RESUMEN
Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E(2) (PGE(2)) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRalpha and -beta) are expressed in cartilage, with LXRbeta being the predominant isoform. Here we show that genetic disruption of Lxrbeta gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE(2) production in articular cartilage treated with IL-1beta, indicating a protective role of LXRbeta in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE(2) production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE(2) synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.
Asunto(s)
Cartílago Articular/metabolismo , Dinoprostona/antagonistas & inhibidores , Receptores Nucleares Huérfanos/agonistas , Osteoartritis/complicaciones , Dolor/metabolismo , Proteínas ADAM/antagonistas & inhibidores , Proteína ADAMTS4 , Animales , Benzoatos/farmacología , Bencilaminas/farmacología , Línea Celular Tumoral , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Ligandos , Receptores X del Hígado , Ratones , Ratones Mutantes , Receptores Nucleares Huérfanos/genética , Receptores Nucleares Huérfanos/fisiología , Osteoartritis/metabolismo , Dolor/etiología , Procolágeno N-Endopeptidasa/antagonistas & inhibidores , Prostaglandina-E Sintasas , RatasRESUMEN
Design, synthesis and structure-activity relationship of a series of biphenylsulfonamido-3-methylbutanoic acid based aggrecanase-1 inhibitors are described. In addition to robust aggrecanase-1 inhibition, these compounds also exhibit potent MMP-13 activity. In cell-based cartilage explants assay compound 48 produced 87% inhibition of proteoglycan degradation at 10 µg/mL. Good pharmacokinetic properties were demonstrated by 46 with a half-life of 6h and bioavailability of 23%.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Compuestos de Bifenilo/farmacología , Procolágeno N-Endopeptidasa/antagonistas & inhibidores , Procolágeno N-Endopeptidasa/metabolismo , Inhibidores de Proteasas/farmacología , Sulfonamidas/farmacología , Proteína ADAMTS4 , Animales , Compuestos de Bifenilo/química , Compuestos de Bifenilo/farmacocinética , Diseño de Fármacos , Humanos , Masculino , Metaloproteinasa 13 de la Matriz/metabolismo , Modelos Moleculares , Osteoartritis/tratamiento farmacológico , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Proteoglicanos/metabolismo , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacocinéticaRESUMEN
Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.
Asunto(s)
Cartílago Articular/metabolismo , Modelos Animales de Enfermedad , Eliminación de Gen , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/genética , Osteoartritis/metabolismo , Proteínas ADAM , Proteína ADAMTS5 , Animales , Dominio Catalítico , Endopeptidasas/química , Endopeptidasas/deficiencia , Endopeptidasas/genética , Endopeptidasas/metabolismo , Exones/genética , Cabeza Femoral , Placa de Crecimiento/metabolismo , Articulaciones/patología , Articulaciones/fisiopatología , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Ratones , Ratones Noqueados , Osteoartritis/genética , Osteoartritis/patología , Osteoartritis/fisiopatología , Proteoglicanos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed. METHODS: TN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA. RESULTS: TN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE2, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA. CONCLUSIONS: TN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.
Asunto(s)
Cartílago Articular/patología , Condrocitos/patología , Matriz Extracelular/metabolismo , Mediadores de Inflamación/metabolismo , Osteoartritis de la Rodilla/patología , Tenascina/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Cartílago Articular/metabolismo , Bovinos , Línea Celular Tumoral , Células Cultivadas , Condrocitos/metabolismo , Matriz Extracelular/patología , Femenino , Humanos , Mediadores de Inflamación/fisiología , Masculino , Persona de Mediana Edad , Osteoartritis de la Rodilla/etiología , Osteoartritis de la Rodilla/metabolismo , Ratas , Ratas Endogámicas Lew , Líquido Sinovial/metabolismo , Tenascina/biosíntesis , Tenascina/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIalpha1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV(356)) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV(356) neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.
