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1.
Sci Rep ; 10(1): 1296, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992827

RESUMO

Mitochondrial mutations and dysfunction have been demonstrated in several age-related disorders including osteoarthritis, yet its relative contribution to pathogenesis remains unknown. Here we evaluated whether premature aging caused by accumulation of mitochondrial DNA mutations in PolgD275A mice predisposes to the development of knee osteoarthritis. Compared with wild type animals, homozygous PolgD275A mice displayed a specific bone phenotype characterized by osteopenia of epiphyseal trabecular bone and subchondral cortical plate. Trabecular thickness was significantly associated with osteocyte apoptosis rates and osteoclasts numbers were increased in subchondral bone tissues. While chondrocyte apoptosis rates in articular and growth plate cartilage were similar between groups, homozygous mitochondrial DNA mutator mice displayed elevated numbers of hypertrophic chondrocytes in articular calcified cartilage. Low grade cartilage degeneration, predominantly loss of proteoglycans, was present in all genotypes and the development of osteoarthritis features was not found accelerated in premature aging. Somatically acquired mitochondrial DNA mutations predispose to elevated subchondral bone turnover and hypertrophy in calcified cartilage, yet additional mechanical or metabolic stimuli would seem required for induction and accelerated progression of aging-associated osteoarthritis.


Assuntos
Senilidade Prematura , Doenças Ósseas Metabólicas , Condrócitos , Polimerase do DNA Mitocondrial , Mutação de Sentido Incorreto , Osteoartrite , Senilidade Prematura/enzimologia , Senilidade Prematura/genética , Senilidade Prematura/patologia , Substituição de Aminoácidos , Animais , Doenças Ósseas Metabólicas/enzimologia , Doenças Ósseas Metabólicas/genética , Doenças Ósseas Metabólicas/patologia , Condrócitos/enzimologia , Condrócitos/patologia , Polimerase do DNA Mitocondrial/genética , Polimerase do DNA Mitocondrial/metabolismo , Hipertrofia , Camundongos , Camundongos Mutantes , Osteoartrite/enzimologia , Osteoartrite/genética , Osteoartrite/patologia
2.
Oxid Med Cell Longev ; 2019: 6404035, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781343

RESUMO

Objective: The integrity of cartilage depends on the correct synthesis of extracellular matrix (ECM) components. In case of insufficient folding of proteins in the endoplasmic reticulum (ER) of chondrocytes, ECM proteins aggregate, ER stress evolves, and the unfolded protein response (UPR) is initiated. By this mechanism, chondrocytes relieve the stress condition or initiate cell death by apoptosis. Especially persistent ER stress has emerged as a pathogenic mechanism in cartilage diseases, such as chondrodysplasias and osteoarthritis. As pharmacological intervention is not available yet, it is of great interest to understand cartilage ER stress in detail and to develop therapeutics to intervene. Methods: ERp57-deficient chondrocytes were generated by CRISPR/Cas9-induced KO. ER stress and autophagy were studied on mRNA and protein level as well as by transmission electron microscopy (TEM) in chondrocyte micromass or cartilage explant cultures of ERp57 KO mice. Thapsigargin (Tg), an inhibitor of the ER-residing Ca2+-ATPase, and 4-Phenylbutyric acid (4-PBA), a small molecular chemical chaperone, were applied to induce or inhibit ER stress. Results: Our data reveal that the loss of the protein disulfide isomerase ERp57 is sufficient to induce ER stress in chondrocytes. 4-PBA efficiently diffuses into cartilage explant cultures and diminishes excessive ER stress in chondrocytes dose dependently, no matter if it is induced by ERp57 KO or stimulation with Tg. Conclusion: ER-stress-related diseases have different sources; therefore, various targets for therapeutic treatment exist. In the future, 4-PBA may be used alone or in combination with other drugs for the treatment of ER-stress-related skeletal disorders in patients.


