Previous studies confirmed that melatonin regulates Runx2 expression but the mechanism is unclear. There is a direct interaction between Runx2 and the vitamin D receptor (VDR). Herein, we observed a direct interaction between melatonin and the VDR but not Runx2 using isothermal titration calorimetry. Furthermore, this direct binding was detected only in the C-terminal ligand binding domain (LBD) of the VDR but not in the N-terminal DNA-binding domain (DBD) or the hinge region. Spectrophotometry indicated that melatonin and vitamin D3 (VD3) had similar uptake rates, but melatonin's uptake was significantly inhibited by VD3 until the concentration of melatonin was obviously higher than that of VD3 in a preosteoblastic cell line MC3T3-E1. GST pull-down and yeast two-hybrid assay showed that the interactive smallest fragments were on the 319-379 position of Runx2 and the N-terminus 110-amino acid DBD of the VDR. Electrophoretic mobility shift assay (EMSA) demonstrated that Runx2 facilitated the affinity between the VDR and its specific DNA substrate, which was further documented by a fluorescent EMSA assay where Cy3 labeled Runx2 co-localized with the VDR-DNA complex. Another fluorescent EMSA assay confirmed that the binding of the VDR to Runx2 was significantly enhanced with an increasing concentrations of the VDR, especially in the presence of melatonin; it was further documented using a co-immunoprecipitation assay that this direct interaction was markedly enhanced by melatonin treatment in the MC3T3-E1 cells. Thus, the VDR is a novel melatonin-binding nuclear receptor, and melatonin indirectly regulates Runx2 when it directly binds to the LBD and the DBD of the VDR, respectively.

中文翻译：

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新型褪黑激素结合核受体的鉴定：维生素D受体。

先前的研究证实褪黑激素调节Runx2表达，但其机制尚不清楚。Runx2与维生素D受体（VDR）之间存在直接的相互作用。在这里，我们观察到了褪黑素和VDR之间的直接相互作用，但使用等温滴定量热法则没有Runx2。此外，仅在VDR的C端配体结合域（LBD）中检测到这种直接结合，而在N端DNA结合域（DBD）或铰链区中未检测到。分光光度法表明褪黑素和维生素D3（VD3）的摄取率相似，但是褪黑素的摄取被VD3显着抑制，直到成骨细胞前细胞系MC3T3-E1中褪黑激素的浓度明显高于VD3。GST下拉和酵母双杂交检测结果表明，相互作用的最小片段位于Runx2的319-379位和VDR的N端110个氨基酸的DBD处。电泳迁移率变动分析（EMSA）表明Runx2促进了VDR及其特异性DNA底物之间的亲和力，这进一步通过荧光EMSA分析得到了证明，其中Cy3标记的Runx2与VDR-DNA复合体共定位。另一种荧光EMSA分析证实，随着VDR浓度的增加，VDR与Runx2的结合显着增强，尤其是在褪黑激素存在的情况下；特别是在褪黑素存在的情况下。使用共同免疫沉淀测定法进一步证明，褪黑激素处理可在MC3T3-E1细胞中显着增强这种直接相互作用。因此，VDR是一种新型的褪黑激素结合核受体，