Inhibition of Smad7 With a Specific Antisense Oligonucleotide Facilitates TGF-β1–Mediated Suppression of Colitis

doi:10.1053/j.gastro.2006.09.016

Gastroenterology

Volume 131, Issue 6, December 2006, Pages 1786-1798

https://doi.org/10.1053/j.gastro.2006.09.016 Get rights and content

Background & Aims: Defective transforming growth factor (TGF)-β1 signaling due to high levels of Smad7 is a feature of inflammatory bowel disease (IBD). In this study, we analyzed the effect of reducing Smad7 levels with antisense oligonucleotide on mouse models of colitis. Methods: Mucosal samples taken from colitic tissue of mice with colitis due to either haptenating reagents (trinitrobenzene sulfonic acid [TNBS] or oxazolone) or to transfer of T cells (SCID transfer colitis) were analyzed for Smad3 and/or Smad7 expression by Western blotting and, in some cases, content of TGF-β1 by enzyme-linked immunosorbent assay. The effect of oral Smad7 antisense oligonucleotide on mucosal inflammation was assessed. Results: TGF-β1 levels were increased in the inflamed tissues of mice with colitis induced by either TNBS or oxazolone. Nevertheless, TGF-β1 did not exert a regulatory effect, probably because TGF-β1 signaling was blocked, as indicated by the presence of reduced Smad3 phosphorylation and high levels of Smad7. Oral administration of Smad7 antisense oligonucleotide to colitic mice restored TGF-β1 signaling via Smad3 and ameliorated inflammation in hapten-induced colitis. In addition, Smad7 antisense oligonucleotide had a therapeutic effect on relapsing TNBS-induced colitis but not on cell-transfer colitis. Conclusions: These data suggest that colitis models associated with high endogenous TGF-β1 levels and defective TGF-β1 signaling due to high levels of Smad7 can be ameliorated by down-regulation of Smad7 and by oral administration of Smad7 antisense oligonucleotide. This may represent a new approach to the control of IBD, particularly during active phases when its Smad7 profile resembles that of hapten-induced colitis.

Section snippets

Induction of TNBS and Oxazolone Colitis

Studies of hapten-induced colitis were performed in 5- to 6-week-old male SJL mice (Harlan Laboratories, S. Pietro al Natisone, UD, Italy), which were maintained in the animal facility at the Istituto Superiore di Sanità (Rome, Italy). The experiments were performed within 7 days of the arrival of the animals. All studies were approved by the Animal Care and Use Committee of Istituto Superiore di Sanità and the Italian Ministry of Health. For induction of colitis, 2.5 mg of TNBS or 6 mg of

TNBS-Induced Colitis Is Characterized by Decreased p-Smad3 and Increased Smad7 Expression

It has been shown previously that T cells isolated from the colon of mice with TNBS-induced colitis and then cultured in the presence or absence of polyclonal stimulants produce only small amounts of TGF-β1.¹⁴ It was nevertheless possible that other cells might be producing TGF-β1 under these circumstances. Accordingly, TGF-β1 levels were measured in extracts of inflamed or control colon mucosa. Somewhat to our surprise, levels of total TGF-β1 were significantly higher in extracts from mice

Discussion

In this study, we took advantage of the accessibility and relative ease of analysis of murine models of mucosal inflammation to extend our previous studies of TGF-β1 signaling in mucosal inflammation using tissues from patients with IBD.⁹ We showed that while levels of TGF-β1 in the inflamed tissue of both Th1-mediated TNBS-induced colitis and Th2-mediated oxazolone-induced colitis are increased, TGF-β1–mediated Smad3 activation does not occur due to the rapid appearance of an inhibitory Smad,

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In physiological conditions, the human gut contains more immune cells than the rest of the body, but no overt tissue damage occurs, because several regulatory mechanisms control the activity of such cells thus preventing excessive and detrimental responses. One such mechanism relies on the action of transforming growth factor (TGF)-β1, a cytokine that targets both epithelial cells and many immune cell types. Loss of TGF-β1 function leads to intestinal pathology in both mice and humans. For instance, disruption of TGF-β1 signaling characterizes the destructive immune-inflammatory response in patients with Crohn’s disease and patients with ulcerative colitis, the major human inflammatory bowel disease (IBD) entities. In these pathologies, the defective TGF-β1-mediated anti-inflammatory response is associated with elevated intestinal levels of Smad7, an antagonist of TGF-β1 signaling. Consistently, knockdown of Smad7 restores TGF-β1 function thereby attenuating intestinal inflammation in patients with IBD as well as in mice with IBD-like colitis. Up-regulation of Smad7 and reduced TGF-β1 signaling occurs also in necrotizing enterocolitis, environmental enteropathy, refractory celiac disease, and cytomegalovirus-induced colitis. In this article, we review the available data supporting the pathogenic role of Smad7 in the gastrointestinal tract and discuss whether and how targeting Smad7 can help attenuate detrimental immuno-inflammatory responses in the gut.
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: Supported by the Fondazione “Umberto Di Mario,” Rome, the Broad Medical Research Program Foundation, and Giuliani, S.p.A, Milan, Italy.

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Basic–alimentary tractInhibition of Smad7 With a Specific Antisense Oligonucleotide Facilitates TGF-β1–Mediated Suppression of Colitis

Section snippets

Induction of TNBS and Oxazolone Colitis

TNBS-Induced Colitis Is Characterized by Decreased p-Smad3 and Increased Smad7 Expression

Discussion

Gastroenterology

Curr Opin Cell Biol

Cell

Mol Cell

Trends Immunol

Inflammatory bowel disease

N Engl J Med

The immunological and genetic basis of inflammatory bowel disease

Nat Rev Immunol

Regulation of immune responses by TGF-β

Annu Rev Immunol

Transforming growth factor-beta 1 knockout miceA mutation in one cytokine gene causes a dramatic inflammatory disease

Am J Pathol

Transforming growth factor β in T cell biology

Nat Rev Immunol

Experimental granulomatous colitis in mice is abrogated by induction of TGF-β-mediated oral tolerance

J Exp Med

Oxazolone colitis: a murine model of T helper cell type 2 colitis treatable with antibodies to interleukin 4

J Exp Med

Basic–alimentary tract
Inhibition of Smad7 With a Specific Antisense Oligonucleotide Facilitates TGF-β1–Mediated Suppression of Colitis