Analysis of vitamin D response element binding protein target genes reveals a role for vitamin D in osteoblast mTOR signaling

Heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 plays a pivotal role in vitamin D receptor (VDR) signaling by acting as a vitamin D response element (VDRE)-binding protein (VDRE-BP). Transcriptional regulation by active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) involves occupancy of VDRE by VDRE-BP or 1,25(OH)2D3 bound-VDR. This relationship is disrupted by over-expression of VDRE-BP and can cause a form of human hereditary vitamin D-resistant rickets (HVDRR). DNA array analyses using B-cells from an HVDRR patient and matched control defined a sub-cluster of genes where 1,25(OH)2D3-regulated transcription was abrogated by over-expression of VDRE-BP. Amongst these, the DNA-damage-inducible transcript 4 (DDIT4), an inhibitor of mammalian target of rapamycin (mTOR) signaling, was also induced by 1,25(OH)2D3 in human osteoblasts. Chromatin immunoprecipitation using 1,25(OH)2D3-treated osteoblasts confirmed that liganded VDR and VDRE-BP compete for binding to the proximal promoter of the DDIT4 gene in a similar fashion to other known 1,25(OH)2D3-target genes. Treatment of osteoblasts with 1,25(OH)2D3 induced DDIT4 expression and suppressed phosphorylated S6K1T389 protein (a downstream target of mTOR). The functional importance of this for 1,25(OH)2D3 responses in osteoblasts was underlined by the fact that siRNA knockdown of DDIT4 expression suppressed antiproliferative and cell growth responses to 1,25(OH)2D3. These data confirm that VDRE-BP is required for normal 1,25(OH)2D3-mediated transcription and cell function in osteoblasts. Conversely over-expression of VDRE-BP exerts a dominant-negative effect on transcription of 1,25(OH)2D3-target genes. Characterization of VDRE-BP action in 1,25(OH)2D3-treated osteoblasts highlights an entirely novel role for vitamin D as a regulator of mTOR – a known ‘master regulator’ of cell function. We performed gene expression microarray analysis in HVDRR EBV-transformed B-cells and control cells in the presence or absence of vitamin D.

异质性核核糖核蛋白（Heterogeneous nuclear ribonucleoprotein，hnRNP）C1/C2作为维生素D反应元件结合蛋白（VDRE-binding protein，VDRE-BP），在维生素D受体（VDR）信号通路中发挥关键调控作用。活化型1,25-二羟维生素D3（1,25(OH)2D3）介导的转录调控，依赖于VDRE-BP结合或1,25(OH)2D3结合的VDR占据维生素D反应元件（VDRE）。VDRE-BP过表达会破坏这一相互作用，进而引发人类遗传性维生素D抵抗性佝偻病（HVDRR）。研究人员通过对HVDRR患者及匹配对照的B细胞进行DNA芯片分析，鉴定出一组基因亚群：其1,25(OH)2D3调控的转录过程会因VDRE-BP过表达被完全阻断。在该亚群基因中，DNA损伤诱导转录因子4（DNA-damage-inducible transcript 4，DDIT4）作为雷帕霉素靶蛋白（mTOR）信号通路的抑制剂，同样可在人成骨细胞中被1,25(OH)2D3诱导表达。通过对1,25(OH)2D3处理的成骨细胞进行染色质免疫沉淀实验，研究人员证实配体结合的VDR与VDRE-BP会以类似其他已知1,25(OH)2D3靶基因的方式，竞争结合DDIT4基因的近端启动子。用1,25(OH)2D3处理成骨细胞可诱导DDIT4表达，并抑制磷酸化S6K1T389蛋白（mTOR下游靶标）的水平。进一步实验表明，通过小干扰RNA（siRNA）敲低DDIT4的表达，会抑制成骨细胞对1,25(OH)2D3的抗增殖及细胞生长应答，这一结果凸显了该机制在成骨细胞1,25(OH)2D3应答中的功能重要性。上述数据证实，VDRE-BP是成骨细胞中正常1,25(OH)2D3介导的转录调控与细胞功能所必需的因子。反之，VDRE-BP过表达会对1,25(OH)2D3靶基因的转录产生显性负调控效应。对1,25(OH)2D3处理的成骨细胞中VDRE-BP作用机制的解析，揭示了维生素D作为mTOR调控因子的全新功能——mTOR是公认的细胞功能“主调控因子”。本研究在存在或缺失维生素D的培养条件下，分别对HVDRR患者EB病毒转化的B细胞及对照细胞开展了基因表达微阵列分析。