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. 2012 Nov;5(6):681-90.
doi: 10.1038/mi.2012.41. Epub 2012 Jun 13.

IFN-γ and TNF-α-induced GBP-1 inhibits epithelial cell proliferation through suppression of β-catenin/TCF signaling

C T Capaldo  1 , N Beeman, R S Hilgarth, P Nava, N A Louis, E Naschberger, M Stürzl, C A Parkos, A Nusrat
Affiliations

IFN-γ and TNF-α-induced GBP-1 inhibits epithelial cell proliferation through suppression of β-catenin/TCF signaling

C T Capaldo (V体育2025版) et al. Mucosal Immunol. 2012 Nov.

Abstract

Proinflammatory cytokines induce guanylate-binding protein 1 (GBP-1) protein expression in intestinal epithelial tissues. GBP-1 has been described as influencing a number of cellular processes important for epithelial homeostasis, including cell proliferation VSports手机版. However, many questions remain as to the role of GBP-1 in intestinal mucosal homeostasis. We therefore sought to investigate the function of proinflammatory cytokine-induced GBP-1 during intestinal epithelial cell proliferation. Through the use of complementary GBP-1 overexpression and small interfering RNA-mediated knockdown studies, we now show that GBP-1 acts to inhibit pro-mitogenic β-catenin/T cell factor (TCF) signaling. Interestingly, proinflammatory cytokine-induced GBP-1 was found to be a potent suppressor of β-catenin protein levels and β-catenin serine 552 phosphorylation. Neither glycogen synthase kinase 3β nor proteasomal inhibition alleviated GBP-1-mediated suppression of cell proliferation or β-catenin/TCF signaling, indicating a non-canonical mechanism of β-catenin inhibition. Together, these data show that cytokine-induced GBP-1 retards cell proliferation by forming a negative feedback loop that suppresses β-catenin/TCF signaling. .

