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Preclinical models of osteochondral defects (OCDs) are fundamental test beds to evaluate treatment modalities before clinical translation. To increase the rigor and reproducibility of translational science for a robust "go or no-go," we evaluated disease progression and pain phenotypes within the whole joint for two OCD rat models with same defect size (1.5 x 0.8 mm) placed either in the trochlea or medial condyle of femur. Remarkably, we only found subtle transitory changes to gaits of rats with trochlear defect without any discernible effect to allodynia. At 8-weeks post-surgery, anatomical evaluations of joint showed early signs of osteoarthritis with EPIC-microCT. For the trochlear defect, cartilage attenuation was increased in trochlear, medial, and lateral compartments of the femur. For condylar defect, increased cartilage attenuation was isolated to the medial condyle of the femur. Further, the medial ossicle showed signs of deterioration as indicated with decreased bone mineral density and increased bone surface area to volume ratio. Thus, OCD in a weight-bearing region of the femur gave rise to more advanced osteoarthritis phenotype within a unilateral joint compartment. Subchondral bone remodeling was evident in both models without any indication of closure of the articular cartilage surface. We conclude that rat OCD, placed in the trochlear or condylar region of the femur, leads to differing severity of osteoarthritis progression. As found herein, repair of the defect with fibrous tissue and subchondral bone is insufficient to alleviate onset of osteoarthritis. Future therapies using rat OCD model should address joint osteoarthritis in addition to repair itself.
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Background: Osteoarthritis (OA) is a debilitating joints disease affecting millions of people worldwide. As OA progresses, chondrocytes experience heightened catabolic activity, often accompanied by alterations in the extracellular environment's osmolarity and acidity. Nevertheless, the precise mechanism by which chondrocytes perceive and respond to acidic stress remains unknown. Recently, there has been growing interest in pH-sensing G protein-coupled receptors (GPCRs), such as GPR68, within musculoskeletal tissues. However, function of GPR68 in cartilage during OA progression remains unknown. This study aims to identify the role of GPR68 in regulation of catabolic gene expression utilizing an in vitro model that simulates catabolic processes in OA. Methods: We examined the expression of GPCR by analyzing high throughput RNA-Seq data in human cartilage isolated from healthy donors and OA patients. De-identified and discarded OA cartilage was obtained from joint arthroplasty and chondrocytes were prepared by enzymatic digestion. Chondrocytes were treated with GPR68 agonist, Ogerin and then stimulated IL1ß and RNA isolation was performed using Trizol method. Reverse transcription was done using the cDNA synthesis kit and the expression of GPR68 and OA related catabolic genes was quantified using SYBR® green assays. Results: The transcriptome analysis revealed that pH sensing GPCR were expressed in human cartilage with a notable increase in the expression of GPR68 in OA cartilage which suggest a potential role for GPR68 in the pathogenesis of OA. Immunohistochemical (IHC) and qPCR analyses in human cartilage representing various stages of OA indicated a progressive increase in GPR68 expression in cartilage associated with higher OA grades, underscoring a correlation between GPR68 expression and the severity of OA. Furthermore, IHC analysis of Gpr68 in murine cartilage subjected to surgically induced OA demonstrated elevated levels of GPR68 in knee cartilage and meniscus. Using IL1ß stimulated in vitro model of OA catabolism, our qPCR analysis unveiled a time-dependent increase in GPR68 expression in response to IL1ß stimulation, which correlates with the expression of matrix degrading proteases suggesting the role of GPR68 in chondrocytes catabolism and matrix degeneration. Using pharmacological activator of GPR68, our results further showed that GPR68 activation repressed the expression of MMPs in human chondrocytes. Conclusions: Our results demonstrated that GPR68 was robustly expressed in human cartilage and mice and its expression correlates with matrix degeneration and severity of OA progression in human and surgical model. GPR68 activation in human chondrocytes further repressed the expression of MMPs under OA pathological condition. These results identify GPR68 as a possible therapeutic target in the regulation of matrix degradation during OA.
