RNF186 regulates EFNB1 (ephrin B1)-EPHB2-induced autophagy in the colonic epithelial cells for the maintenance of intestinal homeostasis

Huazhi Zhang; Zhihui Cui; Du Cheng; Yanyun Du; Xiaoli Guo; Ru Gao; Jianwen Chen; Wanwei Sun; Ruirui He; Xiaojian Ma; Qianwen Peng; Bradley N Martin; Wei Yan; Yueguang Rong; Chenhui Wang

doi:10.1080/15548627.2020.1851496

RNF186 regulates EFNB1 (ephrin B1)-EPHB2-induced autophagy in the colonic epithelial cells for the maintenance of intestinal homeostasis

Autophagy. 2021 Oct;17(10):3030-3047. doi: 10.1080/15548627.2020.1851496. Epub 2020 Dec 17.

Authors

Huazhi Zhang¹, Zhihui Cui¹, Du Cheng², Yanyun Du¹, Xiaoli Guo¹, Ru Gao¹, Jianwen Chen¹, Wanwei Sun¹, Ruirui He¹, Xiaojian Ma¹, Qianwen Peng¹, Bradley N Martin³, Wei Yan⁴, Yueguang Rong⁵, Chenhui Wang^{1

6}

Affiliations

¹ Key Laboratory of Molecular Biophysics of the Ministry of Education, National Engineering Research Center for Nanomedicine, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
² Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan, China.
³ Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA.
⁴ Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Wuhan, China.
⁵ Department of Pathogen Biology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan, China.
⁶ Department of Bioinformatics, Wuhan Institute of Biotechnology, Wuhan, China.

Abstract

Although genome-wide association studies have identified the gene RNF186 encoding an E3 ubiquitin-protein ligase as conferring susceptibility to ulcerative colitis, the exact function of this protein remains unclear. In the present study, we demonstrate an important role for RNF186 in macroautophagy/autophagy activation in colonic epithelial cells and intestinal homeostasis. Mechanistically, RNF186 acts as an E3 ubiquitin-protein ligase for EPHB2 and regulates the ubiquitination of EPHB2. Upon stimulation by ligand EFNB1 (ephrin B1), EPHB2 is ubiquitinated by RNF186 at Lys892, and further recruits MAP1LC3B for autophagy. Compared to control mice, rnf186^-/- and ephb2^-/- mice have a more severe phenotype in the DSS-induced colitis model, which is due to a defect in autophagy in colon epithelial cells. More importantly, treatment with ephrin-B1-Fc recombinant protein effectively relieves DSS-induced mouse colitis, which suggests that ephrin-B1-Fc may be a potential therapy for human inflammatory bowel diseases.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG16L1: autophagy related 16 like 1; ATP: adenosine triphosphate; Cas9: CRISPR associated protein 9; CD: Crohn disease; CQ: chloroquine; Csf2: colony stimulating factor 2; Cxcl1: c-x-c motif chemokine ligand 1; DMSO: dimethyl sulfoxide; DSS: dextran sodium sulfate; EFNB1: ephrin B1; EPHB2: EPH receptor B2; EPHB3: EPH receptor B3; EPHB2^K788R: lysine 788 mutated to arginine in EPHB2; EPHB2^K892R: lysine 892 mutated to arginine in EPHB2; ER: endoplasmic reticulum; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; GWAS: genome-wide association studies; HRP: horseradish peroxidase; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IBD: inflammatory bowel diseases; Il1b: interleukin 1 beta; Il6: interleukin 6; IRGM:immunity related GTPase M; i.p.: intraperitoneally; IPP: inorganic pyrophosphatase; KD: knockdown; KO: knockout; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; NOD2: nucleotide binding oligomerization domain containing 2; PI3K: phosphoinositide 3-kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; RNF186: ring finger protein 186; RNF186^A64T: alanine 64 mutated to threonine in RNF186; RNF186^R179X: arginine 179 mutated to X in RNF186; RPS6: ribosomal protein S6; Tnf: tumor necrosis factor; SQSTM1: sequestosome 1; Ub: ubiquitin; UBE2D2: ubiquitin conjugating enzyme E2 D2; UBE2H: ubiquitin conjugating enzyme E2 H; UBE2K: ubiquitin conjugating enzyme E2 K; UBE2N: ubiquitin conjugating enzyme E2 N; UC: ulcerative colitis; ULK1:unc-51 like autophagy activating kinase 1; WT: wild type.

Keywords: Autophagy; EPHB2; RNF186; ephrin B1; ulcerative colitis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Autophagy* / genetics
Class III Phosphatidylinositol 3-Kinases / metabolism
Colon
Ephrin-B1*
Epithelial Cells / metabolism
Genome-Wide Association Study
Homeostasis
Mice
Receptor, EphB2
Ubiquitin-Protein Ligases* / metabolism

Substances

Efnb1 protein, mouse
Ephrin-B1
Rnf186 protein, mouse
Ubiquitin-Protein Ligases
Class III Phosphatidylinositol 3-Kinases
Ephb2 protein, mouse
Receptor, EphB2

Grants and funding

This investigation was supported by the independent innovation grant from Huazhong University of Science & Technology [grant 2017KFYXJJ163]; and the grant from the National Natural Science Foundation of China [grants 81871280]; and the Junior Thousand Talents Program of China [to C.H.W.].