RESUMO
Type 2 diabetes is linked with increased incidence and severity of osteoarthritis. The purpose of this study was to determine the effect of extracellular glucose within the normal blood glucose and hyperglycemic range on catabolic enzyme production by chondrocytes isolated from osteoarthritic (OA) and macroscopically normal (MN) human cartilage under oxygenated (18.9% oxygen) and hypoxic (1% oxygen) conditions. OA and MN chondrocytes were maintained in 4, 6, 8, or 10 mM glucose for 24 h. Glucose consumption, GLUT1 glucose transporter levels, MMP13 and ADAMTS5 production, and levels of RUNX2, a transcriptional regulator of MMP13, ADAMTS5, and GLUT1, were assessed by enzyme-linked assays, RT-qPCR and/or western blot. Under oxygenated conditions, glucose consumption and GLUT1 protein levels were higher in OA but not MN chondrocytes in 10 mM glucose compared to 4 mM. Both RNA and protein levels of MMP13 and ADAMTS5 were also higher in OA but not MN chondrocytes in 10 mM compared to 4 mM glucose under oxygenated conditions. Expression of RUNX2 was overall lower in MN than OA chondrocytes and there was no consistent effect of extracellular glucose concentration on RUNX2 levels in MN chondrocytes. However, protein (but not RNA) levels of RUNX2 were elevated in OA chondrocytes maintained in 10 mM versus 4 mM glucose under oxygenated conditions. In contrast, neither RUNX2 levels or MMP13 or ADAMTS5 expression were increased in OA chondrocytes maintained in 10 mM compared to 4 mM glucose in hypoxia. Elevated extracellular glucose leads to increased glucose consumption and increased RUNX2 protein levels, promoting production of MMP13 and ADAMTS5 by OA chondrocytes in oxygenated but not hypoxic conditions. These findings suggest that hyperglycaemia may exacerbate chondrocyte-mediated cartilage catabolism in the oxygenated superficial zone of cartilage in vivo in patients with undertreated type 2 diabetes, contributing to increased OA severity.
Assuntos
Proteína ADAMTS5 , Hipóxia Celular , Condrócitos , Glucose , Metaloproteinase 13 da Matriz , Osteoartrite , Humanos , Condrócitos/metabolismo , Condrócitos/patologia , Proteína ADAMTS5/metabolismo , Proteína ADAMTS5/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/genética , Glucose/metabolismo , Glucose/farmacologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/genética , Idoso , Feminino , Oxigênio/metabolismo , Oxigênio/farmacologia , Masculino , Pessoa de Meia-Idade , Células Cultivadas , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genéticaRESUMO
The process of proplatelet formation (PPF) requires coordinated interaction between megakaryocytes (MKs) and the extracellular matrix (ECM), followed by a dynamic reorganization of the actin and microtubule cytoskeleton. Localized fluxes of intracellular calcium ions (Ca2+) facilitate MK-ECM interaction and PPF. Glutamate-gated N-methyl-D-aspartate receptor (NMDAR) is highly permeable to Ca2+. NMDAR antagonists inhibit MK maturation ex vivo; however, there are no in vivo data. Using the Cre-loxP system, we generated a platelet lineage-specific knockout mouse model of reduced NMDAR function in MKs and platelets (Pf4-Grin1-/- mice). Effects of NMDAR deletion were examined using well-established assays of platelet function and production in vivo and ex vivo. We found that Pf4-Grin1-/- mice had defects in megakaryopoiesis, thrombopoiesis, and platelet function, which manifested as reduced platelet counts, lower rates of platelet production in the immune model of thrombocytopenia, and prolonged tail bleeding time. Platelet activation was impaired to a range of agonists associated with reduced Ca2+ responses, including metabotropic like, and defective platelet spreading. MKs showed reduced colony and proplatelet formation. Impaired reorganization of intracellular F-actin and α-tubulin was identified as the main cause of reduced platelet function and production. Pf4-Grin1-/- MKs also had lower levels of transcripts encoding crucial ECM elements and enzymes, suggesting NMDAR signaling is involved in ECM remodeling. In summary, we provide the first genetic evidence that NMDAR plays an active role in platelet function and production. NMDAR regulates PPF through a mechanism that involves MK-ECM interaction and cytoskeletal reorganization. Our results suggest that NMDAR helps guide PPF in vivo.
