Members of CRF family and their receptors: from past to future.

Corticotropin releasing factor (CRF), originally isolated from the mammalian hypothalamus, is a 41 amino acid peptide that plays an important physiological role and is implicated in the pathophysiology of various diseases. In addition to CRF and its related peptides, a large number of small non-peptide CRF analogs have been recently synthesized, some currently in clinical trials having considerable therapeutic potential in the treatment of CRF-related illnesses. CRF and its related peptides exert their multiple actions by interacting with two types of plasma membrane G-protein coupled CRF receptors, the type 1 (CRF(1)) and type 2 (CRF(2)). These receptors, like all GPCRs consist of an amino-terminal extracellular region, a carboxyl-terminal intracellular tail and seven, membrane-spanning segments, connected by alternating intracellular and extracellular loops. This review describes the functional role of CRF receptors and their ligands emphasizing the structural elements that are important for their function and could potentially contribute in the development of future target-based approaches to design new CRF-related drugs which will enrich the pharmaceutical armoire against serious diseases.

The corticotropin-releasing factor (CRF) family of peptides as local modulators of adrenal function.

Corticotropin-releasing factor (CRF), also termed corticotropin-releasing hormone (CRH) or corticoliberin, is the major regulator of the adaptive response to internal or external stresses. An essential component of the adaptation mechanism is the adrenal gland. CRF regulates adrenal function indirectly through the central nervous system (CNS) via the hypothalamic-pituitary-adrenal (HPA) axis and via the autonomic nervous system by way of locus coeruleus (LC) in the brain stem. Accumulating evidence suggests that CRF and its related peptides also affect the adrenals directly, i.e. not through the CNS but from within the adrenal gland where they form paracrine regulatory loops. Indeed, CRF and its related peptides, the urocortins (UCNs: UCN1, UCN2 and UCN3), their receptors CRF type 1 (CRF(1)) and 2 (CRF(2)) as well as the endogenous pseudo-receptor CRF-binding protein (CRF-BP) are all expressed in adrenal cortical, medullary chromaffin and resident immune cells. The intra-adrenal CRF-based regulatory system is complex and depends on the balance between the local concentration of CRF ligands and the availability of their receptors.

Antiproliferative effect of adiponectin on MCF7 breast cancer cells: a potential hormonal link between obesity and cancer.

Adiponectin, a hormone secreted by adipose tissue, circulates at high concentrations in human plasma. Paradoxically, plasma levels of adiponectin are approximately 50% lower in obese than in lean subjects. An association between low plasma levels of adiponectin and higher risk of developing breast and other cancers was recently reported. Obesity and overweight have also been associated with increased mortality from cancer. To test the hypothesis that adiponectin exerts direct antiproliferative and/or pro-apoptotic effects on cancer cells, we used the MCF7 human breast adenocarcinoma cell line. The proliferation rate of the MCF7 cells was measured using the MTT method, while apoptosis was examined by quantifying the DNA fragmentation using an ELISA assay. In addition, adiponectin receptor 1 (AdipoR1) and AdipoR2 mRNA expression was detected using RT-PCR. Adiponectin diminished the proliferation rate of MCF7 cells; this effect was significant after 48-96 hours of treatment. The presence of receptor expression suggested that the effect of adiponectin on cell proliferation was most likely specific and adiponectin receptor-mediated. Adiponectin induced no apoptosis of MCF7 cells over 48 hours. We conclude that adiponectin inhibits proliferation but causes no apoptosis of MCF7 breast cancer cells. These data suggest that adiponectin may represent a direct hormonal link between obesity and cancer.

Corticotropin-releasing factor (CRF) and the urocortins differentially regulate catecholamine secretion in human and rat adrenals, in a CRF receptor type-specific manner.

