Peripheral corticotropin-releasing hormone and urocortin in the control of the immune response

Immunological and cellular stress signals trigger the release of corticotropin-releasing hormone (CRH) from the spleen, thymus and inflamed tissue. In vivo and in vitro studies generally suggest that peripheral, immune CRH has pro-inflammatory effects and acts in a paracrine manner by binding to CRH-R1 and CRH-R2 receptors on neighboring immune cells. However, it now seems likely that some of the suggested pro-inflammatory actions of CRH may be attributed to novel CRH-like peptides or to the related peptide, urocortin, which is also present in immune cells and has especially high affinity for CRH-R2 receptors.     

Corticotropin-releasing hormone skin signaling is receptor-mediated and is predominant in the sebaceous glands.

There is increasing evidence that the sebaceous gland expresses receptors for several neuropeptides and is involved in responses to stress. Among them, corticotropin-releasing hormone (CRH) was currently found to be produced also in the skin. In this study, the distribution of CRH, CRH receptors 1 and 2 (CRH-R1 and CRH-R2), and CRH binding protein (CRH-BP) in cultured human (SZ95) sebocytes was further characterized. Moreover, the effects of CRH and CRH-like peptides on proliferation and inflammatory signaling of CRH receptor-expressing SZ95 sebocytes IN VITRO were investigated. Urocortin (Uct), urotensin and sauvagine are recently described members of the family of structurally related CRH-like peptides, whereas Uct shares a 45% homology with CRH. CRH and Uct inhibited SZ95 sebocyte proliferation with CRH also stimulating interleukin-6 (IL-6) and interleukin-8 (IL-8) release from SZ95 sebocytes. However, CRH had no effect on interleukin-1alpha and interleukin-1beta production in these cells. alpha-Helical-CRF, a CRH antagonistic peptide, annulled the CRH effect on SZ95 sebocyte proliferation and interleukin secretion, while the non-peptidic CRH-R1 selective antagonist antalarmin inhibited the increased production of neutral lipids caused by CRH. In conclusion, CRH, and to a lesser extent Uct, may be involved in signaling of stress pathophysiology in the skin. However, further investigations into the downstream effects of CRH and Uct are required to elucidate the mechanism by which these neuropeptides could establish a stress-related pathophysiological condition in the skin.     

Gonadotropin-releasing hormone (GnRH) positively regulates corticotropin-releasing hormone-binding protein expression via multiple intracellular signaling pathways and a multipartite GnRH response element in alphaT3-1 cells

CRH-binding protein (CRH-BP) binds CRH with high affinity and inhibits CRH-mediated ACTH release from anterior pituitary corticotrope-like cells in vitro. In female mouse pituitary, CRH-BP is localized not only in corticotropes, but is also expressed in gonadotropes and lactotropes. To investigate the functional significance of gonadotrope CRH-BP, we examined the molecular mechanisms underlying GnRH-regulated CRH-BP expression in alphaT3-1 gonadotrope-like cells. CRH-BP is endogenously expressed in alphaT3-1 cells, and quantitative real-time RT-PCR and ribonuclease protection assays demonstrate that GnRH induces a 3.7-fold increase in CRH-BP mRNA levels. GnRH also induces intracellular CRH-BP (2.0-fold) and secreted CRH-BP (5.3-fold) levels, as measured by [125I]CRH:CRH-BP chemical cross-linking. Transient transfection assays using CRH-BP promoter-luciferase constructs indicate that GnRH regulation involves protein kinase C-, ERK- and calcium-dependent signaling pathways and is mediated via a multipartite GnRH response element that includes activator protein 1 and cAMP response element (CRE) sites. The CRE site significantly contributes to GnRH responsiveness, independent of protein kinase A, representing a unique form of multipartite GnRH regulation in alphaT3-1 cells. Furthermore, EMSAs indicate that alphaT3-1 nuclear proteins specifically bind at activator protein 1 and CRE sites. These data demonstrate novel regulation of pituitary CRH-BP, highlighting the importance of the pituitary gonadotrope as a potential interface between the stress and reproductive axes.     

Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors.

