RESUMO
Osteoarthritis (OA) is a degenerative joint disease characterized by irreversible cartilage damage, inflammation and altered chondrocyte phenotype. Transforming growth factor-ß (TGF-ß) signaling via SMAD2/3 is crucial for blocking hypertrophy. The post-translational modifications of these SMAD proteins in the linker domain regulate their function and these can be triggered by inflammation through the activation of kinases or phosphatases. Therefore, we investigated if OA-related inflammation affects TGF-ß signaling via SMAD2/3 linker-modifications in chondrocytes. We found that both Interleukin (IL)-1ß and OA-synovium conditioned medium negated SMAD2/3 transcriptional activity in chondrocytes. This inhibition of TGF-ß signaling was enhanced if SMAD3 could not be phosphorylated on Ser213 in the linker region and the inhibition by IL-1ß was less if the SMAD3 linker could not be phosphorylated at Ser204. Our study shows evidence that inflammation inhibits SMAD2/3 signaling in chondrocytes via SMAD linker (de)-phosphorylation. The involvement of linker region modifications may represent a new therapeutic target for OA.
Assuntos
Condrócitos/metabolismo , Condrócitos/patologia , Osteoartrite/metabolismo , Transdução de Sinais/genética , Proteína Smad2/química , Proteína Smad2/metabolismo , Proteína Smad3/química , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Adulto , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Hipertrofia/metabolismo , Inflamação/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/genética , Osteoartrite/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Domínios Proteicos/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad3/genética , Membrana Sinovial/metabolismo , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Objective: A crucial feature of OA is cartilage degradation. This process is mediated by pro-inflammatory cytokines, among other factors, via induction of matrix-degrading enzymes. Interleukin 37 (IL37) is an anti-inflammatory cytokine and is efficient in blocking the production of pro-inflammatory cytokines during innate immune responses. We hypothesize that IL37 is therapeutic in treating the inflammatory cytokine cascade in human OA chondrocytes and can act as a counter-regulatory cytokine to reduce cartilage degradation in OA. Methods: Human OA cartilage was obtained from patients undergoing total knee or hip arthroplasty. Immunohistochemistry was applied to study IL37 protein expression in cartilage biopsies from OA patients. Induction of IL37 expression by IL1ß, OA synovium-conditioned medium and TNFα was investigated in human OA chondrocytes. Adenoviral overexpression of IL37 followed by IL1ß stimulation was performed to investigate the anti-inflammatory potential of IL37. Results: IL37 expression was detected in cartilage biopsies of OA patients and induced by IL1ß. After IL1ß stimulation, increased IL1ß, IL6 and IL8 expression was observed in OA chondrocytes. Elevated IL37 levels diminished the IL1ß-induced IL1ß , IL6 and IL8 gene levels and IL1ß and IL8 protein levels. In addition to the reduction in pro-inflammatory cytokine expression, IL37 reduced MMP1 , MMP3 , MMP13 and disintegrin and metalloproteinase with thrombospondin motifs 5 gene levels and MMP3 and MMP13 protein levels. Conclusion: IL37 is induced by IL1ß, and IL37 itself reduced IL1ß, IL6 and IL8 production, indicating that IL37 is able to induce a counter-regulatory anti-inflammatory feedback loop in chondrocytes. In addition, IL37 dampens catabolic enzyme expression. This supports IL37 as a potential therapeutic target in OA.
