Mechanism of action of 20-hydroxyvitamin D3 in dermal fibroblasts

描述（申请人提供）：发现CYP 11 A1启动维生素D原代谢为7种Δ-甾体（受到UVB光转化为相应的开环甾类化合物）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，在60 μg/kg剂量下对大鼠和小鼠无毒无钙。这些途径可以在胎盘、肾上腺和表皮角质形成细胞中离体操作。我们还在人血清中检测到20（OH）D3。20（OH）D3在抗增殖、促分化和抗炎试验中至少与1，25（OH）2D 3一样有效，并减弱小鼠中博来霉素诱导的纤维化的发展。然而，在硬皮病的临床前模型中使用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理解。该R21的目的是检验假设，即20（OH）D3直接作用于维生素D受体（VDR）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，一个机械定向的具体目标设计了四个子目标：1。探讨20（OH）D3对皮肤成纤维细胞的抗纤维化作用机制。Subaim 1：我们将研究20（OH）D3抑制真皮成纤维细胞中TGF-β所利用的信号通路。我们将确定这种开环甾类是否抑制TGF-β的其他促纤维化作用，并确定其对每种途径的相对效力; Subaim 2：我们将通过测试20（OH）D3对源自VDR-/-小鼠的成纤维细胞的作用来研究VDR依赖性途径的参与。将使用具有通过RNAi技术沉默的受体的真皮成纤维细胞进行人类的扩增。这些将通过分别使用VDR-GFP和VDRE-LUC构建体定量检测配体诱导的VDR易位至细胞核和VDRE转录活性的激活来补充;子章节3：将检测作用于RORα和RORγ的20（OH）D3将调节成纤维细胞活性的假设。我们将使用生物化学和基于细胞的测定来确定20（OH）D3与RORα和RORγ的相互作用。将使用RORα-和RORγ-/-小鼠的成纤维细胞评价这些受体参与表型调节的情况，并在人真皮成纤维细胞中进一步确认RNAi沉默的受体。对VDR、RORα和γ作用之间的差异和重叠也将通过全基因组RNAseq分析进行检测，并辅以检测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，哪些受RORα或RORγ调节; Subaim 4：我们将检测20（OH）D3的抗纤维化活性是否受C1α和/或C25羟基化的调节。将使用生物化学、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。确定哪些表型治疗通过VDR或RORα和γ由20（OH）D3调节，将允许对适当的KO小鼠进行未来测试，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.