ErbB2 and ErbB3 regulate recovery from dextran sulfate sodium-induced colitis by promoting mouse colon epithelial cell survival

Abstract

ErbB2 and ErbB3 receptor tyrosine kinases are key regulators of proliferation, migration, differentiation and cell survival; however, their roles in gastrointestinal biology remain poorly defined. We hypothesized that ErbB2 and ErbB3 promote colon epithelial cell survival in the context of the wound-healing response following colitis. In this study, mice bearing intestinal epithelial-specific deletion of ErbB2 or ErbB3 were treated with dextran sulfate sodium (DSS). Colon sections were examined for injury, cytokine expression, epithelial cell proliferation and apoptosis. Deletion of epithelial ErbB2 did not affect the extent of intestinal injury in response to DSS, whereas deletion of ErbB3 slightly increased injury. However, the roles of both receptors were more apparent during recovery from DSS colitis, in which ErbB2 or ErbB3 epithelial deletion resulted in greater inflammation and crypt damage during the early reparative period. Moreover, loss of ErbB3 prevented normal epithelial regeneration in the long term, with damage persisting for at least 6 weeks following a single round of DSS. Delayed recovery in mice with epithelial deletion of ErbB2 or ErbB3 was associated with increased colonic expression of tumor necrosis factor alpha and increased epithelial apoptosis. Furthermore, epithelial ErbB3 deletion increased apoptosis at baseline and during DSS injury. Additionally, epithelial cell hyperproliferation during recovery was exacerbated by deletion of either ErbB2 or ErbB3. These results suggest that ErbB2 and ErbB3 have important cytoprotective and reparative roles in the colonic epithelium following injury, by promoting colon epithelial cell survival.

Figure 1. Deletion of colon epithelial ErbB2 or ErbB3 expression in ErbB2 KO^IEC and ErbB3 KO^IEI mice

Epithelial and stromal fractions were isolated from colons of the indicated mice. (A and B) Western blot and immunohistochemical analysis with antibodies against ErbB2 (A) or ErbB3 (B). Omission of primary antibody was used as a control for immunohistochemistry. Actin expression was used as loading control. Brown indicates ErbB2 staining. Red indicates ErbB3 staining, blue indicates DAPI staining. Scale bars, 50 μm. These results are representative of independent analyses of at least three different mice in each group.

Figure 2. Epithelial ErbB2 is required for short-term recovery from DSS-induced colitis

ErbB2 KO^IEC and wild-type controls were treated with 3% DSS for 4 or 7 days (A, C, E), or were treated with 3% DSS for 4 days followed by a recovery period of 3 days or 6 weeks on normal water (B, D, F); n=6–12. (A, B) Representative H&E sections of colons from the indicated mice. Scale bars, 100 μm. (C, D) Colonic injury scores were quantified by a pathologist blinded to the treatment and group. Bars indicate mean value. (E) Body weight loss during DSS administration; values indicate mean±SD. (F) Body weight loss during the 3 day recovery period following 4 days of DSS administration; values indicate mean±SD.

Figure 3. Epithelial ErbB3 is required for short-term and long-term recovery from DSS-induced colitis

ErbB3 KO^IEI (A, B, C, Di, E, F) or ErbB3 KO^IEC (B, Dii) mice and their respective wild-type controls were treated with 3% DSS for 4 or 8 days (A, C, E), or were treated with 3% DSS for 4 days followed by a recovery period of 3 days or 6 weeks on normal water (B, D, F), as indicated; n=7–13. (A, B) Representative H&E sections of colons from the indicated mice. Scale bars, 100 μm. (C, D) Colonic injury scores were quantified by a pathologist blinded to the treatment and group. Bars indicate mean value. (E) Body weight loss during DSS administration; values indicate mean±SD. (F) Body weight loss during the 3 day recovery period following 4 days of DSS administration; values indicate mean±SD.

Figure 4. Colonic TNF-α expression is increased in ErbB2 KO^IEC and ErbB3 KO^IEI mice during recovery from DSS-induced colitis

TNF-α (A, C) and IFN-γ (B, D) mRNA transcript levels were measured by real-time RT-PCR from whole colon RNA of the indicated genotypes and treatment groups. Bars indicate mean expression of transcripts normalized for actin; n=4–7.

Figure 5. Loss of ErbB2 or ErbB3 exacerbates epithelial hyperproliferative response during recovery from DSS-induced colitis

(A, B) Immunostaining for the proliferative marker Ki67 (brown nuclei) in colonic sections of ErbB2 KO^IEC (A), and ErbB3 KO^IEI mice (B) and their respective wild-type controls treated with water, DSS for 4 days or DSS followed by a 3 day recovery period, as indicated. Images are representative of 8–12 mice per group. Scale bars, 100 μm. (C, D) The number of Ki67-positive cells per crypt were quantified in a blinded manner. Bars indicate mean values.

Figure 6. Loss of ErbB2 or ErbB3 increases colonic epithelial apoptosis

(A, B) Apoptotic cells (arrows) in colon sections were detected by TUNEL staining from wild-type or knockout mice treated with water, DSS for 4 days or DSS followed by a 3 day recovery period, as indicated. Images are representative of 8–12 mice per group. Scale bars, 50 μm. (C, D) The number TUNEL-positive cells per 100 crypts were quantified in a blinded manner. Bars indicate mean values.