Assuntos
Condrócitos/metabolismo , Interleucina-1/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite , RNA Mensageiro/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adenoviridae , Western Blotting , Condrócitos/efeitos dos fármacos , Desintegrinas/efeitos dos fármacos , Desintegrinas/genética , Desintegrinas/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-1/genética , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/efeitos dos fármacos , Interleucina-8/genética , Interleucina-8/metabolismo , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/efeitos dos fármacos , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/efeitos dos fármacos , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , RNA Mensageiro/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: To determine the expression of suppressor of cytokine signaling 3 (SOCS-3) in human articular chondrocytes and its functional consequences. METHODS: Chondrocytes were isolated from the cartilage of patients with osteoarthritis (OA), patients with rheumatoid arthritis (RA), and trauma patients and from the healthy cartilage of patients with a femoral neck fracture. The human chondrocyte cell line G6 and primary bovine chondrocytes were used in validation experiments. SOCS-3 messenger RNA (mRNA) expression was measured by quantitative polymerase chain reaction, and SOCS-3 protein levels were determined by Western blotting and immunohistochemical analysis. To ascertain the role of SOCS-3 in the chondrocyte response to interleukin-1ß (IL-1ß) or lipopolysaccharide (LPS), the expression of SOCS3 was either reduced by small interfering RNA or enhanced by viral transduction. RESULTS: The expression of SOCS-3 mRNA (but not that of SOCS-1 mRNA) was significantly enhanced in chondrocytes obtained from OA cartilage (mean ± SD ΔC(t) 3.4 ± 1.0) and RA cartilage (ΔC(t) 3.4 ± 1.4) compared with cartilage obtained from patients with femoral neck fracture (ΔC(t) 5.3 ± 1.2). The expression of SOCS3 correlated significantly with that of other genes known to be expressed in arthritic chondrocytes, such as MMP13 (r = 0.743), ADAMTS4 (r = 0.779), and ADAMTS5 (r = 0.647), and an inverse relationship was observed with COL2A1 (r = -0.561). Up-regulation of SOCS-3 by IL-1 in G6 chondrocytes and its spontaneous expression in OA chondrocytes were reduced by mithramycin, a specific inhibitor of transcription factor Sp-1. Overexpression of SOCS-3 in bovine chondrocytes reduced IL-1- and LPS-induced nitric oxide production and insulin-like growth factor 1-induced proteoglycan synthesis. Interestingly, a similar impairment of function was observed in OA chondrocytes, which was partially restored by SOCS-3 gene knockdown. CONCLUSION: This study demonstrated that both SOCS-3 mRNA and SOCS-3 protein are expressed in human arthritic chondrocytes and affect cellular responses involved in cartilage pathology.
Assuntos
Artrite Reumatoide/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteoartrite do Quadril/metabolismo , Osteoartrite do Joelho/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS5 , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Bovinos , Linhagem Celular , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Feminino , Humanos , Interleucina-1/farmacologia , Masculino , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Pessoa de Meia-Idade , Osteoartrite do Quadril/genética , Osteoartrite do Quadril/patologia , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/patologia , Pró-Colágeno N-Endopeptidase/genética , Pró-Colágeno N-Endopeptidase/metabolismo , Proteoglicanas/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Joint pain severity in arthritic diseases differs between sexes and is often more pronounced in women. This disparity is thought to stem from biological mechanisms, particularly innate immunity, yet the understanding of sex-specific differences in arthritic pain remains incomplete. This study aims to investigate these disparities using an innate immunity-driven inflammation model induced by intra-articular injections of Streptococcus Cell Wall fragments to mimic both acute and pre-sensitized joint conditions. Nociceptive behavior was evaluated via gait analysis and static weight-bearing, and inflammation was evaluated via joint histology and the synovial gene expression involved in immune response. Although acute inflammation and pain severity were comparable between sexes, distinct associations between synovial inflammatory gene expression and static nociceptive behavior emerged. These associations delineated sex-specific relationships with pain, highlighting differential gene interactions (Il6 versus Cybb on day 1 and Cyba/Gas6 versus Nos2 on day 8) between sexes. In conclusion, our study found that, despite similar pain severity between sexes, the association of inflammatory synovial genes revealed sex-specific differences in the molecular inflammatory mechanisms underlying pain. These findings suggest a path towards more personalized treatment strategies for pain management in arthritis and other inflammatory joint diseases.
