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。 在应激系统靶组织水平，我们通过定义糖皮质激素受体基因的新突变和/或缺失导致糖皮质激素受体功能异常或受体减少，阐明了散发性和家族性糖皮质激素耐药的分子病理生理学。我们发现了一例714位氨基酸(GR R714Q)精氨酸到谷氨酰胺杂合子替换的女孩，表现为骨龄加快和轻度阴蒂肿大，表明该患者由于HPA和HPG轴调节失调而导致雄激素作用增强。在同一领域，我们描述了炎症诱导的糖皮质激素抵抗和糖皮质激素分泌不足在急性呼吸窘迫综合征中的作用。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。此外，非编码(NC)RNA生长停滞特异性5(Gas5)，它聚集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。在由HPA和HPG轴的中枢组成的大脑中，我们发现细胞周期蛋白依赖性激酶5(CDK5)通过其激活剂p35/p25与糖皮质激素受体(GR)和盐皮质激素受体(MR)相互作用，差异地调节这些类固醇受体的转录活性。CDK5在中枢神经系统的形态发生和功能中起重要作用，其异常激活与神经退行性疾病的发生有关。内源性糖皮质激素、皮质醇和皮质酮与这些受体结合，在中枢神经系统引起受体特异性的生物学作用，而它们的靶分子之一脑源性生长因子(BDNF)在神经活性、突触可塑性、记忆巩固和情绪变化中发挥关键作用。因此，CDK5的异常激活可能部分通过皮质类固醇受体/BDNF调节神经元的活动，进一步影响HPA的活动，并可能影响HPG轴的活动。我们现在正在开发表达具有特定磷酸化或乙酰化位点的GR突变体的小鼠，以便在动物水平上检验这些GR的表观遗传修饰的作用。 细胞外高渗或渗透应激是陆地生物的主要威胁，因此通过从下丘脑/垂体后叶分泌精氨酸加压素来强烈刺激HPA轴。除了这种全身性反应，它还激活了对细胞外高渗的适应性反应的细胞级联反应。细胞外高渗或渗透应激诱导并激活一种含有Rel同源结构域的转录因子-活化T细胞核因子5(NFAT5)，从而刺激渗透应激反应基因的转录，并导致细胞内小分子有机渗透压的积累，以维持细胞内外的等价性。我们以前报道了淋巴细胞对渗透胁迫作出反应的细胞内信号级联反应，并发现Rho类型的鸟嘌呤核苷酸交换因子BRX或AKAP13对于渗透应激刺激NFAT5的表达是必不可少的，并且是将细胞外高渗信号传递到细胞核的细胞内信号级联的关键组成部分。渗透应激介导的NFAT5的诱导需要BRX的EfG结构域和p38丝裂原激活的激酶(MAPK)，而BRX则通过其C-末端结构域吸引cJun激酶(JNK)相互作用蛋白(JIP)4，JIP 4是p38 MAPK级联和NFAT5激活的支架，将激活的Rho型小G蛋白偶联到p38 MAPK信号通路的组成部分。重要的是，这个信号系统在脾淋巴细胞分化中起着关键作用，而它是大多数器官和组织保护它们免受细胞外高渗和维持其功能的一般机制。为了进一步阐明BRX/NFAT5介导的渗透应激在免疫功能调节中的作用，我们目前正在利用Cre/loxP系统在树突状细胞/单核/巨噬细胞中携带BRX基因特异性缺失的小鼠。

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. At the level of the stress system target tissues, we have elucidated the molecular pathophysiology of sporadic and familial glucocorticoid resistance by defining novel mutations and/or deletions of the glucocorticoid receptor gene leading to abnormally functioning or decreased receptors. We found a girl case with hetrozygotic replacement from arginine to glutamine at amino acid 714 (GR R714Q) who demonstrated accelerated bone age and mild clitolomegaly, indicating that this patient has elevated action of androgen caused by dysregulation of the HPA and HPG axes. In the same area, we have described inflammation-induced glucocorticoid resistance and glucocorticoid secretion insufficiency in the acute respiratory distress syndrome. We have previously reported that CLOCK/BMAL1, the self-oscillating transcription factors that generate circadian rhythms in both 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. Furthermore, 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. In the brain, which consists of central component of the HPA and HPG axis, we found that the cyclin-dependent kinase 5 (CDK5), which plays important roles in the morphogenesis and functions of CNS, and whose aberrant activation is associated with development of neurodegenerative disorders, interacted with both GR and the mineralocorticoid receptor (MR) through its activators p35/p25 and differentially regulated the transcriptional activity of these steroid receptors. Endogenous glucocorticoids, cortisol and corticosterone, bind these receptors and cause receptor-specific biologic actions in CNS, while one of their target molecules, the brain-derived growth factor (BDNF) plays a critical roles in the neurobiability, synaptic plasticity, consolidation of memory and emotional changes. Therefore, aberrant activation of CDK5 may in part regulate neuronal activity through corticosteroid receptors/BDNF, further influencing the activity of the HPA, and possibly, the HPG axis. We are now developing mice expressing GR mutants with specific phosphorylation sites or acetylation sites in order to examine roles of these epigenetic modifications of GR at animal levels. Extracellular hyperosmolarity or osmotic stress is a major threat for land organisms, and thus strongly stimulates the HPA axis through secretion of ariginine vasopression from the hypothalamus/posterior lobe of the pituitary gland. In addition to this systemic response, it also activates a cellular cascade of adaptive response to extracellular hyperosmolarity. Extracellular hyperosmolarity or osmotic stress induces and activates a Rel-homology domain-containing transcription factor, the nuclear factor of activated T-cells 5 (NFAT5), which subsequently stimulates transcription of osmotic stress-responsive genes and causes intracellular accumulation of small organic osmolytes to maintain isotonicity between the inside and outside of the cells. We previously reported an intracellular signaling cascade responsive to osmotic stress in lymphocytes as a model tissue, and found that a Rho-type guanine nucleotide exchange factor (GEF) Brx or AKAP13 is essential for the osmotic stress-stimulated expression of NFAT5, and is a key component of the intracellular signaling cascade transmitting the extracellular hyperosmolarity signal to the nucleus. Osmotic stress-mediated induction of NFAT5 requires the Brx GEF domain and p38 mitogen-activated kinase (MAPK), while Brx in response to osmotic stress attracts through its C-terminal domain the cJun kinase (JNK)-interacting protein (JIP) 4, a scaffold specific to activation of the p38 MAPK cascade and NFAT5, coupling activated Rho-type small G-proteins to components of the p38 MAPK signaling pathway. Importantly, this signaling system plays a critical role in the lymphocyte differentiation in the spleen, while it is a general mechanism for most of the organs and tissues to protect them from extracellular hyperosmolarity and to maintain their functions. To further elucidate roles of Brx/NFAT5-mediated osmotic stress on the regulation of immune function, we are now developing mice carrying specific deletion of the brx gene in dendritic cells/monocytes/macrophages using the Cre/LoxP system.