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Cartílago Articular , Osteoartritis , Humanos , Animales , Ratones , Cartílago Articular/metabolismo , Osteoartritis/genética , Matriz Extracelular/genética , Receptores Acoplados a Proteínas G/genética , Proteínas de Unión al GTP/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Induced pluripotent stem cells (iPSCs) generated from human sources are valuable tools for studying skeletal development and diseases, as well as for potential use in regenerative medicine for skeletal tissues such as articular cartilage. To successfully differentiate human iPSCs into functional chondrocytes, it is essential to establish efficient and reproducible strategies that closely mimic the physiological chondrogenic differentiation process. Here, we describe a simple and efficient protocol for differentiation of human iPSCs into chondrocytes via generation of an intermediate population of mesenchymal progenitors. These methodologies include step-by-step procedures for mesenchymal derivation, induction of chondrogenic differentiation, and evaluation of the chondrogenic marker gene expression. In this protocol, we describe the detailed procedure for successful derivation of mesenchymal progenitor population from human iPSCs, which are then differentiated into chondrocytes using high-density culture conditions by stimulating with bone morphogenetic protein-2 (BMP-2) or transforming growth factor beta-3 (TGFß-3). The differentiated iPSCs exhibit temporal expression of cartilage genes and accumulation of a cartilaginous extracellular matrix in vitro, indicating successful chondrogenic differentiation. These detailed methodologies help effective differentiation of human iPSCs into the chondrogenic lineage to obtain functional chondrocytes, which hold great promise for modeling skeletal development and disease, as well as for potential use in regenerative medicine for cell-based therapy for cartilage regeneration. Key features ⢠Differentiation of human iPSCs into chondrocytes using 3D culture methods. ⢠Uses mesenchymal progenitors as an intermediate for differentiation into chondrocytes.
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Human induced pluripotent stem cells (hiPSCs) hold immense promise in regenerative medicine as they can differentiate into various cell lineages, including adipocytes, osteoblasts, and chondrocytes. Precisely guiding hiPSC-derived mesenchymal progenitor cells (iMSCs) towards specific differentiation pathways is crucial for harnessing their therapeutic potential in tissue engineering, disease modeling, and regenerative therapies. To achieve this, we present a comprehensive and reproducible protocol for effectively differentiating iMSCs into adipocytes and osteoblasts. The differentiation process entails culturing iMSCs in tailored media supplemented with specific growth factors, which act as cues to initiate adipogenic or osteogenic commitment. Our protocol provides step-by-step guidelines for achieving adipocyte and osteoblast differentiation, ensuring the generation of mature and functional cells. To validate the success of differentiation, key assessment criteria are employed. For adipogenesis, the presence of characteristic lipid droplets within the iMSC-derived cells is considered indicative of successful differentiation. Meanwhile, Alizarin Red staining serves as a marker for the osteogenic differentiation, confirming the formation of mineralized nodules. Importantly, the described method stands out due to its simplicity, eliminating the need for specialized equipment, expensive materials, or complex reagents. Its ease of implementation offers an attractive advantage for researchers seeking robust and cost-effective approaches to derive adipocytes and osteoblasts from iMSCs. Overall, this protocol establishes a foundation for exploring the therapeutic potential of hiPSC-derived cells and advancing the field of regenerative medicine. Key features ⢠iMSC derivation in this protocol uses embryonic body formation technique. ⢠Adipogenesis and osteogenesis protocols were optimized for human iPSC-derived iMSCs. ⢠Derivation of iMSC from hiPSC was developed in a feeder-free culture condition. ⢠This protocol does not include human iPSC reprogramming strategies.
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Induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine. The iPSCs exhibit a preference for lineage differentiation to the donor cell type indicating the existence of memory of origin. Although the intrinsic effect of the donor cell type on differentiation of iPSCs is well recognized, whether disease-specific factors of donor cells influence the differentiation capacity of iPSC remains unknown. Using viral based reprogramming, we demonstrated the generation of iPSCs from chondrocytes isolated from healthy (AC-iPSCs) and osteoarthritis cartilage (OA-iPSCs). These reprogrammed cells acquired markers of pluripotency and differentiated into uncommitted mesenchymal-like progenitors. Interestingly, AC-iPSCs exhibited enhanced chondrogenic potential as compared OA-iPSCs and showed increased expression of chondrogenic genes. Pan-transcriptome analysis showed that chondrocytes derived from AC-iPSCs were enriched in molecular pathways related to energy metabolism and epigenetic regulation, together with distinct expression signature that distinguishes them from OA-iPSCs. Our molecular tracing data demonstrated that dysregulation of epigenetic and metabolic factors seen in OA chondrocytes relative to healthy chondrocytes persisted following iPSC reprogramming and differentiation toward mesenchymal progenitors. Our results suggest that the epigenetic and metabolic memory of disease may predispose OA-iPSCs for their reduced chondrogenic differentiation and thus regulation at epigenetic and metabolic level may be an effective strategy for controlling the chondrogenic potential of iPSCs.