Assuntos
Megacariócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Trombocitopenia , Actinas/metabolismo , Animais , Plaquetas/metabolismo , Cálcio , Camundongos , Camundongos Knockout , Receptores de N-Metil-D-Aspartato/genética , Trombocitopenia/genética , Trombopoese/fisiologiaRESUMO
Gout is caused by monosodium urate (MSU) crystal deposition within joints. This leads to acute episodes of inflammation ("gout flares") driven by NLRP3 inflammasome activation in macrophages. Gout flares are frequently present during late night/early morning. The reason for this timing is unclear. Recent evidence suggests the NLRP3 inflammasome is under circadian control. The purpose of this study was to determine whether MSU crystals cause changes in the circadian clock in macrophages leading to time-of-day differences in NLRP3 inflammasome activation. Levels of circadian clock components were measured in undifferentiated "monocytic" and PMA-differentiated "macrophagic" THP-1 cells cultured with/without MSU crystals. Caspase-1 activity was measured to assess NLRP3 inflammasome activity. MSU crystal exposure resulted in minimal effects on clock genes in THP-1 monocytes but BMAL1, CRY1, PER2, and REV-ERBα showed altered expression with reduced protein levels of BMAL1 and REV-ERBα in THP-1 macrophages. REV-ERBα activation or BMAL1 over-expression resulted in reduced MSU crystal-induced caspase-1 activity. BMAL1 knockdown resulted in a further increase in MSU crystal-induced caspase-1 activity, but only at times of day when BMAL1 levels were naturally high. MSU crystal-induced NLRP3 inflammasome activation was greatest at the time of day when BMAL1 levels were naturally low. MSU crystals alter the expression of circadian clock components in THP-1 macrophages leading to loss of BMAL1 and REV-ERBα-mediated repression of NLRP3 inflammasome activity and time-of-day differences in susceptibility to inflammasome activation. Our findings suggest that the nocturnal risk of gout flare is at least partially a consequence of altered circadian control of immune cell function.
Assuntos
Relógios Circadianos , Gota , Humanos , Gota/genética , Inflamassomos/metabolismo , Ácido Úrico/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Relógios Circadianos/genética , Exacerbação dos Sintomas , Macrófagos/metabolismo , Caspases/metabolismo , Interleucina-1beta/metabolismoRESUMO
OBJECTIVES: The prevalence and severity of knee osteoarthritis (OA) are greater in females than males. The purpose of this study was to determine whether there is an underlying difference in the biology of OA chondrocytes between males and females. METHODS: Chondrocytes were obtained following knee arthroplasty from male and female patients with primary OA. Phenotype marker expression, glucose and fat consumption, and rates of glycolysis and oxidative phosphorylation were compared between females and males. RNAi was used to determine the consequences of differential expression of Sry-box transcription factor 9 (SOX9) and PGC1α between males and females. RESULTS: OA chondrocytes from male donors showed elevated ribonucleic acid (RNA) and protein levels of SOX9, elevated COL2A1 protein synthesis, higher glucose consumption, and higher usage of glycolysis compared to females. OA chondrocytes from females had higher PGC1α protein levels, higher fat consumption, and higher oxidative energy metabolism than males. Knockdown of SOX9 reduced expression of COL2A1 to a greater extent in male OA chondrocytes than females whereas knockdown of PGC1α reduced COL2A1 expression in females but not males. Expression of ACAN and the glycolytic enzyme PGK1 was also reduced in males but not females following SOX9 knockdown. CONCLUSIONS: OA chondrocyte phenotype and energy metabolism differ between males and females. Our results indicate transcriptional control of COL2A1 differs between the two. Differences in chondrocyte biology between males and females imply the underlying mechanisms involved in OA may also differ, highlighting the need to consider sex and gender when investigating pathogenesis and potential treatments for OA.
RESUMO
Metabolic syndrome is a risk factor for osteoarthritis. Elevated leptin levels have been implicated as a potential cause of this association. Previous studies have shown that supra-physiological leptin concentrations can induce osteoarthritis-like changes in chondrocyte phenotype. Here, we tested the effects of leptin in the concentration range found in synovial fluid on chondrocyte phenotype. Chondrocytes isolated from macroscopically normal regions of cartilage within osteoarthritic joints from patients undergoing knee arthroplasty, all with body mass index >30 kg/m2 were treated with 2-40 ng/ml leptin for 24 h. Chondrocyte phenotype marker expression was measured by RT-qPCR and western blot. The role of HES1 in mediating the effects of leptin was determined by gene knockdown using RNAi and over-expression using adenoviral-mediated gene delivery. Treatment of chondrocytes with 20 or 40 ng/ml leptin resulted in decreased SOX9 levels and decreased levels of the SOX9-target genes COL2A1 and ACAN. Levels of HES1 were lower and ADAMTS5 higher in chondrocytes treated with 20 or 40 ng/ml leptin. HES1 knockdown resulted in increased ADAMTS5 expression whereas over-expression of HES1 prevented the leptin-induced increase in ADAMTS5. An increase in MMP13 expression was only evident in chondrocytes treated with 40 ng/ml leptin and was not mediated by HES1 activity. High concentrations of leptin can cause changes in chondrocyte phenotype consistent with those seen in osteoarthritis. Synovial fluid leptin concentrations of this level are typically observed in patients with metabolic syndrome and/or women, suggesting elevated leptin levels may form part of the multifactorial network that leads to osteoarthritis development in these patients.
