Mitogen-activated protein kinase-activated protein kinase 2 (MK2) modulates key biological pathways associated with OA disease pathology
Mitogen-activated protein kinase-activated protein kinase 2 (MK2) modulates key biological pathways associated with OA disease pathology
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DOI：
10.1016/j.joca.2008.05.001
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发表时间：
2009-01-01
影响因子：
7
通讯作者：
Newham, P.
中科院分区：
文献类型：
--
作者：
Jones, S. W.;Brockbank, S. M. V.;Newham, P.
目的：研究丝裂原活化蛋白激酶活化蛋白激酶2（MK2）在介导人原发性骨关节炎（OA）软骨细胞对促炎细胞因子的细胞反应中的作用。使用Bioplex测定法测定细胞热休克蛋白（HSP 27）活性。原代CA软骨细胞通过胶原酶消化人关节软骨分离。通过免疫印迹和免疫组织学检测磷酸化MK2。使用阳离子脂质实现了用siRNA转染原代软骨细胞，并通过实时聚合酶链反应测定基因表达。前列腺素E2（PGE 2）和基质金属蛋白酶（MMPs）的产生通过酶联免疫吸附测定法测定。结果：显性阴性MK2的过表达抑制了HSP 27的磷酸化，并显著降低了白细胞介素1（IL-1）β和肿瘤坏死因子（TNF）-α介导的PGE 2在HeLa细胞中的释放。在OA关节软骨和分离的原代OA软骨细胞中检测到磷酸化MK2，其中磷酸化MK2由IL-10诱导。用MK2 siRNA反义转染OA软骨细胞显著降低了基础和IL-10诱导的PGE 2释放。siRNA介导的MK2基因敲减也能显著降低基础和IL-1诱导的MMP 13表达以及MMP 13和MMP 3蛋白的释放，但对MMP 1无影响。结论：MK2在CA人关节软骨和分离的原代人软骨细胞中具有活性，并介导PGE 2、MMP 3和MMP 13的释放。这些发现表明，MK 2通过介导p38激活对PGE 2释放和分解代谢蛋白酶的表达和释放的下游效应，在促成CA痛觉和OA关节结构恶化中发挥作用。(c)2008年国际骨关节炎研究学会。由爱思唯尔有限公司出版。保留所有权利。
Objective: To examine the role of mitogen-activated protein kinase-activated protein kinase 2 (MK2) in mediating the cellular response to pro-inflammatory cytokines in human primary osteoarthritis (OA) chondrocytes.Methods: Delivery of a dominant negative MK2 was achieved in HeLa cells by adenoviral infection. Cellular heat shock protein (HSP27) activity was determined using a Bioplex assay. Primary CA chondrocytes were isolated by collagenase digestion of human articular cartilage. Phosphorylated MK2 was detected by immunoblotting and immunohistology. Transfection of primary chondrocytes with siRNA was achieved using cationic lipid and gene expression determined by real-time polymerase chain reaction. Production of prostaglandin E2 (PGE2) and matrixmetalloproteases (MMPs) was measured by enzyme-linked immunosorbent assay.Results: Over-expression of a dominant negative MK2 inhibited HSP27 phosphorylation and significantly reduced both interleukin 1 (IL-1)beta and tumour necrosis factor (TNF)-alpha mediated release of PGE2 in HeLa cells over a 24 h period. Phosphorylated MK2 was detected in OA articular cartilage and in isolated primary OA chondrocytes, where it was induced by IL-1 P. Transfection of OA chondrocytes with MK2 siRNA antisense significantly reduced both basal and IL-1 0 induced PGE2 release. siRNA mediated MK2 knockdown also significantly reduced both basal and IL-1 induced MMP13 expression and MMP13 and MMP3 protein release but had no effect on MMP1.Conclusions: Our data reveal that MK2 is active in CA human articular cartilage and in isolated primary human chondrocytes and that MK2 mediates the release of PGE2, MMP3 and MMP13. These findings suggest a role for MK2 in contributing to CA algesia and OA joint structural deterioration by mediating the downstream effects of p38 activation on PGE2 release and the expression and release of catabolic proteases. (c) 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.