Pathophysiology of The Hypothalamic-pituitary-adrenal & Gonadal Axes

我们已经证明了几种人类状态的特征是中枢应激系统的过度活动或活动不足，这不仅解释了情绪变化，也解释了患有此类疾病的患者发展、代谢、心血管或自身免疫并发症的倾向。我们目前正在对新发现的非肽、口服CRH 1型受体拮抗剂antalarmin进行临床前研究，这些研究表明，这种拮抗剂可能对大量以应激系统过度活动为特征的状态有用，如抑郁、神经性厌食症和特发性失眠。 我们最近发现，非编码(NC)RNA生长停滞特异性5(Gas5)，它积聚在生长停滞的细胞中，但其生理作用尚不清楚，以及腺苷5‘-单磷酸激活蛋白激酶(AMPK)，它是能量稳态的主要调节者，感知体内的能量消耗，刺激增加燃料摄取和节省外周供应的途径，调节GR的转录活性。前者通过充当诱饵RNA GRE来实现这一点，后者通过磷酸化GR来实现这一点。这些结果表明，HPA轴的生物学行为在靶组织水平上受到营养状态和能量来源的调节，从而影响HPG轴的活动。 我们先前已经报道，在中枢神经系统和外周产生昼夜节律的自振荡转录因子CLOCK/BMAL1节律性地抑制GR诱导的转录活性，表明CLOCK/BMAL1在靶组织中作为糖皮质激素作用的反向负调节因子发挥作用，可能是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，发现时钟对GR转录活动的这种负面调节在人类中也是有效的。作为这个昼夜节律项目的延伸，我们目前正在筛选小鼠卵巢颗粒细胞中以昼夜节律方式调节的microRNAs。MicroRNAs是一种短发夹状RNA，通过影响颗粒细胞的增殖和凋亡以及类固醇的合成，对生殖，特别是颗粒细胞表现出强大的生物学作用。另一方面，颗粒细胞是卵泡正常发育和类固醇激素产生所必需的成分。我们发现从小鼠卵巢获得的原代颗粒细胞表现出几个时钟相关基因的昼夜振荡，如PER1/2和Cry1/2。利用从这些细胞中纯化的总RNA和包含600个已知microRNAs的阵列板，我们现在正在鉴定受昼夜调节的microRNAs。然后，我们将在颗粒细胞中测试它们的生物学意义。 衰老是降低妊娠成功率、最终发展为卵巢衰竭和绝经的一个重要因素，几乎不产生雌激素和孕激素，但这一生理过程背后的生物学机制尚未完全阐明。病理性不孕不育，如营养不良、压力和运动，也有可能与衰老相关性卵巢功能衰竭的部分机制相同。为了检测衰老对卵巢功能的影响，我们再次检测了从小鼠卵巢获得的初级颗粒细胞中microRNAs的表达。我们从2岁的小鼠身上获得了颗粒细胞，并以6-9周龄的幼鼠的细胞为对照，检测了600个microRNAs的表达。一旦我们在老年小鼠的颗粒细胞中发现了显著上调或下调的特定microRNAs，我们将研究它们对类固醇生成的影响，因为我们发现P450侧链裂解酶(P450SCC)和类固醇生成急性调节因子(STAR)的mRNA在老年小鼠卵巢中的表达显著下调(分别下调30倍和5倍)。

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 are currently performing 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 recently 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 the HPA axis are regulated at the level of the 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 are currently screening the microRNAs regulated in a circadian fashion in granulose 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 their proper development and steroid hormone production. We have found that primary granulose cells obtained from mouse ovaries showed 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 are now identifying microRNAs under circadian regulation. We will then test their biologic significance in granulose cells. 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 completely been elucidated 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 microRNAs expression in primary granulosa cells obtained from mouse ovaries. We obtained granulose cells from 2-year old mice and are now testing expression of 600 microRNAs by employing the cells of young mice (6-9 week old) as controls. Once we identify specific microRNAs significantly up- or down-regulated in granulose cells of old mice, we will then examine their effects on steroidogenesis, as we found that mRNA expression of the p450 side chain cleavage enzyme (p450SCC) and the steroidogenic acute regulatory factor (StAR), key molecules for initiating steroidogenesis, were significantly (30- and 5-fold, respectively) down-regulated in ovaries of old mice.