Vitamin D Receptor Coactivators in Keratinocytes

角质形成细胞中的维生素 D 受体共激活剂

• 批准号: 7834026
• 负责人: DANIEL David BIKLE
• 金额: $ 37.19万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-28 至 2010-09-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7834026
• 关键词: Address; Affect; Binding; Complex; DNA-Directed RNA Polymerase; Data; Drug usage; Epidermis; FGR gene; Family; Funding; Genes; Grant; Histones; Immune response; In Vitro; Knowledge; Ligands; Link; Lipids; Mediating; Mediator of activation protein; Modeling; Mus; Names; Nuclear Hormone Receptors; Permeability; Play; Positioning Attribute; Process; Proliferating; Proteins; Regulation; Resistance; Role; Signal Pathway; Skin; Specificity; Steroid Receptors; TRAP Complex; Testing; Thyroid Gland; Transcription Initiation Site; Transferase; VDR interacting protein complex DRIP; Vitamin D; Vitamin D Response Element; Vitamin D3 Receptor; analog; base; gene function; high throughput screening; human RIPK1 protein; in vivo; keratinocyte; keratinocyte differentiation; mouse model; promoter; protein complex; public health relevance; receptor; receptor binding; receptor function; response; restoration; small molecule; thyroid hormone receptor associated protein 220

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.

描述（由申请人提供）：本建议书是作为我们同名的资助RO 1 AR 050023的补充而编制的。 该项目的基础是维生素D受体（VDR）的转录活性受许多辅激活因子和辅阻遏物复合物调节的知识，这些复合物在配体（1，25（OH）2D 3）依赖性（辅激活因子）或抑制性（辅阻遏物）过程中与VDR结合。在角质形成细胞中，主要的共激活因子复合物包括维生素D受体相互作用蛋白（DRIP）复合物（也称为介体或甲状腺受体激活蛋白（TRAP）复合物）和类固醇受体共激活因子（SRC）复合物，它们与DRIP一样具有多个名称。在形成SRC复合物的三种关键SRC蛋白中，仅在角质形成细胞中发现了SRC 2和3，并且在这两种SRC 3中，SRC 3在我们所研究的过程中起主导作用。DRIP复合物跨越从维生素D调节基因的启动子中的维生素D反应元件（VDRE）到转录起始位点的距离，将VDR连接到RNA聚合酶机制。SRC复合物含有组蛋白乙酰转移酶（HAT）活性，并被认为可以打开基因，使转录机制能够与受调节的基因结合。这些辅激活因子复合物在调节角质形成细胞增殖和分化中发挥不同的作用。我们发现DRIP复合物是增殖角质形成细胞中与VDR结合的主要复合物，而SRC 2和3及其相关蛋白是分化角质形成细胞中与VDR结合的主要共激活物。此外，DRIP 205（DRIP复合物中直接结合VDR的蛋白质）在体外和体内增殖的角质形成细胞中大量表达，而SRC 3主要在分化的角质形成细胞和表皮的上层（分化的）中表达。此外，我们已经发现DRIP 205在调节角质形成细胞增殖的wnt/2-连环蛋白信号传导途径的调节中的特定作用，而SRC 3调节1，25（OH）2D 3诱导更多分化功能的能力，例如渗透性屏障形成所需的脂质合成和加工以及由屏障破坏触发的先天免疫应答。这些发现为我们了解一种受体（VDR）和一种配体（1，25（OH）2D 3）如何以顺序和分化特异性方式调节大量基因提供了基础。此外，这些观察结果表明发现小分子的潜力，通过选择性地调节辅激活剂和VDR之间的相互作用，可以表现出调节VDR功能的特异性，而这是配体如1，25（OH）2D 3及其类似物所不能实现的。 我们已经成功地解决了原始提案中的所有目标，并验证了我们的假设，即维生素D调节的角质形成细胞分化过程中依次利用VDR共激活剂DRIP和SRC。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，我们希望超越目前赠款的目标，以测试以下假设。两种主要的共激活因子复合物DRIP和SRC差异性地调节VDR及其配体1，25（OH）2D 3抑制角质形成细胞增殖的能力，同时促进它们的分化，从而产生保护性表皮屏障。我们正在开发在角质形成细胞中选择性缺失DRIP 205和SRC 3的小鼠，并使用高通量筛选来鉴定将差异调节VDR与DRIP 205和SRC 3结合的分子，以测试这一假设。我们提出两个目标。目标1.确定DRIP 205和SRC 3在VDR调节表皮增殖、分化和保护屏障形成的小鼠模型中的选择性作用，其中DRIP 205和SRC 3在角质形成细胞中选择性缺失。目标二。开发选择性抑制DRIP 205和SRC 3对VDR功能调节的方法，使用高通量筛选鉴定对破坏VDR与DRIP 205和SRC 3结合具有选择性的分子，并使用这些药物选择性调节VDR/1，25（OH）2D 3诱导（抑制）维生素D靶基因的能力以及由DRIP 205和SRC 3差异调节的功能。我们预计，在未来一年，这两个目标的进展将使我们处于有利地位，成功恢复对该项目的全额资助。 公共卫生相关性：1，25（OH）2D 3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。我们已经发现，不同的共激活因子，特别是DRIP和SRC，调节VDR的转录活性不同的不同基因，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DRIP 205或SRC 3的小鼠，我们将进一步探索这些差异，以更全面地了解皮肤中VDR的转录活性，并开发选择性调节这些过程的治疗方法。