Regulation of corticotropin-releasing factor receptor type 2beta mRNA via cyclic AMP pathway in A7r5 aortic smooth muscle cells

Corticotropin-releasing factor receptor type 2beta (CRF R2beta) is a member of the Class B heptahelical G protein-coupled receptors. This receptor is positively coupled to adenylate cyclase and is bound preferentially by the CRF-related peptides, urocortin (Ucn), Ucn II and Ucn III. In the rodent, CRF R2beta messenger RNA (mRNA) is expressed in the cardiovascular system, where its levels can be modulated by Ucn. In the present study, we investigated regulation of CRF R2beta levels by Ucn in A7r5 aortic smooth muscle cells. Ribonuclease protection assays show that A7r5 cells expressed the CRF R2beta subtype, which had two isoforms differing in one codon at the junction of exons 3 and 4. Ucn induced accumulation of intracellular cAMP via CRF R2beta in this cell line. In addition to the treatment with Ucn, cAMP agonists or analogues themselves caused a significant decrease in CRF R2beta mRNA levels. Blockade of Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels by H7, a broad protein kinase inhibitor, suggested that a protein kinase pathway might be involved in this regulation. H89, a protein kinase A inhibitor, partially blocked Ucn- or cAMP-induced decreases in CRF R2beta mRNA levels. Thus, Ucn induces intracellular cAMP to downregulate CRF R2beta mRNA expression in A7r5 cells.     

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.     

Regulation of corticotropin-releasing factor receptor type 2beta mRNA by mitogen-activated protein kinases in aortic smooth muscle cells

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors. CRF receptor type 2beta (CRFR2beta) messenger RNA (mRNA) is expressed primarily in the cardiovascular system, where its levels are decreased by urocortin 1 (Ucn1), a novel peptide in the CRF family. In a previous study, we reported that CRFR2beta mRNA levels were partially down-regulated via the cAMP-protein kinase A pathway. This study focused on the involvement of the intracellular mitogen-activated protein (MAP) kinase pathway in the modulation of CRFR2beta mRNA levels. Ribonuclease protection assays showed that decreases in CRFR2beta mRNA levels induced by Ucn1 and cAMP were attenuated by the p38 MAP kinase inhibitor SB202190 or SB203580. This finding suggested that the p38 MAP kinase pathway was involved in this regulation. Anisomycin, a classic p38 kinase activator, increased CRFR2beta mRNA levels in A7r5 cells. This effect of anisomycin was completely reversed by H7, a serine/threonine kinase inhibitor, while both p38 kinase and MAP kinase kinase inhibitors failed to block the increase in CRFR2beta mRNA levels caused by anisomycin. As anisomycin can activate Jun amino terminal kinases, as well as p38 MAP kinase, it is possible that other MAP kinases, such as Jun amino terminal kinases, also contribute to the increase in gene levels. Alternatively, anisomycin may increase CRFR2beta mRNA levels indirectly as a consequence of blocking protein synthesis.     

Urocortins and corticotropin releasing factor type 2 receptors in the hypothalamus and the cardiovascular system

In addition to urocortin (Ucn I), Ucn II and Ucn III were identified as endogenous ligands for corticotropin-releasing factor type 2 receptor (CRF2 receptor). CRF2 receptor is abundantly located in central hypothalamic ventromedial nucleus (VMH) and in peripheral cardiovascular system. In this mini-review, we focused on the roles of these urocortins and CRF2 receptor in the hypothalamus and the cardiovascular system. Ucn II mRNA was increased in the parvocellular part or the magnocellular part of the hypothalamic paraventricular nucleus (PVN) following immobilization stress or 3 days of water deprivation, respectively. Therefore, it is thought that Ucn II may modulate CRF and vasopressin synthesis in the PVN in a paracrine or autocrine fashion through PVN CRF2 receptor. The early and later phases of Ucn I-mediated feeding suppression may be CRF1 and CRF2 receptor-mediated events, respectively. Ucn II decreases food intake at a later phase, beyond 4 h post injection. A large dose of corticosterone increased plasma leptin and insulin levels as well as the levels of CRF2 receptor mRNA. Adrenalectomy, starvation, and immobilization each lowered plasma leptin and insulin levels and were associated with decrements in CRF2 receptor mRNA levels in the VMH. Peripheral injection of leptin increased VMH CRF2 receptor mRNA, as can induce reductions of food intake and body weight, indicating that circulating leptin is involved in the regulation of VMH CRF2 receptor mRNA expression. Therefore, it is also plausible that VMH CRF2 receptor transduces the anorexogenic effects of leptin as well as those of urocortins. The systemic administration of Ucn II decreases mean arterial pressure (arterial vascular tone) and causes tachycardia via vascular CRF2 receptor in rats, similar to the effects of Ucn I. Thus, CRF2 receptor seems to mediate cardioprotective effects of urocortins.     

Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin-GHSR1a system is activated by glucocorticoids in the hypothalamic cells.     

Urocortin 3 expression at baseline and during inflammation in the colon: Corticotropin releasing factor receptors cross-talk

Urocortins (Ucn1-3), members of the corticotropin-releasing factor (CRF) family of neuropeptides, are emerging as potent immunomodulators. Localized, cellular expression of Ucn1 and Ucn2, but not Ucn3, has been demonstrated during inflammation. Here, we investigated the role of Ucn3 in a rat model of Crohn's colitis and the relative contribution of CRF receptors (CRF₁ and CRF₂) in regulating Ucn3 expression at baseline and during inflammation. Ucn3 mRNA and peptide were ubiquitously expressed throughout the GI tract in naïve rats. Ucn3 immunoreactivity was seen in epithelial cells and myenteric neurons. On day 1 of colitis, Ucn3 mRNA levels decreased by 80% and did not recover to baseline even by day 9. Next, we ascertained pro- or anti-inflammatory actions of Ucn3 during colitis. Surprisingly, unlike observed anti-inflammatory actions of Ucn1, exogenous Ucn3 did not alter histopathological outcomes during colitis and neither did it alter levels of pro-inflammatory cytokines IL-6 and TNF-α. At baseline, colon-specific knockdown of CRF₁, but not CRF₂ decreased Ucn3 mRNA by 78%, whereas during colitis, Ucn3 mRNA levels increased after CRF₁ knockdown. In cultured cells, co-expression of CRF₁ + CRF₂ attenuated Ucn3-stimulated intracellular Ca²⁺ peak by 48% as compared to cells expressing CRF₂ alone. Phosphorylation of p38 kinase increased by 250% during colitis and was significantly attenuated after Ucn3 administration. Thus, our results suggest that a balanced and coordinated expression of CRF receptors is required for proper regulation of Ucn3 at baseline and during inflammation.     

Urocortin 1 modulates immunosignaling in a rat model of colitis via corticotropin-releasing factor receptor 2

Urocortins (UCNs) and their receptors are potent immunoregulators in the gastrointestinal (GI) tract, where they can exert both pro- and anti-inflammatory effects. We examined the contribution of Ucn1 and its receptors to the pathogenesis, progression, and resolution of colitis. Trinitrobenzene sulfonic acid was used to induce colitis in rats. Ucn1 mRNA and immunoreactivity (IR) were ubiquitously expressed throughout the GI tract under basal conditions. During colitis, Ucn1 mRNA levels fell below basal levels on day 1 then increased again by day 6, in association with an increase in the number of Ucn1-IR inflammatory cells. Ucn1-IR cells were also numerous in proliferating granulation tissue. In contrast to Ucn1 expression, average phosphorylated ERK1/2 (pERK1/2) expression rose above controls levels on day 1 and was very low on day 6 of colitis. Knockdown of corticotropin-releasing factor 2 (CRF(2)) but not CRF(1) by RNA interference during colitis significantly decreased the macroscopic lateral spread of ulceration compared with uninjected controls or animals with CRF(1) knockdown. After knockdown of CRF(2), but not of CRF(1) during colitis, edema resolution assessed microscopically was slowed, and myeloperoxidase activity remained elevated even at day 6. Ucn1 and TNF-α mRNA peaked earlier, whereas pERK1/2 activation was attenuated after CRF(2) knockdown. Thus we conclude that local CRF(2) and pERK1/2 activation is pivotal for macroscopic spread of colitis and resolution of edema. Elimination of CRF(2), but not CRF(1), results in uncoordinated immune and pERK1/2 signaling responses.     

