Recently, it was reported that 25(OH)D3 (25D3) has physiological bioactivity in certain tissues derived from Cyp27b1 knockout 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 out Cyp27b1 in the mouse renal distal tubular mDCT cell line. Unlike the previously reported mice in which Cyp27b1 was targeted systemically, Cyp27b1 knockout mDCT cells did not produce any measurable 1α,25(OH)2D3 (1,25D3) after 25D3 administration. As was seen with treatment of Cyp27b1 knockout mDCT cells with ≥10-8 M of 1,25D3, the administration of 10-7 M of 25D3 translocated the vitamin D3 receptor (VDR) into the nucleus and promoted the expression of the representative 1,25D3-responsive gene Cyp24a1. 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.

中文翻译：

---

废除Cyp27b1后，25（OH）D3刺激肾小管细胞中维生素D靶基因的表达。

最近，有报道说25（OH）D3（25D3）在Cyp27b1基因敲除小鼠的某些组织中具有生理生物活性。为了研究25D3在肾脏中作为各种促钙物质的信息十字路口的功能，我们采用CRISPR-Cas9系统敲除小鼠肾远端肾小管mDCT细胞系中的Cyp27b1。与先前报道的将Cyp27b1全身性靶向的小鼠不同，Cyp27b1敲除的mDCT细胞在25D3给药后不会产生任何可测量的1α，25（OH）2D3（1,25D3）。如用CYP27b1基因敲除的mDCT细胞用≥10-8M的1,25D3处理所见，施用10-7 M的25D3可以将维生素D3受体（VDR）转移到细胞核中并促进代表性1的表达， 25D3反应基因Cyp24a1。25D3的详尽目标基因谱与1,25D3相似。随后，我们证实了25D3以类似于1,25D3的方式诱导了钙重吸收相关基因calbindin-D9K的表达。我们还发现1,25D3和25D3诱导了megalin基因的表达。染色质免疫沉淀测定法在巨蛋白基因的上游区域鉴定了两个维生素D反应元件，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新的视角。染色质免疫沉淀测定法在巨蛋白基因的上游区域鉴定了两个维生素D反应元件，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新的视角。染色质免疫沉淀测定法在巨蛋白基因的上游区域鉴定了两个维生素D反应元件，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新的视角。