The results presented in this study demonstrate that in the inflamed intestine of patients with IBD, there is marked overexpression of Smad7. This is associated with a reduction in Smad3 phosphorylation, a crucial step in the TGF-β1–mediated signal transduction. In contrast, abundant phosphorylated Smad3 and Smad3/Smad4 complexes are seen in the normal intestinal mucosa, consistent with constitutively active TGF-β1 signaling. We also demonstrate that blocking Smad7 with a specific antisense oligonucleotide restores TGF-β1 signaling and importantly allows TGF-β1 to inhibit proinflammatory cytokine production by isolated mucosal lamina propria mononuclear cells. Finally, in CD tissue, Smad7 antisense restored p-Smad3 and importantly resulted in decreased cytokine production. We believe that this is due to the activity of endogenous TGF-β, given that addition of a neutralizing TGF-β1 to the organ cultures decreased the Smad7 antisense effect on cytokine expression. Together, these results clearly demonstrate that modulating Smad7 expression has profound effects on proinflammatory cytokine production in IBD. Although we only carried out functional studies with CD samples, we believe that the overexpression of Smad7 in UC will also compromise the ability of TGF-β1 to downregulate immune responses in this condition.
TGF-β1 is one of the most widely distributed cytokines that acts on virtually all cell types and mediates highly pleiotropic functions. In particular, TGF-β1 plays an important role in the control of immune homeostasis and prevention of mucosal inflammation (2
). TGF-β1–knockout mice develop a severe multiple organ inflammatory disease, in which the lymphocytic infiltration of the affected organs is associated with increased production of TNF-α and IFN-γ (26
). Abrogation of TGF-β1 signaling in T cells alone is sufficient to break T and B cell homeostasis and induce T cell–mediated inflammatory lesions in various organs, including the intestine (28
). Finally, it has been widely demonstrated that neutralization of TGF-β1 results in the induction and/or amplification of pathogenic responses responsible for the development of experimental colitis resembling either CD or UC (19
). A diminished ability to mount an efficient counterregulatory TGF-β1 response to inflammatory stimuli is therefore believed to be relevant in the pathogenesis of IBD (18
). The diminished Smad3 phosphorylation documented in CD and UC further supports this notion, given that Smad3 activation is essential in the TGF-β1–mediated control of mucosal immunity (10
Smad3 activation can be inhibited through various mechanisms (6
). In this study, however, we focused on the expression of the inhibitory Smad7. In contrast to the TGF-β1R–associated Smads, Smad7 has no carboxy-terminal SSXS sequence and thus does not require any phosphorylation to be active. Smad7 forms a stable complex with activated TGF-β1 RI, thereby blocking the association and phosphorylation of Smad2 and Smad3 (11
). We show that all CD and most UC patients exhibit high levels of Smad7 compared with normal controls. Proteins extracted from both positively selected CD3+
T lymphocytes and non–CD3+
LPMCs from IBD patients showed enhanced Smad7 immunoreactivity on Western blotting in comparison to controls. To the best of our knowledge, this is the first study showing an abnormal expression of Smad7 in a human inflammatory disease. We were also able to show that inhibition of Smad7 protein with Smad7 antisense restored TGF-β1 signaling in LPMCs and enabled exogenous TGF-β1 to inhibit proinflammatory cytokine production; even in inflamed CD tissues, antisense to Smad7 restored TGF-β1 signaling and decreased proinflammatory cytokine production. Although we do not know the mechanisms operating in the tissue, this result nonetheless clearly indicates that Smad7 inhibition may be a novel therapeutic approach in IBD, enabling endogenous TGF-β to dampen local inflammation.
Consistent with our human data, it was recently reported that Smad7 transgenic mice exhibit defective T cell responses to TGF-β1, show markedly greater cytokine production in vitro, and show enhanced antigen-induced airway inflammation (29
). Together, these observations suggest that, despite enhanced TGF-β1 production, defective TGF-β1/Smad signaling contributes to maintaining the chronic inflammatory state in IBD.
The mechanisms/factors that regulate the expression of Smad7 are not fully known. Smad7 was first identified as a gene induced by shear stress in vascular endothelial cells (30
). Neither exposure to TNF-α nor to TGF-β1 altered Smad7 RNA expression in these cells. More recently, Bitzer et al. have shown that activation of NF-κB/RelA by TNF-α, IL-1β, and LPS, but not IFN-γ, increases transcription of the Smad7 gene and elevates intracellular levels of Smad7 protein in fibroblast cell lines (13
). In contrast, Ulloa et al. have demonstrated that activation of the transcription factor STAT1 by IFN-γ enhances Smad7 in human monocytic leukemia U937 cells (14
). It is noticeable that Smad7 is detectable in normal intestine; it will be of interest to determine which cells express this molecule and whether they are unresponsive to TGF-β1. In IBD mucosa, IFN-γ and proinflammatory cytokines are upregulated (15
), so it is likely that these molecules contribute to enhance Smad7 expression in the intestine of patients with IBD and provide a basis for the known antagonism between TGF-β1 and IFN-γ/TNF-α in the regulation of immune cell functions in the gut (18
). In preliminary experiments, however, we have not been able to reduce Smad 7 expression in IBD LPMCs by the addition to the cultures of IFNR-IgG and TNFR-IgG, although negative cytokine neutralization experiments should always be treated with caution. Nor do we believe that these preliminary results exclude a role for both IFN-γ and TNF-α in the regulation of Smad7 in IBD. Given that Smad7 expression is largely dependent on activation of STAT1 and NF-κβ and, in IBD, STAT1 and NF-κβ may be induced by several locally produced molecules, it is possible that blocking IFN-γ or TNF-α is not sufficient to completely inhibit STAT1 and NF-κβ activation and downregulate Smad7. Studies are now in progress to address this issue. Moreover, it is important to point out that regulation of Smad7 induction is a complex phenomenon dependent on the cell type. Therefore we are now carrying on experiments to examine the effect of various cytokines, including IFN-γ and TNF-α, on Smad7 expression in single cell populations isolated from the human intestine.