Asunto(s)
Agrecanos/química , Agrecanos/metabolismo , Serina Endopeptidasas/metabolismo , Factores de Edad , Anciano , Anciano de 80 o más Años , Agrecanos/análisis , Agrecanos/inmunología , Alginatos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cartílago/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Condrocitos/metabolismo , Condrocitos/patología , Progresión de la Enfermedad , Epítopos/química , Epítopos/inmunología , Femenino , Regulación de la Expresión Génica , Ácido Glucurónico , Ácidos Hexurónicos , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Osteoartritis/metabolismo , Osteoartritis/patología , Serina Endopeptidasas/genéticaRESUMEN
Potent 3,4-disubstituted benzofuran P1' MMP-13 inhibitors have been prepared. Selectivity over MMP-2 was achieved through a substituent at the C4 position of the benzofuran P1' moiety of the molecule. By replacing a backbone benzene with a pyridine and valine with threonine, compounds (e.g., 44) with greatly reduced plasma protein binding were also obtained.
Asunto(s)
Benzofuranos/química , Inhibidores de la Metaloproteinasa de la Matriz , Inhibidores de Proteasas/química , Animales , Benzofuranos/síntesis química , Benzofuranos/farmacología , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Unión Proteica , Conejos , Albúmina Sérica/química , Relación Estructura-ActividadRESUMEN
The prevention of aggrecan (a key component of cartilage) cleavage via the inhibition of aggrecanase-1 may provide a unique opportunity to stop the progression of cartilage degradation in osteoarthritis. The evaluation of a series of biphenylsulfonamides resulted in the identification of the ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides analogs (19-21 and 24) with improved Agg-1 inhibition and MMP-2, MMP-13 activity.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/metabolismo , Química Farmacéutica/métodos , Osteoartritis/tratamiento farmacológico , Procolágeno N-Endopeptidasa/antagonistas & inhibidores , Procolágeno N-Endopeptidasa/metabolismo , Sulfonamidas/síntesis química , Proteína ADAMTS4 , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Modelos Químicos , Conformación Molecular , Proteoglicanos/química , Sulfonamidas/farmacologíaRESUMEN
Articular cartilage chondrocytes help in the maintenance of tissue homeostasis and function of the articular joint. Study of primary chondrocytes in culture provides information closely related to in vivo functions of these cells. Limitations in the primary culture of chondrocytes have lead to the development of cells lines that serve as good surrogate models for the study of chondrocyte biology. In this study, we report the establishment and characterization of chondrocyte cell lines, MM-Sv/HP and MM-Sv/HP-2 from mouse articular cartilage. Cells were isolated from mouse femoral head articular cartilage, immortalized and maintained in culture through numerous passages. The morphology of the cells was from fibroblastic to polygonal in nature. Gene expression studies using quantitative PCR (Q-PCR) were performed on cells in monolayer culture and cells embedded in a three-dimensional alginate matrix. Stimulation of cells in monolayer culture with anabolic factor, BMP-2, resulted in increased gene expression of the extracellular matrix molecules, aggrecan and type II collagen and their regulator transcription factor, Sox9. Treatment by pro-inflammatory IL-1 resulted in increased gene expression of catabolic effectors including Aggrecanases (ADAMTS4, ADAMTS5), MMP-13 and nitric oxide synthase (Nos2). Cells in alginate treated with BMP-2 resulted in increased synthesis of proteoglycan which was released into the conditioned media on IL-1 stimulation. Western analysis of conditioned media showed the presence of Aggrecanase-cleaved aggrecan fragments. In summary, MM-Sv/HP and MM-Sv/HP-2 show preservation of important characteristics of articular chondrocytes as examined under multiple culture conditions and would provide a useful reagent in the study of chondrocyte biology.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Cartílago Articular/citología , Cartílago Articular/efectos de los fármacos , Condrocitos/citología , Condrocitos/efectos de los fármacos , Mediadores de Inflamación/farmacología , Interleucina-1/farmacología , Factor de Crecimiento Transformador beta/farmacología , Agrecanos/metabolismo , Alginatos/farmacología , Animales , Biomarcadores/metabolismo , Proteína Morfogenética Ósea 2 , Cartílago Articular/enzimología , Línea Celular Transformada , Condrocitos/enzimología , Endopeptidasas/metabolismo , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Fémur/citología , Fémur/efectos de los fármacos , Humanos , Ratones , FenotipoRESUMEN
