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. 2013 Sep 23;210(10):2041-56.
doi: 10.1084/jem.20122341. Epub 2013 Sep 16.

ER stress transcription factor Xbp1 suppresses intestinal tumorigenesis and directs intestinal stem cells

Lukas Niederreiter  1 , Teresa M J Fritz, Timon E Adolph, Anna-Maria Krismer, Felix A Offner, Markus Tschurtschenthaler, Magdalena B Flak, Shuhei Hosomi, Michal F Tomczak, Nicole C Kaneider, Edina Sarcevic, Sarah L Kempster, Tim Raine, Daniela Esser, Philip Rosenstiel, Kenji Kohno, Takao Iwawaki, Herbert Tilg, Richard S Blumberg, Arthur Kaser
Affiliations

ER stress transcription factor Xbp1 suppresses intestinal tumorigenesis and directs intestinal stem cells

Lukas Niederreiter et al. J Exp Med. .

Abstract

Unresolved endoplasmic reticulum (ER) stress in the epithelium can provoke intestinal inflammation. Hypomorphic variants of ER stress response mediators, such as X-box-binding protein 1 (XBP1), confer genetic risk for inflammatory bowel disease. We report here that hypomorphic Xbp1 function instructs a multilayered regenerative response in the intestinal epithelium. This is characterized by intestinal stem cell (ISC) expansion as shown by an inositol-requiring enzyme 1α (Ire1α)-mediated increase in Lgr5(+) and Olfm4(+) ISCs and a Stat3-dependent increase in the proliferative output of transit-amplifying cells. These consequences of hypomorphic Xbp1 function are associated with an increased propensity to develop colitis-associated and spontaneous adenomatous polyposis coli (APC)-related tumors of the intestinal epithelium, which in the latter case is shown to be dependent on Ire1α. This study reveals an unexpected role for Xbp1 in suppressing tumor formation through restraint of a pathway that involves an Ire1α- and Stat3-mediated regenerative response of the epithelium as a consequence of ER stress. As such, Xbp1 in the intestinal epithelium not only regulates local inflammation but at the same time also determines the propensity of the epithelium to develop tumors.

