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（适用提供）：发现CYP11A1对7替型蛋白D的代谢发现（符合UVB光转化为相应的替耶塞固醇）和顺序的维生素D的生产20（OH）D3和其他水透射率的新型途径，定义了新的液体途径，该杂种的主要是20（OH），这是20（OH），该途径是20（OH），该途径是20（OH）。大鼠和小鼠的无毒和非钙血症的剂量高达60 kg。这些途径可以在plapeta，肾上腺和表皮角质形成细胞中进行离体。我们还检测到人类血清中的20（OH）D3。 20（OH）D3在抗增殖，促差异和抗炎测定中至少具有1,25（OH）2d3的潜力，并减轻了小鼠博来霉素诱导的纤维化的发展。但是，在硬皮病的临床前模型中使用20（OH）D3的主要障碍是对其对真皮成纤维细胞作用的机制缺乏理解。该R21的目的是检验假设，是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） - 依赖性机制抑制纤维纤维细胞活性。为了研究这一假设，一个机械方向的特定目标是设计了四个子象：1。测试皮肤成纤维细胞中20（OH）D3的抗纤维化作用机理。 Subaim 1：我们将研究TGF-在皮肤成纤维细胞中使用的哪些信号通路被20（OH）D3抑制。我们将确定该乳突固醇是否会抑制TGF-的其他纤维化作用，并确定其在每种途径上的相对效力。 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影响来研究VDR依赖性途径的参与。将使用带有RNAi技术沉默的受体的皮肤成纤维细胞进行确认。这些将分别通过使用VDR-GFP和VDRE-LUC构建体的定量测试将配体诱导的VDR转移到核和VDRE转录活性的激活来完成。 Subaim 3：假设20（OH）D3作用于ROR。将测试调节成纤维细胞活动。我们将用ror ror定义20（OH）D3的相互作用？使用生化和基于细胞的测定。这些受体参与表型调节的参与将使用来自Ror的成纤维细胞和ROR？ - / - 小鼠的成纤维细胞，并在人类真皮成纤维细胞中进一步确认，并用RNAi沉默的受体。 vdr上的动作之间的分歧和重叠，ror。还将通过通过测试基因表达和生物信息学分析来补充的整个基因组RNASEQ分析来测试。这将定义哪些表型特征受VDR调节，以及哪些表型特征？ Subaim 4：我们将测试是否在C1 nd/或c25处通过羟基化来调节20（OH）D3的抗纤维化活性。将使用生物化学，基因沉默技术和细胞生物学的技术，并将通过药理学方法来补充。定义哪些表型通过通过VDR或ROR nd调节？到20（OH）D3，将允许对适当的KO小鼠进行未来的测试，以定义受体在体内硬皮病模型中的作用。