Mechanism of action of 20-hydroxyvitamin D3 in dermal fibroblasts

描述（由申请人提供）：CYP11A1的发现启动了维生素D前体到7Δ-steroids的代谢（受UVB光转化为相应的类固醇）和维生素D的顺序羟基化产生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，同时在剂量高达60 μg/kg的大鼠和小鼠中无毒和无钙化。这些途径可以在体外的胎盘、肾上腺和表皮角质形成细胞中运作。我们还在人血清中检测到20(OH)D3。在抗增殖、促分化和抗炎实验中，20(OH)D3至少与125 (OH)2D3一样有效，并能减轻博来霉素诱导的小鼠纤维化的发展。然而，在硬皮病临床前模型中使用20(OH)D3的主要障碍是缺乏对其对真皮成纤维细胞作用机制的了解。本R21的目的是验证20(OH)D3直接作用于维生素D受体(VDR)-或/或视黄酸孤儿受体(ROR)-依赖机制抑制原纤维化成纤维细胞活性的假设。为了研究这一假设，我们设计了一个机械导向的具体目标，包括四个子目标：目的：探讨20(OH)D3对真皮成纤维细胞的抗纤维化作用机制。Subaim 1：我们将研究TGF-ß中真皮成纤维细胞利用的哪些信号通路被20(OH)D3抑制。我们将确定该类固醇是否抑制TGF-ß的其他促纤维化作用，并确定其在各途径上的相对效力；Subaim 2：我们将通过测试20(OH)D3对来自VDR-/-小鼠的成纤维细胞的影响来研究VDR依赖通路的参与。对人类的确认将使用带有RNAi技术沉默受体的真皮成纤维细胞进行。这些将通过定量测试配体诱导的VDR转位到细胞核，以及分别使用VDR- gfp和VDR- luc构建的VDR转录活性的激活来补充。Subaim 3: 20(OH)D3作用于RORα和RORγ调节成纤维细胞活性的假设将被验证。我们将使用生化和基于细胞的分析来定义20(OH)D3与RORα和RORγ的相互作用。这些受体参与一种表型的调节将使用来自RORα-和RORγ-/-小鼠的成纤维细胞进行评估，并在具有RNAi沉默受体的人真皮成纤维细胞中进一步证实。通过全基因组RNAseq分析，并辅以基因表达检测和生物信息学分析，检测VDR、rora和γ作用之间的差异和重叠。这将确定哪些表型性状是由VDR调节的，哪些是由RORα或RORγ调节的；子目标4：我们将测试20(OH)D3的抗纤维化活性是否受C1α和/或C25的羟基化调节。将使用生物化学技术、基因沉默技术和细胞生物学技术，并将进一步辅以药理学方法。确定哪些表型治疗是通过VDR或RORα和γ通过20(OH)D3调节的，将允许在适当的KO小鼠上进行未来的测试，以确定受体在体内硬皮病模型中的作用。

DESCRIPTION (provided by applicant): Discovery of CYP11A1 initiated metabolism of pro-vitamin D to 7Δ-steroids (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 μg/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-ßin dermal fibroblasts are inhibited by 20(OH)D3. We will determine whether this secosteroid inhibits other profibrotic effects of TGF-ß 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 RORα and RORγ will regulate fibroblast activities will be tested. We will define interactions of 20(OH)D3 with RORα and RORγ using biochemical and cell-based assays. Involvement of those receptors in the regulation of a phenotype will be evaluated using fibroblasts from RORα- and RORγ-/- mice with further confirmation in human dermal fibroblasts with receptors silenced by RNAi. Divergence and overlaps between the actions on VDR, RORα and γ 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 RORα or RORγ; Subaim 4: We will test whether antifibrotic activity of 20(OH)D3 is regulated by hydroxylation at C1α and/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 RORα and γ 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.