RESUMO
OBJECTIVE: Dickkopf-3 (Dkk3) is a non-canonical member of the Dkk family of Wnt antagonists and its upregulation has been reported in microarray analysis of cartilage from mouse models of osteoarthritis (OA). In this study we assessed Dkk3 expression in human OA cartilage to ascertain its potential role in chondrocyte signaling and cartilage maintenance. METHODS: Dkk3 expression was analysed in human adult OA cartilage and synovial tissues and during chondrogenesis of ATDC5 and human mesenchymal stem cells. The role of Dkk3 in cartilage maintenance was analysed by incubation of bovine and human cartilage explants with interleukin-1ß (IL1ß) and oncostatin-M (OSM). Dkk3 gene expression was measured in cartilage following murine hip avulsion. Whether Dkk3 influenced Wnt, TGFß and activin cell signaling was assessed in primary human chondrocytes and SW1353 chondrosarcoma cells using qRT-PCR and luminescence assays. RESULTS: Increased gene and protein levels of Dkk3 were detected in human OA cartilage, synovial tissue and synovial fluid. DKK3 gene expression was decreased during chondrogenesis of both ATDC5 cells and humans MSCs. Dkk3 inhibited IL1ß and OSM-mediated proteoglycan loss from human and bovine cartilage explants and collagen loss from bovine cartilage explants. Cartilage DKK3 expression was decreased following hip avulsion injury. TGFß signaling was enhanced by Dkk3 whilst Wnt3a and activin signaling were inhibited. CONCLUSIONS: We provide evidence that Dkk3 is upregulated in OA and may have a protective effect on cartilage integrity by preventing proteoglycan loss and helping to restore OA-relevant signaling pathway activity. Targeting Dkk3 may be a novel approach in the treatment of OA.
Assuntos
Cartilagem Articular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Osteoartrite/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Células Cultivadas , Quimiocinas , Condrogênese/fisiologia , Relação Dose-Resposta a Droga , Regulação para Baixo/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima/fisiologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologiaRESUMO
We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.
