論文の公開元へInflammatory bowel disease-associated ubiquitin ligase RNF183 promotes lysosomal degradation of DR5 and TRAIL- induced caspase activation
概要
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Inflammatory bowel diseaseassociated ubiquitin ligase RNF183
promotes lysosomal degradation
of DR5 and TRAIL-induced caspase
activation
Yan Wu1, Yuka Kimura1, Takumi Okamoto1, Koji Matsuhisa1, Rie Asada2, Atsushi Saito3,
Fumika Sakaue3, Kazunori Imaizumi1* & Masayuki Kaneko1*
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.
Inflammatory bowel disease (IBD) is a group of inflammatory conditions of the colon and small intestine, including Crohn’s disease (CD) and ulcerative colitis (UC)1. IBD results from chronic dysregulation of the mucosal
immune system in the gastrointestinal tract. However, the molecular mechanisms underlying the development and pathophysiology of IBD are not fully understood. Recent study has revealed that expression levels of
RING-finger protein 183 (RNF183), which functions as a ubiquitin ligase and predominantly localises to lysosomes2, in the colon of patients with IBD were 5-fold higher than those in control subjects; in these patients,
RNF183 promoted intestinal inflammation3.
Ubiquitination is mediated by ubiquitin ligase (E3) and repeated to form polyubiquitin chains. Ubiquitin
itself contains seven lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) and the initiator
methionine that can serve as acceptor sites for chain elongation4,5. Ubiquitination has multiple roles not only in
proteasome-mediated protein degradation but also in the targeting of membrane proteins for degradation inside
the lysosome. Ubiquitination provides key signals to membrane proteins for endocytosis and endosomal sorting
into the multivesicular body, which delivers its cargo to the proteolytic interior of the lysosome6,7. There are > 600
putative ubiquitin ligases in the human genome8; however, many have been poorly characterized, particularly
their protein substrates.
1
Department of Biochemistry, Graduate school of Biomedical and Health Sciences, Hiroshima University, Hiroshima,
Japan. 2Department of Medicine, Division of Endocrinology, Metabolism, and Lipid Research, Washington University
School of Medicine, MO, St. Louis, USA. 3Department of Stress Protein Processing, Graduate School of Biomedical
and Health Sciences, Hiroshima University, Hiroshima, Japan. *email: imaizumi@hiroshima-u.ac.jp; mkaneko@
hiroshima-u.ac.jp
Scientific Reports |
(2019) 9:20301 | https://doi.org/10.1038/s41598-019-56748-6
1
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IBD models can be induced in mice by dextran sulphate sodium (DSS) in the drinking water or by a
2,4,6-trinitrobenzene sulfonic acid (TNBS)-ethanol enema, which evoke immune responses and colitis9,10. In
this study, we investigated DSS-induced RNF183 expression in mice colons. In DSS colitis mice, compared
with inflammatory cytokines, RNF183 was expressed at a very early stage and specifically in epithelial cells.
Furthermore, we identified death receptor 5 (DR5) as a substrate of RNF183. DR5, also called tumour necrosis factor (TNF) receptor superfamily member 10B (TNFRSF10B) and TNF-related apoptosis inducing ligand
(TRAIL; also called TNFSF10 and APO-2L) receptor 2 (TRAILR2), is a cell surface receptor of the TNF-receptor
superfamily11. This receptor contains an intracellular death domain and transduces apoptosis signalling through
interaction with caspase-812,13. A previous study showed that DR5 is decreased in the large intestine epithelial
tissue of patients with CD and UC14. Furthermore, DR5 knockout mice are more susceptible to DSS-induced
colitis15. However, the underlying molecular mechanisms of DR5 in IBD remain unclear. Here, we demonstrated
that RNF183 induced K63-linked ubiquitination-mediated lysosomal degradation of DR5 and caspase activation.
Result
RNF183 expression increased in a DSS-induced colitis mouse model. RNF183 mRNA and protein
have been reported to be highly expressed in inflamed colon tissue of patients with UC and CD3. RNF183 expression has also been induced in the mouse colon of TNBS colitis model3. Furthermore, RNF186, a gene closely
related to RNF183, was identified as a disease-susceptibility gene for UC from genome-wide association studies16.
