ESTROGEN REGULATION OF HUMAN INTERLEUKIN 6 GENE PROMOTER

近一半50岁以上的女性(绝经后)将有 骨丢失(骨质疏松症)严重到自发性骨折 腰椎、髋关节或手腕都会出现。雌激素水平降低 绝经后妇女的生殖与骨量丢失有关 雌激素替代疗法是一种主要的治疗方式 保护骨量。促进细胞因子白介素6的产生 成骨细胞(IL-6)通过破骨细胞募集引起骨吸收 和差异化。人成骨样细胞体外产生IL-6的实验研究 细胞(OCs)受促炎细胞因子白介素IL-2刺激 IL-1β和雌激素调节IL-1β对IL-6的影响 信使核糖核酸水平和蛋白质分泌。雌激素的作用机制(S) 细胞因子对人口腔黏膜上皮细胞IL-6基因表达的调控 人们对此知之甚少。雌激素/雌激素受体(ER)被认为是 通过与IL-6基因启动子相互作用间接影响IL-6基因启动子 参与IL-6基因调控的多种转录因子 表情。然而，我们对IL-6基因启动子核苷酸的分析 序列指示另一种潜在机制，通过假定的 远端片段中的雌激素受体/激活蛋白-1结合位点 发起人的身份。 这一提议的假设是雌激素/内质网调节IL-1β- 诱导型IL-6基因在人成骨细胞中的表达 转录因子和/或与IL-6内的ER元件结合 基因启动子。检验这一假设的具体目的是：1) 确定IL-6基因的启动子元件是 IL-1β在人口腔鳞状细胞中的激活。这将通过使用 野生型IL-6基因启动子(Phil-6/Luc+)和一系列缩短的 启动子片段(缺失构建体)。2)确定这些或 不同的区域对应于对雌激素调节有反应的区域。 这将通过治疗IL-1β刺激的OCs来实现 用Phil-6/Luc+或带有17beta- 雌二醇。3)确定与雌激素有关的转录因子 IL-1β诱导的IL-6基因启动子活性的调节。这将是 使用IL-1β和17β的核蛋白提取物来完成 电迁移率移位分析中雌激素刺激的人OCs。 生物医学意义。大约有130万人发生骨折 在美国每年可归因于骨质疏松症， 估计每年耗资100亿美元。身份识别 雌激素调节IL-6基因表达的机制将 加强对骨质疏松症病理生物学的认识 建议未来旨在治疗和预防这种疾病的战略。

Nearly one half of women over the age of 50 (post-menopausal) will have bone loss (osteoporosis) so severe that spontaneous fractures of the lumbar vertebrae, hip, or wrist will occur. Decreased estrogen production by post-menopausal women is associated with loss of bone mass and estrogen replacement therapy is a primary treatment modality to preserve bone mass. Enhanced production of the cytokine interleukin-6 (IL-6) by osteoblasts causes bone resorption via osteoclast recruitment and differentiation. In vitro, IL-6 production by human osteoblast-like cells (OLCs) is stimulated by the pro-inflammatory cytokine interleukin- 1beta (IL-1beta) and estrogen modulates this effect of IL-1beta on IL-6 mRNA levels and protein secretion. The mechanism(s) of estrogen modulation of cytokine-induced IL-6 gene expression in human OLCs is poorly understood. Estrogen/estrogen receptor (ER) is suggested to affect the IL-6 gene promoter indirectly through interaction with various transcription factors involved in regulation of IL-6 gene expression. However, our analysis of the IL-6 gene promoter nucleotide sequence indicates an additional potential mechanism, via a putative estrogen receptor/activator protein-1 binding site in the distal segment of the promoter. The hypothesis of this proposal is that estrogen/ERs modulate IL-1beta- induced-IL-6 gene expression in human osteoblasts via interaction with transcription factors and/or binding to an ER element within the IL-6 gene promoter. The specific aims to test this hypothesis are to: 1) Determine the promoter elements of the IL-6 gene that are required for IL-1beta activation in human OLCs. This will be accomplished by using the wild type IL-6 gene promoter (phIL-6/luc+) and a series of shortened promoter fragments (deletion constructs). 2) Determine if these or different regions correspond to those responsive to estrogen regulation. This will be accomplished by treating IL-1beta-stimulated OLCs transfected with phIL-6/luc+ or deletion constructs with 17beta- estradiol. 3) Identify transcription factors involved in estrogen regulation of IL-1beta-induced-IL-6 gene promoter activity. This will be accomplished using nuclear protein extracts of IL-1beta-and 17beta- estradiol-stimulated human OLCs in electromobility shift assays. Biomedical significance. Approximately 1.3 million bone fractures occur every year in the United States that are attributable to osteoporosis, at an estimated annual cost of 10 billion dollars. Identification of the mechanisms by which estrogen regulates IL-6 gene expression will enhance our understanding of the pathobiology of osteoporosis and suggest future strategies aimed at treating and preventing this disease.