Mechanism of action of 20-hydroxyvitamin D3 in dermal fibroblasts

20-羟基维生素D3对真皮成纤维细胞的作用机制

• 批准号: 9101104
• 负责人: ANDRZEJ T SLOMINSKI
• 金额: $ 18.72万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-06 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9101104
• 关键词: Mechanism; action; 20; hydroxyvitamin; D3

DESCRIPTION (provided by applicant): Discovery of CYP11A1 initiated metabolism of pro-vitamin D to 7�teroids (subject to UVB photoconversion to corresponding secosteroids) and sequential hydroxylation of vitamin D producing 20(OH)D3 and other hydroxyderivatives, defined new metabolic pathways of which the main intermediate, 20(OH)D3, is biologically active, while being nontoxic and noncalcemic in rats and mice at doses as high as 60 �kg. These pathways can operate ex vivo in placenta, adrenal gland and epidermal keratinocytes. We also detected 20(OH)D3 in human serum. 20(OH)D3 is at least as potent as 1,25(OH)2D3 in anti-proliferative, pro-differentiation and anti-inflammatory assays and attenuates development of bleomycin induced fibrosis in mice. However, a major barrier for using 20(OH)D3 in preclinical models of scleroderma is a lack of understanding of the mechanism of its action on dermal fibroblasts. The goal of this R21 is to test hypothesis is that 20(OH)D3 acting directly on vitamin D receptor (VDR)- or/and on retinoic acid orphan receptor (ROR)- dependent mechanisms inhibit profibrotic fibroblast activities. To study this hypothesis one mechanistically oriented specific aim is designed with four subaims: 1. To test the mechanism of antifibrotic action of 20(OH)D3 in dermal fibroblasts. Subaim 1: We will investigate which signaling pathways utilized by TGF-�in dermal fibroblasts are inhibited by 20(OH)D3. We will determine whether this secosteroid inhibits other profibrotic effects of TGF-� and determine its relative potency on each pathway; Subaim 2: We will investigate the involvement of VDR-dependent pathways by testing the effects of 20(OH)D3 on fibroblasts derived from VDR-/- mice. Confirmations for humans will be carried out using dermal fibroblasts with receptors silenced by RNAi technology. These will be complemented by quantitative testing of ligand-induced VDR translocation to the nucleus and activation of VDRE transcriptional activity using VDR-GFP and VDRE-LUC constructs, respectively; Subaim 3: The hypothesis that 20(OH)D3 acting on ROR�nd ROR? will regulate fibroblast activities will be tested. We will define interactions of 20(OH)D3 with ROR�nd ROR? using biochemical and cell-based assays. Involvement of those receptors in the regulation of a phenotype will be evaluated using fibroblasts from ROR�- and ROR?-/- mice with further confirmation in human dermal fibroblasts with receptors silenced by RNAi. Divergence and overlaps between the actions on VDR, ROR�nd ? will also be tested by whole genome RNAseq analysis supplemented by testing gene expression and bioinformatic analysis. This will define which phenotypic traits are regulated by VDR and which by ROR�r ROR?; Subaim 4: We will test whether antifibrotic activity of 20(OH)D3 is regulated by hydroxylation at C1�nd/or C25. Techniques of biochemistry, gene silencing technology and cell biology will be used and will further be supplemented by pharmacological approaches. Defining which phenotypic treats are regulated through VDR or ROR�nd ? by 20(OH)D3, would allow to perform future testing on proper KO mice to define role of the receptor in in vivo scleroderma models.

描述（由申请人提供）：CYP11A1 的发现启动了维生素 D 原代谢为 7°类固醇（经 UVB 光转化为相应的类固醇），并连续羟基化维生素 D 产生 20(OH)D3 和其他羟基衍生物，定义了新的代谢途径其中主要中间体 20(OH)D3 具有生物活性，同时在大鼠和小鼠中无毒且无钙血症，剂量为高达 60 kg。这些途径可以在胎盘、肾上腺和表皮角质形成细胞中发挥作用。我们还检测到人血清中的 20(OH)D3 至少与 1,25(OH) 一样有效。 2D3 在抗增殖、促分化和抗炎测定中可以减弱博来霉素诱导的小鼠纤维化的发展，然而，这是使用的主要障碍。硬皮病临床前模型中的 20(OH)D3 缺乏对其对真皮成纤维细胞的作用机制的了解。该 R21 的目标是检验 20(OH)D3 直接作用于维生素 D 受体 (VDR) 的假设。 - 或/和视黄酸孤儿受体 (ROR) 依赖性机制抑制促纤维化成纤维细胞活性 为了研究这一假设，设计了一个以机械为导向的具体目标，有四个。子目标： 1. 测试 20(OH)D3 在真皮成纤维细胞中的抗纤维化作用机制 子目标 1：我们将研究 TGF-β 在真皮成纤维细胞中利用的哪些信号通路被 20(OH)D3 抑制。这种类固醇是否抑制 TGF-β 的其他促纤维化作用并确定其对每个途径的相对效力：我们将研究以下因素的参与；通过测试 20(OH)D3 对 VDR-/- 小鼠成纤维细胞的影响，将使用经 RNAi 技术沉默受体的真皮成纤维细胞来验证 VDR 依赖性途径。 -分别使用VDR-GFP和VDRE-LUC构建体诱导VDR易位至细胞核并激活VDRE转录活性：Subaim 3的假设；我们将使用生化和基于细胞的检测来确定 20(OH)D3 与 ROR 和 ROR 的相互作用。将使用 ROR�- 和 ROR?-/- 小鼠的成纤维细胞来评估表型的调节，并在受体被 RNAi 沉默的人真皮成纤维细胞中进一步确认。 VDR、ROR 和 ? 作用之间的差异和重叠也将通过全基因组 RNAseq 分析进行测试，并辅以测试基因表达和生物信息学分析，这将确定哪些表型性状受 VDR 调节，哪些表型性状受 ROR?r ROR 调节。 Subaim 4：我们将测试 20(OH)D3 的抗纤维化活性是否受 C1 和/或 C25 的羟基化调节。将使用生物化学、基因沉默技术和细胞生物学，并进一步通过药理学方法进行补充，确定哪些表型治疗是通过 VDR 或 ROR 进行调节的？20(OH)D3 将允许在适当的 KO 小鼠上进行未来的测试。定义受体在体内硬皮病模型中的作用。