Inhibition of Histone Deacetylase 6 by Tubastatin A Attenuates the Progress of Osteoarthritis via Improving Mitochondrial Function

Abstract

Because chondrocytes are the only resident cells in articular cartilage, the steady state of these cells is important for the maintenance of joint function. In various osteoarthritis diseases, chondrocytes undergo a series of pathophysiologic changes, leading to the loss of chondrocytes and the degradation of extracellular matrix (ECM). This study found that Cytoplasmic localized histone deacetylase 6 (HDAC6) is up-regulated on the articular surface in a destabilization of the medial meniscus–induced mouse osteoarthritis model. Because HDAC6 is highly related to the acetylation of tubulin and the function of the microtubule system is closely related to material transport and signal transduction, the relationship between the expression level or activity of HDAC6 and the fate of chondrocytes in vitro and in vivo were confirmed. Primary chondrocytes overexpressing DNA-HDAC6 with plasmid were constructed in vitro, and HDAC6 inhibitor Tubastatin A was selected to inhibit HDAC6 enzyme activity in vivo and in vitro. Subsequently, mitochondrial spatial arrangement, degradation of ECM, and pathological changes in joint were defined. The results indicate that overexpression of HDAC6 causes mitochondrial dysfunction and promotes reactive oxygen species production, leading to degradation of ECM. Tubastatin A treatment after osteoarthritis ameliorates the degradation of cartilage and improves the microenvironment and function of the joint. HDAC6 may be targeted to treat osteoarthritis. Because chondrocytes are the only resident cells in articular cartilage, the steady state of these cells is important for the maintenance of joint function. In various osteoarthritis diseases, chondrocytes undergo a series of pathophysiologic changes, leading to the loss of chondrocytes and the degradation of extracellular matrix (ECM). This study found that Cytoplasmic localized histone deacetylase 6 (HDAC6) is up-regulated on the articular surface in a destabilization of the medial meniscus–induced mouse osteoarthritis model. Because HDAC6 is highly related to the acetylation of tubulin and the function of the microtubule system is closely related to material transport and signal transduction, the relationship between the expression level or activity of HDAC6 and the fate of chondrocytes in vitro and in vivo were confirmed. Primary chondrocytes overexpressing DNA-HDAC6 with plasmid were constructed in vitro, and HDAC6 inhibitor Tubastatin A was selected to inhibit HDAC6 enzyme activity in vivo and in vitro. Subsequently, mitochondrial spatial arrangement, degradation of ECM, and pathological changes in joint were defined. The results indicate that overexpression of HDAC6 causes mitochondrial dysfunction and promotes reactive oxygen species production, leading to degradation of ECM. Tubastatin A treatment after osteoarthritis ameliorates the degradation of cartilage and improves the microenvironment and function of the joint. HDAC6 may be targeted to treat osteoarthritis. Osteoarthritis is a common degenerative joint disease characterized by joint pain and movement disorders. With the aging and obesity of the population, this symptom is more prevalent than in the past few decades.1Jones I.A. Togashi R. Wilson M.L. Heckmann N. Vangsness Jr., C.T. Intra-articular treatment options for knee osteoarthritis.Nat Rev Rheumatol. 2019; 15: 77-90Crossref PubMed Scopus (90) Google Scholar, 2Nelson A.E. Osteoarthritis year in review 2017: clinical.Osteoarthritis Cartilage. 2018; 26: 319-325Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 3Hunter D.J. Bierma-Zeinstra S. Osteoarthritis.Lancet. 2019; 393: 1745-1759Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar Articular cartilage is a highly specialized tissue composed of only one cell type, chondrocytes, which function in dissipating applied forces and lubricate joint movement.4Jay G.D. Elsaid K.A. Kelly K.A. Anderson S.C. Zhang L. Teeple E. Waller K. Fleming B.C. Prevention of cartilage degeneration and gait asymmetry by lubricin tribosupplementation in the rat following anterior cruciate ligament transection.Arthritis Rheum. 