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Figures

Figure 1.
Figure 1.
Xbp1 deletion increases ISC numbers. (A) Animals were injected with BrdU and sacrificed 24 h later. BrdU+ cells per total cells along the crypt–villus axis were counted (n = 3/4; two-tailed Student’s t test). (B) Anti-BrdU IHC of the ileum and colon 24 h after i.p. injection with BrdU (n = 3/4). (C) Similar experiment as A with a 2-h BrdU pulse to assess transit-amplifying cells (n = 4/4; two-tailed Student’s t test). (D) Anti-PCNA IHC of the small intestine (n = 5/5). (E) PCNA+ cells per total cells along the crypt–villus axis were counted (n = 5/5; two-tailed Student’s t test). (F) Olfm4+ ISCs (ISH; red arrowheads) in the small intestine of Xbp1+/+(IEC) and Xbp1−/−(IEC) mice. Lysozyme staining identified fully differentiated Paneth cells (black arrowheads) intermingled with ISCs (n = 3/4). (G) Quantification of Olfm4+ ISCs per 100 crypts (n = 3/4; two-tailed Student’s t test). (H) Sections of Xbp1+/+(IEC) and Xbp1−/−(IEC) mice from C were analyzed for BrdU+ cells at the crypt bottom up to position +4 (n = 4/4). (I) ISC identification by Lgr5 ISH in ileum and colon (Lgr5+ crypts marked by red arrowheads; n = 4/4). Bars: (B, D, and I) 20 µm; (F) 5 µm. (J and K) Analysis of isolated crypt mRNA of Xbp1+/+(IEC) and Xbp1−/−(IEC) mice for transcripts representative of the ISC signature (J; Muñoz et al., 2012) or Paneth cell signature (K; Sato et al., 2011) by RT-pPCR (n = 12/11; Student’s t test). Graphs show mean ± SEM. §, P = 0.0548; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2.
Figure 2.
ISC expansion is dependent on overactivation of Ire1α. (A) Immunoblot of total and phosphorylated Ire1α of indicated genotypes in total epithelial scrapings or after immunoprecipitation (IP) as indicated. Samples were immunoprecipitated with anti-Ire1α pAb H-190 (immunogen: amino acids 371–560) and immunoblotted with anti-Ire1α mAb 14C10 (immunogen: C-terminal fragment) or pAb H-190. Data are representative of four independent experiments. (B) Sections of the indicated genotypes were in situ hybridized for Olfm4 (red arrowheads), stained for lysozyme (black arrowheads), or both (n = 5 per group). Bars, 5 µm. (C) Quantification of Olfm4+ ISCs per 100 crypts (n = 5 per group with combined analysis of duodenum, jejunum, and ileum; one-way ANOVA with Bonferroni post-hoc test). (D) Quantification of Olfm4+ ISCs in Ern1+/+(IEC), Ern1+/−(IEC), and Ern1−/−(IEC) mice (n = 5 per group). (E) Western blot of Ern1+/+(IEC) and Ern1−/−(IEC) (exons 20–21 floxed) mice immunoblotted with mAb 14C10 and pAb H-190 detects a truncated Ire1α protein in Ern1−/−(IEC) epithelial scrapings. (F) Xbp1 mRNA splicing in epithelial scrapings of the indicated genotypes. 171 bp, Xbp1u; 145 bp, Xbp1s. (E and F) Data are representative of two independent experiments. (G) Animals were injected with BrdU and harvested 24 h later to assess epithelial turnover. BrdU+ cells per total cells along the crypt–villus axis were counted (n = 4 per group with combined analysis of duodenum, jejunum, and ileum; one-way ANOVA with Bonferroni post-hoc test). Graphs show mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3.
Figure 3.
Hypomorphic Xbp1 leads to Jak1/Stat3 activation in the intestinal epithelium. (A) Immunoblot for p-Stat3, Stat3, p-Jak1, Jak1, p-Jak2, and Jak2 on epithelial colonic scrapings. Data are representative of more than four independent experiments. (B) IHC localizes p-Stat3 immunoreactivity to IECs in the transit-amplifying zone and villus, but largely spares the crypt bottom (n = 3/3). Boxed areas are shown at higher magnification on the right. Bars, 20 µm. (C) Small intestinal epithelial scrapings from the indicated genotypes were analyzed for p-Stat3, Stat3, p-Jnk, and Jnk. The experiment was performed with four mice per group. (D) Mice were treated with the Stat3 inhibitor S3I-201 or vehicle for 14 d, and BrdU was administered 24 h before harvest. The ratio of BrdU+ cells per total IECs along the crypt–villus axis in the small intestine is presented (n = 4/5/7; one-way ANOVA with Bonferroni post-hoc test). (E) Olfm4+ ISCs counted in the same experiment as in D (n = 2/3/3; one-way ANOVA with Bonferroni post-hoc test). (F) MODE-K.iXbp1 and MODE-K.iCtrl cells were stimulated with the ER stress inducer tunicamycin and analyzed for Stat3 Tyr-705 phosphorylation. Data are representative of two independent experiments. (G) Il6 and Il11 mRNA expression in MODE-K.iXbp1 and MODE-K.iCtrl cells (n = 3; two-sided Student’s t test). Data are representative of two independent experiments. (H) Supernatants from MODE-K.iXbp1 and MODE-K.iCtrl cells were analyzed for IL-6 and IL-11 secretion by ELISA (n = 6; two-sided Student’s t test). (I) MODE-K.iXbp1 and MODE-K.iCtrl cells were incubated with anti–IL-11 and anti–IL-6 mAbs or respective isotype control antibodies, and Stat3 Tyr-705 phosphorylation was analyzed in cell lysates. Data are