DLG2 variants in patients with pubertal disorders.

PURPOSE: Impaired function of gonadotropin-releasing hormone (GnRH) neurons can cause a phenotypic spectrum ranging from delayed puberty to isolated hypogonadotropic hypogonadism (IHH). We sought to identify a new genetic etiology for these conditions. METHODS: Exome sequencing was performed in an extended family with autosomal dominant, markedly delayed puberty. The effects of the variant were studied in a GnRH neuronal cell line. Variants in the same gene were sought in a large cohort of individuals with IHH. RESULTS: We identified a rare missense variant (F900V) in DLG2 (which encodes PSD-93) that cosegregated with the delayed puberty. The variant decreased GnRH expression in vitro. PSD-93 is an anchoring protein of NMDA receptors, a type of glutamate receptor that has been implicated in the control of puberty in laboratory animals. The F900V variant impaired the interaction between PSD-93 and a known binding partner, Fyn, which phosphorylates NMDA receptors. Variants in DLG2 that also decreased GnRH expression were identified in three unrelated families with IHH. CONCLUSION: The findings indicate that variants in DLG2/PSD-93 cause autosomal dominant delayed puberty and may also contribute to IHH. The findings also suggest that the pathogenesis involves impaired NMDA receptor signaling and consequently decreased GnRH secretion.

Involvement of CREB-regulated transcription coactivators (CRTC) in transcriptional activation of steroidogenic acute regulatory protein (Star) by ACTH.

Studies in vivo have suggested the involvement of CREB-regulated transcription coactivator (CRTC)2 on ACTH-induced transcription of the key steroidogenic protein, Steroidogenic Acute Regulatory (StAR). The present study uses two ACTH-responsive adrenocortical cell lines, to examine the role of CRTC on Star transcription. Here we show that ACTH-induced Star primary transcript, or heteronuclear RNA (hnRNA), parallels rapid increases in nuclear levels of the 3 isoforms of CRTC; CRTC1, CRTC2 and CRTC3. Furthermore, ACTH promotes recruitment of CRTC2 and CRTC3 by the Star promoter and siRNA knockdown of either CRTC3 or CRTC2 attenuates the increases in ACTH-induced Star hnRNA. Using pharmacological inhibitors of PKA, MAP kinase and calcineurin, we show that the effects of ACTH on Star transcription and CRTC nuclear translocation depend predominantly on the PKA pathway. The data provides evidence that CRTC2 and CRTC3, contribute to activation of Star transcription by ACTH, and that PKA/CRTC-dependent pathways are part of the multifactorial mechanisms regulating Star transcription.

Genome-Wide Analysis of Glucocorticoid-Responsive Transcripts in the Hypothalamic Paraventricular Region of Male Rats.

Glucocorticoids (GCs) are essential for stress adaptation, acting centrally and in the periphery. Corticotropin-releasing factor (CRF), a major regulator of adrenal GC synthesis, is produced in the paraventricular nucleus of the hypothalamus (PVH), which contains multiple neuroendocrine and preautonomic neurons. GCs may be involved in diverse regulatory mechanisms in the PVH, but the target genes of GCs are largely unexplored except for the CRF gene (Crh), a well-known target for GC negative feedback. Using a genome-wide RNA-sequencing analysis, we identified transcripts that changed in response to either high-dose corticosterone (Cort) exposure for 12 days (12-day high Cort), corticoid deprivation for 7 days (7-day ADX), or acute Cort administration. Among others, canonical GC target genes were upregulated prominently by 12-day high Cort. Crh was upregulated or downregulated most prominently by either 7-day ADX or 12-day high Cort, emphasizing the recognized feedback effects of GC on the hypothalamic-pituitary-adrenal (HPA) axis. Concomitant changes in vasopressin and apelin receptor gene expression are likely to contribute to HPA repression. In keeping with the pleotropic cellular actions of GCs, 7-day ADX downregulated numerous genes of a broad functional spectrum. The transcriptome response signature differed markedly between acute Cort injection and 12-day high Cort. Remarkably, six immediate early genes were upregulated 1 hour after Cort injection, which was confirmed by quantitative reverse transcription PCR and semiquantitative in situ hybridization. This study may provide a useful database for studying the regulatory mechanisms of GC-dependent gene expression and repression in the PVH.

