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1.
FASEB J ; 37(9): e23133, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37566478

RESUMO

Pathways leading to osteoarthritis (OA) are diverse depending on the risk factors involved; thus, developing OA therapeutics has been challenging. Here we report that nuclear protein-1 (Nupr1), a stress-inducible protein/transcription factor, is activated by pathways associated with obesity and aging in chondrocytes. Treatment of human chondrocytes with free fatty acids (palmitate and oleate; a model for high-fat diet/obesity) induced PERK signaling and increased expression of caspase-3, TRB3, and Nupr1. On the other hand, treatment of chondrocytes with menadione (oxidative stress inducer) induced oxidation of IRE1, activated antioxidant response (higher Nrf2 expression), and increased expression of Nupr1 and matrix metalloproteinases. Experimental OA was induced by destabilization of the medial meniscus (DMM) in the knee joints of Nupr1+/+ and Nupr1-/- mice. Loss of Nupr1 expression reduced the severity of cartilage lesions in this model. Together, our findings suggest that Nupr1 is a common factor activated by signaling pathways activated by obesity (ER stress) and age (oxidative stress) and a potential drug target for OA resulting from various risk factors.


Assuntos
Cartilagem Articular , Osteoartrite , Animais , Humanos , Camundongos , Envelhecimento , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Proteínas Nucleares/metabolismo , Obesidade/metabolismo , Osteoartrite/metabolismo
2.
Methods Mol Biol ; 2413: 145-154, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35044662

RESUMO

Immunoprecipitation of protein complexes, also known as co-immunoprecipitation (Co-IP), is a powerful technique to analyze protein-protein interactions. Commercial availability of Dynabeads® Protein A magnetic beads provides a fast, convenient, and efficient method for protein interaction studies by Co-IP followed by immunoblotting (Co-IP-blotting). Recently, the Co-IP-blotting technique helped us to investigate complicated protein interactions/networks involving nuclear protein 1 (Nupr1), a recently discovered regulator of apoptosis in human cartilage cells. The methods and protocols for Co-IP-blotting are reported here in detail.


Assuntos
Imunoprecipitação , Humanos
3.
Sci Rep ; 11(1): 10469, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006989

RESUMO

Reduced knee weight-bearing from prescription or sedentary lifestyles are associated with cartilage degradation; effects on the meniscus are unclear. Rodents exposed to spaceflight or hind limb unloading (HLU) represent unique opportunities to evaluate this question. This study evaluated arthritic changes in the medial knee compartment that bears the highest loads across the knee after actual and simulated spaceflight, and recovery with subsequent full weight-bearing. Cartilage and meniscal degradation in mice were measured via microCT, histology, and proteomics and/or biochemically after: (1) ~ 35 days on the International Space Station (ISS); (2) 13-days aboard the Space Shuttle Atlantis; or (3) 30 days of HLU, followed by a 49-day weight-bearing readaptation with/without exercise. Cartilage degradation post-ISS and HLU occurred at similar spatial locations, the tibial-femoral cartilage-cartilage contact point, with meniscal volume decline. Cartilage and meniscal glycosaminoglycan content were decreased in unloaded mice, with elevated catabolic enzymes (e.g., matrix metalloproteinases), and elevated oxidative stress and catabolic molecular pathway responses in menisci. After the 13-day Shuttle flight, meniscal degradation was observed. During readaptation, recovery of cartilage volume and thickness occurred with exercise. Reduced weight-bearing from either spaceflight or HLU induced an arthritic phenotype in cartilage and menisci, and exercise promoted recovery.


Assuntos
Cartilagem Articular/fisiopatologia , Membro Posterior/fisiopatologia , Articulação do Joelho/fisiopatologia , Osteoartrite do Joelho/fisiopatologia , Fenótipo , Voo Espacial , Animais , Feminino , Glicosaminoglicanos/análise , Masculino , Menisco/química , Menisco/fisiopatologia , Camundongos , Modelos Animais , Suporte de Carga
4.
PLoS One ; 16(2): e0247237, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33617553

