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.
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