IKKa/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation (original) (raw)

2013

Abstract

Background: The non-canonical NF-kB activating kinase IKKa, encoded by CHUK (conserved-helix-loop-helix-ubiquitouskinase), has been reported to modulate pro- or anti- inflammatory responses, cellular survival and cellular differentiation. Here, we have investigated the mechanism of action of IKKa as a novel effector of human and murine chondrocyte extracellular matrix (ECM) homeostasis and differentiation towards hypertrophy. Methodology/Principal Findings: IKKa expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKKaf/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 IKKa 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 IKKa 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 IKKa-deficient chondrocytes. IKKa 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 IKKa-deficient chondrocytes was rescued by a kinase-dead IKKa protein mutant. Conclusions/Significance: IKKa acts independent of its kinase activity to help drive chondrocyte differentiation towards a hypertrophic-like state. IKKa 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. Tgus, our findings are also of potential relevance for defining the onset and/or progression of OA disease.

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