Abstract
Utilizing the Citrobacter rodentium-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 IκBα kinase (IKK)α/β (Ser(176/180)) was elevated during progression and regression of TMCH. Phosphorylation (Ser(32/36)) 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 (Ser(217/221)), ERK1/2 (Thr(202)/Tyr(204)), and p38 (Thr(180)/Tyr(182)) 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, whereas MEK1/2-inhibitor (PD98059) 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 (p65(536)) and Lys(310)-acetylated p65 subunit accompanied functional NF-κB activation during TMCH. RSK-1 phosphorylation at Thr(359)/Ser(363) in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-κB overlapped with p65(536) 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 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-κB in colonic crypts in response to C. rodentium infection.
Highlights
Utilizing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-B activation during progression and regression phases of TMCH
Transmissible murine colonic hyperplasia (TMCH),2 caused by C. rodentium infection, is characterized by significant hyperplasia accompanied by expansion of the proliferative compartment throughout the longitudinal crypt axis [8, 9]
Development of colonic hyperplasia during TMCH is associated with an increased susceptibility to carcinogenesis in response to either a chemical mutagen [10] or in the absence of a functional adenomatous polyposis coli (APC) gene product [35]
Summary
TMCH and Diets—TMCH was induced in Helicobacter-free Swiss-Webster mice (15–20 g; Harlan Sprague-Dawley) by oral inoculation with a 16-h culture of C. rodentium, as described previously [9, 22,23,24,25,26,27,28,29]. For DNA binding assays, relative levels of activated p65 NF-B in nuclear extracts of normal or Citrobacter-infected mouse distal colonic crypts were measured using the TransAM p65 NF-B Chemi Transcription Factor Assay kit from Active Motif (Carlsbad, CA) [27]. Immunofluorescence (IMF) and Immunohistochemistry— IMF or immunohistochemistry studies for anti-LPS as a surrogate to detect the presence of C. rodentium in the colonic mucosa was performed on 5-m-thick paraffin sections from distal colons of normal and TMCH mice (days 6 –34) utilizing the HRP-labeled polymer conjugated to secondary antibody using Envisionϩ System-HRP (3,3Ј-diaminobenzidine; DakoCytomation, Carpinteria, CA) with microwave accentuation as described previously [9, 22, 27, 28]. Controls included either omission of primary antibody or detection of endogenous IgG staining pattern with goat anti-mouse or anti-rabbit IgG (Calbiochem)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have