Positive role of CCAAT/enhancer-binding protein homologous protein, a transcription factor involved in the endoplasmic reticulum stress response in the development of colitis

Abstract

Although recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1alpha, and Caspase-11 (a positive regulator of interleukin-1beta production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1alpha is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1alpha, interleukin-1beta production via the induction of Caspase-11, and intestinal mucosal cell apoptosis.

Figure 1

Development of DSS-induced colitis in CHOP-null mice and WT mice. CHOP-null mice (Chop^−/−) and WT mice were treated with or without 3% DSS for 7 days. Body weight (A) and DAI (B) were measured daily. After 7 days, length of colon (C), colonic MPO activity (D), and colonic TBARS (E) were determined as described in the Materials and Methods. G and H: After 7 days, sections of colonic tissues were prepared and subjected to histological examination (H&E staining) and the damage score and extent of lesion for seven independent sections were determined. F: One of the sections is shown. Values are mean ± SEM (n = 5 to 13). **P < 0.01; *P < 0.05; n.s., not significant.

Figure 2

Expression of CHOP and GRP78 in colonic tissues of CHOP-null mice and WT mice. DSS was administered to CHOP-null mice (Chop^−/−) and WT mice, as described in the legend of Figure 1. Colonic tissues were removed and total RNA was extracted. Samples were subjected to real-time RT-PCR, using a specific primer set for Chop or Grp78. Values were normalized to Gapdh, expressed relative to the control sample (ie, WT mice without DSS treatment), and given as the mean ± SEM (n = 4). A: ** or ^##P < 0.01; *P < 0.05; n.s., not significant. Sections of colonic tissues were prepared and subjected to immunohistochemical analysis with an antibody against CHOP (B) or GRP78 (C). B: The right panel in each column is a ×4 magnified image of the boxed area defined in the left panel. D: Total protein was extracted from colonic tissues and analyzed by immunoblotting with an antibody against CHOP, GRP78, or actin.

Figure 3

Development of TNBS-induced colitis in CHOP-null mice and WT mice. CHOP-null mice (Chop^−/−) and WT mice were intrarectally administered with (TNBS) or without (EtOH) TNBS of 3 mg/mouse (A, C–E) or 8 mg/mouse (B) once and cultivated for the indicated days. Body weight (A) and mouse survival rate (B) were measured daily. After 1 day, sections of colonic tissues were prepared and subjected to histological examination (H&E staining) as described in the legend of Figure 1, C–E. Values are mean ± SEM (n = 6 to 12). **P < 0.01; n.s., not significant.

Figure 4

The mRNA expression of various genes in colonic tissues. DSS was administered to CHOP-null mice (Chop^−/−) and WT mice, as described in the legend of Figure 1. Relative mRNA expression of each gene in colonic tissues was monitored and expressed as described in the legend of Figure 2A. A: IL-1β, TNF-α, IL-6. B: Madcam-1, Vcam-1, Icam-1. C: CD11b, CD49d, L-selectin. D: IL-10. E: Capsase-11. F: Ero-1α, Bcl-2. Values are mean ± SEM (n = 4 to 5). ** or ^##P < 0.01; * or ^#P < 0.05; n.s., not significant.

Figure 5

The mRNA expression of various genes in peritoneal macrophages. Peritoneal macrophages were prepared from CHOP-null mice (Chop^−/−) and WT mice and incubated with 10 ng/ml of LPS for 12 hours (3 hours for the Tnf-α). Relative mRNA expression of each gene in colonic tissues was monitored and expressed as described in the legend of Figure 2A. A:Chop, Grp78. B: Tnf-α. C:CD11b, D: IL-10. E:Caspase-11. F:Ero-1α. Values are mean ± SEM (n = 3). ** or ^##P < 0.01; * or ^#P < 0.05; n.s., not significant.

Figure 6

Involvement of Caspase-11-Caspase-1-IL-1β pathway in CHOP-dependent exacerbation of DSS-induced colitis. DSS was administered to CHOP-null mice (Chop^−/−) and wild-type mice (WT), as described in the legend of Figure 1, A and B. C and D: Peritoneal macrophages were prepared from CHOP-null mice (Chop^−/−) and WT mice and incubated with 10 ng/ml of LPS for 24 hours. A and C: The amount of IL-1β was determined by enzyme-linked immunosorbent assay. B and D: The Caspase-1 activity was measured as described in Materials and Methods. Values are mean ± SEM (n = 3 to 4). **P < 0.01; *P < 0.05; n.s., not significant.

Figure 7

Involvement of infiltration of macrophages in CHOP-dependent exacerbation of DSS-induced colitis. DSS was administered to CHOP-null mice (Chop^−/−) and WT mice, as described in the legend of Figure 1. A: Sections of colonic tissues were prepared and subjected to immunohistochemical analysis with an antibody against CD68. The bottom panel in each column is a ×4 magnified image of the boxed area defined in the middle panel. RAW264 cells were transiently transfected with expression plasmid for CHOP and C/EBP-β and cultured for 12 hours. The relative mRNA expression of each gene was monitored and expressed as described in the legend of Figure 2A. B–D: Values are mean ± SEM (n = 3). **P < 0.01; *P < 0.05; n.s. not significant. E: The structure and sequences of the CD11b promoter are shown.

Figure 8

Involvement of ROS production in CHOP-dependent exacerbation of DSS-induced colitis. DSS was administered to CHOP-null mice (Chop^−/−) and wild-type mice (WT), as described in the legend of Figure 1. A: After administration of POBN, the colons were dissected and subjected to radical adduct ESR spectrum analysis. The intensity of the ESR signal of radical adduct (shown by bars) was determined, expressed relative to the control sample (ie, wild-type mice without DSS treatment), and given as the mean ± SEM (n = 3). B: *P < 0.05; n.s., not significant. C: Peritoneal macrophages were prepared from CHOP-null mice (Chop^−/−) and WT mice and incubated with 10 ng/ml of LPS for 24 hours. D–H: RAW264 cells were transiently transfected with siRNA for CHOP (D–F) or ERO-1α (G, H) or nonsilencing (ns) siRNA (D–H) and incubated with 50 ng/ml of LPS for 24 hours. The production of ROS was monitored by fluorescence-activated cell sorting analysis with H₂DCF, as described in the Materials and Methods (C, D, and G). Relative mRNA expression of the CHOP (E) or Ero-1α (F, H) was monitored and expressed as described in the legend of Figure 2A. Values are mean ± SEM (n = 3). ** or ^##P < 0.01.

Figure 9

Involvement of ROS-induced apoptosis in CHOP-dependent exacerbation of DSS-induced colitis. DSS was administered to CHOP-null mice (Chop^−/−) and WT mice, as described in the legend of Figure 1. Sections of colonic tissues were prepared and subjected to TUNEL assay and DAPI staining. A: The right panel in each column is a ×3 magnified image of the boxed area defined in the left panel. HCT-15 cells were transfected with siRNA for CHOP (siCHOP) or nonsilencing (ns) siRNA and were incubated with indicated concentrations of menadione for 12 hours. B: Relative mRNA expression of the CHOP was monitored as described in the legend of Figure 2A. C: Cell viability was determined using the MTT method. D: The intracellular antioxidant activity was measured as described in the Materials and Methods. Values shown are mean ± SEM (n = 3). ** or ^##P < 0.01; n.s., not significant.