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Células Madre Pluripotentes Inducidas , Osteoartritis , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Transcriptoma , Epigénesis Genética , Cartílago , Diferenciación Celular/genética , Perfilación de la Expresión Génica , Osteoartritis/genética , Osteoartritis/metabolismoRESUMEN
Many metabolic bone diseases arise as a result excessive osteoclastic bone resorption, which has motivated efforts to identify new molecular targets that can inhibit the formation or activity of these bone-resorbing cells. Mounting evidence indicates that the transcription factor Runx1 acts as a transcriptional repressor of osteoclast formation. Prior studies using a conditional knockout approach suggested that Runx1 in osteoclast precursors acts as an inhibitor of osteoclastogenesis; however, the effects of upregulation of Runx1 on osteoclast formation remain unknown. In this study, we investigated the skeletal effects of conditional overexpression of Runx1 in preosteoclasts by crossing novel Runx1 gain-of-function mice (Rosa26-LSL-Runx1) with LysM-Cre transgenic mice. We observed a sex-dependent effect whereby overexpression of Runx1 in female mice increased trabecular bone microarchitectural indices and improved torsion biomechanical properties. These effects were likely mediated by delayed osteoclastogenesis and decreased bone resorption. Transcriptomics analyses during osteoclastogenesis revealed a distinct transcriptomic profile in the Runx1-overexpressing cells, with enrichment of genes related to redox signaling, apoptosis, osteoclast differentiation, and bone remodeling. These data further confirm the antiosteoclastogenic activities of Runx1 and provide new insight into the molecular targets that may mediate these effects.
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Resorción Ósea , Osteoclastos , Animales , Densidad Ósea , Resorción Ósea/genética , Resorción Ósea/metabolismo , Diferenciación Celular/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Femenino , Ratones , Osteogénesis/genética , Ligando RANK/metabolismoRESUMEN
Background: Intervertebral disc (IVD) degeneration is strongly associated with low back pain and is highly prevalent in the elderly population. Hallmarks of IVD degeneration include cell loss and extracellular matrix degradation. The PH domain leucine-rich-repeats protein phosphatase (PHLPP1) is highly expressed in diseased cartilaginous tissues where it is linked to extracellular matrix degradation. This study explored the ability of PHLPP1 deficiency to protect against age-related spontaneous IVD degeneration. Methods: Lumbar IVDs of global Phlpp1 knockout (KO) and wildtype (WT) mice were collected at 5 months (young) and 20 months (aged). Picrosirius red-alcian blue staining (PR-AB) was performed to examine IVD structure and histological score. The expression of aggrecan, ADAMTS5, KRT19, FOXO1 and FOXO3 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human nucleus pulposus (NP) samples were obtained from patients diagnosed with IVD degeneration. PHLPP1 knockdown in human degenerated NP cells was conducted using small interfering RNA (siRNA) transfection. The expression of PHLPP1 regulated downstream targets was analyzed via immunoblot and real time quantitative PCR. Results: Histological analysis showed that Phlpp1 KO decreased the prevalence and severity of age-related IVD degeneration. The deficiency of PHLPP1 promoted the increased expression of NP phenotypic marker KRT19, aggrecan and FOXO1, and decreased levels of ADMATS5 and cell apoptosis in the NP of aged mice. In degenerated human NP cells, PHLPP1 knockdown induced FOXO1 protein levels while FOXO1 inhibition offset the beneficial effects of PHLPP1 knockdown on KRT19 gene and protein expression. Conclusions: Our findings indicate that Phlpp1 deficiency protected against NP phenotypic changes, extracellular matrix degradation, and cell apoptosis in the process of IVD degeneration, probably through FOXO1 activation, making PHLPP1 a promising therapeutic target for treating IVD degeneration.
