Enhanced sensitivity to DSS colitis caused by a hypomorphic Mbtps1 mutation disrupting the ATF6-driven unfolded protein response

Abstract

Here, we describe an N-ethyl-N-nitrosourea (ENU)-induced missense error in the membrane-bound transcription factor peptidase site 1 (S1P)-encoding gene (Mbtps1) that causes enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis. S1P cleaves and activates cAMP response element binding protein/ATF transcription factors, the sterol regulatory element-binding proteins (SREBPs), and other proteins of both endogenous and viral origin. Because S1P has a nonredundant function in the ATF6-dependent unfolded protein response (UPR), woodrat mice show diminished levels of major endoplasmic reticulum chaperones GRP78 (BiP) and GRP94 in the colon upon DSS administration. Experiments with bone marrow chimeric mice reveal a requirement for S1P in nonhematopoietic cells, without which a diminished UPR and colitis develop.

Fig. 1.

The hypopigmented woodrat mutant mice have a point mutation in Mbtps1. (A) The woodrat coat color phenotype at 4 months postpartum. (B) DNA sequence taken from exon 13 of Mbtps1, the gene coding for S1P, in a C57BL/6 WT control (Upper) and a woodrat homozygous mouse (Lower). The mutation is an A-to-G transition that results in a Y-to-C substitution at amino acid 496.

Fig. 2.

Increased susceptibility of woodrat mice to DSS-induced colitis. (A) Percent initial weight of WT and Mbtps1^wrt/wrt mice after administration of 2% DSS for 7 days. C57BL/6 mice receiving 0% DSS served as controls. Each group, n = 9–20. *, P < 0.05; **, P < 0.005, 2-tailed Mann–Whitney test for WT and Mbtps1^wrt/wrt. (B) C57BL/6 and Mbtps1^wrt/wrt were treated with antibiotics in drinking water 5 days before and during the administration of DSS (2%). Weight loss was determined daily, n = 5 each group. (C) Photograph of representative colons and cecums from untreated C57BL/6 mice (top), CD57BL/6 mice treated for 9 days with 2% DSS (middle), and Mbtps1^wrt/wrt mice treated for 9 days with 2% DSS (bottom). (D) Representative photomicrographs of colons from WT (C57BL/6) and Mbtps1^wrt/wrt mice at days 0 (Upper) and 7 (Lower) of DSS administration. (H&E; magnification: 100×.) (E) Concentrations of IL-1β and IL-6 in supernatants from distal colonic explants cultured for 24 h from WT (C57BL/6) and Mbtps1^wrt/wrt mice either before or after 7 days administration of 2% DSS, n = 6–7 each group. *, P < 0.05, Student's t test; n.s., not significant. (F) Plasma FITC-dextran concentrations in WT (C57BL/6) and Mbtps1^wrt/wrt mice 4 h following oral gavage (40 mg/100 g body weight) are shown. Mice with damaged epithelial barrier (C57BL/6 mice, receiving 9 days of 2% DSS) served as a positive control, n = 7–8 for WT and Mbtps1^wrt/wrt; n = 3 for C57BL/6 with 2% DSS; n.s., not significant.

Fig. 3.

Reduced ER stress response in the colon of woodrat mice after DSS administration. Protein extracts from the distal colon of WT (C57BL/6), Mbtps1^+/wrt, and Mbtps1^wrt/wrt mice before (A and B) and after (C and D) administration of 2% DSS in the drinking water for 3 days were analyzed by Western blotting (A and C) and quantitative image analysis (B and D) with GRP78- and GRP94-specific antibodies. β-actin was used as a loading control. n = 6–14 (B), n = 11–14 (D). *, P < 0.05; **, P < 0.005, Student's t test for WT and Mbtps1^wrt/wrt; n.s., not significant.

Fig. 4.

ER stress in nonhematopoietic cells correlates with the colitis phenotype. (A) Protein extracts from the distal colon of chimeric mice treated with 2% DSS for 7 days were analyzed by Western blotting with GRP78- and GRP94-specific antibodies. β-Actin was used as a loading control. Shown are representative extracts from each donor–recipient combination (n = 5). (B) Immunohistochemical detection of GRP78 (brown) in paraffin-embedded distal colonic sections in different BM chimeric mice. (C) Weight loss in BM chimeric mice was measured daily upon administration of 2% DSS in the drinking water. C57BL/6 mice receiving 0% DSS served as controls. n = 5 for WT→woodrat (wrt), woodrat→WT; WT→WT and for woodrat→woodrat. Statistical analyses of differences between groups at given days are shown in Table S6. (D) mRNA was extracted from colonic epithelial cells from WT (C57BL/6) and Mbtps1^wrt/wrt mice. GRP78 and GRP94 expression was examined by quantitative real-time PCR. Expression levels were normalized to β-actin, n = 9–10 each group. **, P < 0.005; ***, P < 0.0005, Student's t test. (E) Tunicamycin was injected i.p. into WT (C57BL/6) and Mbtps1^wrt/wrt mice, and survival was monitored for 96 h, n = 11 each group. ***, P < 0.0005, log-rank test. (F) Two representative colonic sections from the distal part of WT and Mbtps1^wrt/wrt mice (H&E-stained) are shown. (Magnification: B and F, 200×.)