Assuntos
Apoptose/efeitos dos fármacos , Cartilagem/enzimologia , Condrócitos/enzimologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fenilbutiratos/farmacologia , Isomerases de Dissulfetos de Proteínas/deficiência , Animais , Apoptose/genética , Cartilagem/citologia , Linhagem Celular , Condrócitos/citologia , Estresse do Retículo Endoplasmático/genética , Camundongos , Camundongos Knockout , Isomerases de Dissulfetos de Proteínas/metabolismo
3.
Cell Mol Biol (Noisy-le-grand) ; 65(6): 91-95, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31472053

RESUMO

Osteoarthritis (OA) is a degenerative joint disease usually seen in the elderly, which incidence increases with age. Its pathogenesis and underlying mechanism are still unclear. The disease severely affects the physical health and life quality of patients, thereby constituting a huge economic burden to family and society. Luteolin (LUT) is a natural flavonoid with multiple pharmacological properties. Many plants containing LUT have been applied in the treatment of several inflammation-related diseases due the relatively strong anti-inflammatory effects of LUT. The present study investigated the influence of LUT on cell apoptosis and inflammatory reactions in cartilage of OA guinea pigs, and its underlying mechanism. It was found that LUT effectively inhibited proliferation of OA cartilage cells, down-regulated the expressions of JNK and p38MAPK in cartilage cells of OA, and downregulated NO, TNF-α and IL-6. Thus, it alleviated inflammatory reactions, protected cartilage cells, and delayed cartilage degeneration.


Assuntos
Apoptose/efeitos dos fármacos , Condrócitos/enzimologia , Condrócitos/patologia , Luteolina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Osteoartrite/enzimologia , Osteoartrite/patologia , Animais , Osso e Ossos/patologia , Cartilagem/patologia , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Cobaias , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Articulações/patologia , Lipopolissacarídeos , Luteolina/química
4.
J Orthop Res ; 37(11): 2429-2436, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31304988

RESUMO

Knee injuries cause structural damage and acute inflammation that initiates the development of post-traumatic osteoarthritis (PTOA). NADPH oxidase 4 (Nox4), a member of a family of enzymes that generates reactive oxygen species (ROS), plays a pivotal role in normal development of the musculoskeletal system, but may increase ROS production to harmful levels after joint injury. The role of ROS in both normal joint homeostasis and injury is poorly understood, but inhibition of excessive ROS production by Nox4 after joint injury could be protective to the joint, decreasing oxidative stress, and initiation of PTOA. Knee injuries were simulated using inflammatory cytokines in cultured primary human chondrocytes and a non-invasive mouse model of PTOA in C57BL/6N and Nox4 knockout mice. There is an acute decrease in Nox4 activity within 24 h after injury in both systems, followed by a subsequent sustained low-level increase, a novel finding not seen in any other system. Inhibition of Nox4 activity by GKT137831 was protective against early structural changes after non-invasive knee injury in a mouse model. Nox4 knockout mice had significant differences in structural and mechanical properties of bone, providing further evidence for the role of Nox4 in development of joint tissues and biochemical response after joint injury. Nox4 plays a significant role in the acute phase after joint injury, and targeted inhibition of inflammation caused by Nox4 may be protective against early joint changes in the pathogenesis of PTOA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2429-2436, 2019.


Assuntos
Lesões do Ligamento Cruzado Anterior/complicações , Condrócitos/enzimologia , NADPH Oxidase 4/metabolismo , Osteoartrite do Joelho/enzimologia , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Adolescente , Adulto , Animais , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Camundongos Knockout , Pessoa de Meia-Idade , NADPH Oxidase 4/antagonistas & inibidores , NADPH Oxidase 4/genética , Osteoartrite do Joelho/etiologia , Osteoartrite do Joelho/prevenção & controle , Cultura Primária de Células , Pirazóis/farmacologia , Piridinas/farmacologia , Adulto Jovem
6.
Int J Mol Med ; 43(5): 2241-2251, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896805

RESUMO

Osteoarthritis (OA) is the most common type of degenerative joint disease and secreted inflammatory molecules serve a pivotal role in it. Peimine has been reported to have anti­inflammatory activity. In order to investigate the potential therapeutic role of Peimine in OA, mouse articular chondrocytes were treated with IL­1ß and different doses of Peimine in vitro. The data revealed that Peimine not only suppressed IL­1ß­induced production of nitric oxide (NO) and prostaglandin E2, but also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase­2 (COX­2). In addition, Peimine inhibited the IL­1ß­induced mRNA expression of matrix metalloproteinase (MMP)­1, MMP­3, MMP­9, MMP­13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)­4 and ADAMTS­5. Furthermore, Peimine inhibited IL­1ß­induced activation of the mitogen­activated protein kinase (MAPK) pathway. The protective effect of Peimine on IL­1ß­treated chondrocytes was attenuated following activation of the MAPK pathway, as demonstrated by the increased expression levels of MMP­3, MMP­13, ADAMTS­5, iNOS and COX­2 compared with the Peimine group. The in vivo data suggested that Peimine limited the development of OA in the mouse model. In general, the data indicate that Peimine suppresses IL­1ß­induced inflammation in mouse chondrocytes by inhibiting the MAPK pathway, suggesting a promising therapeutic role for Peimine in the treatment of OA.


