Ligand independent signaling by VDR in Keratinocytes

描述（由申请人提供）：1,25-二羟基维生素D3 （1,25(OH)2D3）的经典生理作用是通过确保肠道对膳食钙的充分吸收来维持适当的钙稳态。然而，1,25(OH)2D3及其同源受体维生素D受体（VDR）在皮肤和角化细胞生物学中也起着关键作用。事实上，VDR是使用125 (OH)2D3类似物治疗牛皮癣（一种皮肤增生性疾病）的临床相关靶点。本研究计划的重点是表皮角质形成细胞中的维生素D内分泌系统，特别是利用维生素D内分泌系统的小鼠模型作为工具，探索维生素D在皮肤中的体内作用和确定维生素D作用的潜在机制。特别是，VDR基因敲除小鼠（VDRKO）对化学诱导的皮肤肿瘤发生高度敏感。相比之下，我们的初步数据显示，基因消融CYP27B1（编码肾α -羟化酶（1aOHase）的基因，产生生物活性1,25(OH)2D3激素配体的酶）完全抵抗化学诱导的皮肤肿瘤发生。这些数据以及在这些小鼠模型和人类中发表的关于毛囊循环的数据表明，VDR在角化细胞中具有新的不依赖于配体的作用。未配体VDR在皮肤中肿瘤抑制作用的分子细节是完全未知的。我们的初步体内和体外数据支持一个模型，即VDR-RXR异源二聚体在角化细胞中选择性地在缺乏其1,25(OH)2D3配体的情况下被EGF激活。在缺乏1,25(OH)2D3配体的情况下，EGF处理驱动VDR和RXR的异源二聚化，这反过来促进异源二聚体结合，选择参与多种过程的角质细胞基因，如毛囊循环和保护皮肤组织免受dna损伤剂引发的肿瘤发生。重要的是，这种机制存在于选择性靶基因中，即并非所有已建立的VDR靶基因都受无配体VDR调控。为了了解VDRKO和11OHaseKO小鼠的体内生物学，需要对这类表达受未配体VDR-RXR控制的未定义基因进行定义。为了确定角化细胞中无配体VDR-RXR的直接靶标基因，我们将对小鼠角化细胞和纯化的WT、VDRKO和1aOHaseKO小鼠的膨出干细胞进行基因表达阵列分析。公共卫生相关性：未配体VDR在皮肤中肿瘤抑制作用的分子细节是完全未知的。我们的工作假设表明，在缺乏125 (OH)2D3配体的情况下，角质形成细胞中的EGF信号通路影响VDR，促进VDR- rxr异源二聚化和选择靶基因的转激活。本提案的主要目的是确定角化细胞中未配体VDR-RXR异源二聚体的靶基因。

DESCRIPTION (provided by applicant): The classic physiological role for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is to maintain appropriate calcium homeostasis by ensuring adequate intestinal absorption of dietary calcium. However, 1,25(OH)2D3 and its cognate receptor, the vitamin D receptor (VDR), also have critical roles in the skin and in keratinocyte biology. In fact, VDR is a clinically relevant target for the use of 1,25(OH)2D3 analogs in the treatment of psoriasis, a hyperproliferative disorder of the skin. The focus of this research proposal is on the vitamin D endocrine system in epidermal keratinocytes and, specifically, on the use of murine models of the vitamin D endocrine system as tools to explore the in vivo role and identify potential mechanisms of vitamin D action in the skin. In particular, the VDR knockout mouse (VDRKO) is highly susceptible to chemically-induced skin tumorigenesis. In contrast, our preliminary data show that genetic ablation of CYP27B1, the gene encoding the renal 1alpha- hydroxylase (1aOHase), the enzyme that generates the bioactive 1,25(OH)2D3 hormonal ligand, is completely resistant to chemically-induced skin tumorigenesis. These data as well as published data on hair follicle cycling in these mouse models and in humans, point to a novel ligand-independent role for VDR in keratinocytes. The molecular details involved in the tumor suppressor role for unliganded VDR in the skin are completely unknown. Our preliminary in vivo and in vitro data support a model in which the VDR-RXR heterodimer is activated by EGF in the absence of its 1,25(OH)2D3 ligand selectively in keratinocytes. EGF treatment drives heterodimerization of VDR and RXR in the absence of the 1,25(OH)2D3 ligand and this in turn promotes heterodimer binding to select keratinocyte genes involved in diverse processes such as hair follicle cycling and in protecting skin tissue from tumorigenesis initiated by DNA-damaging agents. Importantly, this mechanism exists for select target genes, i.e., not all established VDR target genes are regulated by unliganded VDR. It is this class of undefined genes whose expression is controlled by unliganded VDR-RXR that need to be defined in order to understand the in vivo biologies of the VDRKO and 11OHaseKO mice. In order to define the genes that are direct targets for unliganded VDR-RXR in keratinocytes, we will use gene expression array analysis of mouse keratinocytes and purified bulge stem cells from WT, VDRKO, and 1aOHaseKO mice. PUBLIC HEALTH RELEVANCE: The molecular details involved in the tumor suppressor role for unliganded VDR in the skin are completely unknown. Our working hypothesis states that EGF signaling in keratinocytes impinges on VDR to promote VDR-RXR heterodimerization and transactivation of select target genes in the absence of the 1,25(OH)2D3 ligand. The main goal of this proposal is to define target genes for unliganded VDR-RXR heterodimers in keratinocytes.