent with previous reports showing that H. pylori infection causes the activation of NF-B (28) and that the activation of this transcription factor is involved in the induction of Cdx2 expression (20, 21). We then showed that NF-B activation is negatively regulated by NOD1 signaling probably because such signaling activates TRAF3 (8, 29). This possibility was in fact supported by in vitro studies in which we showed that transfection of an epithelial cell line with a TRAF3 expression vector down-regulated H. pyloriinduced NF-B activity, whereas TRAF3-siRNA upregulated such activity; in addition, overexpression of TRAF3 greatly inhibited Cdx2 expression especially in H. pyloriinfected cells in which NOD1 signaling (and presumably TRAF3 activation) was enhanced. Finally, the idea that NOD1 signaling inhibits H. pyloriinduced Cdx2 expression by negative regulation of NF-B was supported by in vivo studies in which we showed that prolonged infection with H. pylori in NOD1-deficient mice as compared with that in NOD1-intact mice was accompanied by greatly increased nuclear expression of NF-B p65 associated with greatly decreased expression of TRAF3. Our observation that NOD1 signaling regulates H. pyloriinduced Cdx2 expression in epithelial cells infected by H. pylori in vitro was fully corroborated by in vivo studies in which we showed that NOD1-deficient mice manifest greatly increased intestinal metaplasia both 8 and 12 months after infection. Moreover, the gastric mucosa of NOD1-deficient mice expressed increased levels of Cdx2 and other markers of intestinal metaplasia. Finally, as already mentioned, the gastric mucosa of NOD1-deficient mice displayed molecular changes in NF-B and TRAF3, indicative of increased Cdx2 expression. These studies thus supported the notion that NOD1 signaling regulates Cdx2 in physiologic epithelial cells as well as in epithelial cell lines.
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