Role of LITAF in Inflammatory Processes

我们的长期目标是开创限制炎症过程的有害影响的新方法(如 在保留有益效果的同时(例如，组织修复、消除感染)。这些研究 我们建议在此建立在我们最新成果的基础上。最值得注意的是，我们发现了炎症的转录调节因子 细胞因子，包括关键的细胞因子肿瘤坏死因子-a(TNF-a)。肿瘤坏死因子受到严格监管；其生产过剩 可能是致命的，比如感染性休克综合征。在以前的研究中，我们发现了一种新的转录因子， 脂多糖诱导的肿瘤坏死因子-α(LITAF)调节肿瘤坏死因子基因的表达。最近，我们发现 另一个新发现的转录因子STAT6B与LITAF结合，这个复合体共同增强了LITAF 促炎症细胞因子GRO、IL-1a、RANTES、肿瘤坏死因子-α、干扰素-γ、单核细胞趋化蛋白-1和单核细胞趋化蛋白-2的表达， 血管内皮生长因子和抗炎细胞因子IL-10。这项提案的具体目的是为了测试 假设(1)LITAF刺激炎症反应，与STAT6B一起，这种反应是 (2)STAT6B单独上调血管内皮细胞生长因子，与LITAF共同抑制血管内皮生长因子。 调节LITAF和STAT6B活性的磷酸化-去磷酸化过程的建议鉴定 将有助于理解这些分子的细胞运输(目标1)。最小和特异的DNA 负责与MCP1启动子上的LITAF结合的序列，以及LITAF-STAT6B的作用(目标2)，将允许 鉴定这些蛋白质可能靶向的其他启动子。它还将允许对LITAF的作用进行评估 和STAT6B在各自基因的调控中发挥作用。为了实现这一点，我们将在 人单核细胞趋化蛋白启动子，揭示LITAF控制的DNA结合序列和STAT6B的作用。 将突变启动子连接到报告基因将使我们能够评估这些突变启动子是否未能 被LITAF和LITAF-STAT6B复合体激活。将设计突变的LITAF和STAT6B蛋白(突变蛋白) 鉴定DNA结合和反式激活结构域(目标2)。这些数据将为以下方面提供关键的见解 阐明其他基因中启动子的相互作用。将对STAT6B和VEGF采取类似的方法来确定 最小的血管内皮生长因子启动子序列和STAT6B多肽参与了这些DNA-蛋白质的相互作用。的作用 在同样的背景下，将积极寻求LITAF-STAT6B复合体抑制血管内皮生长因子(目标3)。LITAF的成功 基因敲除小鼠以及我们提出的建立STAT6B缺陷小鼠的建议将建立在AIMS 1和2的结果基础上 3评估LITAF和STAT6B基因产物通过调节细胞因子活性， 在包括牙周炎在内的炎症性疾病的发展中发挥重要作用(AIMS 3)。AIM 4将扩大规模 我们最近的发现将STAT6B与血管内皮生长因子和血管生成联系起来。我们打算测试血管生成是如何被调节的 通过STAT6B和LITAT-STAT6B复合体。我们发现了LITAF和新的蛋白质STAT6B，这些因素 有助于调节细胞因子的转录和血管生成，将成为剖析该复合体的新工具 在各种炎症条件下介导细胞因子表达的机制，包括牙周炎和 舰船编队。我们的目标是开发药理学方法，旨在调节炎症和 血管生成。

Our long term goal is to pioneer new approaches that limit the harmful effects of inflammatory processes (such as those observed in periodontitis), while preserving the beneficial effects (e.g. tissue repair, resolution of infections). The studies we propose here build upon our recent results. Most notably, we discovered transcriptional regulators of inflammatory cytokines, including the pivotal cytokine tumor necrosis factor-a (TNF-a). TNF is tightly regulated; its overproduction can be lethal, as in septic shock syndrome. In previous studies we discovered a new transcription factor, Lipopolysaccharide-lnduced TNF-Alpha Factor (LITAF), which regulates TNF gene expression. More recently, we found another newly identified transcription factor STAT6B that binds to LITAF, and together this complex enhances the expression of a whole group of pro-inflammatory cytokines: GRO, IL-1a, RANTES, TNF-a, IFN-y, MCP-1, and MCP-2, VEGF as well as the anti-inflammatory cytokine IL-10. The Specific Aims of this proposal are designed to test the hypotheses that (1) LITAF stimulates the inflammatory response and together with STAT6B this response is largely amplified and (2) that STAT6B alone upregulates VEGF while together with LITAF, VEGF is inhibited. The proposed identification of the phosphorylation-dephosphorylation processes governing LITAF and STAT6B activity will be instrumental in understanding the cell trafficking of these molecules (Aim 1). The minimal and specific DNA sequence responsible for protein binding to LITAF on MCP1 promoter, and the role of LITAF-STAT6B (Aim 2), will allow identification of other promoters that may be targeted by these proteins. It will also permit evaluation of the role of LITAF and STAT6B in the regulation of their respective genes. To accomplish this, we will engineer specific mutations within the human MCP promoters, to reveal the exact DNA binding sequence controlled by LITAF and the role of STAT6B. Coupling the mutant promoters to reporter genes will allow us to assess whether these mutant promoters fail to be activated by LITAF and by the LITAF-STAT6B complex. Mutant LITAF and STAT6B proteins (muteins) will be designed to identify both the DNA-binding and trans-activation domains (Aim 2). and this data will provide critical insights for elucidating promoter interactions in other genes. A similar approach will be taken for STAT6B and VEGF to determine the minimal VEGF promoter sequence and STAT6B peptide involved in these DNA-protein interactions. The role of LITAF-STAT6B complex in inhibiting VEGF will be actively pursued in the same context (Aim 3). The success of LITAF knockout mice along with our proposal of generating STAT6B-deficient mice will build upon the results of Aims 1 and 2 and 3 to evaluate the hypothesis that the LITAF and STAT6B gene products, through regulation of cytokine activity, play an important role in the development of inflammatory diseases, including periodontitis (Aims 3). Aim 4 will expand our recent finding linking STAT6B to VEGF and angiogenesis. We intend to test how angiogenesis can be modulated by STAT6B and the LITAT-STAT6B complex. Our discovery of LITAF and the novel protein STAT6B, factors that help regulate cytokine transcription and angiogenesis, will serve as new tools to dissect the complex mechanisms that mediate cytokine expression in various inflammatory conditions, including periodontitis and vessel formation. Our goal is to develop pharmacological approaches aimed at modulating inflammation and angiogenesis.