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

紫外线B既损害皮肤，又是7-脱氢胆固醇光化学转化所必需的 维生素D3（D3）其在C25和C1的顺序羟基化产生生物活性1，25（OH）2D 3， 显示出多种多样的活性。据认为，所有这些影响都是由单一的 分子，1，25（OH）2D 3和单一受体，VDR。发现一种替代途径，其中CYP 11 A1 氧化D3的侧链产生20（OH）D3，并进一步代谢为其它下游衍生物， （OH）nD 3挑战了这一教条。20（OH）D3在人体表皮中的浓度高于 25（OH）D3，并且在人血清中为约3 nM。20（OH）D3显示出生物活性，表明它可以作为一种 表皮屏障的内源性调节剂，而其在循环中的存在表明激素功能。 20（OH）D3在药理学剂量（30-60µg/kg）下是非钙的。初始数据和计算机模型显示 它对VDR有偏向性激动作用，对RORα和RORγ有反向激动作用。假设：20（OH）D3 和/或其代谢物直接作用于VDR和/或RORα和RORγ刺激角质形成细胞分化 程序和光保护和修复机制，保护表皮免受UVB诱导的病理。 与1，25（OH）2D 3不同，这些作用不需要在C1α位进行羟基化。的假设 将测试如下：目标1。为了检测VDR、RORα或RORγ在20（OH）D3介导的细胞凋亡中的相对作用， 调节表皮角质形成细胞的增殖和分化程序。Subaim 1：通过 与重组VDR和ROR的配体结合结构域的体外结合，我们将定义 20（OH）D3及其下游代谢物与受体的天然配体比较。然后我们 将应用复杂的基于细胞的模型来测量配体调节的转录活性。这些将是 辅以分子模拟分析。Subaim 2：VDR和ROR在调节中的相对作用 将使用基因沉默技术测试角质形成细胞增殖和分化的影响。这将是 通过对分离自VDR-/-、RORα-/-或RORγ-/-小鼠的角质形成细胞的测试进行补充。Subaim 3：RNA-Seq 用于基因表达，ChIP-Seq用于靶基因上的受体富集，然后是生物信息学 分析以确定替代目标。目标二。探讨20（OH）D3对UVB辐射的防护作用， 人体表皮Subaim 1：测试20（OH）D3作为生存因子的作用。Subaim 2：测试是否 20（OH）D3可减弱UVB诱导的细胞凋亡。Subaim 3：测试20（OH）D3是否可以充当 抗遗传毒性/抗突变剂。Subaim 4：测试RORα或RORγ在皮肤对 UVB使用具有这些受体的基因型-/-、-/+和+/+的小鼠，并与具有缺陷或完整受体的小鼠进行比较。 VDR。目标3。评价20（OH）D3及其代谢产物的表型效应是否需要1α 如针对1，25（OH）2D 3所述进行羟基化。我们将使用CYP 27 B1基因沉默的细胞。这将是 辅以角质形成细胞中CYP 27 B1活性的药理学抑制。

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.