Blocking Smad7 restores TGF-β1 signaling in chronic inflammatory bowel disease
Giovanni Monteleone, … , Howard W. Steer, Thomas T. MacDonald
Giovanni Monteleone, … , Howard W. Steer, Thomas T. MacDonald
Published August 15, 2001
Citation Information: J Clin Invest. 2001;108(4):601-609. https://doi.org/10.1172/JCI12821.
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Article

Blocking Smad7 restores TGF-β1 signaling in chronic inflammatory bowel disease

Abstract

TGF-β1 functions as a negative regulator of T cell immune responses, signaling to target cells using the Smad family of proteins. We show here that Smad7, an inhibitor of TGF-β1 signaling, is overexpressed in inflammatory bowel disease (IBD) mucosa and purified mucosal T cells. Both whole tissue and isolated cells exhibit defective signaling through this pathway, as measured by phospho-Smad3 immunoreactivity. Specific antisense oligonucleotides for Smad7 reduce Smad7 protein expression in cells isolated from patients with IBD, permitting the cells to respond to exogenous TGF-β1. TGF-β1 cannot inhibit proinflammatory cytokine production in isolated lamina propria mononuclear cells from patients with Crohn disease (CD), but inhibition of Smad7 restores TGF-β1 signaling and enables TGF-β1 to inhibit cytokine production. In inflamed mucosal tissue explants from patients with CD, inhibition of Smad7 also restores p-Smad3 and decreases proinflammatory cytokine production, an effect that is partially blocked by anti–TGF-β1. These results show that Smad7 blockade of TGF-β1 signaling helps maintain the chronic production of proinflammatory cytokines that drives the inflammatory process in IBD and that inhibition of Smad7 enables endogenous TGF-β to downregulate this response.

Authors

Giovanni Monteleone, Andrea Kumberova, Nicholas M. Croft, Catriona McKenzie, Howard W. Steer, Thomas T. MacDonald

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(a) Reduced phosphorylation of Smad3 in IBD tissue. Active (phosphorylat...
(a) Reduced phosphorylation of Smad3 in IBD tissue. Active (phosphorylated, p-Smad3) and inactive Smad3 in mucosal samples of normal controls and patients with CD or UC. Total proteins were immunoprecipitated with a specific Smad3 antibody and subsequently incubated with a phosphoserine antibody (upper blot). After detection of p-Smad3, the membrane was stripped and incubated with a second Smad3 antibody (lower blot) to ascertain equivalent loading of the lanes. To investigate interactions between Smad3 and Smad4, proteins were immunoprecipitated with an anti-Smad3 antibody and subsequently incubated with a Smad4 antibody (middle blot). The example is representative of three separate experiments analyzing in total mucosal samples from eight patients with CD, eight with UC, and eight normal controls. The bottom right panel of a shows quantitative analysis of active/inactive Smad3 ratio in colonic mucosal samples from eight controls, eight patients with CD, and 8 with UC, as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units (a.u.). Each point represents the value (a.u.) of active/inactive Smad3 ratio in mucosal samples taken from a single subject. Horizontal bars indicate the mean. The bottom left panel of a shows a lack of phosphorylated-Smad3 (p-Smad3) signal after treatment with protein phosphatase-2A (PP-2A). Total protein was extracted from three normal controls immunoprecipitated with a specific Smad3 antibody and incubated in the absence or presence of PP-2A. Proteins were then incubated with a phosphoserine antibody. After detection of p-Smad3, the membrane was stripped and incubated with a second Smad3 antibody to show that PP-2A does not affect the content of total Smad3 proteins. The example is representative of two separate experiments in which mucosal samples from six normal controls were used. (b) Relation between active/inactive Smad3 ratio and Smad7 in mucosal samples taken from patients with CD (left) and UC (right). Both Smad3 and Smad7 levels were measured by densitometry scanning of Western blots and expressed in arbitrary units (a.u.). The expression of Smad7 is inversely related to the active/inactive Smad3 ratio in both CD (r = 0.818; P < 0.003) and UC (r = 0.616; P < 0.03).