Laboratory of Immunorheumatology and Tissue Regeneration/RAMSES, Rizzoli Orthopedic Research Institute, Bologna, Italy.
PLoS One. 2013 Sep 2;8(9):e73024. doi: 10.1371/journal.pone.0073024. eCollection 2013.
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.
METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.
非规范 NF-κB 激活激酶 IKKα 由 CHUK（保守螺旋-环-螺旋-泛素-激酶）编码，据报道，它可以调节前炎症或抗炎反应、细胞存活和细胞分化。在这里，我们研究了 IKKα 作为人类和鼠类软骨细胞细胞外基质（ECM）稳态和向肥大分化的新型效应物的作用机制。
方法/主要发现：通过逆转录病毒介导的稳定 shRNA 转导和 Cre 重组酶依赖性 Lox P 位点重组，分别在原代人骨关节炎（OA）软骨细胞和源自 IKKα(f/f)：CreERT2 小鼠的未成熟关节软骨细胞（iMACs）中剔除 IKKα 的表达。通过 mRNA 谱分析、定量 RT-PCR 分析、免疫组织化学和免疫印迹鉴定 MMP-10 是软骨细胞中 IKKα 的主要靶标。MMP-13 和总胶原酶活性显著降低，而 TIMP-3（金属蛋白酶组织抑制剂-3）蛋白水平在 IKKα 缺陷型软骨细胞中升高。IKKα 缺失增强了 ECM 完整性，表现为 II 型胶原（COL2）沉积增加，MMP 依赖性 COL2 降解产物缺失。定量抑制.Alizarin 红染色和多个软骨细胞分化效应物（包括 Runx2、Col10a1 和 Vegfa）的表达减少，表明 IKKα 缺失抑制了软骨细胞分化。重要的是，激酶失活的 IKKα 蛋白突变体可挽救 IKKα 缺陷型软骨细胞的分化。
结论/意义：IKKα 通过多种下游靶标（包括 MMP-10 和 TIMP-3 在 mRNA 和转录后水平）发挥作用，促进软骨细胞向肥大样状态分化，独立于其激酶活性。IKKα 正向调节 ECM 重塑，以维持最大的 MMP-13 活性，这是 ECM 重塑导致软骨细胞分化所必需的。软骨细胞是关节软骨中唯一的细胞成分，在组织稳态过程中处于静止状态并维持 ECM 完整性。在 OA 中，软骨细胞重新获得增殖和分化的能力，其激活导致软骨明显退化。因此，我们的研究结果对于确定 OA 疾病的发病和/或进展也具有潜在意义。