N-((8-Hydroxy-5-substituted-quinolin-7-yl)(phenyl)methyl)-2-phenyloxy/amino-acetamide inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared. Selected compounds 10, 14, 25, and 53 show sub-microM ADAMTS-5 potency and good selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase-1), MMP-13, and MMP-12. Compound 53 shows a good balance of potent ADAMTS-5 inhibition, moderate CYP3A4 inhibition and good rat liver microsome stability. This series of compounds represents progress towards selective ADAMTS-5 inhibitors as disease modifying osteoarthritis agents.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Proteínas ADAM/química , Acetamidas/síntesis química , Acetamidas/farmacología , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS5 , Animales , Química Farmacéutica/métodos , Citocromo P-450 CYP3A , Inhibidores del Citocromo P-450 CYP3A , Inhibidores Enzimáticos del Citocromo P-450 , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Microsomas Hepáticos/efectos de los fármacos , Modelos Químicos , Osteoartritis/tratamiento farmacológico , Procolágeno N-Endopeptidasa/metabolismo , RatasRESUMEN
Aggrecanases are ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motifs) proteases capable of primary (patho)physiological cleavage at specific Glu-Xaa bonds within the core protein of the hyaluronan-binding proteoglycan aggrecan. Accumulating evidence suggests that regulation of the activity of one such aggrecanase, ADAMTS-4 (or Aggrecanase-1), involves post-translational C-terminal processing (truncation) which modulates both glycosaminoglycan (GAG)-binding affinity and enzymatic activity. In the present study, we compared the effects of C-terminal truncation on the GAG-binding properties and aggrecanase activity of ADAMTS-5 (Aggrecanase-2) relative to three other ADAMTS family members, ADAMTS-9, ADAMTS-16 and ADAMTS-18. Full-length recombinant human ADAMTS-5 (M(r) approximately 85 kDa; ADAMTS-5p85) underwent autolytic cleavage during expression by CHO/A2 cells, and co-purified with C-terminally truncated (tr) isoforms of M(r) approximately 60 kDa (ADAMTS-5p60 and M(r) approximately 45 kDa (ADAMTS-5p45). All three ADAMTS-5 isoforms bound to sulfated GAGs (heparin and chondroitin sulfate (CS)). An ADAMTS-5p45 structural mimetic, terminating at Phe628 and comprising the catalytic domain, disintegrin-like domain and thrombospondin type I repeat (TSR)-1 domain (designated trADAMTS-5F628), also bound to heparin, and exhibited potent aggrecanase activity toward cleavage sites both in the aggrecan CS-2-attachment region (at Glu1771-Ala1772) and in the interglobular domain (at Glu373-Ala374). Further truncation (deletion of the TSR-1 domain) of ADAMTS-5 significantly reduced aggrecanase activity, although appreciable GAG (heparin)-binding affinity was maintained. Other TSR-1 domain-bearing truncated ADAMTS constructs demonstrating either positive GAG-binding ability (trADAMTS-9F649) or negligible GAG-affinity (trADAMTS-16F647 and trADAMTS-18F650) displayed comparably low aggrecanase activities. Thus, the presence of TSR-1 on truncated ADAMTSs appears to be necessary, but not sufficient, for effective aggrecanase-mediated catalysis of target Glu-Xaa bonds. Similarly, GAG-binding ability, irrespective of the presence of a TSR-1 domain, does not necessarily empower truncated ADAMTSs with proficient aggrecanase activity.
Asunto(s)
Proteínas ADAM/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Endopeptidasas/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Lectinas Tipo C/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteínas ADAMTS , Proteína ADAMTS5 , Proteína ADAMTS9 , Agrecanos , Animales , Células CHO , Bovinos , Cricetinae , Cricetulus , Humanos , Isoformas de Proteínas/metabolismoRESUMEN
Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.
