Regulation of the Human iNOS Gene in Sepsis and Trauma

描述（由申请人提供）： 人诱导型一氧化氮合酶（hiNOS）基因在脓毒症和其他炎症状态期间几乎在每个器官中表达。虽然NO合成在急性炎症期间具有有益作用，但过量的NO产生是有害的。慢性hiNOS表达与NO介导的组织损伤有关，导致糖尿病、神经退行性疾病和某些癌症。我们的实验室已经从细胞因子刺激的肝细胞中克隆了人iNOS基因，并且我们已经表明，精氨酸应答DNA元件位于启动子区上游约5 kb处。我们发现TNF α和IL-1 β通过NF-κ B传递信号，而IFN γ通过与启动子中-5.2和-5.8 kb处的顺式作用元件结合通过Stat-1传递信号，从而为细胞因子协同作用提供了分子基础。AP-1、C/EBP β（KLF）和KLF 6的进一步功能作用已得到证实。重要的是，我们已经确定了涉及NF-κ B抑制因子（NRF），LIP和p53蛋白的hiNOS抑制机制。随后的染色质结构分析，使用DNA酶I映射和体内足迹显示，调节hiNOS转录甚至比原来预期的更复杂，并表现出组织特异性控制的基础和诱导型转录因子。最近，我们已经确定了Wnt β-catenin/Tcf-4信号通路在调节hiNOS表达中的新作用。因此，我们的假设是，hiNOS基因表达的调节需要一个协调的流动的正和负转录因子结合到一个顺式作用的上游增强子区域位于-5.0和-7.0 kb之间的hiNOS启动子。此外，在约0.2kb处已经鉴定了关键的下游启动子区域，其允许奎宁诱导的转录。在这个计划中，我们将追求两个具体的目标，以进一步阐明所涉及的分子机制：目的I：确定负责细胞因子诱导人iNOS基因的转换因子和功能启动子元件。ChIP试验将用于确认上游增强子区域中NF-κ B、Stat-1和AP-1的体内蛋白-DNA相互作用。Ets-1和Oct-1的新作用将通过凝胶迁移和启动子转染研究进行测试。NRF和NF-kB之间的蛋白质-蛋白质相互作用将被追求，以及由NRF/LIP，KLF 6和p53引起的下游控制机制。目的二：探讨WNT β-catenin/TCF-4信号通路对人iNOS表达的调控作用。将确定β-连环蛋白/Tcf-4与两个TBE位点结合的功能作用，以及与其他调控hiNOS表达的转录因子的可能相互作用。所获得的信息将增加我们对hiNOS转录控制的理解，描述信号转导中精氨酸协同作用的新机制，并有助于设计hiNOS表达相关的病理生理疾病状态的治疗策略。

DESCRIPTION (provided by applicant): The human inducible nitric oxide synthase (hiNOS) gene is expressed in nearly every organ during sepsis and other inflammatory conditions. While NO synthesis has beneficial effects during acute inflammation, excessive NO production is harmful. Chronic hiNOS expression has been implicated in NO-mediated tissue damage leading to diabetes, neurodegenerative disorders, and certain cancers. Our laboratory has cloned the human iNOS gene from eytokine-stimulated hepatoeytes, and we have shown that cytokine-responsive DNA elements are located approximately 5 kb upstream in the promoter region. We found that TNFalpha and IL-1beta signal through NF-kappaB, while IFNgamma signals through Stat-1 by binding to cis-acting elements at -5.2 and -5.8 kb in the promoter, thereby providing a molecular basis for cytokine synergy. Further functional roles have been demonstrated for AP-1, C/EBPbeta (LAP), and KLF6. Importantly, we have identified mechanisms for hiNOS repression that involve NF-kappaB repressing factor (NRF), LIP, and p53 proteins. A subsequent chromatin structure analysis using DNAse I mapping and in vivo footprinting revealed that regulation of hiNOS transcription was even more complex than originally anticipated and exhibited tissue-specific control by both basal and inducible transcription factors. Most recently, we have identified a novel role for the Wnt beta-catenin/Tcf-4 signaling pathway in regulating hiNOS expression. Therefore, our hypothesis is that the regulation of hiNOS gene expression requires an orchestrated flow of positive and negative transcription factors binding to a cis-acting upstream enhancer region located between -5.0 and -7.0 kb in the hiNOS promoter. In addition, a crucial downstream promoter region has been identified at -0.2 kb that is permissive for cytokine-induced transcription. In this proposal, we will pursue two specific aims to further elucidate the molecular mechanisms involved: AIM I: TO DEFINE THE TRANSCRIPTION FACTORS AND FUNCTIONAL PROMOTER ELEMENTS RESPONSIBLE FOR CYTOKINE INDUCTION OF THE HUMAN iNOS GENE. ChIP assay will be used to confirm in vivo protein-DNA interactions for NF-kappaB, Stat-1, and AP-1 in the upstream enhancer region. New roles for Ets-1 and Oct-1 will be tested by gel shift and promoter transfection studies. Protein-protein interactions between NRF and NF-kB will be pursued, as well as mechanisms of downstream control elicited by LAP/LIP, KLF6, and p53. AIM II: TO DETERMINE THE ROLE OF THE WNT Beta-CATENIN/TCF-4 SIGNALING PATHWAY IN REGULATING HUMAN iNOS EXPRESSION. A functional role for Beta-catenin/Tcf-4 binding to two TBE sites will be determined, as well as possible interactions with other transcription factors governing hiNOS expression. The information gained will increase our understanding of the control of hiNOS transcription, describe novel mechanisms of cytokine-synergy in signal transduetion, and help in designing therapeutic strategies for pathophysiologic disease states where hiNOS expression is relevant.