Chemical Receptor Interactions In Reproduction And Hormonal Toxicity

生殖和激素毒性中的化学受体相互作用

• 批准号: 9339237
• 负责人: Kenneth S Korach
• 金额: $ 280.5万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9339237
• 关键词: AF2; Adult; Aggressive behavior; Agonist; Androgens; Animals; Anovulation; Atherosclerosis; Atrophic; Binding; Biochemical; Biological; Biological Assay; Biological Models; Bone Density; Breeding; Cardiovascular Physiology; Cardiovascular system; Cells; Chemicals; Complex; DNA Binding; DNA biosynthesis; Data; Development; Disease; Ductal; EGF gene; Endocrine; Endocrine Disruptors; Environmental Estrogen; Epithelial Cells; Estradiol; Estrogen Nuclear Receptor; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogen Receptors; Estrogens; Evaluation; Exhibits; Experimental Models; FOS gene; Failure; Female; Female infertility; Follicular cyst; Fulvestrant; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic Polymorphism; Genetic Transcription; Genistein; Germ Cells; Gonadotropin Receptors; Gonadotropin Releasing Hormone Inhibitor; Gonadotropins; Growth; Gynecomastia; Hormonal; Hormonal Carcinogenesis; Hormone Receptor; Hormone Responsive; Hormones; Human; Hyperplasia; Immune system; In Vitro; Individual; Infertility; Insulin-Like Growth Factor I; Investigation; Ischemia; Lactoferrin; Lavandula; Lesion; Ligand Binding; Ligands; Malignant Neoplasms; Malignant neoplasm of prostate; Mammary Neoplasms; Mammary gland; Mediating; Membrane; Messenger RNA; Modeling; Molecular; Mus; Mutant Strains Mice; Mutation; Neurosecretory Systems; Oncogenes; Ovarian; Ovary; Patients; Pattern; Phenotype; Phosphorylation; Physiological; Physiology; Play; Process; Progesterone Receptors; Property; Protein Biosynthesis; Protein Kinase C; Receptor Gene; Receptor Signaling; Reproduction; Reproductive Physiology; Research; Role; Selective Estrogen Receptor Modulators; Seminiferous tubule structure; Serum; Sex Behavior; Signal Pathway; Signal Transduction; Skeletal system; Somatic Cell; Specificity; Spermatogenesis; Superovulation; System; Tamoxifen; Tea Tree Oil; Testing; Testis; Time; Tissues; Toxic effect; Transactivation; Transplantation; Trees; Tumor Tissue; Tyrosine Kinase Inhibitor; Uterus; Vagina; Xenobiotics; Yeasts; base; corpus luteum; environmental agent; estrogenic; estrogenic endocrine disruptor; estrophilin; folliculogenesis; granulosa cell; in vivo; insulin receptor substrate 1 protein; male; mammary gland development; mouse model; mutant; novel; prepuberty; receptor; receptor function; reproductive; reproductive tract; research study; response; sperm cell; stereochemistry; synergism; transdifferentiation; tumor; xenoestrogen

Estrogen hormone action within target tissues involves the interaction of the hormonal substance with two different receptor proteins termed ER-alpha and ER-beta. The specificity and responsiveness of tissues to hormonal stimulation are governed in most part by the presence and biochemical action of these receptor proteins. Estrogen receptors (ER) are thought to play a crucial role in development, reproduction and normal physiology. We have generated using gene targeting lines of mice homozygous for the disrupted ERa gene (aERKO) and ERb genes (bERKO)and the recent generation of double ERKO mice abERKO) and characterization of phenotypes in the reproductive, cardiovascular, immune, skeletal and neuroendocrine systems are currently underway. RPA analysis detected comparable levels of ER-b mRNA in tissues of aERKO mice suggesting that ER-b expression is not dependent on ERa. aERKO mice were totally unresponsive to uterotropic assays with estradiol, hydroxy TAM, DES. Further support came from the failure of estrogen, EGF or IGF-1 treatment to induce DNA synthesis in aERKO uteri, even though EGF and IGF-1 signaling was shown to be intact by stimulation of c-fos, or IRS-1 protein phosphorylation. Progesterone receptor mRNA was detected in aERKO mice, but not stimulated by estrogen in the uterus, mammary gland and ovary, indicating an estrogen dependent and independent gene regulation. aERKO females are infertile and have hypoplastic uteri and hyperemic ovaries with no apparent corpora lutea. The aERKO ovarian phenotype occurs developmentally and can be reversed by a GnRH antagonist. Ovarian gonadotropin receptor levels, serum estrogen, androgen and LH levels are elevated compared to WT and bERKO females. aERKO but not bERKO females have a loss in estrogen protection from ischemia and atherosclerosis. Findings in bERKO females assessed by continuos breeding studies and superovulation show arrested folliculogenesis and subfertility. Because of this phenotype we are evaluating whether mutations or polymorphism in the ER-beta gene may correlate with clincal cases of female infertility. Analysis of the mammary glands of adult aERKO females showed a primitive ductal rudiment