Comparative Expression Analysis of Stress-Inducible Genes in Murine Immune Cells.

Background: Psychological stress affects the immune system. Upon stress occurrence, glucocorticoid is released that binds to the glucocorticoid receptor and regulates gene expression. Thus, we aimed to examine the stress-induced immunomodulatory mechanisms by investigating the expression patterns of stress-inducible genes in murine immune cells. Methods: BALB/c, C57BL/6, glucocorticoid-receptor congenic mice, and corticotropin-releasing hormone (CRH)-deficient mice were exposed to synthetic glucocorticoid, dexamethasone, or placed under a restraint condition. The expression level of stress-related genes, such as Rtp801, Gilz, Mkp-1, Bnip3, and Trp53inp1 was measured in the immune cells in these mice. Results: Short restraint stress induced Rtp801 and Gilz expressions that were higher in the spleen of BALB/c mice than those in C57BL/6 mice. Mkp-1 expression increased equally in these two strains, despite the difference in the glucocorticoid level. These three genes induced by short restraint stress were not induced in the CRH-deficient mice. In contrast, Bnip3 and Trp53inp1 were only upregulated upon longer restraint events. In the thymus, Trp53inp1 expression was induced upon short restraint stress, whereas Gilz expression constantly increased upon short and repetitive restraint stresses. Conclusion: These results suggest that singular and repetitive bouts of stress lead to differential gene expression in mice and stress-induced gene expression in thymocytes is distinct from that observed in splenocytes. Gilz, Rtp801, and Mkp-1 genes induced by short restraint stress are dependent on CRH in the spleen.

Hyperadrenocorticism of calorie restriction contributes to its anti-inflammatory action in mice.

Calorie restriction (CR), which lengthens lifespan in many species, is associated with moderate hyperadrenocorticism and attenuated inflammation. Given the anti-inflammatory action of glucocorticoids, we tested the hypothesis that the hyperadrenocorticism of CR contributes to its attenuated inflammatory response. We used a corticotropin-releasing-hormone knockout (CRHKO) mouse, which is glucocorticoid insufficient. There were four controls groups: CRHKO mice and wild-type (WT) littermates fed either ad libitum (AL) or CR (60% of AL food intake), and three experimental groups: (a) AL-fed CRHKO mice given corticosterone (CORT) in their drinking water titrated to match the integrated 24-hr plasma CORT levels of AL-fed WT mice, (b) CR-fed CRHKO mice given CORT to match the 24-hr CORT levels of AL-fed WT mice, and (c) CR-fed CHRKO mice given CORT to match the 24-hr CORT levels of CR-fed WT mice. Inflammation was measured volumetrically as footpad edema induced by carrageenan injection. As previously observed, CR attenuated footpad edema in WT mice. This attenuation was significantly blocked in CORT-deficient CR-fed CRHKO mice. Replacement of CORT in CR-fed CRHKO mice to the elevated levels observed in CR-fed WT mice, but not to the levels observed in AL-fed WT mice, restored the anti-inflammatory effect of CR. These results indicate that the hyperadrenocorticism of CR contributes to the anti-inflammatory action of CR, which may in turn contribute to its life-extending actions.

Gastric corticotropin-releasing factor influences mast cell infiltration in a rat model of functional dyspepsia.

Functional gastrointestinal disorders (FGIDs) are characterized by dysregulated gut-brain interactions. Emerging evidence shows that low-grade mucosal inflammation and immune activation contribute to FGIDs, including functional dyspepsia (FD). Stress plays an important role in the onset of FD symptoms. In human subjects with FD, presence of gastric mast cells has been reported, but factors that influence mast cell infiltration remain uncharacterized. Corticotropin-releasing factor (CRF) initiates the body's stress response and is known to degranulate mast cells. In this study, we delineated the role of the CRF system in the pathogenesis of FD in a rat model. Gastric irritation in neonate rat pups with iodoacetamide (IA) was used to induce FD-like symptoms. RNA interference (RNAi) was used to silence gastric CRF expression. Mast cell infiltrate in the stomach increased by 54% in IA-treated rats compared to controls and CRF-RNAi tended to decrease gastric mast cell infiltrate. Sucrose intake decreased in IA-treated rats and mast cell numbers showed a negative association with sucrose intake. IA treatment and transient silencing of gastric CRF increased hypothalamic CRF levels. In IA-treated rats, gastric levels of CRF receptor 2 (CRF2) decreased by ~76%, whereas hypothalamic CRF receptor 1 (CRF1) levels increased. Plasma levels of TNF-α showed a positive correlation with plasma CRF levels. Levels of phosphorylated p38 and ERK1/2 in the stomach showed a positive correlation with gastric CRF levels. Thus, CRF may contribute to low grade inflammation via modulating mast cell infiltration, cytokine levels, MAPK signaling, and the gut-brain axis.