Assuntos
Cartilagem Articular , Síndrome Metabólica , Osteoartrite , Humanos , Feminino , Leptina/farmacologia , Condrócitos/metabolismo , Síndrome Metabólica/metabolismo , Osteoartrite/metabolismo , Fenótipo , Cartilagem Articular/metabolismo , Células CultivadasRESUMO
Peripheral circadian clocks control cell proliferation and survival, but little is known about their role and regulation in megakaryocytic cells. N-methyl-D-aspartate receptor (NMDAR) regulates the central clock in the brain. The purpose of this study was to determine whether NMDAR regulates the megakaryocytic cell clock and whether the megakaryocytic clock regulates cell proliferation and cell death. We found that both the Meg-01 megakaryocytic cell line and native murine megakaryocytes expressed circadian clock genes. Megakaryocyte-directed deletion of Grin1 in mice caused significant disruption of the circadian rhythm pathway at the transcriptional level and increased expression of BMAL1 at the protein level. Similarly, both pharmacological (MK-801) and genetic (GRIN-/-) inhibition of NMDAR in Meg-01 cells in vitro resulted in widespread changes in clock gene expression including increased expression of BMAL1, the core clock transcription factor. BMAL1 overexpression reduced Meg-01 cell proliferation and altered the time-dependent expression of the cell cycle regulators MYC and WEE1, whereas BMAL1 knockdown led to increased cell death in Meg-01-GRIN1-/- cells. Our results demonstrate that NMDAR regulates the circadian clock in megakaryocytic cells and that the circadian clock component BMAL1 contributes to the control of Meg-01 cell proliferation and survival.
Why was the study done? Time of day impacts platelet function and production. Our bodies are informed about external time by the brain, but all other cells including platelet precursors megakaryocytes also have their own clock.Circadian disruption contributes to disorders such as thrombosis (e.g. stroke and heart attacks) and blood cancers (e.g. leukemia). However, the mechanism of circadian control in megakaryocytes remains poorly elucidated.N-methyl-D-aspartate receptor (NMDAR) regulates circadian clock in the brain and is expressed in megakaryocytes, thus we hypothesized that NMDAR also regulates circadian clock in megakaryocytic cells.What did the researchers do and find? We used Meg-01 cell line, its genetically modified version with deleted NMDAR, and data from murine megakaryocytes to determine the NMDAR impact on the clock in these cells.We found that megakaryocytic cells had all the machinery required to maintain their own circadian clock. NMDAR deletion disrupted circadian clock in megakaryocytic cells.Manipulation of circadian clock in Meg-01 cells (through BMAL1 overexpression) impacted proliferation and survival of cells.What do the results mean? Megakaryocytic cells have their own circadian clock regulated by NMDAR, and its disruption impacts cell proliferation.What is the objective influence on the wider field? It is possible that deregulated function of megakaryocytes that occurs in disease can be corrected through the modulation of NMDAR or other components of the cellular circadian clock, thus further studies to develop and test such strategies in disease models are warranted.
Assuntos
Relógios Circadianos , Camundongos , Animais , Relógios Circadianos/genética , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Regulação da Expressão Gênica , Ritmo Circadiano/fisiologia , Proliferação de CélulasRESUMO
Chondrocyte phenotype and energy metabolism are altered in osteoarthritis (OA). However, most studies characterising the change in human chondrocyte behaviour in OA have been conducted in supraphysiological oxygen concentrations. The purpose of this study was to compare phenotype and energy metabolism in chondrocytes from macroscopically normal (MN) and OA cartilage maintained in 18.9% (standard tissue culture), 6% (equivalent to superficial zone of cartilage in vivo) or 1% oxygen (equivalent to deep zone of cartilage in vivo). MMP13 production was higher in chondrocytes from OA compared to MN cartilage in hyperoxia and physoxia but not hypoxia. Hypoxia promoted SOX9, COL2A1 and ACAN protein expression in chondrocytes from MN but not OA cartilage. OA chondrocytes used higher levels of glycolysis regardless of oxygen availability. These results show that differences in phenotype and energy metabolism between chondrocytes from OA and MN cartilage differ depending on oxygen availability. OA chondrocytes show elevated synthesis of cartilage-catabolising enzymes and chondrocytes from MN cartilage show reduced cartilage anabolism in oxygenated conditions. This is relevant as a recent study has shown that oxygen levels are elevated in OA cartilage in vivo. Our findings may indicate that this elevated cartilage oxygenation may promote cartilage loss in OA.
