Joseph A. Majzoub

Characterization and gestational regulation of corticotropin-releasing hormone messenger RNA in human placenta.

Corticotropin-releasing hormone (CRH), a hypothalamic neuropeptide involved in the regulation of ACTH secretion, has been detected by RIA in extracts of human placenta. We wished to determine whether this immunoreactive substance is a product of CRH gene expression in the placenta. We have found authentic human CRH (hCRH) mRNA in human placental tissue that is similar in size to hypothalamic CRH mRNA. Furthermore, the transcriptional initiation site for placental hCRH mRNA is identical to that previously predicted for hypothalamic hCRH mRNA, 23-26 nucleotides downstream from a canonical promoter element. Placental hCRH mRNA increases more than 20-fold in the 5 wk preceding parturition, in parallel with a rise in placental hCRH peptide content. These data strongly suggest that the hCRH gene is expressed in the placenta and that this expression changes dramatically during gestation.

CRH and the immune system

Inflammatory cytokines released during immune system activation can stimulate the hypothalamic-pituitary-adrenal axis and cause increased secretion of corticotropin-releasing hormone (CRH), adrenocorticotropin and glucocorticoids. Identification of CRH peptide and mRNA, as well as its receptors in immune tissues, suggested a role for this peptide as a mediator of the neuroendocrine-immune interactions. Experimental evidence suggests that CRH may modulate the immune and inflammatory responses via two pathways: an antiinflammatory one operated by centrally released CRH, most likely through stimulation of glucocorticoid and catecholamine release, and one proinflammatory, through direct action of peripherally released CRH. This review highlights these concepts. In addition preliminary data on immune activation and inflammatory response in CRH-deficient mice created in our laboratory are discussed.

Urocortin expression in the Edinger-Westphal nucleus is up-regulated by stress and corticotropin-releasing hormone deficiency.

Urocortin is a 40-amino acid mammalian peptide related to CRH and urotensin. The physiological role of urocortin is unknown, but it has been postulated to serve some of the functions previously attributed to CRH. We had earlier found that urocortin messenger RNA (mRNA) expression within the mouse brain is confined to the region of the Edinger-Westphal (EW) nucleus of the midbrain. To further characterize the regulation of the urocortin gene, we first cloned and sequenced the mouse gene, confirming the presence of a single gene in the murine genome. A general survey of mouse tissues using Northern blot analysis revealed the presence of urocortin mRNA only within the midbrain. By in situ hybridization analysis, we found that urocortin mRNA expression in the EW nucleus is responsive to stress, as mRNA levels increased approximately 3-fold after 3 h of restraint. Chronic glucocorticoid treatment, although not affecting basal levels, blocked the stress-induced rise in urocortin mRNA. Using CRH-deficient [knockout (KO)] mice, we examined the effect of combined CRH and glucocorticoid deficiency upon urocortin mRNA expression. As in wild-type (WT) mice, we had previously found that urocortin expression in CRHKO mouse brain was not detected outside of the EW nucleus. However, we found that urocortin expression within the EW of CRHKO mice is up-regulated 2- to 3-fold compared with that in WT mice. This up-regulation is not due to a lack of inhibition by glucocorticoids, as urocortin mRNA levels in the EW nucleus of CRHKO mice did not change after glucocorticoid supplementation. As the EW does not project to any brain regions known to be involved in the behavioral responses to stress, urocortin expressed in this site is unlikely to mediate stress-induced behaviors. On the other hand, as the EW nucleus may play a role in the regulation of the autonomic nervous system and projects to various brain stem nuclei that express the CRH receptor, urocortin originating in the EW may play a role in the regulation of the autonomic nervous system during stress.

A central theory of preterm and term labor: Putative role for corticotropin-releasing hormone

Near the end of human pregnancy the concentration of placental corticotropin-releasing hormone in maternal blood rises exponentially. The rate of elevation of corticotropin-releasing hormone and its duration through time have been linked to the time of onset of labor. Paradoxically, although glucocorticoids are known to inhibit corticotropin-releasing hormone production within the hypothalamic-pituitary-adrenal axis, cortisol actually increases corticotropin-releasing hormone levels in several areas outside the hypothalamus, including the placenta. Placental corticotropin-releasing hormone may be an important component of a system that controls the normal maturation of the fetus and signals the initiation of labor. Abnormal elevations in corticotropin-releasing hormone, which may be a hormonal response to stressors arising in either the mother, placenta, or fetus, may prove to participate in the premature onset of parturition.

Vasopressin-mediated forearm vasodilation in normal humans. Evidence for a vascular vasopressin V2 receptor.

Arginine vasopressin (AVP) is a potent vasopressor and antidiuretic neurohormone. However, when administered intravenously to humans, AVP causes forearm vasodilation. This effect has been attributed to sympathetic withdrawal, secondary to AVP-induced sensitization of baroreceptors. The possibility that AVP also causes forearm vasodilation directly has not been examined. Accordingly, the direct effect of AVP was determined by studying the forearm blood flow (FBF) response to intraarterial (IA) AVP infusion (0.01-1.0 ng/kg per min). Infusion of IA AVP increased FBF (96%) in the infused arm, but not the control arm, in a dose-dependent manner. The role of specific AVP V1 receptors in mediating this FBF response was determined before and after pretreatment with a V1 antagonist (AVP-A). AVP-A alone had no effect on FBF, but coadministration of AVP and AVP-A potentiated the vasodilatory response (223%). IA infusion of the V2 agonist, 1-desamino[8-D-arginine] vasopressin, caused a dose-dependent increase in FBF. These findings suggest that AVP causes direct, dose-dependent vasodilation in the human forearm that may be mediated by V2 vasopressinergic receptors. In contrast, AVP infusion caused digital vasoconstriction that was blocked by AVP-A, whereas dDAVP did not affect digital blood flow. Thus, AVP induces regionally selective vascular effects, with concurrent forearm vasodilation and digital vasoconstriction.

