Immunology and Inflammation

Vangl2 suppresses NF-κB signaling and ameliorates sepsis by targeting p65 for NDP52-mediated autophagic degradation

Jiansen Lu
Jiahuan Zhang
Huaji Jiang
Zhiqiang Hu
Yufen Zhang
Lian He
Jianwu Yang
Yingchao Xie
Dan Wu
Hongyu Li
Ke Zeng
Peng Tan
Qingyue Xiao
Zijing Song
Chenglong Pan
Xiaochun Bai author has email address
Xiao Yu author has email address

Department of Joint Surgery, the Fifth Affiliated Hospital, Southern Medical University, Guangzhou, Guangdong 510515, China
Department of Immunology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China
Department of Clinical Laboratory Medicine, Guangdong Provincial People’s Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong 510515, China
Department of Orthopaedics, Yuebei People’s Hospital Affiliated to Medical College of Shantou University, Shaoguan 512026, China
Department of Pharmacology, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong 518005, China
Institute of Biosciences and Technology, College of Medicine, Texas A&M University, Houston, Texas 77030, USA
Klarman Cell Observatory, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA
Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong 510515, China
Guangdong Provincial Key Lab of Single Cell Technology and Application, Southern Medical University, Guangzhou, Guangdong 510515, China

https://doi.org/10.7554/eLife.87935.3

Open access
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Figures and data

Vangl2 ablation promotes inflammation during LPS treatment.
(A) Transcription levels of Vangl2 in PBMCs from healthy volunteers (healthy control, HC) and sepsis patients were analyzed by real-time PCR (n=8).
(B) Vangl2 mRNA in different organs from mice treated with or without LPS (n≥3).
(C) The survival rates of WT and Vangl2^ΔM mice treated with high-dosage of LPS (30 mg/kg, i.p.) (n≥4).
(D-F) WT and Vangl2^ΔM mice (n≥3) were treated with LPS (30 mg/kg, i.p.). Splenocytes were collected at 9 h after LPS treatment. Cell lysates of CD11b⁺ sorted splenocytes were analyzed by immunoblotting with the indicated antibodies (D). RNAs from splenocytes were isolated and used for expression analysis of Il1b, Tnfa, and Il6 using qPCR (E). Sera were collected at indicated times post LPS treatment and subjected to ELISA analysis of IL-1β, TNF-α, and IL-6 (F).
PBMCs, peripheral blood mononuclear cells; Unsti, unstimulation; LPS, lipopolysaccharide; LN, lymph node; SP, spleen. Data are representative of three independent experiments and are plotted as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001 vs. corresponding control.

Vangl2 negatively regulates LPS-induced NF-κB activation and proinflammatory cytokines.
(A-D) WT and Vangl2-deficient (n≥3) pMAC (A and C) or neutrophils (B and D) were stimulated with LPS (100 ng/ml) for the indicated times. Immunoblot analysis of total and phosphorylated p65, IKKα/β (A and B), and analysis of grey intensity was shown (C and D).
(E-F) WT and Vangl2-deficient (n≥4) pMAC or neutrophils were stimulated with LPS (100 ng/ml) for 6 h. mRNA levels of Il6 and Tnfa were measure by qPCR (E). IL-6 and TNF-α secretion by WT and Vangl2-deficient BMDMs or neutrophils treated with or without LPS for 6 h was measured by ELISA (F).
(G and H) The WT and Vangl2-deficient (n≥3) neutrophils were treated with LPS (1000 ng/ml) for 4 h, and the nuclear translocation of p65 was detected by immunofluorescence (G) (p65, green; DAPI, blue; Scale bar, 50 μm). Percentages of p65 nuclear translocated cells in WT and Vangl2-deficient neutrophils were determined by counting 100 to 150 cells in non-overlapping fields (H).
(I and J) A549 cells were transfected with Flag-tagged Vangl2 plasmid or empty vector, then stimulated with LPS (100 ng/ml) for the indicated times. Immunoblot analysis of total and phosphorylated p65, IKKα/β (I) (representative image), and analysis of grey intensity was shown (J) (n≥3).
pMAC, peritoneal macrophage; Neu, neutrophil; EV, empty vector. Data are representative of three independent experiments and are plotted as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001 vs. corresponding control.