Assuntos
Sinovite , Masculino , Humanos , Camundongos , Feminino , Animais , Sinovite/metabolismo , Dor , Inflamação/complicações , Artralgia , Imunidade InataRESUMO
Background: Osteoarthritis (OA) is a progressive joint disease and a major cause of chronic pain in adults. The prevalence of OA is higher in female patients, who tend to have worse OA outcomes, partially due to pain. The association between joint pain and OA pathology is often inconclusive. Preclinical research studies have largely overlooked sex as a potential determinant in joint pain during OA. This study aimed to investigate the role of sex in joint pain in the collagenase-induced OA (CiOA) model and its link with joint pathology. Methods: Multiple aspects of pain were evaluated during identically executed experiments of CiOA in male and female C57BL/6J mice. Cartilage damage, osteophyte formation, synovial thickness, and cellularity were assessed by histology on day 56. The association between pain and pathology was investigated, disaggregated by sex. Results: Differences in pain behavior between sexes were found in the majority of the evaluated pain methods. Females displayed lower weight bearing ability in the affected leg compared to males during the early phase of the disease, however, the pathology at the end stage was comparable between sexes. In the second cohort, males displayed increased mechanical sensitivity in the affected joint compared to females but also showed more cartilage damage at the end stage of the model. Within this cohort, gait analysis showed varied results. Males used the affected paw less often and displayed dynamic weight-bearing compensation in the early phase of the model. These differences were not observed in females. Other evaluated parameters displayed comparable gait behavior between males and females. A detailed analysis of individual mice revealed that seven out of 10 pain measurements highly correlated with OA histopathology in females (Pearson r range: 0.642-0.934), whereas in males this measurement was only two (Pearson r range: 0.645-0.748). Conclusion: Our data show that sex is a determinant in the link between pain-related behavior with OA features. Therefore, to accurately interpret pain data it is crucial to segregate data analysis by sex to draw the correct mechanistic conclusion.
Assuntos
Osteoartrite , Camundongos , Masculino , Feminino , Animais , Camundongos Endogâmicos C57BL , Osteoartrite/etiologia , Dor/etiologia , Artralgia/complicações , MarchaRESUMO
Osteoarthritis (OA) is characterized by progressive articular cartilage loss. Human mesenchymal stromal cells (MSCs) can be used for cartilage repair therapies based on their potential to differentiate into chondrocytes. However, the joint microenvironment is a major determinant of the success of MSC-based cartilage formation. Currently, there is no tool that is able to predict the effect of a patient's OA joint microenvironment on MSC-based cartilage formation. Our goal was to develop a molecular tool that can predict this effect before the start of cartilage repair therapies. Six different promoter reporters (hIL6, hIL8, hADAMTS5, hWISP1, hMMP13, and hADAM28) were generated and evaluated in an immortalized human articular chondrocyte for their responsiveness to an osteoarthritic microenvironment by stimulation with OA synovium-conditioned medium (OAs-cm) obtained from 32 different knee OA patients. To study the effect of this OA microenvironment on MSC-based cartilage formation, MSCs were cultured in a three-dimensional pellet culture model, while stimulated with OAs-cm. Cartilage formation was assessed histologically and by quantifying sulfated glycosaminoglycan (sGAG) production. We confirmed that OAs-cm of different patients had significantly different effects on sGAG production. In addition, significant correlations were obtained between the effect of the OAs-cm on cartilage formation and promoter reporter outcome. Furthermore, we validated the predictive value of measuring two promoter reporters with an independent cohort of OAs-cm and the effect of 87.5% of the OAs-cm on MSC-based cartilage formation could be predicted. Together, we developed a novel tool to predict the effect of the OA joint microenvironment on MSC-based cartilage formation. This is an important first step toward personalized cartilage repair strategies for OA patients. Impact statement We describe the development of a novel molecular tool to predict if an osteoarthritis joint microenvironment is permissive for cartilage repair or not. Such a tool is of great importance in determining the success of mesenchymal stromal cell-based cartilage repair strategies.