Assuntos
Cevanas/uso terapêutico , Condrócitos/enzimologia , Condrócitos/patologia , Regulação para Baixo , Inflamação/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas ADAMTS/metabolismo , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Sobrevivência Celular/efeitos dos fármacos , Cevanas/farmacologia , Condrócitos/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Inflamação/patologia , Interleucina-1beta , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Osteoartrite/patologia
7.
Bioorg Med Chem ; 27(6): 1034-1042, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30773420

RESUMO

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC50 values and inhibition constants (Ki) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.


Assuntos
Catepsina K/antagonistas & inibidores , Desdiferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Nitrilos/farmacologia , Inibidores de Proteases/farmacologia , Animais , Compostos Aza/síntese química , Compostos Aza/química , Compostos Aza/farmacologia , Catepsina K/metabolismo , Linhagem Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/enzimologia , Desenho de Fármacos , Humanos , Camundongos , Nitrilos/síntese química , Nitrilos/química , Inibidores de Proteases/síntese química , Inibidores de Proteases/química
8.
Nature ; 566(7743): 254-258, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30728500

RESUMO

Osteoarthritis-the most common form of age-related degenerative whole-joint disease1-is primarily characterized by cartilage destruction, as well as by synovial inflammation, osteophyte formation and subchondral bone remodelling2,3. However, the molecular mechanisms that underlie the pathogenesis of osteoarthritis are largely unknown. Although osteoarthritis is currently considered to be associated with metabolic disorders, direct evidence for this is lacking, and the role of cholesterol metabolism in the pathogenesis of osteoarthritis has not been fully investigated4-6. Various types of cholesterol hydroxylases contribute to cholesterol metabolism in extrahepatic tissues by converting cellular cholesterol to circulating oxysterols, which regulate diverse biological processes7,8. Here we show that the CH25H-CYP7B1-RORα axis of cholesterol metabolism in chondrocytes is a crucial catabolic regulator of the pathogenesis of osteoarthritis. Osteoarthritic chondrocytes had increased levels of cholesterol because of enhanced uptake, upregulation of cholesterol hydroxylases (CH25H and CYP7B1) and increased production of oxysterol metabolites. Adenoviral overexpression of CH25H or CYP7B1 in mouse joint tissues caused experimental osteoarthritis, whereas knockout or knockdown of these hydroxylases abrogated the pathogenesis of osteoarthritis. Moreover, retinoic acid-related orphan receptor alpha (RORα) was found to mediate the induction of osteoarthritis by alterations in cholesterol metabolism. These results indicate that osteoarthritis is a disease associated with metabolic disorders and suggest that targeting the CH25H-CYP7B1-RORα axis of cholesterol metabolism may provide a therapeutic avenue for treating osteoarthritis.


Assuntos
Colesterol/metabolismo , Família 7 do Citocromo P450/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Osteoartrite/metabolismo , Esteroide Hidroxilases/metabolismo , Animais , Transporte Biológico , Condrócitos/enzimologia , Condrócitos/metabolismo , Masculino , Camundongos , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Osteoartrite/enzimologia , Osteoartrite/patologia , Oxisteróis/metabolismo , Esteroide Hidroxilases/deficiência , Regulação para Cima
9.
Ann Rheum Dis ; 78(3): 421-428, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30610061

RESUMO

OBJECTIVE: Osteoarthritis (OA) appears to be associated with various metabolic disorders, but the potential contribution of amino acid metabolism to OA pathogenesis has not been clearly elucidated. Here, we explored whether alterations in the amino acid metabolism of chondrocytes could regulate OA pathogenesis. METHODS: Expression profiles of amino acid metabolism-regulating genes in primary-culture passage 0 mouse chondrocytes were examined by microarray analysis, and selected genes were further characterised in mouse OA chondrocytes and OA cartilage of human and mouse models. Experimental OA in mice was induced by destabilisation of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing catabolic regulators. The functional consequences of arginase II (Arg-II) were examined in Arg2-/- mice and those subjected to IA injection of an adenovirus encoding Arg-II (Ad-Arg-II). RESULTS: The gene encoding Arg-II, an arginine-metabolising enzyme, was specifically upregulated in chondrocytes under various pathological conditions and in OA cartilage from human patients with OA and various mouse models. Adenovirus-mediated overexpression of Arg-II in mouse joint tissues caused OA pathogenesis, whereas genetic ablation of Arg2 in mice (Arg2 -/-) abolished all manifestations of DMM-induced OA. Mechanistically, Arg-II appears to cause OA cartilage destruction at least partly by upregulating the expression of matrix-degrading enzymes (matrix metalloproteinase 3 [MMP3] and MMP13) in chondrocytes via the nuclear factor (NF)-κB pathway. CONCLUSIONS: Our results indicate that Arg-II is a crucial regulator of OA pathogenesis in mice. Although chondrocytes of human and mouse do not identically, but similarly, respond to Arg-II, our results suggest that Arg-II could be a therapeutic target of OA pathogenesis.


