title: IKKα/CHUK regulates extracellular matrix remodeling independent of its kinase activity to facilitate articular chondrocyte differentiation.
abstract: BACKGROUND
  The non-canonical NF-κB activating kinase IKKα, encoded by CHUK (conserved-helix-loop-helix-ubiquitous-kinase), has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKα as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy.
  
  
  RESULTS
  IKKα expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKKα(f/f):CreERT2 mice by retroviral-mediated stable shRNA transduction and Cre recombinase-dependent Lox P site recombination, respectively. MMP-10 was identified as a major target of IKKα in chondrocytes by mRNA profiling, quantitative RT-PCR analysis, immunohistochemistry and immunoblotting. ECM integrity, as assessed by type II collagen (COL2) deposition and the lack of MMP-dependent COL2 degradation products, was enhanced by IKKα ablation in mice. MMP-13 and total collagenase activities were significantly reduced, while TIMP-3 (tissue inhibitor of metalloproteinase-3) protein levels were enhanced in IKKα-deficient chondrocytes. IKKα deficiency suppressed chondrocyte differentiation, as shown by the quantitative inhibition of.Alizarin red staining and the reduced expression of multiple chondrocyte differentiation effectors, including Runx2, Col10a1 and Vegfa,. Importantly, the differentiation of IKKα-deficient chondrocytes was rescued by a kinase-dead IKKα protein mutant.
  
  
  CONCLUSIONS
  IKKα acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKα positively modulates ECM remodeling via multiple downstream targets (including MMP-10 and TIMP-3 at the mRNA and post-transcriptional levels, respectively) to maintain maximal MMP-13 activity, which is required for ECM remodeling leading to chondrocyte differentiation. Chondrocytes are the unique cell component in articular cartilage, which are quiescent and maintain ECM integrity during tissue homeostasis. In OA, chondrocytes reacquire the capacity to proliferate and differentiate and their activation results in pronounced cartilage degeneration. Τηυσ, our findings are also of potential relevance for defining the onset and/or progression of OA disease.
authors.affiliation: Laboratory of Immunorheumatology and Tissue Regeneration/RAMSES, Rizzoli Orthopedic Research Institute, Bologna, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Olivotto
authors.forename: Miguel
authors.initials: M
authors.lastname: Otero
authors.forename: Annalisa
authors.initials: A
authors.lastname: Astolfi
authors.forename: Daniela
authors.initials: D
authors.lastname: Platano
authors.forename: Annalisa
authors.initials: A
authors.lastname: Facchini
authors.forename: Stefania
authors.initials: S
authors.lastname: Pagani
authors.forename: Flavio
authors.initials: F
authors.lastname: Flamigni
authors.forename: Andrea
authors.initials: A
authors.lastname: Facchini
authors.forename: Mary B
authors.initials: MB
authors.lastname: Goldring
authors.forename: Rosa Maria
authors.initials: RM
authors.lastname: Borzì
authors.forename: Kenneth B
authors.initials: KB
authors.lastname: Marcu
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D007150:Immunohistochemistry; D053506:Matrix Metalloproteinase 10 / Q000378:metabolism; D053509:Matrix Metalloproteinase 13 / Q000378:metabolism; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D051550:I-kappa B Kinase; D053509:Matrix Metalloproteinase 13; D053506:Matrix Metalloproteinase 10
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