Assuntos
Cartilagem Articular , Hiperóxia , Osteoartrite , Humanos , Condrócitos/metabolismo , Hiperóxia/metabolismo , Osteoartrite/metabolismo , Fenótipo , Cartilagem Articular/metabolismo , Hipóxia/metabolismo , Metabolismo Energético , Oxigênio/metabolismo , Células CultivadasRESUMO
The circadian clock is a specialized cell signalling pathway present in all cells. Loss of clock function leads to tissue degeneration and premature ageing in animal models demonstrating the fundamental importance of clocks for cell, tissue and organism health. There is now considerable evidence that the chondrocyte circadian clock is altered in OA. The purpose of this review is to summarize current knowledge regarding the nature of the change in the chondrocyte clock in OA and the implications of this change for disease development. Expression of the core clock component, BMAL1, has consistently been shown to be lower in OA chondrocytes. This may contribute to changes in chondrocyte differentiation and extracellular matrix turnover in disease. Circadian clocks are highly responsive to environmental factors. Mechanical loading, diet, inflammation and oxidative insult can all influence clock function. These factors may contribute to causing the change in the chondrocyte clock in OA.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Relógios Circadianos , Osteoartrite/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/fisiopatologia , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Condrogênese , Criptocromos/metabolismo , Dieta , Matriz Extracelular/metabolismo , Humanos , Inflamação , Osteoartrite/fisiopatologia , Estresse Oxidativo , Proteínas Circadianas Period/metabolismo , Núcleo Supraquiasmático/metabolismo , Suporte de CargaRESUMO
The FOXO family of forkhead transcription factors have a pivotal role in determining cell fate in response to oxidative stress. FOXO activity can either promote cell survival or induce cell death. Increased FOXO-mediated cell death has been implicated in the pathogenesis of degenerative diseases affecting musculoskeletal tissues. The aim of this study was to determine the conditions under which one member of the FOXO family, FOXO3a, promotes cell survival as opposed to cell death. Treatment of primary human tenocytes with 1 pM hydrogen peroxide for 18 h resulted in increased protein levels of FOXO3a. In peroxide-treated cells cultured in low serum media, FOXO3a inhibited cell proliferation and protected against apoptosis. However in peroxide treated cells cultured in high serum media, cell proliferation was unchanged but level of apoptosis significantly increased. Similarly, in tenocytes transduced to over-express FOXO3a, cell proliferation was inhibited and level of apoptosis unchanged in cells cultured in low serum. However there was a robust increase in cell death in FOXO3a-expressing cells cultured in high serum. Inhibition of cell proliferation in either peroxide-treated or FOXO3a-expressing cells cultured in high serum protected against apoptosis induction. Conversely, addition of a Chk2 inhibitor to peroxide-treated or FOXO3a-expressing cells overrode the inhibitory effect of FOXO3a on cell proliferation and led to increased apoptosis in cells cultured in low serum. This study demonstrates that proliferating cells may be particularly susceptible to the apoptosis-inducing actions of FOXO3a. Inhibition of cell proliferation by FOXO3a may be a critical event in allowing the pro-survival rather than the pro-apoptotic activity of FOXO3a to prevail.
Assuntos
Apoptose/genética , Proliferação de Células/genética , Fatores de Transcrição Forkhead/genética , Tendões/citologia , Apoptose/efeitos dos fármacos , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Células MCF-7 , Oxidantes/farmacologia , Interferência de RNA , Soro/metabolismoRESUMO
UNLABELLED: Cellular senescence is an irreversible side effect of some pharmaceuticals which can contribute to tissue degeneration. OBJECTIVE: To determine whether pharmaceutical glucocorticoids induce senescence in tenocytes. METHODS: Features of senescence (ß-galactosidase activity at pH 6 (SA-ß-gal) and active mammalian/mechanistic target of rapamycin (mTOR) in cell cycle arrest) as well as the activity of the two main pathways leading to cell senescence were examined in glucocorticoid-treated primary human tenocytes. Evidence of senescence-inducing pathway induction in vivo was obtained using immunohistochemistry on tendon biopsy specimens taken before and 7 weeks after subacromial Depo-Medrone injection. RESULTS: Dexamethasone treatment of tenocytes resulted in an increased percentage of SA-ßgal-positive cells. Levels of phosphorylated p70S6K did not decrease with glucocorticoid treatment indicating mTOR remained active. Increased levels of acetylated p53 as well as increased RNA levels of its pro-senescence effector p21 were evident in dexamethasone-treated tenocytes. Levels of the p53 deacetylase sirtuin 1 were lower in dexamethasone-treated cells compared with controls. Knockdown of p53 or inhibition of p53 activity prevented dexamethasone-induced senescence. Activation of sirtuin 1 either by exogenous overexpression or by treatment with resveratrol or low glucose prevented dexamethasone-induced senescence. Immunohistochemical analysis of tendon biopsies taken before and after glucocorticoid injection revealed a significant increase in the percentage of p53-positive cells (p=0.03). The percentage of p21-positive cells also tended to be higher post-injection (p=0.06) suggesting glucocorticoids activate the p53/p21 senescence-inducing pathway in vivo as well as in vitro. CONCLUSION: As cell senescence is irreversible in vivo, glucocorticoid-induced senescence may result in long-term degenerative changes in tendon tissue.
