CRF-binding Protein mRNA Research Articles

Binge Drinking Decreases Corticotropin‐Releasing Factor‐Binding Protein Expression in the Medial Prefrontal Cortex of Mice

Dysregulation of the corticotropin-releasing factor (CRF) system has been observed in rodent models of binge drinking, with a large focus on CRF receptor 1 (CRF-R1). The role of CRF-binding protein (CRF-BP), a key regulator of CRF activity, in binge drinking is less well understood. In humans, single-nucleotide polymorphisms in CRHBP are associated with alcohol use disorder and stress-induced alcohol craving, suggesting a role for CRF-BP in vulnerability to alcohol addiction. The role and regulation of CRF-BP in binge drinking were examined in mice exposed to the drinking in the dark (DID) paradigm. Using insitu hybridization, the regulation of CRF-BP, CRF-R1, and CRF mRNA expression was determined in the stress and reward systems of C57BL/6J mice after repeated cycles of DID. To determine the functional role of CRF-BP in binge drinking, CRF-BP knockout (CRF-BP KO) mice were exposed to 6 cycles of DID, during which alcohol consumption was measured and compared to wild-type mice. CRF-BP mRNA expression was significantly decreased in the prelimbic (PL) and infralimbic medial prefrontal cortex (mPFC) of C57BL/6J mice after 3 cycles and in the PL mPFC after 6 cycles of DID. No significant changes in CRF or CRF-R1 mRNA levels were observed in mPFC, ventral tegmental area, bed nucleus of the stria terminalis, or amygdala after 3 cycles of DID. CRF-BP KO mice do not show significant alterations in drinking compared to wild-type mice across 6 cycles of DID. These results reveal that repeated cycles of binge drinking alter CRF-BP mRNA expression in the mPFC, a region responsible for executive function and regulation of emotion and behavior, including responses to stress. We observed a persistent decrease in CRF-BP mRNA expression in the mPFC after 3 and 6 DID cycles, which may allow for increased CRF signaling at CRF-R1 and contribute to excessive binge-like ethanol consumption.

Corticotropin-releasing factor type-2 receptor and corticotropin-releasing factor-binding protein coexist in rat ventral tegmental area nerve terminals originated in the lateral hypothalamic area.

There is significant functional evidence showing that corticotropin-releasing factor type-2 receptor (CRF2R) and corticotropin-releasing factor-binding protein (CRF-BP) regulate glutamatergic synapses onto ventral tegmental area (VTA) dopaminergic neurons. It has been shown that CRF requires CRF-BP to potentiate N-methyl-D-aspartate receptors in dopaminergic neurons through CRF2R, and that increases glutamate release in cocaine-treated rats through the activation of CRF2R only by agonists with high affinity to CRF-BP. Furthermore, this CRF-mediated increase in VTA glutamate is responsible for stress-induced relapse to cocaine-seeking behaviour. However, there is a lack of anatomical evidence to explain the mechanisms of CRF actions in VTA. Thus, it was studied whether CRF2R and CRF-BP are expressed in VTA nerve terminals, using a synaptosomal preparation devoid of postsynaptic elements. The current results show that both proteins are co-expressed in glutamatergic and γ-aminobutyric acid (GABA)ergic VTA synaptosomes. A main glutamatergic input to the VTA that has been associated to addictive behaviour is originated in the lateral hypothalamic area (LHA). Thus, this study was focused in the LHA-VTA input using orexin as a marker of this input. The results show that CRF2R and CRF-BP mRNA and protein are expressed in the LHA, and that both proteins are present in orexin-positive VTA synaptosomes. The results showing that CRF2R and CRF-BP are expressed in the LHA-VTA input give anatomical support to suggest that this input plays a role in stress-induced relapse to cocaine-seeking behaviour.

The effect of magnesium sulfate on the placental corticotropin-releasing factor (CRF) and CRF binding protein mRNA expression in perfused human placental cotyledon

Objectives: Stress stimuli and inflammation influence the secretion of the placental corticotropin-releasing factor CRF (CRF) that has a significant role in controlling the timing of birth. The CRF-binding protein (CRF-BP) binds CRF with high affinity and inhibits its activity. Magnesium sulfate (MgSO4) has been known to ameliorate maternal, fetal and gestational tissue-associated inflammatory response. We aimed to study the effect of MgSO4 on the CRF and CRF-BP mRNA expression levels in perfused human cotyledon.Methods: Placentas from elective caesarean section were obtained and selected cotyledons were cannulated and dually perfused ex-vivo within 30 min. MgSO4 (7 mg/dl) was added to the maternal reservoir. Each perfusion experiment was conducted for 180 min. At the end of the experiment, RNA was extracted from the perfused cotyledon, and RT-PCR was performed to quantify the expression of CRF and CRF-BP. Human HPRT gene served as a reference gene.Results: Perfusion with MgSO4 (n = 3) induced a significantly lower CRF and higher CRF-BP mRNA expression compared to placentas perfused only with medium (n = 3).Conclusion: In the human placenta, MgSO4 possibly exerts its action through different modulation on the CRF and CRF-BP expression.