Spontaneous and CRH-Induced Excitability and Calcium Signaling in Mice Corticotrophs Involves Sodium, Calcium, and Cation-Conducting Channels.

Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling.

Oxytocin Regulates Stress-Induced Crf Gene Transcription through CREB-Regulated Transcription Coactivator 3.

The major regulator of the neuroendocrine stress response in the brain is corticotropin releasing factor (CRF), whose transcription is controlled by CREB and its cofactors CRTC2/3 (TORC2/3). Phosphorylated CRTCs are sequestered in the cytoplasm, but rapidly dephosphorylated and translocated into the nucleus following a stressful stimulus. As the stress response is attenuated by oxytocin (OT), we tested whether OT interferes with CRTC translocation and, thereby, Crf expression. OT (1 nmol, i.c.v.) delayed the stress-induced increase of nuclear CRTC3 and Crf hnRNA levels in the paraventricular nucleus of male rats and mice, but did not affect either parameter in the absence of the stressor. The increase in Crf hnRNA levels at later time points was parallel to elevated nuclear CRTC2/3 levels. A direct effect of Thr(4) Gly(7)-OT (TGOT) on CRTC3 translocation and Crf expression was found in rat primary hypothalamic neurons, amygdaloid (Ar-5), hypothalamic (H32), and human neuroblastoma (Be(2)M17) cell lines. CRTC3, but not CRCT2, knockdown using siRNA in Be(2)M17 cells prevented the effect of TGOT on Crf hnRNA levels. Chromatin-immunoprecipitation demonstrated that TGOT reduced CRTC3, but not CRTC2, binding to the Crf promoter after 10 min of forskolin stimulation. Together, the results indicate that OT modulates CRTC3 translocation, the binding of CRTC3 to the Crf promoter and, ultimately, transcription of the Crf gene. SIGNIFICANCE STATEMENT: The neuropeptide oxytocin has been proposed to reduce hypothalamic-pituitary-adrenal (HPA) axis activation during stress. The underlying mechanisms are, however, elusive. In this study we show that activation of the oxytocin receptor in the paraventricular nucleus delays transcription of the gene encoding corticotropin releasing factor (Crf), the main regulator of the stress response. It does so by sequestering the coactivator of the transcription factor CREB, CRTC3, in the cytosol, resulting in reduced binding of CRTC3 to the Crf gene promoter and subsequent Crf gene expression. This novel oxytocin receptor-mediated intracellular mechanism might provide a basis for the treatment of exaggerated stress responses in the future.

Pendrin localizes to the adrenal medulla and modulates catecholamine release.

Pendrin (Slc26a4) is a Cl(-)/HCO3 (-) exchanger expressed in renal intercalated cells and mediates renal Cl(-) absorption. With pendrin gene ablation, blood pressure and vascular volume fall, which increases plasma renin concentration. However, serum aldosterone does not significantly increase in pendrin-null mice, suggesting that pendrin regulates adrenal zona glomerulosa aldosterone production. Therefore, we examined pendrin expression in the adrenal gland using PCR, immunoblots, and immunohistochemistry. Pendrin protein was detected in adrenal lysates from wild-type but not pendrin-null mice. However, immunohistochemistry and qPCR of microdissected adrenal zones showed that pendrin was expressed in the adrenal medulla, rather than in cortex. Within the adrenal medulla, pendrin localizes to both epinephrine- and norepinephrine-producing chromaffin cells. Therefore, we examined plasma catecholamine concentration and blood pressure in wild-type and pendrin-null mice under basal conditions and then after 5 and 20 min of immobilization stress. Under basal conditions, blood pressure was lower in the mutant than in the wild-type mice, although epinephrine and norepinephrine concentrations were similar. Catecholamine concentration and blood pressure increased markedly in both groups with stress. With 20 min of immobilization stress, epinephrine and norepinephrine concentrations increased more in pendrin-null than in wild-type mice, although stress produced a similar increase in blood pressure in both groups. We conclude that pendrin is expressed in the adrenal medulla, where it blunts stress-induced catecholamine release.

