Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea.
Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, South Korea.
CKD Research Institute, Yongin-si, Gyeonggido, South Korea.
Division of Mol Medicine of Musculoskeletal Tissue, University Munster, Munster, Germany.
Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea. ysong@snu.ac.kr.
Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, South Korea. ysong@snu.ac.kr.
Medical Research Center, Institute of Human-Environment Interface Biology, Seoul National University, Seoul, South Korea. ysong@snu.ac.kr.
Abstract
BACKGROUND To investigate the effects of inhibiting histone deacetylase (HDAC) 6 on inflammatory responses and tissue-destructive functions of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA).
METHODS FLS from RA patients were activated with interleukin (IL)-1β in the presence of increasing concentrations of M808, a novel specific HDAC6 inhibitor. Production of ILs, chemokines, and metalloproteinases (MMPs) was measured in ELISAs. Acetylation of tubulin and expression of ICAM-1 and VCAM-1 were assessed by Western blotting. Wound healing and adhesion assays were performed. Cytoskeletal organization was visualized by immunofluorescence. Finally, the impact of HDAC6 inhibition on the severity of arthritis and joint histology was examined in a murine model of adjuvant-induced arthritis (AIA).
RESULTS HDAC6 was selectively inhibited by M808. The HDAC6 inhibitor suppressed the production of MMP-1, MMP-3, IL-6, CCL2, CXCL8, and CXCL10 by RA-FLS in response to IL-1β. Increased acetylation of tubulin was associated with decreased migration of RA-FLS. Inhibiting HDAC6 induced cytoskeletal reorganization in RA-FLS by suppressing the formation of invadopodia following activation with IL-1β. In addition, M808 tended to decrease the expression of ICAM-1 and VCAM-1. In the AIA arthritis model, M808 improved the clinical arthritis score in a dose-dependent manner. Also, HDAC6 inhibition was associated with less severe synovial inflammation and joint destruction.
CONCLUSION Inhibiting HDAC6 dampens the inflammatory and destructive activity of RA-FLS and reduces the severity of arthritis. Thus, targeting HDAC6 has therapeutic potential.
Fig. 1M808 is a selective HDAC6 inhibitor. a Selectivity of M808 for HDAC6. Inhibition of HDAC is dependent on the M808 concentration (IC50 values are shown). b Kinetics of M808 were determined by measuring the activity of HDAC6 in the presence of M808. c Cells were incubated with IL-1β and increasing concentrations of M808. Cell viability was then measured. AMC, RHKK-AC-AMC; IC50, the half maximal inhibitory concentration; IL, interleukin; Ki, inhibitory constant; ND, not determined; S, substrate; V, velocityFig. 2Inhibition of HDAC6 reduces the secretion of MMP-1, MMP-3, and IL-6 by fibroblast-like synoviocytes (FLS). a RA-FLS were activated with IL-1β (10 ng/mL) in the presence of HDAC6 inhibitor M808 and tubastatin A. The expression of HDAC6, total and acetylated cortactin, and total and acetylated α-tubulin were examined by Western blotting. A representative blot from three independent experiments is shown. Acetylated cortactin and acetylated tubulin relative to the respective total protein were shown (n = 3). Data represent the mean ± SEM. Groups were compared using a t-test. *p < 0.05 vs. FLS treated with IL-1β only. b FLS from RA patients (n = 6) were pre-treated for 1 h with M808 and then stimulated with IL-1β (10 ng/mL) for 24 h. The production of IL-6, MMP-1, and MMP-3 was measured in an ELISA. Data represent the mean ± SEM. Groups were compared using a t-test. *p < 0.05 vs. FLS treated with IL-1β only. AU, arbitrary unit; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; MMP, metalloproteinases; Tuba A, tubastatin AFig. 3HDAC6 inhibition suppresses migration of fibroblast-like synoviocytes (FLS). a Cell migration assay. RA-FLS (n = 6) were treated for 1 h with M808, followed by stimulation with IL-1β for 72 h. Migration of RA-FLS in the presence of varying concentrations of M808 is depicted. Shown are representative images from six independent experiments (left panel). Original magnification × 40. The number of migrating RA-FLS in each group was compared using a t-test (right panel). b Following treatment with IL-1β in the presence or absence of M808 (5 μM), FLS were stained for F-actin (green) and cortactin (red). Nuclei were stained with DAPI (blue). Immunofluorescence images are representative of five independent experiments; invadopodia (yellow dots) are seen in the merged panels. Bar = 20 μm. The number of invadopodia per cell was compared between groups using a t-test (right panel). Data represented the mean ± SEMFig. 4M808 inhibits chemotaxis and adhesion of inflammatory cells to RA-fibroblast-like synoviocytes (FLS). a RA-FLS (n = 6) were pre-treated with M808 for 1 h, followed by stimulation with IL-1β for 24 h. The amounts of CCL-2, CXCL-8, and CXCL-10 in culture supernatants were measured in an ELISA. b ICAM-1 and VCAM-1 expression by RA-FLS was examined by Western blotting. The image is representative of three independent experiments (left panel). The expression of ICAM-1 and VCAM-1 relative to GAPDH were quantified (middle and right panels). Data represent the mean ± SEM. c, d Adhesion of calcein-labeled U937 and Jurkat cells to RA-FLS after treatment with the HDAC6 inhibitor (n = 6). Data represent the mean ± SEM. *p < 0.05 vs. cells treated with IL-1β. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; Tuba, tubastatin AFig. 5M808 increases body weight and reduces the clinical score of arthritis in an AIA model. a Experimental design. Arthritis was induced in Lewis rats as described in the “Methods” section. Rats received a daily dose of M808 (0, 3, 10, 30, 50, or 100 mg/kg; n = 9, 9, 9, 7, 7, and 8, respectively), starting on the day before CFA injection. b, c Weight (b) and clinical scores over the treatment period are shown. P values were generated using repeated measures ANOVA. d Synovial inflammation, bone erosion, and cartilage damage were graded after histological examination. e Representative histology images. Data represent the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the M808 0 mg/kg group. CFA, complete Freud’s adjuvant
그림 출처: PubMed Central 공개 논문 (PMC8256575)
Conflict of interest statement
The authors declare that they have no competing interests.
