Epithelial Gab1 calibrates RIPK3-dependent necroptosis to prevent intestinal inflammation
Jiaqi Xu, Shihao Li, Wei Jin, Hui Zhou, Tingting Zhong, Xiaoqing Cheng, Yujuan Fu, Peng Xiao, Hongqiang Cheng, Di Wang, Yuehai Ke, Zhinong Jiang, Xue Zhang
Jiaqi Xu, Shihao Li, Wei Jin, Hui Zhou, Tingting Zhong, Xiaoqing Cheng, Yujuan Fu, Peng Xiao, Hongqiang Cheng, Di Wang, Yuehai Ke, Zhinong Jiang, Xue Zhang
View: Text | PDF
Research Article Gastroenterology Inflammation

Epithelial Gab1 calibrates RIPK3-dependent necroptosis to prevent intestinal inflammation

Abstract

As a hallmark of inflammatory bowel disease (IBD), elevated intestinal epithelial cell (IEC) death compromises the gut barrier, activating the inflammatory response and triggering more IEC death. However, the precise intracellular machinery that prevents IEC death and breaks this vicious feedback cycle remains largely unknown. Here, we report that Grb2-associated binder 1 (Gab1) expression is decreased in patients with IBD and inversely correlated with IBD severity. Gab1 deficiency in IECs accounted for the exacerbated colitis induced by dextran sodium sulfate owing to sensitizing IECs to receptor-interaction protein kinase 3–mediated (RIPK3-mediated) necroptosis, which irreversibly disrupted the homeostasis of the epithelial barrier and promoted intestinal inflammation. Mechanistically, Gab1 negatively regulated necroptosis signaling through inhibiting the formation of RIPK1/RIPK3 complex in response to TNF-α. Importantly, administration of RIPK3 inhibitor revealed a curative effect in epithelial Gab1-deficient mice. Further analysis indicated mice with Gab1 deletion were prone to inflammation-associated colorectal tumorigenesis. Collectively, our study defines a protective role for Gab1 in colitis and colitis-driven colorectal cancer by negatively regulating RIPK3-dependent necroptosis, which may serve as an important target to address necroptosis and intestinal inflammation–related disease.

Authors

Jiaqi Xu, Shihao Li, Wei Jin, Hui Zhou, Tingting Zhong, Xiaoqing Cheng, Yujuan Fu, Peng Xiao, Hongqiang Cheng, Di Wang, Yuehai Ke, Zhinong Jiang, Xue Zhang

×

Figure 7

Gab1 blocks T/S/Z-induced necroptosis via binding with RIPK3.

Options: View larger image (or click on image) Download as PowerPoint
Gab1 blocks T/S/Z-induced necroptosis via binding with RIPK3. (A) Contro...
(A) Control or Gab1-knockdown HT29 cells were treated with DMSO, TNF-α+SM164, TNF-α+Z-VAD-FMK, or T/S/Z for 3 hours, and phosphorylation of RIPK1, RIPK3, and MLKL was determined by Western blotting. Quantitative data were shown as mean ± SEM for 3 independent experiments. (B) Control or Gab1-overexpressed HT29 cells were preincubated with 10 μm Nec-1s for 1 hours, followed by DMSO, TNF-α+SM164, TNF-α+Z-VAD-FMK or T/S/Z treatment for 3 hours. Detection of indicated proteins was carried out by Western blotting. Quantitative data were shown as mean ± SEM for 3 independent experiments. (C) HT29 cells were treated with T/S/Z for 3 hours. Total cell lysates were subjected to immunoprecipitation (IP) with anti-RIPK3 antibody or anti-IgG, followed by immunoblotting analysis with anti-Gab1 antibody or anti-RIPK1 antibody. See Supplemental Methods for antibody information and details on other methods. (D) HEK293T cells were co-transfected with Gab1-Flag and RIPK3-Myc for 24 hours, followed by T/S/Z stimulation for 3 hours. Cell lysates were then subjected to IP using anti-Flag antibody or anti-IgG and immunoblotted as indicated. (E and F) HEK293T cells were lysed and the supernatant was used to carry out a GST pull-down assay to detect the interaction between Gab1 and RIPK3. Recombinant GST-fused Gab1 protein was incubated with HEK293T cell lysates with (E) or without RIPK3 overexpression (F) and analyzed by immunoblotting with the anti-RIPK3 antibody. (G) Western blot showing co-IP assay for RIPK1 and RIPK3 interaction in control or Gab1-knockdown HT29 cells after exposure to T/S/Z for 3 hours. (H) HEK293T cells were cotransfected with Gab1-Flag and AURKA-HA for 24 hours. Total cell lysates were subjected to IP using anti-Flag antibody or anti-IgG, then immunoblotted with indicated antibodies. All samples were biologically independent and 3 independent experiments were performed. Statistical analysis was performed using 2-tailed Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001. AURKA, aurora kinase A.