TGF-β-Runx2-Mmp13通路参与骨关节炎发病的机制及干预该通路对骨关节炎的修复作用研究

Human osteoarthritis (OA) is a progressive disease of the joints characterized by degradation of articular cartilage, chondrocyte hypertrophy, subchondral sclerosis and osteophyte formation. TGF-β signaling plays a critical role in controlling chondrocyte differentiation. Inhibition of TGF-β signaling by overexpression of the dominant-negative type II TGF-β receptor (dn-IIR) or by deletion of the Smad3 gene causes severe OA-like phenotype in mice (Serra et al., 1997; Yang et al., 2001). Recent genetic studies also demonstrated that mutations of the Smad3 gene are associated with high incidence of OA in patients (Valdes et al., 2010; van de Laar et al., 2011; van de Laar et al., 2012). Since the genetic mouse models described above have TGF-β signaling deficiency from embryonic stage, the role of TGF-β signaling in OA development at postnatal and adult stages needs to be further determined. Another critical issue is that the key downstream target genes of endogenous TGF-β signaling in articular chondrocytes during OA development remains to be identified. Our preliminary studies demonstrated that chondrocyte-specific deletion of the type II TGF-β receptor (Tgfbr2) at postnatal stage leads to development of a severe OA-like phenotype. In Tgfbr2 cKO mice Runx2 (mRNA and protein) levels were significantly increased. Inhibition of TGF-β signaling upregulated Mmp13 expression in a Runx2-dependent manner. We generated Tgfbr2/Mmp13 double KO mice and demonstrated that deletion of the Mmp13 gene in Tgfbr2 cKO background significantly reversed the OA features observed in Tgfbr2 cKO mice. Based on these observations, we hypothesize that Runx2 plays a critical role in regulation of MMP13 (the key enzyme in matrix degradation) expression and consequent OA development in Tgfbr2 cKO mice. In this study we propose two specific aims to further determine the role of TGF-β signaling in OA development and repair. In Aim 1, we will determine if inhibition of Runx2 expression will reverse OA phenotype induced by Tgfbr2 cKO. We will generate Tgfbr2 cKO/Runx2+/- mice and determine if deletion of one allele of the Runx2 gene in articular chondrocytes will reverse or reduce OA severity caused by Tgfbr2 cKO. In Aim 2, we will determine if addition of TGF-β and MMP13 inhibitor to bone marrow mesenchymal stem cell-derived chondrocytes will enhance the ability of chondrocyte implantation to repair OA cartilage defects caused by Tgfbr2 cKO. Our hypothesis is that addition of TGF-β and MMP13 inhibitor to chondrocyte implantation will prevent articular chondrocyte hypertrophy and articular cartilage degradation and enhance the healing ability of chondrocytes to the OA articular cartilage defects. Our proposed studies will provide novel insights into the mechanism of OA development and potential novel approach for OA articular cartilage repair.

本课题组在前期工作中证实敲除小鼠软骨细胞的II型TGF-β受体基因会导致严重的骨关节炎（OA）表型，关节软骨细胞Runx2表达明显增加；TGF-β信号下调能通过Runx2依赖方式加强Mmp13基因表达；在Tgfbr2基因敲除的基础上进一步敲除Mmp13基因能够逆转小鼠骨关节炎表型。这些提示TGF-β/Runx2/Mmp13通路在骨关节炎发生发展中起重要作用。本研究中，我们将进一步明确OA发生和修复过程中TGF-β-Runx2-MMP13信号通路的作用。一方面，在Tgfbr2基因敲除小鼠的基础上使其Runx2基因杂合性缺失，观察Runx2基因杂合性缺失能否阻止Tgfbr2基因敲除小鼠的OA发生。另一方面，观察软骨细胞移植联合TGF-β或MMP-13抑制剂能否有效修复Tgfbr2基因敲除小鼠OA表型。本研究将为理解骨关节炎的发生机制和探索修复骨关节炎的新方法开辟新思路。

本课题组前期工作提示TGF-β信号下调可能通过 Runx2依赖方式加强 Mmp13 基因表达，进而引起严重骨关节炎表型。.本项目建立软骨细胞特异性Runx2基因敲除小鼠Runx2Agc1CreER，8周龄时给予他莫昔芬腹腔注射，12周龄时行DMM手术制造OA模型。组织学分析结果显示Cre阴性鼠DMM术后8周可观察到OA表型，包括纤维化、裂缝和软骨退化，12周时加重。而Runx2Agc1CreER KO小鼠DMM术后8周时关节软骨缺损较少，12周时Runx2缺失明显减轻DMM诱导的OA进展。OARSI评分系统评估结果显示DMM术后8周时Runx2Agc1CreER KO小鼠并未减弱软骨退变趋势，但随时间进展，术后12周时Runx2Agc1CreER KO小鼠软骨退变较对照组明显减轻。用OsteoMeasure系统通过组织形态计量学对骨关节炎退变进行定量分析，结果显示尽管DMM术后8周时Runx2Agc1CreER KO小鼠和Cre阴性鼠无明显差别，但可观察到Runx2Agc1CreER KO小鼠关节软骨面积增加的趋势。DMM术后12周时，Runx2敲除对DMM诱导的软骨退变具有明显保护作用。Cre阴性鼠DMM术后8周和12周软骨下硬化均可见，但可被Runx2基因敲除逆转。micro-CT数据分析显示DMM术后12周时Cre阴性鼠软骨下骨量增加，但也可被Runx2基因敲除逆转。上述结果说明软骨细胞特异性Runx2基因敲除可改善骨关节炎表型。.免疫组化结果显示，假手术组小鼠关节软骨MMP13表达很少且主要局限于深部临近软骨下骨，Cre阴性对照鼠DMM术后8周和12周时关节软骨MMP13表达增加，而Runx2Agc1CreER KO小鼠MMP13表达的增加在DMM术后8周和12周时均减少，说明敲除Runx2可逆转软骨细胞MMP13表达的增加。4日龄及2月龄Runx2Agc1CreER 小鼠关节软骨qRT-PCR结果显示Runx2Agc1CreER 小鼠关节软骨细胞Runx2、Mmp9、Mmp13、Adamts4和Adamts5均下降，说明软骨细胞特异性Runx2基因敲除可减少关节软骨基质蛋白酶的表达。. 本研究揭示了Runx2对骨关节炎发生和发展的作用，发现抑制软骨细胞Runx2可部分缓解成年小鼠DMM手术诱导的骨关节炎样改变，为骨关节炎的发病机制及治疗提供新的思路和线索。