Urocortins as cardiovascular peptides

Urocortins (Ucn) 1, 2 and 3, human homologues of fish urotensin I, form the corticotropin-releasing factor (CRF) family, together with CRF, urotensin I and sauvagine. Ucn 3 is a novel member of this family and is a specific ligand for CRF type 2 receptor. CRF type 2 receptor is thought to mediate the stress-coping responses, such as anxiolysis, anorexia, vasodilatation, a positive inotropic action on myocardium and dearousal. Endogenous ligands for the CRF type 2 receptor expressed in the cardiovascular tissues, such as the myocardium, have long been unknown. We have shown expression of Ucn 3 as well as Ucn 1 in the human heart. Ucn 3 is also expressed in the kidney, particularly distal tubules. Studies in various rat tissues showed that high concentrations of immunoreactive Ucn 3 were found in the pituitary gland, adrenal gland, gastrointestinal tract, ovary and spleen in addition to the brain, heart and kidney. These observations suggest that Ucn 3 is expressed in various tissues including heart and kidney, and may regulate the circulation in certain aspects of stress and diseases, such as inflammation. Ucn 1 and 3 appear to have important pathophysiological roles in some cardiovascular diseases.     

Urocortin 3/stresscopin in human colon: possible modulators of gastrointestinal function during stressful conditions

Urocortin 3 (Ucn 3) or stresscopin (SCP) is a new member of the corticotropin-releasing factor (CRF) neuropeptide family and is a specific ligand for CRF type 2 receptor (CRF2). CRF receptors are known to be expressed in the gastrointestinal tract and are considered to play pathophysiological roles, for example, in gastrointestinal motility under stress. We, therefore, examined Ucn 3 expression in the normal human large intestine obtained from surgery and autopsy in order to clarify this local response to stress in human intestine. Both immunohistochemistry and mRNA in situ hybridization demonstrated Ucn 3 expression in myenteric and submucosal nervous plexus, in vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) of blood vessels in subserosa, in smooth muscle layers of the large intestine, and in enterochromaffin cells. In contrast to Urocortin 1 (Ucn 1), Ucn 3 was hardly detected in lamina propria (LP) inflammatory cells in colonic mucosa. In addition, immunohistochemistry demonstrated CRF2 expression in myenteric and submucosal nervous plexus, in smooth muscle layers, in VECs, in VSMCs and in lamina propria inflammatory cells. Immunoreactive Ucn 3 was also detected in the large intestine by RIA, with high concentrations detected in the rectum (15.4+/-9.5 pmol/g wet weight, mean+/-SEM, n=3) and sigmoid colon (6.5+/-3.5 pmol/g wet weight, n=5). Reverse-phase HPLC of the human large intestine disclosed peaks eluting in the position of synthetic Ucn 3 or SCP. These findings all suggest that Ucn 3 plays some physiological or pathological roles in the modulation of gastrointestinal functions during stressful conditions in different manners from Ucn 1.     

Urocortin 2 induces tyrosine hydroxylase phosphorylation in PC12 cells

Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.     

Impact of N-terminal domains for corticotropin-releasing factor (CRF) receptor-ligand interactions

The large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(alpha) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfide arrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.     

Endothelin-converting enzyme-1 actions determine differential trafficking and signaling of corticotropin-releasing factor receptor 1 at high agonist concentrations

CRF receptor 1 (CRF(1)), a key neuroendocrine mediator of the stress response, has two known agonists corticotropin-releasing factor (CRF) and urocortin 1 (Ucn1). Here we report that endothelin-converting enzyme-1 (ECE-1) differentially degrades CRF and Ucn1; ECE-1 cleaves Ucn1, but not CRF, at critical residue Arginine-34/35', which is essential for ligand-receptor binding. At near K(D) agonist concentration (30 nm), both Ucn1- and CRF-mediated Ca(2+) mobilization are ECE-1 dependent. Interestingly, at high agonist concentration (100 nm), Ucn1-mediated Ca(2+) mobilization remains ECE-1 dependent, whereas CRF-mediated mobilization becomes independent of ECE-1 activity. At high agonist concentration, ECE-1 inhibition disrupted Ucn1-, but not CRF-induced CRF(1) recycling and resensitization, but did not prolong the association of CRF(1) with β-arrestins. RNA interference-mediated knockdown of Rab suggests that both Ucn1- and CRF-induced CRF(1) resensitization is dependent on activity of Rab11, but not of Rab4. CRF(1) behaves like a class A G protein-coupled receptor with respect to transient β-arrestins interaction. We propose that differential degradation by ECE-1 is a novel mechanism by which CRF(1) receptor is protected from overactivation by physiologically relevant high concentrations of higher affinity ligand to mediate distinct resensitization and downstream signaling.