Corticotropin-releasing factor (CRF) affects catecholamine production both centrally and peripherally. The aim of the present work was to examine the presence of CRF, its related peptides, and their receptors in the medulla of human and rat adrenals and their direct effect on catecholamine synthesis and secretion. CRF, urocortin I (UCN1), urocortin II (UCN2), and CRF receptor type 1 (CRF1) and 2 (CRF2) were present in human and rat adrenal medulla as well as the PC12 pheochromocytoma cells by immunocytochemistry, immunofluorescence, and RT-PCR. Exposure of dispersed human and rat adrenal chromaffin cells to CRF1 receptor agonists induced catecholamine secretion in a dose-dependent manner, an effect peaking at 30 min, whereas CRF2 receptor agonists suppressed catecholamine secretion. The respective effects were blocked by CRF1 and CRF2 antagonists. CRF peptides affected catecholamine secretion via changes of subplasmaliminal actin filament polymerization. CRF peptides also affected catecholamine synthesis. In rat chromaffin and PC12 cells, CRF1 and CRF2 agonists induced catecholamine synthesis via tyrosine hydroxylase. However, in human chromaffin cells, activation of CRF1 receptors induced tyrosine hydroxylase, whereas activation of CRF2 suppressed it. In conclusion, it appears that a complex intraadrenal CRF-UCN/CRF-receptor system exists in both human and rat adrenals controlling catecholamine secretion and synthesis.

Urocortin III, a brain neuropeptide of the corticotropin-releasing hormone family: modulation by stress and attenuation of some anxiety-like behaviours.

Following its discovery 20 years ago, corticotropin-releasing hormone (CRH) has been postulated to mediate both hormonal and behavioural responses to stressors. Here, we characterize and describe a behavioural role for the murine gene, UcnIII, which encodes a recently discovered CRH-related neuropeptide, urocortin III. We found that mouse UcnIII is expressed predominantly in regions of the brain known to be involved in stress-related behaviours, and its expression in the hypothalamus increases following restraint. In addition, we found that intracerebroventricular administration of mUcnIII stimulates behaviours that are associated with reduced anxiety, including exploration of an open field and decreased latency to enter the lit compartment of a dark-light chamber, but has no effect on the elevated-plus maze. Finally, we found that mUcnIII does not exert any effects on the hormonal stress response. Based upon our findings, UcnIII may be an endogenous brain neuropeptide that is modulated by stress and stimulates behaviours associated with reduced anxiety. In this capacity, UcnIII may attenuate stress-related behaviours, which may be useful both to help cope with stressful situations as well as to avoid pathology associated with excessive reaction to stressors.

Lessons from CRH knockout mice.

Corticotropin-releasing hormone (CRH), the major regulator of hypothalamic-pituitary-adrenal (HPA) axis, has a wide spectrum of actions within the central nervous system and the periphery. The development and use of Crh knockout mice (Crh-/-) has been an important tool for addressing the physiologic and pathologic roles of CRH. This review describes the generation and characterization ofCrh -deficient mice as well as the use of these mice to study the role of CRH in maternal and fetal HPA axes development and in the regulation of the adult HPA axis and behavior. The review concludes with information about recently discovered CRH-related peptides and their possible roles in some of the functions thought initially to be mediated by CRH.

Corticotropin-releasing hormone regulates IL-6 expression during inflammation.

Stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by proinflammatory cytokines results in increased release of glucocorticoid that restrains further development of the inflammatory process. IL-6 has been suggested to stimulate the HPA axis during immune activation independent of the input of hypothalamic corticotropin-releasing hormone (CRH). We used the corticotropin-releasing hormone-deficient (Crh(-/-)) mouse to elucidate the effect of CRH deficiency on IL-6 expression and IL-6-induced HPA axis activation during turpentine-induced inflammation. We demonstrate that during inflammation CRH is required for a normal adrenocorticotropin hormone (ACTH) increase but not for adrenal corticosterone rise. The paradoxical increase of plasma IL-6 associated with CRH deficiency suggests that IL-6 release during inflammation is CRH-dependent. We also demonstrate that adrenal IL-6 expression is CRH-dependent, as its basal and inflammation-induced expression is blocked by CRH deficiency. Our findings suggest that during inflammation, IL-6 most likely compensates for the effects of CRH deficiency on food intake. Finally, we confirm that the HPA axis response is defective in Crh(-/-)/IL-6(-/-) mice. These findings, along with the regulation of IL-6 by CRH, support the importance of the interaction between the immune system and the HPA axis in the pathophysiology of inflammatory diseases.

