Lgr4 gene deficiency increases susceptibility and severity of dextran sodium sulfate-induced inflammatory bowel disease in mice - PubMed (original) (raw)
. 2013 Mar 29;288(13):8794-803; discussion 8804.
doi: 10.1074/jbc.M112.436204. Epub 2013 Feb 7.
Yu Qian, Liang Li, Gaigai Wei, Yuting Guan, Hongjie Pan, Xin Guan, Long Zhang, Xiaoling Lu, Yongxiang Zhao, Mingyao Liu, Dali Li
Affiliations
- PMID: 23393138
- PMCID: PMC3610954
- DOI: 10.1074/jbc.M112.436204
Lgr4 gene deficiency increases susceptibility and severity of dextran sodium sulfate-induced inflammatory bowel disease in mice
Shijie Liu et al. J Biol Chem. 2013.
Abstract
Lgr4/Gpr48 is one of the newly identified R-spondins receptors and potentiates Wnt signaling, which regulates intestinal homeostasis. We used a hypomorphic mouse strain to determine the role of Lgr4 in intestinal inflammation and recovery. Intestinal inflammation was induced with dextran sulfate sodium (DSS) followed by a recovery period. Intestinal inflammation symptoms and molecular mechanisms were examined. We found that Lgr4(-/-) mice exhibited dramatically higher susceptibility to and mortality from DSS-induced inflammatory bowel disease than WT mice. Lgr4 deficiency resulted in greatly reduced numbers of either Paneth cells or stem cells in the intestine. During the intestinal regeneration process, cell proliferation but not apoptosis of intestinal epithelial cells was significantly impaired in Lgr4(-/-) mice. When Wnt/β-catenin signaling was reactivated by crossing with APC(min)(/+) mice or by treating with a GSK-3β inhibitor, the number of Paneth cells was partially restored and the mortality caused by DSS-induced inflammatory bowel disease was strikingly reduced in Lgr4-deficient animals. Thus, Lgr4 is critically involved in the maintenance of intestinal homeostasis and protection against inflammatory bowel disease through modulation of the Wnt/β-catenin signaling pathway.
Figures
FIGURE 1.
Deletion of Lgr4 increases susceptibility and mortality to DSS-induced inflammation. Mice were subjected to DSS-induced colitis for 5 days followed by regular drinking water. A–D, survival rate (A), body weight (B), hematocrit values of peripheral blood (C), and mean disease activity index (DAI) (D) were determined. WT, n = 10; Lgr4+/−, n = 6; _Lgr4_−/−, n = 12. Data are means ± S.E. *, p < 0.01; **, p < 0.001. Mice were euthanized, the entire intestine was obtained in both WT and Lgr4 mutant mice. The lengths of small intestines and colons were measured. E, the alteration of the length of both small intestines and colons was analyzed after DSS treatment when compared with regular drinking water control mice. SI, small intestine; CTR, control; DSS, 2.5% DSS in drinking water for 5 days followed by 3 days of regular drinking water. n ≥ 3 for each group. Data are means ± S.E.
FIGURE 2.
More severe intestine inflammation in DSS-induced _Lgr4_−/− mice. A and C, H&E staining revealed more severe disease activity in both colon (A) and jejunum (C) of _Lgr4_−/− mice on day 8. Note more infiltration of leukocytes in Lgr4 mutant mice. Scale bars: 50 μm. B, the histology score was measured. D, colon MPO activity as a measure of neutrophil infiltration. E, real-time PCR analysis revealed the relative expression of cytokines in colonic mucosa of WT and _Lgr4_−/− mice at day 5 of DSS administration. Data are means ± S.E. *, p < 0.01; **, p < 0.001. n ≥ 5. F, mice were reconstituted with donor bone marrow for 8 weeks, and then bowel inflammation was induced by 2.5% DSS (w/v) in drinking water for 5 days followed by normal drinking water; weight loss and survival rate were monitored every day. WT recipient mice (n = 6) and _Lgr4_−/− recipient mice (n = 5) were both studied. Data are means ± S.E.
FIGURE 3.
Impaired proliferation but not apoptosis of intestinal crypts in Lgr4 mutant mice during recovery. A and B, colon (A) and jejunum (B) sections from WT and _Lgr4_−/− mice on day 8 of DSS-induced colitis; proliferation was identified by Ki67 staining (arrowhead). C, the numbers of Ki67-positive cells were analyzed. SI, small intestine. 40–60 crypts per mouse were examined; n = 3 per group. Data are means ± S.E. *, p < 0.01; **, p < 0.001. D and E, cleaved caspase3-positive cells (arrowhead) were detected through IHC with specific antibodies in mice in normal conditions (D) or after treatment with DSS (E). Scale bars: 50 μm. F, statistic indicated the numbers of apoptosis cells in WT and _Lgr4_−/− mice. CTR, control; DSS, 5 days of treatment with DSS followed by 3 days recovery. Data are means ± S.E. ns, not significant.