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Figures

Figure 1
Figure 1
In vitro proinflammatory cytokine treatment of IECs increases GBP-1 expression. A: SKCO15 and T84 colonic epithelial cells were treated with TNF-α/IFN-γ for the times indicated and GBP-1 expression was examined via immunoblotting. B: SKCO15 cells were treated with TNF-β/IFN-γ for 24 hours and then immunostained for GBP-1 (red). Bar = 40μm. C: Frozen tissue sections were obtained from patients with ulcerative colitis or from non-IBD colonic tissues. Non-IBD tissue shows diffuse surface and crypt epithelium GBP-1 signal. Actively inflamed regions show strong staining of GBP-1 (green) in surface and crypt epithelium. Bar = 100μm.
Figure 2
Figure 2
GBP-1 expression attenuates IEC proliferation. A: SK-CO15 cells were treated with TNF-β/IFN-γ for the times indicated and cell proliferation was determined by EdU incorporation (1hr). n=3. + indicates p < 0.05, all samples versus untreated (0 hours) (n.s. not significant, ANOVA). Error bars = SE. B: SKCO15 cells, either treated with TNF-β/IFN-γ (48hrs) or non-treated controls (NT), were transfected with a GBP-1 targeted siRNA (GBP-1 siRNA) or non-silencing siRNA (control siRNA). Immunofluorescence analysis shows silencing of GBP-1 in SKCO15 cells after cytokine treatment. Proliferation was determined by EdU incorporation (green, scale bar = 50μm). C: SKCO15 cells were transiently transfected with a GBP-1 expression plasmid. Immunofluorescence analysis of IEC monolayers for EdU incorporation (green) and GBP-1 (red). ° indicates p < 0.005 versus total nuclei Error bars = SE, n=3.
Figure 3
Figure 3
GBP-1 suppresses β-catenin protein expression. A: SKCO15 cells were treated with TNF-β/IFN-γ for the times indicated. GBP-1 and β-catenin protein levels were assessed by western blot (representative of three independent experiments). B: Desitometric analysis of β-catenin protein levels during cytokine treatment. C: GBP-1 knockdown by siRNA in SKCO15 cells rescues TNF-β/IFN-γ induced loss of β-catenin protein levels compared with non-silencing siRNA controls. D: Overexpression of GBP-1 is sufficient to suppress β-catenin protein levels compared with controls (empty vector) (0.61+/−0.2, n=4. + p<0.05). E: Lentivirus transfected SKCO15 cells stably expressing GBP-1, GTPase deficient GBP-1 (ΔGTPase), or empty control vector were treated with MG132, an inhibitor of proteasome function, for 8 hours. Right panel, relative β-catenin protein levels were determined by densitometry and compared to controls (0.45+/−0.2 o/e GBP-1 vs controls, 0.44+/−0.2 GTPase deficient GBP-1 (ΔGTPase) vs controls n=6, p<0.05, ANOVA, other comparisons are non-significant). F: SKCO15 cells stably overexpressing wild-type GBP-1 (o/e GBP-1), GTPase deficient GBP-1 (ΔGTPase) or empty vector (control). *, long exposure highlights endogenous GBP-1 expression levels in control cells.
Figure 4
Figure 4
GBP-1 expression alters the subcellular localization of β-catenin. A: Immunofluorescence co-staining of β-catenin and GBP-1 in human ulcerative colitis samples (UC). Scale bar = 15μm. White box indicates magnified image area. Scale bare = 1.5μm. B: Subcellular fractionation and western blot analysis of control and GBP-1 stably overexpressing cells. C: SKCO15 cells transiently transfected with GBP-1 and analyzed for nuclear β-catenin levels by pixel intensity analysis (PI, *, p<0.01, control n=114, GBP-1, n=56).
Figure 5
Figure 5
GBP-1 acts to suppresses β-catenin/TCF signaling. A: SKCO15 cells were transiently transfected with a β-catenin/TCF responsive luciferase reporter construct (TOP) or a reporter in which the TCF responsive sites have been mutated (FOP). Cells were further treated with TNF-β/IFN-γ for the times indicated. + indicates p < 0.05 (ANOVA, all others not significant). n=3. Error bars =SE. B: SKCO15 cells were transiently co-transfected with a β-catenin/TCF responsive luciferase reporter construct and GBP-1 targeted siRNA (GBP-1 siRNA) or non-silencing siRNA (control siRNA). Luciferase activity was assessed 24hrs after transfection and TNF-β/IFN-γ treatment. ° indicates p < 0.005, n=3. C: SKCO15 cells were transduced with retrovirus and selected so as to stably express wild type GBP-1 (o/e GBP-1) or GTPase deficient GBP-1 (ΔGTPase), or were transduced with the same virus lacking GBP-1 (control). These cells were then transiently transfected with a β- catenin/TCF responsive luciferase reporter construct and treated with TNF-β/IFN-γ for 24 hours (values =TOP-FOP). + indicates p < 0.01 between control and each cytokine treated sample, ° indicates p < 0.005 GBP-1 and ΔGTPase GBP-1 overexpression versus untreated control cells, * indicates p<0.01 verses cytokine treated control cells. All other comparisons are non-significant (ANOVA, n=3). Immunoblot analyses show that GBP-1 levels in cells stably expressing GBP-1 proteins are similar to levels attained during cytokine treatment (right panel).
Figure 6
Figure 6
GBP-1 acts to suppresses β-catenin/TCF signaling. A: SKCO15 cells were transiently transfected with a β-catenin/TCF responsive luciferase reporter construct (TOP) or a reporter in which the TCF responsive sites have been mutated (FOP, values = TOP-FOP). Cells were further treated with TNF-β/IFN-γ for 48hrs or transiently transfected with full length GBP-1 or the GBP-1 helical domain where indicated. Protein expression was confirmed by western blot (right panel), * indicates p< 0.05 by ANOVA. B. SKCO15 cells were transiently co-transfected with a β-catenin/TCF responsive luciferase reporter construct and GBP-1 or GBP-5 (values = TOP-FOP). Protein expression was confirmed by western blot (right panel), * indicates p< 0.05 by ANOVA.
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