Pathophysiology of The Hypothalamic-pituitary-adrenal & Gonadal Axes

下丘脑-垂体-肾上腺的病理生理学

• 批准号: 8736816
• 负责人: Tomoshige Kino
• 金额: $ 17.47万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 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/8736816
• 关键词: 2 year old; Adrenal Glands; Affect; Aging; Apoptosis; Autoimmune Process; Biogenesis; Biological; Biological Rhythm; Biology; C-terminal; CREB1 gene; Cardiovascular system; Cell Aging; Cells; Circadian Rhythms; Complex; Corticotropin-Releasing Hormone; Cyclic AMP; Development; Disease; Estrogens; Exercise; Fertility; Functional disorder; Gene Targeting; Genes; Glucocorticoid Receptor; Glucocorticoids; Gonadotropins; Hormones; Human; Hyperactive behavior; Hypothalamic structure; In Vitro; Incidence; Infertility; Ligand Binding Domain; Malnutrition; Mediating; Mediator of activation protein; Menopause; Metabolic; MicroRNAs; Mitochondria; Molecular Profiling; Moods; Mus; Neuraxis; Organ; Ovarian; Ovarian Follicle; Ovary; Pathologic; Patients; Phase; Physiological Processes; Physiology; Pituitary Gland; Pregnancy; Production; Progesterone; Publications; Regulation; Reporting; Reproduction; Reproductive system; Respiratory Chain; Role; Signal Transduction; Steroid biosynthesis; Stress; System; Testing; Tissues; Transactivation; Transcript; Transcription Coactivator; Transcription Repressor/Corepressor; Transcriptional Regulation; X Chromosome; age related; aged; cell age; cell growth regulation; clinical application; cofactor; glucocorticoid-induced orphan receptor; granulosa cell; human study; in vivo; mitochondrial dysfunction; ovarian failure; promoter; protein expression; reproductive; screening; steroid hormone; steroid hormone receptor; transcription factor

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. These changes in the central nervous system also influences fertility by affecting various components of the reproductive system. 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 screened 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 their proper development and steroid hormone production. We have found that primary granulosa cells obtained from mouse ovaries showed circadian oscillation of several CLOCK-related genes, such as Per1/2 and Cry1/2. We found that expression of 10% of over 600 known microRNA we screened were in circadian rhythms. We further focused on miR-196a, as it showed the most significant circadian oscillation. We found that its target gene HOX8B, a transcriptional repressor important for the regulation of cell growth and apoptosis, demonstrated diurnal expression through miR-196a. We are now examining importance of our finding in the biologic action of granulosa cells. Aging is an important factor for reducing the chance of successful pregnancy, eventually developing ovarian failure and menopause, 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 microRNA expression in primary granulosa cells obtained from mouse ovaries. We obtained granulosa cells from 2-year old mice and tested expression of 600 microRNAs by employing the cells of young mice (6-9 week old) as controls. In this screening, we found that 37 microRNAs were significantly regulated in aged cells. We focused on miR-503 and -322, as these microRNAs showed the most significant reduction (over 20-fold) in aged granulosa cells and form a gene complex on chromosome X regulated by the shared promoter. We found in the subsequent protein expression profile analysis that several subunits of the ATP synthase, a component of the mitochondrial respiratory chain, were down-regulated by inhibition of these microRNAs, suggesting that miR-503 and -322 are required for mitochondrial activity. Recent publication indicated that these miRNAs down-regulate expression of mitophagy transcripts, such as Nix/Bnip3L, Ulk1, Gabarapl2, Sh3glb1, Atg12, Becn1, and Bcl2l1, thus these microRNAs maintain mitochondrial biogenesis and energy production by suppressing expression of mitophagy-inducing genes. Since reduction of mitochondria is a key feature of aged ovaries with reduced reproductive activity, it is likely that miR-503 and -322 are mediators of mitochondrial dysfunction observed in aged ovaries, and further, underlying factors for increased incidence of infertility in older subjects through modulation of mitochondrial biogenesis/activity. Crosstalk between the gonadotropin-regulating cAMP signaling system and that of the steroid hormone receptors, such as progesterone (PR), estrogen (ER) and glucocorticoid receptor (GR), are important for the regulation of ovarian functions. To elucidate their crosstalk at the transcriptional regulation, we focused on the CREB-regulated transcriptional coactivator 2 (CRTC2), a cofactor known to be specific for CREB (thus cAMP signaling)-mediated transcriptional regulation, and tested its effect on PR- or GR-induced transcriptional activity. We found that CRTC2 functions as a coregulator of GR and PR, stimulating former transcriptional activity, while repressing latter activity. We further found that CRTC2 physically interacts with the ligand-binding domain of PR and GR through its C-terminal portion harboring the transactivation domain in vitro and in vivo. We are now examining biologic importance of this crosstalk in granulosa cells.

