25(OH)D 3 Stimulates the Expression of Vitamin D Target Genes in Renal Tubular Cells When Cyp27b1 Is Abrogated
25(OH)D 3 Stimulates the Expression of Vitamin D Target Genes in Renal Tubular Cells When Cyp27b1 Is Abrogated
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当 Cyp27b1 被废除时，25(OH)D 3 刺激肾小管细胞中维生素 D 靶基因的表达
DOI：
10.1016/j.jsbmb.2020.105593
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Okazaki T.
中科院分区：
文献类型：
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作者：
Kikuyama T;Susa T;Tamamori-Adachi M;Iizuka M;Akimoto M;Okinaga H;Fujigaki Y;Uchida S;Shibata S;Okazaki T.
最近，有报道称25(OH)D3（25D3）在yp27b1敲除小鼠的某些组织中具有生理生物活性。为了研究25D3在肾脏中作为各种钙化物质信息交叉点的功能，我们采用CRISPR-Cas9系统敲除小鼠肾远端小管mDCT细胞系中的cyp27b1。与先前报道的全身靶向cyp27b1的小鼠不同，cyp27b1敲除的mDCT细胞在给予25D3后没有产生任何可测量的1α，25(OH)2D3（1,25 d3）。在cyp27b1基因敲除mDCT细胞中，用≥10−8M的1,25 d3处理可以看到，10- 7m的25D3将维生素d3受体（VDR）转位到细胞核中，并促进了具有代表性的1,25 d3应答基因ecyp24a1的表达。25D3的穷举靶基因谱与1,25d3相似。随后，我们证实25D3诱导钙重吸收相关基因calbinin - d9k的表达，其方式与1,25d3相似。我们还发现1,25 d3和25D3诱导meggalin基因的表达。染色质免疫沉淀法鉴定了megalin基因上游区域的两个维生素D反应元件，这似乎有助于其表达。总之，我们推测25D3刺激VDR靶基因的能力可能为其在某些组织中的作用提供了新的视角。
Recently, it was reported that 25(OH)D3(25D3) has physiological bioactivity in certain tissues derived fromCyp27b1knockout mice. To investigate the function of 25D3 in the kidney as an informational crossroad of various calciotropic substances, we employed the CRISPR-Cas9 system to knock outCyp27b1in the mouse renal distal tubular mDCT cell line. Unlike the previously reported mice in whichCyp27b1was targeted systemically,Cyp27b1knockout mDCT cells did not produce any measurable 1α,25(OH)2D3(1,25D3) after 25D3 administration. As was seen with treatment ofCyp27b1knockout mDCT cells with ≥10−8M of 1,25D3, the administration of 10-7M of 25D3 translocated the vitamin D3receptor (VDR) into the nucleus and promoted the expression of the representative 1,25D3-responsive geneCyp24a1. The exhaustive target gene profiles of 25D3 were similar to those of 1,25D3. Subsequently, we confirmed that 25D3 induced the expression of the calcium reabsorption-related gene calbindin-D9K, in a way similar to 1,25D3. We also found that 1,25D3 and 25D3 induced the expression of the megalin gene. A chromatin immunoprecipitation assay identified two vitamin D response elements in the upstream region of the megalin gene that seemed to contribute to its expression. Together, we surmise that the ability of 25D3 to stimulate VDR target genes may provide a novel perspective for its role in certain tissues.