Assuntos
Cartilagem Articular , Células-Tronco Mesenquimais , Osteoartrite do Joelho , Cartilagem Articular/patologia , Condrócitos/patologia , Condrogênese/fisiologia , Humanos , Osteoartrite do Joelho/patologiaRESUMO
During osteoarthritis (OA), hypertrophy-like chondrocytes contribute to the disease process. TGF-ß's signaling pathways can contribute to a hypertrophy(-like) phenotype in chondrocytes, especially at high doses of TGF-ß. In this study, we examine which transcription factors (TFs) are activated and involved in TGF-ß-dependent induction of a hypertrophy-like phenotype in human OA chondrocytes. We found that TGF-ß, at levels found in synovial fluid in OA patients, induces hypertrophic differentiation, as characterized by increased expression of RUNX2, COL10A1, COL1A1, VEGFA and IHH. Using luciferase-based TF activity assays, we observed that the expression of these hypertrophy genes positively correlated to SMAD3:4, STAT3 and AP1 activity. Blocking these TFs using specific inhibitors for ALK-5-induced SMAD signaling (5 µM SB-505124), JAK-STAT signaling (1 µM Tofacitinib) and JNK signaling (10 µM SP-600125) led to the striking observation that only SB-505124 repressed the expression of hypertrophy factors in TGF-ß-stimulated chondrocytes. Therefore, we conclude that ALK5 kinase activity is essential for TGF-ß-induced expression of crucial hypertrophy factors in chondrocytes.
Assuntos
Condrócitos , Osteoartrite , Condrócitos/metabolismo , Humanos , Hipertrofia/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Fenótipo , Fator de Crescimento Transformador beta/metabolismo , Fatores de Crescimento Transformadores/genética , Fatores de Crescimento Transformadores/metabolismoRESUMO
During osteoarthritis (OA) chondrocytes show deviant behavior resembling terminal differentiation of growth-plate chondrocytes, characterized by elevated MMP-13 expression. The latter is also a hallmark for OA. TGF-beta is generally thought to be a protective factor for cartilage, but it has also displayed deleterious effects in some studies. Recently, it was shown that besides signaling via the ALK5 (activin-like kinase 5) receptor, TGF-beta can also signal via ALK1, thereby activating Smad1/5/8 instead of Smad2/3. The Smad1/5/8 route can induce chondrocyte terminal differentiation. Murine chondrocytes stimulated with TGF-beta activated the ALK5 receptor/Smad2/3 route as well as the ALK1/Smad1/5/8 route. In cartilage of mouse models for aging and OA, ALK5 expression decreased much more than ALK1. Thus, the ALK1/ALK5 ratio increased, which was associated with changes in the respective downstream markers: an increased Id-1 (inhibitor of DNA binding-1)/PAI-1 (plasminogen activator inhibitor-1) ratio. Transfection of chondrocytes with adenovirus overexpressing constitutive active ALK1 increased MMP-13 expression, while small interfering RNA against ALK1 decreased MMP-13 expression to nondetectable levels. Adenovirus overexpressing constitutive active ALK5 transfection increased aggrecan expression, whereas small interfering RNA against ALK5 resulted in increased MMP-13 expression. Moreover, in human OA cartilage ALK1 was highly correlated with MMP-13 expression, whereas ALK5 correlated with aggrecan and collagen type II expression, important for healthy cartilage. Collectively, we show an age-related shift in ALK1/ALK5 ratio in murine cartilage and a strong correlation between ALK1 and MMP-13 expression in human cartilage. A change in balance between ALK5 and ALK1 receptors in chondrocytes caused changes in MMP-13 expression, thereby causing an OA-like phenotype. Our data suggest that dominant ALK1 signaling results in deviant chondrocyte behavior, thereby contributing to age-related cartilage destruction and OA.
Assuntos
Receptores de Activinas Tipo II/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Osteoartrite/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Activinas Tipo II/genética , Envelhecimento/fisiologia , Animais , Cartilagem/enzimologia , Células Cultivadas , Condrócitos/enzimologia , Regulação Enzimológica da Expressão Gênica , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Masculino , Metaloproteinase 13 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Mensageiro/genética , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução de SinaisRESUMO
In traditional tissue engineering, synthetic or natural scaffolds are usually used as removable temporal support, which involves some biotechnology limitations. The concept of "scaffield" approach utilizing the physical fields instead of biomaterial scaffold has been proposed recently. In particular, a combination of intense magnetic and acoustic fields can enable rapid levitational bioassembly of complex-shaped 3D tissue constructs from tissue spheroids at low concentration of paramagnetic agent (gadolinium salt) in the medium. In the current study, the tissue spheroids from human bladder smooth muscle cells (myospheres) are used as building blocks for assembling the tubular 3D constructs. Levitational assembly is accomplished at low concentrations of gadolinium salts in the high magnetic field at 9.5 T. The biofabricated smooth muscle constructs demonstrate contraction after the addition of vasoconstrictive agent endothelin-1. Thus, hybrid magnetoacoustic levitational bioassembly is considered as a new technology platform in the emerging field of formative biofabrication. This novel technology of scaffold-free, nozzle-free, and label-free bioassembly opens a unique opportunity for rapid biofabrication of 3D tissue and organ constructs with complex geometry.
