HNF-4 FUNCTION IN MODELS OF TRAUMATIC INJURY

描述(由申请人提供)： 人体对严重创伤的直接反应，即急性时相反应(APR)，协调各种炎症信号，并在肝脏大量诱导保护蛋白的过程中产生共同的反应。虽然APR是为生存而设计的，但这种反应的严重或长期激活可导致器官衰竭和死亡，因为其诱导不可避免地与正常肝功能的中断有关。有证据表明，APR对稳态肝功能的抑制是其诱导模式的副产品。进一步的证据表明，这是一个高度调控的过程，共享早期的细胞状态，以及一些与增殖反应有关的信号。有证据表明，分化基因的负调控可能是通过肝脏特异性转录因子的小网络的磷酸化而介导的，特别是HNF-4。更好地了解这一过程可能对急性期的更可靠的解决具有潜在的治疗重要性。第一个目的是通过磷酸肽图分析HNF-4的磷酸化发生在哪里，以确定哪些激酶和信号转导途径参与其中。这也将产生原始材料，以改变HNF-4分子的形式，以测试这种磷酸化对APR和再生的重要性。第二个目的是通过诊断染色质免疫沉淀，追踪这种磷酸化对HNF-4的生化活性的影响，即它的DNA结合和位点选择，以及它在细胞内相互作用和活性的变化。第三，利用DNA微阵列，将获得受HNF-4及其修饰调控的基因的全局图像，并将其与早期几个急性阶段诱导的基因进行比较。这将检验hnf-4‘S修饰的重要性，并确定在APR的早期阶段，当正常肝功能回缩，肝脏为随后的大规模诱导做准备时，哪些转录事件是共同的。

DESCRIPTION (provided by applicant): The body's immediate response to serious trauma, denoted the Acute Phase Response (APR), coordinates a wide variety of inflammatory signals, and produces a common response, in the massive induction of protective proteins by the liver. While APR is designed for survival, severe or prolonged activation of this response can lead to organ failure and death, as its induction is inevitably associated with interruption of normal liver function. Evidence suggests that the APR's repression of steady-state liver function is a byproduct of its mode of induction. Further evidence suggests that this is a highly regulated process which shares an early cell state, and some signals with the proliferative response. Evidence is presented that negative regulation of differentiated genes may be mediated through phosphorylation of a small web of liver-specific transcription factors, particularly HNF-4. Better understanding of this process could potentially have therapeutic importance in allowing more reliable resolution of the acute phase. The first aim is to dissect where HNF-4 phosphorylation occurs, by phosphopeptide mapping, to identify what kinases and thus signal transduction pathways are involved. This will also generate original materials, in the form of altered HNF-4 molecules, to test the importance of this phosphorylation to APR and regeneration. The second aim is to trace the effect of this phosphorylation on the biochemical activities of HNF-4, namely its DNA binding and site selection, and changes in its interactions and activities in the cell, by diagnostic chromatin immunoprecipitation. Third, using DNA microarrays, a global picture of the genes regulated by HNF-4 and its modification will be obtained and compared to that from several acute phase inductions at early stages. This will test both the importance of HNF-4's modification, and establish what transcriptional events are common to the early phase of APR, when normal liver function retracts and the liver prepares for subsequent massive induction.