rather than the fully developed ductal tree seen in WT or bERKO mice. aERKO were used as an experimental model system for evaluating the role of ERa in normal physiological endocrine function and hormonal carcinogenesis. Since aERKO mice have no mammary gland development, a test for the role of estrogen receptor in development of mammary tumors was developed. A cross of aERKO with a mammary sensitive mouse line (WNT-1) expressing the WNT-1 oncogene was produced. These data indicate that the WNT-1 oncogene does not require functional estrogen receptor to produce an epithelial cell specific mitogenic response. Additionally, these animals do develop tumors at approximately half the level as WT. These findings would oppose one earlier hypothesis that mammary tumors develop from hormone responsive tissues. aERKO males are also infertile, with a progressive lesion resulting in atrophy of the testes and seminiferous tubule dysmorphogenesis producing decreased spermatogenesis and inactive sperm. Sperm transplantation studies of aERKO males rescues the infertility phenotype indicating the need for ER-alpha was not in the germ cell but in the somatic cells of the male reproductive tract. The findings also clearly demonstrate a requirement for estrogen receptor action in normal male reproductive physiology. Males have reduced bone density and some alterations in cardiovascular function. Phenotypic differences were seen in sex and aggressive behavior in both aERKO males and females compared to the patterns in WT or bERKO mice. In contrast to the aERKO, the bERKO males are fertile with normal sexual behavior. Recent development of a viable abERKO shows a unique ovarian phenotype of transdifferentiation of granulosa to sertoli cellls, suggesting that both ER signal mechanisms must be functional in order to maintain the proper differentiation state of the granulosa cells. Further characterization of the mice and comparison of the individual and double ER gene KO phenotypes will be required to more fully understand the physiological consequences of ER mediated actions and the specific roles of the two different forms of ER in estrogen hormone responsiveness. As a means of extending the ERKO model evaluating the mechanism of environmental estrogen action ERKO mice were treated with genistein which also acts as a tyrosine kinase inhibitor and shows that uterine growth was not stimulated indicating genistein acts through the estrogen receptor for this response. However, suppression of lutenizing hormone (LH) was shown to be as effective in ERKO as WT mice indicating this estrogen response by genistein does not require the receptor as previously thought. Differences in gene responses to hormonal xenobiotics were also seen in some in vitro gene regulation studies. Using transactivation assay system trichlorobiphenol was shown to be more active than estradiol on the lactoferrin gene. Use of this estrogen responsive yeast system also tested the xenobiotic estrogen synergy concept and showed that using a variety of genes that synergy was not apparent and had no universal application to hormone responsive systems. Our cell systems showed that Lavender and Tea tree oils were acting as endocrine disruptors (estrogenic and antiandrogenic) having an influence on young males to devleop prepubertal idiopathic gynecomastia. Development of a second and novel mouse model for studying ER action is the development of functionally specific domain active mutant receptor mouse models. We have developed mouse model for assessign the biological functionality of a DNA binding mutant ER and a mutant mouse harboring the loss of AF2 ligand activated functional domains, but still exressign AF1 functionality. The AF2 mutant ER does not activate to known hormonal agonists like estradiol but shows antagonist reversal to substnaces like Tamoxifen or Fulvestrant. AF2ER mosue models has uncovered that different tissues are responsive to the ER through AF1 versus AF2 functions. Certain chemicals activate the receptor through AF1 compared to AF2 regions, which may explain the divergent actvities of certian ER theapeutics such as Tamoxifen which are shown to have selective estrogen receptor modulator (SERM) actvities. Furthermore, the DNA binding mutant mosue model shows that ER tetherign gene resposnes do not contribute signifcantly to the hormonal physiological resposne. these models will be used for identifying the mechanistic role the different receptor functions play in the development of tissues and biological responsiveness, cancer and the activities of different environmental endocrine disrupting chemicals. Analysis is underway to investigate the activity of a human mutation in a female patient for the estrogen receptor gene. Our initial studies indicate this mutation results in a loss of coactivator binding and decreased activity.