Assuntos
Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/efeitos dos fármacos , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/efeitos dos fármacos , Serina-Treonina Quinases TOR/efeitos dos fármacos , Tendões/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Adulto , Idoso , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dexametasona/uso terapêutico , Feminino , Técnicas de Silenciamento de Genes , Glucocorticoides/uso terapêutico , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Manguito Rotador , Sirtuína 1/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tendinopatia/tratamento farmacológico , Tendões/citologia , Tendões/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/efeitos dos fármacos , beta-Galactosidase/metabolismoRESUMO
Cell surface marker expression is one of the criteria for defining human mesenchymal stem or stromal cells (MSC) in vitro. However, it is unclear if expression of markers including CD73 and CD90 reflects the in vivo origin of cultured cells. We evaluated expression of 15 putative MSC markers in primary cultured cells from periosteum and cartilage to determine whether expression of these markers reflects either the differentiation state of cultured cells or the self-renewal of in vivo populations. Cultured cells had universal and consistent expression of various putative stem cell markers including > 95% expression CD73, CD90 and PDPN in both periosteal and cartilage cultures. Altering the culture surface with extracellular matrix coatings had minimal effect on cell surface marker expression. Osteogenic differentiation led to loss of CD106 and CD146 expression, however CD73 and CD90 were retained in > 90% of cells. We sorted freshly isolated periosteal populations capable of CFU-F formation on the basis of CD90 expression in combination with CD34, CD73 and CD26. All primary cultures universally expressed CD73 and CD90 and lacked CD34, irrespective of the expression of these markers ex vivo indicating phenotypic convergence in vitro. We conclude that markers including CD73 and CD90 are acquired in vitro in most 'mesenchymal' cells capable of expansion. Overall, we demonstrate that in vitro expression of many cell surface markers in plastic-adherent cultures is unrelated to their expression prior to culture.
Assuntos
5'-Nucleotidase , Biomarcadores , Diferenciação Celular , Células-Tronco Mesenquimais , Osteogênese , Fenótipo , Antígenos Thy-1 , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Biomarcadores/metabolismo , Células Cultivadas , Antígenos Thy-1/metabolismo , 5'-Nucleotidase/metabolismo , Periósteo/citologia , Periósteo/metabolismo , Cartilagem/metabolismo , Cartilagem/citologia , Proteínas Ligadas por GPIRESUMO
The periosteum plays a crucial role in bone healing and is an important source of skeletal stem and progenitor cells. Recent studies in mice indicate that diverse populations of skeletal progenitors contribute to growth, homeostasis and healing. Information about the in vivo identity and diversity of skeletal stem and progenitor cells in different compartments of the adult human skeleton is limited. In this study, we compared non-hematopoietic populations in matched tissues from the femoral head and neck of 21 human participants using spectral flow cytometry of freshly isolated cells. High-dimensional clustering analysis indicated significant differences in marker distribution between periosteum, articular cartilage, endosteum and bone marrow populations, and identified populations that were highly enriched or unique to specific tissues. Periosteum-enriched markers included CD90 and CD34. Articular cartilage, which has very poor regenerative potential, showed enrichment of multiple markers, including the PDPN+CD73+CD164+CD146- population previously reported to represent human skeletal stem cells. We further characterized periosteal populations by combining CD90 with other strongly expressed markers. CD90+CD34+ cells sorted directly from periosteum showed significant colony-forming unit fibroblasts (CFU-F) enrichment, rapid expansion, and consistent multi-lineage differentiation of clonal populations in vitro. In situ, CD90+CD34+ cells include a perivascular population in the outer layer of the periosteum and non-perivascular cells closer to the bone surface. CD90+ cells are also highly enriched for CFU-F in bone marrow and endosteum, but not articular cartilage. In conclusion, our study indicates considerable diversity in the non-hematopoietic cell populations in different tissue compartments within the adult human skeleton, and suggests that periosteal progenitor cells reside within the CD90+CD34+ population.