Rapid Glucocorticoid Feedback Inhibition of ACTH Secretion Involves Ligand-Dependent Membrane Association of Glucocorticoid Receptors.

The hypothesis that rapid glucocorticoid inhibition of pituitary ACTH secretion mediates a feedforward/feedback mechanism responsible for the hourly glucocorticoid pulsatility was tested in cultured pituitary cells. Perifusion with 30 pM CRH caused sustained the elevation of ACTH secretion. Superimposed corticosterone pulses inhibited CRH-stimulated ACTH release, depending on prior glucocorticoid clearance. When CRH perifusion started after 2 hours of glucocorticoid-free medium, corticosterone levels in the stress range (1 µM) caused a delayed (25 min) and prolonged inhibition of CRH-stimulated ACTH secretion, up to 60 minutes after corticosterone withdrawal. In contrast, after 6 hours of glucocorticoid-free medium, basal corticosterone levels inhibited CRH-stimulated ACTH within 5 minutes, after rapid recovery 5 minutes after corticosterone withdrawal. The latter effect was insensitive to actinomycin D but was prevented by the glucocorticoid receptor antagonist, RU486, suggesting nongenomic effects of the classical glucocorticoid receptor. In hypothalamic-derived 4B cells, 10 nM corticosterone increased immunoreactive glucocorticoid receptor content in membrane fractions, with association and clearance rates paralleling the effects on ACTH secretion from corticotrophs. Corticosterone did not affect CRH-stimulated calcium influx, but in AtT-20 cells, it had biphasic effects on CRH-stimulated Src phosphorylation, with early inhibition and late stimulation, suggesting a role for Src phosphorylation on the rapid glucocorticoid feedback. The data suggest that the nongenomic/membrane effects of classical GR mediate rapid and reversible glucocorticoid feedback inhibition at the pituitary corticotrophs downstream of calcium influx. The sensitivity and kinetics of these effects is consistent with the hypothesis that pituitary glucocorticoid feedback is part of the mechanism for adrenocortical ultradian pulse generation.

Helix 8 of the ligand binding domain of the glucocorticoid receptor (GR) is essential for ligand binding.

Membrane association of estrogen receptors (ER) depends on cysteine palmitoylation and two leucines in the ligand binding domain (LBD), conserved in most steroid receptors. The role of this region, corresponding to helix 8 of the glucocorticoid receptor (GR) LBD, on membrane association of GR was studied in 4B cells, expressing endogenous GR, and Cos-7 cells transfected EGFP-GR constructs. 4B cells preloaded with radiolabeled palmitic acid showed no radioactivity incorporation into immunoprecipitated GR. Moreover, mutation C683A (corresponding to ER palmitoylation site) did not affect corticosterone-induced membrane association of GR. Mutations L687-690A, L682A, E680G and K685G prevented membrane and also nuclear localization through reduced ligand binding. L687-690A mutation decreased association of GR with heat shock protein 90 and transcriptional activity, without overt effects on receptor protein stability. The data demonstrate that palmitoylation does not mediate membrane association of GR, but that the region 680-690 (helix 8) is critical for ligand binding and receptor function.

Regulation of hypothalamic corticotropin-releasing hormone transcription by elevated glucocorticoids.

