RESUMEN
Osteoclasts are multinucleated, bone-resorbing cells. However, they also digest cartilage during skeletal maintenance, development and in degradative conditions including osteoarthritis, rheumatoid arthritis and primary bone sarcoma. This study explores the mechanisms behind the osteoclast-cartilage interaction. Human osteoclasts differentiated on acellular human cartilage expressed osteoclast marker genes (e.g. CTSK, MMP9) and proteins (TRAP, VNR), visibly damaged the cartilage surface and released glycosaminoglycan in a contact-dependent manner. Direct co-culture with chondrocytes during differentiation increased large osteoclast formation (p < 0.0001) except when co-cultured on dentine, when osteoclast formation was inhibited (p = 0.0002). Osteoclasts cultured on dentine inhibited basal cartilage degradation (p = 0.012). RNA-seq identified MMP8 overexpression in osteoclasts differentiated on cartilage versus dentine (8.89-fold, p = 0.0133), while MMP9 was the most highly expressed MMP. Both MMP8 and MMP9 were produced by osteoclasts in osteosarcoma tissue. This study suggests that bone-resident osteoclasts and chondrocytes exert mutually protective effects on their 'native' tissue. However, when osteoclasts contact non-native cartilage they cause degradation via MMPs. Understanding the role of osteoclasts in cartilage maintenance and degradation might identify new therapeutic approaches for pathologies characterized by cartilage degeneration.
Asunto(s)
Cartílago/enzimología , Condrocitos/enzimología , Dentina/enzimología , Articulaciones/enzimología , Metaloproteinasas de la Matriz/metabolismo , Osteoclastos/enzimología , Cartílago/ultraestructura , Diferenciación Celular , Células Cultivadas , Condrocitos/ultraestructura , Técnicas de Cocultivo , Dentina/ultraestructura , Humanos , Articulaciones/ultraestructura , Metaloproteinasa 8 de la Matriz/genética , Metaloproteinasa 8 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Metaloproteinasas de la Matriz/genética , Osteoclastos/ultraestructura , ProteolisisRESUMEN
Musculoskeletal dysfunctions are highly prevalent due to increasing life expectancy. Consequently, novel solutions to optimize treatment of patients are required. The current major research focus is to develop innovative concepts for single tissues. However, interest is also emerging to generate applications for tissue transitions where highly divergent properties need to work together, as in bone-cartilage or bone-tendon transitions. Finding medical solutions for dysfunctions of such tissue transitions presents an added challenge, both in research and in clinics. This review aims to provide an overview of the anatomical structure of healthy adult entheses and their development during embryogenesis. Subsequently, important scientific progress in restoration of damaged entheses is presented. With respect to enthesis dysfunction, the review further focuses on inflammation. Although molecular, cellular and tissue mechanisms during inflammation are well understood, tissue regeneration in context of inflammation still presents an unmet clinical need and goes along with unresolved biological questions. Furthermore, this review gives particular attention to the potential role of a signaling mediator protein, transforming growth factor beta-activated kinase-1 (TAK1), which is at the node of regenerative and inflammatory signaling and is one example for a less regarded aspect and potential important link between tissue regeneration and inflammation.
Asunto(s)
Huesos/metabolismo , Inflamación/inmunología , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Tendones/metabolismo , Animales , Huesos/enzimología , Cartílago/enzimología , Cartílago/metabolismo , Humanos , Inflamación/enzimología , Inflamación/metabolismo , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Regeneración/efectos de los fármacos , Regeneración/genética , Regeneración/inmunología , Tendones/anatomía & histología , Tendones/embriología , Tendones/enzimologíaRESUMEN
OBJECTIVE: The integrity of cartilage depends on the correct synthesis of extracellular matrix (ECM) components. In case of insufficient folding of proteins in the endoplasmic reticulum (ER) of chondrocytes, ECM proteins aggregate, ER stress evolves, and the unfolded protein response (UPR) is initiated. By this mechanism, chondrocytes relieve the stress condition or initiate cell death by apoptosis. Especially persistent ER stress has emerged as a pathogenic mechanism in cartilage diseases, such as chondrodysplasias and osteoarthritis. As pharmacological intervention is not available yet, it is of great interest to understand cartilage ER stress in detail and to develop therapeutics to intervene. METHODS: ERp57-deficient chondrocytes were generated by CRISPR/Cas9-induced KO. ER stress and autophagy were studied on mRNA and protein level as well as by transmission electron microscopy (TEM) in chondrocyte micromass or cartilage explant cultures of ERp57 KO mice. Thapsigargin (Tg), an inhibitor of the ER-residing Ca2+-ATPase, and 4-Phenylbutyric acid (4-PBA), a small molecular chemical chaperone, were applied to induce or inhibit ER stress. RESULTS: Our data reveal that the loss of the protein disulfide isomerase ERp57 is sufficient to induce ER stress in chondrocytes. 4-PBA efficiently diffuses into cartilage explant cultures and diminishes excessive ER stress in chondrocytes dose dependently, no matter if it is induced by ERp57 KO or stimulation with Tg. CONCLUSION: ER-stress-related diseases have different sources; therefore, various targets for therapeutic treatment exist. In the future, 4-PBA may be used alone or in combination with other drugs for the treatment of ER-stress-related skeletal disorders in patients.
