RESUMO
OBJECTIVE: We and others previously demonstrated that sirtuin 1 (SIRT-1) regulates apoptosis and cartilage-specific gene expression in human chondrocytes and mouse models. This study was undertaken to determine if SIRT-1 enzymatic activity plays a protective role in cartilage homeostasis in vivo, by investigating mice with SIRT-1 mutations to characterize their cartilage. METHODS: Articular cartilage was harvested from the paws and knees of 5- and 6-month-old wild-type (WT) mice and mice homozygous for SIRT-1tm2.1Mcby (SIRT-1y/y), an allele carrying a point mutation that encodes a SIRT-1 protein with no enzymatic activity (y/y mice). Mice ages 2 days old and 6-7 days old were also examined. Mouse joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures. RESULTS: We found that articular cartilage tissue sections from y/y mice of up to 6 months of age contained reduced levels of type II collagen, aggrecan, and glycosaminoglycan compared to sections from WT mice. In contrast, protein levels of matrix metalloproteinase 8 (MMP-8), MMP-9, and MMP-13 were elevated in the cartilage of y/y mice. In addition, chondrocyte apoptosis was elevated in SIRT-1 mutant mice as compared to their WT littermates. Consistent with these observations, protein tyrosine phosphatase 1b was elevated in the y/y mice. CONCLUSION: Our in vivo findings in this animal model demonstrate that mice with defective SIRT-1 also have defective cartilage, with elevated rates of cartilage degradation with age. Hence, normal cartilage homeostasis requires enzymatically active SIRT-1 protein.
Assuntos
Cartilagem/enzimologia , Condrócitos/enzimologia , Homeostase/fisiologia , Osteoartrite/fisiopatologia , Sirtuína 1/metabolismo , Agrecanas , Animais , Técnicas de Cultura de Células , Colágeno Tipo II , Modelos Animais de Doenças , Proteínas da Matriz Extracelular , Feminino , Immunoblotting , Imuno-Histoquímica , Camundongos , Mutação Puntual , Sirtuína 1/genéticaRESUMO
OBJECTIVE: Sirtuin 1 (SirT1) has been implicated in the regulation of human cartilage homeostasis and chondrocyte survival. Exposing human osteoarthritic (OA) chondrocytes to tumor necrosis factor α (TNFα) generates a stable and enzymatically inactive 75-kd form of SirT1 (75SirT1) via cathepsin B-mediated cleavage. Because 75SirT1 is resistant to further degradation, we hypothesized that it has a distinct role in OA, and the present study was undertaken to identify this role. METHODS: The presence of cathepsin B and 75SirT in OA and normal human chondrocytes was analyzed. Confocal imaging of SirT1 was used to monitor its subcellular trafficking following TNFα stimulation. Coimmunofluorescence staining for cathepsin B, mitochondrial cytochrome oxidase subunit IV, and lysosome-associated membrane protein 1 together with SirT1 was performed. Human chondrocytes were tested for apoptosis by fluorescence-activated cell sorter analysis and immunoblotting for caspases 3 and 8. Human chondrocyte mitochondrial extracts were obtained and analyzed for 75SirT1-cytochrome c association. RESULTS: Confocal imaging and immunoblot analyses following TNFα challenge of human chondrocytes demonstrated that 75SirT1 was exported to the cytoplasm and colocalized with the mitochondrial membrane. Consistent with this, immunoprecipitation and immunoblot analyses revealed that 75SirT1 is enriched in mitochondrial extracts and associates with cytochrome c following TNFα stimulation. Preventing nuclear export of 75SirT1 or reducing levels of full-length SirT1 and 75SirT1 augmented chondrocyte apoptosis in the presence of TNFα. Levels of cathepsin B and 75SirT1 were elevated in OA versus normal chondrocytes. Additional analyses showed that human chondrocytes exposed to OA-derived synovial fluid generated the 75SirT1 fragment. CONCLUSION: These data suggest that 75SirT1 promotes chondrocyte survival following exposure to proinflammatory cytokines.
