Abstract
Interleukin-1β (IL-1β) upregulates intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions in osteoarthritis fibroblast-like synoviocytes (OA-FLS) via nuclear factor (NF)-κB-mediated mechanism; enhancement of leukocyte infiltration and upregulation of proinflammatory mediators play a crucial role in OA pathophysiology. MicroRNA (miR)-146a suppresses inflammatory responses by inhibiting NF-κB activity and target gene expression, and epigenetic mechanisms are reportedly involved in miR expression regulation. Here, we aimed to verify the inhibition of ICAM-1/VCAM-1 expression in OA-FLS on denbinobin treatment and to determine whether this inhibition was due to the miR-146a-dependent pathway. We also assessed the epigenetic regulation caused by histone acetyltransferases involved in denbinobin action. Denbinobin attenuated the upregulation of IL-1β-induced ICAM-1/VCAM-1 expression and monocyte adhesion to OA-FLS. The mechanism underlying the inhibitory effects of denbinobin involved miR-146a induction, which in turn inhibited NF-κB signaling. This is because miR-146a inhibitor abrogated the inhibitory effects of denbinobin. Furthermore, histone acetyltransferase inhibitor attenuated the denbinobin-induced upregulation of miR-146a expression and inhibited the acetylation of NF-κB-binding sites located within the miR-146a promoter region. These data suggest that an epigenetic mechanism plays a crucial role in the upregulation of miR-146a expression in response to denbinobin treatment. Our overall findings suggest that denbinobin can be used as a potent anti-inflammatory agent.
Key message
• Denbinobin inhibited IL-1β-induced ICAM-1/VCAM-1 expression and monocyte adhesion to OA-FLS.
• It was due to denbinobin increased miR-146a level, which in turn inhibited NF-κB signaling.
• Our overall findings suggest that denbinobin can be used as a potent anti-inflammatory agent.
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Acknowledgments
This work was supported by grant from the Ministry of Science and Technology of Taiwan (NSC 100-2320-B-002-004-MY3 and MOST 103-2320-B-002-008-MY3).
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K.-S. Shih, J.-P. Liou, Y.-W. Wu, and I.-N. Hsieh contributed equally to this work.
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Supplemental figure 1
The effect of denbinobin on IL-1β-induced ICAM-1, VCAM-1 and NF-κB signals in OA-FLS. (a) OA-FLS were incubated with IL-1β (10 ng/mL) for 1-24 h, and THP-1 labeled with BCECF-AM (10 μM) adhesion to OA-FLS were photographed and quantified. Images represent magnification at 100×. (b) The viability of OA-FLS was evaluated after 12 or 24 h of treatment with denbinobin (0.01-1 μM) by MTT assay. (c) OA-FLS were transfected with miR-146a inhibitor (10 nM) for 24 h, and incubated with denbinobin (1 μM) for another 18 h, followed by treatment with IL-1β (10 ng/mL) for 6 h. Relative expression levels of indicated proteins were determined by western blotting. (d and e) OA-FLS were transfected with miR-146a mimic or inhibitor (10 nM each) for 24 h. IL-1β was then added and incubated for another 30 min. Whole-cell extracts were subjected to western blotting for the indicated proteins. Results are shown as mean ± SEM from three independent experiments; OA-FLS were obtained from eight patients. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the relevant control group. (GIF 149 kb)
Supplemental figure 2
Denbinobin treatment significantly increased miR-146a expression in OA-FLS. OA-FLS were treated with denbinobin (0.01-1 μM) for different periods (1-24 h), and miR-146a levels were measured by real-time PCR. Results represent mean ± SEM from eight independent experiments; OA-FLS were obtained from eight patients. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the relevant control group. (GIF 15 kb)
Supplemental figure 3
Treatment of OA-FLS with IL-1β significantly increased monocyte adhesion by upregulating ICAM-1 and VCAM-1 expression. (a) OA-FLS (2 × 104 cells) were treated with IL-1β (10 ng/mL) for 1–24 h and washed with PBS. THP-1 monocytes (5 × 104 cells) labeled with 10 μM BCECF-AM were then added, and the coculture was continued at 37 °C for 1 h. After incubation, non-adherent cells were removed by gentle washing with PBS, and adherent THP-1 monocytes were photographed and quantified using fluorescence microscopy. Images represent magnification at 100×. (b and c) OA-FLS treated with IL-1β (10 ng/mL) for various periods (1–24 h), and mRNA or protein levels of ICAM-1 and VCAM-1 were determined using real-time PCR (b) and western blot (c). Results in (b) and (c) are shown as median ± SEM from three independent experiments. OA-FLS were obtained from three patients. Kruskal-Wallis test was used to determine the pairs of groups showing statistically significant differences. *p < 0.05, **p < 0.01 and # p < 0.05, ## p < 0.01 compared to the relevant control group. (GIF 103 kb)
Supplemental figure 4