Opioids suppress basal and nicotine-induced catecholamine secretion via a stabilizing effect on actin filaments.

Catecholamine secretion and actin filament disassembly are closely coupled in chromaffin cells. Opioid suppression of catecholamine secretion is fast and transient, both characteristics of actin filament involvement. The aim of the present work was to test the hypothesis that opioids suppress catecholamine secretion via an inhibitory effect on actin filament disassembly. For this purpose we used the PC12 rat pheochromocytoma cell line. Norepinephrine and dopamine were measured by enzyme-linked immunosorbent assay or RIA. Polymerized actin was measured by rhodamine-phalloidin and visualized by confocal laser scanning microscopy. Opioids suppressed basal catecholamine secretion. The onset of this effect was fast and transient, peaking at 2 min, and was reversible by opioid antagonists. Synchronously, opioids suppressed actin filament disassembly; this was also reversible by opioid antagonists. Cytochalasin B prevented the inhibitory effect of opioids on catecholamine secretion. In addition, opioids suppressed the stimulatory effect of nicotine on catecholamine secretion and actin depolymerization. Changes in actin cytoskeleton in neuron-like PC12 cells make them resistant to both effects of opioids, i.e. on catecholamine secretion and actin disassembly. In conclusion, our data suggest that the suppressive effect of opioids on basal and nicotine-induced catecholamine secretion may result from an opioid-provoked stabilization of cortical actin. It also appears that basal catecholamine secretion is associated with opioid-sensitive machinery regulating the continuous formation of short-lived areas of cortical actin filament disassembly.

Circadian rise in maternal glucocorticoid prevents pulmonary dysplasia in fetal mice with adrenal insufficiency.

The hypothalamic-pituitary-adrenal (HPA) axis, including hypothalamic corticotropin-releasing hormone (CRH) and pituitary corticotropin, is one of the first endocrine systems to develop during fetal life, probably because glucocorticoid secretion is necessary for the maturation of many essential fetal organs. Consistent with this, pregnant mice with an inactivating mutation in the Crh gene deliver CRH-deficient offspring that die at birth with dysplastic lungs, which can be prevented by prenatal maternal glucocorticoid treatment. But children lacking the ability to synthesize cortisol (because of various genetic defects in adrenal gland development or steroidogenesis) are not born with respiratory insufficiency or abnormal lung development, suggesting that the transfer of maternal glucocorticoid across the placenta might promote fetal organ maturation in the absence of fetal glucocorticoid production. We used pregnant mice with a normal HPA axis carrying fetuses with CRH deficiency to characterize the relative contributions of the fetal and maternal adrenal to the activity of the fetal HPA axis, and related these findings to fetal lung development. We found that in the presence of fetal adrenal insufficiency, normal fetal lung development is maintained by the transfer of maternal glucocorticoid to the fetus, specifically during the circadian peak in maternal glucocorticoid secretion.

Animal models of CRH deficiency.

Corticotropin-releasing hormone (CRH), the major regulator of hypothalamic-pituitary-adrenal (HPA) axis, was first isolated due to its ability to stimulate the release of adrenocorticotropic hormone from the anterior pituitary. Later, it was also found to have also a wide spectrum of actions within the central nervous system and the periphery. Studies with pharmacological administration of this peptide and/or antagonists and antibody neutralization techniques have yielded important information concerning the physiological relevance of CRH. The development of CRH knockout mice (CRH KO) has been an important tool for addressing the physiologic and pathologic roles of CRH. This review describes the phenotype of CRH-deficient mice, as well as the use of this model to study the roles of CRH on fetal development and postnatal life. The role of CRH in prenatal development and postnatal regulation of the HPA axis, in activation of the reproductive system during stress, and in modulation of the immune function will be discussed. The review concludes with a comparison of CRH KO mice with other models of CRH deficiency.

KAT45 human pheochromocytoma cell line. A new model for the in vitro study of neuro-immuno-hormonal interactions.

