Normal activity of the hypothalamic pituitary adrenal axis, leading to the secretion of glucocorticoids by the adrenal gland, is essential for normal metabolic activity and for survival during challenging situations. Studies under this project have defined the role of the hypothalamic peptides corticotrophin releasing hormone (CRH) and vasopressin (VP) in the regulation of pituitary ACTH, and contributed to elucidating the regulation of the expression of CRH and VP during stress, and the mechanisms of action, topographic distribution, regulation and physiological role of the receptors for these peptides in the pituitary gland and in the brain. CRH coordinates behavioral, autonomic and hormonal responses to stress and is the main regulator of ACTH secretion in acute and chronic conditions. Following CRH release, rapid but transient activation of CRH transcription is required to restore mRNA and peptide levels. Termination of the response is essential to prevent pathology associated with chronic elevation of CRH and glucocorticoid production. Current work is aimed to elucidate the mechanisms controlling negative and positive transcriptional regulation of CRH, as well as the mechanisms responsible to the normal circadian and ultradian pattern of glucocorticoid secretion. This laboratory has reported that cAMP/phospho-CREB signaling is essential but not sufficient to activate CRH transcription. This finding led to the discovery that transcriptional activation of the CRH gene requires the CREB co-activator Transducer Of Regulated CREB activity (TORC). In resting conditions, the co-activator is found in a phosphorylated, inactive state in the cytoplasm. Its activation and nuclear translocation requires protein kinase A (PKA)- mediated inhibition protein kinases mediating TORC phosphorylation. Experiments examining the trafficking of TORC from the cytoplasm to the nucleus of CRH producing neurons, and the effects of over-expression and knock down of TORC on CRH transcription demonstrated that TORC is essential for CRH transcription, and that during stress the co-activator shifts to the nucleus and binds to the CRH promoter as part of a complex with CREB, paralleling the activation of CRH transcription. Future research is planned to examine the importance of the co-activator TORC during physiological regulation of CRH transcription in vivo. Significant effort was invested on the mechanisms regulating TORC activity. In basal conditions, transcription is low because TORC remains in the cytoplasm, inactivated by phosphorylation through Ser/Thr protein kinases of the AMP-dependent protein kinases (AMPK) family, including salt-inducible kinase (SIK). To determine which kinase is responsible for TORC phosphorylation in CRH neurons, we measured AMPK, SIK1 and SIK2 mRNA in the PVN of rats by in situ hybridization. In basal conditions, low levels of the 3 kinases were found in the dorsomedial PVN, consistent with location in CRH neurons. Restraint stress increased SIK1 mRNA levels, while SIK2 and AMPK mRNA showed only minor increases. Overexpression of either SIK1 or SIK2 in 4B cells reduced nuclear TORC2 levels and inhibited forskolin-stimulated CRH transcription. Conversely, the non-selective SIK inhibitor, staurosporine, increased nuclear TORC2 content and stimulated CRH transcription in 4Bcells, and primary neuronal cultures (heteronuclear RNA). Specific shRNA knock down of endogenous SIK2 but not SIK1 induced nuclear translocation of TORC2 and CRH transcription, suggesting that SIK2 mediates TORC inactivation in basal conditions, while induction of SIK1 limits transcriptional activation. Current research aims to test the hypothesis that while SIK2 mediates sequestration of TORC in basal conditions, induction of SIK1 during stimulation of the CRH neuron mediates phosphorylation and nuclear export of TORC, thus contributing to the termination of the transcriptional response. Since increasing evidence indicates that CREB-dependent transcriptional activation of a number of genes requires the co-activator, TORC, and because of the importance of CREB in many brain functions, the topographic distribution of TORC1, 2 and 3 mRNAs in specific regions of the rat forebrain were studied. In situ hybridization analysis showed that TORC1 is the most abundant isoform in most forebrain structures, followed by TORC2 and TORC3. Although high levels of TORC1 were widely distributed in the forebrain, TORC2 was found in discrete nuclei and TORC3 mostly in the ependyma, and pia mater. In the paraventricular nucleus of the hypothalamus, TORC1 and 2 mRNAs were abundant in the parvicellular and magnocellular neuroendocrine compartments, whereas TORC3 expression was low. All three isoform mRNAs were found elsewhere in the hypothalamus, with the most prominent expression of TORC1 in the ventromedial nucleus, TORC2 in the dorsomedial and arcuate nuclei, TORCs 1 and 2 in the supraoptic nucleus, and TORC2 in the suprachiasmatic nucleus. These differential distribution patterns are consistent with complex roles for all three TORC isoforms in diverse brain structures, and provide a foundation for further studies on the mechanisms of CREB/TORC signaling on brain function. While cyclic AMP/PKA-dependent pathways are essential for transcriptional activation of the CRH gene, the main direct regulators of the CRH neuron, norepinephrine and glutamate do not increase cyclic AMP production. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in central control of the HPA axis. Studies using PACAP knockout mice showed that these mice fail to increase CRH mRNA in response to restraint. Furthermore, PACAP can directly stimulate CRH hnRNA in primary cultures of hypothalamic neurons. The role of PACAP mediating cyclic AMP-signaling in the CRH neuron during stress is under current investigation. The activity of the HPA axis is characterized by circadian and ultradian pattern of glucocorticoid secretion with one secretory pulse per hour. Because of increasing evidence for the importance of pulsatility in regulating glucocorticoid-responsive gene transcription, considerable effort during the past year focused on the mechanisms determining pulsatile secretion at the adrenal level. Availability of active steroidogenic acute regulatory protein (StAR) and side chain cleavage cytochrome P450 (P450scc) are rate-limiting steps for steroidogenesis. The relationship between TORC activation and ACTH-induced steroidogenesis were studied in vivo, by examining the time-course of the effect of ACTH injection (4ng, iv) on the transcriptional activity of StAR and P450scc genes and nuclear accumulation of TORC2 in rat adrenal cortex. ACTH produced rapid (5min) and transient increases in plasma corticosterone. This was followed by increases in StAR and P450scc hnRNA levels by 15 min. Concomitantly, ACTH increased nuclear phospho-CREB, and nuclear accumulation of TORC2, with maximal levels at 5 min and returning to basal by 30 min. The decline of nuclear TORC2 paralleled increases in SIK1 expression. The direct temporal relationship between nuclear accumulation of TORC2 and the increase in transcription of steroidogenic proteins, implicates TORC2 in the physiological regulation of steroidogenesis in the adrenal cortex. The delayed induction of SIK1 suggests a role for SIK1 in the declining phase of steroidogenesis. Further studies in methylprednisolone suppressed rats, with reduced plasma corticosterone, and adrenal StAR and P450scc hnRNA, demonstrated that pulsatile but not constant ACTH infusion restored pulsatile steroid secretion as well as StAR and P450scc hnRNA levels, indicating that pulsatile ACTH release is critical for optimal adrenocortical function.
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