2012; 64: 1162-1171Crossref PubMed Scopus (62) Google Scholar During the progression of osteoarthritis, chondrocytes undergo a series of pathophysiologic phenomena, such as inflammation, oxidative stress, and apoptosis, which may lead to a reduction in cell density, abnormal secretory activity, and degradation of the extracellular matrix (ECM) to further promote the process of osteoarthritis.5Goutas A. Syrrou C. Papathanasiou I. Tsezou A. Trachana V. The autophagic response to oxidative stress in osteoarthritic chondrocytes is deregulated.Free Radic Biol Med. 2018; 126: 122-132Crossref PubMed Scopus (20) Google Scholar, 6Khan N.M. Ahmad I. Haqqi T.M. Nrf2/ARE pathway attenuates oxidative and apoptotic response in human osteoarthritis chondrocytes by activating ERK1/2/ELK1-P70S6K-P90RSK signaling axis.Free Radic Biol Med. 2018; 116: 159-171Crossref PubMed Scopus (60) Google Scholar, 7Li Y.S. Xiao W.F. Luo W. Cellular aging towards osteoarthritis.Mech Ageing Dev. 2017; 162: 80-84Crossref PubMed Scopus (32) Google Scholar Therefore, maintaining the cellular homeostasis of chondrocytes in patients with osteoarthritis may be an effective therapeutic strategy. Cytoskeleton is a cellular structure that orchestrates cellular events and is predominantly composed of actin microfilaments, tubulin microtubules, vimentin, and nuclear lamin intermediate filaments.8Benjamin M. Archer C.W. Ralphs J.R. Cytoskeleton of cartilage cells.Microsc Res Tech. 1994; 28: 372-377Crossref PubMed Scopus (102) Google Scholar Among these elements, the microtubule system, which is mainly composed of tubulin, plays an important role in maintaining cell morphology, cell division, signal transduction, and material transportation. Previous studies in several cell types, including fibroblasts,9Diegelmann R.F. Peterkofsky B. Inhibition of collagen secretion from bone and cultured fibroblasts by microtubular disruptive drugs.Proc Natl Acad Sci U S A. 1972; 69: 892-896Crossref PubMed Scopus (231) Google Scholar,10Bauer E.A. Valle K.J. Colchicine-induced modulation of collagenase in human skin fibroblast cultures, I; stimulation of enzyme synthesis in normal cells.J Invest Dermatol. 1982; 79: 398-402Abstract Full Text PDF PubMed Scopus (40) Google Scholar synoviocytes,11Harris Jr., E.D. Krane S.M. Effects of colchicine on collagenase in cultures of rheumatoid synovium.Arthritis Rheum. 1971; 14: 669-684Crossref PubMed Scopus (136) Google Scholar and fetal chondrocytes,12Jansen H.W. Bornstein P. Effects of antimicrotubular agents on glycosaminoglycan synthesis and secretion by embryonic chick cartilage and chondrocytes.Biochim Biophys Acta. 1974; 362: 150-159Crossref PubMed Scopus (32) Google Scholar,13Bodo M. Carinci P. Baroni T. Becchetti E. Bellucci C. Pezzetti F. Giammarioli M. Stabellini G. Arena N. Collagen synthesis and cell growth in chick embryo fibroblasts: influence of colchicine, cytochalasin B and concanavalin A.Cell Biol. Int. 1996; 20: 177-185Crossref PubMed Scopus (6) Google Scholar have found that in vitro drug-induced disruption of the microtubule system exhibits a reduction in the synthesis and secretion of collagen and proteoglycan, suggesting that the regulation of the microtubule system may serve as a potential strategy for curing osteoarthritis. The function of the microtubule system is mainly regulated by posttranslational modifications of tubulin, such as acetylation, tyrosination, polyglutamylation, and polyglycylation.14Janke C. Bulinski J.C. Post-translational regulation of the microtubule cytoskeleton: mechanisms and functions.Nat Rev Mol Cell Biol. 2011; 12: 773-786Crossref PubMed Scopus (550) Google Scholar,15Bigman L.S. Levy Y. Tubulin tails and their modifications regulate protein diffusion on microtubules.Proc Natl Acad Sci U S A. 2020; 117: 8876-8883Crossref PubMed Scopus (10) Google Scholar According to a previous study, acetylation of tubulin is implicated in regulating the stability and function of the microtubule system and thus functions in a variety of cellular activities.16Hubbert C. Guardiola A. Shao R. Kawaguchi Y. Ito A. Nixon A. Yoshida M. Wang X.F. Yao T.P. HDAC6 is a microtubule-associated deacetylase.Nature. 