Normal rodent adrenal chromaffin cells and the PC12 rat pheochromocytoma cell line produce IL-1 cytokines. The role, if any, of these cytokines is currently unknown. In PC12 cells, they induce the expression of the L-AA decarboxylase mRNA, a major step in the biosynthesis of catecholamines. Very little if any of these cytokines are detectable in normal human adrenal medulla, being confined mainly in the 17 alpha-hydroxylase-positive steroid cells of the zona reticularis. The aim of the present work was to find out if human pheochromocytomas produce IL-1 cytokines, in vitro, and to examine what local role they may exert. As a model, we have used the new KAT45 cell line, which emerged spontaneously from a primary human pheochromocytoma cell culture. We have found that the KAT45 cells secrete IL-1 beta at 47.8 +/- 9 pg/mg total cellular protein (n = 7, at 24 hours). IL-1 beta increased the concentration of norepinephrine in the KAT45 culture medium from 24.2 +/- 3.5 micrograms/mg protein (n = 6 controls, at 24 hours), to 33.2 +/- 3.5 (IL-1 beta 10 mg/ml) or to 42.9 +/- 8 (IL-1 beta 30 mg/ml). This effect was blocked by IL-1ra. The KAT45 cells also produce CRH and ACTH. IL-1 beta stimulated the secretion of CRH from 19.2 +/- 4 pg/mg protein (n = 5 at 36 hours) to 38.7 +/- 4, an effect blocked by IL-1ra in excess. IL-1 beta had no effect on ACTH secretion.

KAT45, a noradrenergic human pheochromocytoma cell line producing corticotropin-releasing hormone.

KAT45 cells were derived from a human pheochromocytoma, which also caused ectopic Cushing's syndrome, and developed into a cell line spontaneously after the continuous primary culture of the tumor cells. These human pheochromocytoma cells were compared with the extensively characterized PC12 rat pheochromocytoma cell line. KAT45 cells resembled PC12 cells in morphology, proliferation rate, response to cholinergic stimuli, and the development of dendrite-like projections after exposure to nerve growth factor. They produced norepinephrine and epinephrine in a ratio of 50:1, as opposed to production of dopamine by PC12 cells, in amounts 1 order of magnitude higher compared with PC12. Because of the ectopic Cushing's syndrome in our patient, her normal ACTH level, and the knowledge that PC12 cells and even normal rat chromaffin cells appear to produce CRH, we examined whether KAT45 cells also produced this neuropeptide. Indeed, KAT45 cells released authentic CRH and contained an apparently intact CRH transcript. Nicotine and KCl depolarization stimulated the secretion of CRH, whereas interleukin-1beta, glucocorticoids, and nerve growth factor stimulated its synthesis. In addition to the potential systemic effects of CRH, which in our patient produced ectopic Cushing's syndrome, CRH can exert paracrine effects within normal or tumoral adrenals. We used KAT45 cells as a model for the study of the local role of CRH. CRH affected several parameters of KAT45 cell metabolism, including their proliferation rate, synthesis of catecholamines, and production of POMC-derived peptides. KAT45 cells, in addition to the data they provided regarding the in vitro profile of a human CRH-producing pheochromocytoma, may prove to be a valuable auxiliary to the PC12 cell line.

Comparative study between normal rat chromaffin and PC12 rat pheochromocytoma cells: production and effects of corticotropin-releasing hormone.

The adrenal medulla of several species and some human pheochromocytomas contain CRH. The first aim of the present work was to find out whether normal rat adrenal chromaffin cells and the PC12 rat pheochromocytoma cell line produce CRH in vitro and what regulates its production. CRH was measured and characterized in the media of both types of chromaffin cells under basal conditions and after exposure to K+, nicotine, interleukin-1 beta, and nerve growth factor (NGF). The second aim was to examine the biological effect of exogenous CRH (and of its antagonist) on the production of catecholamines from these two types of cells. Our results are as follows: 1) Both types of chromaffin cells contained and secreted comparable amounts of immunoreactive-CRH under basal conditions and after K(+)-induced depolarization, nicotine, and interleukin-1 beta; 2) the physicochemical characteristics of the immunoreactive-CRH in the cells and the media were identical to the putative CRH peptide on both sieve chromatography and RP-HPLC; 3) synthetic CRH induced the production of catecholamines from both cell types in a dose- and time-dependent manner; this effect was abolished by the antagonist, alpha helical CRH; 4) exposure of PC12 cells to NGF (for 1 week) resulted in their neuronal differentiation and the stimulation of their production of CRH by 30 times and of dopamine by 10 times, compared with parallel controls; this effect of NGF was abolished by alpha helical CRH. In conclusion, our data suggest that the production of CRH by PC12 cells represents the preservation of a normal chromaffin cell characteristic rather than a tumor-induced ectopic phenomenon.

