PMC

ER stress in goblet cells of IRE1β^−/− mice. (A) The ratio of spliced to unspliced XBP1 mRNA is elevated in IRE1β^−/− colon but decreased in IRE1α^−/− colon. RNAs were extracted from two individual colons of wild-type (IRE1β^+/+, IRE1α^+/+), IRE1β^−/−, IRE1α^+/−, or IRE1α^−/− mice. After RT-PCR, the intensity of each band was measured, and the ratio (s/u) of spliced to unspliced XBP1 mRNA was calculated. (B) Increase of BiP in IRE1β^−/− colons. Western blotting was performed with anti-BiP antibody for colon lysates from wild-type (IRE1β^+/+, IRE1α^+/+), IRE1β^−/−, IRE1α^+/−, or IRE1α^−/− mice. Signal intensity was normalized using β-actin, and fold induction relative to the level for the IRE1β^+/+ sample was calculated. (C) Increase of BiP in IRE1β^−/− goblet cells. Cryosections of IRE1β^+/+ or IRE1β^−/− colons were stained with anti-BiP (green) antibody and DAPI (blue). Double-headed arrow indicates the region where BiP-induced goblet cells were prominent. The “+ DIC” image depicts superimposed immunofluorescence and DIC images.

Distribution and stability of MUC2 mRNA in colon. (A) MUC2 mRNA distribution in IRE1β^+/+ and IRE1β^−/− colons. Colon sections (10 μm in thickness) were fixed and then hybridized with a digoxigenin-labeled MUC2 cRNA probe (SI Materials and Methods). MUC2 mRNA appears as dark staining. (B) Stability of MUC2 mRNA in IRE1β^+/+ and IRE1β^−/− colons. Cells collected from IRE1β^+/+ (wild-type) or IRE1β^−/− (knockout) mouse colons were incubated in the medium containing α-amanitin for the indicated times. RNAs were then extracted from the cells and analyzed by quantitative RT-PCR. Relative RNA decay is expressed as MUC2 mRNA/HPRT1 mRNA ratio, and the values at time 0 were set to 1. Data presented are the averages of six independent experiments, with SD indicated by error bars.

The ER is distended only in goblet cells of IRE1α^+/+β^−/− mice (this is the same mouse line shown as IRE1β^−/−, but here we describe IRE1β^−/− as IRE1α^+/+β^−/− to be clearly understandable). Colons from IRE1α^+/+β^+/+ (A, C, and D), IRE1α^+/+β^−/− (B, E, and F), and IRE1α^−/−β^+/+ (G and H) mice were fixed, stained, and observed by electron microscope as described in SI Materials and Methods. High-magnification images revealed that the ER of IRE1α^+/+β^−/− in early-stage goblet cells (E and F) was distended, whereas the ER of IRE1α^+/+β^+/+ (C and D) and IRE1α^−/−β^+/+ (H) goblet cells in the same stage showed normal structure. A series of low-magnification images depicts an entire (A and B) or a part (G) of crypt, and the double-headed arrow indicates the region where ER-distended goblet cells existed. The black and white arrows in A and C and B and E indicate mucin granule and ER, respectively.

IRE1β is expressed and localized in the ER of goblet cells. (A) After deglycosylation, cryosections of mouse colons were stained with the antibody raised against the cytosolic region of IRE1β and Cy3-conjugated anti-guinea pig IgG as the secondary antibody. MUC2 was stained with anti-MUC2 (R-12) and FITC-conjugated anti-goat IgG. MUC2-positive cells (goblet cells) were also stained with anti-IRE1β antibody. DNA was stained with DAPI. Staining in smooth muscle was judged to be nonspecific, because IRE1β^−/− and IRE1β^+/+ smooth muscle both exhibited the same staining pattern. Differential interference contrast (DIC) images are shown, far right. L, intestinal lumen; SM, smooth muscle. (B) Ultrathin sections of mouse colon were treated with anti-IRE1β and gold particle-labeled anti-guinea pig IgG. Gold particles are seen only on the outside of the ER because this antibody was raised against the cytosolic region of IRE1β. Less (Upper) and more magnified images (Lower) are shown. MG, mucin granule; N, nucleus; NM, nuclear membrane.

Aberrant mucin accumulates in the ER in goblet cells of IRE1β^−/− mice. (A) SBA-binding high-molecular-weight aggregates accumulate in IRE1β^−/− colon. Lysates of mucosal epithelia were electroblotted after 8% SDS/PAGE and treated with HRP-conjugated SBA. (B) The high-molecular-weight aggregates contain MUC2. Lysates as in A were electrophoresed, and the indicated region of the stacking gel containing the IRE1β^−/− lysate was cut out for mass spectrometry (LC/MS/MS) analysis. Five peptides identical to sequences in mouse MUC2 were detected. (C) Locations of the five identified peptides in the primary structure of mouse MUC2. Peptides identified by mass spectrometry are indicated as red lines, with numbers corresponding to those in B. Note that the fifth peptide maps to within the 76-kDa C-terminal region that is cleaved in the Golgi apparatus. The epitope of anti-MUC2 antibody H-300 (green line) is also shown. (D) MUC2, with its uncleaved C-terminal region, accumulates in IRE1β^−/− colon. Western blotting with anti-MUC2 antibody H-300 was performed after nonreducing 2–15% gradient SDS/PAGE. High-molecular-weight protein stained intensely in IRE1β^−/− colon lysate. (E) Immunofluorescence staining of MUC2 in mouse colons. Cryosections were stained with chicken anti-MUC2, goat anti-CRT antibodies, and with DAPI. IRE1β^−/− goblet cells show colocalization of MUC2 and calreticulin (CRT) in the region indicated by the double-headed arrow.