Negative glucocorticoid feedback is essential for preventing the deleterious effects of excessive hypothalamic pituitary adrenal axis axis activation, with an important target being CRH transcription in the hypothalamic paraventricular nucleus. The aim of these studies was to determine whether glucocorticoids repress CRH transcription directly in CRH neurons, by examining glucocorticoid effects on glucocorticoid receptor (GR)-CRH promoter interaction and the activation of proteins required for CRH transcription. Immunoprecipitation of hypothalamic chromatin from intact or adrenalectomized rats subjected to either stress or corticosterone injections showed minor association of the proximal CRH promoter with the GR compared with that with phospho-CREB (pCREB). In contrast, the Period-1 (Per1, a glucocorticoid-responsive gene) promoter markedly recruited GR. Stress increased pCREB recruitment by the CRH but not the Per1 promoter, irrespective of circulating glucocorticoids. In vitro, corticosterone pretreatment (30 minutes or 18 hours) only slightly inhibited basal and forskolin-stimulated CRH heteronuclear RNA in primary hypothalamic neuronal cultures and CRH promoter activity in hypothalamic 4B cells. In 4B cells, 30 minutes or 18 hours of corticosterone exposure had no effect on forskolin-induced nuclear accumulation of the recognized CRH transcriptional regulators, pCREB and transducer of regulated CREB activity 2. The data show that inhibition of CRH transcription by physiological glucocorticoids in vitro is minor and that direct interaction of GR with DNA in the proximal CRH promoter may not be a major mechanism of CRH gene repression. Although GR interaction with distal promoter elements may have a role, the data suggest that transcriptional repression of CRH by glucocorticoids involves protein-protein interactions and/or modulation of afferent inputs to the hypothalamic paraventricular nucleus.

C/EBP maintains chromatin accessibility in liver and facilitates glucocorticoid receptor recruitment to steroid response elements.

Mechanisms regulating transcription factor interaction with chromatin in intact mammalian tissues are poorly understood. Exploiting an adrenalectomized mouse model with depleted endogenous glucocorticoids, we monitor changes of the chromatin landscape in intact liver tissue following glucocorticoid injection. Upon activation of the glucocorticoid receptor (GR), proximal regions of activated and repressed genes are remodelled, and these remodelling events correlate with RNA polymerase II occupancy of regulated genes. GR is exclusively associated with accessible chromatin and 62% percent of GR-binding sites are occupied by C/EBPß. At the majority of these sites, chromatin is preaccessible suggesting a priming function of C/EBPß for GR recruitment. Disruption of C/EBPß binding to chromatin results in attenuation of pre-programmed chromatin accessibility, GR recruitment and GR-induced chromatin remodelling specifically at sites co-occupied by GR and C/EBPß. Collectively, we demonstrate a highly cooperative mechanism by which C/EBPß regulates selective GR binding to the genome in liver tissue. We suggest that selective targeting of GR in other tissues is likely mediated by the combined action of cell-specific priming proteins and chromatin remodellers.

Transcriptional regulation of episodic glucocorticoid secretion.

Circadian and ultradian variations of basal glucocorticoid secretion and transient elevations during stress are essential for homeostasis. Using intronic qRT-PCR to measure changes in primary transcript (hnRNA) we have shown that secretory events induced by stress or ACTH injection are followed by episodic increases in transcription of rate limiting steroidogenic proteins, such as steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage and melanocortin receptor associated protein. These transcriptional episodes imply rapid turnover of steroidogenic proteins and the need of de novo synthesis following each secretory event. In addition to episodic ACTH secretion, it is likely that intracellular feedback mechanisms at the adrenal fasciculata level contribute to the generation of episodes of transcription. The time relationship between activation and translocation of the CREB co-activator, transducer of regulated CREB activity (TORC) to the nucleus preceding transcriptional episodes suggest the involvement of TORC in the transcriptional activation of StAR and other steroidogenic proteins.

Six commercially available angiotensin II AT1 receptor antibodies are non-specific.

Commercially available Angiotensin II AT1 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT1 receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT1 receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT1 receptor. However, identical bands were observed in wild-type mice and in AT1A knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT1 receptors or transfected with AT1A receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT1A knock-out and wild-type mice and in 4B cells with or without AT1 receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT1 receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT1 receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT1 receptor physiology in the absence of full antibody characterization.

Rapid genome-scale mapping of chromatin accessibility in tissue.

BACKGROUND: The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples. RESULTS: Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh. CONCLUSION: The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of clinically relevant samples to allow demarcation of regulatory elements of considerable prognostic significance.

Salt-inducible kinase is involved in the regulation of corticotropin-releasing hormone transcription in hypothalamic neurons in rats.