FIGURE 4.
Lgr4 is required for differentiation of Paneth cells. A, whole-mount X-gal staining was performed on the ileum (left) and colon (right) of Lgr4+/− mice; note the granular particles observed in Paneth cells in the enlargement. B, Paneth cells were assessed by IHC staining with anti-lysozyme antibody. C, the number of Paneth cells in mice was analyzed by IHC with anti-lysozyme antibody. 40–60 crypts per mouse were examined; n = 5 per group. Data are means ± S.E. **, p < 0.001. D, quantitative real-time PCR analysis of the expression level of Paneth cell marker genes. Data are means ± S.E. E, ultrastructure of Paneth cells was explored with electron microscopy. Crypt base columnar cells are easily recognized (white asterisks), and Paneth cells are indicated by the white dashed borders.
FIGURE 5.
Lgr4 is required for maintenance of intestinal stem cells in adult mice. A and B, endogenous GFP (green) driven by Lgr5 promoter indicates crypt base columnar stem cells in duodenum (A) or colon (B) of Lgr5GFP/+;Lgr4+/+(left) and Lgr5GFP/+;Lgr4_−/− mice (right). C, FACS analysis demonstrated that the ratio of GFP-positive ISCs was significantly decreased in Lgr5GFP/+;Lgr4_−/− mice. Lgr5+/− percentage was 13.2 ± 1.62% when compared with Lgr5+/−_;Lgr4_−/− (0.61 ± 0.11%). D, duodenum sections from WT or _Lgr4_−/− mice were prepared. Bmi1+ ISCs were detected by IHCs with Bmi1-specific antibodies (arrowhead). E, statistical data indicating the number of Bmi1+ ISCs in WT and _Lgr4_−/− mice. 300 crypts per mouse from four different pairs of WT and _Lgr4_−/− mice were examined. Data are means ± S.E. *, p < 0.01. Scale bars: 50 μm
FIGURE 6.
Down-regulation of Wnt target genes in _Lgr4_−/− mice. A, nuclear β-catenin was observed at the bottom of crypts in WT mice (left) but not in those of _Lgr4_−/− mice (right). B and C, the Wnt/β-catenin targets, c-Myc and CylinD1, were dramatically down-regulated in _Lgr4_−/− mice as detected by IHC. D, mTOR downstream target, phospho-S6 (p-s6), was decreased in Lgr4 mutant mice. Scale bars: 50 μm. E, quantitative real-time PCR analysis of mRNA levels of Wnt target genes in WT and _Lgr4_−/− littermates. Data are means ± S.E.
FIGURE 7.
The phenotypes are partially restored in Wnt/β-catenin reactivated Lgr4 mutant mice. A, in Lgr4_−/−;APCmin_/+ double mutant mice, Wnt target genes were reactivated. The number of cells with active (nucleus-localized) β-catenin was increased in Lgr4_−/−;APCmin_/+ when compared with Lgr4_−/− mice. B, the expression of c-Myc was increased in crypts in Lgr4_−/−;APCmin/+ mice. C, IHC with antibody against lysozyme indicated the recovery of Paneth cells in the epithelium of Lgr4_−/−;APCmin_/+ mice. The right panel presents the microadenoma in small intestines. D, the number of Paneth cells in mice was analyzed by IHC with lysozyme-specific antibody. 40–60 crypts per mouse were examined; n = 3 per group. Data are means ± S.E. *, p < 0.01. E and F, _Lgr4_−/− mice received 2 mg/kg of SB216763 intraperitoneally every day for 5 days and were euthanized on day 8. Sections of the small intestine from mice of the indicated genotypes were stained with β-catenin antibody (E) or lysozyme (F). Scale bars: 50 μm.
FIGURE 8.
Inhibition of GSK-3β activity ameliorates experimental colitis in Lgr4 mutant mice. A, scheme of treatment with SB216763 in _Lgr4_−/− mice during DSS-induced colitis. B and C, _Lgr4_−/− mice were treated with SB216763 and then subjected to 1% DSS-induced colitis for 5 days. Survival rates (B) and body weight changes (C) were monitored. SB, SB216763. n = 6 per group. Data are means ± S.E. *, p < 0.01. D and E, on day 8 of DSS-induced colitis, intestinal sections from mice of WT, _Lgr4_−/−, and _Lgr4_−/− treated with SB216763 were stained with H&E (insets indicated the infiltration of leukocytes) (D), and the histological score was measured (E) (n = 3 per group). Data are means ± S.E. *, p < 0.01. F, IHC with antibodies of β-catenin and c-Myc indicates reactivation in SB216763-treated _Lgr4_−/− mice. Scale bars: 100 μm. G, colon MPO activity was measured for neutrophil infiltration. n = 3 per group. Data are means ± S.E. *, p < 0.01; **, p < 0.001.
References
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