Vangl2 inhibits NF-κB signaling by interacting with p65.
(A-C) Cho (A) or HEK293T cells (B and C) were co-transfected with a NF-κB and TK-Renilla reporter along with increasing amounts of Vangl2 for 18 h, then treated the cells with or without LPS (A, 250 ng/ml), IL-1β (B, 40 ng/ml), or TNF-α (C, 20 ng/ml) for 6 h. NF-κB promoter driven luciferase activity was measured and normalized to the Renilla luciferase activity.
(D) Luciferase activity in HEK293T transfected with plasmids encoding an NF-κB luciferase reporter and TK-Renilla reporter, together with a vector encoding MyD88, IRAK1, TRAF6, IKKα, IKKβ, or p65, along with or without Vangl2 plasmid, was measured at 24 h after transfection and normalized to the Renilla luciferase activity.
(E) HEK293T cells were transfected with plasmids encoding HA-tagged Vangl2 and Flag-tagged p65, followed by immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA. Throughout was the immunoblot analysis of whole-cell lysates without immunoprecipitation.
(F) BMDMs were stimulated with LPS (100 ng/ml) for the indicated times. The cell lysates were subjected to immunoprecipitation with an anti-p65 antibody or control IgG, followed by immunoblotting with indicated antibodies.
(G) The WT and Vangl2-deficient peritoneal macrophages were treated with LPS (1000 ng/ml) for 4 h, and co-localization of p65 and Vangl2 was detected by immunofluorescence (p65, green; Vangl2, red; DAPI, blue; Scale bar, 50 μm).
(H) A structural diagram of Vangl2 as well as schematic representation of Myc-tagged truncation mutants of Vangl2 (top). HEK293T cells were transfected with Flag-tagged p65 and empty vector, Myc-tagged Vangl2 (FL) or Vangl2 truncation mutants. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies (bottom).
(I) HEK293T cells were transfected with Flag-tagged p65 and HA-tagged Vangl2 FL or PkBD truncation. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(J) Luciferase activity in HEK293T cells transfected with an NF-κB luciferase reporter, together with a vector encoding p65, along with the empty vector or with vectors encoding Vangl2 or its truncation mutants. The results are presented relative to Renilla luciferase activity.
IP, immunoprecipitation; WCL, whole-cell lysate. Data are representative of three independent experiments and are plotted as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001 vs. corresponding control. NS, not significant.

Vangl2 promotes the autophagic degradation of p65.
(A) Immunoblot analysis of HEK293T cells transfected with Flag-p65 and increasing amounts of the vector encoding HA-Vangl2 (0, 250, 500, and 1000 ng).
(B) Total RNA from HEK293T cells as in (A) was isolated and measured by semi-quantitative PCR.
(C) HEK293T cells transfected with Flag-p65 and increasing amounts of the vector encoding HA-Vangl2, and the expressions of p65 in nuclear or cytoplasm were detected by immunoblot.
(D) WT and Vangl2-deficient BMDMs were treated with LPS for the indicated times, and the expressions of p65 and Vangl2 were detected by immunoblot.
(E) HEK293T cells were transfected with Flag-p65 and HA-Vangl2 plasmids, and treated with DMSO, MG132 (10 μM), CQ (50 μM), 3-MA (10 mM) or Baf-A1 (0.2 μM) for 6 h. The cell lysates were analyzed by immunoblot.
(F) HEK293T cells were transfected with empty vector (EV) or Flag-Vangl2 plasmid, and treated with rapamycin for the indicated times. The cell lysates were analyzed by immunoblot with indicated antibodies.
(G) WT, ATG5 KO and Beclin1 KO HEK293T cells were transfected with Flag-p65, together with or without HA-Vangl2 plasmids, and then the cell lysates were analyzed by immunoblot with indicated antibodies.
(H and I) Luciferase activity in WT, ATG5 KO (G) and Beclin1 KO (H) HEK293T cells transfected with plasmids encoding an NF-κB luciferase reporter and TK-Renilla reporter, together with p65 plasmid along with increasing amounts of Vangl2, was measured at 24 h after transfection and normalized to the Renilla luciferase activity.
CHX, cycloheximide; 3-MA, 3-methyladenine; CQ, chloroquine; Baf A1, bafilomycin A1. Data are representative of three independent experiments and are plotted as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001 vs. corresponding control. NS, not significant.

