Pituitary CRH-binding protein and stress in female mice.

The CRH-binding protein (CRH-BP) binds CRH with very high affinity and inhibits CRH-mediated ACTH release from anterior pituitary cells in vitro, suggesting that the CRH-BP functions as a negative regulator of CRH activity. Our previous studies have demonstrated sexually dimorphic expression of CRH-BP in the murine pituitary. Basal CRH-BP expression is higher in the female pituitary, where CRH-BP mRNA is detected in multiple anterior pituitary cell types. In this study, we examined stress-induced changes in CRH-BP mRNA and protein expression in mouse pituitary and assessed the in vivo role of CRH-BP in modulating the stress response. Pituitary CRH-BP mRNA was greater than 200-fold more abundant in females than males, and restraint stress increased pituitary CRH-BP mRNA by 11.8-fold in females and 3.2-fold in males as assessed by qRT-PCR. In females, restraint stress increased CRH-BP mRNA levels not only in POMC-expressing cells, but also in PRL-expressing cells. The increase in female pituitary CRH-BP mRNA following stress resulted in significant increases in CRH-BP protein 4-6h after a 30-minute restraint stress as detected by [(125)I]-CRH:CRH-BP cross-linking analyses. Based on this temporal profile, the physiological role of CRH-BP was assessed using a stressor of longer duration. In lipopolysaccharide (LPS) stress studies, female CRH-BP-deficient mice showed elevated levels of stress-induced corticosterone release as compared to wild-type littermates. These studies demonstrate a role for the pituitary CRH-BP in attenuating the HPA response to stress in female mice.

Novel expression of type 1 corticotropin-releasing hormone receptor in multiple endocrine cell types in the murine anterior pituitary.

The CRH family of ligands signals via two distinct receptors, CRH-R1 and CRH-R2. Previous studies localized CRH-R1 and CRH-R2 to a subset of anterior pituitary corticotropes and gonadotropes, respectively. However, numerous studies have indicated that stress and CRH activity can alter the secretion of multiple anterior pituitary hormones, suggesting a broader expression of the CRH receptors in pituitary. To examine this hypothesis, the in vivo expression of CRH-R1 and CRH-R2 mRNA was further characterized in adult mouse pituitary. Quantitative RT-PCR analysis demonstrated that CRH-R1 mRNA is greater than 100-fold more abundant than CRH-R2 mRNA in male and female mouse pituitaries. Dual in situ hybridization analysis identified cell-specific CRH-R1 expression in the anterior pituitary. At least half of the CRH-R1-positive cells expressed proopiomelanocortin-mRNA (50% in females; 70% in males). In females, a significant percentage of the cells expressing CRH-R1 also expressed transcript for prolactin (40%), LHbeta (10%), or TSH (3%), all novel sites of CRH-R1 expression. Similarly in males, a percentage of CRH-R1-positive cells expressed prolactin (12%), LHbeta (13%), and TSH (5%). RT-PCR studies with immortalized murine anterior pituitary cell lines showed CRH-R1 and/or CRH-R2 expression in corticotropes (AtT-20 cells), gonadotropes (alphaT3-1 and LbetaT2 cells), and thyrotropes (alphaTSH cells). Whereas CRH-R1 expression in corticotropes is well established, the presence of CRH-R1 mRNA in a subset of lactotropes, gonadotropes, and thyrotropes establishes these cell types as novel sites of murine CRH-R1 expression and highlights the pituitary as an important site of interaction between the hypothalamus-pituitary-adrenal and multiple endocrine axes.

CRH-BP: the regulation and function of a phylogenetically conserved binding protein.