雌激素在靶组织内的作用涉及激素物质与两种不同的受体蛋白（称为 ER-α 和 ER-β）的相互作用。组织对激素刺激的特异性和反应性在很大程度上取决于这些受体蛋白的存在和生化作用。雌激素受体（ER）被认为在发育、生殖和正常生理学中发挥着至关重要的作用。我们使用 ERa 基因 (aERKO) 和 ERb 基因 (bERKO) 纯合小鼠的基因打靶系和最新一代双 ERKO 小鼠 abERKO) 生成了基因打靶系，并表征了生殖、心血管、免疫、骨骼和神经内分泌系统的表型目前正在进行中。 RPA 分析检测到 aERKO 小鼠组织中 ER-b mRNA 水平相当，表明 ER-b 表达不依赖于 ERa。 aERKO 小鼠对雌二醇、羟基 TAM、DES 的子宫促宫试验完全没有反应。进一步的支持来自于雌激素、EGF 或 IGF-1 治疗未能诱导 aERKO 子宫中的 DNA 合成，尽管 EGF 和 IGF-1 信号传导通过刺激 c-fos 或 IRS-1 蛋白磷酸化而被证明是完整的。在aERKO小鼠中检测到孕激素受体mRNA，但在子宫、乳腺和卵巢中不受雌激素刺激，表明存在雌激素依赖和独立的基因调节。 aERKO 雌性不育，子宫发育不良，卵巢充血，无明显黄体。 aERKO 卵巢表型在发育过程中发生，可以通过 GnRH 拮抗剂逆转。与 WT 和 bERKO 女性相比，卵巢促性腺激素受体水平、血清雌激素、雄激素和 LH 水平升高。 aERKO 而非 bERKO 女性会丧失雌激素对缺血和动脉粥样硬化的保护作用。通过持续育种研究和超排卵评估的 bERKO 雌性发现，卵泡发生受阻且生育力低下。由于这种表型，我们正在评估 ER-β 基因的突变或多态性是否可能与女性不孕的临床病例相关。对成年 aERKO 雌性乳腺的分析显示出原始的导管雏形，而不是在 WT 或 bERKO 小鼠中看到的完全发育的导管树。 aERKO被用作评估ERa在正常生理内分泌功能和激素致癌作用中的作用的实验模型系统。由于 aERKO 小鼠没有乳腺发育，因此开发了一种针对雌激素受体在乳腺肿瘤发育中的作用的测试。产生了 aERKO 与表达 WNT-1 癌基因的乳腺敏感小鼠系 (WNT-1) 的杂交。这些数据表明WNT-1癌基因不需要功能性雌激素受体来产生上皮细胞特异性促有丝分裂反应。此外，这些动物产生肿瘤的水平大约是野生型动物的一半。这些发现推翻了早期的一项假设，即乳腺肿瘤是由激素反应性组织发展而来。 aERKO 雄性也不育，进行性病变导致睾丸萎缩和生精小管畸形，导致精子发生减少和精子不活跃。 aERKO 雄性的精子移植研究挽救了不育表型，表明对 ER-α 的需求不在生殖细胞中，而是在雄性生殖道的体细胞中。研究结果还清楚地证明了正常男性生殖生理学中雌激素受体作用的必要性。男性骨密度降低，心血管功能发生一些改变。与 WT 或 bERKO 小鼠的模式相比，aERKO 雄性和雌性小鼠在性别和攻击行为方面都存在表型差异。与 aERKO 不同，bERKO 雄性具有正常性行为的生育能力。最近开发的可行的 abERKO 显示了颗粒细胞向支持细胞转分化的独特卵巢表型，这表明两种 ER 信号机制必须发挥作用才能维持颗粒细胞的正确分化状态。需要对小鼠进行进一步的表征以及对个体和双 ER 基因 KO 表型进行比较，以更全面地了解 ER 介导作用的生理后果以及两种不同形式的 ER 在雌激素反应中的具体作用。作为扩展 ERKO 模型评估环境雌激素作用机制的一种手段，ERKO 小鼠接受金雀异黄酮治疗，金雀异黄酮也充当酪氨酸激酶抑制剂，并显示子宫生长没有受到刺激，表明金雀异黄素通过雌激素受体发挥作用。然而，黄体生成素 (LH) 的抑制在 ERKO 中被证明与 WT 小鼠一样有效，这表明金雀异黄素的雌激素反应并不像之前认为的那样需要受体。在一些体外基因调控研究中也发现了基因对激素异生素反应的差异。使用反式激活测定系统，三氯联苯酚对乳铁蛋白基因的活性比雌二醇更强。使用这种雌激素响应酵母系统还测试了异生雌激素协同作用概念，并表明使用多种基因，协同作用并不明显，并且对激素响应系统没有普遍应用。我们的细胞系统表明，薰衣草和茶树油充当内分泌干扰物（雌激素和抗雄激素），对年轻男性产生青春期前特发性男性乳房发育症有影响。 用于研究 ER 作用的第二种新型小鼠模型的开发是功能特异性域活性突变受体小鼠模型的开发。我们开发了小鼠模型，用于评估 DNA 结合突变体 ER 的生物学功能，以及缺失 AF2 配体激活功能域但仍表现出 AF1 功能的突变小鼠。 AF2突变体ER不会激活雌二醇等已知的激素激动剂，但对他莫昔芬或氟维司群等物质表现出拮抗剂逆转作用。 AF2ER 小鼠模型发现，不同的组织通过 AF1 和 AF2 功能对 ER 做出反应。与 AF2 区域相比，某些化学物质通过 AF1 区域激活受体，这可能解释了某些 ER 治疗药物（例如他莫昔芬）的不同活性，这些药物被证明具有选择性雌激素受体调节剂 (SERM) 活性。此外，DNA结合突变体mosue模型表明ER tetherign基因响应对激素生理响应没有显着贡献。这些模型将用于确定不同受体功能在组织发育和生物反应性、癌症以及不同环境内分泌干扰化学物质的活动中发挥的机制作用。正在进行分析以调查女性患者雌激素受体基因的人类突变的活性。我们的初步研究表明，这种突变导致共激活剂结合丧失和活性降低。