Assuntos
Engenharia Tecidual , Alicerces Teciduais , Materiais Biocompatíveis , Biotecnologia , Humanos , Campos Magnéticos , Esferoides CelularesRESUMO
IMPACT STATEMENT: Catabolic factors present in a damaged joint inhibit chondrogenic differentiation of mesenchymal stem cells, thereby reducing the chance for successful cartilage formation. By improving stem cell-based cartilage repair with interleukin-37 (IL37), we might be able to inhibit the worsening progression of focal cartilage defects and prevent further development of joint diseases such as osteoarthritis. This will avoid chronic pain and impaired joint mobility for patients and reduce costs for society.
Assuntos
Microambiente Celular , Condrogênese , Citoproteção , Inflamação/patologia , Interleucina-1/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoartrite/patologia , Cartilagem/metabolismo , Cartilagem/patologia , Microambiente Celular/efeitos dos fármacos , Microambiente Celular/genética , Condrogênese/efeitos dos fármacos , Condrogênese/genética , Colágeno/metabolismo , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoartrite/genética , Proteólise/efeitos dos fármacosRESUMO
BACKGROUND: Chondrogenic differentiation of mesenchymal stem cells (MSC) requires transforming growth factor beta (TGFß) signaling. TGFß binds to the type I receptor activin-like kinase (ALK)5 and results in C-terminal SMAD2/3 phosphorylation (pSMAD2/3C). In turn pSMAD2/3C translocates to the nucleus and regulates target gene expression. Inflammatory mediators are known to exert an inhibitory effect on MSC differentiation. In this study we investigated the effect of interleukin 1 ß (IL1ß) on SMAD2/3 signaling dynamics and post-translational modifications. RESULTS: Co-stimulation of MSC with TGFß and IL1ß did not affect peak pSMAD2C levels at 1h post-stimulation. Surprisingly, SMAD3 transcriptional activity, as determined by the CAGA12-luciferase reporter construct, was enhanced by co-stimulation of TGFß and IL1ß compared to TGFß alone. Furthermore, IL1ß stimulation induced CAGA12-luciferase activity in a SMAD dependent way. As SMAD function can be modulated independent of canonical TGFß signaling through the SMAD linker domain, we studied SMAD2 linker phosphorylation at specific threonine and serine residues. SMAD2 linker threonine and serine modifications were observed within 1h following TGFß, IL1ß or TGFß and IL1ß stimulation. Upon co-stimulation linker modified SMAD2 accumulated in the cytoplasm and SMAD2/3 target gene transcription (ID1, JUNB) at 2-4h was inhibited. A detailed time course analysis of IL1ß-induced SMAD2 linker modifications revealed a distinct temperospatial pattern compared to TGFß. Co-stimulation with both factors resulted in a similar kinetic profile as TGFß alone. Nevertheless, IL1ß did subtly alter TGFß-induced pSMAD2C levels between 8 and 24h post-stimulation, which was reflected by TGFß target gene expression (PAI1, JUNB). Direct evidence for the importance of SMAD3 linker modifications for the effect of IL1ß on TGFß signaling was obtained by over-expression of SMAD3 or a SMAD3 linker phospho-mutant. Finally, an inhibitor screening was performed to identify kinases involved in SMAD2/3 linker modifications. We identified TAK1 kinase activity as crucial for IL1ß-induced SMAD2 linker modifications and CAGA12-luciferase activity. CONCLUSIONS: TGFß and IL1ß signaling interact at the SMAD2/3 level in human primary MSC. Down-stream TGFß target genes were repressed by IL1ß independent of C-terminal SMAD2 phosphorylation. We demonstrate that SMAD2/3 linker modifications are required for this interplay and identified TAK1 as a crucial mediator of IL1ß-induced TGFß signal modulation.
