TNF stimulated increased COX-2 levels by 3 h, and expression peaked at ~16 h in these cells (Fig. 2C); 100 ng/ml TNF most strongly induced COX-2 expression (Fig. 2D). These selleck chem inhibitor data confirm that the concentration of TNF that exhibits a strong cytotoxic effect in COX-2?/? cells is the strongest inducer of COX-2 protein expression and that TNF induction of COX-2 expression is similar in colon and gastric epithelial cells. Fig. 2. TNF or EGF stimulates COX-2 expression in YAMC and ImSt cells. YAMC (A) or ImSt (C) cells were treated with TNF (100 ng/ml) or EGF (10 ng/ml) for 0�C24 h. YAMC (B) or IMST (D) cells were treated with TNF (0�C1,000 ng/ml) or EGF (10 ng/ml) … TNF signals through TNFR1 to induce COX-2 expression.

To determine which of the two TNFRs is required for TNF-induced COX-2 expression, we used conditionally immortalized TNFR1?/? or TNFR2?/? MCE to generate TNFR add-back cell lines. TNFR1?/? MCE cells were infected with recombinant retrovirus to introduce a Vec, a ��DD TNFR1 mutant, or WT TNFR1 (Fig. 3A). TNFR2?/? MCE cells were also infected to introduce Vec or WT TNFR2 (Fig. 3B). TNF did not stimulate COX-2 expression in TNFR1?/? Vec or ��DD cells (Fig. 3C); however, TNF induction of COX-2 was rescued in TNFR1 WT cells. TNF and EGF induced expression of COX-2 in TNFR2?/? Vec cells (Fig. 3D). Interestingly, the basal and TNF- and EGF-stimulated COX-2 expression levels were lower in TNFR2 WT cells. The same pattern of COX-2 expression occurs in WT, TNFR1?/?, and TNFR2?/? ImSt cells (Fig. 3E). Thus, TNF induces COX-2 expression through TNFR1 in GI epithelial cells.

Fig. 3. TNF signals through TNF receptor (TNFR) 1 to induce COX-2 expression. Expression of hemagglutinin (HA)-tagged death domain deletion (��DD) mutant TNFR1 and wild-type (WT) TNFR1 in TNFR1?/? MCE cells (A) and WT TNFR2 in TNFR2?/? … TNF stimulation of COX-2 expression requires EGFR kinase activity. We determined whether EGFR has a role in TNF-induced COX-2 expression by assessing COX-2 expression in the presence of a pharmacological EGFR inhibitor or in the absence of EGFR expression. YAMC cells were treated with TNF in the presence or absence of AG-1478, a small-molecule inhibitor of EGFR kinase activity (Fig. 4A). To quantify the effect of AG-1478 on TNF-stimulated COX-2 expression, we performed a load-response Western blot analysis of the cell lysates (Fig. 4B).

GSK-3 AG-1478 reduced TNF-stimulated COX-2 expression to near-basal levels (Fig. 4C). As expected, the inhibitor also blocked EGF-stimulated COX-2 protein expression. In contrast, AG-1478 did not affect basal COX-2 expression. Interestingly, the concentration of TNF that maximally stimulates COX-2 in YAMC and ImSt cells also maximally stimulated transactivation of EGFR in ImSt (Fig. 4D) and YAMC (76) cells.