Research on Pathophysiology and Novel Treatment of Osteoarthritis through Molecular Biological Approach

骨关节炎(OA)是一种非常常见的骨骼疾病，它影响到身体的所有关节。据估计，有1000多万人患有这种疾病。然而，骨性关节炎发病的分子机制尚不清楚，甚至患病率或发病率等基本流行病学指标也尚未阐明。本研究试图通过以下三个独立的项目来阐明骨关节炎的分子背景和检测其治疗靶点：1)利用小鼠模型进行反向遗传学研究：通过造成膝关节不稳定来建立实验性骨关节炎小鼠模型。在这些模型中，关节软骨中X型胶原(Col X)和基质金属蛋白酶-13(MMP13)的表达在骨性关节炎软骨中被诱导(OsteoARICATIONARCE13：632,2005)。由于已知软骨细胞肥大受转录激活剂Runx2正向调节，我们发现Runx2半胱氨酸…抑制软骨破坏。更多的低氧(关节炎大黄54：2462,2006)。与此同时，我们最近发现了一种新的软骨细胞特异性蛋白Carminin，它与小鼠耳廓软骨的异位骨化相关(J Biol Chem 278：48259,2003年)。当在膝关节诱导骨关节炎时，CMR-L关节中的骨赘形成比WT中的要温和得多(Natural Med 12：665,2006)。Runx2和Carminin不足分别阻止了软骨破坏和骨赘形成，而不影响生理骨骼条件，这一事实表明，这些分子可以作为这些独特的OA疾病的临床治疗靶点。2)从人类基因组学角度出发：我们于2005年建立了一项名为ROAD(针对残疾的骨关节炎研究)的大规模全国性临床研究，并创建了一个全面和系统的数据库，包括城市、山区和沿海地区三个队列的临床和遗传信息。我们总共招募了3040名参与者，其中2164名年龄超过60岁(男性：818，74.7岁，女性：1,470，74.0岁。)调查膝关节和腰椎X线骨性关节炎的患病率及其与局部疼痛的关系。两个膝关节的X线骨性关节炎(KL和GT；或=2)患病率男性为47.0%，女性为70.2%，而两侧椎间隙的患病率分别为84.1%和70.7%。女性(OR=3.28)和山区(OR=1.62)膝关节骨关节炎患病率高于男性(OR=2.06)，腰椎骨关节炎女性高于男性(OR=2.06)。3)软骨再生医学的基础研究：我们建立了原创的软骨形成实时监测系统，并证实转录因子SOX9及其共激活因子SOX5和SOX6(SOX三联体)诱导软骨细胞分化的早期阶段和抑制其终末阶段(关节炎Rheum 50：3561,2004)。目前的微阵列分析确定S100A1和S100B是SOX三人组中最有可能的靶分子。荧光素酶报告、电泳迁移率改变和染色质免疫沉淀分析表明，SOX Trio对这两种S100蛋白都有转录诱导作用，并鉴定了5‘端侧翼区域的各自增强子元件。S100A1和S100B作为转录靶点(EMBO Rep，印刷中)介导SOX Trio对软骨细胞终末分化的抑制作用。我们进一步建立了软骨形成的实时荧光监测细胞系ATDC5-S2RD5。利用它，我们鉴定了一个新的软骨形成因子SNX19，它是一种与p38信号转导软骨形成相关的细胞质蛋白。使用该监测细胞系进一步筛选分子将阐明软骨分化的分子网络，从而产生一种新的软骨再生医学。较少

Osteoarthritis (OA), that affects all joints in the body, is a very common skeletal disorder. It is estimated that more than 10 million persons are suffering from the disease. However, the molecular mechanism of OA remains unclarified, and even the basic epidemiologic index like prevalence rate or incidence rate has not yet been elucidated. This research attempted to clarify the molecular backgrounds and detect the therapeutic target of OA by the following three independent projects.1)Reverse genetics through using mouse models : We created experimental OA models in mice by producing instability in the knee joints. In these models, type X collagen (Col X) expression and matrix metalloproteinase-13 (MMP-13) expression were induced in the joint cartilage during OA progression (Osteoarthritis Cartilage 13 : 632, 2005). Since chondrocyte hypertrophy is known to be positively regulated by a transcriptional activator Runx2, we found that cartilage destruction was suppressed by the Runx2 hapl … More oinsufficiency (Arthritis Rheum 54 : 2462, 2006). In the meantime, we recently identified a novel chondrocyte-specific protein, carminerin, which was up-regulated in association with ectopic ossification of the mouse auricular cartilage (J Biol Chem 278 : 48259, 2003). When OA was induced in the knee joint, osteophyte formation was much milder in the Cmr-l-joints than in the WT (Nature Med 12 : 665, 2006). The facts that Runx2 and carminerin insufficiency prevented cartilage destruction and osteophyte formation, respectively, without affecting physiological skeletal conditions suggest that these molecules can clinically be therapeutic targets of these distinct disorders of OA.2)Forward genetics from human genomic approach : We established a large-scale nationwide clinical study called ROAD (research on osteoarthritis against disability) in 2005, and created a comprehensive and systemic database including clinical and genetic information in three cohorts of urban, mountainous and seacoast areas. We recruited 3,040 participants in total, from which 2,164 subjects older than 60 years (men : 818, 74.7 yrs., women : 1,470, 74.0 yrs.) were enrolled for investigation of the prevalence of radiographic OA of knee and lumbar spine, as well as its association with the respective local pain. Prevalence of radiographic OA (KL> or = 2) in either knee joint was 47.0% in men and 70.2% in women, while that in either intervertebral space was 84.1% in men and 70.7% in women. Prevalence of radiographic knee OA was higher in female sex (OR=3.28) and mountainous residence (OR=1.62), whereas that of lumbar OA was higher in male sex (OR=2.06). With the progress of the ROAD study, the underlying environmental and genetic backgrounds will be elucidated.3)Basic study for cartilage regenerative medicine: We established an original real-time monitoring system for chondrogenesis, and identified that a transcription factor SOX9 and its co-activators SOX5 and SOX6 (the SOX trio) induce the early stage of chondrocyte differentiation and suppress its terminal stage (Arthritis Rheum 50 : 3561, 2004). The present microarray analysis identified S100A1 and S100B as the most probable target molecules of the SOX trio. Luciferase-reporter, electrophoretic mobility shift, and chromatin immunoprecipitation analyses revealed the transcriptional induction of both S100 proteins by the SOX trio, and identified the respective enhancer elements in the 5'-end flanking region. S100A1 and S100B mediate the inhibition of terminal differentiation of chondrocytes by the SOX trio as the transcriptional targets (EMBO Rep, in press). We further established a real-time fluorescence monitoring cell line for chondrogenesis ATDC5-S2RD5. Using it, we identified a novel chondrogenic factor SNX19 which was a cytoplasmic protein related to the p38 signaling for chondrogenesis. Further screening of molecules using this monitoring cell line will elucidate the molecular network underlying chondrogenic differentiation leading to a novel cartilage regenerative medicine. Less