Assuntos
Moléculas de Adesão Celular , Células-Tronco , Humanos , Adulto , Camundongos , Animais , Diferenciação Celular , Antígenos CD34 , Biomarcadores , PeriósteoRESUMO
Efforts to develop effective disease-modifying drugs to treat osteoarthritis have so far proved unsuccessful with a number of promising drug candidates from pre-clinical studies failing to show efficacy in clinical trials. It is therefore timely to re-evaluate our current understanding of osteoarthritis pathogenesis and the similarities and differences in disease development between commonly used pre-clinical mouse models and human patients. There is substantial heterogeneity between patients presenting with osteoarthritis and mounting evidence that the pathways involved in osteoarthritis (e.g. Wnt signalling) differ between patient sub-groups. There is also emerging evidence that the pathways involved in osteoarthritis differ between the STR/ort mouse model (the most extensively studied mouse model of spontaneously occurring osteoarthritis) and injury-induced osteoarthritis mouse models. For instance, while canonical Wnt signalling is upregulated in the synovium and cartilage at an early stage of disease in injury-induced osteoarthritis mouse models, this does not appear to be the case in the STR/ort mouse. Such findings may prove insightful for understanding the heterogeneity in mechanisms involved in osteoarthritis pathogenesis in human disease. However, it is important to recognise that there are differences between mice and humans in osteoarthritis pathogenesis. A much more extensive array of pathological changes are evident in osteoarthritic joints in individual mice with osteoarthritis compared to individual patients. There are also specified differences in the pathways involved in disease development. For instance, although increased TGF-ß signalling is implicated in osteoarthritis development in both mouse models of osteoarthritis and human disease, in mice, this is mainly mediated through TGF-ß3 whereas in humans, it is through TGF-ß1. Studies in other tissues have shown TGF-ß1 is more potent than TGF-ß3 in inducing the switch to SMAD1/5 signalling that occurs in osteoarthritic cartilage and that TGF-ß1 and TGF-ß3 have opposing effects on fibrosis. It is therefore possible that the relative contribution of TGF-ß signalling to joint pathology in osteoarthritis differs between murine models and humans. Understanding the similarities and differences in osteoarthritis pathogenesis between mouse models and humans is critical for understanding the translational potential of findings from pre-clinical studies.
Assuntos
Cartilagem Articular , Osteoartrite , Camundongos , Humanos , Animais , Fator de Crescimento Transformador beta1/metabolismo , Cartilagem Articular/patologia , Fator de Crescimento Transformador beta3/metabolismo , Osteoartrite/metabolismo , Modelos Animais de DoençasRESUMO
Expression of key transcriptional regulators is altered in chondrocytes in osteoarthritis (OA). This contributes to an increase in production of cartilage-catabolizing enzymes such as MMP13 and ADAMTS5. RCOR1 and RCOR2, binding partners for the transcriptional repressor REST, have previously been found to be downregulated in OA chondrocytes although their function in chondrocytes is unclear. HES1 is a known REST/RCOR1 target gene and HES1 has been shown to promote MMP13 and ADAMTS5 expression in murine OA chondrocytes. The purpose of this study was to determine whether reduced REST/RCOR levels leads to increased HES1 expression in human OA chondrocytes and whether HES1 also promotes ADAMTS5 and MMP13 expression in these cells. Chondrocytes were isolated from osteoarthritic and adjacent macroscopically normal cartilage obtained from patients undergoing total knee arthroplasty. RNA and protein levels of REST, RCOR1 and RCOR2 were lower, but levels of HES1 higher, in chondrocytes isolated from osteoarthritic compared to macroscopically normal cartilage. Over-expression of either REST, RCOR1 or RCOR2 resulted in reduced HES1 levels in OA chondrocytes whereas knockdown of REST, RCOR1 or RCOR2 led to increased HES1 expression in chondrocytes from macroscopically normal cartilage. In OA chondrocytes, ADAMTS5 and MMP13 expression were reduced following HES1 knockdown, but further enhanced following HES1 over-expression. Levels of phosphorylated CaMKII were higher in chondrocytes from OA cartilage consistent with previous findings that HES1 only promotes gene transcription in the presence of active CaMKII. These findings identify the REST/RCOR/HES1 pathway as a contributing factor leading to increased ADAMTS5 and MMP13 expression in OA chondrocytes.
