The Marlene & Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD, USA.
J Bone Miner Res. 2012 Apr;27(4):913-25. doi: 10.1002/jbmr.1504.
The fat-soluble prohormone cholecalciferol (Vitamin D3) is a precursor of the circulating 25-OH Vitamin D3, which is converted by 1α-hydroxylase to the biologically active 1,25-OH Vitamin D3. Active Vitamin D3 interacts with the Vitamin D receptor (VDR), a transcription factor that plays an important role in calcium mobilization and bone formation. RUNX2 is a DNA-binding transcription factor that regulates target genes important in bone formation, angiogenesis, and cancer metastasis. Using computer-assisted drug design (CADD) and a microtiter plate-based DNA-binding enzyme-linked immunosorbent assay (D-ELISA) to measure nuclear RUNX2 DNA binding, we have found that Vitamin D3 prohormones can modulate RUNX2 DNA binding, which was dose-dependent and sensitive to trypsin, salt, and phosphatase treatment. Unlabeled oligonucleotide or truncated, dominant negative RUNX2 proteins were competitive inhibitors of RUNX2 DNA binding. The RUNX2 heterodimeric partner, Cbfβ, was detected in the binding complexes with specific antibodies. Evaluation of several RUNX2:DNA targeted small molecules predicted by CADD screening revealed a previously unknown biological activity of the inactive Vitamin D3 precursor, cholecalciferol. Cholecalciferol modulated RUNX2:DNA binding at nanomolar concentrations even in cells with low VDR. Cholecalciferol and 25-OH Vitamin D3 prohormones were selective inhibitors of RUNX2-positive endothelial, bone, and breast cancer cell proliferation, but not of cells lacking RUNX2 expression. These compounds may have application in modulating RUNX2 activity in an angiogenic setting, in metastatic cells, and to promote bone formation in disease-mediated osteoporosis. The combination CADD discovery and D-ELISA screening approaches allows the testing of other novel derivatives of Vitamin D and/or transcriptional inhibitors with the potential to regulate DNA binding and biological function.
脂溶性前体胆钙化醇（维生素 D3）是循环 25-羟维生素 D3 的前体，后者可被 1α-羟化酶转化为具有生物活性的 1,25-羟维生素 D3。活性维生素 D3 与维生素 D 受体（VDR）相互作用，VDR 是一种转录因子，在钙动员和骨形成中发挥重要作用。RUNX2 是一种 DNA 结合转录因子，可调节骨形成、血管生成和癌症转移的重要靶基因。我们使用计算机辅助药物设计（CADD）和基于微量滴定板的 DNA 结合酶联免疫吸附测定（D-ELISA）来测量核 RUNX2 DNA 结合，发现维生素 D3 前体可以调节 RUNX2 DNA 结合，这种结合具有剂量依赖性，并且对胰蛋白酶、盐和磷酸酶处理敏感。未标记的寡核苷酸或截断的、显性负性 RUNX2 蛋白是 RUNX2 DNA 结合的竞争性抑制剂。特异性抗体检测到 RUNX2 异二聚体伴侣 Cbfβ 存在于结合复合物中。对 CADD 筛选预测的几种针对 RUNX2:DNA 的小分子进行评估，揭示了先前未知的无活性维生素 D3 前体胆钙化醇的生物学活性。胆钙化醇以纳摩尔浓度调节 RUNX2:DNA 结合，即使在 VDR 水平低的细胞中也是如此。胆钙化醇和 25-羟维生素 D3 前体是 RUNX2 阳性内皮细胞、骨细胞和乳腺癌细胞增殖的选择性抑制剂，但对缺乏 RUNX2 表达的细胞没有作用。这些化合物可能在调节血管生成环境、转移细胞中的 RUNX2 活性以及促进疾病介导的骨质疏松症中的骨形成方面具有应用。CADD 发现与 D-ELISA 筛选方法相结合，可用于测试其他新型维生素 D 和/或转录抑制剂衍生物，以潜在调节 DNA 结合和生物学功能。
