Differential requirements for IKKα and IKKβ in the terminal differentiation of primary human osteoarthritic chondrocytes

Objective—Osteoarthritic chondrocytes behave in an intrinsically de-regulated manner characterized by the chronic loss of healthy cartilage and inappropriate differentiation to a hypertrophic-like state. IKKβ and IKKα are essential kinases that activate NF-κB transcription factors, which regulate cellular differentiation and inflammation. This study reveals differential roles for each IKK in chondrocyte differentiation and hypertrophy. Methods—Expression of IKKα or IKKβ were ablated in primary human chondrocytes by retrotransduction of specific shRNAs. Micromass cultures that faithfully undergo chondrogenesis to the terminal hypertrophic stage were established and ECM anabolism and remodelling were investigated by biochemical, immunohistochemical and ultrastructural techniques. Cellular parameters of hypertrophy (i.e. proliferation, viability and size) were also analyzed. Results—Extracellular matrix remodelling and mineralization, processes characteristic of terminally differentiated hypertrophic cells, were defective upon IKKα or IKKβ loss. Silencing IKKβ markedly enhanced glycosaminoglycan accumulation, in conjunction with increased Sox9 expression. IKKα ablation dramatically enhanced Col2 deposition independent of Sox9 protein levels but instead in association with RUNX-2 suppression. Moreover IKKα deficient cells retained the phenotypes of pre-hypertrophic-like cells as evidenced by their smaller size and faster proliferation prior to micromass seeding along with the enhanced viability of their differentiated micromasses. Conclusions—IKKα and IKKβ exert differential roles in ECM remodeling and endochondral ossification, events characteristic of hypertrophic chondrocytes and also factors often complicating osteoarthritis. Because IKKα's effects were more profound and pleotrophic in nature our observations suggest that exacerbated IKKα activity may be responsible for at least part of the characteristic abnormal phenotypes of osteoarthritic chondrocytes.