Opioids inhibit dopamine secretion from PC12 rat pheochromocytoma cells in a naloxone-reversible manner.

Opioids inhibit the release of catecholamines in the nervous system. Normal adrenal chromaffin cells produce delta opioids and they respond to them by suppressing the release of their catecholamines. Chromaffin cell tumors, the pheochromocytomas, produce mainly kappa opioids. The aim of this work was: (a) to test if pheochromocytomas retain the response of normal chromaffin cell catecholamines to delta opioids and to naloxone (a general opioid antagonist), and (b) to test if kappa opioids exert any specific effect on catecholamine release from these tumors. Since we have previously shown that, in common with human pheochromocytomas, the PC12 rat pheochromocytoma cells express the prodynorphin gene and secret its kappa opioid products, we used these cells to examine the effect of several opioid agonists and of naloxone on basal, nicotine-, and KCl-induced dopamine release. Dopamine is the main PC12 catecholamine. We have found that the specific kappa opioid agonist U-69593 inhibited the release of dopamine in a dose-dependent manner (IC50=0.5 x 10(-8)M). Under basal conditions the mean concentration of dopamine in the culture media was 11.25 +/- 0.57 ng/mg of total cellular protein (n=13). A 30 min exposure to U-69593 at 10(-6) M suppressed basal dopamine release to 58 +/- 2% (n=7) of controls. A 12 hr pre-incubation with U-69593 caused the same degree of suppression. The effect of the synthetic kappa opioid agonist dynorphin A was indistinguishable from that of U-69593. DADLE (a mu and delta synthetic opioid agonist) was significantly less effective in suppressing dopamine release (IC50=10(-7)M). The concentration of dopamine following exposure to 10-6 M of DADLE for 30 min was 74 +/- 5% of the controls (n=4). The mu opioid agonist DAGO was ineffective. The suppressive effect of all opioid agonists was blocked by naloxone suggesting that conventional opioid receptors were involved.

Kappa opioids exert a strong antiproliferative effect on PC12 rat pheochromocytoma cells.

Pheochromocytomas synthesize several types of opioids and their receptors. Opioids affect the proliferation rate of normal and tumoral cells. We have previously shown that the PC12 rat pheochromocytoma cells synthesize multiple opioids. The aim of the present work was to study the effect of opioids on the proliferation of these pheochromocytoma cells. Thus, the effect of several opioid agonists and antagonists was examined on basal and EGF-induced PC12 cell proliferation. The kappa opioid agonists dynorphin A, U-69593, and U-50488 suppressed basal proliferation in a dose-dependent manner. The effect of kappa opioids was blocked by the general opioid antagonist naloxone and the selective kappa antagonist nor-binaltorphimine. Furthermore, both opioid antagonists given alone had a strong stimulatory effect, a findings suggesting that the proliferation of PC12 cells is under tonic inhibition by locally produced kappa opioids. Finally, the mu-opioid agonist DAGO and the delta and mu agonists DADLE and DSLET were ineffective.

PC12 cells as a model to study the effects of opioids on normal and tumoral adrenal chromaffin cells.

Normal adrenal chromaffin cells produce delta opioid peptides while at the same time they have mainly kappa opioid receptors. This paper describes our date regarding the expression of the prodynorphin gene, the precursor of a family of endogenous kappa opioid ligands, in the PC12 rat pheochromocytoma cell line, and the effects of synthetic kappa opioid agonists and of Naloxone on various aspects of PC12 cell function including their secretion of catecholamines, proliferation and differentiation. This is the first part of a series of projects aimed at studying, (a) the conditions under which the prodynorphin gene is expressed in normal adrenomedullary cells, and (b) the physiological role of the endogenous kappa opioids in the physiology of the adrenal medulla.