INDUCTION OF ACUTE PHASE PROTEIN BIOSYNTHESIS

急性时相反应由大量的防御和适应机制组成，这些机制代表了一种具有重要生物学意义的现象。我们的长期目标是阐明在肝细胞中诱导主要的人类急性时相蛋白C反应蛋白(CRP)的机制。IL-6通过C/EBP和STAT3转录因子诱导人肝癌细胞株Hep 3B中CRP的转录。我们最近发现，Re1蛋白p50以两种意想不到的方式参与了IL-6诱导的CRP的表达：1)p50以两种意想不到的方式与CRP启动子近端C/EBP结合位点重叠的非共识kappaB结合形成与C/EBP物种的三元复合体，这是IL-6诱导CRP所必需的。2)在IL-6存在的情况下，p50与邻近STAT3反应元件的非共识kappaB位点的结构性结合被STAT3结合所取代。这些结果不能反映典型的NFkappaB活性：1)过表达的pp65抑制了CRP的表达；2)在EMSA复合体中未发现p65的表达。这些观察结果提出了我们的工作假设，即非经典的RE1家族成员，包括p50，在近端启动子的两个位置影响IL-6激活的转录因子：在C/EBP结合位点通过稳定C/EBP蛋白的结合而在STAT3结合位点通过影响该因子与其同源位点的结合而影响该因子的结合。我们的具体目的是描述在CRP诱导过程中介导转录因子相互作用的精确分子机制：1.确定p50影响以-53为中心的关键C/EBP结合位点的CRP表达的机制。我们将确定p50与这个区域的结合是否会影响C/EBPbeta在这个位点结合的亲和力和稳定性，并将确定这些因素是否在物理上相互作用。我们将确定p50是否刺激内源性CRP基因的表达，并定义介导其活性的p50和p65结构域。II.明确p50影响STAT3结合位点C反应蛋白表达的机制。II.确定p50影响STAT3结合部位CRP表达的机制，以及增加p50与其结合部位的亲和力是否改变CRP的表达。研究IL-6和IL-1β在调节近端C反应蛋白启动子表达中的作用。IV.研究C反应蛋白启动子-256~-157之间主要反应区的细胞因子反应元件。这些研究有可能对当前非常感兴趣的转录因子，特别是NFkappaB蛋白相互作用影响自然启动子和基因表达的机制做出重大贡献。

The acute phase response consists of a large number of defensive and adaptive mechanisms that represent a phenomenon of great biologic importance. Our long-term goals have to delineate the mechanisms that mediate induction of the major human acute phase protein C-reactive protein (CRP) in hepatocytes. IL-6 induces transcription of CRP in the human hepatoma cell line Hep 3B through C/EBP and STAT3 transcription factors. We recently found that the re1 protein p50 participates in IL-6induced CRP expression in two unexpected ways: 1) p50 binds to a non-consensus kappaB site overlapping the proximal C/EBP binding site of the CRP promoter to form ternary complexes with C/EBP species, which are required for CRP induction by IL-6. 2) Constitutive binding of p50 to a non-consensus kappaB site adjacent to the STAT3 response element was displaced by STAT3 binding in the presence of IL-6. These findings did not reflect classic NFkappaB activity; 1) CRP expression was inhibited by over-expressed pp65 and 2) p65 was not found in EMSA complexes. These observations give rise to our working hypotheses, that non-classic re1 family members, including p50, affect IL-6 activated transcription factors at two sites in the proximal promoter: at the C/EBP binding site by stabilizing binding of C/EBP proteins and at the STAT3 binding site, by affecting binding of this factor to its cognate site. Our specific aims, which are designed to delineate the precise molecular mechanisms that mediate transcription factor interactions in CRP induction, are: I. To define the mechanisms by which p50 influences CRP expression at the critical C/EBP binding site centered at -53. We will determine whether binding of p50 to this region affects affinity and stability of C/EBPbeta for binding at this site and will determine whether these factors physically interact. We will determine if p50 stimulates expression of the endogenous CRP gene and will define the domains of p50 and p65 that mediate their activities. II. To define the mechanisms by which p50 influences CRP expression at the STAT3 binding site. II. To define the To define the mechanisms by which p50 influence CRP expression at the STAT3 binding site and whether increasing p50 affinity for its site alters CRP expression. III. To delineate the specific roles played by IL-6 and IL-1beta in regulating expression on the proximal CRP promoter. IV. To investigate the cytokine response elements in the major response region between -256 and -157 of the CRP promoter. These studies have the potential of contributing substantially to an area of great current interest delineation of the mechanisms by which transcription factors, particularly NFkappaB proteins, interact to influence natural promoters and gene expression.