Asunto(s)
Apoptosis/efectos de los fármacos , Cartílago/enzimología , Condrocitos/enzimología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fenilbutiratos/farmacología , Proteína Disulfuro Isomerasas/deficiencia , Animales , Apoptosis/genética , Cartílago/citología , Línea Celular , Condrocitos/citología , Estrés del Retículo Endoplásmico/genética , Ratones , Ratones Noqueados , Proteína Disulfuro Isomerasas/metabolismoRESUMEN
Growth plate cartilage contributes to the generation of a large variety of shapes and sizes of skeletal elements in the mammalian system. The removal of cartilage and how this process regulates bone shape are not well understood. Here we identify a non-bone-resorbing osteoclast subtype termed vessel-associated osteoclast (VAO). Endothelial cells at the bone/cartilage interface support VAOs through a RANKL-RANK signalling mechanism. In contrast to classical bone-associated osteoclasts, VAOs are dispensable for cartilage resorption and regulate anastomoses of type H vessels. Remarkably, proteinases including matrix metalloproteinase-9 (Mmp9) released from endothelial cells, not osteoclasts, are essential for resorbing cartilage to lead directional bone growth. Importantly, disrupting the orientation of angiogenic blood vessels by misdirecting them results in contorted bone shape. This study identifies proteolytic functions of endothelial cells in cartilage and provides a framework to explore tissue-lytic features of blood vessels in fracture healing, arthritis and cancer.
Asunto(s)
Cartílago/enzimología , Endotelio/enzimología , Osteoclastos/fisiología , Osteogénesis , Péptido Hidrolasas/metabolismo , Animales , Resorción Ósea , Huesos/irrigación sanguínea , Huesos/citología , Cartílago/metabolismo , Endotelio/metabolismo , Placa de Crecimiento/anatomía & histología , Ratones Endogámicos C57BL , Osteoclastos/clasificación , Osteoclastos/metabolismoRESUMEN
BACKGROUND/AIM: Primary bone neoplasms include osteosarcomas (OS), chondrosarcomas (CS), and giant cell tumors (GCT). Nicotinamide phosphoribosyl transferase (NAMPT) catalyzes the rate-limiting step of nicotinamide adenine dinucleotide synthesis and is increased in multiple tumor types. In malignancies, NAMPT expression often correlates positively with tumor grade, chemotherapy resistance, and metastatic potential. MATERIALS AND METHODS: Tissue microarray was used to examine NAMPT expression in benign bone and cartilage, GCTs, OS, and different CS grades. RESULTS: For the first time, we showed that NAMPT expression was increased in GCTs and OS compared to benign bone, and in CS compared to benign cartilage. Its expression also increased with higher CS grade. CONCLUSION: Our data indicate that NAMPT plays a role in bone sarcomas and GCTs, and its higher expression may contribute to increased tumor aggressiveness.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Óseas/enzimología , Huesos/enzimología , Cartílago/enzimología , Condrosarcoma/enzimología , Citocinas/análisis , Nicotinamida Fosforribosiltransferasa/análisis , Osteosarcoma/enzimología , Neoplasias Óseas/patología , Huesos/patología , Cartílago/patología , Condrosarcoma/patología , Tumor Óseo de Células Gigantes/enzimología , Tumor Óseo de Células Gigantes/patología , Humanos , Inmunohistoquímica , Clasificación del Tumor , Osteosarcoma/patología , Análisis de Matrices Tisulares , Regulación hacia ArribaRESUMEN
Human mesenchymal stem cell (hMSC)-based chondrogenesis is a key process used to develop tissue engineered cartilage constructs from stem cells, but the resulting constructs have inferior biochemical and biomechanical properties compared to native articular cartilage. Transforming growth factor ß containing medium is commonly applied to cell layers of hMSCs, which aggregate upon centrifugation to form 3-D constructs. The aggregation process leads to a high cell density condition, which can cause nutrient limitations during long-term culture and, subsequently, inferior quality of tissue engineered constructs. Our objective is to modulate the aggregation process by targeting RhoA/ROCK signaling pathway, the chief modulator of actomyosin contractility, to enhance the end quality of the engineered constructs. Through ROCK inhibition, repression of cytoskeletal tension in chondrogenic hMSCs was achieved along with less dense aggregates with enhanced transport properties. ROCK inhibition also led to significantly increased cartilaginous extracellular matrix accumulation. These findings can be used to create an improved microenvironment for hMSC-derived tissue engineered cartilage culture. We expect that these findings will ultimately lead to improved cartilaginous tissue development from hMSCs.
