Vitamin D Receptor Coactivators in Keratinocytes

描述(由申请人提供)：本建议书是为补充我们的同名拨款RO1 AR050023而准备的。这个项目的基础是知道维生素D受体(VDR)的转录活性受一些辅助激活物和辅助抑制物复合体的调节，这些复合体在配体(1，25(OH)2D3)依赖的(辅助激活物)或抑制(辅助抑制物)的过程中与VDR结合。在角质形成细胞中，主要的辅活化子复合体包括维生素D受体相互作用蛋白(DRIP)复合体(也称为介体或甲状腺受体激活蛋白{TRAP}复合体)和类固醇受体辅活化子(SRC)复合体，它们和DRIP一样有多种名称。在角质形成细胞中，对SRC复合体的形成起关键作用的3种SRC蛋白中，仅发现了SRC 2和3，其中SRC3在我们所研究的过程中起主导作用。水滴复合体跨越了从维生素D调节基因启动子中的维生素D反应元件(VDRE)到转录起始点的距离，将VDR与RNA聚合酶机制联系起来。SRC复合体含有组蛋白乙酰转移酶(HAT)活性，被认为打开了基因，使转录机制能够与被调控的基因结合。这些共激活复合体在调节角质形成细胞的增殖和分化中发挥着不同的作用。我们发现，在增殖的角质形成细胞中，水滴复合体是与VDR结合的主要复合体，而在分化的角质形成细胞中，SRC2和3及其相关蛋白是与VDR结合的主要共激活因子。此外，DRIP205是水滴复合体中直接与VDR结合的蛋白，在体外和体内增殖的角质形成细胞中大量表达，而SRC3主要在分化的角质形成细胞和表皮的上层(分化)表达。此外，我们发现DRIP205在调节WNT/2-catenin信号通路调节角质形成细胞增殖中具有特定的作用，而SRC3调节1，25(OH)2D3诱导更多分化功能的能力，如通透性屏障形成所需的脂质合成和加工，以及屏障被破坏所触发的先天免疫反应。这些发现为我们理解一个受体(VDR)和一个配体(1，25(OH)2D3)如何以顺序和分化特异性的方式调节大量基因提供了基础。此外，这些观察表明，有可能发现小分子，通过选择性地调节共激活子和VDR之间的相互作用，可以在调节VDR功能方面表现出特异性，这是1，25(OH)2D3及其类似物等配体无法实现的。我们已经成功地解决了最初提案中的所有目标，并验证了我们的假设，即在维生素D调节的角质形成细胞分化过程中，VDR辅助激活剂Drop和SRC被顺序利用。本竞争性修订申请中的初步数据部分将证明这一事实。在接下来的一年里，我们希望超越当前赠款的目标，测试以下假设。两个主要的共激活复合体，水滴和SRC，不同地调节VDR及其配体1，25(OH)2D3抑制角质形成细胞增殖的能力，同时促进角质形成细胞的分化，从而形成保护性的表皮屏障。我们正在开发在角质形成细胞中选择性缺失DRIP205和SRC3的小鼠，并使用高通量筛选来识别将差异调节VDR与DRIP205和SRC3结合的分子，以检验这一假说。我们提出了两个目标。目的1.在角质形成细胞中DRIP205和SRC3选择性缺失的小鼠模型中，确定DRIP205和SRC3在VDR调节表皮增殖、分化和保护性屏障形成中的选择性作用。目的：通过高通量筛选，建立选择性抑制DRIP205和SRC3调节VDR功能的方法，以确定选择性破坏VDR与DRIP205和SRC3结合的分子，并利用这些药物选择性地调节VDR/1，25(OH)2D3诱导(抑制)受DRIP205和SRC3差异调控的维生素D靶基因和功能的能力。我们预计，来年在这两个目标方面取得的进展将使我们处于成功恢复对该项目的全额供资的有利地位。 与公共健康相关：1，25(OH)2D3及其受体VDR调节角质形成细胞分化和功能的机制尚不清楚。我们发现，不同的共激活因子，特别是水滴和SRC，针对不同的基因调节VDR的转录活性，从而选择性地影响角质形成细胞中的不同功能。通过开发缺乏DRIP205或SRC3的小鼠，我们将进一步探索这些差异，以努力更全面地了解VDR在皮肤中的转录活性，并开发有选择地调节这些过程的治疗方法。

DESCRIPTION (provided by applicant): This proposal has been prepared as a supplement to our grant RO1 AR050023 of the same name. The basis for this project is the knowledge that the transcriptional activity of the vitamin D receptor (VDR) is regulated by a number of coactivator and corepressor complexes, which bind to the VDR in a ligand (1,25(OH)2D3) dependent (coactivators) or inhibited (corepressors) process. In the keratinocyte the major coactivator complexes include the vitamin D receptor interacting protein (DRIP) complex (also known as Mediator or Thyroid Receptor Activating Protein {TRAP} complex) and the steroid receptor coactivator (SRC) complexes, which like DRIP have multiple names. Of the three SRC proteins critical for formation of SRC complexes, only SRC 2 and 3 have been found in keratinocytes, and of these two SRC3 plays the dominant role in the processes we have examined. The DRIP complex spans the distance from vitamin D response elements (VDRE) in the promoters of vitamin D regulated genes to the transcription start site, linking the VDR to the RNA polymerase machinery. The SRC complexes contain histone acetyl transferase (HAT) activity and are thought to open up the gene to enable the transcriptional machinery to bind to the gene being regulated. These coactivator complexes play different roles in regulating keratinocyte proliferation and differentiation. We found that the DRIP complex is the main complex binding to VDR in the proliferating keratinocyte, whereas SRC2 and 3 and their associated proteins are the major coactivators binding to VDR in the differentiated keratinocyte. Furthermore, DRIP205, the protein in the DRIP complex that binds directly to VDR, is