Impaired diurnal adrenal rhythmicity restored by constant infusion of corticotropin-releasing hormone in corticotropin-releasing hormone-deficient mice.

The normal pattern of daily glucocorticoid production in mammals requires circadian modulation of hypothalamicpituitary-adrenal axis activity. To assess both the factors responsible for imparting this diurnal profile and its physiologic importance, we have exploited corticotropin-releasing hormone (CRH)-deficient mice generated by homologous recombination in embryonic stem cells. CRH-deficient mice have lost normal circadian variations in plasma ACTH and glucocorticoid while maintaining normal circadian locomotor activity. Constant peripheral infusion of CRH produced marked diurnal excursions of plasma glucocorticoid, indicating that CRH acts in part as a permissive factor for other circadian modulators of adrenocortical activity. The presence of atrophic adrenals in CRH-deficient mice without an overt deficit in basal plasma ACTH concentration suggests that the diurnal increase in ACTH is essential to maintain normal adrenal function.

Expression of the mouse corticotropin-releasing hormone gene in vivo and targeted inactivation in embryonic stem cells.

Corticotropin-releasing hormone (CRH), one of the primary regulators of the hypothalamic-pituitary-adrenal (HPA) axis, exhibits abnormal regulation in pathologic states such as depression and anorexia nervosa. Analysis of the role of CRH in regulation of the HPA axis would be facilitated by the creation of animal models in which CRH gene structure and function could be manipulated. We have determined the DNA sequence of the mouse CRH gene. Using a highly sensitive reverse transcription-polymerase chain reaction method, we have found expression of CRH mRNA in adrenal, ovary, testis, gut, heart, anterior pituitary, lung, and spleen, in addition to cerebral cortex and hypothalamus. Within the spleen, CRH mRNA is localized specifically to T-lymphocytes. We mapped the chromosomal location of mouse CRH via interspecific mouse backcrosses to chromosome 3, which is not the site of any naturally occurring mutations consistent with CRH deficiency. Because of this, we inactivated a CRH allele in mouse embryonic stem (ES) cells by homologous recombination with a mutant mouse CRH gene lacking the entire coding region of preproCRH. Mice chimeric for each of two ES clones with an inactivated CRH allele are being used to generate animals with complete CRH deficiency.

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.

Loss of function of the melanocortin 2 receptor accessory protein 2 is associated with mammalian obesity.

Accessory to Obesity? Melanocortin receptors are a family of cell membrane receptors that control diverse physiological functions. Mutations in the gene encoding melanocortin 4 receptor (MC4R) are a cause of familial early-onset obesity. Asai et al. (p. 275) studied the function of an accessory protein for MC4R signaling, MRAP2, and found that mice genetically deficient in MRAP2 develop severe obesity. Sequencing of MRAP2 in unrelated, severely obese humans revealed one individual with a clearly disruptive genetic variant, suggesting that MRAP2 mutations might also be a rare cause of human obesity. In a zebrafish model, Sebag et al. (p. 278) studied two paralogs of the MRAP2 accessory protein, one of which enhanced MC4R responsiveness to α–melanocyte-stimulating hormone, which regulates feeding and growth. Disruption of a protein required for effective signaling by a melanocortin receptor causes severe obesity in mice. Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors in vitro. To investigate the physiological role of brain-expressed melanocortin 2 receptor accessory protein 2 (MRAP2), we characterized mice with whole-body and brain-specific targeted deletion of Mrap2, both of which develop severe obesity at a young age. Mrap2 interacts directly with melanocortin 4 receptor (Mc4r), a protein previously implicated in mammalian obesity, and it enhances Mc4r-mediated generation of the second messenger cyclic adenosine monophosphate, suggesting that alterations in Mc4r signaling may be one mechanism underlying the association between Mrap2 disruption and obesity. In a study of humans with severe, early-onset obesity, we found four rare, potentially pathogenic genetic variants in MRAP2, suggesting that the gene may also contribute to body weight regulation in humans.

Placental corticotropin-releasing hormone: Function and regulation

Corticotropin-releasing hormone is a neuropeptide placentally expressed among mammals only in primates. Its expression increases as much as 100 times during the last 6 to 8 weeks of pregnancy and is paradoxically stimulated by glucocorticoids. Increasing evidence suggests that placental corticotropin-releasing hormone may have evolved in primates to stimulate fetal adrenocorticotropin release and adrenal steroidogenesis, thus satisfying the high demand for synthesis of dehydroepiandrosterone, the predominant source of placental estradiol. Concomitant stimulation by placental corticotropin-releasing hormone of fetal cortisol and dehydroepiandrosterone would couple the glucocorticoid effects on fetal organ maturation with the timing of parturition, an obvious benefit in postnatal survival.