Studies in mammalian skin have shown expression of the genes for corticotropin-releasing hormone (CRH) and the related urocortin peptide, with subsequent production of the respective peptides. Recent molecular and biochemical analyses have further revealed the presence of CRH receptors (CRH-Rs). These CRH-Rs are functional, responding to CRH and urocortin peptides (exogenous or produced locally) through activation of receptor(s)-mediated pathways to modify skin cell phenotype. Thus, when taken together with the previous findings of cutaneous expression of POMC and its receptors, these observations extend the range of regulatory elements of the hypothalamic-pituitary-adrenal axis expressed in mammalian skin. Overall, the cutaneous CRH/POMC expression is highly reactive to common stressors such as immune cytokines, ultraviolet radiation, cutaneous pathology, or even the physiological changes associated with the hair cycle phase. Therefore, similar to its central analog, the local expression and action of CRH/POMC elements appear to be highly organized and entrained, representing general mechanism of cutaneous response to stressful stimuli. In such a CRH/POMC system, the CRH-Rs may be a central element.     

Corticotropin-releasing hormone-binding protein in primates

In humans, placental corticotropin-releasing hormone (CRH) production has been linked to the determination of gestational length, and a late gestational fall in CRH-binding protein (CRH-BP) has been linked to the onset of parturition. Expression of placental CRH mRNA is limited to primates, and only in man has a circulating CRH-BP been described. As the fall in CRH-BP in late gestation has been associated with parturition in humans, we sought to determine whether a CRH-BP circulated in the plasma of other primates. It is unclear whether maternal plasma CRH concentrations are elevated in New World monkeys and prosimians. We have therefore performed CRH plasma measurements in the blood of pregnant marmosets, in several species of lemur, and in pregnant and fetal rhesus monkeys as a positive control. Using gel chromatography, CRH-BP was detected in the human, gorilla, chimpanzee, orangutan, gibbon, macaque, squirrel monkey, and marmoset, but was absent in the mandrill, spider monkey, and lemur. CRH was detected in the plasma of pregnant marmosets and rhesus monkeys. CRH was also detected in the fetal rhesus monkey, but at lower concentrations than in maternal plasma. CRH immunoreactivity was not detectable in the plasma of pregnant lemurs or in extracts of lemur placenta. In conclusion, a circulating binding protein for CRH exists in all species of apes but occurs variably among New World and Old World monkeys and is absent in lemurs. The variable occurrence of the CRH-BP does not support a role for this protein in the mechanism of parturition in primates. Maternal CRH is elevated in the pregnant marmoset and rhesus, and may play a role in the pregnancy of New and Old World monkeys.     

Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1alpha and R2beta CRH receptor subtypes and stimulation of Gq-proteins

CRH and CRH-related peptides such as urocortin mediate their actions in the human myometrium via activation of two distinct classes of CRH receptors, R1 and R2. These heptahelical receptors are able to stimulate a number of different intracellular signals; one key mediator of G protein-activated intracellular signaling is the cascade of p42/p44, mitogen-activated protein kinase (MAPK). We therefore hypothesized that activation of MAPK might mediate CRH and or/urocortin actions in the myometrium. In cultured human pregnant myometrial cells, urocortin but not CRH was able to induce MAPK phosphorylation and activation, suggesting that in the human myometrium these two peptides have distinct actions and biological roles. To identify the particular receptor subtypes mediating this phenomenon, all known CRH receptors present in the human myometrial cells were stably expressed individually in HEK293 and CHO cells, and their ability to activate MAPK was tested. The R1alpha and R2beta, but not the R1beta, R1c, or R1d, receptor subtypes were able to mediate urocortin-induced MAPK activation. The signaling components were further investigated; activation of Gs, Go, or Gi proteins did not appear to be involved, but activation of Gq with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation correlated with MAPK phosphorylation. Studies on Gq protein activation using [alpha-32P]-GTP-gamma-azidoanilide and IP3 production in cells expressing the R1alpha or R2beta CRH receptors demonstrated that urocortin was 10 times more potent than CRH. Moreover, urocortin (UCN) generated peak responses that were 50-70% greater than CRH in activating the Gq protein and stimulating IP3 production. In conclusion, UCN acting thought multiple receptor subtypes can stimulate myometrial MAPK via induction of the Gq/phospholipase C/IP3/PKC pathway, whereas CRH-induced activation of this pathway appears to be insufficient to achieve MAPK activation.     

