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, 9 (1), 20301

Inflammatory Bowel Disease-Associated Ubiquitin Ligase RNF183 Promotes Lysosomal Degradation of DR5 and TRAIL-induced Caspase Activation

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Inflammatory Bowel Disease-Associated Ubiquitin Ligase RNF183 Promotes Lysosomal Degradation of DR5 and TRAIL-induced Caspase Activation

Yan Wu et al. Sci Rep.

Abstract

RNF183 is a ubiquitin ligase containing RING-finger and transmembrane domains, and its expression levels are increased in patients with inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, and in 2,4,6-trinitrobenzene sulfonic acid-induced colitis mice. Here, we further demonstrate that RNF183 was induced to a greater degree in the dextran sulfate sodium (DSS)-treated IBD model at a very early stage than were inflammatory cytokines. In addition, fluorescence-activated cell sorting and polymerase chain reaction analysis revealed that RNF183 was specifically expressed in epithelial cells of DSS-treated mice, which suggested that increased levels of RNF183 do not result from the accumulation of immune cells. Furthermore, we identified death receptor 5 (DR5), a member of tumour necrosis factor (TNF)-receptor superfamily, as a substrate of RNF183. RNF183 mediated K63-linked ubiquitination and lysosomal degradation of DR5. DR5 promotes TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis signal through interaction with caspase-8. Inhibition of RNF183 expression was found to suppress TRAIL-induced activation of caspase-8 and caspase-3. Thus, RNF183 promoted not only DR5 transport to lysosomes but also TRAIL-induced caspase activation and apoptosis. Together, our results provide new insights into potential roles of RNF183 in DR5-mediated caspase activation in IBD pathogenesis.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
RNF183 expression in the dextran sulfate sodium (DSS)-induced colitis mouse model. (a) Changes in body weight and (b) colon length between the control (water) and DSS-treated mice after 5 days exposure. Asterisks represent significant differences (n = 6; Student’s t-test, *p < 0.05). (c) Haematoxylin-eosin (HE) staining of mouse colon tissue exposed to control (water) and 3.5% DSS for 5 days ( × 20 magnification). (d) Expression of RNF183 and RNF186 mRNA in whole colon tissues between control and 3.5% DSS-treated mice at 5 days. (e) The mRNA levels of inflammatory markers in whole colon tissues between control and 3.5% DSS-treated mice at 5 days. The expression levels were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) Asterisks represent significant differences (n = 6; paired t-test, *p < 0.05, **p < 0.01, ***p < 0.001).
Figure 2
Figure 2
RNF183 expression at the early stage of DSS-induced colitis. (a) HE staining of colon tissues of DSS-treated mice at 0, 1, and 3 days ( × 20 magnification). (b) The expressions of RNF183, TNF-α, and IL-1β mRNA in DSS-treated mice at 0, 1, and 3 days. The expression levels were determined by qRT-PCR. Asterisks represent significant differences (n = 5; one-way analysis of variance (ANOVA), followed by Dunnett’s test, *p < 0.05, **p < 0.01; 0 day vs. 1 and 3 days).
Figure 3
Figure 3
RNF183 expression in colonic epithelial cells of DSS-treated mice. (a) Epcam (CD326) and (b) Ptprc (CD45) mRNA levels in isolated colonic epithelial and immune cells, respectively. Isolated colonic cells from drinking water (DW)- and DSS-treated mice at 5 days were labelled with anti-CD326 (PE) and anti-CD45 (FITC) antibodies and sorted by fluorescence-activated cell sorting. The expression levels in sorted cells were determined by qRT-PCR. (c) RNF183 and RNF186 mRNA levels in colonic epithelial and immune cells from DW- and DSS-treated mice at 5 days. The expression levels in sorted cells were determined by qRT-PCR. Asterisks represent significant differences (n = 5; paired t-test, ***p < 0.001). nd: not detected.