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Sexual dimorphism of the skeleton is well documented. At maturity, the male skeleton is typically larger and has a higher bone density than the female skeleton. However, the underlying mechanisms for these differences are not completely understood. In this study, we examined sexual dimorphism in the formation of osteoclasts between cells from female and male mice. We found that the number of osteoclasts in bones was greater in females. Similarly, in vitro osteoclast differentiation was accelerated in female osteoclast precursor (OCP) cells. To further characterize sex differences between female and male osteoclasts, we performed gene expression profiling of cultured, highly purified, murine bone marrow OCPs that had been treated for 3 days with macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). We found that 125 genes were differentially regulated in a sex-dependent manner. In addition to genes that are contained on sex chromosomes, transcriptional sexual dimorphism was found to be mediated by genes involved in innate immune and inflammatory response pathways. Furthermore, the NF-κB-NFATc1 axis was activated earlier in female differentiating OCPs, which partially explains the differences in transcriptomic sexual dimorphism in these cells. Collectively, these findings identify multigenic sex-dependent intrinsic difference in differentiating OCPs, which results from an altered response to osteoclastogenic stimulation. In humans, these differences could contribute to the lower peak bone mass and increased risk of osteoporosis that females demonstrate relative to males. © 2021 American Society for Bone and Mineral Research (ASBMR).
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Osteoclastos , Caracteres Sexuales , Animales , Células de la Médula Ósea , Diferenciación Celular , Células Cultivadas , Femenino , Factor Estimulante de Colonias de Macrófagos , Masculino , Ratones , Factores de Transcripción NFATC , Osteogénesis , Ligando RANKRESUMEN
Intervertebral disc (IVD) disease (IDD) is a complex, multifactorial disease. While various aspects of IDD progression have been reported, the underlying molecular pathways and transcriptional networks that govern the maintenance of healthy nucleus pulposus (NP) and annulus fibrosus (AF) have not been fully elucidated. We defined the transcriptome map of healthy human IVD by performing single-cell RNA-sequencing (scRNA-seq) in primary AF and NP cells isolated from non-degenerated lumbar disc. Our systematic and comprehensive analyses revealed distinct genetic architecture of human NP and AF compartments and identified 2,196 differentially expressed genes. Gene enrichment analysis showed that SFRP1, BIRC5, CYTL1, ESM1 and CCNB2 genes were highly expressed in the AF cells; whereas, COL2A1, DSC3, COL9A3, COL11A1, and ANGPTL7 were mostly expressed in the NP cells. Further, functional annotation clustering analysis revealed the enrichment of receptor signaling pathways genes in AF cells, while NP cells showed high expression of genes related to the protein synthesis machinery. Subsequent interaction network analysis revealed a structured network of extracellular matrix genes in NP compartments. Our regulatory network analysis identified FOXM1 and KDM4E as signature transcription factor of AF and NP respectively, which might be involved in the regulation of core genes of AF and NP transcriptome.
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Anillo Fibroso/fisiología , Núcleo Pulposo/fisiología , Transcripción Genética/genética , Matriz Extracelular/genética , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Humanos , Disco Intervertebral/fisiología , Degeneración del Disco Intervertebral/genética , Desplazamiento del Disco Intervertebral/genética , RNA-Seq/métodos , Transducción de Señal/genética , Transcriptoma/genéticaRESUMEN
Intervertebral disc (IVD) degeneration (IDD) is a multifactorial physiological process which is often associated with lower back pain. Previous studies have identified some molecular markers associated with disc degeneration, which despite their significant contributions, have provided limited insight into the etiology of IDD. In this study, we utilized a network medicine approach to uncover potential molecular mediators of IDD. Our systematic analyses of IDD associated with 284 genes included functional annotation clustering, interaction networks, network cluster analysis and Transcription factors (TFs)-target gene network analysis. The functional enrichment and protein-protein interaction network analysis highlighted the role of inflammatory genes and cytokine/chemokine signaling in IDD. Moreover, sub-network analysis identified significant clusters possessing organized networks of 24 cytokine and chemokine genes, which may be considered as key modulators for IDD. The expression of these genes was validated in independent microarray datasets. In addition, the regulatory network analysis identified the role of multiple transcription factors, with RUNX1 being a master regulator in the pathogenesis of IDD. Our analyses highlighted the role of cytokine genes and interacting pathways in IDD and further improved our understanding of the genetic mechanisms underlying IDD.
