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BACKGROUND: Previously, fragments from Sirtuin 1 (SIRT1) were identified in preclinical and clinical samples to display an increase in serum levels for N-terminal (NT) SIRT1 vs. C-terminal (CT) SIRT1, indicative of early signs of OA. Here we tested NT/CT SIRT1 levels as well as a novel formulated sandwich assay to simultaneously detect both domains of SIRT1 in a manner that may inform us about the levels of full-length SIRT1 in the circulation (flSIRT1) of clinical cohorts undergoing knee joint distraction (KJD). METHODS: We employed an indirect ELISA assay to test NT- and CT-SIRT1 levels and calculated their ratio. Further, to test flSIRT1 we utilized novel antibodies (Ab), which were validated for site specificity and used in a sandwich ELISA method, wherein the CT-reactive served as capture Ab, and its NT-reactive served as primary detection Ab. This method was employed in human serum samples derived from a two-year longitudinal study of KJD patients. Two-year clinical and structural outcomes were correlated with serum levels of flSIRT1 compared to baseline. RESULTS: Assessing the cohort, exhibited a significant increase of NT/CT SIRT1 serum levels with increased osteophytes and PIIANP/CTX-II at baseline, while a contradictory increase in NT/CT SIRT1 was associated with less denuded bone, post-KJD. On the other hand, flSIRT1 exhibited an upward trend in serum level, accompanied by reduced denuded bone for 2-year adjusted values. Moreover, 2 year-adjusted flSIRT1 levels displayed a steeper linear regression for cartilage and bone-related structural improvement than those observed for NT/CT SIRT1. CONCLUSIONS: Our data support that increased flSIRT1 serum levels are a potential molecular endotype for cartilage-related structural improvement post-KJD, while NT/CT SIRT1 appears to correlate with osteophyte and PIIANP/CTX-II reduction at baseline, to potentially indicate baseline OA severity.
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Osteoartritis de la Rodilla , Sirtuina 1 , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Biomarcadores/sangre , Cartílago Articular/patología , Cartílago Articular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Articulación de la Rodilla/diagnóstico por imagen , Articulación de la Rodilla/patología , Estudios Longitudinales , Osteoartritis de la Rodilla/sangre , Osteoartritis de la Rodilla/cirugía , Sirtuina 1/sangreRESUMEN
Objective: Previous studies have shown that the cleavage of Sirt1 contributes to the development of osteoarthritis (OA). In fact, OA was effectively abrogated by the intra-articular (IA) administration of two compounds, one blocking Sirt1 cleavage (CA074me) and the other activating Sirt1 (SRT1720), using a post-traumatically induced model (PTOA) in young female mice. In this study, we attempted to understand if this local treatment is effective in preventing age-associated OA (AOA) progression and symptoms. Design: A group of 17-month-old female C57BL/6J mice were IA administered with CA074me and/or SRT1720 or their combination. Joint histopathological analysis and bone histomorphometry were carried out, with an assessment of knee mechanical hyperalgesia. A serum analysis for NT/CT Sirt1 was carried out along with immunohistochemistry for articular cartilage to detect p16INK4A or γH2A.X. Similarly, meniscal cartilage was monitored for Lef1 and Col1a1 deposition. The data were compared for young female mice subjected to post-traumatic OA (PTOA). Results: Similar to PTOA, combination-treated AOA exhibited improved knee hyperalgesia, yet structural improvements were undetected, corresponding to unchanged NT/CT Sirt1 serum levels. Both AOA and PTOA exhibited unchanged staining for nuclear p16INK4A or γH2A.X and lacked a correlation with OA severity. Contrarily to PTOA, the combination treatment with AOA did not exhibit a local reduction in the Lef1 and Col1 targets. Conclusions: When targeting Sirt1 cleavage, the PTOA and AOA models exhibited a similar pain response to the combination treatment; however, they displayed diverse structural outcomes for joint-related damage, related to Lef1-dependent signaling. Interestingly, nuclear p16INK4A was unaffected in both models, regardless of the treatment's effectiveness. Finally, these findings highlight the variations in the responses between two highly researched OA preclinical models, reflecting OA pathophysiology heterogeneity and variations in gender-related drug-response mechanisms.