Assuntos
Arginase/fisiologia , Artrite Experimental/enzimologia , Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Osteoartrite/enzimologia , Animais , Artrite Experimental/induzido quimicamente , Modelos Animais de Doenças , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Osteoartrite/induzido quimicamente , Regulação para Cima
10.
J Cell Physiol ; 234(5): 6042-6053, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30500068

RESUMO

Chondrocyte apoptosis has been implicated as a major pathological osteoarthritis (OA) change in humans and experimental animals. We evaluate the ability of miR-186 on chondrocyte apoptosis and proliferation in OA and elucidate the underlying mechanism concerning the regulation of miR-186 in OA. Gene expression microarray analysis was performed to screen differentially expressed messenger RNAs (mRNAs) in OA. To validate the effect of miR-186 on chondrocyte apoptosis, we upregulated or downregulated endogenous miR-186 using mimics or inhibitors. Next, to better understand the regulatory mechanism for miR-186 governing SPP1, we suppressed the endogenous expression of SPP1 by small interfering RNA (siRNA) against SPP1 in chondrocytes. We identified SPP1 is highly expressed in OA according to an mRNA microarray data set GSE82107. After intra-articular injection of papain into mice, the miR-186 is downregulated while the SPP1 is reciprocal, with dysregulated PI3K-AKT pathway in OA cartilages. Intriguingly, miR-186 was shown to increase chondrocyte survival, facilitate cell cycle entry in OA chondrocytes, and inhibit chondrocyte apoptosis in vitro by modulation of pro- and antiapoptotic factors. The determination of luciferase activity suggested that miR-186 negatively targets SPP1. Furthermore, we found that the effect of miR-186 suppression on OA chondrocytes was lost when SPP1 was suppressed by siRNA, suggesting that miR-186 affected chondrocytes by targeting and depleting SPP1, a regulator of PI3K-AKT pathway. Our findings reveal a novel mechanism by which miR-186 inhibits chondrocyte apoptosis in OA by interacting with SPP1 and regulating PI3K-AKT pathway. Restoring miR-186 might be a future therapeutic strategy for OA.


Assuntos
Apoptose , Artrite Experimental/enzimologia , Condrócitos/enzimologia , Articulações/enzimologia , MicroRNAs/metabolismo , Osteoartrite/enzimologia , Osteopontina/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/genética , Artrite Experimental/patologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Proliferação de Células , Condrócitos/patologia , Bases de Dados Genéticas , Regulação para Baixo , Humanos , Articulações/patologia , Masculino , Camundongos , MicroRNAs/genética , Células NIH 3T3 , Osteoartrite/induzido quimicamente , Osteoartrite/genética , Osteoartrite/patologia , Osteopontina/genética , Papaína , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , Transdução de Sinais
11.
J Cell Physiol ; 234(6): 9156-9167, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30311192