Chronic CRH depletion from GABAergic, long-range projection neurons in the extended amygdala reduces dopamine release and increases anxiety.

The interplay between corticotropin-releasing hormone (CRH) and the dopaminergic system has predominantly been studied in addiction and reward, while CRH-dopamine interactions in anxiety are scarcely understood. We describe a new population of CRH-expressing, GABAergic, long-range-projecting neurons in the extended amygdala that innervate the ventral tegmental area and alter anxiety following chronic CRH depletion. These neurons are part of a distinct CRH circuit that acts anxiolytically by positively modulating dopamine release.

Lack of CRH Affects the Behavior but Does Not Affect the Formation of Short-Term Memory.

Corticotropin-releasing hormone (CRH) is involved in modification of synaptic transmission and affects spatial discrimination learning, i.e., affects the formation of memory in long-term aspect. Therefore, we have focused on CRH effect on short-term memory. We have used stress task avoidance (maze containing three zones: entrance, aversive, and neutral) and compared the behavior and short-term memory in wild-type mice and mice lacking CRH (CRH KO) experiencing one 120-min session of restraint stress. As control, non-stressed animals were used. As expected, the animals that experienced the stress situation tend to spend less time in the zone in which the restraint chamber was present. The animals spent more time in the neutral zone. There were significant differences in number of freezing bouts in the aversive and entrance zones in CRH KO animals. CRH KO control animals entered the neutral zone much more faster than WT control and spent more time immobile in the neutral zone than WT control. These data give evidence that lacking of CRH itself improves the ability of mice to escape away from potentially dangerous area (i.e., those in which the scent of stressed animal is present).

The Effect of Acute and Repeated Stress on CRH-R1 and CRH-R2 mRNA Expression in Pituitaries of Wild Type and CRH Knock-Out Mice.

The activation of the HPA axis is the endocrine measure of stress responsiveness that is initiated by corticotropin-releasing hormone (CRH). CRH exerts its effects via CRHR1 and CRH-R2 receptors coupled to the cAMP signaling system and this process involves transcription factor cAMP-responsive element-binding protein (CREB).This study investigated the role of CRH and the possible involvement of CREB in gene regulation of CRH receptor, under basal conditions and after stress application in the pituitary. We used wild type (wt +/+) controls and CRH knock-out (CRH-KO -/-) male mice. Using CRH-deficient mice, we were able to investigate the consequences of the lack of the CRH on the expression of CRH receptors and transcriptional regulation mediated by CREB. We estimated the effect of acute (IMO 1×) and repeated (IMO 7×) restraint stressors lasting 30 and 120 min on the expression of mRNA CREB, CRH-R1, and CRH-R2 by qPCR. We found very significant difference in the expression of these peptides under the effect of single and repeated stress in control and CRH-KO mice. Our results indicate that both CRH receptors and CREB might be involved in the regulation of stress response in the pituitary of mice. We propose that regulation of the stress response may be better understood if more were known about the mechanisms of CRH receptor signal transduction and involvement of CREB system.

Role of Corticotropin-Releasing Factor in Cerebellar Motor Control and Ataxia.

