HNF-4 FUNCTION IN MODELS OF TRAUMATIC INJURY

描述（由申请人提供）：机体对严重创伤的直接反应，称为急性期反应（Acute Phase Response， APR），协调多种炎症信号，并在肝脏大量诱导保护蛋白的过程中产生共同反应。虽然APR是为生存而设计的，但严重或长时间的激活会中断正常的体内平衡功能，可能导致一些危重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，这是一个高度调控的过程，共享早期细胞类型发育的重要途径，以及与增殖反应相关的一些信号。有证据表明，分化基因的损伤诱导调节可能是通过肝脏特异性转录因子的磷酸化介导的，特别是HNF-4。更好地了解调节这一过程的机制对支持急性期反应具有重要的治疗意义。第一个目标是剖析HNF-4磷酸化发生的位置。这将通过磷酸肽作图来完成。我们还将确定启动apr的激酶和信号转导途径。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。第二个目标是追踪这种磷酸化对HNF-4生化活性的影响，它的DNA结合和位点选择，以及它在细胞中的相互作用和活动的变化。这将使用诊断性染色质免疫沉淀进行。第三个目标将使用DNA微阵列，生成受伤前后受HNF-4调控的基因的图像。急性期诱导的体外模型也将用于直接操纵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的功能机制，并检验其修饰的重要性。它将确定APR早期阶段共有的转录事件，此时正常的肝功能被修改，肝脏为随后的蛋白质生产大规模调节做准备。

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.