Asunto(s)
Cartílago Articular/metabolismo , Glicoproteínas/metabolismo , Animales , Bovinos , Glicoproteínas/química , Glicoproteínas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Insulin-like growth factor-I (IGF-I) is thought to play an important role in skeletal growth and development through its mitogenic and anabolic effects on epiphyseal growth plate chondrocytes. The bone morphogenetic proteins (BMPs) have been shown to promote endochondral osteogenesis, and some members of the BMP family, including BMP-2 and BMP-9, have anabolic effects on chondrocyte metabolism. We tested the hypothesis that BMP-2 and BMP-9 interact with IGF-I to modulate growth plate chondrocyte mitotic activity. IGF-I, but neither BMP-2 nor BMP-9, stimulated chondrocyte DNA synthesis. However, both BMP-2 and BMP-9 augmented the mitogenic action of IGF-I. BMP-2, but not BMP-9 increased IGF-I binding to growth plate chondrocytes in kinetic studies. In affinity labeling studies, 125I-IGF-I predominantly labeled an Mr approximately 135-kDa moiety, consistent with the alpha subunit of the type 1 IGF receptor and an Mr approximately 250-kDa moiety consistent with the type 2 IGF receptor. 125I-IGF-I labeling also appeared at Mr approximately 43 kDa, consistent with 125I-IGF-I binding to insulin-like growth binding protein-3. Treatment of chondrocytes with BMP-2, but not with BMP-9, increased the intensity of the Mr approximately 135-kDa band and decreased the intensity of the Mr approximately 43-kDa band. Taken together, these data suggest that the BMPs may modulate the action of IGF-I via the type 1 IGF receptor and/or IGF binding proteins.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Condrocitos/metabolismo , Placa de Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/farmacología , Células CHO , Cartílago Articular/citología , Bovinos , Células Cultivadas , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Factor 2 de Diferenciación de Crecimiento , Placa de Crecimiento/citología , Radioisótopos de Yodo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Members of the bone morphogenetic protein (BMP) group of the TGF-beta superfamily have been shown to enhance matrix synthesis and maintain cartilage phenotype in long-term culture. These proteins have also been shown to augment cartilage repair in vivo, and may be of potential therapeutic benefit in the treatment of damaged articular cartilage. The present study was undertaken to examine the effects of BMP-9 on the metabolism of juvenile and adult bovine cartilage in vitro, and to compare the effects to those produced by two previously characterized BMPs: BMP-2 and 13 (CDMP-2). BMP-9 lead to a 7-8-fold stimulation of proteoglycan synthesis at the highest concentration tested, and a 6.4-fold stimulation of collagen synthesis at a concentration of 50 ng/mL in juvenile cartilage. BMP-2 also lead to a 7-8-fold increase in proteoglycan synthesis at the highest concentration tested, and was able to induce collagen synthesis 6.4-fold, but at a concentration of 1000 ng/mL. Proteoglycans isolated from BMP-9 treated cartilage exhibited an increased hydrodynamic size possibly due to increased glycosaminoglycan substitution or decreased C-terminal proteolysis. Consistent with the idea of limited C-terminal proteolysis, BMP-9 treatment lead to a significant reduction in the turnover rate of proteoglycans in juvenile explants. Interestingly, all three BMPs were unable to induce a measurable anabolic response in adult cartilage explants.
Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Cartílago/efectos de los fármacos , Factores de Edad , Animales , Proteína Morfogenética Ósea 2 , Receptores de Proteínas Morfogenéticas Óseas , Cartílago/metabolismo , Bovinos , Factor 2 de Diferenciación de Crecimiento , Proteoglicanos/biosíntesis , Receptores de Factores de Crecimiento/análisis , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Members of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family share common structural features including a disintegrin domain, a zinc metalloprotease domain, and at least one thrombospondin motif. Aberrant expression of several of these proteins has led to an understanding of their role in human disease; however, a link to function for many has not yet been made. One such uncharacterized family member, ADAMTS-8, shares significant protein sequence homology with a subgroup of ADAMTSs that includes ADAMTS-1, ADAMTS-4, ADAMTS-5, and ADAMTS-15. Each of these proteases has been shown to cleave 'aggrecanase-susceptible' site(s) within the extracellular matrix (ECM) proteoglycan aggrecan, and ADAMTS-4 and ADAMTS-5 have been postulated to play a role in the depletion of articular cartilage in osteoarthritic disease. Based on sequence relationships, in the present study we examined the ability of ADAMTS-8 to exhibit 'aggrecanase' activity. A neoepitope monoclonal antibody (MAb; AGG-C1; anti-NITEGE373) was developed and used to demonstrate the ability of ADAMTS-8 to cleave aggrecan at the aggrecanase-susceptible Glu373-Ala374 peptide bond. In addition, expression analyses demonstrated the presence of ADAMTS-8 mRNA transcripts in normal and osteoarthritic human cartilage.