RESUMO

Mechanical stress plays a key role in regulating cartilage degradation in osteoarthritis (OA). The aim of this study was to evaluate the effects and mechanisms of mechanical stress on articular cartilage. A total of 80 male Sprague-Dawley rats were randomly divided into eight groups (n = 10 for each group): control group (CG), OA group (OAG), and CG or OAG subjected to low-, moderate-, or high-intensity treadmill exercise (CL, CM, CH, OAL, OAM, and OAH, respectively). Chondrocytes were obtained from the knee joints of rats; they were cultured on Bioflex 6-well culture plates and subjected to different durations of cyclic tensile strain (CTS) with or without exposure to interleukin-1ß (IL-1ß). The results of the histological score, immunohistochemistry, enzyme-linked immunosorbent assay, and western-blot analyses indicated that there were no differences between CM and CG, but OAM showed therapeutic effects compared with OAG. However, CH and OAH experienced more cartilage damage than CG and OAG, respectively. CTS had no therapeutic effects on collagen II of normal chondrocytes, which is consistent with findings after treadmill exercise. However, CTS for 4 hr could alleviate the chondrocyte damage induced by IL-1ß by activating AMP-activated protein kinase (AMPK) phosphorylation and suppressing nuclear translocation of nuclear factor (NF)-κB p65. Our findings indicate that mechanical stress had no therapeutic effects on normal articular cartilage and chondrocytes; mechanical stress only caused damage with excessive stimulation. Still, moderate biomechanical stress could reduce sensitization to the inflammatory response of articular cartilage and chondrocytes through the AMPK/NF-κB signaling pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Artrite Experimental/prevenção & controle , Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Terapia por Exercício , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Artrite Experimental/enzimologia , Artrite Experimental/patologia , Artrite Experimental/fisiopatologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Cartilagem Articular/fisiopatologia , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Interleucina-1beta/farmacologia , Masculino , Fosforilação , Ratos Sprague-Dawley , Corrida , Transdução de Sinais , Estresse Mecânico
12.
Matrix Biol ; 78-79: 32-46, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-29425696

RESUMO

The story of hyaluronan in articular cartilage, pericellular hyaluronan in particular, essentially is also the story of aggrecan. Without properly tethered aggrecan, the load bearing function of cartilage is compromised. The anchorage of aggrecan to the cell surface only occurs due to the binding of aggrecan to hyaluronan-with hyaluronan tethered either to a hyaluronan synthase or by multivalent binding to CD44. In this review, details of hyaluronan synthesis are discussed including how HAS2 production of hyaluronan is necessary for normal chondrocyte development and matrix assembly, how an abundance or deficit of pericellular hyaluronan alters chondrocyte metabolism, and whether hyaluronan size matters or changes with aging or disease. The biomechanical role and matrix assembly function of hyaluronan in addition to the functions of hyaluronidases are discussed. The turnover of hyaluronan is considered including mechanisms by which its turnover, at least in part, is mediated by endocytosis by chondrocytes and regulated by aggrecan degradation. Differences between turnover and clearance of newly synthesized hyaluronan and aggrecan versus the half-life of hyaluronan remaining within the inter-territorial matrix of cartilage are discussed. The release of neutral pH-acting hyaluronidase activity remains one unanswered question concerning the loss of cartilage hyaluronan in osteoarthritis. Signaling events driven by changes in hyaluronan-chondrocyte interactions may involve a chaperone function of CD44 with other receptors/cofactors as well as the changes in hyaluronan production functioning as a metabolic rheostat.


Assuntos
Condrócitos/citologia , Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , Animais , Condrócitos/enzimologia , Condrócitos/metabolismo , Endocitose , Matriz Extracelular/metabolismo , Meia-Vida , Humanos
13.
J Tissue Eng Regen Med ; 13(4): 537-545, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30552734

RESUMO

Plasma fibronectin (pFN) plays a crucial role in wound healing by binding to integrins and inducing cell migration. It is known to induce the migration and proliferation of mesenchymal progenitor cells in vitro, which play a key role during microfracture in cartilage repair. Endogenous chondrocytes from the native cartilage of the defect rim might aid in cartilage repair. In this study, the effect of pFN on proliferation, migration, and differentiation was tested on human articular chondrocytes. Results showed that treatment with pFN increased the migration of chondrocytes in a range of 1-30 µg/ml as tested with no effect on proliferation. TGFß3-induced chondrogenesis was not affected by pFN. Especially, gene expression of matrix metalloproteinases was not increased by pFN. Plasma FN fragmentation due to storage conditions could be excluded by SDS-PAGE. Moreover, bioactivity of pFN did not alter during storage at 4°C and 40°C for up to 14 days. Taken together, pFN induces the migration but not proliferation of human articular chondrocytes with no inhibitory effect on chondrogenic differentiation. Additionally, no loss of activity or fragmentation of pFN was observed after lyophilization and storage, making pFN an interesting bioactive factor for chondrocyte recruitment.


