Department of Genetics, Faculty of Biology, University of Barcelona, IBUB, CIBERER, Barcelona, Spain.
J Bone Miner Res. 2011 May;26(5):1133-44. doi: 10.1002/jbmr.293.
LRP5 is an osteoporosis susceptibility gene. Association analyses reveal that individual single-nucleotide polymorphisms (SNPs) determine variation in bone mineral density (BMD) among individuals as well as fracture risk. In a previous study, we identified a lumbar spine BMD-associated SNP, rs312009, located in the LRP5 5' region. A RUNX2 binding site was identified in this region by gel-shift experiments. Here we test the functionality of this SNP and examine whether RUNX2 is indeed a regulator of LRP5 expression. Gene reporter assays were used to test rs312009 functionality. Bioinformatic predictive tools and gel-shift and gene reporter assays were used to identify and characterize additional RUNX2 binding elements in the 3.3-kb region upstream of LRP5. Allelic differences in the transcriptional activity of rs312009 were observed in two osteoblastic cell lines, the T allele being a better transcriber than the C allele. RUNX2 cotransfection in HeLa cells revealed that the LRP5 5' region responded to RUNX2 in a dose-dependent manner and that the previously identified RUNX2 binding site participated in this response. Also, RUNX2 inhibition by RNAi led to nearly 60% reduction of endogenous LRP5 mRNA in U-2 OS cells. Four other RUNX2 binding sites were identified in the 5' region of LRP5. Luciferase experiments revealed the involvement of each of them in the RUNX2 response. The allelic differences observed point to the involvement of rs312009 as a functional SNP in the observed association. To our knowledge, this is the first time that the direct action of RUNX2 on LRP5 has been described. This adds evidence to previously described links between two important bone-regulating systems: the RUNX2 transcription-factor cascade and the Wnt signaling pathway.
LRP5 是骨质疏松症的易感基因。关联分析显示，个体单核苷酸多态性（SNP）决定了个体骨密度（BMD）的变化以及骨折风险。在之前的研究中，我们鉴定了一个位于 LRP5 5'区的腰椎 BMD 相关 SNP(rs312009)。凝胶迁移实验鉴定出该区域存在一个 RUNX2 结合位点。在此，我们测试了该 SNP 的功能，并且研究了 RUNX2 是否确实是 LRP5 表达的一个调控因子。使用基因报告基因检测来测试 rs312009 的功能。生物信息学预测工具、凝胶迁移和基因报告基因检测用于鉴定和描述 LRP5 上游 3.3kb 区域的其他 RUNX2 结合元件。在两个成骨细胞系中观察到 rs312009 的转录活性的等位基因差异，T 等位基因比 C 等位基因具有更好的转录活性。在 HeLa 细胞中转染 RUNX2 显示，LRP5 5'区以剂量依赖的方式对 RUNX2 作出反应，并且之前鉴定的 RUNX2 结合位点参与了这种反应。此外，用 RNAi 抑制 RUNX2 导致 U-2 OS 细胞中内源性 LRP5 mRNA 减少近 60%。在 LRP5 的 5'区还鉴定出另外四个 RUNX2 结合位点。荧光素酶实验揭示了它们每一个都参与了 RUNX2 的反应。所观察到的等位基因差异表明 rs312009 作为一个功能性 SNP 参与了观察到的关联。据我们所知，这是首次描述 RUNX2 对 LRP5 的直接作用。这为先前描述的两个重要的骨调节系统之间的联系提供了证据：RUNX2 转录因子级联和 Wnt 信号通路。