Activation of CRH transcription requires phosphorylation of cAMP response element-binding protein (CREB) and translocation of the CREB coactivator, transducer of regulated CREB activity (TORC) from cytoplasm to nucleus. 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 SIK1 and SIK2 mRNA in the hypothalamic paraventricular nucleus of rats by in situ hybridization. In basal conditions, low mRNA levels of the two kinases were found in the dorsomedial paraventricular nucleus, consistent with location in CRH neurons. One hour of restraint stress increased SIK1 mRNA levels, whereas SIK2 mRNA showed only minor increases. In 4B hypothalamic neurons, or primary cultures, SIK1 mRNA (but not SIK2 mRNA) was inducible by the cAMP stimulator, forskolin. Overexpression of either SIK1 or SIK2 in 4B cells reduced nuclear TORC2 levels (Western blot) and inhibited forskolin-stimulated CRH transcription (luciferase assay). Conversely, the nonselective SIK inhibitor, staurosporine, increased nuclear TORC2 content and stimulated CRH transcription in 4Bcells and primary neuronal cultures (heteronuclear RNA). Unexpectedly, in 4B cells specific short hairpin RNA knockdown of endogenous SIK2 but not SIK1 induced nuclear translocation of TORC2 and CRH transcription, suggesting that SIK2 mediates TORC inactivation in basal conditions, whereas induction of SIK1 limits transcriptional activation. The study provides evidence that SIK represses CRH transcription by inactivating TORC, providing a potential mechanism for rapid on/off control of CRH transcription.

The molecular physiology of CRH neurons.

Corticotropin releasing hormone (CRH) is essential for stress adaptation by mediating hypothalamic-pituitary-adrenal (HPA) axis, behavioral and autonomic responses to stress. Activation of CRH neurons depends on neural afferents from the brain stem and limbic system, leading to sequential CRH release and synthesis. CRH transcription is required to restore mRNA and peptide levels, but termination of the response is essential to prevent pathology associated with chronic elevations of CRH and HPA axis activity. Inhibitory feedback mediated by glucocorticoids and intracellular production of the repressor, Inducible Cyclic AMP Early Repressor (ICER), limit the magnitude and duration of CRH neuronal activation. Induction of CRH transcription is mediated by the cyclic AMP/protein kinase A/cyclic AMP responsive element binding protein (CREB)-dependent pathways, and requires cyclic AMP-dependent nuclear translocation of the CREB co-activator, Transducer of Regulated CREB activity (TORC). This article reviews current knowledge on the mechanisms regulating CRH neuron activity.

The distribution of messenger RNAs encoding the three isoforms of the transducer of regulated cAMP responsive element binding protein activity in the rat forebrain.

Increasing evidence indicates that the cAMP responsive element binding protein (CREB)-dependent transcriptional activation of a number of genes requires the CREB co-activator: transducer of regulated CREB activity (TORC). Because of the central importance of CREB in many brain functions, we examined the topographic distribution of TORC1, 2, and 3 mRNAs in specific regions of the rat forebrain. In situ hybridisation analysis revealed that TORC1 is the most abundant isoform in most forebrain structures, followed by TORC2 and TORC3. All three TORC isoforms were found in a number of brain nuclei, the ventricular ependyma and pia mater. 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. The relative expression of TORC isoforms was confirmed by quantitative reverse transcriptase-polymerase chain reaction analysis in the hippocampus and hypothalamus. 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 signalling on brain function.

ACTH-dependent ultradian rhythm of corticosterone secretion.