RESUMO

Increased intake of dietary saturated fatty acids has been linked to obesity and the development of Osteoarthritis (OA). However, the mechanism by which these fats promote cartilage degradation and the development of OA is not clearly understood. Here, we report the effects of consumption of common dietary saturated and unsaturated fatty acids, palmitate and oleate, respectively, on body weight, metabolic factors, and knee articular cartilage in a mouse model of diet-induced obesity. Mice fed on a diet rich in saturated or unsaturated fatty acid gained an equal amount of weight; however, mice fed a palmitate diet, but not a control or oleate diet, exhibited more cartilage lesions and increased expression of 1) unfolded protein response (UPR)/endoplasmic reticulum (ER) stress markers including BIP, P-IRE1α, XBP1, ATF4, and CHOP; 2) apoptosis markers CC3 and C-PARP; and 3) negative cell survival regulators Nupr1 and TRB3, in knee articular cartilage. Palmitate-induced apoptosis was confirmed by TUNEL staining. Likewise, dietary palmitate was also increased the circulatory levels of classic proinflammatory cytokines, including IL-6 and TNF-α. Taken together, our results demonstrate that increased weight gain is not sufficient for the development of obesity-linked OA and suggest that dietary palmitate promotes UPR/ER stress and cartilage lesions in mouse knee joints. This study validates our previous in vitro findings and suggests that ER stress could be the critical metabolic factor contributing to the development of diet/obesity induced OA.


Assuntos
Cartilagem Articular/efeitos dos fármacos , Ácidos Graxos/efeitos adversos , Articulação do Joelho/efeitos dos fármacos , Palmitatos/efeitos adversos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Cartilagem Articular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Dieta/efeitos adversos , Articulação do Joelho/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/induzido quimicamente , Osteoartrite/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Cartilage ; 13(2_suppl): 1442S-1455S, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-32940061

RESUMO

OBJECTIVE: Meniscus injury and the hypoxia-inducible factor (HIF) pathway are independently linked to osteoarthritis pathogenesis, but the role of the meniscus HIF pathway remains unclear. We sought to identify and evaluate HIF pathway response in normal and osteoarthritic meniscus and to examine the effects of Epas1 (HIF-2α) insufficiency in mice on early osteoarthritis development. METHODS: Normal and osteoarthritic human meniscus specimens were obtained and used for immunohistochemical evaluation and cell culture studies for the HIF pathway. Meniscus cells were treated with pro-inflammatory stimuli, including interleukins (IL)-1ß, IL-6, transforming growth factor (TGF)-α, and fibronectin fragments (FnF). Target genes were also evaluated with HIF-1α and HIF-2α (Epas1) overexpression and knockdown. Wild-type (n = 36) and Epas1+/- (n = 30) heterozygous mice underwent destabilization of the medial meniscus (DMM) surgery and were evaluated at 2 and 4 weeks postoperatively for osteoarthritis development using histology. RESULTS: HIF-1α and HIF-2α immunostaining and gene expression did not differ between normal and osteoarthritic meniscus. While pro-inflammatory stimulation significantly increased both catabolic and anabolic gene expression in the meniscus, HIF-1α and Epas1 expression levels were not significantly altered. Epas1 overexpression significantly increased Col2a1 expression. Both wild-type and Epas1+/- mice developed osteoarthritis following DMM surgery. There were no significant differences between genotypes at either time point. CONCLUSION: The HIF pathway is likely not responsible for osteoarthritic changes in the human meniscus. Additionally, Epas1 insufficiency does not protect against osteoarthritis development in the mouse at early time points after DMM surgery. The HIF pathway may be more important for protection against catabolic stress.


Assuntos
Menisco , Osteoartrite , Animais , Condrócitos/metabolismo , Hipóxia/metabolismo , Hipóxia/patologia , Meniscos Tibiais/patologia , Menisco/metabolismo , Camundongos , Osteoartrite/metabolismo
6.
Aging Dis ; 11(5): 1091-1102, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33014525

RESUMO

Aging is a major risk factor for the development of osteoarthritis (OA). One hallmark of aging is loss of proteostasis resulting in increased cellular stress and cell death. However, its effect on the development of OA is not clear. Here, using knee articular cartilage tissue from young and old cynomolgus monkeys (Macaca fascicularis), we demonstrate that with aging there is loss of molecular chaperone expression resulting in endoplasmic reticulum (ER) stress and cell death. Chondrocytes from aged articular cartilage showed decreased expression of molecular chaperones, including protein disulfide isomerase, calnexin, and Ero1-like protein alpha, and increased immunohistochemical staining for ER stress markers (phosphorylated IRE1 alpha, spliced X-box binding protein-1, activating transcription factor 4 and C/EBP homologous protein), and apoptotic markers [cleaved caspase 3 and cleaved poly(ADP-ribose) polymerase], suggesting that decreased expression of molecular chaperone during aging induces ER stress and chondrocyte apoptosis in monkey articular cartilage. Apoptosis induced by aging-associated ER stress was further confirmed by TUNEL staining. Aged monkey cartilage also showed increased expression of nuclear protein 1 (Nupr1) and tribbles related protein-3 (TRB3), known regulators of apoptosis and cell survival pathways. Treatment of cultured monkey chondrocytes with a small molecule chemical chaperone, 4-phenylbutyric acid (PBA, a general ER stress inhibitor) or PERK Inhibitor I (an ER stress inhibitor specifically targeting the PERK branch of the unfolded protein response pathway), decreased the expression of ER stress and apoptotic markers and reduced the expression of Nupr1 and TRB3. Consistent with the above finding, knockdown of calnexin expression induces ER stress and apoptotic markers in normal human chondrocytes in vitro. Taken together, our study clearly demonstrates that aging promotes loss of proteostasis and induces ER stress and chondrocyte apoptosis in articular cartilage. Thus, restoring proteostasis using chemical/molecular chaperone or ER stress inhibitor could be a therapeutic option to treat aged-linked OA.