Assuntos
Condrócitos , Osteoartrite , Humanos , Camundongos , Animais , Condrócitos/metabolismo , Metaloproteinase 13 da Matriz/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Osteoartrite/metabolismo , RNA/metabolismo , Células Cultivadas , Fatores de Transcrição HES-1/metabolismo , Proteína ADAMTS5/genética , Proteína ADAMTS5/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Correpressoras/metabolismoRESUMO
Accumulating evidence in tissues suggests an interconnection between circadian clocks and redox regulation. Diurnal variations in antioxidant levels, circadian rhythms of antioxidant enzyme activity, and differences in oxidative stress markers at different times of the day all indicate that oxidative stress responses follow a circadian rhythm. Disruptions of circadian rhythms are linked to a number of age-related diseases, including those in the eye. Typically, ocular tissues contain a robust antioxidant defence system to maintain redox balance and minimise oxidative stress and damage. The lens, in particular, contains remarkably high levels of the antioxidant glutathione (GSH). However, with advancing age, GSH levels deplete, initiating a chain of biochemical events that ultimately result in protein aggregation, light scattering, and age-related cataracts. While there is evidence that the lens exhibits circadian rhythms in the synthesis and release of melatonin, little is known about the regulation or function of timekeeping mechanisms in the lens. Since circadian rhythms are disrupted with age, and the depletion of GSH in the lens is a known initiating factor in the development of age-related cataracts, understanding the mechanisms involved in regulating GSH levels may lead to the future development of approaches to manipulate the clock to restore GSH levels and redox balance in the lens, and protect the lens from cataracts.
RESUMO
The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 -/- cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 -/- cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 -/- cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.
Assuntos
Eritrócitos/fisiologia , Megacariócitos/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Apoptose/genética , Sistemas CRISPR-Cas , Cálcio/metabolismo , Sinalização do Cálcio , Carcinogênese , Diferenciação Celular , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/genética , Homeostase , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Receptores de N-Metil-D-Aspartato/genética , TrombopoeseRESUMO
In osteoarthritis, chondrocytes undergo a phenotype shift characterised by reduced expression of SOX9 (sry-box 9) and increased production of cartilage-degrading enzymes, e.g. MMP13 (matrix metalloproteinase 13) and ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5). The chondrocyte clock is also altered. Specifically, the peak level of PER2 is elevated, but peak level of BMAL1 reduced in osteoarthritic chondrocytes. The purpose of this study was to determine whether increased PER2 expression causes disease-associated changes in chondrocyte activity and to identify whether known risk factors for osteoarthritis induce changes in PER2 and BMAL1 expression. Primary human chondrocytes isolated from macroscopically normal cartilage were serum-starved overnight then re-fed with serum-replete media with/without interleukin 1ß (IL-1ß) (10 ng/mL), hydrogen peroxide (100 µM) or basic calcium phosphate (BCP) crystals (50 µg/mL). Peak level of BMAL1 was lower, whereas PER2 levels remained elevated for longer, in chondrocytes treated with IL-1ß, hydrogen peroxide or BCP crystals compared to untreated cells. Levels of SOX9 were lower, whereas levels of ADAMTS5 and MMP13 were higher, in chondrocytes exposed to any of the three treatments compared to untreated cells. Knockdown of PER2 using siRNA partially abrogated the effects of each treatment on chondrocyte phenotype marker expression. Similarly, in chondrocytes isolated from osteoarthritic cartilage PER2 knockdown was associated with increased SOX9, reduced ADAMTS5 and reduced RNA and protein levels of MMP13 indicating partial mitigation of the osteoarthritic phenotype. Conversely, further ablation of BMAL1 expression in osteoarthritic chondrocytes resulted in a further reduction in SOX9 and increase in MMP13 expression. Overexpression of PER2 in the H5 chondrocyte cell line led to increased ADAMTS5 and MMP13 and decreased SOX9 expression. Localised inflammation, oxidative stress and BCP crystal deposition in osteoarthritic joints may contribute to disease pathology by inducing changes in the chondrocyte circadian clock.
Assuntos
Condrócitos/metabolismo , Relógios Circadianos , Proteínas Circadianas Period/metabolismo , Condrócitos/efeitos dos fármacos , Relógios Circadianos/efeitos dos fármacos , Ritmo Circadiano/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/tratamento farmacológico , RNA/metabolismoRESUMO
Due to their ability to mimic the actions of mammalian estrogens, soy phytoestrogens have been proposed as potential therapeutic agents to aid in preventing postmenopausal bone loss. In vitro, phytoestrogens promote osteoblastogenesis and inhibit osteoclastogenesis. Although a relatively large number of intervention studies have been undertaken in animals and humans, the efficacy of phytoestrogens as bone-protective agents in vivo remains unclear. Differences in the bioactivities of individual phytoestrogens, differences in phytoestrogen metabolism and bioavailability within different study populations, and imprecise reporting of the dose of phytoestrogens administered in intervention studies may have contributed to the disparity in study findings.
