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A novel pathogenic role of the ER chaperone GRP78/BiP in rheumatoid arthritis.

ABSTRACT: An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum (ER). In this study, we found that ER stress-associated gene signatures were highly expressed in rheumatoid arthritis (RA) synoviums and synovial cells. Proinflammatory cytokines, such as TNF and IL-1?, increased the expression of GRP78/BiP, a representative ER chaperone, in RA synoviocytes. RA synoviocytes expressed higher levels of GRP78 than osteoarthritis (OA) synoviocytes when stimulated by thapsigargin or proinflammatory cytokines. Down-regulation of Grp78 transcripts increased the apoptosis of RA synoviocytes while abolishing TNF- or TGF-?-induced synoviocyte proliferation and cyclin D1 up-regulation. Conversely, overexpression of the Grp78 gene prevented synoviocyte apoptosis. Moreover, Grp78 small interfering RNA inhibited VEGF(165)-induced angiogenesis in vitro and also significantly impeded synoviocyte proliferation and angiogenesis in Matrigel implants engrafted into immunodeficient mice. Additionally, repeated intraarticular injections of BiP-inducible factor X, a selective GRP78 inducer, increased synoviocyte proliferation and angiogenesis in the joints of mice with experimental OA. In contrast, mice with Grp78 haploinsufficiency exhibited the suppression of experimentally induced arthritis and developed a limited degree of synovial proliferation and angiogenesis. In summary, this study shows that the ER chaperone GRP78 is crucial for synoviocyte proliferation and angiogenesis, the pathological hallmark of RA.

SUBMITTER: Yoo SA

PROVIDER: S-EPMC3328363 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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A novel pathogenic role of the ER chaperone GRP78/BiP in rheumatoid arthritis.

Yoo Seung-Ah SA You Sungyong S Yoon Hyung-Ju HJ Kim Dong-Ho DH Kim Hyun-Sook HS Lee Kyungho K Ahn Jin Hee JH Hwang Daehee D Lee Amy S AS Kim Ki-Jo KJ Park Yune-Jung YJ Cho Chul-Soo CS Kim Wan-Uk WU

The Journal of experimental medicine 20120319 4

An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum (ER). In this study, we found that ER stress-associated gene signatures were highly expressed in rheumatoid arthritis (RA) synoviums and synovial cells. Proinflammatory cytokines, such as TNF and IL-1β, increased the expression of GRP78/BiP, a representative ER chaperone, in RA synoviocytes. RA synoviocytes expressed higher levels of GRP78 than osteoarthritis (OA) synoviocytes when stimula ...[more]

PMID: 22430489

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Project description:IntroductionAnti-citrullinated protein/peptide antibodies (ACPAs) are highly specific to rheumatoid arthritis (RA) patients and are thought to have a close relationship with the pathogenesis of arthritis. Several proteins, including fibrinogen, vimentin, and alpha-enolase, were reported as ACPA-target antigens, and their importance in RA pathogenesis was widely proposed. We identified citrullinated immunoglobulin binding protein (citBiP) as another ACPA target in RA patients and examined its pro-inflammatory role in arthritis.MethodsWe measured the levels of anti-citBiP, anti-BiP, and anti-cyclic citrullinated peptide (CCP) antibodies in the serum of RA patients (n = 100), systemic lupus erythematosus (SLE) patients (n = 60), and healthy controls (n = 30) using ELISA and immunoblotting. Epitope mapping was performed using 27 citBiP-derived peptides. In the mouse study, after DBA/1J mice were immunized with BiP or citBiP, serum titers of ACPAs were measured by ELISA and immunohistochemistry. The development of collagen-induced arthritis (CIA) was observed in BiP- or citBiP-pre-immunized mice.ResultsThe serum levels of anti-BiP and anti-citBiP antibodies were significantly increased in RA patients, although only anti-BiP antibodies were slightly increased in SLE patients. Interestingly, anti-citBiP antibody levels were higher than anti-BiP antibody levels in 72% of RA patients, whereas no significant increase in anti-citBiP antibody levels was detected in SLE patients and healthy controls. The serum levels of anti-CCP antibodies were correlated with those of anti-citBiP antibodies in RA patients (R2 = 0.41). Several citrulline residues of citBiP were determined to be major epitopes of anti-citBiP antibodies, one of which showed cross-reactivity with CCP. Immunization of DBA/1J mice with citBiP induced several kinds of ACPAs, including anti-CCP and anti-citrullinated fibrinogen antibodies. Pre-immunization with citBiP exacerbated CIA, and anti-CCP antibody levels were increased in citBiP-pre-immunized CIA mice.ConclusionsCitBiP is a newly described ACPA target that may play a pro-inflammatory role in arthritis.

