A Novel Regulation of the Phase II Enzyme Estrogen Sulfotransferase

说明(由申请人提供)：雌激素磺基转移酶(EST或SULT1E1)是一种II相结合酶，以其磺化和灭活雌激素(一种抗炎激素)的活性而闻名。这是因为磺化雌激素不能结合和激活雌激素受体。EST已被证明受核受体的转录调控。然而，目前还不清楚EST是否以及如何受到炎症刺激的调节，以及EST的炎症反应调节的结果是什么。雌激素的平衡合成和代谢密切地调节着雌激素的动态平衡。雌激素失活的一个关键代谢途径是通过EST介导的硫化作用。然而，EST的表达和调控是否会影响宿主对炎性刺激的敏感性还没有得到测试。我们的初步结果表明：1)细菌脂多糖(LPS)治疗或盲肠结扎穿孔(CLP)脓毒症模型小鼠能及时诱导EST在肝脏中特异性表达和活性。 内毒素诱导的EST与抑制雌激素诱导的子宫上皮细胞增殖和雌激素反应基因的表达以及降低循环中雌二醇水平有关；4)内毒素诱导的EST不依赖于核受体。相反，在Toll样受体4基因敲除(TLR4-/-)小鼠中，Est的CLP诱导被取消；5)Est是一个潜在的NF-κB靶基因，在Est基因启动子中已预测出几个可能的NF-kB结合位点；6)Est对炎症的调节在雄性小鼠中是保守的，在人类肝细胞中也是如此；7)Est-/-小鼠受到内毒素应答的炎症反应减弱，可能是由于雌激素活性增加。基于这些观察结果，我们假设EST受到炎性刺激的调节；反过来，EST的表达和调节影响宿主对炎性刺激的敏感性。具体地说，我们假设：1)内毒素和CLP诱导EST的表达，从而剥夺雌激素；2)TLR4是EST炎症反应调节所必需的，EST是核因子-kB的转录靶点；3)EST消融可以减轻小鼠的炎症反应，而转基因EST在肝脏中的过表达使小鼠对内毒素和CLP诱导的炎症反应敏感。我们提出了三个特定的目标来验证我们的假设：1)确定炎症刺激是否以EST依赖的方式剥夺雌激素；2)确定炎症刺激调节EST表达的分子机制；3)确定EST消融是否减弱小鼠的炎症反应，而转基因EST在肝脏中的过表达使小鼠对内毒素和CLP诱导的炎症反应敏感。据我们所知，这项研究是第一次尝试评估EST对炎症刺激的调节以及这种调节对雌激素稳态的影响。这项拟议的研究具有医学意义，特别是在妇女健康方面。我们推测，炎症和EST的相互调节可能代表了一种尚待探索的炎症内分泌调节机制。阐明这一机制可能为炎症相关疾病的防治提供新的治疗靶点。

DESCRIPTION (provided by applicant): Estrogen sulfotransferase (EST or SULT1E1) is a phase II conjugating enzyme best known for its activity in sulfonating and deactivating estrogens, an anti-inflammatory hormone. This is because sulfonated estrogens fail to bind to and activate the estrogen receptor. EST has been shown to be transcriptionally regulated by nuclear receptors. However, it is unclear whether and how EST is regulated by inflammatory stimuli and what is the consequence of inflammation responsive regulation of EST. The estrogen homeostasis is tightly regulated by balanced synthesis and metabolism of estrogens. A critical metabolic pathway to deactivate estrogens is through EST-mediated sulfation. However, it has not been tested whether the expression and regulation of EST affect the host's sensitivity to inflammatory stimuli. Our preliminary results showed that: 1) Treatment of mice with bacterial lipopolysaccharide (LPS) or subjected mice to the cecal ligation and puncture (CLP) model of sepsis induced the expression and activity of EST specifically in the liver in time- and dose-dependent manner; 2) Depletion of liver Kupffer cells attenuated the EST induction by LPS; 3) The EST induction in LPS-treated mice was associated with an inhibition of estrogen-induced uterine epithelial proliferation and estrogen responsive gene expression, as well as a decreased circulating level of estradiol; 4) The LPS induction of EST appeared to be nuclear receptor- independent. In contrast, the CLP induction of EST was abolished in Toll-like receptor 4 knockout (TLR4-/-) mice; 5) EST is a potential NF-κB target gene, and several putative NF-kB binding sites have been predicted in the EST gene promoter; 6) The inflammatory regulation of EST was conserved in male mice, as well as in human liver cells; 7) EST-/- mice were attenuated from an LPS-responsive inflammatory response, likely due to increased estrogen activities. Based on these observations, we hypothesize that EST is subject to the regulation by inflammatory stimuli; and reciprocally, the expression and regulation of EST influence the host's sensitivity to inflammatory stimuli. Specifically, we hypothesize that: 1) LPS and CLP induce the expression of EST, and consequently deprive estrogens; 2) TLR4 is required for the inflammatory responsive regulation of EST, and EST is a transcriptional target of NF-kB; and 3) EST ablation attenuates mice from, whereas transgenic overexpression of EST in the liver sensitizes mice to, LPS- and CLP-induced inflammatory responses. We propose three specific aims to test our hypotheses: 1) To determine whether the inflammatory stimuli deprive estrogens in an EST-dependent manner; 2) To determine the molecular mechanism by which the inflammatory stimuli regulate the expression of EST; and 3) To determine whether EST ablation attenuates mice from, whereas transgenic overexpression of EST in the liver sensitizes mice to, LPS- and CLP-induced inflammatory response. To our knowledge, this study represents the first attempt to evaluate the regulation of EST in response to inflammatory stimuli and the implications of this regulation in estrogen homeostasis. The proposed study is of medical significance, especially in women's health. We reason that the reciprocal regulation of inflammation and EST may represent a yet to be explored mechanism of endocrine regulation of inflammation. Elucidation of this mechanism may provide a novel therapeutic target for the prevention and treatment of inflammation related diseases.