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Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Redes Reguladoras de Genes , Degeneración del Disco Intervertebral/genética , Citocinas/genética , Citocinas/metabolismo , HumanosRESUMEN
BACKGROUND: Chondroclasts and osteoclasts have been previously identified as the cells capable of resorbing mineralized cartilage and bone matrices, respectively. While both cell types appear morphologically similar, contain comparable ultrastructural features, and express tartrate-resistant acid phosphatase (TRAP), however, no information is available about the genomic similarities and differences between osteoclasts and chondroclasts. METHODS: To address this question, we laser captured homogeneous populations of TRAP-positive cells that interact with bone (osteoclasts) and TRAP-positive cells that interact with mineralized cartilage (chondroclasts) on the same plane from murine femoral fracture callus sections. We then performed a global transcriptome profiling of chondroclasts and osteoclasts by utilizing a mouse genome Agilent GE 4X44K V2 microarray platform. Multiple computational approaches and interaction networks were used to analyze the transcriptomic landscape of osteoclasts and chondroclasts. RESULTS: Our systematic and comprehensive analyses using hierarchical clustering and principal component analysis (PCA) demonstrate that chondroclasts and osteoclasts are transcriptionally distinct cell populations and exhibit discrete transcriptomic signatures as revealed by multivariate analysis involving scatter plot, volcano plot, and heatmap analysis. TaqMan qPCR was used to validate the microarray results. Intriguingly, the functional enrichment and integrated network analyses revealed distinct Gene Ontology terms and molecular pathways specific to chondroclasts and osteoclasts and further suggest that subsets of metabolic genes were specific to chondroclasts. Protein-protein interaction (PPI) network analysis showed an abundance of structured networks of metabolic pathways, ATP synthesis, and proteasome pathways in chondroclasts. The regulatory network analysis using transcription factor-target gene network predicted a pool of genes including ETV6, SIRT1, and ATF1 as chondroclast-specific gene signature. CONCLUSIONS: Our study provides an important genetic resource for further exploration of chondroclast function in vivo. To our knowledge, this is the first demonstration of genetic landscape of osteoclasts from chondroclasts identifying unique molecular signatures, functional clustering, and interaction network.
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Fosfatasa Ácida , Osteoclastos , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Animales , Huesos/metabolismo , Cartílago/metabolismo , Ratones , Osteoclastos/metabolismo , TranscriptomaRESUMEN
Intervertebral disc degeneration (IDD) is a public health dilemma as it is associated with low back and neck pain, a frequent reason for patients to visit the physician. During IDD, nucleus pulposus (NP), the central compartment of intervertebral disc (IVD) undergo degeneration. Stem cells have been adopted as a promising biological source to regenerate the IVD and restore its function. Here, we describe a simple, two-step differentiation strategy using a cocktail of four factors (LDN, AGN, FGF, and CHIR) for efficient derivation of notochordal cells from human embryonic stem cells (hESCs). We employed a CRISPR/Cas9 based genome-editing approach to knock-in the mCherry reporter vector upstream of the 3' untranslated region of the Noto gene in H9-hESCs and monitored notochordal cell differentiation. Our data show that treatment of H9-hESCs with the above-mentioned four factors for 6 days successfully resulted in notochordal cells. These cells were characterized by morphology, immunostaining, and gene and protein expression analyses for established notochordal cell markers including FoxA2, SHH, and Brachyury. Additionally, pan-genomic high-throughput single cell RNA-sequencing revealed an efficient and robust notochordal differentiation. We further identified a key regulatory network consisting of eight candidate genes encoding transcription factors including PAX6, GDF3, FOXD3, TDGF1, and SOX5, which are considered as potential drivers of notochordal differentiation. This is the first single cell transcriptomic analysis of notochordal cells derived from hESCs. The ability to efficiently obtain notochordal cells from pluripotent stem cells provides an additional tool to develop new cell-based therapies for the treatment of IDD.
