Role of Selenium in Development and Health

硒是人类和其他哺乳动物饮食中必需的微量营养素。硒对健康有许多益处，包括预防癌症和心脏病以及人类免疫缺陷病毒（HIV）阳性患者获得性免疫缺陷综合征（艾滋病）的进展。近年来，已经进行了大量的人体临床试验，以评估硒在预防癌症、延缓艾滋病进展等方面的作用，耗资数十亿美元，但对硒在哺乳动物分子水平上如何发挥这些健康益处知之甚少。我们提出硒的健康益处主要是由于它作为含硒氨基酸硒半胱氨酸（Sec）存在于硒蛋白中。因此，我们的项目重点是：1)建立小鼠模型来评估硒和硒蛋白在癌症预防和发展中的作用；2)表征和阐明各种硒蛋白的功能及其在癌症预防和发展中的作用；3)确定Sec生物合成和结合到蛋白质中的方法。本文讨论的项目探讨了我们在各种小鼠模型的发展研究，以确定硒在癌症和发展中的作用。在过去的一年里，我们专注于完成所有关于小鼠模型特征的研究（见下文）。我们之前启动了硒蛋白15 （Sep15）敲除小鼠的体内致癌研究，以检验我们的体外研究结果，阐明这种硒蛋白在癌症中的作用。我们研究了Sep15小鼠结肠化学诱导的异常隐窝灶（ACF）的形成，并与杂合子和野生型产仔对照进行了比较，以及饲粮硒对ACF形成的影响。我们的研究结果表明，Sep15基因敲除小鼠可以防止ACF的形成，而ACF的形成似乎与膳食硒无关。此外，我们的研究小组此前曾报道，从小鼠内皮细胞中选择性去除硒代半胱氨酸tRNA基因（Trsp）是胚胎致死的。为了研究谷胱甘肽过氧化物酶基因（gpx4）和硫氧还蛋白还原酶1 （tr1）在内皮细胞发育中的重要性，我们制造了内皮特异性敲除小鼠，编码gpx4或tr1基因的靶向缺失。gpx4内皮敲除小鼠是胚胎致死性的，其表型几乎概括了之前报道的Trsp内皮敲除胚胎的所有变化。简而言之，两个胚胎（trsp和gpx4内皮敲除）都显示出明显的中枢神经系统异常，大脑中有广泛的坏死区域。两种胚胎血液中有核红细胞均多于对照胚胎。然而，trsp内皮敲除胚胎的胸腺要么不存在，要么杂乱无章，而gpx4胚胎的胸腺与对照胚胎的胸腺没有差异。tr1内皮敲除无致死性，但e18天胚胎胸腺组织紊乱，淋巴细胞成熟程度低于对照组。我们现在正在研究TR1内皮敲除小鼠是否在胸腺或其他免疫相关组织中有问题，与具有完全功能TR1基因的对照小鼠相比。我们还研究了对乙酰氨基酚（APAP）对肝脏中编码TR1基因敲除的小鼠的影响，因为APAP肝毒性（APAP- h）是美国药物性肝衰竭的最常见原因。由于谷胱甘肽的消耗在APAP肝损伤中起主要作用，并且GSH在肝损伤中的许多潜在代谢已被阐明，因此研究了TR1（哺乳动物细胞中也是主要抗氧化剂）在APAP- h中的作用以及该硒酶与谷胱甘肽系统的相互关系。肝脏特异性TR1敲除小鼠（tr1KOliv）用肝毒性剂量的APAP治疗。令人惊讶的是，tr1KOliv小鼠对APAP毒性具有抗性，因为用于证明肝毒性的组织学和肝酶与未治疗的小鼠没有显着差异。然而，接受治疗的对照组小鼠表现出广泛的肝脏损伤。结果强烈提示tr1KOliv小鼠主要通过NRF2激活进行外源解毒。这些发现为apap诱导的肝损伤提供了新的见解，并为预防和/或改善药物性肝损伤提供了新的治疗靶点。此外，为了研究TR1在肝癌中的作用，将肝脏TR1敲除小鼠暴露于肝癌致癌物二乙基亚硝胺（DEN）中。我们发现肝脏特异性TR1敲除小鼠比对照小鼠更容易发生肿瘤（肝腺瘤、肝癌和肝胆管细胞腺瘤）。18只基因敲除小鼠中有16只出现肿瘤，而对照组的19只小鼠中只有2只出现肿瘤。此外，我们发现nrf2调节基因在TR1缺失的肝脏中上调，包括几种谷胱甘肽转移酶和硒蛋白，谷胱甘肽过氧化物酶2 (GPx2)，在肿瘤中甚至进一步上调。GPx2在肝脏肿瘤中的作用目前正在使用人肝癌细胞系（Hep G2）进行体外评估。我们之前已经证明，硒蛋白通过靶向巨噬细胞中trsp的去除，从而在巨噬细胞中发挥适当的巨噬细胞功能。此外，我们发现巨噬细胞中TR1在脂多糖（LPS）刺激下上调，并且这种刺激依赖于p38信号。我们也开始通过开发和表征巨噬细胞特异性TR1敲除小鼠来研究TR1在巨噬细胞功能中的作用。虽然这只小鼠没有明显的表型，但我们目前正在研究硫氧还蛋白依赖性炎症信号的可能改变。在过去的一年里，我们完成了皮肤表皮细胞条件敲除GPx4和TR1的研究。这两种蛋白都是细胞ROS水平的重要调节因子，敲除它们会导致胚胎死亡。因此，为了阐明这些蛋白在皮肤中的体内作用，我们建立了条件敲除小鼠模型来检测GPx4和TR1在皮肤功能和发育中的作用。虽然在皮肤中靶向去除TR1没有观察到明显的表型变化，但角化细胞中GPx4的时空破坏通过诱导COX-2表达调节了体内毛囊发育和体外角化细胞增殖。这种消融产生了表型改变的后代，这种变化在毛囊形态发生的早期阶段更为明显，证实了GPx4在毛囊发育中的重要性。有趣的是，这些研究首次表明，在角质形成细胞中，GPx4的缺乏由GPx1和TR1补偿，从而确立了GPx4作为皮肤抗氧化剂的重要性。我们利用loxP-Cre技术建立了乳腺TR1条件敲除小鼠模型，并正在扩大群体以阐明TR1在乳腺癌中的作用。我们将比较TR1乳腺敲除小鼠和对照小鼠的乳腺癌发病率，以评估TR1在这种恶性肿瘤的过程中是否具有抗癌或促癌基因的作用。我们将研究tnf - α对乳腺荷瘤控制和TR1敲除小鼠的影响，以阐明TR1在肿瘤凋亡途径中的作用。我们还将在乳腺癌荷瘤小鼠的饮食中加入亚硒酸钠，以确定亚硒酸钠对体内小鼠模型中TR1缺陷癌细胞的高度特异性细胞毒性作用。

Selenium is an essential micronutrient in the diet of humans and other mammals. Many health benefits have been attributed to selenium including preventing cancer and heart disease and the progression of acquired immunodeficiency syndrome (AIDS) in human immunodeficiency virus (HIV)-positive patients. Numerous human clinical trails have been undertaken in recent years to assess the role of this element in cancer prevention, delaying the progression of AIDS, etc., at a cost of billions of dollars, but little is known about how selenium acts at the molecular level in mammals to exert these many health benefits. We proposed that the health benefits of selenium are due largely to its presence in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec). Our program therefore focuses on: 1) developing mouse models to assess the role of selenium and selenoproteins in cancer prevention and development, 2) characterizing and elucidating the function of various selenoproteins and their roles in cancer