Asunto(s)
Cartílago/enzimología , Condrogénesis , Matriz Extracelular/metabolismo , Células Madre Mesenquimatosas/enzimología , Transducción de Señal , Quinasas Asociadas a rho/antagonistas & inhibidores , Transporte Biológico Activo , Cartílago/citología , Células Cultivadas , Citoesqueleto/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos , Quinasas Asociadas a rho/metabolismoRESUMEN
Recent findings demonstrate that leptin plays a significant role in chondrocyte and osteoblast differentiation. However, the mechanisms by which leptin acts on cartilage endplate (CEP) cells to give rise to calcification are still unclear. The aim of this study was to evaluate the effects of leptin that induced mineralization of CEP cells in vitro and in vivo. We constructed a rat model of lumbar disc degeneration and determined that leptin was highly expressed in the presence of CEP calcification. Rat CEP cells treated with or without leptin were used for in vitro analysis using RT-PCR and Western blotting to examine the expression of osteocalcin (OCN) and runt-related transcription factor 2 (Runx2). Both OCN and Runx2 expression levels were significantly increased in a dose- and time-dependent manner. Leptin activated ERK1/2 and STAT3 phosphorylation in a time-dependent manner. Inhibition of phosphorylated ERK1/2 using targeted siRNA suppressed leptin-induced OCN and Runx2 expression and blocked the formation of mineralized nodules in CEP cells. We further demonstrated that exogenous leptin induced matrix mineralization of CEP cells in vivo. We suggest that leptin promotes the osteoblastic differentiation of CEP cells via the MAPK/ERK signal transduction pathway and may be used to investigate the mechanisms of disc degeneration.
Asunto(s)
Cartílago/enzimología , Cartílago/patología , Degeneración del Disco Intervertebral/enzimología , Degeneración del Disco Intervertebral/patología , Leptina/farmacología , Sistema de Señalización de MAP Quinasas , Osteogénesis/efectos de los fármacos , Animales , Calcificación Fisiológica/efectos de los fármacos , Cartílago/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Modelos Animales de Enfermedad , Vértebras Lumbares/efectos de los fármacos , Vértebras Lumbares/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Placa Motora/efectos de los fármacos , Placa Motora/patología , Osteocalcina/metabolismo , Fosforilación/efectos de los fármacos , Ratas Sprague-Dawley , Factor de Transcripción STAT3/metabolismoRESUMEN
Cartilage originates from mesenchymal cell condensations that differentiate into chondrocytes of transient growth plate cartilage or permanent cartilage of the articular joint surface and trachea. MicroRNAs fine-tune the activation of entire signaling networks and thereby modulate complex cellular responses, but so far only limited data are available on miRNAs that regulate cartilage development. Here, we characterize a miRNA that promotes the biosynthesis of a key component in the RAF/MEK/ERK pathway in cartilage. Specifically, by transcriptome profiling we identified miR-322 to be upregulated during chondrocyte differentiation. Among the various miR-322 target genes in the RAF/MEK/ERK pathway, only Mek1 was identified as a regulated target in chondrocytes. Surprisingly, an increased concentration of miR-322 stabilizes Mek1 mRNA to raise protein levels and dampen ERK1/2 phosphorylation, while cartilage-specific inactivation of miR322 in mice linked the loss of miR-322 to decreased MEK1 levels and to increased RAF/MEK/ERK pathway activation. Such mice died perinatally due to tracheal growth restriction and respiratory failure. Hence, a single miRNA can stimulate the production of an inhibitory component of a central signaling pathway to impair cartilage development.
