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，影响身体的所有关节。据估计，有1 000多万人患有这种疾病。然而，OA的分子机制仍不清楚，甚至流行病学的基本指标，如患病率或发病率尚未阐明。本研究试图通过以下三个独立的项目来阐明OA的分子背景和检测OA的治疗靶点。1）利用小鼠模型进行反向遗传学研究：通过在小鼠膝关节中产生不稳定性来建立实验性OA模型。在这些模型中，在OA进展期间在关节软骨中诱导X型胶原（Col X）表达和基质金属蛋白酶-13（MMP-13）表达（骨关节炎Carcinoma 13：632，2005）。由于软骨细胞肥大被认为是由转录激活因子Runx 2正调控的，我们发现软骨破坏被Runx 2 hapl抑制。 关于我们 不充分性（Arthritis Rheum 54：2462，2006）。同时，我们最近鉴定了一种新的软骨细胞特异性蛋白，carminerin，其与小鼠耳软骨的异位骨化相关上调（J Biol Chem 278：48259，2003）。当在膝关节中诱导OA时，Cmr-l-关节中的骨赘形成比WT中温和得多（Nature Med 12：665，2006）。Runx 2和carminerin不足分别防止软骨破坏和骨赘形成而不影响生理骨骼状况的事实表明，这些分子可以在临床上成为这些不同OA疾病的治疗靶标。2）来自人类基因组方法的正向遗传学：我们建立了一个名为ROAD的大规模全国性临床研究2005年，该研究所开展了一项名为“骨关节炎防治残疾研究”的研究，并建立了一个全面和系统的数据库，其中包括城市、山区和沿海地区三个群体的临床和遗传信息。我们总共招募了3，040名参与者，其中2，164名60岁以上的受试者（男性：818，74.7岁，妇女：1 470 74.0岁）入组研究膝关节和腰椎放射学OA的患病率及其与相应局部疼痛的相关性。膝关节X线骨关节炎（KL ≥ 2）的患病率男性为47.0%，女性为70.2%，而椎间间隙的患病率男性为84.1%，女性为70.7%。女性（OR=3.28）和山区居民（OR=1.62）膝关节放射学OA患病率较高，男性（OR=2.06）腰椎放射学OA患病率较高。随着ROAD研究的深入，其潜在的环境和遗传背景将得到进一步的阐明。3）软骨再生医学基础研究：我们建立了一个独创的软骨再生实时监测系统，并鉴定了一个转录因子SOX 9及其共激活因子SOX 5和SOX 6（SOX trio）诱导软骨细胞分化的早期阶段并抑制其终末阶段（Arthritis Rheum 50：3561，2004）。目前的微阵列分析确定S100 A1和S100 B为SOX三人组最可能的靶分子。荧光素酶报告，电泳迁移率变化，染色质免疫沉淀分析揭示了两个S100蛋白的转录诱导SOX三重奏，并确定了各自的增强子元件在5 '端侧翼区。S100 A1和S100 B通过作为转录靶标的SOX三重体介导软骨细胞终末分化的抑制（EMBO Rep，出版中）。我们进一步建立了一个实时荧光监测软骨形成的细胞系ATDC 5-S2 RD 5。利用它，我们鉴定了一种新的软骨形成因子SNX 19，它是一种与软骨形成的p38信号相关的细胞质蛋白。使用该监测细胞系的分子的进一步筛选将阐明导致新的软骨再生医学的成软骨分化的分子网络。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