abundantly expressed in proliferating keratinocytes in vitro and in vivo, whereas SRC3 is expressed primarily in differentiated keratinocytes and in the upper (differentiated) layers of the epidermis. Moreover, we have found a specific role for DRIP205 in the regulation of wnt/2-catenin signaling pathways regulating keratinocyte proliferation, whereas SRC3 regulates the ability of 1,25(OH)2D3 to induce more differentiated functions such as lipid synthesis and processing required for permeability barrier formation and the innate immune response triggered by disruption of the barrier. These findings provide a basis by which we can understand how one receptor (VDR) and one ligand (1,25(OH)2D3) can regulate a large number of genes in a sequential and differentiation specific fashion. Furthermore, these observations indicate the potential for discovering small molecules that by selectively modulating the interactions between the coactivators and VDR can manifest specificity in regulating VDR function not achievable by ligands such as 1,25(OH)2D3 and its analogs. We have successfully addressed all aims in the original proposal and verified our hypothesis that the VDR coactivators DRIP and SRC are sequentially utilized during vitamin D regulated keratinocyte differentiation. The preliminary data section in this competitive revision application will demonstrate that fact. During the coming year we want to move beyond the aims of the current grant to test the following hypothesis. The two main coactivator complexes, DRIP and SRC, differentially modulate the ability of VDR and its ligand 1,25(OH)2D3 to inhibit keratinocyte proliferation, while promoting their differentiation leading to a protective epidermal barrier. We are developing mice in which DRIP205 and SRC3 are selectively deleted in keratinocytes and using high throughput screening to identify molecules that will differentially regulate VDR binding to DRIP205 and SRC3 to test this hypothesis. We propose 2 aims. Aim 1. Determine the selective roles of DRIP205 and SRC3 in VDR regulation of epidermal proliferation, differentiation, and protective barrier formation in mouse models in which DRIP205 and SRC3 are selectively deleted in keratinocytes. Aim 2. Develop the means to selectively inhibit DRIP205 and SRC3 modulation of VDR function using high throughput screening to identify molecules selective for disruption of the binding of VDR to DRIP205 and SRC3 and use these drugs to selectively modulate the ability of VDR/1,25(OH)2D3 to induce (suppress) vitamin D target genes and functions differentially regulated by DRIP205 and SRC3. We anticipate that progress in these two aims over the coming year will put us in excellent position for a successful restoration of full funding for this project. PUBLIC HEALTH RELEVANCE: The mechanisms by which 1,25(OH)2D3 and its receptor VDR regulate keratinocyte differentiation and function remain unclear. We have discovered that different coactivators, DRIP and SRC in particular, regulate the transcriptional activity of VDR differently for different genes, and thus selectively affect different functions in the keratinocyte. By developing mice lacking either DRIP205 or SRC3, we will further explore these differences in an effort to more fully understand VDR transcriptional activity in the skin and develop means of selectively regulating these processes therapeutically.