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.

靶组织中的雌激素激素作用涉及激素物质与称为ER-Alpha和ER-Beta的两种不同受体蛋白的相互作用。组织对激素刺激的特异性和反应性在大多数方面受这些受体蛋白的存在和生化作用的控制。雌激素受体（ER）被认为在发育，繁殖和正常生理学中起着至关重要的作用。我们使用纯合子的基因靶向线生成了纯合子的ERA基因（Aerko）和ERB基因（Berko）（Berko）以及近来的双Erko小鼠Aberko）以及在生殖，心血管，免疫，免疫，骨骼和神经洋顿蛋白系统中的表征。 RPA分析检测到Aerko小鼠组织中的ER-B mRNA水平可比水平，这表明ER-B表达不依赖ERA。 Aerko小鼠对雌二醇，羟基TAM，des完全没有反应对子宫型测定。进一步的支持来自雌激素，EGF或IGF-1治疗因诱导Aerko子宫中DNA合成的失败，尽管通过刺激C-FOS或IRS-1蛋白磷酸化，EGF和IGF-1信号传导被证明是完整的。在Aerko小鼠中检测到孕激素受体mRNA，但在子宫，乳腺和卵巢中未被雌激素刺激，表明雌激素依赖性和独立的基因调节。 Aerko女性是不育的，具有肿瘤性子宫和多血症的卵巢，没有明显的Corpora lutea。 Aerko卵巢表型在发育中发生，可以由GNRH拮抗剂逆转。与WT和Berko雌性相比，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，但没有Berko女性在缺血和动脉粥样硬化中雌激素保护损失。通过连续育种研究和超排除评估的Berko雌性中的发现表明，卵泡发生和不足。由于这种表型，我们正在评估ER-BETA基因中的突变或多态性是否可能与女性不育症的crincal病例相关。对成年Aerko雌性的乳腺的分析显示出原始的导管含量，而不是在WT或Berko小鼠中看到的完全发育的导管树。 Aerko用作一种实验模型系统，以评估ERA在正常生理内分泌功能和激素癌变中的作用。由于Aerko小鼠没有乳腺发育，因此开发了雌激素受体在乳腺肿瘤发育中的作用的测试。产生了表达Wnt-1癌基因的乳腺敏感小鼠系（Wnt-1）的Aerko十字。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有丝分裂反应。此外，这些动物确实会出现肿瘤的水平，如WT的一半。这些发现将反对以前的假设，即乳腺肿瘤是由激素反应性组织发展而来的。 Aerko雄性也不育，进行性病变导致睾丸萎缩和生精的小管变形症，产生了精子发生和无活性精子的降低。 Aerko雄性的精子移植研究挽救了不育表型，表明需要Er-α在生殖细胞中，而是在男性生殖道的体细胞中。这些发现还清楚地表明了对正常男性生殖生理学中雌激素受体作用的要求。雄性骨密度降低，心血管功能有所改变。与WT或Berko小鼠的模式相比，在性行为和女性的性行为和攻击行为中，表型差异在性别和侵略性行为中都存在。与Aerko相反，Berko雄性具有正常的性行为。有生存的阿伯科的最新发展显示了颗粒向Sertoli细胞转分解的独特卵巢表型，这表明这两种ER信号机制都必须起作用，以维持颗粒细胞的适当分化状态。将需要进一步表征小鼠的进一步表征，并需要更充分地了解ER介导的作用的生理后果以及ER两种不同形式的ER在雌激素激素反应性中的特定作用。作为扩展评估环境雌激素作用机理Erko小鼠机制的ERKO模型的一种手段，用染料木黄酮处理，该模型也充当酪氨酸激酶抑制剂，并表明未刺激子宫生长，表明雌激素受体通过雌激素受体起作用。然而，表明抑制黄体激素（LH）在ERKO中与WT小鼠一样有效，表明染料碱性的雌激素反应不需要以前认为的受体。在某些体外基因调节研究中，还观察到了基因对激素异种生物学的反应差异。使用反式激活测定系统三氯苯酚比乳铁蛋白基因上的雌二醇更活跃。这种雌激素反应性酵母系统的使用还测试了异源雌激素协同概念，并表明使用多种基因表明协同作用并不明显，并且没有普遍应用于激素反应式系统。我们的细胞系统表明，薰衣草和茶树油充当内分泌破坏者（雌激素和抗雄激素），对年轻男性对德夫勒普前植物学特发性妇科症具有影响。 用于研究ER作用的第二种新型小鼠模型的开发是功能特定域活动突变受体小鼠模型的发展。我们已经开发了小鼠模型，以评估DNA结合突变体ER和具有AF2配体激活功能域损失的突变小鼠的生物学功能，但仍具有外观AF1功能。 AF2突变体不会激活诸如雌二醇之类的已知激素激动剂，而是表现出拮抗剂的逆转为他莫昔芬或富伏剂等替代品。 AF2ER MOSUE模型发现，不同的组织通过AF1与AF2功能对ER有反应。与AF2区相比，某些化学物质通过AF1激活受体，这可能解释了诸如他莫昔芬的证书的差异行为，这些作用是他具有选择性雌激素受体调节剂（SERM）的作用。此外，DNA结合突变体Mosue模型表明，ER链接基因resposes并不能显着促进激素生理疗法。这些模型将用于识别不同受体功能在组织发展和生物反应性，癌症以及不同环境内分泌破坏化学物质的活性中起作用的机械作用。正在进行分析以研究雌激素受体基因的女性患者中人类突变的活性。我们的初步研究表明，这种突变导致共激活因子结合和活性降低。