Assuntos
Cartilagem Articular/citologia , Diferenciação Celular , Movimento Celular , Condrócitos/citologia , Fibronectinas/sangue , Adulto , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Feminino , Fibronectinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , Proteoglicanas/metabolismo
14.
Biomed Pharmacother ; 106: 1169-1174, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30119184

RESUMO

Osteoarthritis (OA) is a degenerative joint disease that is commonly accompanied by inflammation. Scoparone is a biologically active constituent isolated from Artemisia capillaris and possesses anti-inflammatory activity. However, the effect of scoparone on inflammatory response in OA has not been authenticated. The aim of this study was to evaluate the role of scoparone in OA in vitro. Our results showed that IL-1ß treatment significantly inhibited the cell viability of chondrocytes, whereas the inhibition effect was attenuated by scoparone in a dose-dependent manner. IL-1ß also efficiently induced the production of nitric oxide (NO), prostaglandin E2 (PGE2), MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 in chondrocytes. However, scoparone dose-dependently suppressed the induction. In addition, scoparone repressed IL-1ß-induced the expression of iNOS and COX-2 in chondrocytes. Furthermore, the activation of the PI3K/Akt/NF-κB pathway induced by IL-1ß was diminished by scoparone treatment. Taken together, these findings indicated that scoparone inhibited the expression of inflammatory mediators in IL-1ß-induced chondrocytes via regulating the PI3K/Akt/NF-κB pathway. Thus, scoparone may be used as a new therapeutic agent for the treatment of OA.


Assuntos
Anti-Inflamatórios/farmacologia , Condrócitos/efeitos dos fármacos , Cumarínicos/farmacologia , Mediadores da Inflamação/metabolismo , Interleucina-1beta/farmacologia , NF-kappa B/metabolismo , Osteoartrite do Joelho/tratamento farmacológico , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas ADAMTS/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/enzimologia , Condrócitos/patologia , Relação Dose-Resposta a Droga , Humanos , Metaloproteinases da Matriz Secretadas/metabolismo , Pessoa de Meia-Idade , Osteoartrite do Joelho/enzimologia , Osteoartrite do Joelho/patologia , Fosforilação , Transdução de Sinais/efeitos dos fármacos
15.
Biomed Pharmacother ; 107: 433-439, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30103115

RESUMO

Osteoarthritis (OA) is a common articular disease that features cartilage loss and destruction. It has been confirmed that inflammation plays major roles in the progression of osteoarthritis. Caffeic acid (CA), a key dietary nutrient commonly found in coffee, has shown its anti-inflammatory properties in various inflammation diseases. However, the effects of CA in osteoarthritis remain explored. Here we investigated the effects of CA on IL-1ß induced increased expression of inflammatory factors as well as the degradation of Collagen II and aggrecan in rat chondrocytes. CA prevented the cartilage damage induced by IL-1ß in vivo organ culture of articular cartilage. Besides, the IL-1ß induced increased production of inflammation factors such as iNOS and COX2 could be inhibited by CA. Additionally, CA could also suppress IL-1ß induced expression of cartilage matrix catabolic enzymes such as ADAMTS5 and MMPs. Moreover, CA could prevent IL-1ß induced degradation of Collagen II and aggrecan in chondrocytes. Furthermore, CA inhibited NF-κB activity and the activation of JNK pathway. This study reveals that CA inhibits IL-1ß induced inflammation responses through suppression of NF-κB and MAPK related JNK signaling pathways. These results demonstrate that CA may provide new avenues for osteoarthritis treatment in future.


Assuntos
Ácidos Cafeicos/farmacologia , Cartilagem Articular/patologia , Condrócitos/patologia , Inflamação/patologia , Interleucina-1beta/efeitos adversos , Substâncias Protetoras/farmacologia , Proteína ADAMTS5/metabolismo , Agrecanas/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Colágeno Tipo II/metabolismo , Ciclo-Oxigenase 2/metabolismo , Interleucina-1beta/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos , Ratos Sprague-Dawley
16.
Int J Exp Pathol ; 99(3): 113-120, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-30073722

RESUMO

Precartilaginous stem cells (PSCs) are adult stem cells which could self-renew or differentiate into chondrocytes to promote bone growth. In this study, we aimed to understand the role of transforming growth factor-ß1 (TGF-ß1) in precartilaginous stem cell (PSC) differentiation and to study the mechanisms that underlie this role. We purified PSCs from the neonatal murine perichondrial mesenchyme using immunomagnetic beads, and primary cultured them. Their phenotype was confirmed by the PSC marker fibroblast growth factor receptor-3 (FGFR-3) overexpression. TGF-ß1 was added to induce PSCs differentiation. TGF-ß1 increased mRNA expression of chondrogenesis-related genes (collagen type II, Sox 9 and aggrecan) in the cultured PSCs. This was abolished by TGF-ß receptor II (TGFRII) and Casein kinase 1 epsilon (CK1ε) lentiviral shRNA depletion. Meanwhile, we found that TGF-ß1 induced CK1ε activation, glycogen synthase kinase-3ß (GSK3ß) phosphorylation and ß-catenin nuclear translocation in the mouse PSCs, which was almost completely blocked by TGFRII and CK1ε shRNA knockdown. Based on these results, we suggest that TGF-ß1 induces CK1ε activation to promote ß-catenin nuclear accumulation, which then regulates chondrogenesis-related gene transcription to eventually promote mouse PSC differentiation.


