Chong Ka-Wing, Chanalaris Anastasios, Burleigh Annika, Jin Huilin, Watt Fiona E, Saklatvala Jeremy, Vincent Tonia L
Kennedy Institute of Rheumatology and University of Oxford, London, UK.
Arthritis Rheum. 2013 Sep;65(9):2346-55. doi: 10.1002/art.38039.
The articular cartilage is known to be highly mechanosensitive, and a number of mechanosensing mechanisms have been proposed as mediators of the cellular responses to altered mechanical load. These pathways are likely to be important in tissue homeostasis as well as in the pathogenesis of osteoarthritis. One important injury-activated pathway involves the release of pericellular fibroblast growth factor 2 (FGF-2) from the articular cartilage. Using a novel model of murine cartilage injury and surgically destabilized joints in mice, we examined the extent to which FGF-2 contributes to the cellular gene response to injury.
Femoral epiphyses from 5-week-old wild-type mice were avulsed and cultured in serum-free medium. Explant lysates were Western blotted for phospho-JNK, phospho-p38, and phospho-ERK or were fixed for immunohistochemical analysis of the nuclear translocation of p65 (indicative of NF-κB activation). RNA was extracted from injured explants, rested explants that had been stimulated with recombinant FGF-2 or FGF-18, or whole joints from either wild-type mice or FGF-2(-/-) mice. Reverse transcription-polymerase chain reaction was performed to examine a number of inflammatory response genes that had previously been identified in a microarray analysis.
Murine cartilage avulsion injury resulted in rapid activation of the 3 MAP kinase pathways as well as NF-κB. Almost all genes identified in murine joints following surgical destabilization were also regulated in cartilage explants upon injury. Many of these genes, including those for activin A (Inhba), tumor necrosis factor-stimulated gene 6 (Tnfaip6), matrix metalloproteinase 19 (Mmp19), tissue inhibitor of metalloproteinases 1 (Timp1), and podoplanin (Pdpn), were significantly FGF-2 dependent following injury to cartilage in vitro and to joint tissues in vivo.
FGF-2-dependent gene expression occurs in vitro and in vivo in response to cartilage/joint injury in mice.
已知关节软骨具有高度的机械敏感性，并且已经提出了多种机械传感机制作为细胞对机械负荷改变的反应的介质。这些途径可能在组织稳态以及骨关节炎的发病机制中起重要作用。一条重要的损伤激活途径涉及关节软骨中细胞周围成纤维细胞生长因子2（FGF-2）的释放。使用一种新型的小鼠软骨损伤模型和小鼠手术性关节不稳定模型，我们研究了FGF-2在多大程度上促成细胞对损伤的基因反应。
从5周龄野生型小鼠的股骨骨骺处撕脱并在无血清培养基中培养。将外植体裂解物进行蛋白质免疫印迹检测磷酸化JNK、磷酸化p38和磷酸化ERK，或者固定用于p65核转位的免疫组织化学分析（指示NF-κB激活）。从受伤的外植体、用重组FGF-2或FGF-18刺激的静止外植体或野生型小鼠或FGF-2基因敲除小鼠的整个关节中提取RNA。进行逆转录-聚合酶链反应以检测先前在微阵列分析中鉴定的一些炎症反应基因。
小鼠软骨撕脱损伤导致3条丝裂原活化蛋白激酶途径以及NF-κB的快速激活。在手术性关节不稳定后在小鼠关节中鉴定出的几乎所有基因在软骨外植体损伤时也受到调控。这些基因中的许多，包括激活素A（Inhba）、肿瘤坏死因子刺激基因6（Tnfaip6）、基质金属蛋白酶19（Mmp19）、金属蛋白酶组织抑制剂1（Timp1）和血小板反应蛋白1（Pdpn）的基因，在体外软骨损伤和体内关节组织损伤后显著依赖FGF-2。
在小鼠中，FGF-2依赖性基因表达在体外和体内对软骨/关节损伤产生反应。
