Figure 1.

Characterization of NF-κB site and NF-κB in mucin MUC2 induction by P. aeruginosa. (A) Human MUC2 regulatory regions (base pairs −2864 to +14, −1627 to +14, and −1308 to +14) were subcloned upstream of a luciferase reporter gene in pGL2 basic vector and transfected into HM3 cells. (B) Human MUC2 regulatory regions (base pairs −1628 to −1307, −1528 to −1307, and −1430 to −1307) were subcloned upstream of a TK-32 promoter luciferase vector (named as −1.6/−1.3TK, −1.5/−1.3TK, and −1.4/−1.3TK, respectively) and transfected into HM3 cells. In both A and B, transfected cells were treated with either P. aeruginosa culture supernatants or vehicle for 6 hr prior to cell lysis. Luciferase activity was then assessed in P. aeruginosa-treated and nontreated cells. Nuclear proteins were incubated with a probe consisting of the ³²P-labeled double-stranded oligonucleotides corresponding to the human MUC2 regulatory region base pairs −1458/−1430 in the absence (C) or presence of various mutant unradiolabeled oligonucleotides (M1 to M4) as indicated (D), or preincubated with antibodies to NF-κB p65, NF-κB p50, c-Rel, or C/EBP (E), and were subjected to EMSA. Probe incubation was with nuclear extracts (10 μg) from HM3 cells either treated or untreated with P. aeruginosa culture supernatant as indicated at the top. (F) Human MUC2 regulatory regions (base pairs −1528 to −1430) containing the wild type (WT) or various mutated sites within the region −1458/−1430 as indicated above were subcloned upstream of the TK-32 promoter in a luciferase vector (named as M1 to M4)) and transfected into HM3 cells. Transfected cells were treated with either P. aeruginosa culture supernatants or vehicle for 6 hr prior to cell lysis. Luciferase activity was then assessed in P. aeruginosa-treated and untreated cells. (G) Effect of NF-κB inhibitor CAPE. A human MUC2 construct p-2864luc was transfected into HM3 cells. Transfected cells were pretreated with or without CAPE (15 μg/ml) for 1 hr before P. aeruginosa treatment as indicated. In all the experiments shown above, transfections were carried out in triplicate. Values are the means ± SD; n = 3. Similar results were observed in another MUC2-expressing epithelial cell line, NCIH292.