INDUCTION OF ACUTE PHASE PROTEIN BIOSYNTHESIS

急性期反应由大量的防御和适应机制组成，这些机制代表了具有重要生物学意义的现象。我们的长期目标是阐明介导肝细胞中主要的人类急性期蛋白C反应蛋白（CRP）诱导的机制。IL-6通过C/EBP和STAT 3转录因子诱导人肝癌细胞系Hep 3B中CRP的转录我们最近发现，re 1蛋白p50以两种意想不到的方式参与IL-6诱导的CRP表达：1）p50与CRP启动子近端C/EBP结合位点重叠的非共有kappaB位点结合，与C/EBP形成三元复合物，这是IL-6诱导CRP所必需的。2)在IL-6存在下，p50与STAT 3反应元件附近的非共有kappaB位点的组成性结合被STAT 3结合所取代。这些发现并不反映经典的NF κ B活性; 1）CRP表达被过度表达的pp 65抑制，2）在EMSA复合物中未发现p65。这些观察结果引起了我们的工作假设，即非经典的re 1家族成员，包括p50，影响IL-6激活的转录因子在两个位点的近端启动子：在C/EBP结合位点，通过稳定C/EBP蛋白的结合和在STAT 3结合位点，通过影响该因子的结合，其同源位点。我们的具体目标，这是为了描绘精确的分子机制，介导转录因子相互作用的CRP诱导，是：I。确定p50影响CRP在以-53为中心的关键C/EBP结合位点表达的机制。我们将确定p50与该区域的结合是否影响C/EBP β在该位点结合的亲和力和稳定性，并确定这些因子是否物理相互作用。我们将确定p50是否刺激内源性CRP基因的表达，并确定p50和p65介导其活性的结构域。二.明确p50影响STAT 3结合位点CRP表达的机制。二.确定p50影响CRP在STAT 3结合位点表达的机制，以及增加p50对其位点的亲和力是否会改变CRP的表达。三.阐明IL-6和IL-1 β在调节近端CRP启动子表达中的特定作用。 四.研究CRP启动子-256 ~-157之间主要反应区的细胞因子反应元件。这些研究有可能大大有助于一个领域的巨大的当前利益划定的机制，转录因子，特别是NF κ B蛋白，相互作用，影响天然启动子和基因表达。

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.