prevention and development, and 3) identifying the means by which Sec is biosynthesized and incorporated into protein. The project discussed herein examines our research on the development of various mouse models for determining the role of selenium in cancer and development. In the past year, we have focused on completing all of our studies on characterizing our mouse models (see below). We previously initiated in vivo carcinogenesis studies involving the selenoprotein 15 (Sep15) knockout mice to examine our in vitro findings elucidating the role of this selenoproteins in cancer. We examined the formation of chemically-induced aberrant crypt foci (ACF) in the colon of Sep15 mice compared to heterozygous and wild type litter mate controls and the influence of dietary selenium on the formation of ACF. Our results demonstrated that Sep15 knockout mice are protected against ACF formation, which seemed independent of dietary selenium. In addition, it has been previously reported by our group that selective removal of the selenocysteine tRNA gene (Trsp) from mouse endothelial cells is embryonic lethal. In order to study the importance of gluthathione peroxidase gene (gpx4) and thioredoxin reductase 1 (tr1) in the development of endothelial cells, we generated endothelial specific knockout mice encoding the targeted loss of either the gpx4 or tr1 gene. gpx4 endothelial knockout mice are embryonic lethal, showing a phenotype that recapitulate almost all the changes previously reported on Trsp endothelial knockout embryo. Briefly, both embryos (trsp and gpx4 endothelial knockout) showed marked central nervous system abnormalities with extensive necrotic areas in the brain. Also both of them presented more nucleated erythrocytes in the blood than the control embryos. However, the thymus of trsp endothelial knockout embryos was either not present or disorganized whereas the thymus of gpx4 embryos showed no difference with the thymus of the control embryos. tr1 endothelial knockout is not lethal, but the thymus in the E 18 days embryos is disorganized and the lymphocytes are less mature than in the thymus of control mice. We are now examining whether the adult TR1 endothelial knockout mice have problems in their thymus or other immune-related tissues compared to control mice with a fully functional TR1 gene. We have also examined the effect acetaminophen (APAP) on mice encoding a TR1 knockout in liver as APAP hepatotoxicity (APAP-H) is the most common cause of drug-induced liver failure in the United States. Since the depletion of glutathione plays a major role in APAP liver damage and much of the underlying metabolism of GSH in liver damage has been elucidated, the role of TR1, also a major antioxidant in mammalian cells, in APAP-H, and the interrelationship of this selenoenzyme with the glutathione system were examined. Liver-specific TR1 knockout mice (tr1KOliv) were treated with a hepatotoxic dose of APAP. Surprisingly, tr1KOliv mice were resistant to APAP toxicity as histology and liver enzymes used for demonstrating hepatotoxicity were not significantly different from untreated mice. Control, treated mice, however, showed extensive liver damage. The results strongly suggest that tr1KOliv mice are primed for xenobiotic detoxification primarily through NRF2 activation. The findings