Asunto(s)
Cartílago Articular/enzimología , Endopeptidasas/metabolismo , Metaloendopeptidasas/metabolismo , Proteínas ADAM , Proteína ADAMTS9 , Agrecanos , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Células CHO , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Lectinas Tipo C , Metaloendopeptidasas/genética , Metaloendopeptidasas/inmunología , Metaloendopeptidasas/aislamiento & purificación , Osteoartritis/metabolismo , Reacción en Cadena de la Polimerasa , Proteoglicanos/metabolismo , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
OBJECTIVE: Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA). METHODS: Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis. RESULTS: LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage. CONCLUSION: Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.
Asunto(s)
Cartílago Articular/patología , Glicoproteínas/uso terapéutico , Osteoartritis/patología , Osteoartritis/prevención & control , Proteínas Recombinantes/uso terapéutico , Animales , Cartílago Articular/efectos de los fármacos , Cartílago Articular/lesiones , Adhesión Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Glicoproteínas/administración & dosificación , Glicoproteínas/farmacología , Inyecciones Intraarticulares , Masculino , Distribución Aleatoria , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/patología , Resultado del TratamientoRESUMEN
OBJECTIVE: Protein kinase Czeta (PKCzeta), an atypical PKC, has been found to be transcriptionally up-regulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKCzeta in interleukin-1beta (IL-1beta)-induced NF-kappaB signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation. METHODS: Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKCzeta. Western blot analysis was used to evaluate phosphorylation of PKCzeta and NF-kappaB. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production. RESULTS: Phosphorylation of PKCzeta and NF-kappaB was induced by IL-1beta treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKCzeta suppressed IL-1beta-induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1beta-induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKCzeta and NO production. Furthermore, small interfering RNA- or short hairpin RNA-mediated knockdown of PKCzeta mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. CONCLUSION: Our results show that PKCzeta is involved in the regulation of IL-1beta-induced NF-kappaB signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKCzeta could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA.
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Proteínas ADAM/metabolismo , Condrocitos/metabolismo , Interleucina-1beta/fisiología , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Osteoartritis/metabolismo , Procolágeno N-Endopeptidasa/metabolismo , Proteína Quinasa C/metabolismo , Proteína ADAMTS4 , Células Cultivadas , Condrocitos/patología , Endopeptidasas/metabolismo , Humanos , Óxido Nítrico/metabolismo , Osteoartritis/patología , Fosforilación , ARN Mensajero/metabolismo , Transducción de Señal/fisiologíaRESUMEN
5'-Phenyl-3'H-spiro[indoline-3,2'-[1,3,4]thiadiazol]-2-one inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared via commercially available starting materials. Selected compounds 23, 33-35 show sub-micromolar ADAMTS-5 potency and strong SAR trends with selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase-1), MMP12, and MMP13. This series of compounds represents progress toward a selective ADAMTS-5 inhibitor as a disease modifying osteoarthritis drug.
