Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 May;9(5):480-5.
doi: 10.1038/embor.2008.37. Epub 2008 Mar 28.

Synoviolin promotes IRE1 ubiquitination and degradation in synovial fibroblasts from mice with collagen-induced arthritis

Beixue Gao  1 , Sang-Myeong Lee, An Chen, Jinping Zhang, Donna D Zhang, Krishnaswamy Kannan, Robert A Ortmann, Deyu Fang
Affiliations

Synoviolin promotes IRE1 ubiquitination and degradation in synovial fibroblasts from mice with collagen-induced arthritis

Beixue Gao et al. EMBO Rep. 2008 May.

Abstract

The E3 ubiquitin ligase synoviolin (SYVN1) functions as an anti-apoptotic factor that is responsible for the outgrowth of synovial cells during the development of rheumatoid arthritis. The molecular mechanisms underlying SYVN1 regulation of cell death are largely unknown. Here, we report that elevated SYVN1 expression correlates with decreased levels of the protein inositol-requiring enzyme 1 (IRE1)-a pro-apoptotic factor in the endoplasmic reticulum (ER)-stress-induced apoptosis pathway-in synovial fibroblasts from mice with collagen-induced arthritis (CIA). SYVN1 interacts with and catalyses IRE1 ubiquitination and consequently promotes IRE1 degradation. Suppression of SYVN1 expression in synovial fibroblasts from CIA mice restores IRE1 protein expression and reverses the resistance of ER-stress-induced apoptosis of CIA synovial fibroblasts. These results show that SYVN1 causes the overgrowth of synovial cells by degrading IRE1, and therefore antagonizes ER-stress-induced cell death.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Decreased IRE1 protein expression in synovial fibroblasts from mice with collagen-induced arthritis. (A) The expression of SYVN1, IRE1, CHOP, ATF6 and actin in the lysates of synovial fibroblasts from normal and CIA mice were determined by western blotting. (B) The band densities of each protein in (A) were quantified and shown. (C–F) Total RNA from synovial cells was purified and reverse transcribed into complementary DNA using oligo-dT primers. The cDNA levels of (C) SYVN1, (D) ire1, (E) chop and (F) ATF6 were determined by real-time PCR. Error bars represent data from three independent experiments. Student's t-test was used for statistical analysis; *P>0.05; **P<0.05; ***P<0.001. CIA, collagen-induced arthritis; IRE1, inositol-requiring enzyme 1; SYVN1, synoviolin.
Figure 2
Figure 2
Synoviolin interacts with IRE1. (A) Flag-tagged SYVN1 and IRE1 expression plasmids were co-transfected into human embryonic kidney 293 cells. IRE1 protein in the lysates of transfected cells was immunoprecipitated with an IRE1 antibody. The interaction of SYVN1 with IRE1 was detected with a Flag antibody (top panel). The same membrane was reprobed with an IRE1 antibody (middle panel). The expression levels of SYVN1 in the whole-cell lysates were determined using a Flag antibody (bottom panel). (B) SYVN1 protein was immunoprecipitated from the lysate of synovial cells using anti-SYVN1 or normal rabbit IgG. The interaction of IRE1 (top panel) or CHOP (middle panel) with SYVN1 was detected with IRE1 and CHOP antibodies, respectively. The same membrane was reprobed with a SYVN1 antibody (bottom panel). IRE1, inositol-requiring enzyme 1; SYVN1, synoviolin.
Figure 3
Figure 3
Synoviolin induces ubiquitination of IRE1. (A) Different combinations of IRE1, HA-Ub and SYVN1 expression plasmids were co-transfected into human embryonic kidney 293 cells. IRE1 proteins in the lysates of transfected cells were immunoprecipitated with an IRE1 antibody. The conjugation of ubiquitin onto IRE1 was detected with an HA antibody (top panel). The same membrane was reprobed with an IRE1 antibody (middle panel). The protein expression of SYVN1 in the whole-cell lysates was detected with a SYVN1 antibody (bottom panel). (B) Synovial fibroblasts from normal mice were transfected with SYVN1 short interfering RNA expression