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，它会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明确。本研究通过以下三个独立项目，试图厘清OA的分子背景，检测OA的治疗靶点。1)通过小鼠模型的反向遗传学：我们通过在小鼠膝关节产生不稳定性来建立实验性OA模型。在这些模型中，骨性关节炎进展过程中诱导关节软骨中X型胶原（Col X）表达和基质金属蛋白酶-13 （MMP-13）表达（Osteoarthritis cartilage 13: 632, 2005）。由于已知软骨细胞肥大受到转录激活因子Runx2的正向调节，我们发现软骨破坏受到Runx2基因的抑制。（Arthritis Rheum 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，carminerin，它与小鼠耳软骨异位骨化有关（J Biol Chem 278: 48259, 2003）。当膝关节发生骨关节炎时，cmr -l关节的骨赘形成要比WT轻得多（Nature Med 12: 665, 2006）。Runx2和carminin不足分别阻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，这些分子在临床上可以成为这些不同oa疾病的治疗靶点。2005年，我们在全国范围内建立了大规模的临床研究ROAD (research on osteoarthritis against disability)，建立了包括城市、山区和沿海地区三个队列的临床和遗传信息的全面、系统的数据库。我们共招募了3040名参与者，其中2164名年龄在60岁以上（男性：818,74.7岁）。（女性：1470人，74.0岁）纳入研究膝关节和腰椎骨性关节炎的发病率及其与各自局部疼痛的关系。双膝关节骨性关节炎（KL> or = 2）的发生率男性为47.0%，女性为70.2%；双椎间隙骨性关节炎的发生率男性为84.1%，女性为70.7%。女性（OR=3.28）和山区居民（OR=1.62）患膝关节骨性关节炎的比例较高，而男性患腰椎骨性关节炎的比例较高（OR=2.06）。随着ROAD研究的进展，潜在的环境和遗传背景将得到阐明。3)软骨再生医学基础研究：我们建立了软骨形成的原始实时监测系统，发现转录因子SOX9及其共激活因子SOX5和SOX6 （SOX三重奏）诱导软骨细胞早期分化并抑制其终末期分化（Arthritis Rheum 50: 3561, 2004）。目前的微阵列分析确定S100A1和S100B是SOX三人组中最可能的目标分子。荧光素酶报告蛋白、电泳迁移率转移和染色质免疫沉淀分析揭示了SOX三组对这两种S100蛋白的转录诱导作用，并在5'端侧翼区鉴定了各自的增强子元件。S100A1和S100B介导了SOX三人组作为转录靶点对软骨细胞终末分化的抑制（EMBO Rep, in press）。我们进一步建立了ATDC5-S2RD5软骨形成实时荧光监测细胞系。利用它，我们发现了一种新的软骨形成因子SNX19，这是一种与软骨形成的p38信号相关的细胞质蛋白。利用这种监测细胞系进一步筛选分子将阐明软骨分化的分子网络，从而形成一种新的软骨再生医学。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