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Diferenciación Celular/genética , Células Madre Embrionarias Humanas/metabolismo , Degeneración del Disco Intervertebral/genética , Transcriptoma/genética , Biomarcadores/metabolismo , Proteínas Fetales/genética , Factores de Transcripción Forkhead/genética , Proteínas Ligadas a GPI/genética , Redes Reguladoras de Genes/genética , Factor 3 de Diferenciación de Crecimiento/genética , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes Inducidas , Péptidos y Proteínas de Señalización Intercelular/genética , Disco Intervertebral/crecimiento & desarrollo , Degeneración del Disco Intervertebral/patología , Proteínas de Neoplasias/genética , Notocorda/crecimiento & desarrollo , Notocorda/metabolismo , Núcleo Pulposo/crecimiento & desarrollo , Núcleo Pulposo/metabolismo , Factor de Transcripción PAX6/genética , Regeneración/genética , Factores de Transcripción SOXD/genética , Análisis de la Célula Individual , Proteínas de Dominio T Box/genéticaRESUMEN
Autophagy is very critical for multiple cellular processes. Autophagy plays a critical role in bone cell differentiation and function.
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Autofagia/fisiología , Remodelación Ósea/fisiología , Huesos/citología , Osteogénesis/fisiología , Animales , Diferenciación Celular/fisiología , Homeostasis/fisiología , Humanos , Osteoblastos/citología , Osteoclastos/citología , Osteocitos/citologíaRESUMEN
OBJECTIVE: Cytokine expression is tightly regulated posttranscriptionally, but high levels of interleukin-6 (IL-6) in patients with osteoarthritis (OA) indicate that regulatory mechanisms are disrupted in this disorder. The enzyme ZCCHC6 (zinc-finger CCHC domain-containing protein 6; TUT-7) has been implicated in posttranscriptional regulation of inflammatory cytokine expression, but its role in OA pathogenesis is unknown. The present study was undertaken to investigate whether ZCCHC6 directs the expression of IL-6 and influences OA pathogenesis in vivo. METHODS: Human and mouse chondrocytes were stimulated with recombinant IL-1ß. Expression of ZCCHC6 in human chondrocytes was knocked down using small interfering RNAs. IL-6 transcript stability was determined by actinomycin D chase, and 3'-uridylation of microRNAs was determined by deep sequencing. Zcchc6-/- mice were produced by gene targeting. OA was surgically induced in the knee joints of mice, and disease severity was scored using a semiquantitative grading system. RESULTS: ZCCHC6 was markedly up-regulated in damaged cartilage from human OA patients and from wild-type mice with surgically induced OA. Overexpression of ZCCHC6 induced the expression of IL-6, and its knockdown reduced IL-6 transcript stability and IL-1ß-induced IL-6 expression in chondrocytes. Reintroduction of Zcchc6 in Zcchc6-/- mouse chondrocytes rescued the IL-1ß-induced IL-6 expression. Knockdown of ZCCHC6 reduced the population of micro-RNA 26b (miR-26b) with 3'-uridylation by 60%. Zcchc6-/- mice with surgically induced OA produced low levels of IL-6 and exhibited reduced cartilage damage and synovitis in the joints. CONCLUSION: These findings indicate that ZCCHC6 enhances IL-6 expression in chondrocytes through transcript stabilization and by uridylating miR-26b, which abrogates repression of IL-6. Inhibition of IL-6 expression and significantly reduced OA severity in Zcchc6-/- mice identify ZCCHC6 as a novel therapeutic target to inhibit disease pathogenesis.
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Silenciador del Gen , Interleucina-6/metabolismo , Nucleotidiltransferasas/metabolismo , Osteoartritis/genética , Índice de Severidad de la Enfermedad , Animales , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Articulación de la Rodilla/metabolismo , Ratones , ARN Nucleotidiltransferasas , ARN Interferente Pequeño/metabolismo , Regulación hacia ArribaRESUMEN
Nrf2, a redox regulated transcription factor, has recently been shown to play a role in cartilage integrity but the mechanism remains largely unknown. Osteoarthritis (OA) is a multifactorial disease in which focal degradation of cartilage occurs. Here, we studied whether Nrf2 exerts chondroprotective effects by suppressing the oxidative stress and apoptosis in IL-1ß stimulated human OA chondrocytes. Expression of Nrf2 and its target genes HO-1, NQO1 and SOD2 was significantly high in OA cartilage compared to normal cartilage and was also higher in damaged area compared to smooth area of OA cartilage of the same patient. Human chondrocytes treated with IL-1ß resulted in robust Nrf2/ARE reporter activity, which was inhibited by pretreatment with antioxidants indicating that Nrf2 activity was due to IL-1ß-induced ROS generation. Ectopic expression of Nrf2 significantly suppressed the IL-1ß-induced generation of ROS while Nrf2 knockdown significantly increased the basal as well as IL-1ß-induced ROS levels in OA chondrocytes. Further, Nrf2 activation significantly inhibited the IL-1ß-induced activation of extrinsic and intrinsic apoptotic pathways as determined by inhibition of DNA fragmentation, activation of Caspase-3,-8,-9, cleavage of PARP, release of cytochrome-c, suppression of mitochondrial dysfunction and mitochondrial ROS production in OA chondrocytes. Nrf2 over-expression in OA chondrocytes increased the expression of anti-apoptotic proteins while pro-apoptotic proteins were suppressed. Importantly, Nrf2 over-expression activated ERK1/2 and its downstream targets-ELK1, P70S6K and P90RSK and suppressed the IL-1ß-induced apoptosis whereas inhibition of ERK1/2 activation abrogated the protective effects of Nrf2 in OA chondrocytes. Taken together, our data demonstrate that Nrf2 is a stress response protein in OA chondrocytes with anti-oxidative and anti-apoptotic function and acts via activation of ERK1/2/ELK1-P70S6K-P90RSK signaling axis. These activities of Nrf2 make it a promising candidate for the development of novel therapies for the management of OA.