Assuntos
Células-Tronco Adultas/efeitos dos fármacos , Caseína Quinase Iépsilon/metabolismo , Diferenciação Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Receptor do Fator de Crescimento Transformador beta Tipo II/agonistas , Fator de Crescimento Transformador beta1/farmacologia , beta Catenina/metabolismo , Células-Tronco Adultas/enzimologia , Agrecanas/genética , Agrecanas/metabolismo , Animais , Animais Recém-Nascidos , Caseína Quinase Iépsilon/genética , Diferenciação Celular/genética , Células Cultivadas , Condrócitos/enzimologia , Condrogênese/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos Endogâmicos C57BL , Técnicas de Transferência Nuclear , Fenótipo , Fosforilação , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
J Biol Chem ; 293(31): 12259-12270, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-29929979

RESUMO

Certain dysregulated chondrocyte metabolic adaptive responses such as decreased activity of the master regulator of energy metabolism AMP-activated protein kinase (AMPK) promote osteoarthritis (OA). Metabolism intersects with epigenetic and transcriptional responses. Hence, we studied chondrocyte ATP-citrate lyase (ACLY), which generates acetyl-CoA from mitochondrial-derived citrate, and modulates acetylation of histones and transcription factors. We assessed ACLY in normal and OA human knee chondrocytes and cartilages by Western blotting and immunohistochemistry, and quantified acetyl-CoA fluorometrically. We examined histone and transcription factor lysine acetylation by Western blotting, and assessed histone H3K9 and H3K27 occupancy of iNOS, MMP3, and MMP13 promoters by chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR). We analyzed iNOS, MMP3, MMP13, aggrecan (ACAN), and Col2a1 gene expression by RT-qPCR. Glucose availability regulated ACLY expression and function, nucleocytosolic acetyl-CoA, and histone acetylation. Human knee OA chondrocytes exhibited increased ACLY activation (assessed by Ser-455 phosphorylation), associated with increased H3K9 and H3K27 acetylation. Inhibition of ACLY attenuated IL-1ß-induced transcription of iNOS, MMP3, and MMP13 by suppressing acetylation of p65 NF-κB, H3K9, and H3K27, blunted release of NO, MMP3, and MMP13, and also reduced SOX9 acetylation that promoted SOX9 nuclear translocation, leading to increased aggrecan and Col2a1 mRNA expression. ACLY is a novel player involved in regulation of cartilage matrix metabolism. Increased ACLY activity in OA chondrocytes increased nucleocytosolic acetyl-CoA, leading to increased matrix catabolism via dysregulated histone and transcription factor acetylation. Pharmacologic ACLY inhibition in OA chondrocytes globally reverses these changes and stimulates matrix gene expression and AMPK activation, supporting translational investigation in OA.


Assuntos
ATP Citrato (pro-S)-Liase/metabolismo , Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Matriz Extracelular/enzimologia , Osteoartrite do Joelho/enzimologia , ATP Citrato (pro-S)-Liase/genética , Acetilcoenzima A/metabolismo , Acetilação , Agrecanas/genética , Agrecanas/metabolismo , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Histonas/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/metabolismo
18.
Biomed Pharmacother ; 102: 1099-1104, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29710527

RESUMO

Excessive expression of matrix metalloproteinases (MMPs) induced by pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) has been associated with destruction of the articular cartilage matrix in chondrocytes from patients with osteoarthritis (OA). Among the MMPs, MMP-1, MMP-3 and MMP-13 participate in the degradation of type II collagen, the main component of the extracellular matrix in articular cartilage. Sodium butyrate is a bacterial metabolite used in the treatment of inflammatory diseases. However, the effects of sodium butyrate on the expression of MMPs and degradation of type II collagen has not been explored. In the current study, for the first time, we aimed to determine whether sodium butyrate influences IL-1ß-induced degradation of type II collagen in human chondrocytes in the articular cartilage matrix. Our results indicate that sodium butyrate significantly abrogated IL-1ß-induced up-regulation of MMP-1, MMP-3, and MMP-13 at both the gene and protein levels. In addition, treatment with sodium butyrate suppressed expression of the endogenous tissue inhibitors TIMP-1 and TIMP-2. Notably, sodium butyrate attenuated IL-1ß-induced degradation of type II collagen. Mechanistically, we found that sodium butyrate abolished the activation of NF-κB by inhibiting phosphorylation of IKK, IκBα, and NF-κB p65. Based on these observations, we conclude that butyrate may have potential as a therapeutic agent for OA.


