This study delineates an important pathway of LPS-induced COX-2 upregulation through EGFR transactivation, suggesting a novel role of EGFR in enterocyte homeostasis and, potentially, in the protection against NEC. Although the pathogenesis of NEC continues to be explored, the unifying hypothesis includes mucosal injury of the small intestine, followed by bacterial translocation and an exaggerated inflammatory response to endotoxin (LPS) 
. LPS binds Toll-like receptor 4 (TLR4), leading to activation of nuclear factor (NF)-κB and subsequent proinflammatory cytokine release by enterocytes and other cells 
. Despite this proinflammatory mechanism, the role of LPS in inflammatory disorders of the intestine is not entirely clear. Whereas LPS has been shown to reduce enterocyte migration and proliferation via TLR4, which may impair intestinal healing 
, LPS-induced COX-2 expression stimulates proliferation of colonocytes and repair of colonic epithelium 
. Regarding the pathophysiology of NEC, several reports have demonstrated the role of TLR4 as causative for the disease 
, and yet Grishin and colleagues reported LPS stimulation of COX-2 was protective in experimental NEC 
. Our data help to explain this dichotomy by showing that LPS can induce COX-2 expression in enterocytes through EGFR transactivation. Although our data suggests that LPS and EGF can induce COX-2 through separate pathways, we cannot exclude a converging pathway downstream.
One goal of this study was to identify signaling pathways that mediate COX-2 induction after LPS transactivation of EGFR. By using specific inhibitors of EGFR kinase activity we established that induction of COX-2 by LPS involves EGFR transactivation. Our data also demonstrate that transactivation of EGFR by LPS is dependent on p38 and MMP activity. Once EGFR is transactivated, further induction of COX-2 depends on activation of ERK and Src, two downstream targets of EGFR. Whereas both ERK and Src are required for induction of COX-2 by LPS via transactivation of EGFR, they appear to act in distinct parallel pathways ().
Proposed mechanism of LPS-induced COX-2 stimulation via EGFR transactivation.
We speculate that MMPs are involved in transactivation of EGFR by LPS, but none of these phosphotyrosines on EGFR couple to COX-2 induction. In this case Src may actually be involved in transactivation of EGFR phosphotyrosines that do couple to COX-2 activation, but these phosphotyrosines are not visible to the phosphotyrosine antibody that we used in this study.
Grishin and colleagues previously reported an essential role of the p38 MAPK in COX-2 upregulation in enterocytes 
. Our data show that p38 is required for LPS induced transactivation of EGFR, but not for subsequent EGFR-mediated COX-2 induction. Thus, inflammatory upregulation of COX-2 in the intestine is more complex than previously thought and may involve p38-dependent and p38-independent pathways.
Our central finding is the critical involvement of EGFR in LPS-induced COX-2 expression in enterocytes. EGFR is abundantly expressed in premature intestinal epithelial cells 
and EGFR and its ligands have long been recognized as protective factors in NEC 
. The significance of MAPK-independent EGFR signaling in LPS-induced wound repair has been previously described in airway epithelial cells 
. Interestingly, the effects of LPS on wound healing were dose-dependent 
, which is yet another illustration of the delicate balance between injury and repair that characterizes the complex molecular pathways in epithelium-microbial interactions. The effects of LPS and EGF on EGFR signaling may also be time-dependent. While prolonged receptor stimulation with LPS did not increase COX-2 expression compared to 24 hour pretreatment alone, sustained stimulation for 6 hours with EGF enhanced the COX-2 signal by Western blot (data not shown).
Inhibition of LPS-stimulated enterocyte proliferation by the selective COX-2 inhibitor Celecoxib, and by the selective EGFR kinase inhibitor AG1478 implies a possible role of EGFR-mediated induction of COX-2 in epithelial restitution. Based on our data, we propose a role of EGFR transactivation by LPS in the inflammatory upregulation of COX-2 in the intestine.