Blocking Smad7 restores TGF-β1 signaling in chronic inflammatory bowel disease (original) (raw)
Smad7 in active lesions of patients with IBD. The TGF-β1/Smad signaling system is notable for an autoinhibitory loop that involves Smad7 (11, 12). Total proteins extracted from intestinal mucosal samples were first analyzed by Western blotting for Smad7. Smad7 protein expression was increased in all patients with active CD and most UC patients compared with normal controls (Figure 1a). Elevated levels of Smad7 were seen in IBD regardless of whether proteins were extracted from surgically derived mucosal tissues or endoscopic biopsies with the same degree of inflammation. Patients with active CD had a median of 7.17 densitometry arbitrary units (range, 3.9–16.08) and patients with UC a median of 4.985 (range, 1.9–8.22). Both CD and UC patients exhibited levels of Smad7 significantly higher than those found in normal controls (median, 1.06; range, 0.5–2.5) (P < 0.0003 and 0.0006, respectively) (Figure 1a, middle panel). Increased expression of Smad7 was also seen in the mucosal samples of patients with indeterminate colitis and pouchitis (data not shown).
Smad 7 in IBD. (a) Representative expression of Smad7 (upper blot) and β-actin (lower blot) protein in colonic mucosal samples taken from three normal controls, three patients with active CD, and three with UC. The example is representative of five separate experiments analyzing in total mucosal samples from eight patients with CD, eight with UC, and eight normal controls. Quantitative data are shown in the middle panel as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units (a.u.). Each point represents the value (a.u.) of Smad7 in mucosal samples taken from a single subject. Horizontal bars indicate the median value. Shown in the bottom panel is expression of Smad7 protein in both CD3-depleted LPMCs and positively selected CD3+ T lymphocytes (T-LPL) from intestinal mucosal samples taken from three patients with CD, three patients with UC, and three normal controls. The example is representative of three separate experiments analyzing Smad7 in both CD3-depleted LPMCs and CD3+ T-LPLs from five patients with CD, four with UC, and six normal controls. (b) Expression of Smad7 (upper blot) and β-actin (lower blot) protein in CD: effect of disease activity. Colonic mucosal biopsy specimens were taken from two normal controls and from inflamed (I) and uninflamed (U) areas of three patients with CD. Arrows indicate Smad7 and β-actin detected by specific polyclonal antibodies. The example is representative of two separate experiments using in total samples from four controls and five CD patients.
Five patients with CD and three with UC had paired biopsy specimens taken from endoscopically abnormal (gross lesions) and endoscopically normal areas. Smad7 was expressed at high levels in all areas of endoscopically abnormal mucosa compared with endoscopically normal mucosa (Figure 1b). The expression of Smad7 in normal mucosal areas of patients with IBD was not different from that seen in normal controls.
In IBD tissue, both positively selected CD3+ T lymphocytes and CD3-depleted LPMCs contained high levels of Smad7 compared with those of controls (Figure 1a, bottom panel).
Downregulation of phosphorylated Smad3 in active lesions of patients with IBD. Phosphorylation of Smad3 by the activated TGF-β1 RI is an important step in the initiation of TGF-β1 signal transduction (5, 6, 10). We therefore analyzed Smad3 phosphorylation in the intestinal mucosa of patients with IBD. Total proteins extracted from intestinal mucosal samples were immunoprecipitated with anti-Smad3 and subsequently incubated with a phosphoserine antibody. In mucosal samples from normal controls, abundant phosphorylated Smad3 was seen. In contrast, Smad3 phosphorylation was markedly reduced in mucosal samples from CD and, to a lesser degree, UC patients (Figure 2a). p-Smad3 was also detected in mucosal samples from patients with diverticular disease (data not shown).
(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).
To demonstrate that the phosphoserine antibody specifically recognizes phosphorylated residues, total proteins extracted from normal controls and immunoprecipitated with Smad3 antibody were incubated with or without PP-2A. Treatment with PP-2A abolished the p-Smad3 signal with no change in the total Smad3 protein content (Figure 2a, bottom left panel).
Analysis of active (phosphorylated)/inactive Smad3 ratios showed a significantly decrease in patients with active CD and UC compared with controls (P < 0.0001 and P < 0.0032, respectively) (Figure 2a, bottom right panel). Once activated, Smad3 associates with Smad4 and translocate to the nucleus (5, 6). To investigate whether downregulation of active Smad3 was associated with a reduced expression of Smad3-bound Smad4, proteins were immunoprecipitated with anti-Smad3 and then probed with a Smad4 antibody. Mucosal samples taken from active IBD exhibited a reduced expression of Smad3-bound Smad4 in comparison to that observed in normal controls (Figure 2a). There was a clear inverse relationship in individual samples between the expression of Smad7 and the amount of p-SMAD3 (Figure 2b).
