The Vitamin D Receptor: Ligand-Dependent and Independent Actions

描述（由申请人提供）：本提案中的研究重点是确定VDR的配体依赖和独立作用的分子基础，使用皮肤作为模型系统。与VDR突变的人类一样，VDR基因缺失的小鼠也会出现脱发。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、正常头发循环和角质形成细胞中典型Wnt （cWnt）信号传导所必需的。干扰cWnt信号也会损害KSC功能。研究VDR与典型Wnt信号效应物相互作用的研究表明，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-catenin结合域。我们将研究削弱VDR-Lef1相互作用在角化细胞中的功能后果，并确定这两种蛋白之间的相互作用是直接的，还是涉及其他因素。虽然角化细胞特异性表达的VDR转基因可以挽救VDR缺失小鼠的毛发周期缺陷，但组成型活性的-catenin转基因却不能，使VDR处于cWnt信号激活的水平或下游。cWnt信号在WT小鼠中诱导毛囊生长（生长期），而在VDR-/-小鼠中不诱导Shh和Gli1 mRNA。此外，Shh、Gli1和经典cWnt靶基因c-myc在VDR-/-小鼠皮肤中的表达受损。虽然Shh在原代角质形成细胞中不表达，但在VDR-/-角质形成细胞中Gli1和c-myc mRNA水平降低，并通过VDR转染恢复。ChIP分析表明，VDR与Gli1调控序列相互作用。对WT、VDR-/-和Lef1-/-小鼠角质形成细胞的研究将证实在染色质完整的情况下，未配体的VDR和Lef1与Gli1的调控区域相互作用，是基础和wnt3a诱导的Gli1基因表达所必需的。我们还将研究HH通路的激活是否会诱导VDR-/-小鼠的增生。VDR-/-小鼠在伤口愈合方面也表现出缺陷。初步研究指出巨噬细胞募集和血管侵袭缺陷，这在维生素D缺乏的WT小鼠中也观察到，表明这些反映了VDR的配体依赖作用受损。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的哪些作用依赖于配体。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。

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.