7.
FASEB J ; 34(4): 5818-5826, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32124494

RESUMO

Mice fed a high-fat diet (HFD) become obese and develop osteoarthritis (OA)-like lesions, including chondrocyte apoptosis, in the knee joints. However, the mechanism by which HFD/obesity induces chondrocyte apoptosis is not clearly understood. In the present study, male mice were fed a low-fat diet (LFD, 10% kcal), HFD (45% kcal), or a HFD administered with 0.5 g/kg bodyweight of 4-phenyl butyric acid (PBA, a small chaperone known to ease endoplasmic reticulum [ER] stress), via the drinking water. At the end of the 18-week study, stifle (knee) joints from all animals were collected, fixed, paraffin embedded, and sectioned. Immunostaining of joints from the HFD group showed increased expression of ER stress and apoptotic markers and increased expression of nuclear protein 1 and tribbles related protein-3 compared to the LFD group. Mice on HFD also showed higher percentage of chondrocyte death, lower chondrocyte numbers per cartilage area, and thickening of subchondral bone. Administration of PBA alleviated all of the HFD-induced symptoms. Our study demonstrated that HFD induces ER stress to promote chondrocyte death and subchondral bone thickening, which could be relieved by alleviating ER stress via PBA administration, suggesting that ER stress could play an important role in obesity-linked OA and could be targeted for OA therapeutics.


Assuntos
Apoptose , Condrócitos/patologia , Dieta Hiperlipídica/efeitos adversos , Estresse do Retículo Endoplasmático , Articulação do Joelho/patologia , Osteoartrite/patologia , Animais , Condrócitos/metabolismo , Articulação do Joelho/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/etiologia
8.
Biosci Rep ; 39(2)2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30674641

RESUMO

Obesity, a major risk factor for the development of osteoarthritis (OA), is associated with increased circulating levels of free fatty acids (FFA). However, the role of these FFAs in OA pathophysiology is not clearly understood. In the present study, we found that palmitate treatment of human primary articular chondrocytes increased the expression of ER stress markers [activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP)] and apoptosis markers [cytochrome c and cleaved caspase-3 (CC3)]. Palmitate treatment also increased the expression of Nuclear protein 1 (Nupr1) and tribbles related protein 3 (TRB3), which are known negative regulators of cell survival pathways. Knockdown of Nupr1 or CHOP expression inhibited palmitate mediated increased expression of TRB3 and CC3, indicating that Nupr1 and CHOP cooperate to regulate cell survival and apoptotic pathways in human chondrocytes. Nupr1 knockdown had no effect on CHOP expression whereas CHOP knockdown abolished the palmitate-mediated Nupr1 expression, indicating that CHOP is functional upstream to Nupr1 in this pathway. Moreover, overexpression of Nupr1 markedly increased the basal expression of pro-apoptotic molecules, including cytochrome c and CC3. Taken together, our study demonstrates that Nupr1 plays a crucial role in palmitate-induced apoptosis in human chondrocytes and Nupr1 as a potential novel drug target for the treatment of OA.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Condrócitos/metabolismo , Condrócitos/patologia , Proteínas de Neoplasias/metabolismo , Palmitatos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cartilagem Articular/citologia , Caspase 3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Condrócitos/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Neoplasias/genética , Osteoartrite/etiologia , Palmitatos/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
9.
J Biol Chem ; 293(42): 16376-16389, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30190325