2002; 417: 455-458Crossref PubMed Scopus (1606) Google Scholar As a member of the histone deacetylase family, histone deacetylase 6 (HDAC6) does not bind to histones like other HDACs do but selectively binds to α-tubulin and other substrates in the cytoplasm.16Hubbert C. Guardiola A. Shao R. Kawaguchi Y. Ito A. Nixon A. Yoshida M. Wang X.F. Yao T.P. HDAC6 is a microtubule-associated deacetylase.Nature. 2002; 417: 455-458Crossref PubMed Scopus (1606) Google Scholar,17Zheng Z. Zhou Y. Ye L. Lu Q. Zhang K. Zhang J. Xie L. Wu Y. Xu K. Zhang H. Xiao J. Histone deacetylase 6 inhibition restores autophagic flux to promote functional recovery after spinal cord injury.Exp Neurol. 2020; 324: 113138Crossref PubMed Scopus (9) Google Scholar Studies on the overexpression of HDAC6 in macrophages and epithelial cells found that the overexpression of HDAC6 significantly induced the production of reactive oxygen species (ROS) and inflammatory cascade through the up-regulation of NADPH oxidase expression and activity.18Youn G.S. Lee K.W. Choi S.Y. Park J. Overexpression of HDAC6 induces pro-inflammatory responses by regulating ROS-MAPK-NF-kappaB/AP-1 signaling pathways in macrophages.Free Radic Biol Med. 2016; 97: 14-23Crossref PubMed Scopus (68) Google Scholar,19Wang J. Zhao L. Wei Z. Zhang X. Wang Y. Li F. Fu Y. Liu B. Inhibition of histone deacetylase reduces lipopolysaccharide-induced-inflammation in primary mammary epithelial cells by regulating ROS-NF-small ka, CyrillicB signaling pathways.Int Immunopharmacol. 2018; 56: 230-234Crossref PubMed Scopus (8) Google Scholar Recent studies found that the selective HDAC6 inhibitor ricolinostat (ACY-1215) not only inhibits expression of matrix-degrading proteases, including matrix metallopeptidase (MMP)-1 and MMP-13 in chondrocytes, but also prevents aberrant subchondral bone formation through down-regulating vascular endothelial growth factor expression in osteoblasts.20Li L. Liu F. Huang W. Wang J. Wan Y. Li M. Pang Y. Yin Z. Ricolinostat (ACY-1215) inhibits VEGF expression via PI3K/AKT pathway and promotes apoptosis in osteoarthritic osteoblasts.Biomed Pharmacother. 2019; 118: 109357Crossref PubMed Scopus (10) Google Scholar,21Cheng C. Shan W. Huang W. Ding Z. Cui G. Liu F. Lu W. Xu J. He W. Yin Z. ACY-1215 exhibits anti-inflammatory and chondroprotective effects in human osteoarthritis chondrocytes via inhibition of STAT3 and NF-kappaB signaling pathways.Biomed Pharmacother. 2019; 109: 2464-2471Crossref PubMed Scopus (16) Google Scholar Nevertheless, in view of the off-target effect and multitarget effect of inhibitors, whether HDAC6 interferes with the generation of ROS and participates in the disease deterioration during the progression of osteoarthritis remains to be further explored. This study focuses on the variation of HDAC6 expression level in chondrocytes in a mouse model of destabilization of the medial meniscus (DMM). Subsequently, plasmid-loading DNA-HDAC6 or the optimized HDAC6 inhibitor Tubastatin A was applied to overexpress HDAC6 or block HDAC6 activity to observe the changes in the fate of the chondrocytes. Finally, the mice were injected intraperitoneally with Tubastatin A to confirm its effect on progression of osteoarthritis, aiming to confirm HDAC6 as a potential therapeutic target for osteoarthritis. A total of 48 healthy 8-week–old, male, C57BL/6OlaCnc mice (20 to 25 g) were used for experiments. Sample size was not determined statistically before experimentation. Samples were not randomized. No criteria were used to include or exclude date points or participants. The animals were housed in the OptiMICE Rotary Experimental Animal Cage System (catalog number C89100; Animal Care Systems Inc., Centennial, CO) under standard conditions, including adequate temperature and humidity (60%) and a 12-hour light/12-hour dark cycle. The mice had free access to water and food. 