The Vitamin D Receptor: Ligand-Dependent and Independent Actions

维生素 D 受体：配体依赖性和独立作用

基本信息

批准号：
8884188
负责人：
Marie Demay
金额：
$ 4.47万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8884188
关键词：
Address Alopecia Binding Binding Sites Biological Assay Biological Models Biology Blood Vessels C-terminal COS-7 Cell Cell Count Cell physiology Chromatin Consensus Cutaneous DNA Binding Domain Defect Diabetes Mellitus Disease Dominant-Negative Mutation Erinaceidae Exhibits Familial hypophosphatemic bone disease Gelshift Analysis Gene Expression Gene Expression Profiling Goals Growth Hair Hair follicle structure Hereditary Disease Human Impaired wound healing Inflammatory Investigation Knockout Mice Ligands Messenger RNA Molecular Mus Mutagenesis Mutation Nucleic Acid Regulatory Sequences Pathway interactions Phase Play Production Proteins Public Health Recombinant Proteins Regulation Reporter Response Elements Role Signal Pathway Signal Transduction Skin Staging Stem cells System Time Transfection Transgenes Vascular Endothelial Growth Factors Vitamin D Vitamin D Deficiency Vitamin D3 Receptor Wound Healing base c-myc Genes cytokine human disease in vivo keratinocyte macrophage mouse model mutant novel promoter receptor function response restoration

项目摘要

DESCRIPTION (provided by applicant): Investigations in this proposal are focused on identifying the molecular basis for the ligand dependent and independent actions of the VDR, using the skin as a model system. Like humans with VDR mutations, VDR null mice develop alopecia. We have demonstrated that the ligand-independent actions of the VDR are required for hair follicle keratinocyte stem cell (KSC) function, normal hair cycling and canonical Wnt (cWnt) signaling in keratinocytes. Interfering with cWnt signaling also impairs KSC function. Studies examining the interactions of the VDR with effectors of canonical Wnt signaling demonstrate that the VDR interacts with Lef1 via its DNA binding domain and that the VDR interacting domain of Lef1 is independent of its �-catenin binding domain. We will examine the functional consequences of impairing VDR-Lef1 interactions in keratinocytes and determine if the interaction between these two proteins is direct, or involves additional factors. While keratinocyte-specific expression of a VDR transgene rescues the hair cycle defect in VDR null mice, a constitutively active �-catenin transgene does not, placing the VDR at the level of, or downstream from activation of cWnt signaling. Initiation of hair follicle growth (anagen) by cWnt signaling induces Shh and Gli1 mRNA in WT mice but not in VDR-/- mice. Furthermore, the expression of Shh, Gli1 and the classic cWnt target gene c-myc, is impaired in the skin of VDR-/- mice. Although Shh is not expressed in primary keratinocytes, Gli1 and c-myc mRNA levels are reduced in VDR-/- keratinocytes and restored by VDR transfection. ChIP analyses demonstrate that the VDR interacts with Gli1 regulatory sequences. Studies in keratinocytes from WT, VDR-/- and Lef1-/- mice will address the hypothesis that the unliganded VDR and Lef1 interact with regulatory regions of Gli1 in the context of intact chromatin and are required for basal and Wnt3a-induced expression of this gene. We will also examine if activation of the HH pathway induces anagen in VDR-/- mice. The VDR-/- mice also exhibit a defect in wound healing. Preliminary studies point to defects in macrophage recruitment and vascular invasion, which are also observed in vitamin D deficient WT mice, demonstrating that these reflect impaired ligand-dependent actions of the VDR. We will identify the cellular and molecular basis for the abnormalities observed and determine which actions of the VDR required for wound repair are ligand dependent. The goal of these studies is not to study skin biology per se, but rather to characterize the cellular and molecular basis for in vivo findings to identify novel actions of the liganded and unliganded VDR.

描述（由申请人提供）：本提案的研究重点是确定 VDR 配体依赖性和独立作用的分子基础，使用皮肤作为模型系统，与具有 VDR 突变的人类一样，VDR 缺失小鼠也会出现脱发。已经证明，VDR 的配体独立作用是毛囊角质形成细胞干细胞 (KSC) 功能、正常毛发周期和角质形成细胞中经典 Wnt (cWnt) 信号传导所必需的。干扰 cWnt 信号传导也会损害 KSC 功能，研究检查了 VDR 与经典 Wnt 信号传导效应子的相互作用，表明 VDR 通过其 DNA 结合域与 Lef1 相互作用，并且 Lef1 的 VDR 相互作用域独立于其β-连环蛋白结合。我们将检查角质形成细胞中 VDR-Lef1 相互作用受损的功能后果，并确定这两种蛋白质之间的相互作用是直接的还是涉及其他因素。 VDR 转基因的角质形成细胞特异性表达可挽救 VDR 缺失小鼠的毛发周期缺陷，而组成型活性β-连环蛋白转基因则不能，将 VDR 置于毛囊生长启动的水平或下游。生长期）通过cWnt信号传导在WT小鼠中诱导Shh和Gli1 mRNA，但在VDR-/-小鼠中不诱导此外，Shh、Gli1和经典的表达。 cWnt 靶基因 c-myc 在 VDR-/- 小鼠皮肤中受损，尽管 Shh 在原代角质形成细胞中不表达，但 VDR-/- 角质形成细胞中的 Gli1 和 c-myc mRNA 水平降低，并通过 VDR ChIP 转染恢复。分析表明，VDR 与 Gli1 调节序列相互作用，对 WT、VDR-/- 和 Lef1-/- 小鼠的角质形成细胞进行的研究将解决以下假设：未配体的 VDR 和 Lef1 在完整染色质的背景下与 Gli1 的调节区域相互作用，并且是该基因的基础和 Wnt3a 诱导表达所必需的。我们还将检查 HH 途径的激活是否会诱导 VDR-/- 小鼠的毛发生长初期。 -/- 小鼠还表现出伤口愈合缺陷，在维生素 D 缺乏的 WT 小鼠中也观察到了这一缺陷，表明这些缺陷反映了巨噬细胞募集和血管侵袭的缺陷。 VDR 的配体依赖性作用受损。我们将确定观察到的异常的细胞和分子基础，并确定伤口修复所需的 VDR 的哪些作用是配体依赖性的。这些研究的目的不是研究皮肤生物学本身。而是表征体内发现的细胞和分子基础，以确定配体和未配体 VDR 的新作用。