Asunto(s)
Cartílago/embriología , Cartílago/enzimología , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas , MicroARNs/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión/genética , Sistemas CRISPR-Cas/genética , Condrocitos/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Placa de Crecimiento/metabolismo , Hemicigoto , Homeostasis , MAP Quinasa Quinasa 1/genética , Masculino , Ratones Transgénicos , MicroARNs/genética , Organogénesis/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , TransfecciónRESUMEN
OBJECTIVE: Ficus deltoidea Jack (mistletoe fig) is an ornamental plant found in various parts of the world and used as traditional herbal medicine in some countries. This study investigated the potential use of F deltoidea leaf extract to mitigate osteoarthritis (OA) in ovariectomized (estrogen-deficient postmenopausal model) rats and the mechanisms involved. Diclofenac was used for comparison. METHODS: Sprague-Dawley female rats (12 weeks old) were divided randomly into five groups (nâ=â6): healthy; nontreated OA; OAâ+âdiclofenac (5âmg/kg); OAâ+âextract (200âmg/kg); and OAâ+âextract (400âmg/kg). Two weeks after bilaterally ovariectomy, OA was induced by intra-articular injection of monosodium iodoacetate into the right knee joints. After 28 days of treatment, the rats were evaluated for knee OA via physical (radiological and histological observations), biochemical, enzyme-linked immunosorbent assay, and gene expression analysis, for inflammation and cartilage degradation biomarkers. RESULTS: The osteoarthritic rats treated with the extract, and diclofenac showed significant reduction of cartilage erosion (via radiological, macroscopic, and histological images) compared with untreated osteoarthritic rats. The elevated serum interleukin-1ß, prostaglandin E2, and C-telopeptide type II collagen levels in osteoarthritic rats were significantly reduced by F deltoidea leaf extract comparable to diclofenac. The extract significantly down-regulated the interleukin-1ß, prostaglandin E2 receptor, and matrix metalloproteinase-1 mRNA expressions in the osteoarthritic cartilages, similar to diclofenac. CONCLUSIONS: F deltoidea leaf extract mitigated postmenopausal osteoarthritic joint destruction by inhibiting inflammation and cartilage degradation enzymes, at an effective extract dose equivalent to about 60âmg/kg for humans. The main bioactive compounds are probably the antioxidative flavonoids vitexin and isovitexin.
Asunto(s)
Ficus/química , Osteoartritis/prevención & control , Extractos Vegetales/administración & dosificación , Hojas de la Planta/química , Posmenopausia , Animales , Antiinflamatorios no Esteroideos , Apigenina/administración & dosificación , Cartílago/efectos de los fármacos , Cartílago/enzimología , Cartílago/patología , Colágeno Tipo II/sangre , Diclofenaco/administración & dosificación , Dinoprostona/sangre , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Femenino , Inflamación/prevención & control , Interleucina-1beta/sangre , Interleucina-1beta/genética , Metaloproteinasa 1 de la Matriz/genética , Osteoartritis/inducido químicamente , Osteoartritis/patología , Ovariectomía , Fragmentos de Péptidos/sangre , Fitoterapia , Ratas , Ratas Sprague-Dawley , Receptores de Prostaglandina E/genéticaRESUMEN
Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation.