Cerebellar ataxia, characterized by motor incoordination, postural instability, and gait abnormality [1-3], greatly affects daily activities and quality of life. Although accumulating genetic and non-genetic etiological factors have been revealed [4-7], effective therapies for cerebellar ataxia are still lacking. Intriguingly, corticotropin-releasing factor (CRF), a peptide hormone and neurotransmitter [8, 9], is considered a putative neurotransmitter in the olivo-cerebellar system [10-14]. Notably, decreased levels of CRF in the inferior olive (IO), the sole origin of cerebellar climbing fibers, have been reported in patients with spinocerebellar degeneration or olivopontocerebellar atrophy [15, 16], yet little is known about the exact role of CRF in cerebellar motor coordination and ataxia. Here we report that deficiency of CRF in the olivo-cerebellar system induces ataxia-like motor abnormalities. CRFergic neurons in the IO project directly to the cerebellar nuclei, the ultimate integration and output node of the cerebellum, and CRF selectively excites glutamatergic projection neurons rather than GABAergic neurons in the cerebellar interpositus nucleus (IN) via two CRF receptors, CRFR1 and CRFR2, and their downstream inward rectifier K+ channel and/or hyperpolarization-activated cyclic nucleotide-gated (HCN) channel. Furthermore, CRF promotes cerebellar motor coordination and rescues ataxic motor deficits. The findings define a previously unknown role for CRF in the olivo-cerebellar system in the control of gait, posture, and motor coordination, and provide new insight into the etiology, pathophysiology, and treatment strategy of cerebellar ataxia.

Loss of hypothalamic corticotropin-releasing hormone markedly reduces anxiety behaviors in mice.

A long-standing paradigm posits that hypothalamic corticotropin-releasing hormone (CRH) regulates neuroendocrine functions such as adrenal glucocorticoid release, whereas extra-hypothalamic CRH has a key role in stressor-triggered behaviors. Here we report that hypothalamus-specific Crh knockout mice (Sim1CrhKO mice, created by crossing Crhflox with Sim1Cre mice) have absent Crh mRNA and peptide mainly in the paraventricular nucleus of the hypothalamus (PVH) but preserved Crh expression in other brain regions including amygdala and cerebral cortex. As expected, Sim1CrhKO mice exhibit adrenal atrophy as well as decreased basal, diurnal and stressor-stimulated plasma corticosterone secretion and basal plasma adrenocorticotropic hormone, but surprisingly, have a profound anxiolytic phenotype when evaluated using multiple stressors including open-field, elevated plus maze, holeboard, light-dark box and novel object recognition task. Restoring plasma corticosterone did not reverse the anxiolytic phenotype of Sim1CrhKO mice. Crh-Cre driver mice revealed that PVHCrh fibers project abundantly to cingulate cortex and the nucleus accumbens shell, and moderately to medial amygdala, locus coeruleus and solitary tract, consistent with the existence of PVHCrh-dependent behavioral pathways. Although previous, nonselective attenuation of CRH production or action, genetically in mice and pharmacologically in humans, respectively, has not produced the anticipated anxiolytic effects, our data show that targeted interference specifically with hypothalamic Crh expression results in anxiolysis. Our data identify neurons that express both Sim1 and Crh as a cellular entry point into the study of CRH-mediated, anxiety-like behaviors and their therapeutic attenuation.

Stress increases GABAergic neurotransmission in CRF neurons of the central amygdala and bed nucleus stria terminalis.

Corticotrophin Releasing Factor (CRF) is a critical stress-related neuropeptide in major output pathways of the amygdala, including the central nucleus (CeA), and in a key projection target of the CeA, the bed nucleus of the stria terminalis (BnST). While progress has been made in understanding the contributions and characteristics of CRF as a neuropeptide in rodent behavior, little attention has been committed to determine the properties and synaptic physiology of specific populations of CRF-expressing (CRF(+)) and non-expressing (CRF(-)) neurons in the CeA and BnST. Here, we fill this gap by electrophysiologically characterizing distinct neuronal subtypes in CeA and BnST. Crossing tdTomato or channelrhodopsin-2 (ChR2-YFP) reporter mice to those expressing Cre-recombinase under the CRF promoter allowed us to identify and manipulate CRF(+) and CRF(-) neurons in CeA and BnST, the two largest areas with fluorescently labeled neurons in these mice. We optogenetically activated CRF(+) neurons to elicit action potentials or synaptic responses in CRF(+) and CRF(-) neurons. We found that GABA is the predominant co-transmitter in CRF(+) neurons within the CeA and BnST. CRF(+) neurons are highly interconnected with CRF(-) neurons and to a lesser extent with CRF(+) neurons. CRF(+) and CRF(-) neurons differentially express tonic GABA currents. Chronic, unpredictable stress increase the amplitude of evoked IPSCs and connectivity between CRF(+) neurons, but not between CRF(+) and CRF(-) neurons in both regions. We propose that reciprocal inhibition of interconnected neurons controls CRF(+) output in these nuclei.

