title: Differential requirements for IKKalpha and IKKbeta in the differentiation of primary human osteoarthritic chondrocytes.
abstract: OBJECTIVE
  Osteoarthritic (OA) chondrocytes behave in an intrinsically deregulated manner, characterized by chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKalpha and IKKbeta are essential kinases that activate NF-kappaB transcription factors, which in turn regulate cell differentiation and inflammation. This study was undertaken to investigate the differential roles of each IKK in chondrocyte differentiation and hypertrophy.
  
  
  METHODS
  Expression of IKKalpha or IKKbeta was ablated in primary human chondrocytes by retro-transduction of specific short-hairpin RNAs. Micromass cultures designed to reproduce chondrogenesis with progression to the terminal hypertrophic stage were established, and anabolism and remodeling of the extracellular matrix (ECM) were investigated in the micromasses using biochemical, immunohistochemical, and ultrastructural techniques. Cellular parameters of hypertrophy (i.e., proliferation, viability, and size) were also analyzed.
  
  
  RESULTS
  The processes of ECM remodeling and mineralization, both characteristic of terminally differentiated hypertrophic cells, were defective following the loss of IKKalpha or IKKbeta. Silencing of IKKbeta markedly enhanced accumulation of glycosaminoglycan in conjunction with increased SOX9 expression. Ablation of IKKalpha dramatically enhanced type II collagen deposition independent of SOX9 protein levels but in association with suppressed levels of runt-related transcription factor 2. Moreover, IKKalpha-deficient cells retained the phenotype of cells in a pre-hypertrophic-like state, as evidenced by the smaller size and faster proliferation of these cells prior to micromass seeding, along with the enhanced viability of their differentiated micromasses.
  
  
  CONCLUSIONS
  IKKalpha and IKKbeta exert differential roles in ECM remodeling and endochondral ossification, which are events characteristic of hypertrophic chondrocytes and also complicating factors often found in OA. Because the effects of IKKalpha were more profound and pleotrophic in nature, our observations suggest that exacerbated IKKalpha activity may be responsible, at least in part, for the characteristic abnormal phenotypes of OA chondrocytes.
authors.affiliation: Istituti Ortopedici Rizzoli, Bologna, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Olivotto
authors.forename: Rosa Maria
authors.initials: RM
authors.lastname: Borzi
authors.forename: Roberta
authors.initials: R
authors.lastname: Vitellozzi
authors.forename: Stefania
authors.initials: S
authors.lastname: Pagani
authors.forename: Annalisa
authors.initials: A
authors.lastname: Facchini
authors.forename: Michela
authors.initials: M
authors.lastname: Battistelli
authors.forename: Marianna
authors.initials: M
authors.lastname: Penzo
authors.forename: Xiang
authors.initials: X
authors.lastname: Li
authors.forename: Flavio
authors.initials: F
authors.lastname: Flamigni
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Falcieri
authors.forename: Andrea
authors.initials: A
authors.lastname: Facchini
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Marcu
mesh_terms: D002454:Cell Differentiation / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000201:enzymology*; D024043:Collagen Type II / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D005109:Extracellular Matrix / Q000378:metabolism; D020868:Gene Silencing; D006609:High Mobility Group Proteins / Q000235:genetics; D006801:Humans; D006984:Hypertrophy; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology*; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics
chemical_list: D024043:Collagen Type II; D050659:Core Binding Factor Alpha 1 Subunit; D006609:High Mobility Group Proteins; D012333:RNA, Messenger; C493709:RUNX2 protein, human; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D014157:Transcription Factors; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human