Assuntos
Interleucina-1beta/genética , MAP Quinase Quinase Quinases/genética , Proteína Smad2/genética , Proteína Smad3/genética , Fator de Crescimento Transformador beta/genética , Diferenciação Celular/genética , Condrogênese/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Interleucina-1beta/administração & dosagem , Interleucina-1beta/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/administração & dosagem , Fator de Crescimento Transformador beta/metabolismoRESUMO
Apocynin, an inhibitor of NADPH-oxidase, is known to partially reverse the inflammation-mediated cartilage proteoglycan synthesis in chondrocytes. More recently, it was reported that apocynin prevents cyclooxygenase (COX)-2 expression in monocytes. The present study aimed to investigate whether these in vitro features of apocynin could be confirmed in vivo. In a mouse model of zymosan-induced acute arthritis apocynin was administered orally (0, 3.2, 16 and 80 microg/ml in the drinking water) and the effects on cartilage proteoglycan synthesis were monitored. In a mouse model of zymosan-induced inflammation of the ears apocynin was administered orally (14 mg/kg/day by gavage) and the effects on ear swelling and ex vivo produced prostaglandin E2 (PGE2) by lipopolysaccharide (LPS)-stimulated blood cells were measured. In this study, ibuprofen was used as a positive control (50 mg/kg/day by gavage) and animals received vehicle as a negative control. Apocynin dose-dependently reversed the inhibition of proteoglycan synthesis in articular cartilage of the arthritic joint. A statistically significant increase in proteoglycan synthesis was found at a dose of 80 microg/ml apocynin. Apocynin did not affect the proteoglycan synthesis of the control knee joints. Apocynin significantly decreased the zymosan-induced ear swelling at 1, 2 and 4 h (hours) after zymosan injection versus the vehicle treated group at 14 mg/kg/day. The ex vivo production of PGE2 by LPS-stimulated blood cells was significantly decreased after in vivo apocynin treatment. Ibuprofen decreased ear swelling at the same time-points as apocynin and inhibited the ex vivo produced PGE2. In conclusion, the present study confirmed two important features of apocynin in vivo: (1) oral administration of apocynin can partially reverse the inflammation-induced inhibition of cartilage proteoglycan synthesis, and (2) oral administration of apocynin has COX inhibitory effects similar to the non-steroidal anti-inflammatory drug (NSAID) ibuprofen. Therefore, apocynin might be of potential use during the treatment of chronic inflammatory joint diseases like osteoarthritis or rheumatoid arthritis.
Assuntos
Acetofenonas/farmacologia , Cartilagem Articular/efeitos dos fármacos , Inflamação/prevenção & controle , NADPH Oxidases/antagonistas & inibidores , Proteoglicanas/biossíntese , Acetofenonas/administração & dosagem , Administração Oral , Animais , Artrite/induzido quimicamente , Artrite/metabolismo , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Dinoprostona/biossíntese , Ingestão de Líquidos , Otopatias/induzido quimicamente , Otopatias/prevenção & controle , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Feminino , Inflamação/induzido quimicamente , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , ZimosanRESUMO
INTRODUCTION: Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor ß (TGFß) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFß receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. MATERIALS & METHODS: ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFß1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. RESULTS: ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFß receptors did not clearly associate with chondrogenic induction. TGFß increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFß. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFß-induced chondrogenesis. CONCLUSION: ALK5 as well as ALK1 are required for TGFß-induced chondrogenic differentiation of BMSCs, and TGFß not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors.