Asunto(s)
Acetilcolinesterasa/deficiencia , Apnea/fisiopatología , Huesos/embriología , Butirilcolinesterasa/deficiencia , Cartílago/embriología , Mesodermo/fisiología , Errores Innatos del Metabolismo/fisiopatología , Osteogénesis/fisiología , Acetilcolinesterasa/fisiología , Animales , Bencenamina, 4,4'-(3-oxo-1,5-pentanodiil)bis(N,N-dimetil-N-2-propenil-), Dibromuro/farmacología , Bencenamina, 4,4'-(3-oxo-1,5-pentanodiil)bis(N,N-dimetil-N-2-propenil-), Dibromuro/toxicidad , Huesos/enzimología , Huesos/patología , Butirilcolinesterasa/fisiología , Cartílago/enzimología , Cartílago/patología , Embrión de Pollo , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/toxicidad , Condrogénesis/efectos de los fármacos , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/fisiología , Ratones , Ratones Noqueados , Nicotina/farmacología , Nicotina/toxicidad , Técnicas de Cultivo de Órganos , Receptor Nicotínico de Acetilcolina alfa 7/efectos de los fármacos , Receptor Nicotínico de Acetilcolina alfa 7/fisiologíaRESUMEN
AIMS: The molecular mechanism of osteoarthritis (OA) has never been understood clearly, but it has been suggested that imbalance of degradation and synthesis in cartilage contribute to the underlying mechanisms of OA. In this study, we investigated the effectiveness in the cartilage metabolism of the artichoke extract that includes the compound cynaropicrin. MAIN METHODS: We evaluated the efficacy of the artichoke extract or cynaropicrin in the cartilage metabolism factors and NF-κB signaling activity stimulated by inflammatory cytokine in chondrogenic cell lines, OUMS-27 and SW1353, using qRT-PCR, immunofluorescence and immunoblotting. KEY FINDINGS: We initially found that an artichoke extract and cynaropicrin both inhibited the increase of cartilage degradation factor MMP13 and further decreased the synthesis factor aggrecan induced by TNF-α in OUMS-27. In addition, cynaropicrin suppressed the enhancement of master regulator HIF-2α on cartilage degradation and further reduced the master regulator Sox9 on cartilage synthesis induced by TNF-α. We observed that cynaropicrin suppresses NF-κB signaling, which controls HIF-2α and Sox9. Since, HIF-2α is induced by p65 (RelA), we evaluated the effect of cynaropicrin and observed that it suppressed the nuclear transport of p65 (RelA) by inhibiting phosphorylation of IκBα. Moreover, cynaropicrin not only suppressed TNF-α stimulation, it had a similar effect on IL-1ß stimulation. No significant cytotoxicity with cynaropicrin was observed. SIGNIFICANCE: These finding suggest that cynaropicrin is an effective substance that can improve the balance of cartilage metabolism, by altering the equilibrium of cartilage degradation and synthesis induced by multiple mediators know to contribute to OA.
Asunto(s)
Cartílago/metabolismo , Lactonas/metabolismo , Sesquiterpenos/metabolismo , Agrecanos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cartílago/enzimología , Línea Celular , Humanos , Hidrólisis , Metaloproteinasa 13 de la Matriz/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción SOX9/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The single nucleotide polymorphism (SNP) rs835487 is associated with hip osteoarthritis (OA) at the genome-wide significance level and is located within CHST11, which codes for carbohydrate sulfotransferase 11. This enzyme post-translationally modifies proteoglycan prior to its deposition in the cartilage extracellular matrix. Using bioinformatics and experimental analyses, our aims were to characterise the rs835487 association signal and to identify the causal functional variant/s. Database searches revealed that rs835487 resides within a linkage disequilibrium (LD) block of only 2.7 kb and is in LD (r2 ≥ 0.8) with six other SNPs. These are all located within intron 2 of CHST11, in a region that has predicted enhancer activity and which shows a high degree of conservation in primates. Luciferase reporter assays revealed that of the seven SNPs, rs835487 and rs835488, which have a pairwise r2 of 0.962, are the top functional candidates; the haplotype composed of the OA-risk conferring G allele of rs835487 and the corresponding T allele of rs835488 (the G-T haplotype) demonstrated significantly different enhancer activity relative to the haplotype composed of the non-risk A allele of rs835487 and the corresponding C allele of rs835488 (the A-C haplotype) (p < 0.001). Electrophoretic mobility shift assays and supershifts identified several transcription factors that bind more strongly to the risk-conferring G and T alleles of the two SNPs, including SP1, SP3, YY1 and SUB1. CHST11 was found to be upregulated in OA versus non-OA cartilage (p < 0.001) and was expressed dynamically during chondrogenesis. Its expression in adult cartilage did not however correlate with rs835487 genotype. Our data demonstrate that the OA susceptibility is mediated by differential protein binding to the alleles of rs835487 and rs835488, which are located within an enhancer whose target may be CHST11 during chondrogenesis or an alternative gene.