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Condrocitos/fisiología , Factor 2 Relacionado con NF-E2/metabolismo , Osteoartritis/metabolismo , Apoptosis , Hidrolasas de Éster Carboxílico/metabolismo , Caspasas/metabolismo , Células Cultivadas , Humanos , Interleucina-1beta/inmunología , Sistema de Señalización de MAP Quinasas , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Regulación hacia Arriba , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Osteoarthritis (OA) is the most common joint disease and the leading cause of chronic disability in middle-aged and older populations worldwide. The development of disease modifying therapy for OA is in its infancy largely because the regulatory mechanisms for the molecular effectors of OA pathogenesis are poorly understood. Recent studies identified epigenetic events as a critical regulator of molecular players involved in the induction and development of OA. Epigenetic mechanisms include DNA methylation, non-coding RNA and histone modifications. The aim of this review is to briefly highlight the recent advances in the epigenetics of cartilage and potential of HDACs (Histone deacetylases) inhibitors in the therapeutic management of OA. We summarize the recent studies utilizing HDAC inhibitors as potential therapeutics for inhibiting disease progression and preventing the cartilage destruction in OA. HDACs control normal cartilage development and homeostasis and understanding the impact of HDACs inhibitors on the disease pathogenesis is of interest because of its importance in affecting overall cartilage health and homeostasis. These findings also shed new light on cartilage disease pathophysiology and provide substantial evidence that HDACs may be potential novel therapeutic targets in OA.
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Inhibidores de Histona Desacetilasas/uso terapéutico , Osteoartritis/tratamiento farmacológico , Osteoartritis/genética , Animales , Epigénesis Genética , HumanosRESUMEN
MicroRNAs, a group of small, noncoding RNAs that post-transcriptionally regulate gene expression, play important roles in chondrocyte function and in the development of osteoarthritis. We characterized the dynamic repertoire of the chondrocyte miRNome and miRISC-associated miRNome by deep sequencing analysis of primary human chondrocytes. IL-1ß treatment showed a modest effect on the expression profile of miRNAs in normal and osteoarthritis (OA) chondrocytes. We found a number of miRNAs that showed a wide range of sequence modifications including nucleotide additions and deletions at 5' and 3' ends; and nucleotide substitutions. miR-27b-3p showed the highest expression and miR-140-3p showed the highest number of sequence variations. AGO2 RIP-Seq analysis revealed the differential recruitment of a subset of expressed miRNAs and isoforms of miRNAs (isomiRs) to the miRISC in response to IL-1ß, including miR-146a-5p, miR-155-5p and miR-27b-3p. Together, these results reveal a complex repertoire of miRNAs and isomiRs in primary human chondrocytes. Here, we also show the changes in miRNA composition of the miRISC in primary human chondrocytes in response to IL-1ß treatment. These findings will provide an insight to the miRNA-mediated control of gene expression in the pathogenesis of OA.
