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.

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Nicolaides, Nicolas C; Charmandari, Evangelia; Chrousos, George P et al. (2014) Circadian endocrine rhythms: the hypothalamic-pituitary-adrenal axis and its actions. Ann N Y Acad Sci 1318:71-80
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Nader, Nancy; Bachrach, Bert E; Hurt, Darrell E et al. (2010) A novel point mutation in helix 10 of the human glucocorticoid receptor causes generalized glucocorticoid resistance by disrupting the structure of the ligand-binding domain. J Clin Endocrinol Metab 95:2281-5

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