The activity of the hypothalamic-pituitary-adrenal axis is characterized by an ultradian pulsatile pattern of glucocorticoid secretion. Despite increasing evidence for the importance of pulsatility in regulating glucocorticoid-responsive gene transcription, little is known about the mechanism underlying the pulsatility of glucocorticoid synthesis and release. We tested the hypothesis that pulsatile ACTH release is critical for optimal adrenocortical function. Hypothalamic-pituitary-adrenal activity was suppressed by oral methylprednisolone, and ACTH (4 ng/h) was infused for 24h either as a constant infusion or in 5-min pulses at hourly intervals. Control methylprednisolone-treated rats had very low plasma corticosterone (CORT) levels with undetectable pulses and also had steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage (P450scc) heteronuclear RNA levels reduced to approximately 50% of that seen in untreated animals. Pulsatile but not constant ACTH infusion restored pulsatile CORT secretion, and this was accompanied by parallel rises in StAR and P450scc heteronuclear RNA levels during the rising phase of the CORT pulse, which then fell during the falling phase. The pulsatile pattern of StAR and P450scc was paralleled by pulsatile transcription of the melanocortin 2 receptor accessory protein. Pulsatile ACTH activation of the adrenal cortex not only is critical for the secretion of CORT but also induces episodic transcription of the rate-limiting enzymes necessary for physiological steroidogenic responses. Because constant infusion of identical amounts of ACTH did not activate CORT secretion, pulsatility of ACTH provides a more effective signaling system for the activation of adrenocortical activity.

HPA axis responsiveness to stress: implications for healthy aging.

The major neuroendocrine response mediating stress adaptation is activation of the hypothalamic pituitary adrenal axis, with stimulation of corticotropin releasing hormone (CRH) and vasopressin (VP) from parvocellular neurons of the hypothalamic paraventricular nucleus, leading to stimulation of pituitary ACTH secretion and increases in glucocorticoid secretion from the adrenal cortex. Basal production and transient increases during stress of glucocorticoids and its hypothalamic regulators are essential for neuronal plasticity and normal brain function. While activation of the HPA axis is essential for survival during stress, chronic exposure to stress hormones can predispose to psychological, metabolic and immune alterations. Thus, prompt termination of the stress response is essential to prevent negative effects of inappropriate levels of CRH and glucocorticoids. This review addresses the regulation of HPA axis activity with emphasis on the mechanisms of termination of CRH transcription, which is a critical step in this process. In addition, the actions by which glucocorticoids, CRH and VP can affect the aging process will be discussed.

Regulation of the hypothalamic-pituitary-adrenal axis by neuropeptides.

The major endocrine response to stress occurs via activation of the hypothalamic-pituitary-adrenal (HPA) axis, leading ultimately to increases in circulating glucocorticoids, which are essential for the metabolic adaptation to stress. The major players in the HPA axis are the hypothalamic neuropeptide, corticotropin releasing hormone (CRH), the pituitary hormone adrenocorticotropic hormone, and the negative feedback effects of adrenal glucocorticoids. In addition, a number of other neuropeptides, including vasopressin (VP), angiotensin II, oxytocin, pituitary adenylate cyclase activating peptide, orexin and cholecystokinin, and nesfatin can affect HPA axis activity by influencing the expression and secretion of CRH, and also by modulating pituitary corticotroph function or adrenal steroidogenesis. Of these peptides, VP co-secreted with CRH from axonal terminals in the external zone of the median eminence plays a prominent role by potentiating the stimulatory effect of CRH and by increasing the number of pituitary corticotrophs during chronic challenge. Although the precise role and significance of many of these neuropeptides in regulating HPA axis activity requires further investigation, it is likely that they are part of a multifactorial system mediating the fine tuning of HPA axis activity during adaptation to a variety of physiological and stressful conditions.

Fos proteins are not prerequisite for osmotic induction of vasopressin transcription in supraoptic nucleus of rats.

While it is well known that osmotic stimulation induces the expression of Fos family members in the supraoptic nucleus (SON), it is unclear whether the induced protein products are involved in the regulation of the gene transcription of arginine vasopressin (AVP). In the present study, we examined the in vivo correlation between changes in AVP gene transcription and expression of the various Fos family members in the SON after acute osmotic stimuli. The data demonstrated that the peak of AVP transcription (measured by intronic in situ hybridization) observed 15min after an injection of hypertonic saline preceded the expression of Fos proteins, which became detectable at 30min and peaked at 120min. Electrophoretic mobility shift assay showed that the expressed Fos proteins bound to the composite AP-1/CRE-like site in the AVP promoter. These data suggest that Fos proteins in the SON induced by acute osmotic stimuli could affect AVP gene transcription by binding to the AVP promoter, but they are not prerequisite for the induction of AVP gene transcription.