Corticotropin‐releasing factor binding protein within the ventral tegmental area is expressed in a subset of dopaminergic neurons

Corticotropin-releasing factor (CRF) and related peptides play a role in mediating neuronal effects of stress. These peptides mediate stress responses by their interactions with the CRF receptors and the CRF-binding protein (CRF-BP). Because the CRF-BP is implicated in neurotransmission within the ventral tegmental area (VTA), we investigated whether the CRF-BP is expressed in VTA neurons. By in situ hybridization, we detected cellular expression of CRF-BP mRNA in the VTA; no such expression was seen in neighboring substantia nigra pars compacta (SNC) or substantia nigra pars reticulata. By double in situ hybridization, we determined that VTA neurons with CRF-BP mRNA coexpressed transcripts encoding either tyrosine hydroxylase [TH; a marker for dopamine (DA) neurons] or glutamic acid decarboxylase [GAD; synthesizing enzyme of gamma-aminobutyric acid (GABA)]. Neurons with CRF-BP mRNA represented 25% of the total population of TH-expressing neurons and 28% of the total population of GAD-expressing neurons, indicating that discrete subpopulations of dopaminergic and GABAergic neurons are present in the VTA. Within the total population of neurons containing CRF-BP mRNA, 70% coexpressed TH mRNA and only 27% coexpressed GAD mRNA. As far as we are aware, we provide the first anatomical evidence that a molecule, CRF-BP, is encoded by DAergic neurons of the VTA but not by those of the SNC. We propose, based on the observation that the majority of VTA neurons expressing CRF-BP mRNA are DAergic, that in the VTA interactions of CRF-BP with CRF, or with CRF-related peptides, are likely to be mediated predominantly by DAergic neurons.

Labor (Term and Preterm) Is Associated With Changes in the Placental mRNA Expression of Corticotrophin-Releasing Factor

Because maternal plasma corticotrophin-releasing factor (CRF) levels increase during the last weeks of pregnancy and at parturition, the present study evaluated whether placental mRNA expression of CRF and CRF-binding protein (CRF-BP) are modified in preterm delivery. A group of 30 women with singleton pregnancies were enrolled in the study. A placental tissue specimen was collected from pregnant women (1) at term after cesarean delivery (39.3 +/- 0.1 gestational weeks; n = 10), (2) at term after spontaneous vaginal delivery (40.1 +/- 0.2 gestational weeks; n = 10), or (3) at preterm delivery (32.4 +/- 0.4 gestational weeks; n = 10). Changes of placental mRNA expression were evaluated by real-time quantitative reverse transcriptase polymerase chain reaction analysis. Placental CRF mRNA expression at term (P < .001) and preterm delivery (P < .001) was significantly higher than in cesarean delivery and highest in preterm placentas. With respect to CRF-BP, no significant difference in placental mRNA expression was observed among samples collected after term or preterm delivery and cesarean delivery. The present study showed for the first time that both term and preterm labor are associated with increased expression of placental CRF but not CRF-BP mRNA.

Corticotropin-releasing factor (CRF), but not corticosterone, increases basolateral amygdala CRF-binding protein

Corticotropin-releasing factor (CRF) is a key mediator of the behavioral, autonomic, and endocrine responses to stress. CRF binds two receptors and a CRF-binding protein (CRF-BP), which may inactivate or modulate the actions of CRF at its receptors. The amygdala is an important anatomical substrate for CRF and contains CRF, its receptors, and CRF-BP. Our previous studies demonstrated that acute stress increases basolateral amygdala (BLA) CRF-BP mRNA. However, factors that may be responsible for this increase remain unclear. Both CRF and corticosterone are released during stress and are known to increase CRF-BP in vitro. However, the effects of these agents in vivo on brain CRF-BP have not been studied. Therefore, we examined the effects of CRF and corticosterone administration on BLA CRF-BP mRNA in rats. The findings demonstrate that intracerebroventricular CRF (5 μg) significantly increases BLA CRF-BP mRNA 9 h post-infusion, a time point consistent with that observed for the effects of acute stress-induced increases in CRF-BP. In contrast, injection of corticosterone at a dose mimicking acute stress (6.5 mg/kg sc) failed to increase BLA CRF-BP mRNA 9 h post-injection. Surprisingly, two different CRF antagonists failed to block CRF-induced increases in CRF-BP mRNA. These results suggest that CRF, but not corticosterone, may be responsible for stress-induced increases in BLA CRF-BP gene expression. Furthermore, this effect appears to be mediated by mechanisms other than the identified CRF receptors.

