REGULATION OF ORNITHINE DECARBOXYLASE EXPRESSION IN FIBROBLASTS

亚精胺和亚精胺的生物合成受到调控 通过关键酶鸟氨酸脱羧酶(ODC)和一成不变 在动物细胞被各种刺激激活时增强。这个 ODC mRNA水平受转录或转录后调控。 转录上，取决于细胞类型。信号转导 在转录水平上控制ODC基因的途径多种多样， 包括涉及蛋白激酶A(PKA)、蛋白激酶C(PKC)、 蛋白酪氨酸激酶和类固醇激素受体。我们目前 已经确定了ODC启动子的两个区域，它们对 受调控的转录。区域A的中心位置相对于 转录起始位置，位于TATAA附近的位置 Box；该元件与cAMP调节的序列具有很强的相似性 生长抑素基因(“Cre”)的元件，并结合cAMP调节的 CREB因子，当以纯化的重组蛋白或在细胞中存在时 萃取物。Cre样位点突变强烈抑制基础水平 由ODC启动子驱动的转录。B区富含43个BP的GC 区域位于-92BP到-134BP之间。删除此区域， 保持A区不变，对神经元的基础活动几乎没有影响 启动子，但干扰PKA或佛波酯的调节。这些 结果提示，B区在ODC的调控中起着重要作用 通过PKA和PKC途径转录。这项提议的一个主要目标是 是通过对B区的详细突变分析来验证这一假设， 不同突变对基因调控的影响相互关联 启动子与蛋白质结合的变化。由此鉴定出的蛋白质 分析将是孤立的，并针对其监管 属性，目的是了解这些DNA结合是如何 蛋白质与信号转导途径在功能上相互作用。这个 炎症还上调成纤维细胞中ODC mRNA的水平 中介物IL-1。这一规定的机制将在#年进行研究。 细节，如果是在转录水平，细胞因子反应 ODC基因中的元素将被识别出来。如果这些元素是 与已发现的不同，与它们结合的蛋白质将是 特色化的。受其他感兴趣的细胞因子的调节，如肿瘤坏死因子- 阿尔法和IL-6，将以相同的方式进行检测。

Biosynthesis of the polyamines, spermidine and spermine, is regulated through the key enzyme ornithine decarboxylase (ODC) and is invariably enhanced upon activation of animal cells by a variety of stimuli. The level of ODC mRNA is regulated either transcriptionally or post- transcriptionally, depending on the cell type. The signal transduction pathways that control the ODC gene at the transcriptional level are varied, including those involving protein kinase A (PKA), protein kinase C (PKC), protein tyrosine kinases, and steroid hormone receptors. We at present have identified two regions of the ODC promoter that are important for regulated transcription. Region A is centered at -45 bp relative to the site of transcriptional initiation, in a position adjacent to the TATAA box; this element shows strong sequence similarity to the cAMP-regulated element of the somatostatin gene ("CRE"), and binds the cAMP-regulated factor, CREB, when presented as purified recombinant protein or in cell extracts. Mutation of the CRE-like site strongly depresses basal level transcription driven by the ODC promoter. Region B is a 43-bp GC-rich region located between -92 bp and -134 bp. Deletion of this region, leaving Region A intact, has little or no effect on basal activity of the promoter, but interferes with regulation by PKA or phorbol esters. These results suggest that Region B plays an important role in regulation of ODC transcription by the PKA and PKC pathways. A major goal of this proposal is to test this hypothesis by detailed mutational analysis of Region B, correlating the influence of various mutations on regulation of the promoter with changes in protein binding. Proteins identified by this analysis will be isolated and characterized with regard to their regulatory properties, with the objective of understanding how these DNA-binding proteins interact functionally with the signal transduction pathways. The level of ODC mRNA is also upregulated in fibroblasts by the inflammatory mediator IL-1. The mechanism of this regulation will be examined in detail, and if it is at the transcriptional level, cytokine-responsive elements will be identified in the ODC gene. If these elements are different from those already found, proteins binding to them will be characterized. Regulation by other cytokines of interest, such as TNF- alpha and IL-6, will be examined in the same way.