Tau protein phosphorylation in diverse brain areas of normal and CRH deficient mice: up-regulation by stress.

Tau protein misfolding is a pathological mechanism, which plays a critical role in the etiopathogenesis of neurodegeneration. However, it is not entirely known what kind of stimuli can induce the misfolding. It is believed that physical and emotional stresses belong to such risk factors. Although the influence of stress on the onset and progression of Alzheimer's disease (AD) has already been proposed, the molecular links between stresses and AD are still unknown. We have therefore focused our attention on determination of the influence of acute immobilization stress (IMO) in normal mice and mice deficient in corticotropin-releasing hormone (CRH). Specifically, we have analyzed levels of hyperphosphorylated tau proteins, bearing the AD-specific phospho-epitopes (AT-8, pT181, and PHF-1), which are implicated in the pathogenesis of AD. We found that IMO induces transient hyperphosphorylation of tau proteins regardless of continuation of the stimulus. Concerning tau modifications, detailed analysis of the mouse brain revealed that neurons in different brain regions including frontal cortex, temporal cortex, hippocampal C1 and CA3 regions, dentate gyrus as well as nucleus basalis Meynert, and several brainstem nuclei such as locus coeruleus but also raphe nucleus and substantia nigra respond similarly to IMO. The strongest tau protein phosphorylation was observed after 30 min of IMO stress. Stress lasting for 120 min led either to the disappearance of tau hyperphosphorylation or to the induction of a second wave of hyperphosphorylation. Noteworthy is the magnitude of pathological phosphorylation of tau protein in CRH and glucocorticoids deficient mice, being much lower in comparison to that observed in wild-type animals, which suggests a critical role of CRH in the pathogenesis of AD. Thus, our results indicate that hyperphosphorylation of tau protein induced by stress may represent the pathogenic event upstream of tau protein misfolding, which leads to progression or eventually initiation of neurodegeneration. The data show that CRH plays an important role in stress induced hyperphosphorylation of tau protein, which might be either a direct effect of CRH innervations in the brain or an effect mediated via the hypothalamo-pituitary-adrenal axis.

A novel role of peripheral corticotropin-releasing hormone (CRH) on dermal fibroblasts.

Corticotropin-releasing hormone, or factor, (CRH or CRF) exerts important biological effects in multiple peripheral tissues via paracrine/autocrine actions. The aim of our study was to assess the effects of endogenous CRH in the biology of mouse and human skin fibroblasts, the primary cell type involved in wound healing. We show expression of CRH and its receptors in primary fibroblasts, and we demonstrate the functionality of fibroblast CRH receptors by induction of cAMP. Fibroblasts genetically deficient in Crh (Crh-/-) had higher proliferation and migration rates and compromised production of IL-6 and TGF-ß1 compared to the wildtype (Crh+/+) cells. Human primary cultures of foreskin fibroblasts exposed to the CRF(1) antagonist antalarmin recapitulated the findings in the Crh-/- cells, exhibiting altered proliferative and migratory behavior and suppressed production of IL-6. In conclusion, our findings show an important role of fibroblast-expressed CRH in the proliferation, migration, and cytokine production of these cells, processes associated with the skin response to injury. Our data suggest that the immunomodulatory effects of CRH may include an important, albeit not explored yet, role in epidermal tissue remodeling and regeneration and maintenance of tissue homeostasis.

Resilience to social stress coincides with functional DNA methylation of the Crf gene in adult mice.

DNA methylation regulates gene transcription and has been suggested to encode psychopathologies derived from early life stress. We found that methylation regulated the expression of the Crf (also known as Crh) gene and that chronic social stress in adult mice induced long-term demethylation of this genomic region. Demethylation was observed only in the subset of defeated mice that displayed social avoidance and site-specific knockdown of Crf attenuated the stress-induced social avoidance.

