Abstract
Necroptosis of intestinal epithelial cells has been indicated to play an important role in the pathogenesis of inflammatory bowel disease (IBD). The identification of dysregulated proteins that can regulate necroptosis in dextran sulfate sodium (DSS)-induced colitis is the key to the rational design of therapeutic strategies for colitis. Through tandem mass tag (TMT)-based quantitative proteomics, HtrA2 was found to be downregulated in the colon of DSS-treated mice. UCF-101, a specific serine protease inhibitor of HtrA2, significantly alleviated DSS-induced colitis as indicated by prevention of body weight loss and decreased mortality. UCF-101 decreased DSS-induced colonic inflammation, prevented intestinal barrier function loss and inhibited necroptosis of intestinal epithelial cells. In vitro, UCF-101 or silencing of HtrA2 decreased necroptosis of HT-29 and L929 cells. UCF-101 decreased phosphorylation of RIPK1 and subsequent phosphorylation of RIPK3 and MLKL during necroptosis. Upon necroptotic stimulation, HtrA2 translocated from mitochondria to cytosol. HtrA2 directly interacted with RIPK1 and promoted its degradation during a specific time phase of necroptosis. Our findings highlight the importance of HtrA2 in regulating colitis by modulation of necroptosis and suggest HtrA2 as an attractive target for anti-colitis treatment.
Highlights
Inflammatory bowel diseases (IBD), namely Crohn’s disease and ulcerative colitis, affect about 3.7 million people in the USA and Europe[1]
HtrA2 is downregulated in colons of dextran sulfate sodium (DSS)-treated mice To identify important proteins that are dysregulated in the inflamed colon that could be used as novel potential therapeutic targets, we utilized a DSS-induced colitis mouse model for quantitative proteomics
HtrA2 contributes to necroptosis by degrading RIPK1 To find the target of HtrA2, we examined the effect of UCF-101 on important factors involved in necroptosis, including RIPK1, RIPK3, mixed lineage kinase domain like pseudokinase (MLKL), and their phosphorylated status
Summary
Inflammatory bowel diseases (IBD), namely Crohn’s disease and ulcerative colitis, affect about 3.7 million people in the USA and Europe[1]. Upon TNF-α stimulation, RIPK1, FADD, and CYLD are recruited to TNFR1 to form a protein complex. When caspase-8 activity is absent or inhibited, the phosphorylated RIPK1 regulates the formation of a necrosome that consists of RIPK1, RIPK3, and MLKL13. MLKL is recruited to the RIPK1/RIPK3 complex and phosphorylated by p-RIPK3. Phosphorylated MLKL forms oligomers and translocates to the intracellular plasma membrane where it binds to phosphatidylinositol lipids and cardiolipin, leading to the formation of pores and disrupting cellular membrane integrity[16]. As an essential factor for necroptosis in the context of TNF-α, RIPK1 is reported to regulate necroptosis positively in a kinase-dependent manner, and negatively in a kinaseindependent manner whereby the scaffolding function of the RHIM domain prevents ZBP1 from activating RIPK3 and represses necroptosis[14,17,18]
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