provide new insights into APAP-induced liver damage and suggest new therapeutic targets for prevention and/or amelioration of drug-induced liver damage. Furthermore, to examine the role of TR1 in liver cancer, liver TR1 knockout mice were exposed to the liver carcinogen, diethylnitrosamine, (DEN). We found that liver-specific TR1 knockout mice developed tumors (hepatic adenomas, hepatic carcinomas and hepatocholangiocellular adenomas) much more readily than control mice. 16 of 18 knockout mice developed tumors compared to 2 of 19 of control mice. In addition, we found an upregulation of Nrf2-modulated genes in the TR1 deficient livers, including several of the glutathione transferases and the selenoprotein, glutathione peroxidase 2 (GPx2), which was even further up-regulated in tumors. The role of GPx2 in liver tumors is currently being evaluated in vitro using a human liver carcinoma cell line (Hep G2). We have previously shown that selenoproteins play a role in proper macrophage function by targeting the removal of trsp, and thus all selenoproteins, in macrophage. In addition, we have found that TR1 is up-regulated in macrophage upon stimulation with lipopolysaccharide (LPS) and that this stimulation is dependent upon p38 signaling. We have also begun examining the role of TR1 in macrophage function by developing and characterizing a macrophage-specific TR1 knockout mouse. Although this mouse has no overt phenotype, we are currently accessing possible alterations in thioredoxin-dependent inflammatory signaling. In the past year, we completed our studies on conditional knockout of GPx4 and TR1 in epidermal cells in skin. Both these proteins are important regulators of cellular ROS levels and knocking them out results in embryonic lethality. Hence, to elucidate the in vivo role of these proteins in skin, we generated conditional knockout mouse models to examine the role of GPx4 and TR1 in skin function and development. Though no obvious phenotypic changes were observed for targeted removal of TR1 in skin, spatio-temporal disruption of GPx4 in keratinocytes modulated hair follicle development in vivo and keratinocyte proliferation in vitro through induction of COX-2 expression. This ablation generated progeny with an altered phenotype, with the variations being more evident in early stages of hair follicle morphogenesis, substantiating the importance of GPx4 in hair follicle development. Interestingly, these studies show for the first time that in keratinocytes, the lack of GPx4 is compensated by GPx1 and TR1, establishing the importance of GPx4 as an antioxidant in skin. We have established a breast TR1 conditional knockout mouse model using loxP-Cre technology and are expanding the population to elucidate the role of TR1 in breast cancer. We will compare the incidence of breast cancer in TR1 breast knockout and control mice to assess whether TR1 has a role as an anti- or pro-cancer gene in the process of this malignancy. We will examine the effect of TNF-alpha on breast tumor-bearing control and TR1 knockout mice to elucidate the involvement of TR1 in the cancer apoptotic pathway. We also will introduce sodium selenite into the diet of breast tumor-bearing mice to determine the highly specific cytotoxic effect of selenite on TR1 deficient cancer cells in an in vivo mouse model.