Analysis of mechanism of the bone complication in diabetes mellitus by reactivation of the gene expression through oxidative stress

为了阐明糖尿病骨并发症的发生机制，本研究以氧化应激为研究对象，采用小鼠骨髓基质细胞系进行体外实验。甲基乙二醛(MG)诱导的氧化应激可上调破骨细胞分化因子(RANKL)基因的表达，同时上调SFRP-4基因的表达，而MG则相反地抑制RANKL拮抗剂OPG基因的表达。此外，我们还观察到氧化应激抑制了Wnt/β-catenin信号转导通路。在SFRP-4基因启动子区域的分析中，我们发现在TATA-box上游的5个碱基上存在高度甲基化的双胞嘧啶-鸟嘌呤序列(CPGS)，这是由于甲基胞嘧啶结合蛋白2(MeCP2)的募集下调了SFRP-4基因的转录。我们观察到氧化应激条件下TATA盒结合蛋白(TBP)与TATA盒的粘附性增强。这些发现阐明了氧化应激诱导的SFRP-4基因表达恢复的部分机制。接下来，我们设计了体内研究，在这些研究中，我们从组织形态上比较了从药物诱导的糖尿病小鼠和健康小鼠获得的长骨，并观察了糖尿病小鼠股骨的骨小梁减少。此外，我们最初建立了SFRP-4基因敲除小鼠，并观察到该动物在自然过程中由于衰老而对骨量减少具有抵抗力。这些研究表明，急性氧化应激通过RANKL信号通路促进骨吸收，而在轻度和持久的氧化应激如衰老中，sfpr-4在骨质减少和最终骨质疏松发生之前通过Wnt/β-catenin信号抑制骨代谢的周转。

To elucidate the mechanism of diabetic bone complication, we focused on the oxidative stress and performed in vitro studies by using mouse bone marrow stromal cell line. Oxidative stress induced by methylglyoxal (MG) treatment upregulated secreted Frizzled-related protein 4 (sFRP-4) gene expression as well that of osteoclast differentiating factor (RANKL) gene while MG treatment suppressed the expression of Osteoprotegerin (OPG, a RANKL antagonist) gene in reciprocal manner. Furthermore we observed oxidative stress suppressed Wnt/ β -catenin signal transduction pathway. In the analysis of sFRP-4 gene promoter region, we found highly methylated two-tandem cytosine-guanine sequences (CpGs) at 5 bases upstream of TATA-box following to downregulation of sFRP-4 gene transcription through methylcytosine binding protein 2 (MeCP2) recruitment. We observed enhanced adhesion of TATA-box binding protein (TBP) to TATA-box under the condition of oxidative stress. These findings elucidated a part of the mechanisms of restoration in sFRP-4 gene expression induced by oxidative stress. We next designed in vivo studies, in which we histomorphologically compared long bones obtained from drug-induced diabetic mice and healthy mice, and we observed reduction of trabecular bones in diabetic mouse femur. Moreover, we originally established sFRP-4 knock-out mice and observed that this animal was resistant to osteopenia due to senescence in natural course. These studies suggest that acute oxidative stress promotes bone resorption through RANKL signaling whereas, in mild and persistent oxidative stress as senescence, sFPR-4 represses turnover of bone metabolism through Wnt/ β-catenin signaling prior to the onset of osteopenia and ultimately osteoporosis