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B. and a HDAC6 PubMed Scopus Google Scholar The results of indicate that the acetylation level of α-tubulin in a and a and In cellular that Tubastatin A exhibits Therefore, of Tubastatin A was applied in in vitro experiments. with the HDAC6 overexpression the treatment of Tubastatin A the of tubulin in primary by HDAC6 overexpression and a more of cellular in with the of tubulin acetylation for mitochondrial spatial was used to the mitochondrial and in primary chondrocytes overexpressing HDAC6 with or Tubastatin A. The results indicate that the overexpression of HDAC6 not to to the cell like the and but to in the Tubastatin A treatment significantly of the cell with the HDAC6 overexpression and In the results of of ROS that the of Tubastatin A the ROS level by HDAC6 overexpression to a level to that of the and the A and indicate that the overexpression of HDAC6 in primary chondrocytes results in a of the expression of and collagen and a up-regulation of the expression level of MMP-13 with the and the treatment of Tubastatin A the effect by HDAC6 overexpression The results the effect of HDAC6 on degradation and mitochondrial in primary chondrocytes in vitro. Tubastatin A were injected mice intraperitoneally to observe the effect of inhibition of HDAC6 enzyme activity on the osteoarthritis model. by and and that the osteoarthritis exhibits cartilage surface tissue and tissue with the treatment with Tubastatin A to inhibit enzyme activity of HDAC6 inhibits degradation of cartilage surface tissue and tissue A and B with on the system and system also that the treatment of Tubastatin A significantly after osteoarthritis and of the knee in mice for that induced with the the of Tubastatin A significantly and the after A and B of the in knee joint in mice found that treatment with Tubastatin A significantly joint The results of the of the ROS with a in the joint and the glycosaminoglycan in the cartilage tissue indicate with the causes a in ROS level but glycosaminoglycan the of Tubastatin A in the osteoarthritis model significantly the level of ROS and the of glycosaminoglycan in the joint and are the only resident cells of articular cartilage, and the steady state of their is of for the maintenance of articular cartilage This study found that the expression of HDAC6 in cells of the articular surface in the mouse osteoarthritis model. HDAC6 is a localized histone deacetylase that deacetylase activity and Z. Zhou Y. Ye L. Lu Q. Zhang K. Zhang J. Xie L. Wu Y. Xu K. Zhang H. Xiao J. Histone deacetylase 6 inhibition restores autophagic flux to promote functional recovery after spinal cord injury.Exp Neurol. 2020; 324: 113138Crossref PubMed Scopus (9) Google C. K. M. S. the between and cell signaling by acetylation and 26: PubMed Scopus Google Scholar The acetylation of tubulin by deacetylase activity of HDAC6 is of for the regulation of the function of the microtubule Tubulin acetylation is in material signal transduction, and cell is that acetylation of tubulin transport of the spatial of T. M. T. Lee H. J. T. S. spatial of promotes of the 14: PubMed Scopus Google J.R. K.J. and on Cell Biol. PubMed Scopus Google Scholar Lee Y. Yao T.P. in mitochondrial and Cell Biol. PubMed Scopus Google Scholar that and bind HDAC6 to In a in cells mitochondrial activity as the primary HDAC6 as a of J. X. R. Wang Q. Wu Z. S.C. M. Li H. of of mitochondrial function by a in PubMed Scopus Google Scholar the effect of HDAC6 on in the of osteoarthritis remains to be to the role of chondrocytes in the articular primary chondrocytes overexpressing HDAC6 were with a plasmid in vitro. the of the expression level of the HDAC6 protein and the of acetylation of α-tubulin were overexpressing their the cell and the primary The regulation of activity, and stability of was in mitochondrial and cell apoptosis, and human Y. Zhang Z. H. of mitochondrial and Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar was that mitochondrial including variation in mitochondrial activity or is implicated in the of osteoarthritis and may cartilage E. P. F. J. activity is in osteoarthritic human articular Rheum. PubMed Scopus Google I. C. The role of in osteoarthritis.Nat Rev Rheumatol. 