Figure 4
Figure 4
Interaction of DR5 with RNF183 and its ubiquitination. (a) Co-immunoprecipitation of RNF183 and DR5. Cell lysates from HEK293 cells stably expressing FLAG-tagged RNF183 were immunoprecipitated with anti-FLAG antibody, and the immune complexes were analysed by western blotting with anti-DR5 or anti-FLAG antibodies. IP; immunoprecipitation. The full-length blots are presented in Supplementary Fig. S7. (b) Ubiquitination of DR5 by RNF183. HEK293 cells stably expressing V5-tagged RNF183 were transfected with HA-tagged ubiquitin. At 36 h after transfection, the cells were incubated with or without 10 μM MG132 or 100 μM chloroquine (CQ) for 12 h. Cell lysates were immunoprecipitated with anti-DR5 antibody, and the immune complexes were analysed by western blotting with anti-HA or anti-DR5 antibodies. The full-length blots are presented in Supplementary Fig. S8. (c) Quantitative graph of data from (b). Asterisks represent significant differences (n = 5; paired t-test, *p < 0.05). (d) K48- and K63-linked ubiquitination of DR5. HEK293 cells stably expressing V5-tagged RNF183 or RNF182 or mock-transfected cells were transfected with HA-tagged ubiquitin K48 or K63 mutants. Cell lysates were immunoprecipitated with anti-DR5 antibody. The full-length blots are presented in Supplementary Fig. S9.
Figure 5
Figure 5
Involvement of RNF183 in the localization and degradation of DR5. (a) Effect of RNF183 overexpression on DR5 subcellular localization. HeLa cells transfected with EmGFP-tagged RNF183 or EmGFP alone were subjected to immunofluorescence staining with DR5 and LAMP1 antibodies (grey, EmGFP; green, DR5; red, LAMP1). (b) Effect of RNF183 knockdown on DR5 protein levels. RNF183-siRNA or non-target control (NC)-siRNA were transfected into HEK293 cells stably expressing V5-tagged RNF183. Cell lysates were subjected to western blotting with DR5 and V5 antibodies. The full-length blots are presented in Supplementary Fig. S10. (c) Quantitative graph of data from (b). Asterisks represent significant differences (n = 3; paired t-test, ***p < 0.001). (d) Cell surface expression level of DR5. HEK293 cells expressing V5-tagged RNF183 were treated with 100 μM CQ for 12 h. Then, cells were labeled with PE-conjugated anti-DR5 antibody. (e) DR5 protein levels in colonic epithelial cells from DW- and DSS-treated mice at 5 days. Cell lysates were subjected to western blotting with anti-DR5 and anti-β-actin antibodies. The full-length blots are presented in Supplementary Fig. S11. (f) Quantitative graph of data from (e). Asterisks represent significant differences (n = 6; paired t-test, **p < 0.01).
Figure 6
Figure 6
Involvement of RNF183 in TRAIL-induced apoptosis. (a) Effect of RNF183 knockdown on TRAIL-induced caspase activation. RNF183-siRNA was transfected into HEK293 cells stably expressing V5-tagged RNF183. At 30 h after transfection, TRAIL (100 ng/mL) was added and cells were incubated for 2.5, 5, and 7.5 h. Cell lysates were subjected to western blotting with anti-cleaved caspase-3, cleaved caspase-8 antibodies, and anti-caspase-3 antibody (for procaspase-3). The full-length blots are presented in Supplementary Fig. S12. (b,c) Effect of RNF183 overexpression on TRAIL-induced caspase activation. HEK293 cells expressing mock or V5-tagged RNF183 were treated with TRAIL for 5 h. Cells were subjected to immunofluorescence staining with cleaved caspase-3, cleaved caspase-8 antibodies (red), and DAPI (blue). (d,e) Quantitative graph of data from (b,c). Asterisks represent significant differences (n = 3; Student’s t-test, *p < 0.05, **p < 0.01). (f) Apoptotic cell death was evaluated by Annexin V assay. HEK293 cells expressing mock or RNF183 were treated with TRAIL (300 ng/ml) for 3 h. The percentage of apoptotic cells was determined by FITC-annexin-V/propidium iodide (PI) staining.

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