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Condrocitos/química , Condrocitos/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , MicroARNs/análisis , Complejo Silenciador Inducido por ARN/análisis , Células Cultivadas , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Interleucina-1beta/metabolismo , MicroARNs/genéticaRESUMEN
BACKGROUND/OBJECTIVE: Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1ß. METHODS: The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. RESULTS: BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1ß induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that BME activated autophagy in chondrocytes via inhibition of mammalian target of rapamycin (mTOR) pathway. Of importance is our finding that BME-mediated suppression of IL-1ß induced expression of IL-6, MMP-3, -9, and -13 was autophagy dependent and was abrogated by inhibition of autophagy. CONCLUSION: The above results show that the Butea monosperma (Lam.) extract has strong potential to activate autophagy and suppress IL-1ß induced expression of IL-6 and MMP-3, -9 and -13 in human OA chondrocytes. This study shows that BME or compounds derived from BME can be developed as safe and effective chondroprotective agent(s) that function by activating autophagy to suppress the expression of inflammatory and catabolic factors associated with OA pathogenesis.
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Butea , Condrocitos/metabolismo , Interleucina-1beta/farmacología , Interleucina-6/biosíntesis , Metaloproteinasas de la Matriz/biosíntesis , Osteoartritis/metabolismo , Anciano , Autofagia/efectos de los fármacos , Autofagia/fisiología , Condrocitos/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Flores , Expresión Génica , Humanos , Interleucina-6/genética , Metaloproteinasa 13 de la Matriz/biosíntesis , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/biosíntesis , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasas de la Matriz/genética , Persona de Mediana Edad , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacologíaRESUMEN
Wogonin has recently been shown to possess anti-inflammatory and chondroprotective properties and is of considerable interest due to its broad pharmacological activities. The present study highlights that Wogonin binds DNA and exerts chondroprotective effects in vitro. Wogonin showed strong binding with chondrocytes genomic DNA in vitro. The mode of binding of Wogonin to genomic-DNA was assessed by competing Wogonin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. EtBr fluorescence reduced significantly with increase in Wogonin concentration suggesting possible DNA intercalation of Wogonin. Further, in silico molecular docking of Wogonin on mammalian DNA also indicated possible intercalation of Wogonin with DNA. The denaturation and FRET studies revealed that Wogonin prevents denaturation of DNA strands and provide stability to genomic DNA against a variety of chemical denaturants. The cellular uptake study showed that Wogonin enters osteoarthritis chondrocytes and was mainly localized in the nucleus. Wogonin treatment to OA chondrocytes protects the fragmentation of genomic DNA in response to IL-1ß as evaluated by DNA ladder and TUNEL assay. Treatment of chondrocytes with Wogonin resulted in significant suppression of IL-1ß-mediated induction of ROS. Further, Wogonin exhibited protective potential through potent suppression of extrinsic and intrinsic apoptotic pathways and induction of anti-apoptotic proteins in IL-1ß-stimulated osteoarthritis chondrocytes. Our data thus suggest that DNA intercalation by Wogonin may result in the stabilization of genomic DNA leading to protective activity.
Asunto(s)
Condrocitos/efectos de los fármacos , ADN/metabolismo , Flavanonas/farmacología , Sustancias Intercalantes/farmacología , Interleucina-1beta/farmacología , Sustancias Protectoras/farmacología , Apoptosis/efectos de los fármacos , Sitios de Unión , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , ADN/química , Flavanonas/química , Flavanonas/metabolismo , Flavonoides/química , Flavonoides/farmacología , Transferencia Resonante de Energía de Fluorescencia , Humanos , Sustancias Intercalantes/química , Sustancias Intercalantes/metabolismo , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , Osteoartritis/metabolismo , Osteoartritis/patología , Sustancias Protectoras/química , Sustancias Protectoras/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
This article contains data related to the article "Wogonin, a plant derived small molecule exerts potent anti-inflammatory and chondroprotective effects through activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes" (Khan et al. 2017) [1]. The data are related to effects of Wogonin on the viability and IL-1ß-stimulated activation of NF-κB and ERK1/2, JNK1/2 and p38 MAPKs in human OA chondrocytes. Gene expression data representing the chondrogenic phenotype and the efficiency of Nrf2 knockdown in monolayer culture of human OA chondrocytes were shown. Moreover, mass spectrometric calibration curve of Wogonin used to quantify the intracellular uptake were also presented. The data are presented in the form of figures and significance of these has been given in the research article (Khan et al. 2017) [1].