2011; PubMed Scopus Google Scholar Nevertheless, is a of on the spatial of in the of osteoarthritis. mitochondrial dysfunction after osteoarthritis mitochondrial remains to be the role of HDAC6 in chondrocytes after osteoarthritis, the HDAC6 inhibitor Tubastatin A was selected to with the enzyme activity of The in vitro results indicate that the treatment of primary chondrocytes overexpressing HDAC6 with Tubastatin A significantly the acetylation of the number of expression level of the spatial of and ROS Because studies have that mitochondrial function degradation of cartilage after X. Q. X. L. Wu J. Zhang W. Shao H. P. Liu F. Wang F. of of inhibits cartilage matrix via 2018; PubMed Scopus Google Cell and apoptosis in osteoarthritic PubMed Scopus Google Scholar was and found that the overexpression of HDAC6 induced the degradation of of collagen and and of the of Tubastatin A the In in vivo that inhibition of HDAC6 activity by of Tubastatin A with in the mouse osteoarthritis model inhibit cartilage of and joint and Because studies have found that of osteoarthritis model mice treatment effect on the progression of Q. X. L. S. Zhang Z. X. Ye B. Wan S. The ameliorates through the of the pathway in a rat model of knee 2019; PubMed Scopus Google Scholar the is not the degradation of cartilage, the matrix protein glycosaminoglycan in cartilage was to confirm the effect of Tubastatin A on the degradation of the in the osteoarthritis In view of the relationship between mitochondrial dysfunction and ROS production and the role of ROS in the of R. Jr., K. M.L. The of of in vivo in osteoarthritic a role in Rheum. PubMed Scopus Google Scholar the of ROS was by in and found that Tubastatin A treatment after significantly ROS results that HDAC6 may the mitochondrial spatial to mitochondrial thus the degradation of cartilage and deterioration of the microenvironment after osteoarthritis. number of studies have found that changes also cartilage and promote the of R.F. Ageing and the of osteoarthritis.Nat Rev Rheumatol. 2016; 12: PubMed Scopus Google Scholar Because of the important role of mitochondrial dysfunction and oxidative stress in HDAC6 may also serve as a target to in the regulation of mitochondrial homeostasis in osteoarthritis induced by Nevertheless, HDAC6 not only promotes tubulin but also participates in the the effect of HDAC6 on chondrocytes in the of osteoarthritis only on its deacetylase activity not HDAC6 improves the spatial of remains Because the inhibition of HDAC6 activity to promote the of autophagic M. K. M. I. C. A. N. J. inhibition improves autophagic and function to loss of in a mouse model of Med. 2018; PubMed Scopus Google Scholar this inhibition may promote to The inhibition of HDAC6 may also on the mitochondrial signal pathway to promote mitochondrial J. X. R. Wang Q. Wu Z. S.C. M. Li H. of of mitochondrial function by a in PubMed Scopus Google Scholar this study that the expression of HDAC6 is in chondrocytes during the osteoarthritis and the up-regulation of HDAC6 is with the abnormal of inhibition of HDAC6 activity by Tubastatin A improves the of to mitochondrial function through α-tubulin and the number of which may to degradation of the and the progression of osteoarthritis Therefore, HDAC6 is as a of mitochondrial function and a potential target for treatment of osteoarthritis and the of Tubastatin A to treat osteoarthritis. the in are not as

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