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Cartílago Articular , Osteoartritis , Sirtuina 1 , Animales , Femenino , Ratones , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Hiperalgesia , Ratones Endogámicos C57BL , Osteoartritis/tratamiento farmacológico , Osteoartritis/etiología , Sirtuina 1/efectos de los fármacosRESUMEN
Critical maxillofacial bone fractures do not heal spontaneously, thus, often there is a need to facilitate repair via surgical intervention. Gold standard approaches, include the use of autologous bone graft, or devices supplemented with osteogenic growth factors and bone substitutes. This research aimed to employ a critical size calvaria defect model, to determine if the addition of chondrocytes to collagen-containing bone graft substitute, may expedite bone repair. As such, using a critical size rat calvaria defect, we implanted a collagen scaffold containing bone graft substitute (i.e., Bone graft scaffold, BG) or BG supplemented with costal chondrocytes (cBG). The rats were subjected to live CT imaging at 1, 6, 9, and 12 weeks following the surgical procedure and sacrificed for microCT imaging of the defect site. Moreover, serum markers and histological evaluation were assessed to determine osseous tissue regeneration and turnover. Live CT and microCT indicated cBG implants displayed expedited bone repair vs, BG alone, already at 6 weeks post defect induction. cBG also displayed a shorter distance between the defect edges and greater mineral apposition distance compared to BG. Summerizing, the data support the addition of chondrocytes to bone substitute, accelerates the formation of new bone within a critical size defect.
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Sustitutos de Huesos , Condrocitos , Ratas , Animales , Andamios del Tejido , Cráneo , Colágeno , Osteogénesis , Regeneración ÓseaRESUMEN
Cancer cells have an altered transcriptome, which contributes to their abnormal behavior. Many tumors have high levels of kinetochore genes, which play important roles in genome stability. This overexpression could be utilized to destabilize cancer cell genomes, however this has not been proven specifically. We investigated the link between kinetochore gene overexpression, chromosomal number variations (CNVs) and genomic instability. Data on RNA expression and CNV from 12 different cancer types were evaluated using information theory. In all cancer types, we looked at the relationship between RNA expression and CNVs. Kinetochore gene expression was found to be substantially linked with CNV levels. In all cancer types, with the exception of thyroid cancer, highly expressed kinetochore genes were enriched in the most dominant cancer-specific co-expression subnetworks characterizing the largest patient subgroups. Except for thyroid cancer, kinetochore inner protein CENPA was among the transcripts most strongly associated with CNV values in all cancer types studied, with significantly higher expression levels in patients with high CNVs than in patients with low CNVs. CENPA function was investigated further in cell models by transfecting genomically stable (HCT116) and unstable (MCF7 and HT29) cancer cell lines using CENPA overexpression vectors. This overexpression increased the number of abnormal cell divisions in the stable cancer cell line HCT116 and, to a lesser extent, in the unstable cell lines MCF7 and HT29. Overexpression improved anchorage-independent growth properties of all cell lines. Our findings suggest that overexpression of kinetochore genes in general, and CENPA in particular, can cause genomic instability and cancer progression.
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In cranial flat bone fractures, spontaneous bone repair will occur only when the fracture ends are in close contact. However, in cases wherein bone discontinuity is extensive, surgical interventions are often required. To this end, autologous bone is harvested and surgically integrated into the site of fracture. Here we propose to use cartilage, as an alternative autologous source, to promote cranial fracture repair. The advantage of this approach is the potential reduction in donor site morbidity, likely due to the avascular and aneural nature of cartilage. As a first step we attempted to induce cartilage mineralization in vitro, using micromass primary chondrocyte cultures, incubated with BMP2 and/or WISP1, which were examined histologically following a 3-week culture period. Next, chondrocyte seeded collagen scaffolds were evaluated in vitro for expression profiles and ALP activity. Finally, chondrocyte-seeded collagen scaffolds were implanted in a Lewis rats 8 mm critical calvaria defect model, which was imaged via live CT for 12 weeks until sacrifice. End points were analyzed for microCT, histology, and serum levels of bone related markers. Micromass cultures exhibited an osseous inducing trend following WISP1 administration, which was maintained in chondrocyte seeded scaffolds. Accordingly, in vivo analysis was carried out to assess the impact of WISP1-pretreated chondrocytes (WCS) versus untreated chondrocytes (UCS) in calvaria defect model and compared to untreated control comprised of a defect-associated blood clot (BC) or empty collagen scaffold (CS) implant. Live CT and microCT exhibited higher mineralization volumes in critical defect implanted with UCS, with some structural improvements in WCS. Histological analysis exhibited higher anabolic bone formation in WCS and trabecular bone was detected in WCS and UCS groups. Chondrocytes implanted into critical cranial defect expedite the formation of native-like osseous tissue, especially after WISP1 priming in culture. Ultimately, these data support the use of autologous chondrocytes to repair critical maxillofacial defects.