Cortistatin modulates the expression and release of corticotrophin releasing hormone in rat brain. Comparison with somatostatin and octreotide

Cortistatin (CST) is an endogenous neuropeptide characterized by remarkable structural and functional resemblance to somatostatin (SST), both peptides sharing the ability to bind and activate all five SST receptor subtypes. Evidence is also available showing that CST exerts biological activities independently from SST, perhaps via the activation of specific receptors that remain to be fully characterized at present. Here we have investigated the effects of CST on the gene expression and release of corticotrophin releasing hormone (CRH) from rat hypothalamic and hippocampal explants; moreover, we compared the effects of CST with those of SST and octreotide (OCT) in these models. We found that: (i) CST inhibits the expression and release of CRH from rat hypothalamic and hippocampal explants under basal conditions as well as after CRH stimulation by well known secretagogues; (ii) SST does not modify basal CRH secretion from the hypothalamus or the hippocampus, while it is able to reduce KCl-stimulated CRH release from both brain areas; (iii) OCT inhibits both basal and KCl-induced CRH secretion from rat hypothalamic explants, while it has no effect on CRH release from the hippocampus, either under basal conditions or after stimulation by high K(+) concentrations; (iv) at variance with CST; SST and OCT have not effect whatsoever on veratridine-induced CRH release from the hypothalamus. In conclusion the present findings provide in vitro evidence in support of the hypothesis that CST plays a role in the regulation of endocrine adaptive responses to stress.     

Cardiovascular actions of CRH and urocortin: an update

Urocortin is a potent regulator of cardiac function, with actions that are prolonged in experimental animals. These changes are mediated via binding to CRH receptors found in peripheral tissues. The diversity of actions of urocortin on behaviour, appetite, inflammation and the cardiovascular system suggest that this peptide may be an endogenous factor mediating actions previously attributed to CRH. The present review will focus on the recent understanding of mechanisms mediating the cardiovascular actions of urocortin and CRH reported to date.     

Corticotropin-releasing hormone receptors

Corticotropin-releasing hormone (CRH) and related peptides (urocortins, sauvagine, urotensin) play a central role in the co-ordination of autonomic, behavioural, cardiovascular, immune and endocrine responses to stressful stimuli. Their actions are mediated through activation of two types of G-protein-coupled receptors encoded by separate genes. In this review we focus on the diverse structural and functional characteristics of the family of CRH-like peptides and their receptors.     

Localization and physiological roles of urocortin

Urocortin, a 40 amino acid peptide, is a corticotropin-releasing factor (CRF) related peptide, and can bind to all three types of CRF receptors (CRF type 1, type 2a and type 2b receptors) with higher affinities for these receptors than CRF. Immunoreactivity of urocortin is widely distributed in central nervous, digestive, cardiovascular, reproductive, immune and endocrine systems. Urocortin plays important roles in appetite-suppression, immunomodulation, steroidogenesis in the ovary, maintenance of the placental function, labor, and cardioprotection via CRF receptors. Although urocortin has potent adrenocorticotropin (ACTH) releasing activity in vitro, endogenous urocortin does not act on pituitary ACTH secretion in vivo.     

Rat cerebral cortex corticotropin-releasing hormone receptors: evidence for receptor coupling to multiple G-proteins

The wide distribution of corticotrophin-releasing hormone (CRH) receptors in brain and periphery appear to be important in integrating the responses of the brain, endocrine and immune systems to physiological, psychological and immunological stimuli. The type 1 receptors are highly expressed throughout the cerebral cortex, a region involved in cognitive function and modulation of stress responses, where they are coupled to the adenylyl cyclase system. Using techniques that analyse receptor-mediated guanine-nucleotide binding protein (G-proteins) activation, we recently demonstrated that expressed type 1alpha CRH receptors are capable of activating multiple G-proteins, which suggests that CRH can regulate multiple signalling pathways. In an effort to characterize the intracellular signals generated by CRH in the rat cerebral cortex we sought to identify G-proteins activated by CRH in a physiological membrane environment. Rat cerebral cortical membrane suspensions were analysed for the ability of CRH to stimulate incorporation of [alpha-32P]-GTP-gamma-azidoanilide to various G-protein alpha-chains. Our results show that CRH receptors are coupled to and activate at least five different G-proteins (Gs, Gi, Gq/11, Go and Gz) with subsequent stimulation of at least two intracellular signalling cascades. In addition, the photoaffinity experiments indicated that the CRH receptors preferentially activate the 45 kDa form of the Gs alpha-protein. This data may help elucidate the intracellular signalling pathways mediating the multiple actions of CRH especially under different physiological conditions.     

