[Show abstract][Hide abstract] ABSTRACT: Epiregulin (EPI) and amphiregulin (AR) are epidermal growth factor receptor (EGFR) ligands implicated in mucosal repair and tumorigenesis. We have shown that Toll-like receptor 4 (TLR4) induces intestinal epithelial cell (IEC) proliferation by activating EGFR through AR expression. We examined whether TLR4 differentially regulates expression of EGFR ligands in response to mucosal injury. The human IEC line SW480 was examined expression of EGFR ligands, EGFR phosphorylation, and proliferation in response to lipopolysaccharide (LPS). Small-interfering RNA (siRNA) was used to block TLR4. Neutralizing antibodies to EGFR ligands were used to examine inhibition of LPS-dependent EGFR activation. Acute colitis and recovery were examined in the mice given 2.5% dextran sodium sulfate (DSS). Colonic secretion of EPI and AR was analyzed by enzyme-linked immunosorbent assay. LPS selectively induces EPI and AR but not other EGFR ligands. LPS induced early EPI mRNA expression between 30 min and 24 h. The neutralizing antibodies to EPI and AR prevented activation of EGFR by LPS. LPS induces IEC proliferation (200%, P=0.01) in 24 h but blocking EPI and AR significantly decreased proliferation. In vivo, mucosal EPI and AR expression are significantly decreased in TLR4(-/-) mice (P=0.02) compared to wild-type mice during acute colitis. EPI and AR exhibit different kinetics in response to mucosal damage: EPI expression is upregulated acutely at day 7 of DSS, but falls during recovery at day 14. By contrast, a sustained upregulation of AR expression is seen during mucosal injury and repair. We show that TLR4 regulates EPI and AR expression and that both these EGFR ligands are necessary for optimal proliferation of IEC. The diverse kinetics of EPI and AR expression suggest that they function in distinct roles with respect to acute injury vs repair. Our results highlight the role of bacterial sensing for IEC homeostasis and may lead to targeted therapy for mucosal healing and prevention of tumorigenesis.
[Show abstract][Hide abstract] ABSTRACT: We have previously found that TLR4-deficient (TLR4-/-) mice demonstrate decreased expression of mucosal PGE 2 and are protected against colitis-associated neoplasia. However, it is still unclear whether PGE 2 is the central factor downstream of TLR4 signaling that promotes intestinal tumorigenesis. To further elucidate critical downstream pathways involving TLR4-mediated intestinal tumorigenesis, we examined the effects of exogenously administered PGE 2 in TLR4-/- mice to see if PGE 2 bypasses the protection from colitis-associated tumorigenesis.
Mouse colitis-associated neoplasia was induced by azoxymethane (AOM) injection followed by two cycles of dextran sodium sulfate (DSS) treatment. Two different doses of PGE 2 (high dose group, 200 microg, n = 8; and low dose group, 100 microg, n = 6) were administered daily during recovery period of colitis by gavage feeding. Another group was given PGE 2 during DSS treatment (200 microg, n = 5). Inflammation and dysplasia were assessed histologically. Mucosal Cox-2 and amphiregulin (AR) expression, prostanoid synthesis, and EGFR activation were analyzed.
In control mice treated with PBS, the average number of tumors was greater in WT mice (n = 13) than in TLR4-/- mice (n = 7). High dose but not low dose PGE 2 treatment caused an increase in epithelial proliferation. 28.6% of PBS-treated TLR4-/- mice developed dysplasia (tumors/animal: 0.4 +/- 0.2). By contrast, 75.0% (tumors/animal: 1.5 +/- 1.2, P < 0.05) of the high dose group and 33.3% (tumors/animal: 0.3 +/- 0.5) of the low dose group developed dysplasia in TLR4-/- mice. Tumor size was also increased by high dose PGE 2 treatment. Endogenous prostanoid synthesis was differentially affected by PGE 2 treatment during acute and recovery phases of colitis. Exogenous administration of PGE 2 increased colitis-associated tumorigenesis but this only occurred during the recovery phase. Lastly, PGE 2 treatment increased mucosal expression of AR and Cox-2, thus inducing EGFR activation and forming a positive feedback mechanism to amplify mucosal Cox-2.
These results highlight the importance of PGE 2 as a central downstream molecule involving TLR4-mediated intestinal tumorigenesis.