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Osteoarthritis (OA) is characterized by progressive, irreversible erosion of articular cartilage accompanied by severe pain and immobility. This study aimed to assess the effect and mechanism of action of HU308, a selective cannabinoid receptor type 2 (CB2) agonist, in preventing OA-related joint damage. To test the assumption that HU308 could prevent OA-related joint damage, Cnr2 null mice and wild type (WT) mice were aged to reach 20 months and analyzed for joint structural features. OA was induced in WT mice via a post-traumatic procedure or aging, followed by HU308 local (intra-articular) or systemic (intraperitoneal) administration, respectively. Additional analyses of time and dose courses for HU308 were carried out in human primary chondrocytes, analyzed by RNA sequencing, RT-PCR, chromatin immunoprecipitation, and immunoblotting. Our results showed that Cnr2 null mice exhibited enhanced age-related OA severity and synovitis compared to age-matched WT mice. Systemic administration of HU308 to 16-month-old mice improved pain sensitivity and maintained joint integrity, which was consistent with the intra-articular administration of HU308 in post-traumatic OA mice. When assessing human chondrocytes treated with HU308, we uncovered a dose- and time-related increase in ACAN and COL2A1 expression, which was preceded by increased SOX9 expression due to pCREB transcriptional activity. Finally, transcriptomic analysis of patient-derived human chondrocytes identified patient subpopulations exhibiting HU308-responsive trends as judged by enhanced SOX9 expression, accompanied by enriched gene networks related to carbohydrate metabolism. Collectively, the results showed that HU308 reduced trauma and age-induced OA via CB2-pCREB dependent activation of SOX9, contributing to augmented gene networks related to carbohydrate metabolism. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
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Cannabinoides , Cartílago Articular , Osteoartritis , Humanos , Ratones , Animales , Anciano , Osteoartritis/tratamiento farmacológico , Osteoartritis/genética , Osteoartritis/metabolismo , Cannabinoides/farmacología , Dolor/metabolismo , Ratones Noqueados , Metabolismo de los Hidratos de Carbono , Condrocitos/metabolismo , Cartílago Articular/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/farmacologíaRESUMEN
BACKGROUND: In a previous report, we have identified the cannabinoid receptor 2 (CB2) agonist HU308 to possess a beneficial effect in preventing age and trauma-induced osteoarthritis (OA) in mice. The effects of HU308 were largely related to the capacity of this compound to induce cartilage anabolism which was dependent on the CREB/SOX9 axis, and exhibited pro-survival and pro-proliferative hallmarks of articular cartilage following treatment. Here, we utilized the novel cannabinoid-fenchone CB2 agonists (1B, 1D), which were previously reported to render anti-inflammatory effects in a zymosan model. METHODS: Initially, we assessed the selectivity of CB2 using a Gs-protein receptor cAMP potency assay, which was also validated for antagonistic effects dependent on the Gi-protein receptor cAMP pathway. Based on EC50 values, 1D was selected for a zymosan inflammatory pain model. Next, 1D was administered in two doses intra-articularly (IA), in a post-traumatic medial meniscal tear (MMT, Lewis rats) model, and compared to sham, vehicle, and a positive control consisting of fibroblast growth factor 18 (FGF18) administration. The histopathological assessment was carried out according to the Osteoarthritis Research Society International (OARSI) guidelines for rat models following 28 days post-MMT. RESULTS: The G protein receptor assays confirmed that both 1B and 1D possess CB2 agonistic effects in cell lines and in chondrocytes. Co-administering a CB2 antagonists to 25 mg/kg 1D in a paw inflammatory pain model abolished 1D-related anti-swelling effect and partially abolishing its analgesic effects. Using an MMT model, the high dose (i.e., 24 µg) of 1D administered via IA route, exhibited reduced cartilage damage. Particularly, this dose of 1D exhibited a 30% improvement in cartilage degeneration (zonal/total tibial scores) and lesion depth ratios (44%), comparable to the FGF18 positive control. Synovitis scores remained unaffected and histopathologic evaluation of subchondral bone damage did not suggest that 1D treatment changed the load-bearing ability of the rats. Contrary to the anabolic effect of FGF18, synovial inflammation was observed and was accompanied by increased osteophyte size. CONCLUSION: The structural histopathological analysis supports a disease-modifying effect of IA-administered 1D compound without any deleterious effects on the joint structure.
