HEME OXYGENASE-1 GENE EXPRESSION IN ENDOTOXIC SHOCK

内毒素休克是严重革兰氏病的危及生命的后果- 以血管平滑肌细胞为特征的阴性感染 放松和严重的低血压。一氧化氮(NO)，通过 诱导型一氧化氮合酶(INOS)途径是其中的主要贡献者 进程。然而，我们实验室最近的调查表明 血红素加氧酶(HO)，一种产生一氧化碳(CO)的酶 在血红素代谢过程中，也可能参与了 内毒素休克的血流动力学妥协。诱导型HO(HO-1)为 接受内毒素的大鼠血管组织中显著增加， 和非选择性HO活性抑制剂消除内毒素诱导 低血压。此外，HO-1mRNA的诱导也处于 基因转录。转录因子与转录因子的相互作用之外 它们的DNA结合域，一个合适的染色质环境是 对基因转录很重要。通过改变染色质结构， 结构蛋白将转录因子组装成增强子 很复杂。一种这样的结构蛋白，高迁移率基团蛋白 (HMG)-I(Y)，已被证明促进协同结合 转录因子转化成一种复合体，这种复合体可以有效地 协同提高基因转录。来自我们的初步数据 实验室研究表明，与HO-1一样，HMG-I(Y)的mRNA和蛋白在 IL-1β和内毒素对体外培养血管平滑肌细胞的作用 活着。此外，HMG-I(Y)与HO-1的5‘侧翼序列结合 吉恩。在本申请中，我们建议测试HO-1在 内毒素休克，并研究其调控的分子机制。 血管平滑肌细胞中HO-1的转录诱导。 提出了三个具体目标。目的1：确定HO-1的作用 内毒素引起的低血压和通过产生缺陷小鼠而死亡 在HO-1基因中。我们假设HO-1基因的缺失将 预防体内内毒素引起的低血压和死亡。目标2：实现 识别特定的DNA序列(顺式作用元件) 对血管平滑肌细胞中HO-1基因的诱导很重要，以及 染色质在HO-1基因调控中的作用 抄写。我们假设顺式作用的相互作用 在一个综合环境中，具有反式作用因素的要素 HO-1基因转录。最后，目标3：确定和描述 那些DNA结合蛋白(反式作用因子)，与 与HMG-I(Y)结合，对HO-1基因的诱导具有重要调控作用 IL-1β在血管平滑肌细胞中的转录。这些 研究应该为HO-1的调控提供新的见解，并且 内毒素休克的发病机制。

Endotoxic shock is a life-threatening consequence of severe gram- negative infection characterized by vascular smooth muscle cell relaxation and severe hypotension. Nitric oxide (NO), through the inducible NO synthase (iNOS) pathway, is a major contributor in this process. However, recent investigations from our laboratory suggest that heme oxygenase (HO), an enzyme that generates carbon monoxide (CO) in the course of heme metabolism, may also be involved in the hemodynamic compromise of endotoxic shock. Inducible HO (HO-1) is dramatically increased in vascular tissue from rats receiving endotoxin, and non-selective inhibitors of HO activity abrogate endotoxin-induced hypotension. In addition, the induction of HO-1 mRNA is at the level of gene transcription. Beyond an interaction of transcription factors with their DNA binding domains, an appropriate chromatin environment is important for gene transcription. By changing chromatin structure, architectural proteins assemble transcription factors into an enhancer complex. One such architectural protein, high mobility groups protein (HMG)-I(Y), has been shown to promote cooperative binding of transcription factors into a complex that efficiently and synergistically increases gene transcription. Preliminary data from our laboratory show that like HO-1, HMG-I(Y) mRNA and protein is induced in vascular smooth muscle cells by IL-1 beta in vitro and by endotoxin in vivo. Furthermore, HMG-I(Y) binds to 5' flanking sequence of the HO-1 gene. In this application we propose to test the role of HO-1 in endotoxic shock, and to study the molecular mechanisms regulating the transcriptional induction of HO-1 in vascular smooth muscle cells. Three specific aims are proposed. Aim 1: To determine the role of HO-1 in endotoxin-induced hypotension and death by generating mice deficient in the HO-1 gene. We hypothesize that deletion of the HO-1 gene will prevent endotoxin-induced hypotension and death in vivo. Aim 2: To identify the specific DNA sequences (cis-acting elements) that are important for HO-1 gene induction in vascular smooth muscle cells, and to determine the role of chromatin in regulating HO-1 gene transcription. We hypothesize that the interaction of cis-acting elements with trans-acting factors, in an integrated setting, mediates HO-1 gene transcription. Finally, Aim 3: To identify and characterize those DNA-binding proteins (trans-acting factors), that in conjunction with HMG-I(Y), are important for regulating the induction of HO-1 gene transcription by IL-1 beta in vascular smooth muscle cells. These studies should provide new insight into the regulation of HO-1, and the pathogenesis of endotoxic shock.