To further analyze TGF-β1/Smad signaling in IBD tissue, LPMCs extracted from normal or IBD patients were stimulated with TGF-β1, and both total and phosphorylated Smad3 analyzed by Western blotting. p-Smad3 signal was detected in unstimulated normal LPMCs and enhanced by TGF-β1 stimulation (Figure 3). In contrast, p-Smad3 immunoreactivity was barely detectable in unstimulated LPMCs from both CD and UC and not increased by TGF-β1 stimulation (Figure 3).
Stimulation of CD and UC LPMCs with TGF-β1 does not result in enhanced p-Smad3. LPMCs isolated from the colon of one normal control, one CD patient, or one UC patient were cultured in the presence or absence of TGF-β1 (1 ng/ml) for 1 hour. Phosphorylated (top blot, p-Smad3) and total (bottom blot) Smad3 were analyzed in proteins extracted from LPMCs as indicated in Figure 2. The example is representative of four separate experiments, in each of which, LPMCs from one normal control, one patient with CD, and one patient with UC were analyzed. In all cases, LPMCs from normal patients showed endogenous p-Smad3 that was enhanced by TGF-β1, whereas in all IBD patients, endogenous p-Smad3 was barely detectable after TGF-β1 stimulation. The bottom panel shows quantitative analysis of active/inactive Smad3 ratio in TGF-β1–stimulated LPMCs from four controls, four patients with CD, and four 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 LPMC samples taken from a single subject. Horizontal bars indicate the mean.
Inhibition of Smad7 enables cells to respond to TGF-β1 and downregulates proinflammatory cytokines in IBD. To examine whether Smad7 is directly responsible for inhibiting TGF-β1/Smad3 signaling in IBD, we analyzed the IBD LPMC response to TGF-β1 after treating the cells with specific Smad7 antisense or sense oligonucleotides. Treatment of both CD and UC LPMCs with the Smad7 antisense but not with the sense oligonucleotide inhibited Smad7 expression (Figure 4a), and this was associated with the restoration of Smad3 phosphorylation in response to TGF-β1 (Figure 4b). Densitometric analysis of the Western blots in five CD patients and five UC patients showed clearly that the antisense markedly reduced Smad7 protein and also markedly increased p-Smad3 in the same cells. Taken together, these observations indicate that in both CD and UC intestinal mucosa, overexpression of Smad7 is associated with defective TGF-β1 signaling.
Antisense to Smad7 restores TGF-β1 signaling in both CD and UC LPMCs. (a) Treatment of CD and UC LPMCs with a specific Smad7 antisense but not a sense oligonucleotide inhibits Smad7 expression. CD and UC LPMCs were cultured in the absence (U, unstimulated) or presence of a specific Smad7 antisense (AS) or control sense (S) oligonucleotide for 24 hours. Arrows indicate Smad7 detected by a specific polyclonal antibody. The example is representative of three separate experiments analyzing in total LPMCs from five CD or five UC patients. Quantitative data are shown in the right panel as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units (a.u.). Each point represents the value (a.u.) of Smad7 in LPMCs taken from a single subject. Horizontal bars indicate the median value. (b) Inhibition of Smad7 restores the TGF-β1–induced Smad3 phosphorylation in both CD and UC LPMCs. LPMCs were cultured in the absence (U, unstimulated) or presence of a specific Smad7 antisense (AS) or control sense (S) oligonucleotide, and then stimulated with 1 ng/ml TGF-β1 for 1 hour. The right panel shows quantitative analysis of active/inactive Smad3 ratio in LPMC from five patients with CD and five 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 LPMCs taken from a single subject. Horizontal bars indicate the mean.