Denbinobin downregulated ICAM-1 and VCAM-1 expression in OA-FLS and inhibited monocyte adhesion. (a) OA-FLS (2 × 104) were incubated with 1 μM denbinobin for 18 h, then stimulated with IL-1β (10 ng/mL) for 6 h. After washing with PBS, THP-1 monocytes (5 × 104) labeled with BCECF-AM (10 μM) were added to OA-FLS for 1 h, and THP-1 monocyte adhesion was photographed and evaluated using fluorescence microscopy. (b and c) OA-FLS incubated with or without denbinobin (0.01–1 μM) for 18 h and treated with IL-1β (10 ng/mL) for 6 h, and mRNA or protein levels of ICAM-1 and VCAM-1 were determined using real-time PCR (b) and western blotting (c). (d) OA-FLS viability was determined after 12 h or 24 h of treatment with 0.01–1 μM of denbinobin using the MTT assay (compared to the control group). Results in a–c represent median ± SEM from three independent experiments; Kruskal-Wallis test was used to determine the pairs of groups showing statistically significant differences. OA-FLS were obtained from four patients. *p < 0.05 and # p < 0.05 compared to the relevant control group; **p < 0.01 compared to the indicated group. (GIF 116 kb)
Supplemental figure 5
Denbinobin inhibited the expression of adhesion molecules by upregulating miR-146a expression in OA-FLS. (a) OA-FLS were treated with denbinobin at the indicated concentrations for different periods (1–24 h), and miR-146a levels were measured using real-time PCR. (b) OA-FLS were transfected with an miR-146a inhibitor for 24 h prior to treatment with denbinobin (1 μM) for another 18 h, followed by treatment with IL-1β (10 ng/mL) for 6 h. Whole-cell extracts were subjected to western blotting for the indicated proteins. (c) OA-FLS treated as explained in (b), washed with PBS, and then incubated with BCECF-AM-labeled THP-1 monocytes for 1 h. Subsequently, THP-1 monocyte adhesion was photographed and evaluated using fluorescence microscopy. Results in (a) and (c) represent median ± SEM from three independent experiments; Kruskal-Wallis test was used to determine the pairs of groups showing statistically significant differences. OA-FLS were obtained from four patients. *p < 0.05, **p < 0.01 compared to the relevant control group; ## p < 0.01 compared to the indicated group. (GIF 126 kb)
Supplemental figure 6
Denbinobin upregulated miR-146a expression by regulating NF-κB-binding sites located within the miR-146a promoter region. (a and d) OA-FLS treated with 1 μM denbinobin for different periods as indicated in A or incubated with or without anacardic acid (50 μM) for 1 h prior to denbinobin (1 μM) treatment for another 12 h (d). Subsequently, nuclear proteins were isolated to determine the inhibition of HAT activity using a commercial kit. (b) Inhibition of HDAC activity in HeLa nuclear extracts by denbinobin was measured using a HDAC activity assay kit. Trichostatin A (TSA; 20 μM) was used as a positive control. (c and e) OA-FLS cells treated as explained in (d), histone H3 acetylation levels at the NF-κB-binding sites I and II located within the miR-146a promoter region were analyzed using a chromatin immunoprecipitation assay (c), and miR-146a expression was determined using real-time PCR (e). Results are shown as mean median ± SEM from three independent experiments; Kruskal-Wallis test was used to determine the pairs of groups showing statistically. OA-FLS were obtained from three patients. *p < 0.05, **p < 0.01 compared to the relevant control group; # p < 0.05, ## p < 0.01 compared to the indicated group. (GIF 73 kb)
Supplemental figure 7
Denbinobin inhibited NF-κB signaling and inflammatory mediator production via miR-146a regulation. (a and b) OA-FLS were transfected with an miR-146a mimic or inhibitor (10 nM each) for 24 h, and the cells were then incubated with denbinobin (0.01–1 μM) for 24 h. IL-1β (10 ng/mL) was then added, and incubation was continued for 30 min. Relative expression levels of IRAK-1 and TRAF6 (a), phospho-IKK, -p65, and -IκB or total proteins (b) were determined using western blotting. (c) OA-FLS were transfected with an miR-146a inhibitor for 24 h prior to treatment with denbinobin (0.1–1 μM) for another 24 h, followed by treatment with IL-1β (10 ng/mL) for 24 h. Supernatants were assayed for IL-6 and PGE2 (c). Results in (c) are shown as median ± SEM from three independent experiments; Kruskal-Wallis test was used to determine the pairs of groups showing statistically. OA-FLS were obtained from three patients. *p < 0.05 and # p < 0.05 compared to the indicated group. (GIF 102 kb)
Supplemental figure 8
Evaluation of ICAM-1, VCAM-1 expressions under mock vector transfection, and determining transfection efficiency in OA-FLS. (A) OA-FLS were transfected with mock vector or miR-146a mimic for 24 h prior to treatment with IL-1β (10 ng/mL) for 6 h. Whole-cell extracts were subjected to western blotting for the indicated proteins. (B) OA-FLS were transfected with pGFP plasmid for 24 h, then transfection efficiency was estimated as percentage of GFP positive cells. (GIF 39 kb)
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Yang, CR., Shih, KS., Liou, JP. et al. Denbinobin upregulates miR-146a expression and attenuates IL-1β-induced upregulation of ICAM-1 and VCAM-1 expressions in osteoarthritis fibroblast-like synoviocytes. J Mol Med 92, 1147–1158 (2014). https://doi.org/10.1007/s00109-014-1192-8
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DOI: https://doi.org/10.1007/s00109-014-1192-8