We now describe the identification of a novel and potentially important link between TLR4 signaling and activation of the AKT-GSK3β pathway leading to the inhibition of enterocyte proliferation, an effect that plays a role in the pathogenesis of NEC. The current findings are consistent with previous work indicating that enterocyte proliferation may be inhibited after endotoxin exposure18, 19
, yet extend these observations by providing both mechanistic insights and physiological relevance. In support of the relevance of the current findings to the pathogenesis of NEC, we now show that TLR4 activation – which we and others have previously shown to be important in NEC pathogenesis8, 20
- leads to decreased β-catenin and increased GSK3β expression in the intestinal mucosa in both human an murine NEC, while inhibition of TLR4 signaling in enterocytes in vivo reverses these effects and restores levels of enterocyte proliferation in experimental NEC. It is interesting to note that other diseases of intestinal inflammation are associated with impaired enterocyte proliferation, including various models of colitis that mimic inflammatory bowel disease in humans21, 22
. Given the observation that TLR4 expression has been shown to be increased in specimens obtained from regions of active ulcerative colitis in humans23
, it is tempting to speculate that by inhibition of enterocyte proliferation – perhaps via effects on β-catenin signaling - TLR4 activation could contribute to persistent mucosal injury in these inflammatory intestinal diseases also.
With further respect to NEC pathogenesis, we note that it has recently been reported that in the newborn intestine, vaginal delivery and LPS exposure lead to LPS unresponsiveness by the primary enterocytes24
. Based upon our recent discovery that the hypoxia and endotoxemia that characterize NEC leads to persistent upregulation of intestinal TLR48
, we now propose that part of the mechanism of NEC reflects the inability of the intestine to down-regulate TLR4 signaling in order to become tolerant to the luminal bacteria. This would be expected to lead to exaggerated TLR4 signaling upon bacterial colonization, resulting in impaired enterocyte proliferation and persistent susceptibility to mucosal injury as seen in NEC. These findings expand the current understanding of the role of TLR4 in intestinal homeostasis, while also providing novel insights into the molecular mechanisms that lead to NEC.
How can the current findings be reconciled with the observation that patients with chronic intestinal inflammation have an increased propensity to develop intestinal tumors, suggesting that enterocyte proliferation may be increased at least transiently during states of chronic intestinal injury? First, we point out that the current findings may be specific for the newborn small intestine, as TLR4 activation did not decrease enterocyte proliferation in either adult intestine or newborn colon. Second, it is well established that downstream mediators of TLR4 signaling – including the transcription factor NFkB and associated molecules - have established roles in the development of tumors, in part via transcription of oncogenes25
, which could lead to induction of tumors after chronic intestinal inflammation. And third, while we now demonstrate that TLR4 signaling in enterocytes is responsible for the inhibition of enterocyte proliferation in vivo, it is quite possible that in conditions of colitis that favor the development of tumors, the effects of TLR4 signaling may occur via hematopoietic cells and not on enterocytes. In support of this possibility, Abreu et al has recently used TLR4 wild-type and mutant chimeric mice to show that TLR4 signaling in leukocytes plays a key role in colitis associated tumor formation26
. We now propose that in the intestinal inflammatory environment, TLR4 activation in different cell types leads to disparate effects: in newborn enterocytes, an inhibition of cell turnover is observed leading to mucosal injury, while in the setting of chronic NFkB activation – potentially via effects on leukocytes - tumorogenesis may occur. It is also likely that the extent and duration of inflammation, the age of the individual and the location of the inflammation (colon vs. ileum) play important roles in determining whether enterocyte proliferation is impaired, as we now show, as opposed to the more long term models of colitis in which tumors may form27
In summary, we now provide evidence for a novel link between TLR4 and the GSK3β-β-catenin signaling pathway in enterocytes, and show that the interaction of these signaling pathways plays a key role in the pathogenesis of NEC via impaired enterocyte proliferation. The mechanisms by which this pathway is regulated in vivo may open up future directions of study regarding both the pathogenesis and potential treatment of devastating diseases such as NEC.