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ALTERATIONS IN THE CONTRACTILE PROPERTIES OF ILEAL SMOOTH MUSCLE EXPOSED TO CLOSTRIDIUM DIFFICILE TOXINS: THE INVOLVEMENT OF HIF-1alpha
E Ihara1,2, S Hirota1,2, K Fines1,2, P Beck2, JA MacDonald1,2
1Smooth Muscle and 2Gastrointestinal Research Groups, University of Calgary, Calgary, Alberta
INTRODUCTION: Clostridium difficile (C. diff.) infections cause colitis with associated diarrhea that can progress to toxic megacolon and sepsis, often leading to death. Upon clinical examination, C. diff.-associated colitis often resembles that which is caused by ischemia. C. diff. toxin A (TcdA) and toxin B (TcdB) can trigger immune responses that generate a cytokine milieu that may be responsible for the ischemia-like epithelial response and the altered motility associated with C. diff. infections. Thus we hypothesized that C. diff. toxin-induced inflammation activates protective hypoxic signaling pathways that control the immune response and intestinal motility during C. diff. infections.
METHODS: To study the effect of C. diff. toxins on intestinal epithelial cells Caco-2 cells were exposed to toxin and the expression of HIF-1alpha was determined with real-time RT-PCR and Western blotting. HIF-1alpha-DNA binding was examined using EMSA to evaluate the putative induction of transcription events. To investigate the role of HIF-1alpha in the regulation of intestinal motility upon exposure to C. diff. toxins, ileal loops created in wild-type and epithelium-targeted HIF-1alpha null mice (HIF-1alpha(Ep–/–)) were treated with crude C. diff. toxin (containing both TcdA and TcdB) for 4 hr. Ileal smooth muscle was then removed and contractions evoked by carbachol and KCl were examined.
RESULTS: HIF-1alpha mRNA levels were significantly elevated upon exposure to crude C. diff. toxin (0.1 – 50 µg/mL). Furthermore, HIF-1alpha protein levels and HIF-1alpha-DNA binding were increased in response to crude C. diff. toxin (100 µg/mL). Injection of the crude toxin into ileal loops of both wild-type and HIF-1alpha (Ep–/–) mice led to an augmentation of KCl- and carbachol-induced contractions. However, the toxin-induced hyperresponsiveness to carbachol observed in the wild-type ilium was significantly attenuated in the presence of Y-27632 (10 µM) a selective inhibitor of Rho-kinase, an effect not observed in the HIF-1alpha (Ep–/–) mice.
CONCLUSION: Exposure of intestinal epithelium to C. diff. toxins can stimulate the HIF-1alpha pathway which may increase the contribution of Rho-kinase to cholinergic contractions of ileal smooth muscle.