Effect of a small molecule inhibitor of nuclear factor-kappaB nuclear translocation in a murine model of arthritis and cultured human synovial cells

Abstract

A small cell-permeable compound, dehydroxymethylepoxyquinomicin (DHMEQ), does not inhibit phosphorylation and degradation of IkappaB (inhibitor of nuclear factor-kappaB [NF-kappaB]) but selectively inhibits nuclear translocation of activated NF-kappaB. This study aimed to demonstrate the antiarthritic effect of this novel inhibitor of the NF-kappaB pathway in vivo in a murine arthritis model and in vitro in human synovial cells. Collagen-induced arthritis was induced in mice, and after onset of arthritis the mice were treated with DHMEQ (5 mg/kg body weight per day). Using fibroblast-like synoviocyte (FLS) cell lines established from patients with rheumatoid arthritis (RA), NF-kappaB activity was examined by electrophoretic mobility shift assays. The expression of molecules involved in RA pathogenesis was determined by RT-PCR, ELISA, and flow cytometry. The proliferative activity of the cells was estimated with tritiated thymidine incorporation. After 14 days of treatment with DHMEQ, mice with collagen-induced arthritis exhibited decreased severity of arthritis, based on the degree of paw swelling, the number of swollen joints, and radiographic and histopathologic scores, compared with the control mice treated with vehicle alone. In RA FLS stimulated with tumor necrosis factor-alpha, activities of NF-kappaB components p65 and p50 were inhibited by DHMEQ, leading to suppressed expression of the key inflammatory cytokine IL-6, CC chemokine ligand-2 and -5, matrix metalloproteinase-3, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. The proliferative activity of the cells was also suppressed. This is the first demonstration of an inhibitor of NF-kappaB nuclear translocation exhibiting a therapeutic effect on established murine arthritis, and suppression of inflammatory mediators in FLS was thought to be among the mechanisms underlying such an effect.

Figure 1

Clinical effect of NF-κB inhibitor DHMEQ on collagen-induced arthritis in DBA/1J mice. After onset of arthritis, animals were treated with 100 μg/day dehydroxymethylepoxyquinomicin (DHMEQ; ■; n = 10) or vehicle (□; n = 10). (a) Sum of the thickness of the right and the left hind paws of each mouse after 2 weeks of treatment. Each paw was measured twice and the average was plotted. (b) Sum of the number of swollen joints (described in the Materials and methods section) in the right and the left hind paws of each mouse after 2 weeks of treatment. Each paw was counted twice and the average was plotted. Maximum possible number is 32 per mouse. (c) Change in body weight of each mouse during the first week of treatment. Horizontal bars represent the mean. NF-kB, nuclear factor-κB.

Figure 2

Effect of DHMEQ on radiographic findings in collagen-induced arthritis in mice. (a) A representative radiograph of the left metatarsophalangeal (MTP) joints of a mouse treated with dehydroxymethylepoxyquinomicin (DHMEQ), which shows small bone erosions, and (b) that of a control mouse, which shows remarkable soft tissue swelling and large bone erosions. (c) Soft tissue swelling and (d) bone erosions of bilateral second, third and fourth MTP joints observed in the radiographs were scored as described in the Materials and methods section. Values are expressed as the mean ± standard deviation of the total scores of 10 mice in each group, determined by five independent observers.

Figure 3

Effect of DHMEQ on histopathologic findings in collagen-induced arthritis in mice. (a) A representative specimen of the metatarsophalangeal (MTP) joint of a dehydroxymethylepoxyquinomicin (DHMEQ)-treated mouse, showing almost normal findings, and (b) that of a control mouse showing remarkable cell infiltration in the synovium and bone destruction accompanied by pannus invasion into the marrow space. The severity of (c) synovitis and (d) bone destruction in the specimens were scored as described in the Materials and methods section. Values are expressed as the mean ± standard deviation of the total scores of 10 mice in each group, determined by five independent observers.

