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

• 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
• 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

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（由申请方提供）：本提案中的研究重点是使用皮肤作为模型系统，确定VDR的配体依赖性和独立性作用的分子基础。与具有VDR突变的人类一样，VDR缺失小鼠发生脱发。我们已经证明，VDR的配体非依赖性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的典型Wnt（cWnt）信号传导所必需的。干扰cWnt信号传导也损害KSC功能。检查VDR与经典Wnt信号传导效应物的相互作用的研究表明，VDR通过其DNA结合结构域与Lef 1相互作用，并且Lef 1的VDR相互作用结构域不依赖于其β-连环蛋白结合结构域。我们将研究损害角质形成细胞中VDR-Lef 1相互作用的功能后果，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。 虽然VDR转基因的角质形成细胞特异性表达挽救了VDR缺失小鼠中的毛发周期缺陷，但组成型活性β-连环蛋白转基因不能，将VDR置于cWnt信号传导激活的水平或下游。通过cWnt信号传导启动毛囊生长（生长期）诱导WT小鼠中的Shh和Gli 1 mRNA，但不在VDR-/-小鼠中。此外，Shh、Gli 1和经典cWnt靶基因c-myc的表达在VDR-/-小鼠的皮肤中受损。虽然Shh在原代角质形成细胞中不表达，但Gli 1和c-myc mRNA水平在VDR-/-角质形成细胞中降低，并通过VDR转染恢复。ChIP分析表明VDR与Gli 1调控序列相互作用。对WT、VDR-/-和Left 1-/-小鼠角质形成细胞的研究将解决以下假设：未配体的VDR和Left 1在完整染色质的情况下与Gli 1的调节区相互作用，并且是该基因的基础和Wnt 3a诱导表达所需的。我们还将检查HH通路的激活是否诱导VDR-/-小鼠的生长期。VDR-/-小鼠也表现出伤口愈合缺陷。初步研究指出巨噬细胞募集和血管侵袭的缺陷，这也在维生素D缺乏的WT小鼠中观察到，表明这些反映了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，并确定创伤修复所需的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，以确定配体和非配体VDR的新作用。

项目成果

Phosphate regulates embryonic endochondral bone development.

• DOI: 10.1002/jcb.22302
• 发表时间: 2009-10-15
• 期刊: JOURNAL OF CELLULAR BIOCHEMISTRY
• 影响因子: 4
• 作者: Zalutskaya, Alena A.;Cox, Megan K.;Demay, Marie B.
• 通讯作者: Demay, Marie B.

The vitamin D receptor, the skin and stem cells.

维生素 D 受体、皮肤和干细胞。

• DOI: 10.1016/j.jsbmb.2010.01.015
• 发表时间: 2010
• 期刊: The Journal of steroid biochemistry and molecular biology
• 作者: Luderer,HilaryF;Demay,MarieB
• 通讯作者: Demay,MarieB

Analysis of vitamin D-dependent calcium-binding protein messenger ribonucleic acid expression in mice lacking the vitamin D receptor.

• DOI: 10.1210/endo.139.3.5803
• 发表时间: 1998-03
• 期刊: Endocrinology
• 影响因子: 4.8
• 作者: Yan Chun Li;A. Pirro;M. Demay

Evaluation of keratinocyte proliferation and differentiation in vitamin D receptor knockout mice.

• DOI: 10.1210/endo.141.6.7515
• 发表时间: 2000-06
• 期刊: Endocrinology
• 影响因子: 4.8
• 作者: Yoshiyuki Sakai;M. Demay

Highlights from the 19th Workshop on Vitamin D in Boston, March 29-31, 2016.

2016 年 3 月 29 日至 31 日在波士顿举行的第 19 届维生素 D 研讨会要点。

• DOI: 10.1016/j.jsbmb.2017.06.005
• 发表时间: 2017
• 期刊: The Journal of steroid biochemistry and molecular biology
• 作者: Christakos,Sylvia;White,JohnH;Hewison,Martin;Welsh,JoEllen;Lips,Paul;Bouillon,Roger;Demay,MarieB
• 通讯作者: Demay,MarieB