Assuntos
Receptores de Activinas Tipo II/genética , Receptores de Ativinas/genética , Medula Óssea/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/fisiologia , Proteínas Serina-Treonina Quinases/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Fator de Crescimento Transformador beta/genética , Células da Medula Óssea/fisiologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/fisiologia , Condrogênese/fisiologia , Colágeno Tipo II/genética , Regulação para Baixo/fisiologia , Expressão Gênica/fisiologia , Humanos , Receptor do Fator de Crescimento Transformador beta Tipo I , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: To rescue chondrogenic differentiation of human mesenchymal stem cells (hMSCs) in osteoarthritic conditions by inhibition of protein kinases. METHODS: hMSCs were cultured in pellets. During early chondrogenic differentiation, these were exposed to osteoarthritic synovium-conditioned medium (OAS-CM), combined with the Janus kinase (JAK)-inhibitor tofacitinib and/or the transforming growth factor ß-activated kinase 1 (TAK1)-inhibitor oxozeaenol. To evaluate effects on chondrogenesis, the glycosaminoglycan (GAG) content of the pellets was measured at the time that chondrogenesis was manifest in control cultures. Moreover, mRNA levels of matrix molecules and enzymes were measured during this process, using real-time polymerase chain reaction (RT-PCR). Initial experiments were performed with hMSCs from a fetal donor, and results of these studies were confirmed with hMSCs from adult donors. RESULTS: Exposure to OAS-CM resulted in pellets with a much lower GAG content, reflecting inhibited chondrogenic differentiation. This was accompanied by decreased mRNA levels of aggrecan, type II collagen, and Sox9, and increased levels of matrix metalloproteinase (MMP)1, MMP3, MMP13, ADAMTS4, and ADAMTS5. Both tofacitinib (JAK-inhibitor) and oxozeaenol (TAK1 inhibitor) significantly increased the GAG content of the pellets in osteoarthritis (OA)-like conditions. The combination of both protein kinase inhibitors showed an additive effect on GAG content. In agreement with this, in the presence of OAS-CM, both tofacitinib and oxozeaenol increased mRNA expression of sox9. The expression of aggrecan and type II collagen was also up-regulated, but this only reached significance for aggrecan after TAK1 inhibition. Both inhibitors decreased the mRNA levels of MMP1, 3, and 13 in the presence of OAS-CM. Moreover, oxozeaenol also significantly down-regulated the mRNA levels of aggrecanases ADAMTS4 and ADAMTS5. When combined, the inhibitors caused additive reduction of OA-induced MMP1 mRNA expression. Counteraction of OAS-CM-induced inhibition of chondrogenesis by these protein kinase inhibitors was confirmed with hMSCs of two different adult donors. Both tofacitinib and oxozeaenol significantly improved GAG content in cell pellets from these adult donors. CONCLUSIONS: Tofacitinib and oxozeaenol partially prevent the inhibition of chondrogenesis by factors secreted by OA synovium. Their effects are additive. This indicates that these protein kinase inhibitors can potentially be used to improve cartilage formation under the conditions occurring in osteoathritic, or otherwise inflamed, joints.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Janus Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/antagonistas & inibidores , Células-Tronco Mesenquimais/patologia , Osteoartrite/patologia , Inibidores de Proteínas Quinases/farmacologia , Adulto , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/crescimento & desenvolvimento , Cartilagem Articular/patologia , Feto/citologia , Humanos , Janus Quinases/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Células-Tronco Mesenquimais/enzimologia , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Fatores de Tempo , Zearalenona/farmacologiaRESUMO
BACKGROUND: Both Wnt signaling and TGF-ß signaling have been implicated in the regulation of the phenotype of many cell types including chondrocytes, the only cell type present in the articular cartilage. A changed chondrocyte phenotype, resulting in chondrocyte hypertrophy, is one of the main hallmarks of osteoarthritis. TGF-ß signaling via activin-like kinase (ALK)5, resulting in Smad 2/3 phosphorylation, inhibits chondrocyte hypertrophy. In contrast, TGF-ß signaling via ALK1, leading to Smad 1/5/8 phosphorylation, has been shown to induce chondrocyte hypertrophy. In this study, we investigated the capability of Wnt3a and WISP1, a protein downstream in canonical Wnt signaling, to skew TGF-ß signaling in chondrocytes from the protective Smad 2/3 towards the Smad 1/5/8 pathway. RESULTS: Stimulation with Wnt3a, either alone or in combination with its downstream protein WISP1, decreased TGF-ß-induced C-terminal phosphorylation of Smad 2/3. In addition, both Wnt3a and WISP1 increased Smad 1/5/8 phosphorylation at the C-terminal domain in both murine and human chondrocytes. DKK-1, a selective inhibitor of canonical Wnt signaling, abolished these effects. TGF-ß signaling via Smad 2/3, measured by the functional CAGA12-Luc reporter construct activity, was decreased by stimulation with Wnt3a in accordance with the decrease in Smad 2/3 phosphorylation found on Western blot. Furthermore, in vivo overexpression of the canonical Wnt8a decreased Smad 2/3 phosphorylation and increased Smad 1/5/8 phosphorylation. CONCLUSIONS: Our data show that canonical Wnt signaling is able to skew TGF-ß signaling towards dominant signaling via the ALK1/Smad 1/5/8 pathway, which reportedly leads to chondrocyte hypertrophy. In this way canonical Wnts and WISP1, which we found to be increased during experimental osteoarthritis, may contribute to osteoarthritis pathology.
