Pathophysiology of The Hypothalamic-pituitary-adrenal & Gonadal Axes

我们已经证明，几种人类状态的特征是中枢应激系统的活动过度或活动减退，这不仅解释了情绪变化，而且还解释了患有此类疾病的患者发展发育，代谢，心血管或自身免疫并发症的倾向。我们完成了临床前研究与新发现的非肽类，口服，CRH 1型受体拮抗剂，antalarmin，这表明，这样的拮抗剂可能是有用的，在大量的状态，其特征在于过度活跃的应激系统，如抑郁症，神经性厌食症和特发性失眠。 我们还发现非编码（nc）RNA生长停滞特异性5（Gas 5），其在生长停滞细胞中积累，但其生理作用尚不清楚，以及腺苷5'一磷酸活化蛋白激酶（AMPK），能量稳态的主要调节剂，感知体内能量消耗并刺激增加燃料摄入并节省外周供应的途径，调节转录活性的GR。前者完成这作为一个诱饵RNA GRE，后者通过磷酸化的GR。这些结果表明，HPA轴的生物学行为的调节在靶组织水平的营养状态和可用性的能源，随后影响HPG轴的行动。 我们以前曾报道过，CLOCK/BMAL 1，自振荡转录因子，产生昼夜节律在中枢神经系统和外周，有节奏地抑制GR诱导的转录活性，表明CLOCK/BMAL 1的功能作为一个反相负调节糖皮质激素的作用在靶组织中，可能通过拮抗昼夜波动的循环糖皮质激素的生物作用。我们进行了一项人体研究，发现CLOCK对GR转录活性的负调控在人体中也有功能。作为这个昼夜节律项目的延伸，我们筛选了小鼠卵巢颗粒细胞中以昼夜节律方式调控的microRNA。MicroRNA是短发夹样RNA，其通过影响颗粒细胞的增殖和凋亡以及类固醇生成而对生殖，特别是颗粒细胞表现出强烈的生物学作用。另一方面，颗粒细胞是卵泡的组成部分，是卵母细胞正常发育和类固醇激素产生所必需的。我们发现，从小鼠卵巢获得的初级颗粒细胞中的几个时钟相关基因，如Per 1/2和Cry 1/2的昼夜振荡。使用从这些细胞中纯化的总RNA和含有600种已知microRNA的阵列板，我们发现检测的10%的miRNAs处于昼夜节律波动中，其中miR 196 a表现出最显著的变化。在基于计算机/网站的搜索中，几个Hox基因的mRNA/蛋白质表达可能受到该miRNA的调控。其中，HoxA 5的mRNA表达呈典型的昼夜节律性，HoxA 5是一种在颗粒细胞中表达的转录因子，具有已知的活性以支持颗粒细胞对促黄体生成素的反应。我们现在正在进行几个系列的实验，以研究miR 196 a及其昼夜节律对颗粒细胞活动的影响，重点是HoxA 5。 衰老是降低成功妊娠机会的重要因素，最终发展为卵巢衰竭和绝经，几乎不产生雌激素和孕激素，但这一生理过程的生物学机制尚未完全阐明。病理性不孕症，如营养不良，压力和运动，也可能是衰老依赖性卵巢功能衰竭的部分机制。为了检查衰老对卵巢功能的影响，我们再次检查了从小鼠卵巢获得的初级颗粒细胞中的microRNA表达：我们从2岁的小鼠获得颗粒细胞，并通过使用年轻小鼠（6-9周龄）的细胞作为对照来检查其600种microRNA的表达。我们发现，年轻卵巢中最高表达的miR 503和miR 322的表达在老年颗粒细胞中显著下调（减少超过90%）。我们正在通过蛋白质组学分析来检测这些miRNAs的靶蛋白。我们现在正在准备一项动物研究，在这项研究中，我们使用化学修饰的miRNA抑制剂敲除小鼠中的这些miRNA，以检查它们对排卵和生育的作用。

We have demonstrated that several human states are characterized by hyperactivity or hypoactivity of the central stress system, which explains not only mood changes but also the propensity of patients with such disorders to develop developmental, metabolic, cardiovascular or autoimmune complications. We completed preclinical studies with the newly discovered nonpeptide, oral, CRH type 1 receptor antagonist, antalarmin, which show that such an antagonist may be useful in a large number of states characterized by hyperactivity of the stress system, such as depression, anorexia nervosa and idiopathic insomnia. We also found that the noncoding (nc) RNA growth arrest-specific 5 (Gas5), which accumulates in growth-arrested cells, but whose physiologic roles are not known as yet, and the adenosine 5' monophosphate-activated protein kinase (AMPK), a master regulator of energy homeostasis, sensing energy depletion inside the body and stimulating pathways that increase fuel uptake and save on peripheral supplies, regulated transcriptional activity of the GR. The former accomplished this by acting as a decoy RNA GRE, the latter by phosphorylating the GR. These results indicate that the biologic actions of HPA axis are regulated at the level of target tissues by the nutritional state and availability of energy resources, subsequently influencing the action of HPG axis. We have previously reported that CLOCK/BMAL1, the self-oscillating transcription factors that generate circadian rhythms both in the central nervous system and periphery, rhythmically repressed GR-induced transcriptional activity, indicating that CLOCK/BMAL1 functions as a reverse phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing the biologic actions of diurnally fluctuating circulating glucocorticoids. We performed one human study and revealed that this negative regulation on GR transcriptional activity by CLOCK was also functional in humans. As an extension of this circadian rhythm project, we have screened the microRNAs regulated in a circadian fashion in granulosa cells of mouse ovaries. MicroRNAs are short hairpin-like RNAs that demonstrate strong biological actions on reproduction, and specifically, granulosa cells by influencing proliferation and apoptosis, as well as steroidogenesis of these cells. Granulosa cells, on the other hand, are components of ovarian follicles required for proper development of oocytes and steroid hormone production. We have found that primary granulosa cells obtained from mouse ovaries were in circadian oscillation of several CLOCK-related genes, such as Per1/2 and Cry1/2. Using total RNAs purified from these cells and the array plates containing 600 known microRNAs, we found that 10% of the miRNAs examined was under circadian fluctuation among which miR196a showed the most significant changes. In computer/website-based search, mRNA/protein expression of several Hox genes may be regulated by this miRNA. Among them, mRNA expression of the HoxA5, a transcription factor expressed in the granulosa cells with known activity to support their responsiveness to the luteinizing hormone, was in typical circadian rhythmicity. We are now performing several lines of experiments to examine impact of miR196a and its circadian rhythms on granulosa cell activities by focusing on HoxA5. Aging is an important factor for reducing the chance of successful pregnancy, eventually developing ovarian failure and menopause with virtually no production of estrogens and progestins, but the biological mechanisms underlying this physiologic process have not been elucidated completely as yet. It is also possible that pathologic infertility, such as by malnutrition, stress and exercise, might share part of the mechanisms responsible for aging-dependent ovarian failure. To examine impact of aging on ovarian functions, we again examined microRNA expression in primary granulosa cells obtained from mouse ovaries: We obtained granulosa cells from 2-year old mice, and examined their expression of 600 microRNAs by employing the cells of young mice (6-9 week old) as controls. We identified that expression of miR503 and miR322, which are among the most highly expressed miRNAs in young ovaries, was significantly down regulated in aged granulosa cells (by over 90% reduction). We are now examining target protein of these miRNAs by using the proteomic analysis. We are now preparing one animal study in which we knockdown these miRNAs in mice using chemically modified miRNA inhibitors to examine their roles on ovulation and fertility.