Fos expression in tyrosine hydroxylase containing hypothalamic neurons in CRH-KO mice: effect of immobilization stress.

OBJECTIVE: Little is known about the response of tyrosine hydroxylase (TH) containing hypothalamic neurons to stress in corticoliberine deficient (CRH-KO) mice. This study was aimed to extend this issue and reveal the data leading to a better understanding of physiological/anatomical plasticity of hypothalamic TH cells in response to acute immobilization stress (IMO) as well as of possible of CRH body deficiency contribution in the regulation of TH cells during stress. We examined the topographic distribution of TH protein immunolabeled perikarya in selected hypothalamic structures including the paraventricular (PVN), supraoptic (SON), periventricular (PeVN), arcuate (ArcN), dorsomedial (DMN), and ventromedial (VMN) nuclei and extrahypothalamic zona incerta (ZI) in CRH-KO and wild type (WT) mice. METHODS: The animals were perfused with fixative 120 min after a single IMO stress. The brains were removed, cryo-sectioned throughout the hypothalamus and Fos-TH co-localizations were processed immunohistochemically. Fos protein was visualized by diaminobenzidine (DAB) intensified with nickel ammonium sulphate, while TH cells were labeled only with DAB chromogen. The evaluation of Fos-TH co-labeled perikarya was performed with the use of computerized Leica light microscope and expressed as the percentage of total amount of TH labeled cells. RESULTS: From the qualitative point of view, the present data indicate similar anatomical distribution of TH immunoreactive perikarya in all brain structures investigated in both WT and CRH-KO mice, while from the quantitative point of view only TH cells in the DMN of CRH-KO mice showed a trend for increased activation by IMO. CONCLUSIONS: In several hypothalamic structures the basic population of TH neurons was not affected by the absence of endogenous CRH. Based on the data of this study it can also be assumed that despite of the presence of direct reciprocal connections between PVN and DMN neurons, PVN CRH neurons possibly are not participating in the regulation of TH neurons in the DMN during IMO stress. KEYWORDS: Hypothalamic nuclei - Fos-immunohistochemistry - Tyrosine hydroxylase - Immobilization stress - CRH knockout mice.

Corticotropin-releasing hormone deficiency is associated with reduced local inflammation in a mouse model of experimental colitis.

CRH, the hypothalamic component of the hypothalamic-pituitary adrenal axis, attenuates inflammation through stimulation of glucocorticoid release, whereas peripherally expressed CRH acts as a proinflammatory mediator. CRH is expressed in the intestine and up-regulated in patients with ulcerative colitis. However, its pathophysiological significance in intestinal inflammatory diseases has just started to emerge. In a mouse model of acute, trinitrobenzene sulfonic acid-induced experimental colitis, we demonstrate that, despite low glucocorticoid levels, CRH-deficient mice develop substantially reduced local inflammatory responses. These effects were shown by histological scoring of tissue damage and neutrophil infiltration. At the same time, CRH deficiency was found to be associated with higher serum leptin and IL-6 levels along with sustained anorexia and weight loss, although central CRH has been reported to be a strong appetite suppressor. Taken together, our results support an important proinflammatory role for CRH during mouse experimental colitis and possibly in inflammatory bowel disease in humans. Moreover, the results suggest that CRH is involved in homeostatic pathways that link inflammation and metabolism.

Muscarinic receptors are affected by corticotropin-releasing hormone and c-fos gene disruptions: is there a mutual connection to adrenoceptors?

In the last decade, progress in gene disruption technology has allowed the study of the effects of the single-gene knockout (KO) on different molecules involved in the signaling cascade activated via muscarinic receptors. Many KO mice targeting muscarinic receptors have been developed, that is, all (M1-M5) muscarinic receptor KO mice (Wess, 2003) and acetylcholinesterase (AChE) KO mice(Xie et al., 2000). Recently, we have shown that these (AChE-/-) mice not only reveal changes in the number of muscarinic receptors in the heart, lung, cortex, and cerebellum but also in the number of adrenoceptors (Teplicky et al., 2004). Next, we studied whether the disruption of corticotropin-releasing hormone (CRH) or c-Fos could affect the properties of muscarinic receptors and adrenoceptors in the lungs and hearts of mice. The effects of immobilization stress in CRH KO animals were also studied.