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Osteoartritis , Ratas , Ratones , Animales , Zimosan , Ratas Endogámicas Lew , Osteoartritis/metabolismo , Dolor/patologíaRESUMEN
Cartilage mineralization is a tightly controlled process, imperative for skeletal growth and fracture repair. However, in osteoarthritis (OA), cartilage mineralization may impact the joint range of motion, inflict pain, and increase chances for joint effusion. Here we attempt to understand the link between inflammation and cartilage mineralization by targeting Sirtuin 1 (SIRT1) and lymphoid enhancer binding factor 1 (LEF1), both reported to have contrasting effects on cartilage. We find that inflammatory-dependent cleavage of SIRT1 or its cartilage-specific genetic ablation, directly enhanced LEF1 expression accompanied by a catabolic response. Applying a posttraumatic OA (PTOA) model to cartilage-specific Sirt1 nulls displayed severe OA, which was accompanied by synovitis, meniscal mineralization, and osteophyte formation of the lateral joint compartment. Alternatively, cartilage-specific Lef1 nulls presented reduced lateral mineralization, OA severity, and local pain. Differential gene expression analysis revealed that Lef1 ablation reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Toll-like receptor (Tlr) pathways, while enhancing SRY-Box transcription factor 9 (Sox9) and cartilaginous extracellular matrix genes. The results support a link between inflammation and Lef1-dependent cartilage mineralization, mediated by the inactivation of Sirt1. By ablating Lef1 in a PTOA model, the structural and pain-related phenotypes of OA were reduced, in part, by preventing cartilage mineralization of the lateral joint compartment, partially manifested by meniscal tissue mineralization. Overall, these data provide a molecular axis to link between inflammation and cartilage in a PTOA model.
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Calcinosis , Cartílago Articular , Osteoartritis , Sinovitis , Calcinosis/metabolismo , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Humanos , Inflamación , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Osteoartritis/genética , Osteoartritis/metabolismo , Dolor , Sinovitis/genética , Sinovitis/patologíaRESUMEN
Mammalian oocyte quality reduces with age. We show that prior to the occurrence of significant aneuploidy (9M in mouse), heterochromatin histone marks are lost, and oocyte maturation is impaired. This loss occurs in both constitutive and facultative heterochromatin marks but not in euchromatic active marks. We show that heterochromatin loss with age also occurs in human prophase I-arrested oocytes. Moreover, heterochromatin loss is accompanied in mouse oocytes by an increase in RNA processing and associated with an elevation in L1 and IAP retrotransposon expression and in DNA damage and DNA repair proteins nuclear localization. Artificial inhibition of the heterochromatin machinery in young oocytes causes an elevation in retrotransposon expression and oocyte maturation defects. Inhibiting retrotransposon reverse-transcriptase through azidothymidine (AZT) treatment in older oocytes partially rescues their maturation defects and activity of the DNA repair machinery. Moreover, activating the heterochromatin machinery via treatment with the SIRT1 activating molecule SRT-1720, or overexpression of Sirt1 or Ezh2 via plasmid electroporation into older oocytes causes an upregulation in constitutive heterochromatin, downregulation of retrotransposon expression, and elevated maturation rates. Collectively, our work demonstrates a significant process in oocyte aging, characterized by the loss of heterochromatin-associated chromatin marks and activation of specific retrotransposons, which cause DNA damage and impair oocyte maturation.
