Role of LITAF in Inflammatory Processes

我们的长期目标是开拓新的方法，限制炎症过程的有害影响（如 在牙周炎中观察到），同时保持有益效果（例如，组织修复、感染消退）。研究 我们建议在此以我们最近的成果为基础。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，包括关键细胞因子肿瘤坏死因子-α（TNF-α）。TNF受到严格的调控，它的过度生产 可能是致命的比如感染性休克综合症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TNF-α因子（LITAF），调节TNF基因表达。最近，我们发现 另一种新鉴定的转录因子STAT 6 B与LITAF结合，这种复合物共同增强了 一整组促炎细胞因子的表达：GRO、IL-1 α、RANTES、TNF-α、IFN-γ、MCP-1和MCP-2， VEGF以及抗炎细胞因子IL-10。本提案的具体目标旨在测试 假设（1）LITAF刺激炎症反应，并且与STAT 6 B一起， 很大程度上扩增，和（2）单独的STAT 6 B上调VEGF，而与LITAF一起，VEGF被抑制。 LITAF和STAT 6 B活性的磷酸化-去磷酸化过程的鉴定 将有助于理解这些分子的细胞运输（目标1）。最小和特定的DNA 负责与MCP 1启动子上的LITAF蛋白结合的序列，以及LITAF-STAT 6 B（Aim 2）的作用，将允许 鉴定可能被这些蛋白质靶向的其他启动子。它还将允许评估LITAF的作用 和STAT 6 B在各自基因的调节中的作用。为了实现这一目标，我们将在 人MCP启动子，以揭示由LITAF控制的确切DNA结合序列和STAT 6 B的作用。 将突变启动子与报告基因偶联将使我们能够评估这些突变启动子是否不能被表达。 由LITAF和LITAF-STAT 6 B复合物激活。将设计突变LITAF和STAT 6 B蛋白（突变蛋白） 以鉴定DNA结合和反式激活结构域（Aim 2）。这些数据将提供关键的见解， 阐明其他基因中的启动子相互作用。对于STAT 6 B和VEGF将采取类似的方法来确定 最小的VEGF启动子序列和STAT 6 B肽参与这些DNA-蛋白质相互作用。的作用 LITAF-STAT 6 B复合物在抑制VEGF中的作用将在相同的背景下被积极追求（目的3）。LITAF的成功 敲除小鼠沿着我们提出的产生STAT 6 B缺陷小鼠的方案将建立在目标1和2的结果之上 和3评估LITAF和STAT 6 B基因产物，通过调节细胞因子活性， 在包括牙周炎在内的炎性疾病的发展中起重要作用（目的3）。Aim 4将扩大 我们最近的发现将STAT 6 B与VEGF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， STAT 6 B和LITAT-STAT 6 B复合物。我们发现LITAF和新型蛋白STAT 6 B， 有助于调节细胞因子转录和血管生成，将作为新的工具，剖析复杂的 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 血管形成我们的目标是开发旨在调节炎症的药理学方法， 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.