我们已经证明，几种人类状态的特征是中枢应激系统的过度活跃或低活跃，这不仅解释了情绪变化，还解释了患有此类疾病的患者发生发育、代谢、心血管或自身免疫并发症的倾向。中枢神经系统的这些变化还通过影响生殖系统的各个组成部分来影响生育能力。 我们之前报道过，CLOCK/BMAL1（在中枢神经系统和外周产生昼夜节律的自振荡转录因子）有节奏地抑制 GR 诱导的转录活性，表明 CLOCK/BMAL1 作为糖皮质激素的反相负调节因子发挥作用可能通过拮抗昼夜波动的循环糖皮质激素的生物作用，在靶组织中发挥作用。我们进行了一项人体研究，结果表明 CLOCK 对 GR 转录活性的负调控在人类中也发挥作用。作为该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节的 microRNA。 MicroRNA 是短发夹状 RNA，通过影响这些细胞的增殖和凋亡以及类固醇生成，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需的成分。我们发现从小鼠卵巢获得的原代颗粒细胞显示出几个与 CLOCK 相关的基因的昼夜振荡，例如 Per1/2 和 Cry1/2。我们发现，在我们筛选的 600 多种已知 microRNA 中，有 10% 的表达符合昼夜节律。我们进一步关注 miR-196a，因为它显示出最显着的昼夜节律振荡。我们发现其靶基因 HOX8B（一种对调节细胞生长和凋亡很重要的转录抑制因子）通过 miR-196a 表现出昼夜表达。我们现在正在研究我们的发现对颗粒细胞生物学作用的重要性。 衰老是降低成功怀孕机会、最终导致卵巢衰竭和更年期的重要因素，但这一生理过程背后的生物学机制尚未完全阐明。营养不良、压力和运动等引起的病理性不孕也可能与衰老依赖性卵巢功能衰竭的部分机制相同。为了研究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原代颗粒细胞中的 microRNA 表达。我们从 2 岁小鼠身上获取颗粒细胞，并以幼鼠（6-9 周龄）的细胞作为对照，测试了 600 个 microRNA 的表达。在这次筛选中，我们发现 37 个 microRNA 在衰老细胞中受到显着调节。我们重点关注 miR-503 和 -322，因为这些 microRNA 在衰老的颗粒细胞中表现出最显着的减少（超过 20 倍），并在 X 染色体上形成受共享启动子调节的基因复合体。我们在随后的蛋白质表达谱分析中发现，ATP合酶（线粒体呼吸链的一个组成部分）的几个亚基通过抑制这些microRNA而下调，这表明miR-503和-322是线粒体活性所必需的。最近的出版物表明，这些 miRNA 下调线粒体自噬转录本的表达，例如 Nix/Bnip3L、Ulk1、Gabarapl2、Sh3glb1、Atg12、Becn1 和 Bcl2l1，因此这些 miRNA 通过抑制线粒体自噬诱导基因的表达来维持线粒体生物合成和能量产生。 。由于线粒体减少是衰老卵巢生殖活动降低的一个关键特征，因此 miR-503 和 -322 很可能是在衰老卵巢中观察到的线粒体功能障碍的介质，并且进一步是通过以下方式导致老年受试者不孕发生率增加的潜在因素：线粒体生物发生/活性的调节。 促性腺激素调节 cAMP 信号系统与类固醇激素受体（例如孕酮 (PR)、雌激素 (ER) 和糖皮质激素受体 (GR)）之间的串扰对于卵巢功能的调节非常重要。为了阐明它们在转录调控中的串扰，我们重点研究了 CREB ​​调控的转录辅激活因子 2 (CRTC2)，这是一种已知对 CREB（因此 cAMP 信号传导）介导的转录调控具有特异性的辅因子，并测试了其对 PR 或 GR 的影响。 -诱导转录活性。我们发现 CRTC2 作为 GR 和 PR 的共调节因子发挥作用，刺激前者的转录活性，同时抑制后者的活性。我们进一步发现 CRTC2 在体外和体内通过其含有反式激活结构域的 C 端部分与 PR 和 GR 的配体结合结构域发生物理相互作用。我们现在正在研究颗粒细胞中这种串扰的生物学重要性。