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Heterocromatina , Retroelementos , Animales , Heterocromatina/genética , Heterocromatina/metabolismo , Mamíferos/genética , Meiosis , Ratones , Oocitos/metabolismo , Oogénesis , Retroelementos/genética , Sirtuina 1/metabolismoRESUMEN
Pre-pubertal oocytes are still dormant. They are arrested in a GV state and do not undergo meiotic divisions naturally. A multitude of molecular pathways are changed and triggered upon initiation of puberty. It is not yet clear which epigenetic events occur in oocytes upon pubertal transition, and how significant these epigenetic events may be. We evaluated epigenetic marker levels in mouse pre-pubertal and post-pubertal female oocytes. In addition, we evaluated H3K9me2 levels in human oocytes collected from fertility preservation patients, comparing the levels between pre-pubertal patients and post-pubertal patients. The chromatin structure shows a lower number of chromocenters in mouse post-pubertal oocytes in comparison to pre-pubertal oocytes. All heterochromatin marker levels checked (H3K9me2, H3K27me3, H4K20me1) significantly rise across the pubertal transition. Euchromatin markers vary in their behavior. While H3K4me3 levels rise with the pubertal transition, H3K27Ac levels decrease with the pubertal transition. Treatment with SRT1720 [histone deacetylase (HDAC) activator] or overexpression of heterochromatin factors does not lead to increased heterochromatin in pre-pubertal oocytes. However, treatment of pre-pubertal oocytes with follicle-stimulating hormone (FSH) for 24 h - changes their chromatin structure to a post-pubertal configuration, lowers the number of chromocenters and elevates their histone methylation levels, showing that hormones play a key role in chromatin regulation of pubertal transition. Our work shows that pubertal transition leads to reorganization of oocyte chromatin and elevation of histone methylation levels, thus advancing oocyte developmental phenotype. These results provide the basis for finding conditions for in-vitro maturation of pre-pubertal oocytes, mainly needed to artificially mature oocytes of young cancer survivors for fertility preservation purposes.
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Older age is associated with reduced immune function. Our aim was to study how age affects the development of apical periodontitis (AP). AP was induced in two age groups of mice (young vs. adult). Histological samples were stained by Hematoxylin Eosin, Brown and Brenn, and Tartrate-Resistant Acid Phosphatase. In addition, the samples were scanned by Micro-Computerized-Tomography (micro-CT) to evaluate apical constriction and periapical lesion size. Cell density in the periapical region was computationally assessed. Moreover, lesion immune cell populations were characterized by flow cytometry and immunofluorescence. The young group presented more canals with necrotic radicular pulp compared to the adults. There was no difference in bacteria location in the canals between the groups. Apical constriction size was larger in the young mice compared to the adults. The periapical cell density was higher in the young group, while the dominant immune cells in the lesions were neutrophils, which also exhibited the highest young/adult ratio. Immunofluorescence demonstrated neutrophils in the lesion. More osteoclasts were present in the lesions of the young mice, in correlation to the higher volume of bone resorption in this group. Overall, we conclude that the immune reaction to AP stimuli was attenuated in the adult mice compared to the young.
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Envejecimiento , Resorción Ósea/patología , Osteoclastos/patología , Periodontitis Periapical/patología , Animales , Recuento de Células/métodos , Modelos Animales de Enfermedad , Ratones , Neutrófilos/patología , Periodontitis Periapical/microbiología , Ligando RANK/metabolismoRESUMEN
OBJECTIVE: Periodontitis is one the most common chronic inflammatory conditions, resulting in destruction of tooth-supporting tissues and leading to tooth loss. Porphyromonas gingivalis activates host macrophages to secrete pro-inflammatory cytokines and elicit tissue damage, in part by inducing NF-kappa-B transactivation. Since NFκB transactivation is negatively regulated by the Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase enzyme Sirt1, we sought to assess if RAW264.7 macrophages exposed to P. gingivalis demonstrate impaired Sirt1 activity, to ultimately induce a pro-inflammatory response. METHODS: RAW264.7 macrophages were incubated with heat- killed P. gingivalis for 2, 4, 8, and 24 h. Stimulated RAW264.7 were assessed for TNFα expression via PCR, ELISA, and ChIP analysis. Following the activation of RAW264.7 macrophages, immunoblot analysis was executed to detect modifications in Sirt1 and the NFκB subunit RelA that is essential for NFκB transcriptional activity. RESULTS: TNFα expression was elevated 4 h after exposure to P. gingivalis. ChIP confirmed that RelA was enriched in the mouse TNFα promoter 4 h following stimulation, which correlated with the increased TNFα mRNA levels. Preceding TNFα expression, we detected Phosphoserine 536 and acetylated lysine 310 of RelA after 2 hours exposure with P. gingivalis. Moreover, reduced Sirt1 activity was associated with its cleavage in RAW264.7 protein extracts, after 2 hours of P. gingivalis exposure. Blocking TLR2/4 signaling prevented Sirt1 cleavage, loss of deacetylase activity, and TNFα secretion, while co-administering CA074Me (a cathepsin B inhibitor) with P. gingivalis reduced RelA promoter enrichment, resulting in impaired TNFα expression. CONCLUSIONS: Together, the results suggest that P. gingivalis induces TNFα expression, at least in part, by enhancing cleavage of Sirt1 via a TLR-dependent signaling circuit.
