Mechanism of action and function of novel secosteroid 20(OH)D3 in the skin

中波紫外线B会损害皮肤，也是7-脱氢胆固醇光化学转化所必需的 至维生素D3(D3)。它在C25和C1位的顺序羟基化产生生物活性1，25(OH)2D3，该活性 展示了各种各样的多效性活动。人们认为，所有这些效应都是由单一的 分子1，25(OH)2D3和单一受体VDR。发现Cyp11a1的另一条途径 氧化D3的侧链产生20(OH)D3并进一步代谢到其他下游衍生物， (哦)ND3，挑战这一教条。20(OH)D3在人体表皮中的浓度高于 25(OH)D3，人血清中的~3 nm。20(OH)D3显示出生物活性，提示它可以作为一种 表皮屏障的内源性调节剂，而它在循环中的存在表明荷尔蒙功能。 20(OH)D3在药理剂量(30-60微克/公斤)下不含钙质。初步数据和计算机模型显示 它可以作为VdR的偏向激动剂和RoRα和RoRγ的反向激动剂。假设：20(OH)D3 和/或其代谢产物直接作用于VdR和/或RoRα和RoRγ促进角质形成细胞分化 程序和光保护和修复机制，以保护表皮免受UVB诱导的病理。 与1，25(OH)2D3不同，这些作用不需要它在C1α位置上的羟基化。假说 将进行如下测试：目的1.测试VdR、RoRα或RoRγ在20(OH)D3介导的过程中的相对作用 表皮角质形成细胞增殖和分化程序的调控。苏巴伊姆1：穿入 体外结合重组VDR和RORs的配基结合域我们将定义 20(OH)D3及其下游与受体的代谢产物与其天然配体的比较。那我们 将应用复杂的基于细胞的模型来测量配体调节的转录活动。这些将是 辅以分子模型分析。Subaim 2：VDR和RORS在调节中的相对作用 角质形成细胞的增殖和分化将使用基因沉默技术进行测试。这将是 此外，还对从Vdr-/-、RoRα-/-或RoRγ-/-小鼠分离的角质形成细胞进行了测试。Subaim 3：RNA-Seq 用于基因表达和CHIP-SEQ用于目标基因上的受体浓缩，然后是生物信息学 分析以确定替代目标。目的2.确定20(OH)D3对UVB辐射的防护作用 人类的表皮。Subaim 1：测试20(OH)D3作为生存因子的作用。Subaim 2：测试 20(OH)D3可抑制中波紫外线诱导的细胞凋亡。Subaim 3：测试20(OH)D3是否可以作为 抗遗传毒性/抗突变药物。Subaim 4：测试RoRα或RoRγ在皮肤对 UVB使用这些受体基因-/-、-/+和+/+的小鼠，并与缺陷或完整的小鼠进行比较 VDR。目的3.评价20(OH)D3及其代谢物的表型效应是否需要1α 羟基化反应如1，25(OH)2D3所述。我们将使用带有沉默的CYP27B1基因的细胞。这将是 辅以药物对角质形成细胞中细胞色素P27B1活性的抑制。

Ultraviolet B both damages the skin and is required for photochemical transformation of 7-dehydrocholesterol to vitamin D3 (D3). Its sequential hydroxylation at C25 and C1 generates biologically active 1,25(OH)2D3 that displays a variety of pleiotropic activities. It was believed that all of these effects are mediated by single molecule, 1,25(OH)2D3, and single receptor, VDR. Discovery of an alternative pathway in which CYP11A1 oxidizes the side chain of D3 producing 20(OH)D3 with its further metabolism to other downstream-derivatives, (OH)nD3, challenged this dogma. 20(OH)D3 is detectable in the human epidermis at concentration higher than 25(OH)D3, and in human serum at ~3nM. 20(OH)D3 shows biological activities suggestive that it can act as an endogenous regulator of epidermal barrier, while its presence in circulation suggests hormonal functions. 20(OH)D3 is noncalcemic at pharmacological doses (30-60µg/kg). Initial data and computer modeling indicate that it can act as biased agonist on VDR and reverse agonist on RORα and RORγ. Hypothesis: 20(OH)D3 and/or its metabolites acting directly on VDR and/or RORα and RORγ stimulate keratinocyte differentiation program and photoprotective and repair mechanisms that protect epidermis against UVB-induced pathology. These effects would not require its hydroxylation in position C1α, in contrast to 1,25(OH)2D3. The hypothesis will be tested as follows: Aim 1. To test the relative role of VDR, RORα, or RORγ in 20(OH)D3 mediated regulation of the proliferation and differentiation programs in epidermal keratinocytes. Subaim 1: Through in vitro binding to the ligand-binding domain of recombinant VDR and RORs we will define relative interactions of 20(OH)D3 and its downstream metabolites with the receptors in comparison to their native ligands. Then we will apply complex cell-based models to measure ligands modulated transcriptional activities. These will be supplemented by molecular modeling analyses. Subaim 2: The relative roles of VDR and RORs in regulation of keratinocytes proliferation and differentiation will be tested using gene silencing technology. This will be complemented by tests on keratinocytes isolated from VDR-/-, RORα-/- or RORγ-/- mice. Subaim 3: RNA-Seq for gene expression and ChIP-Seq for receptor enrichment on target genes followed by bioinformatics analyses to identify alternative targets. Aim 2. To define protective role of 20(OH)D3 against UVB radiation in the human epidermis. Subaim 1: Testing the role of 20(OH)D3 as a survival factor. Subaim 2: Testing whether 20(OH)D3 attenuates UVB induced apoptosis. Subaim 3: Testing whether 20(OH)D3 can act as an antigenotoxic/antimutagenic agent. Subaim 4: Testing the role of RORα or RORγ in skin responses to the UVB using mice with genotype -/-,-/+ and +/+ for these receptors and comparing with mice with defective or intact VDR. Aim 3. To evaluate whether the phenotypic effects of 20(OH)D3 and its metabolites require 1α hydroxylation as described for 1,25(OH)2D3. We will use cells with silenced CYP27B1 gene. This will be complemented by pharmacological inhibition of the CYP27B1 activity in keratinocytes.