Figure 4

Inhibition by DHMEQ of NF-κB in rheumatoid arthritis fibroblast-like synoviocytes. Nuclear extracts were obtained from unstimulated and tumor necrosis factor (TNF)-α-stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and nuclear factor-κB (NF-κB) DNA-binding activity was examined by electrophoretic mobility shift assays. For supershift assays, the DNA-protein mixture was incubated with antibodies to p65, p50, or p52 before electrophoresis. To confirm the specificity of the assay, 100-fold excess of unlabeled NF-κB probe was included as a competitor. To assess whether dehydroxymethylepoxyquinomicin (DHMEQ) inhibits NF-κB activation in RA FLS, the cells were incubated with DHMEQ for 20 minutes before the stimulation with TNF-α. Data shown are representative of three independent experiments.

Figure 5

Effect of DHMEQ on inflammatory mediator mRNA expression by RA FLS stimulated with TNF-α. (a) Five rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) cell lines (#1–#5) obtained from different patients were stimulated with tumor necrosis factor (TNF)-α in the presence or absence of 10 μg/ml dehydroxymethylepoxyquinomicin (DHMEQ) and mRNA expression of CC chemokine ligand (CCL)2, CCL5, IL-6, IL-1β, matrix metalloproteinase (MMP)-3, and vascular endothelial cell growth factor (VEGF) was examined by RT-PCR. (b) Densitometric analysis of these results. Intensity of each band was normalized relative to that of β-actin in the same lane, and the mean ± standard deviation of the five cell lines are shown. *P < 0.05 versus T. D, DHMEQ; T, TNF-α.

Figure 6

Suppressive effect of DHMEQ on inflammatory mediator production by RA-FLS at the protein level. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were stimulated with tumour necrosis factor (TNF)-α in the presence or absence of 10 μg/ml dehydroxymethylepoxyquinomicin (DHMEQ), and levels of secreted CC chemokine ligand (CCL)2, CCL5, IL-6, and matrix metalloproteinase (MMP)-3 in the culture supernatants were measured using ELISA. Values are expressed as the mean ± standard deviation of three independent experiments.

Figure 7

Suppression of ICAM-1 expression by DHMEQ. Shown, using flow cytometry, is suppression of intercellular adhesion molecule (ICAM)-1 expressed on tumor necrosis factor (TNF)-α-stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) by dehydroxymethylepoxyquinomicin (DHMEQ). Cells were preincubated for 20 minutes with 10 μg/ml DHMEQ or vehicle. TNF-α stimulated or unstimulated RA FLS were incubated with isotype-matched control IgG or anti-ICAM-1 antibody, followed by phycoerythrin-labeled second antibody. (a) Representative data are shown, along with (b) the means ± standard deviation of ICAM-1-positive cells in four independent experiments.

Figure 8

Suppression of VCAM-1 expression by DHMEQ. Suppression of vascular cell adhesion molecule (VCAM)-1 expressed on tumor necrosis factor (TNF)-α-stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) by dehydroxymethylepoxyquinomicin (DHMEQ). Flow cytometric analysis was carried out (as in Fig. 7) except that anti-VCAM-1 antibody was used. (a) Representative data are shown, along with (b) the means ± standard deviation of VCAM-1-positive cells in three independent experiments.

Figure 9

Suppression of proliferative activity of RA FLS by DHMEQ. Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) were stimulated with tumor necrosis factor (TNF)-α or unstimulated in the presence or absence of dehydroxymethylepoxyquinomicin (DHMEQ), cultured for 48 hours, and incorporation of [³H]thymidine during the last 24 hours was measured. Values are expressed as mean ± standard deviation of triplicate measurements. Data shown are representative of three independent experiments. *P < 0.01, **P < 0.001. cpm, counts/minute.

Figure 10

Cytotoxicity of DHMEQ. Significant cytotoxicity was not observed in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) treated with dehydroxymethylepoxyquinomicin (DHMEQ). Cells were stimulated with 5 ng/ml tumor necrosis factor (TNF)-α and incubated in serum-free medium for 14 hours with 0–10 μg/ml DHMEQ or with the apoptosis inducer staurosporin (1 μmol/l), and Cy3-labeled annexin V binding cells were measured by flow cytometry. Data shown are representative of three independent experiments. MFI, mean fluorescence intensity.