Assuntos
Conservadores da Densidade Óssea/farmacologia , Osso e Ossos/efeitos dos fármacos , Glycine max/química , Osteoporose Pós-Menopausa/prevenção & controle , Fitoestrógenos/farmacologia , Animais , Osso e Ossos/metabolismo , Ensaios Clínicos como Assunto , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Humanos , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/epidemiologiaRESUMO
Newly described lipoxygenase (LOX)-generated lipid mediators, that is, resolvins and protectins as well as lipoxins, are both anti-inflammatory and proresolving. We aimed to determine whether these lipid mediators are present in bone marrow and whether their lipidomic profiles are altered following ovariectomy or dietary supplementation with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) ethyl esters. Female rats were ovariectomised or sham-operated. Shams and one ovariectomised group received a diet devoid of omega-3 long-chain polyunsaturated fatty acids. The remaining ovariectomised rats received either 0.5 g EPA or DHA ethyl ester/kg body weight/day for 4 months. Bone marrow was analyzed using both GC to determine fatty acid composition and mediator lipidomics by LC/MS/MS profiling for the presence of LOX-pathway lipid mediators derived from arachidonic acid (AA), EPA, and DHA. LOX-derived products including lipoxins, resolvin D1, resolvin E1, and protectin D1 were identified in bone marrow by the presence of diagnostic ions in their corresponding MS-MS spectra. The proportion of AA relative to DHA and of AA-derived relative to DHA-derived mediators in bone marrow was higher in ovariectomised compared to sham-operated rats. DHA or EPA ethyl ester supplementation increased the percentage of DHA and EPA in bone marrow and increased the proportion of LOX mediators biosynthesized from DHA or EPA, respectively. Given the potent bioactivities of the lipoxins, resolvins, and protectins, the presence and changes in profile postovariectomy and with EPA and DHA ethyl ester supplementation may be of interest in bone marrow function and as a potential source of these mediators in vivo.
Assuntos
Ácidos Graxos Ômega-3/metabolismo , Inflamação/patologia , Lipídeos/fisiologia , Ovariectomia , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/administração & dosagem , Ácido Eicosapentaenoico/análogos & derivados , Ácido Eicosapentaenoico/metabolismo , Ácidos Graxos Ômega-3/administração & dosagem , Feminino , Inflamação/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos/análise , Lipoxinas , Lipoxigenase/metabolismo , Espectrometria de Massas , RatosRESUMO
Bone-protective effects of combined treatment with long chain polyunsaturated fatty acids (LCPUFAs) and estrogenic compounds following ovariectomy have previously been reported. Recent evidence suggests the n-3 LCPUFA docosahexaenoic acid (DHA, 22:6n-3) is particularly bone-protective. The aim of this study was to determine whether combined treatment with DHA and estrogenic compounds has a beneficial effect on bone mass in ovariectomized (OVX) rats. Rats were randomized into 9 groups and either ovariectomized (8 groups) or sham-operated (1 group). Using a 2 x 4 factorial design approach, OVX animals received either no estrogenic compound, genistein (20 mg/kg body weight/day), daidzein, (20 mg/kg body weight/day) or 17 beta-estradiol (1 microg/day) with or without DHA (0.5 g/kg body weight/day) for 18 weeks. Bone mineral content (BMC), area (BA), and density (BMD), plasma osteocalcin and IL-6 concentrations, and red blood cell (RBC) fatty acid composition were measured. Femur BMC was significantly greater in animals treated with DHA or 17 beta-estradiol than in ovariectomized controls. Plasma carboxylated osteocalcin was significantly higher in DHA-treated animals and total osteocalcin significantly lower in 17 beta-estradiol-treated animals compared with ovariectomized controls. There were significant interactions between treatment with estrogenic compounds and DHA for femur BMC, plasma IL-6 concentration, and RBC fatty acid composition. Combined treatment with 17beta-estradiol+DHA was more effective than either treatment alone at preserving femur BMC and lowering circulating concentrations of pro-inflammatory IL-6. The percentage of n-3 LCPUFAs in RBCs was significantly greater in animals receiving 17 beta-estradiol+DHA compared with either treatment alone. There was no beneficial effect of combined DHA and phytoestrogen treatment on bone. Results from this study raise the possibility that co-treatment with 17 beta-estradiol and DHA may allow a lower dose of 17 beta-estradiol to be used to provide the same bone-protective effects as when 17 beta-estradiol is administered alone.