Corticotropin Releasing Hormone-Binding Protein (CRH-BP), a 37 kDa secreted glycoprotein, binds both CRH and urocortin with high affinity and is structurally unrelated to the CRH receptors. CRH-BP orthologues have been identified in multiple invertebrate and vertebrate species. It is strongly conserved throughout evolution, suggesting the maintenance of a structural conformation necessary for biological activity. CRH-BP is an important modulator of CRH activity; it inhibits CRH-induced ACTH secretion from pituitary corticotropes and may exert similar actions at central sites of CRH release. While the function of CRH-BP is thought to be primarily inhibitory, recent studies indicate that novel functional roles may exist in both the brain and pituitary. Regulation of CRH-BP expression by stress and metabolic factors are consistent with in vivo models of altered CRH-BP expression. Positive regulation of pituitary CRH-BP by reproductive hormones suggests that additional interactions between the stress and reproductive axes may exist. While recent research has focused on the evolutionary conservation, expanded sites of expression, regulation and in vivo function of CRH-BP, a more complete understanding of the central and peripheral functions of CRH-BP and its mechanisms of action will help elucidate its potential role in the etiology or treatment of disorders of CRH dysregulation.

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.

Alpha-melanophore-stimulating hormone in the brain, cranial placode derivatives, and retina of Xenopus laevis during development in relation to background adaptation.

The amphibian Xenopus laevis can adapt the color of its skin to the light intensity of the background. A key peptide in this adaptation process is alpha-melanophore-stimulating hormone (alpha-MSH), which is derived from proopiomelanocortin (POMC) and released by the endocrine melanotrope cells in the pituitary pars intermedia. In this study, the presence of alpha-MSH in the brain, cranial placode derivatives, and retina of developing Xenopus laevis was investigated using immunocytochemistry, to test the hypothesis that POMC peptide-producing neurons and endocrine cells have a common embryonic origin and a common function, i.e., controlling each other's activities and/or being involved in the process of physiological adaptation. The presence of alpha-MSH-positive cells in the suprachiasmatic nucleus, ventral hypothalamic nucleus, epiphysis, and endocrine melanotrope and corticotrope cells, which are all involved in regulation of adaptation processes, has been detected from stage 37/38 onward. This is consistent with the presumed common origin of these cells, the anterior neural ridge (ANR) of the neural-plate-stage embryo. The olfactory epithelium and the otic and epibranchial ganglia also contain alpha-MSH, indicating that these placodal derivatives originate from a common placodal domain continuous with the ANR. Furthermore, we demonstrate the presence of alpha-MSH in a subpopulation of retinal ganglion cells (RGCs), which is possibly also derived from the ANR. Immunoreactivity for alpha-MSH in RGCs that are located in the dorsal part of the retina is dependent on the background light intensity, suggesting that these cells are involved in the regulation of background adaptation. Taken together, the results support the hypothesis that POMC peptide-producing cells have a common embryonic origin and are involved in adaptation processes.

Regulation of neurons in the suprachiasmatic nucleus of Xenopus laevis.

In the amphibian Xenopus laevis, suprachiasmatic melanotrope-inhibiting neurons (SMINs) play an important role in the regulation of the background adaptation process. In this study, we investigated the innervation of the SMINs at the light- and electron- microscopical level. Immunocytochemistry in combination with confocal laser scanning microscopy revealed co-existence of neuropeptide Y (NPY) and synaptobrevin in spots in the direct vicinity of the SMINs, suggesting the existence of NPY-containing synapses on these cells. At the ultrastructural level, the SMINs showed a high degree of plasticity, containing more electron-dense vesicles and a larger extent of RER in white- than in black-adapted animals. In black-adapted animals, symmetric synapses (Gray type II) were observed on the soma of the SMINs, suggesting an inhibitory input to these cells. The synaptic profiles contained electron-lucent and electron-dense vesicles, indicating the involvement of both a classical neurotransmitter and a neuropeptide (possibly NPY) in this input. In white-adapted animals, synapses were only found at some distance from the SMIN somata. Our findings indicate a striking plasticity of the innervation of the SMINs in relation to background adaptation and support the hypothesis that the SMINs are innervated by NPY-containing interneurons that inhibit SMIN activity in black-adapted animals.