There is a large body of evidence that intestinal inflammation is in part dependent on the balance between inflammatory cytokines, especially IFN-γ, and TGF-β1 activity (18–22). Therefore, we examined whether restoring TGF-β1 signaling by Smad7 antisense resulted in an inhibition of inflammatory cytokines. Given that in preliminary experiments we determined that IFN-γ was produced at high levels in CD but not UC in comparison to controls, we focused on CD samples. CD LPMCs pretreated with medium alone or Smad7 antisense or sense oligonucleotides were stimulated with SEB in the presence or absence of graded doses of TGF-β1. LPMCs pretreated with or without oligonucleotides and then cultured in medium alone showed only low numbers of transcripts for both IFN-γ (4,293 ± 839/μg RNA in LPMCs pretreated with medium; 5,748 ± 958/μg RNA in LPMCs pretreated with sense; and 1,239 ± 543/μg RNA in LPMCs pretreated with antisense) and TNF-α (3,939 ± 1,245/μg RNA in LPMCs pretreated with medium; 4,983 ± 981/μg RNA in LPMCs pretreated with sense; and 2,019 ± 765/μg RNA in LPMCs pretreated with antisense). Stimulation of LPMCs with SEB resulted in a large increase in the number of transcripts for both IFN-γ and TNF-α (P < 0.0001), and there was no effect of the oligonucleotides on this response (Figure 5). The addition of graded doses of TGF-β1 to CD LPMCs precultured in medium alone or with sense oligonucleotides had only a marginal effect on TNF-α and IFN-γ transcripts, although there was modest inhibition at the highest dose used (Figure 5; P ≤ 0.05) (Table 1). In contrast, CD LPMCs pretreated with the Smad7 antisense oligonucleotide showed markedly reduced TNF-α and IFN-γ transcripts at all doses of TGF-β1 tested (Figure 5; P ≤ 0.002) (Table 1). The ability of TGF-β1 to inhibit cytokine production of LPMCs from three normal controls was also studied (Figure 5, insets). Unstimulated LPMCs cultured in medium alone or with the sense or antisense oligonucleotides showed less than 1,000 TNF-α and IFN-γ transcripts per microgram of RNA. After SEB stimulation in the presence of oligonucleotides, there was a 10- to 12-fold increase in the number of cytokine transcripts. This was inhibited in a dose-dependent fashion by TGF-β1, regardless of whether the cells had been pretreated with sense or antisense oligonucleotides.
Antisense to Smad7 enables TGF-β1 to inhibit CD LPMC proinflammatory cytokine production. CD LPMCs were cultured in the absence or presence of a specific Smad7 antisense or sense oligonucleotide for 24 hours. The cells were washed with PBS, resuspended in RPMI 1640 supplemented with HL-1 and cultured with or without SEB in the presence or absence of graded doses of TGF-β1 for 12 hours. In CD LPMCs pretreated with the Smad7 antisense oligonucleotide, as little as 0.1 ng/ml of TGF-β1 significantly decreased transcripts for both IFN-γ and TNF-α (P < 0.002). In CD LPMCs precultured with medium or sense Smad7 oligonucleotide, only a modest decrease in IFN-γ and TNF-α transcripts was seen with 10 ng/ml TGF-β1 (P ≤ 0.05). Data are expressed as mean ± SEM of four separate experiments, in each of which, LPMCs from one CD patient were used. The inset shows that, as expected, in LPMCs from normal controls, TGF-β1 dose-dependently inhibited SEB-stimulated cytokine transcripts, regardless of whether LPMCs were pretreated or not with oligonucleotides. Data are expressed as mean ± SEM of three separate experiments, in each of which, LPMCs from one normal control were used.
Percentage of inhibition of SEB-stimulated IFN-γ and TNF-α RNA transcripts in CD LPMCs by TGF-β1A
We next used ex vivo organ culture of inflamed mucosal samples from CD patients to determine whether antagonizing Smad7 also reduced proinflammatory cytokines in tissue. After 40 hours of culture in medium or with the sense oligonucleotide, Smad7 was readily detectable; p-Smad3 was present but barely detectable; and TNF-α and IFN-γ proteins were clearly present in the tissue (Figure 6a). Smad7 antisense reduced Smad7 protein and also resulted in increased p-Smad3; importantly, it also reduced TNF-α and IFN-γ in the tissues (Figure 6a). To confirm these results, we analyzed IFN-γ protein in the organ culture supernatants. After 40 hours in medium alone, the amount of IFN-γ in the supernatants was 439 ± 221 pg/mg tissue. With the sense oligonucleotide, there was no decrease (421 ± 181.2 pg/mg tissue). However with antisense to Smad7, IFN-γ concentrations decreased 60% and 80% in all samples to a mean of 108 ± 38.7 pg/mg tissue (P < 0.02), also showing that the cytokine Western blotting did reflect cytokine concentrations in the supernatants. Finally, to examine whether the downregulation of cytokines in ex vivo organ culture of inflamed mucosal samples from CD treated with the Smad7 antisense was due to the endogenous TGF-β1, Smad7 antisense was added to the organ cultures with or without a neutralizing TGF-β1 antibody. Anti–TGF-β1 had no effect on the decreased expression of Smad 7 protein induced by the antisense oligonucleotide (Figure 6b). However in the presence of the antibody, there was clearly less endogenous p-Smad3 in the tissue, and cytokine expression remained elevated.
(a) Inhibition of Smad7 protein by a specific Smad7 antisense oligonucleotide in CD mucosal tissue results in increased p-Smad3 and decreased tissue TNF-α and IFN-γ. The example is representative of four separate experiments, in each of which inflamed mucosal tissue from one CD patient was used. Identical results were seen in each patient. (b) Addition of a neutralizing TGF-β1 antibody decreases but does not abrogate the Smad7 antisense effect on both p-Smad3 and cytokine expression. The example is representative of three separate experiments, in each of which, inflamed mucosal tissue from one CD patient was used. Identical results were seen in each patient.