IKKα/CHUK Regulates Extracellular Matrix Remodeling Independent of Its Kinase Activity to Facilitate Articular Chondrocyte Differentiation

Eleonora Olivotto,Flavio Flamigni,Rosa Maria Borzì,Andrea Facchini,Annalisa Astolfi,Miguel Otero,Daniela Platano,Annalisa Facchini,Kenneth B Marcu,Stefania Pagani,Mary B Goldring,Florence Ruggiero

doi:10.1371/journal.pone.0073024

Abstract

BackgroundThe 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.Methodology/Principal FindingsIKKα expression was ablated in primary human osteoarthritic (OA) chondrocytes and in immature murine articular chondrocytes (iMACs) derived from IKKαf/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/SignificanceIKKα 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.

Highlights

Cells differentiate in response to environmental signals from their neighbors and from extracellular matrix (ECM) effectors
To determine if the loss of Col10a1 and Runx2 expression in IKKa KO immature murine articular chondrocytes (iMACs) was indicative of a clear deficiency in terminal differentiation capacity, we evaluated the same cells for calcium deposition, a defining feature of chondrocyte hypertrophy
We observed that the effects of IKKa on ECM remodeling and subsequent aspects of chondrocyte differentiation towards a hypertrophic-like state are evolutionarily conserved between human OA and murine articular chondrocytes (ACs), and the functional effects of IKKa in chondrocytes are not linked to unique properties of cells originating from OA diseased cartilage (Figure 1 and Figure S1)

Summary

Introduction

Cells differentiate in response to environmental signals from their neighbors and from extracellular matrix (ECM) effectors. During endochondral ossification hypertrophic chondrocytes undergo dramatic, stressassociated ECM remodeling, which has been suggested as a ‘‘developmental model’’ to grasp the contributions of exacerbated environmental stresses in the onset and progression of osteoarthritis (OA) [4,5,6,7,8,9] This concept is supported by findings in early OA cartilage lesions revealing up-regulation of chondrocyte differentiation-related genes, and by in vivo studies showing that alterations in ECM structural integrity or in effectors of progression to hypertrophy can lead to OA pathology [10]. ELF3, an epithelial cell-specific ETS family transcription factor, is subject to NF-kB-dependent IL-1b signaling in chondrocytes wherein it suppresses COL2A1 gene expression [20] and directly activates MMP13 transcription [21] Another direct target of the canonical NF-kB signaling, HIF2a, plays a central role in OA cartilage, where it interconnects inflammatory ECM degradative processes with chondrocyte hypertrophy, controlling the expression of MMP13, NOS2 and VEGF among other factors [22,23]. 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

Methods

Results

Conclusion