Figure 1.
Decreased number of mature goblet cells in the large intestine of adult Oasis−/− mice.
(A) Peptide features of mouse OASIS and ATF6. (B) RT-PCR analysis of Oasis mRNA in various tissues from 8-week-old mice. Oasis mRNA was highly expressed in the large intestine. (C) Western blotting of OASIS in the large intestine from 8-week-old mice. Both full-length OASIS (full OASIS) and the N-terminal OASIS (N-OASIS) were detected in the large intestine. (D) HE (upper panels) and PAS (lower panels) staining of the large intestines from 8-week-old WT and Oasis−/− mice. In the Oasis−/− large intestine, the number of mature goblet cells containing large mucosal granules was markedly decreased compared with that in WT mice. (E and F) Quantification of (E) PAS-positive cells and (F) areas of PAS-positive cells in (D) (n = 9). Values represent the means ± s.d. ***P<0.001.
Figure 2.
Increased susceptibility to DSS-induced colitis in Oasis−/− mice.
(A) HE staining of the large intestines from 12-week-old WT and Oasis−/− mice. (B) Survival rates of WT and Oasis−/− mice that received 3.5% DSS for 10 days. Note that the mortality of Oasis−/− mice was observed at 2 days earlier than that of WT mice (n = 9). (C) Body weight changes of WT and Oasis−/− mice. After administration of DSS, Oasis−/− mice showed severe loss of body weight compared with that of WT mice (n = 9). (D) WT and Oasis−/− large intestines exposed to 3.5% DSS for 5 days. The large intestine of Oasis−/− mice exposed to 3.5% DSS was shortened and bleeding, and there was a reduction in the number of fecal pellets. (E) Quantification of colon length in (D) (n = 7). (F) HE (upper panels) and PAS (lower panels) staining of large intestines from WT and Oasis−/− mice exposed to 3.5% DSS for 5 days. Note that the Oasis−/− large intestine exhibited mucosal damage, degeneration of the mucosal epithelium, a decrease in the number of goblet cells, and an increase of crypt loss compared with those in WT large intestines. (G) Higher magnification of HE staining in (F). Arrowheads show inflammatory cells. The lamina propria of the Oasis−/− large intestine showed severe infiltration of inflammatory cells including macrophages and neutrophils. (H) Histological scores of colitis in WT and Oasis−/− mice that received 3.5% DSS for 5 days (n = 6). The scoring was carried out as described in the EXPERIMENTAL PROCEDURES. (I) RT-PCR analysis of inflammatory cytokines in WT and Oasis−/− large intestinal mucosa exposed to 3.5% DSS for 5 days (n = 5). The expression levels of inflammatory cytokines were higher in Oasis−/− mice than those in WT mice. Values represent the means ± s.d. *P<0.05; ***P<0.001.
Figure 3.
ER stress is accelerated in Oasis−/− mice with DSS-induced colitis.
(A) In situ hybridization of ER stress markers Bip (upper panels) and Chop (lower panels) in the large intestinal mucosa of WT and Oasis−/− mice exposed to 3.5% DSS for 5 days. In WT mice, both ER stress markers were mainly observed in the basal crypt. In contrast, these markers were expressed in both the basal and apical crypts of Oasis−/− mice. (B) The number of Bip- and Chop-positive cells per crypt in (A) (n = 5). The number of cells positive for each ER stress marker was increased by about 1.5-fold in the Oasis−/− large intestinal mucosa compared with that in WT mice. (C) RT-PCR analysis of ER stress markers Bip and Chop in the large intestine of WT and Oasis−/− mice exposed to 3.5% DSS for 5 days (n = 5). (D) Western blotting of cleaved caspase-12 and -3 in the large intestinal mucosa of WT and Oasis−/− mice exposed to 3.5% DSS for 5 days. (E) TUNEL staining of the large intestinal mucosa in WT and Oasis−/− mice exposed to 3.5% DSS for 5 days. (F) The number of TUNEL-positive cells per 10 crypts in (E) (n = 4). The number of TUNEL-positive cells was increased in the apical portion of the mucosa in Oasis−/− mice. Values represent the means ± s.d. *P<0.05.
Figure 4.
TUDCA alleviates DSS-induced colitis.
All the Oasis−/− mice received 3.5% DSS and some were given TUDCA (+TUDCA) and others were given the same volume of PBS (vehicle) daily by oral administration for 5 days. (A) HE (upper panels) and PAS (lower panels) staining of the large intestinal mucosa of Oasis−/− mice exposed to 3.5% DSS and TUDCA or the vehicle. (B) Higher magnification of HE staining in (A). Arrowheads show inflammatory cells. (C) Histological scores of control and TUDCA-treated Oasis−/− mice that received 3.5% DSS (n = 4). The pathological findings were markedly improved in Oasis−/− mice treated with TUDCA. (D) RT-PCR analysis of Bip and Chop in the large intestinal mucosa of Oasis−/− mice. The expression levels of these ER stress markers in the large intestinal mucosa of Oasis−/− mice were decreased by treatment with TUDCA. (E) Quantification of the expression levels of Bip and Chop in (D) (n = 4). (F) RT-PCR analysis of inflammatory cytokines in the large intestinal mucosa of Oasis−/− mice exposed to 3.5% DSS and TUDCA or the vehicle for 5 days (n = 4). Note that the expression levels of inflammatory cytokines were decreased in Oasis−/− mice that received TUDCA. (G) Western blotting of cleaved caspase-12 and -3 in Oasis−/− large intestinal mucosa exposed to 3.5% DSS and TUDCA or the vehicle for 5 days. (H) TUNEL staining of the large intestinal mucosa in Oasis−/− mice that received 3.5% DSS and TUDCA or the vehicle. (I) The number of TUNEL-positive cells in (H) (n = 4). The number of TUNEL-positive cells was decreased in Oasis−/− mice treated with TUDCA. Values represent the means ± s.d. *P<0.05; **P<0.01.
Figure 5.
Acceleration of inflammatory responses by ER stress.
(A and B) RT-PCR analysis of (A) Bip and (B) Chop in LS174T human colon carcinoma cells treated with ER stress inducer tunicamycin (Tm) and TUDCA for 24 h (n = 4). The expression levels of both Bip and Chop were downregulated by TUDCA. (C–E) RT-PCR analysis of (C) Tnfα, (D) IL-1, and (E) IL-6 in LS174T cells treated with Tm and TUDCA (n = 4). Note that the expression levels of these inflammatory cytokines were upregulated in LS174T cells treated with Tm, and downregulated by treatment with TUDCA. (F) Luciferase assay using LS174T cells transfected with the p-Luc reporter plasmid containing the NF-κB binding sequence. Relative activities were increased by treatment of LS174T cells with Tm, and decreased by treatment with TUDCA (n = 4). Values represent the means ± s.d. *P<0.05; **P<0.01; ***P<0.001.