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

新型secosteroid 20(OH)D3在皮肤中的作用机制和功能

• 批准号: 9914825
• 负责人: ANDRZEJ T SLOMINSKI
• 金额: $ 32.67万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9914825
• 关键词: 7-dehydrocholesterol; Acids; Adrenal Glands; Affect; Agonist; Anti-Inflammatory Agents; Apoptosis; Attenuated; Binding; Bioinformatics; Biological; Blood Circulation; CYP11A1 gene; CYP27B1 gene; Calcitriol; Calcium; Cell Differentiation process; Cells; ChIP-seq; Cholecalciferol; Clinical; Complement; Complex; Computer Models; DNA Repair; Defect; Development; Dose; Endocrine; Epidermis; Fibroblasts; Gene Chips; Gene Silencing; Genes; Genetic Complementation Test; Genetic Transcription; Genotype; Goals; Heterozygote; Homeostasis; Hormonal; Human; Hydroxylation; In Vitro; Laboratories; Ligand Binding Domain; Ligands; Measures; Mediating; Metabolic; Metabolism; Modeling; Mus; Nuclear; Orphan; Outcome; Oxides; Pathology; Pathway interactions; Pharmacology; Phenotype; Positioning Attribute; Process; Radiation Protection; Recombinants; Regulation; Role; Secosteroids; Serum; Side; Skin; Suggestion; Technology; Testing; Tretinoin; UVB induced; Ultraviolet B Radiation; Vitamin D; Vitamin D3 Receptor; analog; base; data modeling; immunoregulation; keratinocyte; keratinocyte differentiation; melanocyte; molecular modeling; novel; orphan nuclear receptor ROR-gamma; prevent; programs; receptor; repaired; response; single molecule; skin barrier; stressor; transcriptome sequencing; ultraviolet

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.

紫外线 B 既损伤皮肤，又是 7-脱氢胆固醇光化学转化所必需的 其在 C25 和 C1 处连续羟基化生成具有生物活性的 1,25(OH)2D3。 人们相信所有这些效应都是由单一介导的。 分子 1,25(OH)2D3 和单一受体 VDR 发现 CYP11A1 的替代途径。 氧化 D3 的侧链，生成 20(OH)D3，并进一步代谢为其他下游衍生物， (OH)nD3，挑战了这一法则。20(OH)D3 在人体表皮中的浓度高于 25(OH)D3，并且在人血清中浓度约为 3nM 20(OH)D3 显示出生物活性，表明它可以充当 表皮屏障的内源性调节剂，而其在循环中的存在表明荷尔蒙功能。 初始数据和计算机模型表明，20(OH)D3 在药理学剂量 (30-60 µg/kg) 下不会产生钙血症。 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/或其代谢物直接作用于VDR和/或RORα和RORγ刺激角质形成细胞分化 程序以及光保护和修复机制，保护表皮免受 UVB 引起的病变。 与 1,25(OH)2D3 的假设相反，这些作用不需要其在 C1α 位置上的羟基化。 将进行如下测试： 目标 1. 测试 VDR、RORα 或 RORγ 在 20(OH)D3 介导中的相对作用 Subaim 1：通过 in 调节表皮角质形成细胞的增殖和分化程序。 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及其天然配体的比较。 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。 辅以分子模型分析 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情况。 通过对从 VDR-/-、RORα-/- 或 RORγ-/- 小鼠分离的角质形成细胞进行测试来补充：RNA-Seq。 用于基因表达和 ChIP-Seq 用于目标基因的受体富集，然后进行生物信息学 目标 2. 确定 20(OH)D3 对 UVB 辐射的保护作用。 Subaim 1：测试 20(OH)D3 作为生存因子的作用。 20(OH)D3 减弱 UVB 诱导的细胞凋亡。Subaim 3：测试 20(OH)D3 是否可以充当细胞凋亡抑制剂。 Subaim 4：测试 RORα 或 RORγ 在皮肤反应中的作用。 UVB 使用具有这些受体基因型 -/-、-/+ 和 +/+ 的小鼠，并与有缺陷或完整的小鼠进行比较 VDR。目标 3. 评估 20(OH)D3 及其代谢物的表型效应是否需要 1α 如 1,25(OH)2D3 所示，我们将使用具有沉默 CYP27B1 基因的细胞。 通过对角质形成细胞中 CYP27B1 活性的药理抑制来补充。