RESUMO

Reactive oxygen species (ROS), in particular H2O2, regulate intracellular signaling through reversible oxidation of reactive protein thiols present in a number of kinases and phosphatases. H2O2 has been shown to regulate mitogen-activated protein kinase (MAPK) signaling depending on the cellular context. We report here that in human articular chondrocytes, the MAPK family member c-Jun N-terminal kinase 2 (JNK2) is activated by fibronectin fragments and low physiological levels of H2O2 and inhibited by oxidation due to elevated levels of H2O2 The kinase activity of affinity-purified, phosphorylated JNK2 from cultured chondrocytes was reversibly inhibited by 5-20 µm H2O2 Using dimedone-based chemical probes that react specifically with sulfenylated cysteines (RSOH), we identified Cys-222 in JNK2, a residue not conserved in JNK1 or JNK3, as a redox-reactive site. MS analysis of human recombinant JNK2 also detected further oxidation at Cys-222 and other cysteines to sulfinic (RSO2H) or sulfonic (RSO3H) acid. H2O2 treatment of JNK2 resulted in detectable levels of peptides containing intramolecular disulfides between Cys-222 and either Cys-213 or Cys-177, without evidence of dimer formation. Substitution of Cys-222 to alanine rendered JNK2 insensitive to H2O2 inhibition, unlike C177A and C213A variants. Two other JNK2 variants, C116A and C163A, were also resistant to oxidative inhibition. Cumulatively, these findings indicate differential regulation of JNK2 signaling dependent on H2O2 levels and point to key cysteine residues regulating JNK2 activity. As levels of intracellular H2O2 rise, a switch occurs from activation to inhibition of JNK2 activity, linking JNK2 regulation to the redox status of the cell.


Assuntos
Condrócitos/metabolismo , Cisteína/metabolismo , Peróxido de Hidrogênio/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Células Cultivadas , Fibronectinas , Humanos , Peróxido de Hidrogênio/farmacologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
10.
Biochem Biophys Res Commun ; 502(3): 370-374, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-29852167

RESUMO

Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy.


Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Menisco/citologia , Menisco/metabolismo , Ácido Palmítico/metabolismo , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Leupeptinas/farmacologia , Menisco/efeitos dos fármacos , Osteoartrite/etiologia , Ácido Palmítico/farmacologia , Fenilbutiratos/farmacologia , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Suínos
11.
Arthritis Rheumatol ; 68(1): 117-26, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26314228

RESUMO

OBJECTIVE: Oxidative posttranslational modifications of intracellular proteins can potentially regulate signaling pathways relevant to cartilage destruction in arthritis. In this study, oxidation of cysteine residues to form sulfenic acid (S-sulfenylation) was examined in osteoarthritic (OA) chondrocytes and investigated in normal chondrocytes as a mechanism by which fragments of fibronectin (FN-f) stimulate chondrocyte catabolic signaling. METHODS: Chondrocytes isolated from OA and normal human articular cartilage were analyzed using analogs of dimedone that specifically and irreversibly react with protein S-sulfenylated cysteines. Global S-sulfenylation was measured in cell lysates with and without FN-f stimulation by immunoblotting and in fixed cells by confocal microscopy. S-sulfenylation in specific proteins was identified by mass spectroscopy and confirmed by immunoblotting. Src activity was measured in live cells using a fluorescence resonance energy transfer biosensor. RESULTS: Proteins in chondrocytes isolated from OA cartilage were found to have elevated basal levels of S-sulfenylation relative to those of chondrocytes from normal cartilage. Treatment of normal chondrocytes with FN-f induced increased levels of S-sulfenylation in multiple proteins, including the tyrosine kinase Src. FN-f treatment also increased the levels of Src activity. Pretreatment with dimedone to alter S-sulfenylation function or with Src kinase inhibitors inhibited FN-f-induced production of matrix metalloproteinase 13. CONCLUSION: These results demonstrate for the first time the presence of oxidative posttranslational modification of proteins in human articular chondrocytes by S-sulfenylation. Due to the ability to regulate the activity of a number of cell signaling pathways, including catabolic mediators induced by fibronectin fragments, S-sulfenylation may contribute to cartilage destruction in OA and warrants further investigation.


Assuntos
Cartilagem Articular/citologia , Condrócitos/metabolismo , Cisteína/metabolismo , Osteoartrite/metabolismo , Oxirredução , Ácidos Sulfênicos/metabolismo , Quinases da Família src/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Condrócitos/efeitos dos fármacos , Cicloexanonas/farmacologia , Feminino , Fibronectinas/farmacologia , História Antiga , Humanos , Immunoblotting , Espectrometria de Massas , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/metabolismo , Microscopia Confocal , Pessoa de Meia-Idade , Fragmentos de Peptídeos/farmacologia , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Quinases da Família src/efeitos dos fármacos
12.
Sci Transl Med ; 7(278): 278ra32, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25761888

