Novel Changes in NF-κB Activity during Progression and Regression Phases of Hyperplasia ROLE OF MEK, ERK, AND p38

Utilizing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-kappa B activation during progression (days 6 and 12 post-infection) and regression (days 20-34 post-infection) phases of TMCH. NF-kappa B activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-kappa B activity at days 27 and 34, however, remained 2-3-fold higher than uninfected control. Expression of the downstream target gene CXCL-1/KC in the crypts correlated with NF-kappa B activation kinetics. Phosphorylation of cellular I kappa B alpha kinase (IKK)alpha/beta (Ser(176/180)) was elevated during progression and regression of TMCH. Phosphorylation (Ser(32/36)) and degradation of I kappa B alpha, however, contributed to NF-kappa B activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser(217/221)), ERK1/2 (Thr(202)/Tyr(204)), and p38 (Thr(180)/Tyr(182)) paralleled IKK alpha/beta kinetics at days 6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK, and p38 significantly blocked NF-kappa B activity in vitro, whereas MEK1/2-inhibitor (PD98059) also blocked increases in MEK1/2, ERK1/2, and IKK alpha/beta thereby inhibiting NF-kappa B activity in vivo. Cellular and nuclear levels of Ser(536)-phosphorylated (p65(536)) and Lys(310)-acetylated p65 subunit accompanied functional NF-kappa B activation during TMCH. RSK-1 phosphorylation at Thr(359)/Ser(363) in cellular/nuclear extracts and co-immuno-precipitation with cellular p65-NF-kappa B overlapped with p65(536) kinetics. Dietary pectin (6%) blocked NF-kappa B activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-kappa B activation persisted despite the lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. The MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-kappa B in colonic crypts in response to C. rodentium infection.