representative of three independent experiments. (J) MODE-K.iXbp1 and MODE-K.iCtrl cells were co-silenced with Jak1-, Jak2-, Jak3-, and Tyk2-specific or scrambled siRNAs, and Stat3 Tyr-705 phosphorylation was analyzed. Data are representative of four independent experiments. (K) IECs of Xbp1+/+(IEC) and Xbp1−/−(IEC) mice were analyzed for p–NF-κB p65 and total NF-κB p65. Data are representative of two independent experiments. (L) The NF-κB inhibitor BAY 11-7082 or vehicle was administered to Xbp1+/+(IEC) and Xbp1−/−(IEC) mice for 2 wk, and Stat3 Tyr-705 phosphorylation was analyzed in small IEC scrapings. Data are representative of three independent experiments. (A, C, F, and I–L) Gapdh was used as a loading control. (M) Mice were treated with the Jnk inhibitor SP600125 or vehicle for 14 d, and Olfm4+ ISCs were counted (n = 5/6/7; one-way ANOVA with Bonferroni post-hoc test). (N) The ratio of BrdU+ cells per total IECs along the crypt–villus axis in the small intestine 24 h after BrdU administration is analyzed in the same experiment as in M (n = 5/6/7; one-way ANOVA with Bonferroni post-hoc test). Graphs show mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 4.
Figure 4.
Xbp1 suppresses CAC. (A) Schematic overview of the AOM/DSS CAC model. (B) Representative H&E staining of colon of Xbp1+/+(IEC) and Xbp1−/−(IEC) at day 61 of AOM/DSS colitis. Individual tumors are highlighted by red dashed lines (one of three individual experiments; n = 4/3; total n = 13/10). (C and D) Tumor number (C) and area (D) in Xbp1+/+(IEC) and Xbp1−/−(IEC) at day 61 of AOM/DSS colitis (one of three individual experiments; n = 4/3; total n = 13/10; two-sided Student’s t test). (E, top) Atypical regenerative foci (white dashed lines) identified on H&E stainings on day 15 of AOM/DSS colitis. Number of lesions per colon is shown. (bottom) Staining for p53 was analyzed by IHC (arrowheads, p53+ nuclei; HPF, high power field; n = 4/4; two-sided Student’s t test). (F) ROS in colonic epithelium before (n = 6/7; two-sided Student’s t test) and on day 15 of AOM/DSS (n = 5/5; two-sided Student’s t test). (G) Detection of aneuploidy by analysis of DNA content with propidium iodide in isolated colonic IECs (n = 3/3; one-sided Student’s t test; M1 = G0/G1; M2 = G2/S; M3 = aneuploid). (H) IHC on day 15 of the AOM/DSS model localizes increased p-Stat3 immunoreactivity to IECs (n = 4/4). Bars: (B) 2 mm; (E [top] and H) 50 µm; (E, bottom) 20 µm. (I) Western blot for p-Stat3, Stat3, p-Jak1, and Jak1 on colonic IEC scrapings on day 15 of the AOM/DSS model. Gapdh was used as a loading control. Data are representative of two independent experiments. Graphs show mean ± SEM. §, P = 0.1024; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 5.
Figure 5.
Epithelial Xbp1 suppresses tumor burden in Apcmin mice. (A) Representative macroscopic pictures of the colon with rectal tumors in the indicated genotypes analyzed at age 15 wk (n = 13/10). Boxed areas are shown at higher magnification on the right. (B) Representative H&E-stained sections, with tumors highlighted by dotted lines (n = 13/10). (C) Quantification of tumor numbers per mouse along the intestinal tract (n = 13/10; two-sided Student’s t test). (D) Peripheral blood count of the indicated genotypes at age 15 wk (n = 13/10; two-sided Student’s t test). (E) Ileal and colonic tumor counts in the indicated genotypes stratified by size of tumors (n = 13/10; two-sided Student’s t test). (F) Tumors from colons of Xbp1+/+(IEC);Apcmin and Xbp1−/−(IEC);Apcmin mice were microdissected, and mRNA expression of the indicated targets was analyzed by qPCR (n = 5/5; two-tailed Student’s t test). (G) Olfm4+ ISCs (red arrowheads; ISH) and lysozyme+ Paneth cells (black arrowheads; IHC) in the indicated genotypes (n = 4/4). Bars: (A) 5 mm; (B) 100 µm; (G) 5 µm. (H) Number of Olfm4+ ISCs per 100 crypts in Xbp1+/+(IEC);Apcmin and Xbp1−/−(IEC);Apcmin small intestines. The occasional presence of crypts with lysozyme+ mature Paneth cells among the vast majority of crypts with lysozyme− Paneth cell remnants in Xbp1−/−(IEC);Apcmin mice allowed crypt-specific stratification of Olfm4+ cell enumeration in lysozyme+ and lysozyme− crypts (n = 4/4; two-sided Student’s t test). Graphs show mean ± SEM. §, P = 0.0519; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 6.
Figure 6.
Increased tumor formation in Xbp1−/−(IEC);Apcmin mice is dependent on Ire1α. (A) Representative macroscopic pictures of colonic tumors of the indicated genotypes analyzed at age 15 wk (n = 14/10). Boxed areas are shown at higher magnification on the right. Bars, 5 mm. (B) Enumeration of tumors in the indicated genotypes at 15 wk of age. Mean tumor numbers ± SEM along the intestinal tract are shown (n = 14/10; two-sided Student’s t test). (C and D) Ileal (C) and colonic (D) tumor counts stratified by tumor size (n = 14/10; two-sided Student’s t test). Graphs show mean ± SEM.
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