Assuntos
Receptores de Ativinas Tipo I/metabolismo , Transdução de Sinais , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Receptores de Activinas Tipo II , Animais , Proteínas de Sinalização Intercelular CCN/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrócitos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/metabolismoRESUMO
OBJECTIVE: Wnt signaling pathway proteins are involved in embryonic development of cartilage and bone, and, interestingly, developmental processes appear to be recapitulated in osteoarthritic (OA) cartilage. The present study was undertaken to characterize the expression pattern of Wnt and Fz genes during experimental OA and to determine the function of selected genes in experimental and human OA. METHODS: Longitudinal expression analysis was performed in 2 models of OA. Levels of messenger RNA for genes from the Wnt/beta-catenin pathway were determined in synovium and cartilage, and the results were validated using immunohistochemistry. Effects of selected genes were assessed in vitro using recombinant protein, and in vivo by adenoviral overexpression. RESULTS: Wnt-induced signaling protein 1 (WISP-1) expression was strongly increased in the synovium and cartilage of mice with experimental OA. Wnt-16 and Wnt-2B were also markedly up-regulated during the course of disease. Interestingly, increased WISP-1 expression was also found in human OA cartilage and synovium. Stimulation of macrophages and chondrocytes with recombinant WISP-1 resulted in interleukin-1-independent induction of several matrix metalloproteinases (MMPs) and aggrecanase. Adenoviral overexpression of WISP-1 in murine knee joints induced MMP and aggrecanase expression and resulted in cartilage damage. CONCLUSION: This study included a comprehensive characterization of Wnt and Frizzled gene expression in experimental and human OA articular joint tissue. The data demonstrate, for the first time, that WISP-1 expression is a feature of experimental and human OA and that WISP-1 regulates chondrocyte and macrophage MMP and aggrecanase expression and is capable of inducing articular cartilage damage in models of OA.
Assuntos
Artrite Experimental/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Osteoartrite do Quadril/metabolismo , Osteoartrite do Joelho/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Artrite Experimental/genética , Proteínas de Sinalização Intercelular CCN , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/enzimologia , Endopeptidases/biossíntese , Expressão Gênica , Membro Posterior/patologia , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/farmacologia , Articulações/metabolismo , Articulações/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Metaloproteinases da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas/genética , Osteoartrite do Quadril/genética , Osteoartrite do Joelho/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/farmacologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/genética , Membrana Sinovial/metabolismoRESUMO
Osteoarthritis has as main characteristics the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. In this study enhanced expression of TGF-beta1 and -beta3 was detected in developing osteophytes and articular cartilage during murine experimental osteoarthritis. To determine the role of endogenous TGF-beta on osteophyte formation and articular cartilage, TGF-beta activity was blocked via a scavenging soluble TGF-beta-RII. Our results clearly show that inhibition of endogenous TGF-beta nearly completely prevented osteophyte formation. In contrast, treatment with recombinant soluble TGF-beta-RII markedly enhanced articular cartilage proteoglycan loss and reduced the thickness of articular cartilage. In conclusion, we show for the first time that endogenous TGF-beta is a crucial factor in the process of osteophyte formation and has an important function in protection against cartilage loss.