Expression profiles and functional analyses of Wnt-related genes in human joint disorders

Abstract

Rheumatoid arthritis (RA) and osteoarthritis (OA) are joint disorders that cause major public health problems. Previous studies of the etiology of RA and OA have implicated Wnt genes, although the exact nature of their involvement remains unclear. To further clarify the relationship between RA, OA, and the Wnt gene family, gene expression analyses were performed on articular cartilage, bone, and synovial tissues in knee joints taken from RA, OA, and nor-mal/control patients. Cytokine assays were also performed in cells transfected with Wnt-7b, a member of the gene family most closely linked to RA and OA. Of the human Wnt genes, real-time PCR analysis revealed significant up-regulation of Wnt-7b in OA cartilage and RA synovium. In situ hybridization and immunohistochemistry also revealed that Wnt-7b was present in articular cartilage, bone, and synovium of RA samples and in osteophytes, articular cartilage, bone marrow, and synovium of OA samples. The levels of the cytokines tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 were significantly increased in RA synovium and Wnt-7b-transfected normal synovial cells when compared with normal samples. These results point to the potential involvement of Wnt signaling in the pathobiology of both RA and OA.

Figure 1

Comprehensive analyses of Wnt genes, FRZB, LRP5, and β-catenin in human control samples, cartilage, and synovium in RA (A) and cartilage and synovium in OA (B) by real-time PCR. The template concentration is determined by the Ct value. GAPDH gene is used as an internal control. The abundance of each Wnt transcript is expressed as ΔCt, which is the difference between Ct (GAPDH) and Ct (target gene). The baseline of ΔCt is set at −20, so that more mRNA expressions correspond to longer bars. Expression pattern of Wnt genes by real-time PCR in RA, OA cartilage and synovium. C: Summary of the results in A and B. Wnt-7b in OA cartilage, and RA synovium, Wnt-10a in RA cartilage were significantly up-regulated, compared with control samples. Wnt-3 in RA cartilage and synovium, Wnt-3a in OA synovium, Wnt-5b in OA cartilage and synovium, Wnt-9a in RA cartilage, Wnt-9b in OA synovium, Wnt-10b in OA cartilage and RA synovium, Wnt-11 in OA cartilage and RA synovium, FRZB in OA synovium, and RA cartilage and synovium were significantly down-regulated compared with control samples.

Figure 2

Clinical data of WBC count, CRP (mg/L), and Hct (%). Blue dots represent the data from control, pink dots from OA, and yellow dots from RA synovium. Wnt-11 expression significantly decreased as CRP increased in RA and OA patients, compared with control patients.

Figure 3

Histological appearance of Wnt-7b by in situ hybridization (ISH) and immunohistochemistry (IHC). Cross-section at 100× magnification is in the left panel and 400× magnification section is in the right panel. Higher-magnification images corresponding to the box areas in each figure. In RA samples, ISH and IHC show that Wnt-7b is present at osteoclast-like cells of articular cartilage (A, arrows), bone marrow stromal cells or hematopoietic cells of bone marrow (B, arrows), and fibroblastic cells and vessel walls of the synoviums (C, arrows). In OA samples, ISH and IHC reveal that Wnt-7b is expressed in multinucleated osteoclast-like cells in osteophytes (D, arrows), osteoclast-like cells in the articular cartilage (E, arrows), and fibroblastic cells and vessel walls of synovium (F, arrows).

Figure 4

Western blot analysis of Wnt-7b using primary synovial cells in control, RA, OA patients, and normal synovial cells after transfection of Wnt-7b (tWNT-7B). The expression of Wnt-7b was significantly increased in RA and tWNT-7B, compared with the control and OA samples (A). β-Tubulin protein levels obtained by Western blotting was used as a positive control (B).

Figure 5

The expression changes of TNF-α, IL-1β, and IL-6 in primary synovial cells from RA, OA patients, and normal synovial cells after transfection of Wnt-7b (tWNT-7B). Quantitation of cytokine levels in primary synovial cells was measured by chemiluminescence imaging device in the OA, RA, and tWNT-7B. The score in the controls (the expression in empty vector transfected cells) was used as a standard (fold change of controls = 1). The expression of TNF-α, IL-1β, and IL-6 in RA, and tWNT-7B was twofold to fourfold changes than that of OA. The expression of TNF-α was significantly higher than that of IL-1β and IL-6 in each group (P < 0.05).