Asunto(s)
Condrogénesis/genética , Predisposición Genética a la Enfermedad , Intrones , Osteoartritis de la Cadera/genética , Polimorfismo de Nucleótido Simple , Sulfotransferasas/genética , Alelos , Animales , Cartílago/enzimología , Cartílago/patología , Línea Celular Tumoral , Biología Computacional , Bases de Datos de Ácidos Nucleicos , Estudio de Asociación del Genoma Completo , Haplotipos , Humanos , Osteoartritis de la Cadera/enzimología , Osteoartritis de la Cadera/patología , Sulfotransferasas/metabolismoRESUMEN
I was given the honor of delivering the 2015 Lifetime Membership Award lecture at the International Proteolysis Society's annual meeting held in Penang, Malaysia in October 2015. It gave me an opportunity to look back on how I started my research on matrix metalloproteinases (MMPs) and how I continued to work on these proteinases for the next 42 years. This is a series of sketches from the personal journey that I took with MMPs, starting from the purification of metalloproteinases, cloning, structural studies, then to a more recent encounter, endocytic regulation of matrix-degrading metalloproteinases.
Asunto(s)
Bioquímica/historia , Metaloproteinasas de la Matriz/metabolismo , Cartílago/enzimología , Clonación Molecular , ADN Complementario/genética , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Metaloproteinasas de la Matriz/química , Metaloproteinasas de la Matriz/genéticaRESUMEN
BACKGROUND: To better understand the complex mechanisms of bone formation it is fundamental that genes central to signaling/regulatory pathways and matrix formation are identified. Cell systems were used to analyze genes differentially expressed during extracellular matrix mineralization and bhmt3, coding for a betaine-homocysteine S-methyltransferase, was shown to be down-regulated in mineralizing gilthead seabream cells. METHODS: Levels and sites of bhmt3 expression were determined by qPCR and in situ hybridization throughout seabream development and in adult tissues. Transcriptional regulation of bhmt3 was assessed from the activity of promoter constructs controlling luciferase gene expression. Molecular phylogeny of vertebrate BHMT was determined from maximum likelihood analysis of available sequences. RESULTS: bhmt3 transcript is abundant in calcified tissues and localized in cartilaginous structures undergoing endo/perichondral ossification. Promoter activity is regulated by transcription factors involved in bone and cartilage development, further demonstrating the central role of Bhmt3 in chondrogenesis and/or osteogenesis. Molecular phylogeny revealed the explosive diversity of bhmt genes in neoteleost fish, while tissue distribution of bhmt genes in seabream suggested that neoteleostean Bhmt may have undergone several steps of sub-functionalization. CONCLUSIONS: Data on bhmt3 gene expression and promoter activity evidences a novel function for betaine-homocysteine S-methyltransferase in bone and cartilage development, while phylogenetic analysis provides new insights into the evolution of vertebrate BHMTs and suggests that multiple gene duplication events occurred in neoteleost fish lineage. GENERAL SIGNIFICANCE: High and specific expression of Bhmt3 in gilthead seabream calcified tissues suggests that bone-specific betaine-homocysteine S-methyltransferases could represent a suitable marker of chondral ossification.
