INTERLEUKIN-1 GENE REGULATION IN UTERINE SMOOTH MUSCLE

子宫平滑肌中 INTERLEUKIN-1 基因的调控

• 批准号: 2205749
• 负责人: BRIAN D WILCOX
• 金额: $ 15.09万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2205749
• 关键词: biological signal transduction; gel mobility shift assay; gene deletion mutation; genetic promoter element; genetic regulation; immunocytochemistry; in situ hybridization; interleukin 1; laboratory rat; myometrium; nuclear runoff assay; nucleic acid sequence; pregnancy; progesterone; protein biosynthesis; reporter genes; second messengers; serotonin; serotonin inhibitor; transcription factor; uterus

The long-term goals of this application are to characterize the mechanisms by which serotonin and progesterone regulate the production of interleukin-1 in the myometrial smooth muscle cells of the uterus. Preliminary studies conducted by the applicant demonstrate that depletion of serotonin from myometrial cell cultures results in a significant decrease in IL-1alpha mRNA. Conversely, replenishment of the indoleamine results in an induction of IL-1alpha mRNA. Selective serotonin receptor antagonists are able to block this induction while selective agonists fully mimic the effect of the native hormone. Nuclear run-on analysis demonstrates that the increase in steady state levels of IL-1alpha mRNA are accompanied by an increase in IL-1alpha transcription. Similarly, the addition of cycloheximide has neither a positive nor negative effect on induction, consistent with a direct effect on transcription as a major regulator of levels of IL-1alpha mRNA. The inductive effect of serotonin can be mimicked by phorbol esters, indicating the possible involvement of protein kinase C; regardless of the mode of induction, however, increases in IL-1alpha mRNA can be completely blocked by the addition of progesterone or glucocorticoids. We propose to isolate the 5'-flanking, and hence, putative promoter region of the rat IL-1alpha gene. Following sequencing, a detailed deletion and mutation analysis of this region will be conducted utilizing the promoter ligated into a reporter gene construct and transiently transfected into primary cultures of rat myometrial smooth muscle cells. These studies will establish the specific regions of the promoter that are necessary for serotonin and progesterone responsiveness. Similarly, studies will be conducted to elucidate the second messenger pathways utilized in these cells to accomplish the reciprocal regulation of IL-Ia mRNA by serotonin and progesterone. Included will be studies focused on elucidating the ability of IL-1 to auto-regulate levels of its own mRNA. Finally, we will investigate the production and regulation of uterine IL-Ia in vivo, using in situ hybridization and immunohistochemical techniques during pregnancy and post-partum involution. These studies should not only shed new light on this newly discovered mode of regulation of IL-1, but should also help us to better understand the role of IL- 1alpha in a number of important biological and pathological phenomenon that occur in the uterus during pregnancy and involution.

该应用的长期目标是表征机制 5-羟色胺和孕酮调节的产生 子宫肌层平滑肌细胞中的白介素-1。 申请人进行的初步研究证明了耗尽 从肌层细胞培养物中的5-羟色胺导致显着 减少IL-1Alpha mRNA。相反，补充吲哚美碱 导致诱导IL-1Alpha mRNA。选择性5-羟色胺受体 拮抗剂能够阻止这种诱导，而选择性激动剂 完全模仿天然激素的作用。核跑步分析 证明IL-1Alpha mRNA的稳态水平的增加 伴随着IL-1Alpha转录的增加。同样， 添加环己酰亚胺对 诱导，与对转录的直接影响一致 IL-1Alpha mRNA水平的调节剂。 5-羟色胺的诱导作用 可以通过佛波酯模仿，表明可能参与 蛋白激酶C；但是，无论归纳方式如何增加 在IL-1Alpha中，可以通过添加完全阻止 孕酮或糖皮质激素。我们建议隔离5'飞机， 因此，大鼠IL-1Alpha基因的推定启动子区域。下列的 对该区域的测序，详细的缺失和突变分析将 使用连接到报告基因构建体的启动子进行进行 并瞬时转染大鼠肌层光滑的一级培养物 肌肉细胞。这些研究将确定 羟色胺和孕激素反应性所需的启动子。 同样，将进行研究以阐明第二使者 在这些单元中使用的途径来完成相互调节 5-羟色胺和孕酮的IL-IA mRNA。其中包括研究 专注于阐明IL-1自动调节其水平的能力 自己的mRNA。最后，我们将研究 子宫IL-IA在体内，使用原位杂交和免疫组织化学 怀孕期间的技术和产后相关性。这些研究 不仅应该在这种新发现的法规模式下开发新的灯光 IL-1的作品，但也应该帮助我们更好地了解IL的作用 1α在许多重要的生物学和病理现象中 这在怀孕和涉及期间的子宫中发生。