Vangl2 enhances the recognition of p65 by cargo receptor NDP52.
(A) HEK293T cells transfected with a vector expressing HA-Vangl2 along with the empty vector or vector encoding Flag-p62/NDP52/NBR1/Nix. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(B) HEK293T cells transfected with a vector expressing HA-p65 along with the empty vector or vector encoding Flag-p62/NDP52/NBR1/Nix. The cell lysates were subjected to immuno-precipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(C) HEK293T cells were transfected with HA-p65 together with Flag-NDP52 or Flag-p62, as well as with empty vector or Myc-Vangl2. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(D) WT, NDP52 KO and p62 KO HEK293T cells were transfected with a vector expressing HA-p65 along with the empty vector or vector encoding Flag-Vangl2. The cell lysates were immunoblotted with the indicated antibodies.
(E) WT and Vangl2-deficient BMDMs were stimulated with LPS (100 ng/ml) for the indicated times. The cell lysates were subjected to immunoprecipitation with an anti-p65 antibody or control IgG and immunoblotted with the indicated antibodies.
(F) WT and NDP52 KO HEK293T cells were transfected with a vector expressing HA-p65 along with the empty vector or vector encoding Flag-Vangl2. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(G) Luciferase activity in WT and NDP52 KO HEK293T cells transfected with plasmids encoding NF-κB luciferase reporter and TK-Renilla reporter, together with p65 plasmid along with increasing amounts of Vangl2 plasmid, was measured at 24 h after transfection.
(H) WT and NDP52 KO HEK293T were treated with CHX for the indicated times. The cell lysates were immunoblotted with the indicated antibodies.
Data are representative of three independent experiments and are plotted as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001 vs. corresponding control. NS, not significant.

Vangl2 increases the K63-linked ubiquitination of p65.
(A) WT and Vangl2-deficient BMDMs were stimulated with LPS (100 ng/ml) for the indicated times. The cell lysates were subjected to immunoprecipitation with an anti-p65 antibody or control IgG and immunoblotted with the indicated antibodies.
(B) HEK293T cells were transfected with Flag-p65, Myc-Vangl2, HA-Ub or HA-K63 plasmids with the indicated combinations for 24 h and then treated with CQ for 8 h. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(C) HEK293T cells were transfected with Flag-p65, Myc-Vangl2 and HA-Ub/K63/K11/K27/K33/K48 plasmids with the indicated combinations for 24 h and then treated with CQ and Baf-A1 for 8 h. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(D) HEK293T cells were transfected with a vector expressing Flag-p65 and HA-K63 along with Scramble or Vangl2 siRNA. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
Data are representative of three independent experiments.

Vangl2 recruits PDLIM2 to ubiquitinate p65.
(A) HEK293T cells were transfected with the indicated siRNA, NF-κB reporter plasmids together with HA-Vangl2, Flag-p65 or the control vector as indicated for 24 h, and then subjected to luciferase assay and immunoblotting analysis.
(B and C) BMDMs were transfected with Pdlim2 or Scramble siRNA along with the empty vector or vector encoding Flag-Vangl2, stimulated with LPS (100 ng/ml) for 6 h, then analyzed by qPCR for Il6 (B) and Il1b (C) expression.
(D) HEK293T cells transfected with HA-PDLIM2 along with the empty vector or vector encoding Flag-Vangl2. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(E) HEK293T cells were transfected with Flag-p65, HA-PDLIM2 and Myc-Vangl2 plasmids with the indicated combinations for 24 h and then treated with CQ and Baf-A1 for 8 h. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(F) HEK293T cells were transfected with Flag-p65, HA-PDLIM2 and Myc-Vangl2 plasmids with the indicated combinations for 24 h. The cell lysates were immunoblotted with the indicated antibodies.
(G) HEK293T cells were transfected with Flag-p65, HA-K63 and Myc-Vangl2 plasmids, the expression of E3 ubiquitin ligase was interfered with Pdlim2 siRNA and then treated with CQ and Baf-A1 for 8 h. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(H) HEK293T cells were transfected with Pdlim2 or Scramble siRNA, along with or without HA-PDLIM2, then treated with CQ and Baf-A1 for 8 h. The cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblotted with the indicated antibodies.
(I) A schematic model to illustrate how Vangl2-PDLIM2-NDP52-p65 axis negatively regulates NF-κB activation. During LPS stimulation, Vangl2 expression is up-regulated, thus constituting a negative feedback loop to regulate NF-κB activation. In detail, Vangl2 functions as an adaptor protein to recruit an E3 ubiquitin ligase PDLIM2 to increase K63-linked ubiquitination of p65 and promotes NDP52-mediated p65 degradation through selective autophagy, resulting in ameliorating sepsis and suppressing production of pro-inflammatory cytokines.
Data are representative of three independent experiments and are plotted as the mean ±SD. *p<0.05, **p<0.01, ***p<0.001 vs. corresponding control. NS, not significant.

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