Differential requirements for IKKα and IKKβ 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. IKK␣ and IKK␤ are essential kinases that activate NF-B 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 IKK␣ or IKK␤ was ablated in primary human chondrocytes by retrotransduction 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 IKK␣ or IKK␤. Silencing of IKK␤ markedly enhanced accumulation of glycosaminoglycan in conjunction with increased SOX9 expression. Ablation of IKK␣ dramatically enhanced type II collagen deposition independent of SOX9 protein levels but in association with suppressed levels of runt-related transcription factor 2. Moreover, IKK␣-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.Conclusion. IKK␣ and IKK␤ 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 IKK␣ were more profound and pleotrophic in nature, our observations suggest that exacerbated IKK␣ activity may be responsible, at least in part, for the characteristic abnormal phenotypes of OA chondrocytes.

CXC chemokine ligand 12a enhances chondrocyte proliferation and maturation during endochondral bone formation

“…In metatarsal organ culture and primary chondrocyte culture in vitro, the loss of function of p65, a major component of the canonical NF-kB pathway, attenuated longitudinal bone growth due to decreased chondrocyte proliferation and differentiation, whereas gain of function with p65 overexpression led to the opposite result 45 . In addition, the inhibition of IKKa resulted in the arrest of chondrocyte maturation at the pre-hypertrophic stage in micromass culture of articular chondrocytes 46 . All these findings indicate that NF-kB contributes to chondrocyte proliferation and hypertrophic differentiation, and our data suggest that the CXCL12a/CXCR4 axis can be another major upstream regulator of NF-kB signaling in chondrocyte.…”

In vitro study of inhibitory millimeter wave treatment effects on the TNF-α-induced NF-κB signal transduction pathway

“…IKKß is mainly involved in inflammatory and innate immune responses, mediating the rapid response of recruited immune cells to inflammation and injury through NF-κB signalling (25). In response to TNF-·, TAK1 recruits TNFR1 through RIP to construct the fusion protein, TAK1-DD, through the binding of TAK1 to the RIP death domain, which in turn activates the IKK complex.…”

Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis

“…Human OA chondrocytes are subject to differential control by the NFκB-activating inhibitor of κB kinases (IKKα and IKKβ [Olivotto et al 2008], as well as by MMP-13 itself [BorzÌ et al 2010]. Phenotypic modifications induced by IKKα and IKKβ knockdown are associated with changes in the expression and cellular localization of regulators including Runx2, β-catenin, and Sox9 [BorzÌ et al 2010].…”