RESUMO

Early mobilization of critically ill patients with the acute respiratory distress syndrome (ARDS) has emerged as a therapeutic strategy that improves patient outcomes, such as the duration of mechanical ventilation and muscle strength. Despite the apparent efficacy of early mobility programs, their use in clinical practice is limited outside of specialized centers and clinical trials. To evaluate the mechanisms underlying mobility therapy, we exercised acute lung injury (ALI) mice for 2 days after the instillation of lipopolysaccharides into their lungs. We found that a short duration of moderate intensity exercise in ALI mice attenuated muscle ring finger 1 (MuRF1)-mediated atrophy of the limb and respiratory muscles and improved limb muscle force generation. Exercise also limited the influx of neutrophils into the alveolar space through modulation of a coordinated systemic neutrophil chemokine response. Granulocyte colony-stimulating factor (G-CSF) concentrations were systemically reduced by exercise in ALI mice, and in vivo blockade of the G-CSF receptor recapitulated the lung exercise phenotype in ALI mice. Additionally, plasma G-CSF concentrations in humans with acute respiratory failure (ARF) undergoing early mobility therapy showed greater decrements over time compared to control ARF patients. Together, these data provide a mechanism whereby early mobility therapy attenuates muscle wasting and limits ongoing alveolar neutrophilia through modulation of systemic neutrophil chemokines in lung-injured mice and humans.


Assuntos
Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/terapia , Terapia por Exercício , Músculo Esquelético/patologia , Neutrófilos/metabolismo , Condicionamento Físico Animal , Síndrome de Emaciação/patologia , Lesão Pulmonar Aguda/sangue , Lesão Pulmonar Aguda/complicações , Animais , Quimiocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Inflamação/patologia , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Atividade Motora , Proteínas Musculares/metabolismo , Atrofia Muscular/patologia , Alvéolos Pulmonares/metabolismo , Alvéolos Pulmonares/patologia , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Fatores de Tempo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/metabolismo , Síndrome de Emaciação/sangue , Síndrome de Emaciação/complicações
13.
Arthritis Rheumatol ; 66(5): 1266-71, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24497499

RESUMO

OBJECTIVE: Nuclear protein 1 (Nupr1) is a stress-inducible protein that is involved in gene transcription. The present study was undertaken to determine whether chondrocytes express Nupr1 and whether Nupr1 regulates matrix metalloproteinase 13 (MMP-13) expression. METHODS: Paraffin-embedded cartilage sections from normal human and osteoarthritic (OA) cartilage were immunostained using anti-Nupr1 antibody. To measure Nupr1 expression, total RNA was isolated from joint tissue obtained 8 weeks after surgery from young (12-week-old) and older (12-month-old) mice that underwent destabilization of the medial meniscus (DMM) to induce OA. Human chondrocytes were stimulated with 1-10 ng/ml interleukin-1ß (IL-1ß), 25 µM tert-butyl-hydroperoxide (tBHP), or 2 µM thapsigargin, and Nupr1 expression was analyzed by quantitative polymerase chain reaction. In addition, chondrocytes were transfected with small interfering RNA to knock down Nupr1 expression and then stimulated overnight with IL-1ß. After incubation, the conditioned medium was collected and MMP levels measured. RESULTS: Increased Nupr1 immunostaining was noted in human OA cartilage compared to normal cartilage. Expression was also increased in joint tissue from 12-month-old mice that underwent DMM surgery compared to sham-operated controls. Stimulation of chondrocytes with IL-1ß induced a 2-fold increase in Nupr1 messenger RNA (mRNA) within 1 hour, with the increase peaking to 4-fold at 6 hours. Treatment of chondrocytes with tBHP to induce oxidative stress increased Nupr1 mRNA expression by >2-fold; treatment with thapsigargin to induce endoplasmic reticulum stress did not produce a similar effect. Knockdown of Nupr1 inhibited IL-1ß-mediated induction of MMP-13. CONCLUSION: Nupr1 is expressed in cartilage, and its levels are increased in OA. Nupr1 expression is required for IL-1ß-mediated expression of MMP-13. These findings provide evidence of a novel pathway for regulation of IL-1ß-mediated production of MMPs in chondrocytes.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Proteínas de Neoplasias/metabolismo , Osteoartrite/metabolismo , Animais , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Humanos , Interleucina-1beta/farmacologia , Camundongos , Osteoartrite/patologia , Estresse Oxidativo/efeitos dos fármacos , Tapsigargina/farmacologia , terc-Butil Hidroperóxido/farmacologia
14.
Arthritis Res Ther ; 14(1): R23, 2012 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-22289259

