REGULATION OF ORNITHINE DECARBOXYLASE EXPRESSION IN FIBROBLASTS

生物合成的多胺，亚精胺和精胺，是受管制的 通过关键酶鸟氨酸脱羧酶（ODC）， 在动物细胞被各种刺激激活后增强。 的 ODC mRNA的水平在转录或转录后受到调节。 在转录上，取决于细胞类型。 的信号转导 在转录水平上控制ODC基因的途径是多种多样的， 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， 调节转录。 区域A相对于区域B以-45bp为中心。 转录起始位点，在邻近TATAA的位置 框中;该元件显示出与cAMP调节的 生长抑素基因（“CRE”）的元件，并结合cAMP调节的 当以纯化的重组蛋白或在细胞中呈递时， 萃取物 CRE样位点的突变强烈降低基础水平 由ODC启动子驱动的转录。 区域B是一个43-bp的富含GC的 位于-92 bp和-134 bp之间的区域。 删除该区域， 保留区域A完整，对细胞的基础活性几乎没有或没有影响。 启动子，但干扰PKA或佛波醇酯的调节。 这些 结果表明，区域B在ODC的调节中起重要作用 通过PKA和PKC途径转录。 该提案的一个主要目标是 是通过对区域B进行详细的突变分析来验证这一假设， 关联各种突变对调节细胞内蛋白质的影响， 启动子与蛋白质结合的变化。 通过这种方法鉴定的蛋白质 分析将被隔离，并根据其监管特点进行表征。 性质，目的是了解这些DNA结合 蛋白质在功能上与信号转导途径相互作用。 的 成纤维细胞中的ODC mRNA水平也被炎性细胞因子上调。 介体IL-1。 这一规定的机制将在 如果它是在转录水平上，那么它是一种对尼古丁敏感的基因。 将在ODC基因中鉴定元件。 如果这些元素是 与已经发现的不同，与它们结合的蛋白质将被 表征了 其他感兴趣的细胞因子如TNF-α的调节 α和IL-6将以相同的方式进行检查。

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.