plasmids (lane 1) or control plasmids (lane 2). Two days after transfection, GFP+ cells were sorted, reseeded and then transfected with HA-Ub expression plasmid. IRE1 ubiquitination in those transfected cells was determined as described in (A) (top panel). The expression levels of IRE1 (second panel), SYVN1 (third panel) and GFP (bottom panel) in the whole-cell lysates were analysed by western blotting. (C) IRE1 and HA-Ub expression plasmids were co-transfected with SYVN1 or its C307A (CA) mutant. IRE1 ubiquitination in the transfected cells was determined as described in (A). (D) IRE1 expression plasmids were co-transfected with SYVN1 or SYVN1/CA mutant. SYVN1 proteins in the lysates of transfected cells were immunoprecipitated with a Flag antibody and the interacting IRE1 was detected with an IRE1 antibody (top panel). The same membrane was reprobed with a SYVN1 antibody (middle panel). The expression levels of IRE1 in the whole-cell lysates were used as controls (bottom panel). GFP, green fluorescent protein; HA, haemagglutinin; IRE1, inositol-requiring enzyme 1; SYVN1, synoviolin; WT, wild type.
Figure 4
Figure 4
Synoviolin promotes IRE1 degradation. (A) IRE1 and ATF6 expression plasmids were co-transfected with or without SYVN1 or SYVN1/C307A (CA) expression plasmids into human embryonic kidney 293 cells. Transfected cells were treated with cycloheximide (CHX) for different periods, as indicated. The protein stability of IRE1 (top panels) and ATF6 (bottom panels) was determined by western blotting. (B) The band densities of western blotting were quantified using Phosphorimager software. Error bars represent data from three independent experiments (mean±s.d.). The half-lives (t1/2) of IRE1 were calculated. IRE1, inositol-requiring enzyme 1; SYVN1, synoviolin.
Figure 5
Figure 5
Suppression of synoviolin expression in synovial cells of mice with collagen-induced arthritis restores IRE1 protein expression. (A) SYVN1 siRNA expression plasmids or control (CTL) plasmids were transfected into synovial fibroblasts from normal and CIA mice. Three days after transfection, GFP-positive cells were sorted and the expression of SYVN1 (top panel) and IRE1 (middle panel) was determined by SYVN1 and IRE1 antibodies, respectively. The protein levels of actin were analysed as loading controls (bottom panel). (B,C) Synovial fibroblasts, transfected with either (B) control plasmids or (C) SYVN1 siRNA expression plasmids, were treated with or without tunicamycin (TM; 10 μg/ml) for 18 h. Apoptosis was determined by annexin V staining by using flow cytometry. (D) Synovial fibroblasts from normal and CIA mice were transfected with or without IRE1 expression plasmids. Three days after transfection, cells were treated with TM or dimethyl sulphoxide (DMSO) as a control. The percentages of apoptotic cells were analysed as described in (B) and (C). Error bars (mean±s.d.) represent data from three independent experiments. Student's t-test was used for statistical analysis. CIA, collagen-induced arthritis; IRE1, inositol-requiring enzyme 1; siRNA, short interfering RNA; SYVN1, synoviolin.
See this image and copyright information in PMC

References

    1. Amano T et al. (2003) Synoviolin/Hrd1, an E3 ubiquitin ligase, as a novel pathogenic factor for arthropathy. Genes Dev 17: 2436–2449 - PMC - PubMed
    1. Bordallo J, Plemper RK, Finger A, Wolf DH (1998) Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol Biol Cell 9: 209–222 - PMC - PubMed
    1. Brummelkamp TR, Bernards R, Agami R (2002) A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550–553 - PubMed
    1. Denic V, Quan EM, Weissman JS (2006) A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation. Cell 126: 349–359 - PubMed
    1. Friedlander R, Jarosch E, Urban J, Volkwein C, Sommer T (2000) A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nat Cell Biol 2: 379–384 - PubMed

Publication types