RESUMO

INTRODUCTION: Obesity is one of the major risk factors for the development of osteoarthritis (OA). Although the mechanical factors appear to be critical, recent studies have suggested a role for adipokines in cartilage degradation. Chondrocytes from osteoarthritic cartilage respond poorly to insulin-like growth factor-1 (IGF-1) and the molecular mechanism(s) involved is not clearly understood. The purpose of the present study was to determine the role of extracellular nicotinamide phosphoribosyltransferase (eNAMPT/visfatin), a newly described adipokine, in regulating IGF-1 function in chondrocytes. METHODS: Human articular chondrocytes isolated from normal ankle cartilage were pretreated with eNAMPT (0.1 to 5.0 µg/ml) overnight followed by stimulation with IGF-1 (50 ng/ml) for 24 hours, and proteoglycan synthesis was measured by [35S]sulfate incorporation. Chondrocytes were pretreated with eNAMPT overnight followed by IGF-1 for 10 minutes, and the cell lysates were immunoblotted for various signaling proteins that are activated by IGF-1 using phosphospecific antibodies. In addition, chondrocytes were pretreated with mitogen-activated protein kinase kinase inhibitor (U0126) prior to stimulation with eNAMPT and IGF-1. RESULTS: Pretreatment of chondrocytes with eNAMPT inhibited IGF-1-stimulated proteoglycan synthesis in a dose-dependent manner. Treatment of chondrocytes with eNAMPT inhibited IGF-1-induced phosphorylation of signaling molecules, including insulin receptor substrate-1 and AKT. Interestingly, pretreatment of chondrocytes with eNAMPT did not inhibit IGF-1-mediated phosphorylation of the IGF-1 receptor; however, it stimulated a sustained phosphorylation of the extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) signaling pathway. Inhibition of the ERK/MAPK signaling pathway restored IGF-1-mediated insulin receptor substrate-1 and AKT phosphorylation. CONCLUSIONS: Our study demonstrates that eNAMPT/visfatin inhibits IGF-1 function in articular chondrocytes by activating the ERK/MAPK pathway independent of the IGF-1 receptor. Since eNAMPT levels are elevated in the synovial fluid of OA patients, the signaling pathway activated by eNAMPT could contribute to IGF-1 resistance in OA.


Assuntos
Condrócitos/efeitos dos fármacos , Fator de Crescimento Insulin-Like I/farmacologia , Nicotinamida Fosforribosiltransferase/farmacologia , Proteoglicanas/biossíntese , Transdução de Sinais/efeitos dos fármacos , Adipocinas/farmacologia , Butadienos/farmacologia , Cartilagem Articular/citologia , Células Cultivadas , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Immunoblotting , Proteínas Substratos do Receptor de Insulina/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nitrilas/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Biochim Biophys Acta ; 1822(4): 600-6, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22266138

RESUMO

S100 proteins are low molecular weight calcium binding proteins expressed in vertebrates. The family constitutes 21 known members that are expressed in several tissues and cell types and play a major role in various cellular functions. Uniquely, members of the S100 family have both intracellular and extracellular functions. Several members of the S100 family (S100A1, S100A2, S100A4, S1008, S100A9, S100A11, and S100B) have been identified in human articular cartilage, and their expression is upregulated in diseased tissue. These S100 proteins elicit a catabolic signaling pathway via receptor for advanced glycation end products (RAGE) in cartilage and may promote progression of arthritis. This review summarizes our current understanding of the role of S100 proteins in cartilage biology and in the development of arthritis.


Assuntos
Artrite/fisiopatologia , Cartilagem/fisiologia , Proteínas S100/fisiologia , Animais , Humanos
16.
J Biol Chem ; 285(41): 31517-24, 2010 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-20685652

RESUMO

S100A4, a member of the S100 family of proteins, plays an important role in matrix remodeling by up-regulating the expression of matrix metalloproteinases (MMPs). We have previously shown that S100A4 is overexpressed in diseased cartilage and that extracellular S100A4 stimulates MMP-13 production, a major type II collagen-degrading enzyme, via activation of receptor for advanced glycation end product signaling. In the present study, using human articular chondrocytes, we show that intracellular S100A4 translocated into the nucleus upon interleukin-1ß (IL-1ß) stimulation and translocation required post-translational modification of S100A4 by the sumo-1 protein. Two sumoylation sites were identified on the S100A4 molecule, Lys(22) and Lys(96). Mutation of these lysine residues abolished the ability of S100A4 to be sumoylated and to translocate into the nucleus. Blocking of sumoylation and nuclear transport of S100A4 inhibited the IL-1ß-induced production of MMP-13. Nuclear S100A4 was bound to the promoter region of MMP-13 in IL-1ß-treated cells. Thus, we demonstrate a novel mechanism for sumoylated S100A4 as a regulator of expression of the MMP-13 gene.