Corticotropin-releasing hormone receptor (CRHR)1 and CRHR2 are both trafficking and signaling receptors for urocortin

Transport of urocortin, a potent satiety peptide, occurs at the blood-brain barrier of the mouse. Endocytosis of urocortin by the cerebral microvessel endothelial cells composing the blood-brain barrier is a rate-limiting step of this transport, but the cellular mechanisms involved have not been fully elucidated. The presence of both CRH receptors R1 and R2 in isolated cerebral microvessels shown in this study suggested that both subtypes might mediate urocortin transport. The roles of these two receptors in the endocytosis and signal transduction of urocortin were tested by overexpression studies in human embryonic kidney 293 cells. Both receptors led to a significant increase of binding and endocytosis of radiolabeled urocortin. CRHR1-mediated urocortin endocytosis was blocked by astressin (antagonist for both CRHRs), whereas CRHR2-mediated urocortin endocytosis was also blocked by antisauvagine 30 (selective CRHR2beta antagonist). Chlorpromazine, filipin, and nystatin had no effect on urocortin endocytosis, indicating the lack of significant involvement of clathrin or caveolae membrane microdomains. Both CRHR1 and CRHR2 were able to mediate the ligand-induced increase of cAMP production, suggesting that the overexpressed receptors were biologically active. Elevation of intracellular cAMP by forskolin or dibutyryl-cAMP, however, did not show acute modulation of the binding and endocytosis of urocortin. Despite the substantial intracellular degradation of endocytosed urocortin in cells overexpressing either CRHR1 or CRHR2, intact urocortin could be exocytosed during the 1-h study interval. We conclude that both CRHR1 and CRHR2 play a facilitatory role in the non-clathrin-, non-caveolae-mediated endocytosis and intracellular signal transduction of this potent peptide.     

Involvement of CRH receptors in urocortin-induced hyperthermia

The actions of individual corticotropin-releasing hormone (CRH) receptor (CRHR1 and CRHR2) were studied on the hyperthermia caused by urocortin 1, urocortin 2 and urocortin 3 in rats. Urocortin 1, urocortin 2 or urocortin 3 was injected into the lateral brain ventricle in conscious rats and the colon temperature was measured at different times following injection, up to 6h. In order to study the possible role of CRH receptors, the animals were treated with a urocortins together with the urocortin receptor inhibitors CRF 9-41, antalarmin and astressin 2B to influence the action of urocortins in initiating hyperthermia. Urocortin 1 at a dose of 2microg caused an increase in colon temperature, maximal action being observed in body temperature at 3h. CRH 9-41 and antalarmin, CRHR1 receptor antagonists, prevented the urocortin-induced increase in colon temperature while astressin 2B (CRHR2 receptor antagonist) was ineffective. Urocortin 2 at a dose of 2microg showed a byphasic action in increase in colon temperature having the first peak between 30 min and 1h and the second peak at 4h following treatment. CRF (9-41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 2. Urocortin 3 in a dose of lmicrog increased colon temperature; the maximal effect was observed at 2h. CRF (9-41) and antalarmin was ineffective while astressin 2B fully blocked the action of urocortin 3. The results demonstrated that urocortin 1, 2 or 3 when injected into the lateral brain ventricle caused increases in body temperature is mediated by urocortin receptors. The action of urocortin 1 is mediated by CRHR1 receptor, while in the action of urocortin 2 and urocortin 3 CRHR2 receptor is involved.     

Corticotropin releasing hormone and related peptides can act as bioregulatory factors in human keratinocytes

Summary Following previous findings in human skin of the functional expression of genes for the corticotropin releasing hormone (CHR) receptor type 1 (CRH-R1) and CRH itself, we searched for local phenotypic effects for peptides related to CRH. We now report that CRH, sauvagine, and urocortin inhibit proliferation of human HaCaT keratinocytes in a dose-dependent manner. The peptides produced variable cyclic adenosine 3′∶5′-monophosphate stimulation with CRH having the highest potency. Binding of iodine 125 CRH to intact keratinocytes was inhibited by increasing doses of CRH, sauvagine, or urocortin, all showing equal inhibitory potency. Immunocytochemistry identified CRH-R1 immunoreactivity in HaCaT keratinocytes. In conclusion, CRH (exogenous or produced locally) and the related urocortin and sauvagine peptides can modify human keratinocyte phenotype through a receptor-mediated pathway.     

CRH-deficient mice have a normal anorectic response to chronic stress.

Many studies have implicated corticotropin-releasing hormone (CRH) as a mediator of stress-induced decreases in food intake. However, urocortin, sauvagine, and urotensin, other members of the family of CRH-like molecules, have also been shown to be potent inhibitors of food intake. This raises the possibility that a CRH-related molecule might also be responsible for stress-induced anorexia. We therefore examined the effects of three chronic stressors, repetitive daily restraint, turpentine abscess, and surgical stress, upon food intake in wildtype and CRH-deficient mice created by targeted inactivation of the CRH gene. We have found that both genotypes have similar basal food intake which initially decreases to the same degree following initiation of each stress paradigm. Food intake also recovers following the same time course and to the same degree in both genotypes. Therefore, CRH is not necessary for decreases in food-intake induced by the chronic stressors examined in this study.