Asunto(s)
Betaína-Homocisteína S-Metiltransferasa/metabolismo , Cartílago/enzimología , Condrogénesis , Proteínas de Peces/metabolismo , Osteogénesis , Dorada/metabolismo , Animales , Betaína-Homocisteína S-Metiltransferasa/genética , Línea Celular , Clonación Molecular , Evolución Molecular , Proteínas de Peces/genética , Regulación Enzimológica de la Expresión Génica , Filogenia , Regiones Promotoras Genéticas , Dorada/genética , Transcripción Genética , TransfecciónRESUMEN
AIMS: Chordomas and chondrosarcomas are malignant mesenchymal tumours with overlapping morphological and immunohistochemical (IHC) characteristics. Our aim was to evaluate the IHC expression of α-methylacyl-CoA racemase (AMACR/P504S), ß-catenin and E-cadherin in chordomas relative to chondrosarcomas and assess the utility of these markers for differential diagnosis. METHODS: Archival sections of 18 chordomas, 19 chondrosarcomas and 10 mature cartilage samples were immunostained and scored for AMACR, ß-catenin and E-cadherin and the relative differential capacity of each marker was calculated. In addition, AMACR mRNA level was assessed in 5 chordomas by RT-PCR and evaluated by comparative CT method. RESULTS: AMACR and ß-catenin stained 88.9% and 94.1% of the chordomas respectively, 21.1% and 10.5% of the chondrosarcomas correspondingly and none of the mature cartilage samples. E-cadherin stained positively 82.4% of the chordomas, 36.8% of the chondrosarcomas and 42.9% of the mature cartilage cases. Both AMACR and ß-catenin showed statistically significant difference between chordomas and chondrosarcomas (p < 0.001 for both), unlike E-cadherin. AMACR was detected at the mRNA level. CONCLUSIONS: AMACR is expressed in most of the chordomas but only in a minority of chondrosarcomas. AMACR may serve as IHC marker of chordoma with differentiating ability comparable to that of ß-catenin.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Condroma/enzimología , Condrosarcoma/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Racemasas y Epimerasas/biosíntesis , Cartílago/enzimología , Cartílago/patología , Condroma/patología , Condrosarcoma/patología , Femenino , Humanos , MasculinoRESUMEN
OBJECTIVES: To investigate the effects of all-trans retinoic acid (ATRA) on arthritis and the expressions of inflammatory cytokines and cartilage damage related proteases of the collagen-induced arthritis model (CIA) rats in vivo. METHODS: The CIA model of rheumatoid arthritis was induced with C2 and incomplete Freund's adjuvant. The rats were randomly divided into control group, CIA model group and two ATRA dose groups (ATRA 0.50 mg/kg group and ATRA 1.00 mg/kg group). ATRA were given three times per week for six weeks in ATRA groups. Morphological changes, arthritis index (AI) scores, the semi-quantitative scores of pathology damage, the protein expressions of cartilage damage related proteases and the serum levels of TNF-α, IL-17A, IFN-γ, IL-4, IL-10 were observed. RESULTS: The AI scores of ATRA groups were similar to CIA model group ( P<0.05). Apparent morphological disorders in knee and ankle joints were observed in the CIA model group and ATRA 1.00 mg/kg group. The structure of knee joint was improved slightly in ATRA 0.50 mg/kg group. The serum levels of TNF-α, IFN-γ and IL-17A were decreased in both ATRA groups; ATRA also can increase the serum level of IL-4. Compared to CIA model group, the protein expressions of ADAMTS-4, MMP3, MMP1 were decreased in both ATRA groups ( P<0.05). CONCLUSIONS: ATRA, which was able to inhibit pro-inflammatory cytokines secretion, could correct the imbalance of Th1/Th2 and Th17/Treg. ATRA also can reduce the expressions of cartilage damage related proteases, which proved that ATRA may have a beneficial effect on rheumatoid arthritis.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Cartílago/enzimología , Citocinas/sangre , Péptido Hidrolasas/metabolismo , Tretinoina/farmacología , Animales , Artritis Experimental/inducido químicamente , Artritis Reumatoide , RatasRESUMEN