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Periodontitis , Porphyromonas gingivalis , Animales , Lipopolisacáridos/farmacología , Macrófagos , Ratones , FN-kappa B , Sirtuina 1 , Factor de Necrosis Tumoral alfaRESUMEN
OBJECTIVE: Previous work has established that the deacetylase sirtuin-1 (SIRT1) is cleaved by cathepsin B in chondrocytes subjected to proinflammatory stress, yielding a stable but inactive N-terminal (NT) polypeptide (75SIRT1) and a C-terminal (CT) fragment. The present work examined if chondrocyte-derived NT-SIRT1 is detected in serum and may serve as an investigative and exploratory biomarker of osteoarthritis (OA). METHODS: We developed a novel ELISA assay to measure the ratio of NT to CT of SIRT1 in the serum of human individuals and mice subjected to post-traumatic OA (PTOA) or age-dependent OA (ADOA). We additionally monitored NT/CT SIRT1 in mice subject to ADOA/PTOA followed by senolytic clearance. Human chondrosenescent and non-senescent chondrocytes were exposed to cytokines and analysed for apoptosis and NT/CT SIRT1 ratio in conditioned medium. RESULTS: Wild-type mice with PTOA or ADOA of moderate severity exhibited increased serum NT/CT SIRT1 ratio. In contrast, this ratio remained low in cartilage-specific Sirt1 knockout mice despite similar or increased PTOA and ADOA severity. Local clearance of senescent chondrocytes from old mice with post-traumatic injury resulted in a lower NT/CT ratio and reduced OA severity. While primary chondrocytes exhibited NT/CT ratio increased in conditioned media after prolonged cytokine stimulation, this increase was not evident in cytokine-stimulated chondrosenescent cells. Finally, serum NT/CT ratio was elevated in humans with early-stage OA. CONCLUSIONS: Increased levels of serum NT/CT SIRT1 ratio correlated with moderate OA in both mice and humans, stemming at least in part from non-senescent chondrocyte apoptosis, possibly a result of prolonged inflammatory insult.
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Biomarcadores/sangre , Osteoartritis/patología , Sirtuina 1/sangre , Animales , Cartílago Articular/metabolismo , Cartílago Articular/patología , Senescencia Celular/fisiología , Condrocitos/metabolismo , Condrocitos/patología , Humanos , Ratones , Osteoartritis/sangreRESUMEN
In contrast to normal regenerating tissue, resistance to Fas- and FasL-positive T cell-induced apoptosis were detected in myofibroblasts from fibrotic-lungs of humans and mice following bleomycin (BLM) exposure. In this study we show, decreased FLIP expression in lung-tissues with resolution of BLM-induced fibrosis and in isolated-lung fibroblasts, with decreased resistance to apoptosis. Using a FLIP-expression vector or a shFLIP-RNA, we further confirmed the critical need for FLIP to regain/lose susceptibility of fibrotic-lung myofibroblast to Fas-induced apoptosis. Our study further show that FLIP is regulated by SIRT1 (Sirtuin 1) deacetylase. Chimeric mice, with SIRT1-deficiency in deacetylase domain (H355Y-Sirt1y/y), specifically in mesenchymal cells, were not only protected from BLM-induced lung fibrosis but, as assessed following Ku70 immunoprecipitation, had also decreased Ku70-deacetylation, decreasedKu70/FLIP complex, and decreased FLIP levels in their lung myofibroblasts. In addition, myofibroblasts isolated from lungs of BLM-treated miR34a-knockout mice, exposed to a miR34a mimic, which we found here to downregulate SIRT1 in the luciferase assay, had a decreased Ku70-deacetylation indicating decrease in SIRT1 activity. Thus, SIRT1 may mediate, miR34a-regulated, persistent FLIP levels by deacetylation of Ku70 in lung myofibroblasts, promoting resistance to cell-death and lung fibrosis.
