Zhang Dongdong, Park Bo Mi, Kang Myengmo, Nam HeeJin, Kim Eun Jin, Bae ChuHyun, Lim Sung Kil
Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Republic of Korea; Division of Endocrinology & Metabolism, Department of Internal Medicine, Affiliated Yantai Hospital of Binzhou Medical University, Yantai, PR China.
Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seoul, Republic of Korea.
Biochem Biophys Res Commun. 2016 Apr 8;472(3):471-6. doi: 10.1016/j.bbrc.2016.01.178. Epub 2016 Feb 1.
Sclerostin, encoded by the Sost gene, is mainly produced by osteocytes in bone and antagonizes the Wnt/β-catenin signaling pathway, which is a requisite for bone formation. Currently, human anti-sclerostin antibodies are being tested in phase III clinical trials. In addition, serum sclerostin levels are reported to be associated with bone mineral density and fracture risk in normal individuals; however, the correlation between serum sclerostin and bone mass remains controversial. To study the effects of the continuous exposure of exogenous sclerostin on bone, a ΦC31 integrase system, which has the characteristics of site-specificity and efficiency, was applied for the delivery of the Sost gene in this study. We injected Sost-attB plasmid with or without ΦC31 integrase plasmid into the mouse tail vein using a hydrodynamic-based method. The site-specific integration of the Sost gene into the mouse genome was confirmed by examining a pseudo-attP site on the hepatic genomic DNA. Sclerostin was expressed in the hepatocytes, secreted into the blood flow, and maintained at high concentrations in the mice with both Sost-attB plasmid and ΦC31 integrase plasmid injections, which was observed by serial measurement. Moreover, the mice with long-term high levels of serum sclerostin showed trabecular bone loss on micro-CT analysis. Peripheral B cell populations were not affected. Our results suggested that sclerostin could be expressed in the liver and sustained successfully at high levels in the blood by using the ΦC31 integrase system, leading to trabecular bone loss. These findings may help to further ascertain the effects of sclerostin introduced exogenously on the skeleton.
由Sost基因编码的硬化蛋白主要由骨中的骨细胞产生，并拮抗Wnt/β-连环蛋白信号通路，而该信号通路是骨形成所必需的。目前，人抗硬化蛋白抗体正在进行III期临床试验。此外，据报道，正常个体的血清硬化蛋白水平与骨矿物质密度和骨折风险相关；然而，血清硬化蛋白与骨量之间的相关性仍存在争议。为了研究外源性硬化蛋白持续暴露对骨的影响，本研究应用了具有位点特异性和高效性的ΦC31整合酶系统来递送Sost基因。我们采用基于流体动力学的方法将携带或不携带ΦC31整合酶质粒的Sost-attB质粒注入小鼠尾静脉。通过检测肝脏基因组DNA上的假attP位点，证实了Sost基因在小鼠基因组中的位点特异性整合。通过连续测量观察到，硬化蛋白在肝细胞中表达，分泌到血流中，并在同时注射Sost-attB质粒和ΦC31整合酶质粒的小鼠中维持在高浓度。此外，微CT分析显示，长期血清硬化蛋白水平高的小鼠出现小梁骨丢失。外周B细胞群体未受影响。我们的结果表明，通过使用ΦC31整合酶系统，硬化蛋白可以在肝脏中表达并在血液中成功维持在高水平，导致小梁骨丢失。这些发现可能有助于进一步确定外源性引入的硬化蛋白对骨骼的影响。