Assuntos
Ácido Butírico/farmacologia , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Proteólise/efeitos dos fármacos , Proteínas ADAMTS/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Humanos , Interleucina-1beta/farmacologia , Metaloproteinases da Matriz/metabolismo , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Regulação para Cima/efeitos dos fármacos
19.
Osteoarthritis Cartilage ; 26(8): 1110-1117, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29803826

RESUMO

OBJECTIVES: The chondrocytes' pericellular matrix acts as a mechanosensor by sequestering growth factors that are bound to heparan sulfate (HS) proteoglycans. Heparanase is the sole mammalian enzyme with HS degrading endoglycosidase activity. Here, we aimed to ascertain whether heparanase plays a role in modulating the anabolic or catabolic responses of human articular chondrocytes. METHODS: Primary chondrocytes were incubated with pro-heparanase and catabolic and anabolic gene expression was analyzed by quantitative polymerase chain reaction (PCR). MMP13 enzymatic activity in the culture medium was measured with a specific fluorescent assay. Extracellular regulated kinase (ERK) phosphorylation was evaluated by Western blot. Human osteoarthritis (OA) cartilage was assessed for heparanase expression by reverse-transcriptase PCR, by Western blot and by a heparanase enzymatic activity assay. RESULTS: Cultured chondrocytes rapidly associated with and activated pro-heparanase. Heparanase induced the catabolic genes MMP13 and ADAMTS4 and the secretion of active MMP13, and down-regulated the anabolic genes ACAN and COL2A1. PG545, a HS-mimetic, inhibited the effects of heparanase. Heparanase expression and enzymatic activity were demonstrated in adult human osteoarthritic cartilage. Heparanase induced ERK phosphorylation in cultured chondrocytes and this could be inhibited by PG545, by fibroblast growth factor 2 (FGF2) neutralizing antibodies and by a FGF-receptor inhibitor. CONCLUSIONS: Heparanase is active in osteoarthritic cartilage and induces catabolic responses in primary human chondrocytes. This response is due, at least in part, to the release of soluble growth factors such as FGF2.


Assuntos
Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Glucuronidase/metabolismo , Osteoartrite/enzimologia , Adulto , Western Blotting , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Int J Mol Sci ; 19(5)2018 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-29747385

RESUMO

Osteoarthritis (OA) is the most common and increasing joint disease worldwide. Current treatment for OA is limited to control of symptoms. The purpose of this study was to determine the effect of specificity protein 1 (SP1) inhibitor Mithramycin A (MitA) on chondrocyte catabolism and OA pathogenesis and to explore the underlying molecular mechanisms involving SP1 and other key factors that are critical for OA. Here, we show that MitA markedly inhibited expressions of matrix-degrading enzymes induced by pro-inflammatory cytokine interleukin-1β (IL-1β) in mouse primary chondrocytes. Intra-articular injection of MitA into mouse knee joint alleviated OA cartilage destruction induced by surgical destabilization of the medial meniscus (DMM). However, modulation of SP1 level in chondrocyte and mouse cartilage did not alter catabolic gene expression or cartilage integrity, respectively. Instead, MitA significantly impaired the expression of HIF-2α known to be critical for OA pathogenesis. Such reduction in expression of HIF-2α by MitA was caused by inhibition of NF-κB activation, at least in part. These results suggest that MitA can alleviate OA pathogenesis by suppressing NF-κB-HIF-2α pathway, thus providing insight into therapeutic strategy for OA.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Osteoartrite/tratamento farmacológico , Plicamicina/análogos & derivados , Animais , Cartilagem Articular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Condrócitos/metabolismo , Progressão da Doença , Indução Enzimática/efeitos dos fármacos , Interleucina-1beta/farmacologia , Articulações/patologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Osteoartrite/enzimologia , Osteoartrite/patologia , Plicamicina/administração & dosagem , Plicamicina/farmacologia , Plicamicina/uso terapêutico , Fator de Transcrição Sp1/metabolismo
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