HNF-4 FUNCTION IN MODELS OF TRAUMATIC INJURY

描述（由申请人提供）：身体对严重创伤的即时反应，表示为急性期反应（APR），协调各种炎症信号，并在肝脏大量诱导保护性蛋白中产生共同反应。虽然APR是为生存而设计的，但严重或长期激活并中断正常的稳态功能，可能导致某些重症患者的器官衰竭和死亡。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，这是一个高度调节的过程，其共享对早期细胞类型发育重要的途径，并且具有与增殖反应相关的一些信号。有证据表明，损伤诱导的分化基因的调节可能是通过肝脏特异性转录因子，特别是HNF-4的磷酸化介导的。更好地理解调节这一过程的机制将在有利地支持急性期反应方面具有治疗重要性。第一个目标将剖析HNF-4磷酸化发生的位置。这将通过磷酸肽图谱进行。我们还将确定激酶，从而参与启动APR的信号转导途径。这将产生原始材料，以改变的HNF-4分子的形式，以测试这种磷酸化对APR的重要性。第二个目的是追踪这种磷酸化对HNF-4的生化活性，其DNA结合和位点选择的影响，以及它在细胞中的相互作用和活动的变化。这将使用诊断染色质免疫沉淀法进行。第三个目标将使用DNA微阵列，以产生损伤前后HNF-4调控的基因的图像。急性期诱导的体外模型也将用于允许直接操纵HNF-4并测量其对急性期转录事件的影响。这些研究将阐明HNF-4功能的机制，并测试HNF-4修饰的重要性。它将建立什么转录事件是共同的早期阶段的APR，当正常的肝功能被修改，肝脏准备随后的大规模调制蛋白质的生产。

DESCRIPTION (provided by applicant): The body's immediate reaction 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 the APR is designed for survival, severe or prolonged activation with its interruption of normal homeostatic functions, likely contributes to organ failure and death in some critically ill patients. 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 pathways important to early cell type development, and with some signals associated with the proliferative response. Evidence is presented that injury induced regulation of differentiated genes may be mediated through phosphorylation of liver-specific transcription factors, particularly HNF-4. Better understanding of the mechanisms regulating this process would have therapeutic importance in advantageous support of the acute phase response. The first aim will dissect where on HNF-4 phosphorylation occurs. This will be done by phosphopeptide mapping. We will also identify the kinases and thus the signal transduction pathways that are involved in initiating the APR. This will generate original materials, in the form of altered HNF-4 molecules, to test the importance of this phosphorylation to the APR. The second aim is to trace the effect of this phosphorylation on the biochemical activities of HNF-4, its DNA binding and site selection, and changes in its interactions and activities in the cell. This will be carried out using diagnostic chromatin immunoprecipitation. The third Aim will use DNA microarrays, to produce a picture of the genes regulated by HNF-4 before and after injury. An in vitro model of acute phase induction will also be used to allow direct manipulation of HNF-4 and to measure its effects on acute phase transcriptional events. These studies will elucidate the mechanisms of HNF-4 functions and test, the importance of HNF-4's modifications. It will establish what transcriptional events are common to the early phase of the APR, when normal liver function is modified, and the liver prepares for subsequent massive modulation of protein production.