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

Abstract Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-κB activation during progression (days 6 and 12 post-infection) and regression (days 20-34 post-infection) phases of TMCH. NF-κB activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-κB 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-κB activation kinetics. Phosphorylation of cellular IKKα/β (Ser176/180) was elevated during progression and regression of TMCH. Phosphorylation (Ser32/36) and degradation of IκBα however, contributed to NF-κB activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204) and p38 (Thr180/Tyr182) paralleled IKKα/β kinetics at days-6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK and p38 significantly blocked NF-κB activity in vitro while MEK1/2-inhibitor (PD 98059) also blocked increases in MEK1/2, ERK1/2 and IKKα/β thereby inhibiting NF-κB activity in vivo. Cellular and nuclear levels of Ser-536-phosphorylated (p65536) and Lys-310-acetylated p65 subunit accompanied functional NF-κB activation during TMCH. RSK-1 phosphorylation at Thr359/Ser363 in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-κB overlapped with p65536 kinetics. Dietary pectin (6%) blocked NF-κB activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-κB activation persists despite lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-κB in colonic crypts in response to CR infection.