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: 8833514
• 负责人: ARNOLD E POSTLETHWAITE
• 金额: $ 0.57万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-18 至 2015-04-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8833514
• 关键词: Adjustment Disorders; Adrenal Glands; Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Attenuated; Biochemical; Biochemistry; Bioinformatics; Biological Assay; Biological Factors; Bleomycin; CYP11A1 gene; Cell Lineage; Cell Nucleus; Cells; Cellular biology; Cholecalciferol; Cholesterol Monooxygenase (Side-Chain-Cleaving); Chronic; Complement; Dermal; Development; Disease; Dose; Fibroblasts; Fibrosis; Future; Gene Expression; Gene Silencing; Genome; Goals; Health; Human; Hydroxylation; Ligands; Metabolic Pathway; Metabolism; Modeling; Mus; Orphan; Pathway interactions; Patients; Phase I Clinical Trials; Phenotype; Placenta; Positioning Attribute; Pre-Clinical Model; Preclinical Testing; Production; RNA Interference; Rattus; Regulation; Relative (related person); Role; Scleroderma; Secosteroids; Serum; Signal Pathway; Skin; Steroids; Techniques; Technology; Testing; Tretinoin; Ultraviolet B Radiation; Vitamin D; Vitamin D3 Receptor; base; design; in vitro activity; in vivo; keratinocyte; novel; preclinical study; receptor; trait

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 μkg. 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-β1 in dermal fibroblasts are inhibited by 20(OH)D3. We will determine whether this secosteroid inhibits other profibrotic effects of TGF-β1 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 RORγ 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 RORγ 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（由适用提供）：发现CYP11A1对7Δ类固醇的代谢发现（易于UVB光转移至相应的替代类固醇），并顺序羟基化的维生素D生产20（OH）D3和其他羟基源性，定义了新的综合路径，该疗程为20（oh），oh is ohologiatient ohologiatient of 3 ohologiatient ohologiatient of 3 ohologiated ohologiatientiation of 3 ohologiatiential of 3 oh oh oh oh oh oh oh oh oh oh。大鼠和小鼠的无毒和非钙血症的剂量高达60μkg。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-β1在皮肤成纤维细胞中使用的哪些信号传导途径被20（OH）D3抑制。我们将确定该乳突类固醇是否抑制TGF-β1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：将测试对RORα和RORγ作用的20（OH）D3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测定法定义20（OH）D3与RORα和RORγ的相互作用。这些受体参与表型的调节，将使用来自RORα-和RORγ-/ - 小鼠的成纤维细胞进行评估，并在人类真皮成纤维细胞中进一步确认，并用RNAi沉默的受体。通过测试基因表达和生物信息学分析补充的整个基因组RNASEQ分析，还将测试对VDR，RORα和RORγ的作用之间的分歧和重叠。这将定义哪些表型性状受VDR调节，哪些由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的羟基化调节。生物化学，基因沉默技术和细胞生物学的技术将被使用，并将通过药物方法进一步补充。通过20（OH）D3来定义哪些表型治疗通过VDR或RORα和RORγ调节，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。