Assuntos
Núcleo Celular/metabolismo , Condrócitos/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Interleucina-1beta/metabolismo , Metaloproteinase 13 da Matriz/biossíntese , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas S100/metabolismo , Proteína SUMO-1/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Substituição de Aminoácidos , Núcleo Celular/genética , Células Cultivadas , Condrócitos/citologia , Colágeno Tipo II/biossíntese , Colágeno Tipo II/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , Metaloproteinase 13 da Matriz/genética , Mutação de Sentido Incorreto , Regiões Promotoras Genéticas/fisiologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/genética , Proteína SUMO-1/genética
17.
Arthritis Rheum ; 60(3): 792-800, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19248116

RESUMO

OBJECTIVE: S100A4 has been shown to be increased in osteoarthritic (OA) cartilage and to stimulate chondrocytes to produce matrix metalloproteinase 13 (MMP-13) through activation of the receptor for advanced glycation end products (RAGE). The aim of this study was to examine the mechanism of S100A4 secretion by chondrocytes. METHODS: Human articular chondrocytes isolated from ankle cartilage were stimulated with 10 ng/ml of interleukin-1beta (IL-1beta), IL-6, IL-7, or IL-8. Cells were pretreated with either a JAK-3 inhibitor, brefeldin A, or cycloheximide. Immunoblotting with phospho-specific antibodies was used to determine the activation of signaling proteins. Secretion of S100A4 was measured in conditioned media by immunoblotting, and MMP-13 was measured by enzyme-linked immunosorbent assay. RESULTS: Chondrocyte secretion of S100A4 was observed after treatment with IL-6 or IL-8 but was much greater in cultures treated with equal amounts of IL-7 and was not observed after treatment with IL-1beta. IL-7 activated the JAK/STAT pathway, with increased phosphorylation of JAK-3 and STAT-3, leading to increased production of S100A4 and MMP-13. Overexpression of a dominant-negative RAGE construct inhibited the IL-7-mediated production of MMP-13. Pretreatment of chondrocytes with a JAK-3 inhibitor or with cycloheximide blocked the IL-7-mediated secretion of S100A4, but pretreatment with brefeldin A did not. CONCLUSION: IL-7 stimulates chondrocyte secretion of S100A4 via activation of JAK/STAT signaling, and then S100A4 acts in an autocrine manner to stimulate MMP-13 production via RAGE. Since both IL-7 and S100A4 are up-regulated in OA cartilage and can stimulate MMP-13 production by chondrocytes, this signaling pathway could contribute to cartilage destruction during the development of OA.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Interleucina-7/fisiologia , Janus Quinases/metabolismo , Proteínas S100/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/fisiologia , Brefeldina A/farmacologia , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/patologia , Cicloeximida/farmacologia , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100
18.
Arthritis Rheum ; 60(2): 492-500, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19180501

RESUMO

OBJECTIVE: The chondrocyte response to insulin-like growth factor 1 (IGF-1) is reduced with aging and in osteoarthritis (OA). IGF-1 signals through the phosphatidylinositol 3-kinase/Akt pathway. TRB3, a tribbles homolog, has been shown to inhibit IGF-1-mediated activation of Akt in HEK 293 cells. This study was undertaken to determine if TRB3 is expressed in chondrocytes, and whether the chondrocyte response to IGF-1 is reduced by TRB3. METHODS: Human articular cartilage was obtained from normal tissue donors and from patients with OA at the time of knee replacement surgery. TRB3 was assessed in the tissue samples by reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry. Overexpression of TRB3 was induced by transient transfection to determine the effects of TRB3 on cell survival and proteoglycan synthesis. RESULTS: TRB3 messenger RNA was detected in normal human chondrocytes. TRB3 protein levels were low in cells from normal cartilage but significantly increased in cells from OA cartilage. Incubation with 2 agents that induce endoplasmic reticulum stress, tunicamycin and thapsigargin, increased TRB3 levels in normal cells. Overexpression of TRB3 inhibited Akt phosphorylation and reduced chondrocyte survival and proteoglycan synthesis. CONCLUSION: These results are the first to demonstrate that TRB3 is present in human chondrocytes, and that the level of TRB3 is increased in OA cartilage and in isolated OA chondrocytes. Because it is an inhibitor of Akt activation, elevated TRB3 production could play a role in the increased cell death and reduced response to IGF-1 observed in OA cartilage.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Condrócitos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Osteoartrite do Joelho/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteoglicanas/biossíntese , Proteínas Repressoras/metabolismo , Adulto , Idoso de 80 Anos ou mais , Articulação do Tornozelo , Cartilagem Articular/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Criança , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Inibidores Enzimáticos/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteoglicanas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Repressoras/genética , Tapsigargina/farmacologia , Tunicamicina/farmacologia , Adulto Jovem
19.
J Physiol ; 581(Pt 2): 457-66, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17347267