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Endopeptidasas/metabolismo , Indoles/química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Tiadiazoles/química , Estructura Molecular , Inhibidores de Proteasas/síntesis química , Compuestos de Espiro/síntesis química , Relación Estructura-Actividad , Tiadiazoles/síntesis química , Tiadiazoles/farmacologíaRESUMEN
OBJECTIVE: To identify potential molecular mediators and biomarkers for osteoarthritis (OA), through comparative proteomic analysis of articular cartilage tissue obtained from normal donors without OA (n = 7) and patients with OA (n = 7). METHODS: The proteomic analyses comprised extraction of soluble proteins from cartilage, separation of the protein mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel digestion, and subsequent nano-liquid chromatography-tandem mass spectrometry analysis in conjunction with a database search for protein identification and semiquantitation. RESULTS: A total of 814 distinct proteins were identified with high confidence from 14 samples; 420 of these proteins were detected with > or = 3 unique peptides in at least 4 samples from the same group. Using stringent criteria, 59 proteins were found to be differentially expressed in OA cartilage. Gene Ontology and Ingenuity pathway analysis tools were used to characterize these proteins into functional categories. One of the up-regulated proteins, HtrA1, a serine protease, was detected at high levels in cartilage. CONCLUSION: Altered protein expression in the disease state is associated with many aspects of the pathogenesis of OA, such as increased proteolysis, lipid metabolism, immune response, and decreased signal transduction. To our knowledge, this is the first time that a large portion of these proteins and their expression patterns were identified in cartilage, thus providing new insights for finding novel pathologic mediators and biomarkers of OA.
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Biomarcadores/metabolismo , Cartílago Articular/metabolismo , Osteoartritis/metabolismo , Proteómica/métodos , Anciano , Anciano de 80 o más Años , Cartílago Articular/patología , Cromatografía Liquida , Biología Computacional , Electroforesis en Gel de Poliacrilamida , Humanos , Persona de Mediana Edad , Osteoartritis/patología , Solubilidad , Espectrometría de Masas en TándemRESUMEN
OBJECTIVE: To phenotypically characterize ADAMTS-4- and ADAMTS-5-double-knockout mice, and to determine the effect of deletion of ADAMTS-4 and ADAMTS-5 on the progression of osteoarthritis (OA) in mice. METHODS: Mice lacking the catalytic domain of ADAMTS-4 and ADAMTS-5 were crossed to generate ADAMTS-4/5-double-knockout animals. Twelve-week-old and 1-year-old male and female ADAMTS-4/5-double-knockout mice were compared with age- and sex-matched wild-type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS-4/5-double-knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS-4/5-double-knockout mice were treated with interleukin-1 (IL-1) plus retinoic acid ex vivo, to examine proteoglycan degradation. RESULTS: There were no genotype-related phenotype differences between ADAMTS-4/5-double-knockout and WT mice through 1 year of age, with the exception that female ADAMTS-4/5-double-knockout mice had a lower mean terminal body weight at the 12-week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS-4/5-double-knockout mice compared with WT mice. Following stimulation of cartilage explants with IL-1 plus retinoic acid, aggrecanase-mediated degradation in ADAMTS-4/5-double-knockout mice was ablated, to a level comparable with that in ADAMTS-5-knockout mice. CONCLUSION: Dual deletion of ADAMTS-4 and ADAMTS-5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS-4/5-double-knockout mice were comparable with those observed with deletion of ADAMTS-5 alone.
Asunto(s)
Proteínas ADAM/genética , Osteoartritis de la Cadera/fisiopatología , Osteoartritis de la Rodilla/fisiopatología , Procolágeno N-Endopeptidasa/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Proteína ADAMTS5 , Agrecanos/metabolismo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Genotipo , Articulación de la Cadera/enzimología , Articulación de la Cadera/patología , Inestabilidad de la Articulación/patología , Inestabilidad de la Articulación/fisiopatología , Articulación de la Rodilla/enzimología , Articulación de la Rodilla/patología , Masculino , Ratones , Ratones Noqueados , Osteoartritis de la Cadera/patología , Osteoartritis de la Rodilla/patología , Fenotipo , Procolágeno N-Endopeptidasa/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
5-Benzylidene-2-thioxo-thiazolidin-4-one inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared via commercially available starting materials. The identified compounds show micromolar ADAMTS-5 potency and demonstrate SAR trends for both the aryl group and thioxothiazolidinone zinc chelator. This series of compounds represents steps toward a metalloprotease inhibitor as a disease-modifying osteoarthritis drug.