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Bleomicina/efectos adversos , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/antagonistas & inhibidores , Regulación hacia Abajo , MicroARNs/genética , Fibrosis Pulmonar/terapia , ARN Interferente Pequeño/farmacología , Sirtuina 1/metabolismo , Acetilación/efectos de los fármacos , Animales , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Técnicas de Inactivación de Genes , Humanos , Autoantígeno Ku/metabolismo , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones , Especificidad de Órganos , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismoRESUMEN
The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.
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Inflamación/inducido químicamente , Periodontitis Periapical/inducido químicamente , Animales , Inflamación/patología , Ratones , Periodontitis Periapical/patologíaRESUMEN
The Growth Advantage in Stationary Phase (GASP) phenomenon, described in bacteria, reflects the genetic adaptation of bacteria to stress, including starvation, for a long time. Unlike in stationary phase where no cell division occurs, GASP harbors active cell division, concurrent with genetic adaptation. Here we show that GASP occurs also in eukaryotes. Two strains of Saccharomyces cerevisiae (Sc404 and Sc424) have been isolated from 2-year-old sealed bottles of beer. These strains presented advantage in survival and growth over the parent during stress. The differences between the strains are irreversible and therefore genetic in origin rather than epigenetic. Direct competition assays show that Sc404 and Sc424 outcompete the parent in direct competition. DNA sequencing shows changes of the genome: the TOR complexes are mutated, and DNA repair gene mutations confer a mutator phenotype. The differences between the strains are reflected in a difference in taste between beers brewed from them.
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Ancient fermented food has been studied based on recipes, residue analysis, and ancient-DNA techniques and reconstructed using modern domesticated yeast. Here, we present a novel approach based on our hypothesis that enriched yeast populations in fermented beverages could have become the dominant species in storage vessels and their descendants could be isolated and studied today. We developed a pipeline of yeast isolation from clay vessels and screened for yeast cells in beverage-related and non-beverage-related ancient vessels and sediments from several archaeological sites. We found that yeast cells could be successfully isolated specifically from clay containers of fermented beverages. The findings that genotypically the isolated yeasts are similar to those found in traditional African beverages and phenotypically they grow similar to modern beer-producing yeast strongly suggest that they are descendants of the original fermenting yeast. These results demonstrate that modern microorganisms can serve as a new tool in bio-archaeology research.IMPORTANCE So far, most of the study of ancient organisms has been based mainly on the analysis of ancient DNA. Here we show that it is possible to isolate and study microorganisms-yeast in this case-from ancient pottery vessels used for fermentation. We demonstrate that it is highly likely that these cells are descendants of the original yeast strains that participated in the fermentation process and were absorbed into the clay matrix of the pottery vessels. Moreover, we characterized the isolated yeast strains, their genomes, and the beer they produced. These results open new and exciting avenues in the study of domesticated microorganisms and contribute significantly to the fields of bio- and experimental archaeology that aim to reconstruct ancient artifacts and products.
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Arqueología/métodos , Fósiles/microbiología , Sedimentos Geológicos/microbiología , Técnicas Microbiológicas/métodos , Levaduras/aislamiento & purificación , GenotipoRESUMEN
The C-terminus of SIRT1 can be cleaved by cathepsin B at amino acid H533 to generate a lower-functioning, N-terminally intact 75â kDa polypeptide (75SIRT1) that might be involved in age-related pathologies. However, the mechanisms underlying cathepsin B docking to and cleavage of SIRT1 are unclear. Here, we first identified several 75SIRT1 variants that are augmented with aging correlatively with increased cathepsin B levels in various mouse tissues, highlighting the possible role of this cleavage event in age-related pathologies. Then, based on H533 point mutation and structural modeling, we generated a functionally intact ΔSIRT1 mutant, lacking the internal amino acids 528-543 (a predicted C-terminus loop domain), which exhibits resistance to cathepsin B cleavage in vitro and in cell cultures. Finally, we showed that cells expressing ΔSIRT1 under pro-inflammatory stress are more likely to undergo caspase 9- dependent apoptosis than those expressing 75SIRT1. Thus, our data suggest that the 15-amino acid predicted loop motif embedded in the C-terminus of SIRT1 is susceptible to proteolytic cleavage by cathepsin B, leading to the formation of several N-terminally intact SIRT1 truncated variants in various aging mouse tissues.This article has an associated First Person interview with the first author of the paper.
