Background & aims: Transforming growth factor (TGF)-beta1 is one of the most powerful endogenous negative regulators of inflammation. In patients with inflammatory bowel disease, despite abundant local TGF-beta1, there is a failure of TGF-beta-mediated negative regulation of nuclear factor kappaB activation and proinflammatory cytokine production because of increased intracellular expression of the endogenous inhibitor of TGF-beta1 signaling, Smad7. In this study, we examined the molecular mechanism underlying the induction of Smad7 in the human gut.
Methods: Whole intestinal mucosal and lamina propria mononuclear cell samples were analyzed for Smad7 by real-time polymerase chain reaction and Western blotting. Smad7 ubiquitination and acetylation, and interaction of Smad7 with the intrinsic histone acetyltransferase, p300, were examined by immunoprecipitation and Western blotting. The effect of p300 silencing on Smad7 expression was determined in Crohn's disease lamina propria mononuclear cells.
Results: We showed that Smad7 is not transcriptionally regulated in human gut but that its increase in patients with inflammatory bowel disease is due to posttranscriptional acetylation and stabilization by p300, which prevents Smad7 ubiquitination and degradation in the proteasome. Hence, Smad7 protein in cells from normal gut is ubiquitinated and rapidly degraded. In contrast, in inflamed gut, Smad7 is acetylated and not ubiquitinated, is not degraded, and can be decreased by short interfering RNA to p300.
Conclusions: These results identify posttranslational protein modification as of importance in chronic gut inflammation in humans.