Thus, we examined whether RNF183 and RNF186 expressions are increased in another IBD model. An acute
colitis model was established by 3.5% DSS in the drinking water for 5 days. Significant reduction in body weight
(Fig. 1a) and shortening in colon length (Fig. 1b) were observed in DSS-treated mice after 5 days of exposure.
Concomitantly, the colon tissue of the DSS-treated mice showed loss of crypts and goblet cells and mucus layer and
substantial neutrophil infiltration into the lamina propria (Fig. 1c), which indicated successful establishment of
the IBD model with DSS. The mRNA levels of RNF183 and inflammatory markers were compared between water
controls and DSS-treated mice by using quantitative reverse transcription polymerase chain reaction (qRT-PCR).
RNF183 mRNA level was significantly increased in DSS-treated mice compared with that in control mice,
whereas RNF186 was not increased (Fig. 1d). TNF-α, IL-1β, and IL-6 mRNA levels were markedly upregulated
in the DSS-treated mice group (Fig. 1e).
RNF183 mRNA expression increased at an early stage of DSS-induced colitis. Our results indicate the possibility that DSS-induced RNF183 expression is caused by a secondary inflammatory response. To
determine when RNF183 is expressed in DSS-induced colitis, we investigated RNF183 expression at an early
stage of DSS-induced colitis. To compare the progression of pathological conditions and expression of genes, the
disease activity index (DAI) was used to assess DSS colitis severity, as performed in previous studies17,18. Health
status was investigated at 1, 3, and 5 days, including weight of the animal, stool consistency, and presence of rectal
bleeding (Table 1). DSS-treated mice at 5 days had the highest clinical scores. These assessed parameters gradually increased with increasing duration of DSS treatment. The colonic tissue of the DSS-treated mice at 1 and
3 days was observed by haematoxylin-eosin (HE) staining (Fig. 2a). DSS-treated mice at 3 days showed modest
inflammatory infiltration, whereas those at 1 day did not show any change. We examined RNF183, TNF-α, and
IL-1β mRNA levels after 1 and 3 days of DSS exposure by using qRT-PCR. RNF183 mRNA expression was significantly elevated at 1 day, whereas TNF-α and IL-1β mRNA levels were not significantly changed at 1 day (Fig. 2b).
This quick response of RNF183 without signs of significant inflammatory responses suggests that DSS-induced
RNF183 expression does not result from a secondary inflammatory response caused by increased inflammatory
cytokines.
RNF183 was expressed in colonic epithelial cells of DSS-treated mice. It remains unclear where
DSS-induced RNF183 localises because there is no specific anti-RNF183 antibody. To determine which colonic
cells express RNF183, we isolated epithelial and immune cells from colon tissues of the DSS-treated mice by using
fluorescence-activated cell sorting. CD326 (Epithelial cell adhesion molecule, EpCAM) and CD45 (receptor-type
tyrosine-protein phosphatase C, PTPRC) fluorescent antibodies were used for sorting of epithelial cells (Fig. 3a)
and immune cells (Fig. 3b), respectively; we checked their corresponding gene expression by using qRT-PCR.
qRT-PCR analysis of sorted cells revealed that the RNF183 mRNA levels in DSS-treated CD326-positive epithelial cells were significantly increased relative to those in CD326-positive non-treated control cells, consistent
with the experimental result on the whole colon tissues (Fig. 3c). However, RNF183 in CD45-positive cells was
not detected in both DSS-treated and non-treated control cells (Fig. 3c), although CD45-positive immune cells
were successfully isolated from colon tissues (Fig. 3b). Thus, these results indicate that RNF183 was selectively
expressed in colonic epithelial cells, but not immune cells.
Identification of DR5 as a substrate of RNF183. To elucidate the role of RNF183 in IBD, we identified RNF183-interacting proteins. We performed a mass spectrometric analysis of proteins that were
co-immunoprecipitated with RNF183 from human embryonic kidney HEK293 cells engineered to stably
express RNF183. DR5 was identified as a candidate for RNF183 substrate (number of detected peptides, 1; peptide cover rate, 3.86%). DR5 is a cell surface receptor of the TNF-receptor superfamily that binds TRAIL and
activates caspase-8. Next, we confirmed the interaction between RNF183 and DR5 by co-immunoprecipitation. ...
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