Expression and function of wingless and frizzled homologs in rheumatoid arthritis

Abstract

Rheumatoid arthritis (RA) is accompanied by synovial inflammation, proliferation, and cartilage destruction. The reasons the activation of synovial fibroblasts often persists despite antiinflammatory therapy are not known. One possibility is that the synovial membrane becomes gradually repopulated with immature mesenchymal and bone marrow cells with altered properties. To explore this hypothesis, we have investigated the expression in RA synovial tissues of various embryonic growth factors from the wingless (wnt) and frizzled (fz) families, which have been implicated in cell-fate determination in both bone marrow progenitors and limb-bud mesenchyme. Reverse transcriptase-PCR analysis revealed expression of five wnt (wnt1, 5a, 10b, 11, and 13) and three fz (fz2, 5, and 7) isoforms in RA synovial tissues. Osteoarthritis synovial tissues expressed much less wnt5a and fz5. Northern blotting confirmed the overexpression of wnt5a and fz5 in RA synovial tissues, in comparison to a panel of normal adult tissues. Compared with normal synovial fibroblasts, cultured RA fibroblast-like synoviocytes expressed higher levels of IL-6, IL-8, and IL-15. Transfection of normal fibroblasts with a wnt5a expression vector reproduced this pattern of cytokine expression and stimulated IL-15 secretion. These results suggest that the unusual phenotypic properties of RA fibroblasts may be attributable partly to their replacement with primitive fibroblast-like synoviocytes with characteristics of immature bone marrow and mesenchymal cells. Clear delineation of the signaling pathway(s) initiated by the wnt5a/fz5 ligand-receptor pair in the RA synovium may yield new targets for therapeutic intervention.

Figure 1

RT-PCR analysis of various wnt and fz isoforms in RA and OA tissue specimens. Total RNA was extracted from five RA and five OA synovial tissue samples. RT-PCR was performed by using 1 μg of RNA from each sample with different primers for the specified wnt and fz isoforms as well as β-actin. (A) fz2, (B) fz5, (C) wnt1, (D) wnt5a, (E) wnt10b, (F) wnt13, and (G) β-actin. The PhiX 174 DNA standard is shown in the first lane of each gel.

Figure 2

Expression of fz5, wnt5a, and β-actin in RA tissue samples and adult tissues by Northern analysis. A–C show the expression of β-actin, fz5, and wnt5a in RA tissue specimens. D–F show the expression of the same genes in 12 adult tissues that are peripheral blood leukocyte, lung, placenta, small intestine, liver, kidney, spleen, thymus, colon, skeletal muscle, heart, and brain (lanes 1–12, respectively, in D–F). (A) Northern blot showing 2.3-kb β-actin-specific band in three RA tissue specimens; (B) 5-kb fz5-specific band in three RA tissue specimens; (C) lanes 1–3, 5-kb wnt5a-specific band in three RA specimens; lane 4, wnt5a-specific band in fetal fibroblast as a positive control. (D) Northern blot showing 2.3-kb β-actin-specific band in 12 different adult tissue specimens; (E) 5-kb fz5-specific band in the 12 adult tissue Northern blot; (F) result of wnt5a probe hybridization by using the same Northern blot. Specific activity of the probes used for hybridization and RNA concentrations of the tissue samples was the same for each analysis. The adult multiple tissue Northern blot was stripped and reprobed during the course of the experiments, and β-actin hybridization was performed last of all.

Figure 3

RT-PCR analysis to identify expression of wnt5a, IL-6, IL-8, and IL-15 in RA fibroblasts compared with normal synovial fibroblasts. Lane 1, DNA standard PhiX174; lane 2, wnt5a-specific RT-PCR product from rheumatoid synovial fibroblast (RA 508); lanes 3–5 and 6, IL-6-, IL-8-, IL-15-, and G3PDH-specific RT-PCR products from RA 508; lanes 7–11, wnt5a-, IL-6-, IL-8-, IL-15-, and G3PDH-specific RT-PCR products from RA 498; lanes 12–16, wnt5a-, IL-6-, IL-8-, IL-15-, and G3PDH-specific RT-PCR products in normal synovial fibroblasts.

Figure 4

RT-PCR experiment to analyze IL-6, IL-8, and IL-15 expression in wnt5a-transfected (transient) normal synovial fibroblasts and determination of transfection efficiency. (A) Lane 1, DNA standard PhiX174; lanes 2–5, IL-6-, IL-8-, IL-15-, and G3PDH-specific RT-PCR products from wnt5a-pcDNA3-transfected normal synovial fibroblasts; lanes 6–9, IL-6-, IL-8-, IL-15-, and G3PDH- specific RT-PCR products from empty vector transfected normal synovial fibroblasts; lanes 10–13, IL-6-, IL-8-, IL-15-, and G3PDH-specific RT-PCR products from untransfected normal synovial fibroblasts. (B) Bar graph showing fold increase in IL-6, IL-8, and IL-15 gene expression (measured by PCR product intensity) on transfection of normal synovial fibroblasts with wnt5a expression construct. Fold increase with wnt5a is compared with the effects produced by transfection by the empty vector. This represents an average of five different experiments. For each experiment, the same amounts of RNA were used from the wnt5a- and empty vector-transfected cells. G3PDH expression was used an internal control. (C) Western blot of lysate from wnt5a-HA-transfected synovial fibroblasts. Lane 1, ≈60 kDa wnt5a-HA protein from wnt5a-HA-transfected synovial fibroblasts; lanes 2 and 3, no band observed from untransfected synovial fibroblasts.

Figure 5

(A) RT-PCR analysis of IL-6, IL-8, and IL-15 in wnt5a and pcDNA3 stable transfectants. Lane 1, DNA standard PhiX174; lanes 2–5, IL-6-, IL-8-, IL-15-, and G3PDH-specific PCR products from wnt5a stable transfectants; lanes 6–9, IL-6-, IL-8-, IL-15-, and G3PDH-specific PCR products obtained from pcDNA3 stable transfectants; lane10, wnt5a-specific PCR product (using wnt5a-specific and pcDNA3-bovine growth hormone-specific primers) obtained from wnt5a stable transfectant. (B) IL-15 protein expression in synovial fibroblasts. Bar graph showing fold difference in IL-15 protein levels in cell supernatants of RA, wnt5a-transfected, and normal synovial fibroblasts. This represents the average of three experiments. The baseline level of expression was estimated to be 60 pg/μg total protein by comparison with a known standard.