Mechanisms of Estrogen Repression of TNF-a Transcription

描述(申请人提供)：骨质疏松症是导致老年妇女死亡和残疾的主要原因之一。骨质疏松症每年导致68,000人死亡，相当于乳腺癌和所有妇科癌症死亡人数的总和。预防骨质疏松症最有效的策略之一是在绝经开始时补充雌激素。不幸的是，长期服用雌激素会导致不良反应，包括增加乳腺癌和子宫内膜癌的发病率。显然，雌激素保留了对骨骼的有益作用，但不会对乳腺或子宫内膜细胞产生增殖作用，有可能成为预防骨质疏松症的一线药物。发现更安全和更有选择性的雌激素的一种方法是剖析雌激素激活和抑制基因转录的分子机制。与转录激活相反，人们对抑制的机制知之甚少。我们研究了雌激素和植物雌激素如何抑制肿瘤坏死因子(TNF-α)启动子，因为肿瘤坏死因子-α通过刺激破骨细胞向骨吸收而导致骨质疏松症。在这些研究的基础上，我们假设雌激素受体(ERbeta)在抑制基因转录方面比ERα更有效，并且ER和辅助激活蛋白的激活功能表面是雌激素介导的抑制所必需的。虽然这些发现是新的和重要的，但它们的解释是有限的，因为它们是在瞬时转基因细胞中与报告基因相连的肿瘤坏死因子-α启动子完成的。这项建议的目的是将这些观察扩展到天然的肿瘤坏死因子-α基因，并进一步探索内质网抑制基因转录的基本分子机制。在这项提案中，我们将使用染色质免疫沉淀分析来表征在天然的肿瘤坏死因子-α启动子上发生的蛋白质-蛋白质相互作用，这些蛋白质发生在雌激素受体、转录因子，如c-jun和NFKB，以及由四环素可诱导启动子控制的稳定转染ERpha或ERbeta的骨细胞中的p160和p300共调节蛋白之间。我们假设，识别参与抑制的蛋白质，并确定这些因素如何相互作用，是开发抑制选择性雌激素的关键。我们认为抑制选择性雌激素有可能成为预防骨质疏松症的一线药物，因为我们假设这些雌激素可以预防骨质疏松症，但不会促进乳腺癌或子宫内膜癌。

DESCRIPTION (provided by applicant): Osteoporosis is one of the leading causes of mortality and disability in older women. Osteoporosis causes 68,000 deaths per year, which is equivalent to the number of deaths from breast cancer and all gynecological cancers combined. One of the most effective strategies to prevent osteoporosis is to replace estrogen at the onset of menopause. Unfortunately, prolonged treatment with estrogen leads to adverse effects, including an increased incidence of breast and endometrial cancer. Clearly, estrogens that retain their beneficial effects on bone, but do not elicit a proliferative effect on breast or endometrial cells, have the potential to become first-line drugs to prevent osteoporosis. One approach to discover safer and more selective estrogens is to dissect the molecular mechanisms whereby estrogens activate and repress gene transcription. In contrast to transcriptional activation, very little is known about the mechanisms of repression. We studied how estrogens and phytoestrogens repress the tumor necrosis factor (TNF-alpha) promoter, because TNF-a causes osteoporosis by stimulating osteoclasts to resorb bone. Based on these studies, we hypothesized that estrogen receptor (ERbeta) is more effective than ERalpha at repressing gene transcription, and that the activation function-2 surface of ERs and coactivator proteins are required for estradiol-mediated repression. Whereas these findings are novel and important, their interpretation is limited, because they were done with the TNF-alpha promoter linked to reporter genes in transient transfected cells. The goals of this proposal are to extend these observations to the native TNF-alpha gene, and to further probe the basic molecular mechanisms whereby ERs repress gene transcription. In this proposal we will use chromatin immunoprecipitation assays to characterize the protein-protein interactions that occur at the native TNF-alpha promoter between estrogen receptors, transcription factors, such as c-jun and NFKB, and p160 and p300 coregulatory proteins in bone cells that are stably transfected with ERalpha or ERbeta controlled by a tetracycline-inducible promoter. We hypothesize that identifying the proteins involved in repression, and determining how these factors interact with each other is key to developing repression-selective estrogens. We believe that repression-selective estrogens have the potential to become first-line drugs for preventing osteoporosis, because we hypothesize that these estrogens will prevent osteoporosis, but will not promote breast or endometrial cancer.