The effects of fullerene C60 (FC60) on the level of free radical and destruction processes were studied in rats with experimental adjuvant arthritis (AA). It was shown the protective effect of FC60 during AA. The effect was accompanied by an increase of the antioxidant enzymes activity, superoxide dismutase in the liver (15.96 ± 0.38 µmol/kg x s) and in the kidneys (5.36 ± 0.27 µmol/kg x s) and catalase in the kidneys (9.56 ± 0.78 µmol/kg x s) and in the heart (2.26 ± 0.41 µmol/kg x s) in comparison to control group (43.83± 5.69%; 54.55 ± 6.18%; 11.68 ± 0.52 µmol/kg x s; 3.43 ± 0.47 µmol/kg x s; 4.77 ± 0.5 µmol/kg x s; 0.98 ± 0.12 µmol/kg x s accordingly). It was shown a protective effect of FC60 during AA directed on the depression of the destructive processes in connective tissue that was expressed through the reduction of the total collagenolitic activity level in cartilage (10.05 ± 0.06 µmol/g/min) and bone (11.21 ± 0.04 µmol/g/min) tissues, free hydroxyproline contents (1.54 ± 0.04 µg/ml) and alkaline phasphatase activity (1.24 ± 0,14 µmol/l x sec) in comparison to control group (11.91 ± 0.49 µmol/g/min; 13.19 ± 0.15 µmol/g/min; 2.25 ± 0.07 µg/ ml; 2.19 ± 0.24 µmol/l x sec accordingly). Taken together, these results accentuate the perspective of future investigations of action FC60 during rheumatoid arthritis as a feasible therapeutic agent.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/tratamiento farmacológico , Depuradores de Radicales Libres/farmacología , Radicales Libres/antagonistas & inhibidores , Fulerenos/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Artritis Experimental/metabolismo , Artritis Experimental/patología , Huesos/efectos de los fármacos , Huesos/enzimología , Cartílago/efectos de los fármacos , Cartílago/enzimología , Catalasa/metabolismo , Radicales Libres/metabolismo , Hidroxiprolina/metabolismo , Articulaciones/efectos de los fármacos , Articulaciones/metabolismo , Articulaciones/patología , Riñón/efectos de los fármacos , Riñón/enzimología , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
We report the synthesis, the antioxidant and the inhibitory activity (IC50) on metalloproteinases (MMPs) 3 and 13 of 2-benzo[d]isothiazolylimino-5-benzylidene-4-thiazolidinones. Their potential as protective agents in osteoarthritis (OA) was evaluated by biological assays on chondrocytes cultures, stimulated by IL-1ß. The chondroprotective capability, related both to antioxidant activity and to inhibition of MMPs, was studied in vitro, by determining nitric oxide production and glycosaminoglycans release. Moreover, selected derivatives 1h and 1g was studied for nuclear factor kappaB (NF-κB) inhibition by Western Blot analysis and for MMP-3 protein release using ELISA test. The structure-activity relationship of tested compounds demonstrates a favorable effect of the para substitution on the 5-benzilydene ring. Compound 1g shows a potent and selective activity on MMP-3 versus MMP-13. Accordingly, 1g possesses high antioxidant effect, NO lowering and GAGs restoring capability and also reduces the production of MMPs and NF-κB expression. Thus 1g can be considered as new potential chondroprotective agents.
Asunto(s)
Cartílago/efectos de los fármacos , Condrocitos/efectos de los fármacos , Tiazolidinas/química , Cartílago/enzimología , Cartílago/patología , Células Cultivadas , Condrocitos/enzimología , Condrocitos/patología , Humanos , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/metabolismo , Osteoartritis/enzimología , Osteoartritis/patología , Osteoartritis/prevención & control , Sustancias Protectoras/química , Sustancias Protectoras/farmacología , Sustancias Protectoras/uso terapéutico , Tiazolidinas/farmacología , Tiazolidinas/uso terapéuticoRESUMEN
Non-destructive detection of cartilage-degrading activities represents an advance in osteoarthritis (OA) research, with implications in studies of OA pathogenesis, progression, and intervention strategies. Matrix metalloproteinases (MMPs) are principal cartilage degrading enzymes that contribute to OA pathogenesis. MMPSense750 is an in-vivo fluorimetric imaging probe with the potential to continuously and non-invasively trace real-time MMP activities, but its use in OA-related research has not been reported. Our objective is to detect and characterize the early degradation activities shortly after cartilage or joint injury with MMPSense750. We determined the appropriate concentration, assay time, and linear range using various concentrations of recombinant MMPs as standards. We then quantified MMP activity from cartilage explants subjected to either mechanical injury or inflammatory cytokine treatment in-vitro. Finally, we performed in-vivo MMP imaging of a mouse model of post-traumatic OA. Our in-vitro results showed that the optimal assay time was highly dependent on the MMP enzyme. In cartilage explant culture media, mechanical impact or cytokine treatment increased MMP activity. Injured knees of mice showed significantly higher fluorescent signal than uninjured knees. We conclude that MMPSense750 detects human MMP activities and can be used for in-vitro study with cartilage, as well as in-vivo studies of knee injury, and can offering real-time insight into the degradative processes that occurring within the joint before structural changes become evident radiographically.