Assuntos
Apoptose/efeitos dos fármacos , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteoartrite do Joelho/metabolismo , Sirtuína 1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Idoso , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Catepsina B/genética , Catepsina B/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Citocromos c/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Expressão Gênica , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Osteoartrite do Joelho/patologia , Osteoartrite do Joelho/cirurgia , Sirtuína 1/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
OBJECTIVE: The protein deacetylase SirT1 positively regulates cartilage-specific gene expression, while the proinflammatory cytokine tumor necrosis factor α (TNFα) negatively regulates these same genes. This study was undertaken to test the hypothesis that SirT1 is adversely affected by TNFα, resulting in altered gene expression. METHODS: Cartilage-specific gene expression, SirT1 activity, and results of chromatin immunoprecipitation analysis at the α2(I) collagen enhancer site were determined in RNA, protein extracts, and nuclei of human osteoarthritic chondrocytes left untreated or treated with TNFα. Protein extracts from human chondrocytes transfected with epitope-tagged SirT1 that had been left untreated or had been treated with TNFα were analyzed by immunoblotting with SirT1 and epitope-specific antibodies. The 75-kd SirT1-reactive protein present in TNFα-treated extracts was identified by mass spectroscopy, and its amino-terminal cleavage site was identified via Edman sequencing. SirT1 activity was assayed following an in vitro cathepsin B cleavage reaction. Cathepsin B small interfering RNA (siRNA) was transfected into chondrocytes left untreated or treated with TNFα. RESULTS: TNFα-treated chondrocytes had impaired SirT1 enzymatic activity and displayed 2 forms of the enzyme: a full-length 110-kd protein and a smaller 75-kd fragment. The 75-kd SirT1 fragment was found to lack the carboxy-terminus. Cathepsin B was identified as the TNFα-responsive protease that cleaves SirT1 at residue 533. Reducing cathepsin B levels via siRNA following TNFα exposure blocked the generation of the 75-kd SirT1 fragment. CONCLUSION: These data indicate that TNFα, a cytokine that mediates joint inflammation in arthritis, induces cathepsin B-mediated cleavage of SirT1, resulting in reduced SirT1 activity. This reduced SirT1 activity correlates with the reduced cartilage-specific gene expression evident in these TNFα-treated cells.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteoartrite/metabolismo , Sirtuína 1/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Osteoartrite/genética , Sirtuína 1/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: The protein deacetylase SirT1 inhibits apoptosis in a variety of cell systems by distinct mechanisms, yet its role in chondrocyte death has not been explored. We undertook the present study to assess the role of SirT1 in the survival of osteoarthritic (OA) chondrocytes in humans. METHODS: SirT1, protein tyrosine phosphatase 1B (PTP1B), and PTP1B mutant expression plasmids as well as SirT1 small interfering RNA (siRNA) and PTP1B siRNA were transfected into primary human chondrocytes. Levels of apoptosis were determined using flow cytometry, and activation of components of the insulin-like growth factor receptor (IGFR)/Akt pathway was assessed using immunoblotting. OA and normal knee cartilage samples were subjected to immunohistochemical analysis. RESULTS: Expression of SirT1 in chondrocytes led to increased chondrocyte survival in either the presence or the absence of tumor necrosis factor alpha/actinomycin D, while a reduction of SirT1 by siRNA led to increased chondrocyte apoptosis. Expression of SirT1 in chondrocytes led to activation of IGFR and the downstream kinases phosphatidylinositol 3-kinase, phosphoinosite-dependent protein kinase 1, mTOR, and Akt, which in turn phosphorylated MDM2, inhibited p53, and blocked apoptosis. Activation of IGFR occurs at least in part via SirT1-mediated repression of PTP1B. Expression of PTP1B in chondrocytes increased apoptosis and reduced IGFR phosphorylation, while down-regulation of PTP1B by siRNA significantly decreased apoptosis. Examination of cartilage from normal donors and OA patients revealed that PTP1B levels are elevated in OA cartilage in which SirT1 levels are decreased. CONCLUSION: For the first time, it has been demonstrated that SirT1 is a mediator of human chondrocyte survival via down-regulation of PTP1B, a potent proapoptotic protein that is elevated in OA cartilage.