RESUMO

Cells that are metabolically active and in a high degree of differentiation and proliferation require cobalamin (Cbl: vitamin B(12)) and they obtain it from the circulation bound to transcobalamin (TC) via the transcobalamin receptor (TC-R). This study has investigated the plasma membrane dynamics of TC-R expression in polarized human intestinal epithelial Caco-2 cells using techniques of pulse-chase labelling, domain-specific biotinylation and cell fractionation. Endogenously synthesized TC-R turned over with a half-life (T(1/2)) of 8 h following its delivery to the basolateral plasma membrane (BLM). The T(1/2) of BLM delivery was 15 min and TC-R delivered to the BLM was endocytosed and subsequently degraded by leupeptin-sensitive proteases. However, about 15% of TC-R endocytosed from the BLM was transcytosed (T(1/2), 45 min) to the apical membranes (BBM) where it underwent endocytosis and was degraded. TC-R delivery to both BLM and BBM was inhibited by Brefeldin A and tunicamycin, but not by wortmannin or leupeptin. Colchicine inhibited TC-R delivery to BBM, but not BLM. At steady state, apical TC-R was associated with megalin and both these proteins were enriched in an intracellular compartment which also contained Rab5 and transferrin receptor. These results indicate that following rapid delivery to both plasma membrane domains of Caco-2 cells, TC-R undergoes constitutive endocytosis and degradation by leupeptin-sensitive proteases. TC-R expressed in apical BBM complexes with megalin during its transcytosis from the BLM.


Assuntos
Membrana Celular/metabolismo , Polaridade Celular , Endocitose , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/metabolismo , Biotinilação , Brefeldina A/farmacologia , Células CACO-2 , Fracionamento Celular , Membrana Celular/efeitos dos fármacos , Colchicina/farmacologia , Endocitose/efeitos dos fármacos , Endossomos/metabolismo , Células Epiteliais/efeitos dos fármacos , Glicosilação , Complexo de Golgi/metabolismo , Meia-Vida , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Cinética , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Peptídeo Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico , Receptores da Transferrina/metabolismo , Moduladores de Tubulina/farmacologia , Tunicamicina/farmacologia , Proteínas rab5 de Ligação ao GTP/metabolismo
20.
Arthritis Rheum ; 54(9): 2901-11, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16948116

RESUMO

OBJECTIVE: S100 proteins have been implicated in various inflammatory conditions, including arthritis. The aims of this study were to determine whether chondrocytes produce S100A4 and whether S100A4 can stimulate the production of matrix metalloproteinase 13 (MMP-13) by articular chondrocytes via receptor for advanced glycation end products (RAGE)-mediated signaling. METHODS: The expression of chondrocyte S100A4 was analyzed by immunohistochemistry using normal and osteoarthritic (OA) cartilage and by immunoblotting of chondrocyte cell lysates. RAGE signaling was examined by stimulating chondrocytes with S100A4 and monitoring for the activation of MAP kinases and NF-kappaB. Production of MMP-13 was determined in the conditioned medium. A pulldown assay using biotin-labeled S100A4 was used to demonstrate binding to RAGE. RESULTS: S100A4 expression was detected in human articular chondrocytes by immunoblotting and appeared to increase in the cell lysates from OA tissue. Marked positive immunostaining for S100A4 was also noted in sections of human cartilage with changes due to OA. Stimulation of chondrocytes with S100A4 increased the phosphorylation of Pyk-2, MAP kinases, and activated NF-kappaB, followed by increased production of MMP-13 in the conditioned medium. This signaling was inhibited in cells pretreated with soluble RAGE, advanced glycation end product-bovine serum albumin, or the antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin, or by overexpression of a dominant-negative RAGE construct. A pulldown assay showed that S100A4 binds to RAGE in chondrocytes. CONCLUSION: This is the first study to demonstrate that S100A4 binds to RAGE and stimulates a RAGE-mediated signaling cascade, leading to increased production of MMP-13. Since both S100A4 and RAGE are up-regulated in OA cartilage, this signaling pathway could contribute to cartilage degradation in OA.


Assuntos
Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Colagenases/genética , Proteínas S100/metabolismo , Produtos Finais de Glicação Avançada/fisiologia , Humanos , Imuno-Histoquímica , Metaloproteinase 13 da Matriz , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína A4 de Ligação a Cálcio da Família S100 , Soroalbumina Bovina/fisiologia , Transdução de Sinais
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