Urocortin 2 modulates glucose utilization and insulin sensitivity in skeletal muscle.

Skeletal muscle is the principal tissue responsible for insulin-stimulated glucose disposal and is a major site of peripheral insulin resistance. Urocortin 2 (Ucn 2), a member of the corticotropin-releasing factor (CRF) family, and its cognate type 2 CRF receptor (CRFR2) are highly expressed in skeletal muscle. To determine the physiological role of Ucn 2, we generated mice that are deficient in this peptide. Using glucose-tolerance tests (GTTs), insulin-tolerance tests (ITTs), and hyperinsulinemic euglycemic glucose clamp studies, we demonstrated that mice lacking Ucn 2 exhibited increased insulin sensitivity and were protected against fat-induced insulin resistance. Administration of synthetic Ucn 2 to mutant mice before the GTTs and ITTs restored blood glucose to WT levels. Administration of a CRFR2 selective antagonist to WT mice resulted in a GTT profile that mirrored that of Ucn 2-null mice. Body composition measurements of Ucn 2-null mice on a high-fat diet demonstrated decreases in fat and increases in lean tissue compared with WT mice. We propose that null mutant mice display increased glucose uptake in skeletal muscle through the removal of Ucn 2-mediated inhibition of insulin signaling. In keeping with these data, Ucn 2 inhibited insulin-induced Akt and ERK1/2 phosphorylation in cultured skeletal muscle cells and C2C12 myotubes. These data are consistent with the hypothesis that Ucn 2 functions as a local negative regulator of glucose uptake in skeletal muscle and encourage exploration of the possibility that suppression of the Ucn 2/CRFR2 pathway may provide benefits in insulin-resistant states such as type 2 diabetes.

Glucocorticoid-deficient corticotropin-releasing hormone knockout mice maintain glucose requirements but not autonomic responses during repeated hypoglycemia.

Glucocorticoids have been implicated in hypoglycemia-induced autonomic failure but also contribute to normal counterregulation. To determine the influence of normal and hypoglycemia-induced levels of glucocorticoids on counterregulatory responses to acute and repeated hypoglycemia, we compared plasma catecholamines, corticosterone, glucagon, and glucose requirements in male wild-type (WT) and glucocorticoid-deficient, corticotropin-releasing hormone knockout (CRH KO) mice. Conscious, chronically cannulated, unrestrained WT and CRH KO mice underwent a euglycemic (Prior Eu) or hypoglycemic clamp (Prior Hypo) on day 1 followed by a hypoglycemic clamp on day 2 (blood glucose both days, 65 +/- 1 mg/dl). Baseline epinephrine and glucagon were similar, and norepinephrine was elevated, in CRH KO vs. WT mice. CRH KO corticosterone was almost undetectable (<1.5 microg/dl) and unresponsive to hypoglycemia. CRH KO glucose requirements were significantly higher during day 1 hypoglycemia despite epinephrine and glucagon responses that were comparable to or greater than those in WT. Hyperinsulinemic euglycemia did not increase hormones or glucose requirements above baseline. On day 2, Prior Hypo WT had significantly higher glucose requirements and significantly lower corticosterone and glucagon responses. Prior Hypo and Prior Eu CRH KO mice had similar day 2 glucose requirements. However, Prior Hypo CRH KO mice had significantly lower day 2 epinephrine and norepinephrine vs. Prior Eu CRH KO and tended to have lower glucagon than on day 1. We conclude that glucocorticoid insufficiency in CRH KO mice correlates with 1) impaired counterregulation during acute hypoglycemia and 2) complex effects after repeated hypoglycemia, neither preventing decreased hormone responses nor worsening glucose requirements.

Hipoglucemia debida a insuficiencia suprarrenal secundaria a déficit de CRH / Hypoglycemia due to adrenal insufficiency secondary to corticotropin-releasing hormone deficiency

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