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原到7Δ-类固醇的代谢(经过UVB光转化为相应的Secosterid)和随后的维生素D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，同时在大鼠和小鼠中以高达60μg/kg的剂量无毒和无钙化。这些途径可以在体外作用于胎盘、肾上腺和表皮角质形成细胞。我们还检测到人血清中的20(OH)D3。20(OH)D3在抗增殖、促分化和抗炎试验中的效力至少与1，25(OH)2D3相当，并可减轻博莱霉素诱导的小鼠纤维化的发展。然而，在硬皮病的临床前模型中使用20(OH)D3的一个主要障碍是缺乏对其对真皮成纤维细胞作用机制的了解。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/或维甲酸孤儿受体(ROR)依赖的机制，抑制促纤维化成纤维细胞的活性。为了研究这一假说，设计了一个机械定向的特异性目标，包括四个亚目的：1.测试20(OH)D3在真皮成纤维细胞中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转化生长因子-？我们将确定这种Secosterid是否抑制了转化生长因子-B的其他促纤维化作用，并确定其在每个途径上的相对效力；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依赖的途径的参与。人类的确认将使用真皮成纤维细胞进行，受体通过RNAi技术沉默。此外，还将分别使用VDR-GFP和VDRE-Luc构建物对配体诱导的VDR移位到细胞核和激活VDRE转录活性进行定量测试；Subaim 3：将检验20(OH)D3作用于RoRα和RoRγ将调节成纤维细胞活性的假设。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。这些受体在表型调控中的作用将使用来自RoRα-和RoRγ-/-小鼠的成纤维细胞进行评估，并在受体被RNA干扰沉默的人皮肤成纤维细胞中得到进一步证实。对Vdr、RoRα和γ的作用之间的差异和重叠也将通过全基因组RNAseq分析以及基因表达测试和生物信息学分析来测试。这将定义哪些表型性状受VdR调节，哪些受RoRα或RoRγ调节；Subaim 4：我们将测试20(OH)D3的抗纤维化活性是否受C1α和/或C25羟化的调节。将使用生物化学、基因沉默技术和细胞生物学技术，并将进一步辅之以药理学方法。确定哪些表型治疗是通过vdr或rorα和γ由20(OH)D3调节的，将允许在适当的KO小鼠上进行未来的测试，以确定该受体在活体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.