title: 5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes.
abstract: Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing the maturation program seen in growth plate chondrocytes. Only during pathologic conditions, such as in osteoarthritis, are maturational constraints lost causing recapitulation of the process that occurs during endochondral ossification. With the aim of establishing a model to identify regulatory mechanisms that suppress AC hypertrophy, we examined the capability of 5-azacytidine (Aza) to have an impact on the maturational program of these cells. Primary ACs do not spontaneously express markers of maturation and are refractory to treatment by factors that normally regulate chondrocyte maturation. However, following exposure to Aza, ACs (i) were induced to express type X collagen (colX), Indian hedgehog, and alkaline phosphatase and (ii) showed altered colX and AP expression in response to bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta (TGF-beta), and parathyroid hormone-related protein (PTHrP). Since Aza unmasked responsiveness of ACs to BMP-2 and TGF-beta, we examined the effect of Aza treatment on signaling via these pathways by assessing the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the Smad2 and 3-degrading ubiquitin E3 ligase Smurf2. Aza-treated ACs displayed less Smad2 and 3 and increased Smad1, 5, and Smurf2 protein and showed a loss of TGF-beta signaling on the P3TP-luciferase reporter. Suggesting that Aza-induction of Smurf2 may be responsible for the loss of Smad2 and 3 protein via this pathway, immunoprecipitation and metabolic labeling experiments confirmed that Aza accelerated the ubiquitination and degradation of these targets. Overall, Aza-treated ACs represent a novel model for the study of mechanisms that regulate maturational potential of articular cartilage, with the data suggesting that maturation of these cells may be due to up-regulation of Smad1 and 5 coupled with a Smurf2-dependent degradation of Smad2 and 3 and loss of TGF-beta signaling.
authors.affiliation: Center for Musculoskeletal Research, Department of Orthopaedics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA. michael_zuscik@urmc.rochester.edu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Zuscik
authors.forename: Jonathan F
authors.initials: JF
authors.lastname: Baden
authors.forename: Qiuqian
authors.initials: Q
authors.lastname: Wu
authors.forename: Tzong J
authors.initials: TJ
authors.lastname: Sheu
authors.forename: Edward M
authors.initials: EM
authors.lastname: Schwarz
authors.forename: Hicham
authors.initials: H
authors.lastname: Drissi
authors.forename: Regis J
authors.initials: RJ
authors.lastname: O'Keefe
authors.forename: J Edward
authors.initials: JE
authors.lastname: Puzas
authors.forename: Randy N
authors.initials: RN
authors.lastname: Rosier
mesh_terms: D000818:Animals; D001374:Azacitidine / Q000494:pharmacology*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D002645:Chickens; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D053823:Hedgehog Proteins; D015398:Signal Transduction / Q000187:drug effects*; D051785:Smad Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D004268:DNA-Binding Proteins; D053823:Hedgehog Proteins; D051785:Smad Proteins; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D025801:Ubiquitin; D001374:Azacitidine