Investigating a pathogenic role for TXNDC5 in rheumatoid arthritis

Abstract

Introduction: Expression 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.

Method: Expression 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.

Results: Real-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.

Conclusions: TXNDC5 is up-regulated in synovial tissues of RA patients. TXNDC5 has a genetic effect on the risk of RA and AS.

Figure 1

Immunodetection of TXNDC5 in synovial membranes from patients with RA, OA and AS. (A) Immunolocalization of TXNDC5 in synovial membranes. The left lane indicates results of immunohistochemistry, and the right lane indicates results of immunofluorescent labeling. Original magnification: 100×. Arrows indicate the upper layer of synovial membranes. (B) Semi-quantitative analysis of immunofluorescent signals of TXNDC5. TXNDC5 had significantly higher expression in the synovial tissue of RA patients compared to the synovial tissues of OA and AS patients. AS, ankylosing spondylitis; OA, osteoarthritis; RA, rheumatoid arthritis.

Figure 2

Quantitative analysis of TXNDC5 expression. (A) TXNDC5 at molecular weight of 50 kDa was detected in synovial tissues of RA, OA and AS patients using Western blot analysis. Sample loading was normalized using GADPH at molecular weight of 37 kDa. (B) TXNDC5 expression was semi-quantitatively analyzed by normalizing the signal density of TXNDC5 to that of GADPH. (C) TXNDC5 mRNA expression was measured in synovial tissues using real time PCR. The expression was normalized to that of β-actin. TXNDC5 had significantly higher expression in the synovial tissue of RA patients compared to the synovial tissues of OA and AS patients. AS, ankylosing spondylitis; OA, osteoarthritis; RA, rheumatoid arthritis.

Figure 3

Serum levels of TXNDC5 and anti-TXNDC5 antibody in patients with arthritic diseases and healthy controls. TXNDC5 levels are represented by OD values of absorbance at 405 nm and are expressed as the mean ± standard error of the mean. (A) A sandwich ELISA detected increased level of TXNDC5 in blood samples from RA patients compared to samples from OA, AS and SLE patients, as well as from healthy controls. (B) An ELISA indicated that levels of anti-TXNDC5 antibodies were not significantly different among blood samples from RA, OA and AS patients and the healthy controls. AS, ankylosing spondylitis; OA, osteoarthritis; RA, rheumatoid arthritis.

Figure 4

LD plot of the 96 genotyped SNPs in the TXNDC5 gene. (A) Linkage disequilibrium in the RA group. (B) Linkage disequilibrium in the AS group. Red areas representing higher levels of LD. Blue areas represent LD comparisons with low confidence of estimation. Dark triangles represent haplotype blocks. Numbers in squares are D' values. AS, ankylosing spondylitis; RA, rheumatoid arthritis; SNP, single nucleotide polymorphism.