Assuntos
Apoptose/fisiologia , Condrócitos/patologia , Osteoartrite/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptores de Somatomedina/metabolismo , Sirtuína 1/metabolismo , Idoso , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Regulação para Baixo/fisiologia , Humanos , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Sirtuína 1/genética , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
OBJECTIVE: We previously demonstrated that Sirt1 regulates apoptosis in cartilage in vitro. Here we attempt to examine in vivo cartilage homeostasis, using Sirt1 total body knockout (KO) mice. METHOD: Articular cartilage was harvested from hind paws of 1-week and 3-week-old mice carrying wild type (WT) or null Sirt1 gene. Knees of Sirt1 haploinsufficient mice also were examined, at 6 months. Joint cartilage was processed for histologic examination or biochemical analyses of chondrocyte cultures. RESULTS: We found that articular cartilage tissue sections from Sirt1 KO mice up to 3 weeks of age exhibited low levels of type 2 collagen, aggrecan, and glycosaminoglycan content. In contrast, protein levels of MMP-13 were elevated in the Sirt1 KO mice, leading to a potential increase of cartilage breakdown, already shown in the heterozygous mice. Additional results showed elevated chondrocyte apoptosis in Sirt1 KO mice, as compared to WT controls. In addition to these observations, PTP1b (protein tyrosine phosphatase b) was elevated in the Sirt1 KO mice, in line with previous reports. CONCLUSION: The findings from this animal model demonstrated that Sirt1 KO mice presented an altered cartilage phenotype, with an elevated apoptotic process and a potential degradative cartilage process.
Assuntos
Cartilagem Articular/química , Osteoartrite/genética , Animais , Apoptose , Doenças das Cartilagens/genética , Doenças das Cartilagens/metabolismo , Doenças das Cartilagens/patologia , Cartilagem Articular/patologia , Condrócitos/fisiologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Homeostase , Camundongos , Camundongos Knockout , Osteoartrite/metabolismo , Osteoartrite/patologia , FenótipoRESUMO
Cartilage oligomeric matrix protein (COMP) is a component of cartilage, synovium, ligament, and tendon, yet its normal function is largely unknown. To identify its function we have expressed it in 293 and HeLa cell lines and in primary human chondrocytes. We find that COMP protects these cells against death, either in the presence or absence of tumor necrosis factor alpha and is able to block activation of caspase 3, a critical effector caspase. This effect appears to be mediated by the IAP (inhibitor of apoptosis protein) family of anti-apoptotic proteins because the levels of XIAP, survivin, cIAP1 and cIAP2 are significantly elevated in the COMP-expressing cells and down-regulation of survivin and XIAP protein levels by small interfering RNAs blocks the ability of COMP to enhance survival. The mRNAs for most of the IAP family members were not increased by COMP, indicating that a translational/post-translational mechanism was involved in their induction. However, in both HeLa cells and chondrocytes, COMP induced survivin mRNA by 5-fold. Thus survivin is the first gene identified to be up-regulated transcriptionally by COMP. The carboxyl-terminal half of the protein comprising the type 3 repeats and the RGD sequence (CaCTD domain) was sufficient to promote survival and to elevate the IAPs. Further, an RGD peptide was able to block the prosurvival effect of COMP and the induction of XIAP and survivin, indicating that survival is likely mediated through integrin signaling. These data point to a new role for COMP in protecting cells against death.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/fisiologia , Proteína de Matriz Oligomérica de Cartilagem , Caspase 3/genética , Caspase 3/metabolismo , Caspase 6/genética , Caspase 6/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular , Células Cultivadas , Condrócitos/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/fisiologia , Glicoproteínas/genética , Glicoproteínas/fisiologia , Células HeLa , Humanos , Immunoblotting , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/fisiologia , Proteínas Matrilinas , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Survivina , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologiaRESUMO
SirT1 is an NAD-dependent histone deacetylase that regulates gene expression, differentiation, development, and organism life span. Here we investigate the function of SirT1 in human chondrocytes derived from osteoarthritic patients. Elevation of SirT1 protein levels or activity in these chondrocytes led to a dramatic increase in cartilage-specific gene expression, whereas a reduction in SirT1 levels or activity significantly lowered cartilage gene expression. SirT1 associated with the cartilage-specific transcription factor Sox9, enhancing transcription from the collagen 2(alpha1) promoter in a Sox9-dependent fashion. Consistent with this association, SirT1 was targeted to the collagen 2(alpha1) enhancer and promoter, which in turn recruited the coactivators GCN5, PGC1alpha, and p300. This led to elevated marks of active chromatin within the promoter; that is, acetylated histone K9/K14 and histone H4K5 as well as trimethylated histone H3K4. Finally, alterations in the NAD salvage pathway enzyme nicotinamide phosphoribosyltransferase led to changes in NAD levels, SirT activity, and cartilage-specific gene expression in human chondrocytes. SirT1, nicotinamide phosphoribosyltransferase, and NAD may, therefore, provide a positive function in human cartilage by elevating expression of genes encoding cartilage extracellular matrix.