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Catepsina B/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Sirtuina 1/química , Sirtuina 1/metabolismo , Animales , Senescencia Celular/fisiología , Biología Computacional , Femenino , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Transgénicos , Dominios y Motivos de Interacción de Proteínas/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Sirtuina 1/genéticaRESUMEN
Reduced SIRT1 activity and levels during osteoarthritis (OA) promote gradual loss of cartilage. Loss of cartilage matrix is accompanied by an increase in matrix metalloproteinase (MMP) 13, partially because of enhanced LEF1 transcriptional activity. In this study, we assessed the role of SIRT1 in LEF1-mediated MMP13 gene expression in human OA chondrocytes. Results showed that MMP13 protein levels and enzymatic activity decreased significantly during SIRT1 overexpression or activation by resveratrol. Conversely, MMP13 gene expression was reduced in chondrocytes transfected with SIRT1 siRNA or treated with nicotinamide (NAM), a sirtuin inhibitor. Chondrocytes challenged with IL-1ß, a cytokine involved in OA pathogenesis, enhanced LEF1 protein levels and gene expression, resulting in increased MMP13 gene expression; however, overexpression of SIRT1 during IL-1ß challenge impeded LEF1 levels and MMP13 gene expression. Previous reports showed that LEF1 binds to the MMP13 promoter and transactivates its expression, but we observed that SIRT1 repressed LEF1 protein and mRNA expression, ultimately reducing LEF1 transcriptional activity, as judged by luciferase assay. Finally, mouse articular cartilage from Sirt1-/- presented increased LEF1 and MMP13 protein levels, similar to human OA cartilage. Thus, demonstrating for the first time that SIRT1 represses MMP13 in human OA chondrocytes, which appears to be mediated, at least in part, through repression of the transcription factor LEF1, a known modulator of MMP13 gene expression.-Elayyan, J., Lee, E.-J., Gabay, O., Smith, C. A., Qiq, O., Reich, E., Mobasheri, A., Henrotin, Y., Kimber, S. J., Dvir-Ginzberg, M. LEF1-mediated MMP13 gene expression is repressed by SIRT1 in human chondrocytes.
Asunto(s)
Condrocitos/metabolismo , Regulación de la Expresión Génica/fisiología , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Sirtuina 1/metabolismo , Proteína ADAMTS4/genética , Proteína ADAMTS4/metabolismo , Animales , Cartílago Articular , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Factor de Unión 1 al Potenciador Linfoide/genética , Metaloproteinasa 13 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 8 de la Matriz/genética , Metaloproteinasa 8 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Osteoartritis/metabolismo , Sirtuina 1/genéticaRESUMEN
Changes in the content of aggrecan, an essential proteoglycan of articular cartilage, have been implicated in the pathophysiology of osteoarthritis (OA), a prevalent age-related, degenerative joint disease. Here, we examined the effect of SOX9 acetylation on ACAN transactivation in the context of osteoarthritis. Primary chondrocytes freshly isolated from degenerated OA cartilage displayed lower levels of ACAN mRNA and higher levels of acetylated SOX9 compared with cells from intact regions of OA cartilage. Degenerated OA cartilage presented chondrocyte clusters bearing diffused immunostaining for SOX9 compared with intact cartilage regions. Primary human chondrocytes freshly isolated from OA knee joints were cultured in monolayer or in three-dimensional alginate microbeads (3D). SOX9 was hypo-acetylated in 3D cultures and displayed enhanced binding to a -10 kb ACAN enhancer, a result consistent with higher ACAN mRNA levels than in monolayer cultures. It also co-immunoprecipitated with SIRT1, a major deacetylase responsible for SOX9 deacetylation. Finally, immunofluorescence assays revealed increased nuclear localization of SOX9 in primary chondrocytes treated with the NAD SIRT1 cofactor, than in cells treated with a SIRT1 inhibitor. Inhibition of importin ß by importazole maintained SOX9 in the cytoplasm, even in the presence of NAD. Based on these data, we conclude that deacetylation promotes SOX9 nuclear translocation and hence its ability to activate ACAN.