Project description:BackgroundHuman BiP is traditionally regarded as a major endoplasmic reticulum (ER) chaperone performing a number of well-described functions in the ER. In recent years it was well established that this molecule can also be located in other cell organelles and compartments, on the cell surface or be secreted. Also novel functions were assigned to this protein. Importantly, BiP protein appears to be involved in cancer and rheumatoid arthritis progression, autoimmune inflammation and tissue damage, and thus could potentially be used for therapeutic purposes. In addition, a growing body of evidence indicates BiP as a new therapeutic target for the treatment of neurodegenerative diseases. Increasing importance of this protein and its involvement in critical human diseases demands new source of high quality native recombinant human BiP for further studies and potential application. Here we introduce yeast Saccharomyces cerevisiae as a host for the generation of human BiP protein.ResultsExpression of a full-length human BiP precursor in S. cerevisiae resulted in a high-level secretion of mature recombinant protein into the culture medium. The newly discovered ability of the yeast cells to recognize, correctly process the native signal sequence of human BiP and secrete this protein into the growth media allowed simple one-step purification of highly pure recombinant BiP protein with yields reaching 10 mg/L. Data presented in this study shows that secreted recombinant human BiP possesses native amino acid sequence and structural integrity, is biologically active and without yeast-derived modifications. Strikingly, ATPase activity of yeast-derived human BiP protein exceeded the activity of E. coli-derived recombinant human BiP by a 3-fold.ConclusionsS. cerevisiae is able to correctly process and secrete human BiP protein. Consequently, resulting recombinant BiP protein corresponds accurately to native analogue. The ability to produce large quantities of native recombinant human BiP in yeast expression system should accelerate the analysis and application of this important protein.

Project description:IntroductionExpression of TXNDC5, which is induced by hypoxia, stimulates cell proliferation and angiogenesis. Our previous study detected increased TXNDC5 expression in the synovial tissues of rheumatoid arthritis (RA) patients using proteomic methods. The current study investigated a pathogenic role for TXNDC5 in RA.MethodExpression of TXNDC5 in synovial membranes was quantitatively analyzed by immunohistochemistry, Western blotting and real-time polymerase chain reaction (PCR). Serum TXNDC5 levels and serum anti-TXNDC5 antibody levels were determined using sandwich enzyme-linked immunosorbent assay (ELISA). A total of 96 single nucleotide polymorphisms (SNPs) in or near the TXNDC5 gene were genotyped using custom-designed Illumina 96-SNP VeraCode microassay. Allele frequencies and genotype frequencies of SNPs were assessed using a case-control design in a cohort of 267 Chinese patients with RA, 51 patients with ankylosing spondylitis (AS) and 160 healthy controls. Additional genotyping of 951 patients with RA and 898 healthy controls was performed for four SNPs (rs2277105, rs369086, rs443861 and rs11962800) using the TaqMan method.ResultsReal-time PCR, Western blotting and immunohistochemistry detected significantly higher TXNDC5 expression in the synovial tissues of RA patients compared to samples from patients with osteoarthritis (OA) or AS. ELISA detected significantly higher levels of TXNDC5 in the blood of RA patients compared to OA, AS and systemic lupus erythematosus patients, and healthy controls. ELISA did not detect significantly different levels of anti-TXNDC5 antibody in the blood of RA, OA and AS patients and healthy controls. A total of 9 SNPs (rs9505298, rs41302895, rs1225936, rs1225938, rs372578, rs443861, rs408014, rs9392189 and rs2743992) showed significant association with RA, while 16 SNPs (rs1044104, rs1225937, rs1225938, rs372578, rs89715, rs378963, rs1225944, rs1225947, rs1238994, rs369086, rs408014, rs368074, rs1225954, rs1225955, rs13209404 and rs3812162) showed significant association with AS. Taqman SNP assay demonstrated that rs443861 has an association with RA, which correlates with the microassay results.ConclusionsTXNDC5 is up-regulated in synovial tissues of RA patients. TXNDC5 has a genetic effect on the risk of RA and AS.

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A novel pathogenic role of the ER chaperone GRP78/BiP in rheumatoid arthritis.

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A novel pathogenic role of the ER chaperone GRP78/BiP in rheumatoid arthritis.

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