Decreased Amygdala CRF-Binding Protein mRNA in Post-Mortem Tissue from Male but not Female Bipolar and Schizophrenic Subjects

Stressful life events are commonly associated with the onset and maintenance of psychopathology and much research has focused on the role of the corticotropin-releasing factor (CRF) system in mediating psychopathology. Since CRF serves to integrate the stress response, it is possible that the CRF system plays a role as a neurochemical linkage between stress and psychopathology. CRF-binding protein (CRF-BP) is thought to modulate CRF activity by decreasing its actions. Therefore, in some psychopathological states, alterations in CRF-BP function may contribute to dysregulation of the CRF system. Since the amygdala CRF system mediates stress- and anxiety-related behaviors and alterations in amygdala function are associated with psychopathology, we examined amygdala CRF-BP gene expression in post-mortem brains from subjects with major depression, bipolar disorder, and schizophrenia as well as in controls. In addition to characterizing the anatomic distribution of CRF-BP mRNA in the human amygdala and medial temporal lobe region, we found a significant decrease in CRF-BP mRNA levels in the basolateral amygdala of male bipolar and male schizophrenic subjects and the lateral amygdala of male bipolar subjects. These results raise the possibility that men with decreased amygdala CRF-BP may be more vulnerable to the effects of stress exposure on the etiology or maintenance of bipolar disorder or schizophrenia.

Stress elevates corticotropin-releasing factor (CRF) and CRF-binding protein mRNA levels in rainbow trout (Oncorhynchus mykiss)

The objectives of this study were to characterize rainbow trout (Oncorhynchus mykiss) corticotropin-releasing factor (CRF)-binding protein (CRF-BP) cDNA and to examine the variations in CRF-BP and CRF mRNA levels in response to different intensities of stress. Trout were physically disturbed by a single or three consecutive periods of chasing until exhaustion followed by 2 h of recovery. The pituitary CRF-BP and preoptic area CRF1 mRNA contents were significantly increased only after repeated chasing events. Physical disturbance increased plasma cortisol levels with the largest change occurring in the group of trout that were exposed to repeated chasing events. Trout were also individually isolated in 120 l tanks or confined to 1.5 l boxes for 4, 24 or 72 h. CRF-BP mRNA levels in confined fish were greater than those of isolated fish at 72 h although there were no differences compared with the control group. CRF1 mRNA levels in the preoptic area were greater and remained elevated for a longer period in confined compared with isolated trout. Isolation led to a transient increase in plasma cortisol levels, but the higher cortisol values developed in the confined fish suggest that this treatment was more stressful than isolation. These results demonstrate that the intensity and duration of stress are important factors regulating CRF and CRF-BP mRNA levels in rainbow trout. We hypothesize that pituitary CRF-BP is involved in regulating the activity of the stress axis, possibly by reducing access to CRF1 receptors in the corticotropes.

Distribution of Corticotropin‐Releasing Factor Binding Protein‐Immunoreactivity in the Rat Hypothalamus: Association With Corticotropin‐Releasing Factor‐, Urocortin 1‐ and Vimentin‐Immunoreactive Fibres

Corticotropin-releasing factor binding protein (CRF-BP) is a 37-kDa protein with high affinity binding sites for both corticotropin-releasing factor (CRF) and urocortin 1. Previous studies have examined the distribution of CRF-BP mRNA and peptide within the central nervous system. Due to the predominant cortical localisation, very little is known about CRF-BP in subcortical structures including the hypothalamus. The present study employed immunohistochemistry to characterise the distribution of CRF-BP-like-immunoreactive (-ir) cells and fibres in the rat hypothalamus. Bipolar and multipolar CRF-BP-ir neurones were scattered throughout the rostro-caudal extent of the hypothalamus. Distinct clusters of CRF-BP-ir neurones were identified in the anterior and posterior parvocellular and dorsal cap subdivisions of the paraventricular nucleus (PVN), as well as in the dorsal hypothalamic area, dorsomedial hypothalamic nucleus (DMN), ventral premammillary nucleus and zona incerta. CRF-BP-ir fibres extending from the third ventricle were found in the mediobasal hypothalamus and within the arcuate nucleus-median eminence region. Double immunostaining together with confocal microscopy demonstrated that the CRF-BP-immunostained fibres within the mediobasal hypothalamus coincided with vimentin immunostaining indicating that CRF-BP-ir is present within tanycytes. To define the relationship between CRF-BP-ir cells and endogenous ligands for CRF-BP, double immunohistochemistry was performed to examine possible sites within the hypothalamus where CRF- or urocortin 1-ir fibres innervate regions that contain CRF-BP-ir cell bodies. CRF-BP-ir cell bodies typically coincided with dense CRF-ir, but not urocortin 1-ir fibre innervation. CRF-ir fibre innervation was moderate to high within the anterior and posterior parvocellular subdivisions of the PVN, the dorsal cap of the PVN, DMN and the zona incerta; all regions that contained CRF-BP-ir cell populations. These studies demonstrate that, within the hypothalamus, CRF-BP-ir cells and fibres are concentrated within a circuitry known to be involved in mediating neuroendocrine and autonomic responses to stress.

The effects of acute stress on the regulation of central and basolateral amygdala CRF-binding protein gene expression

Corticotropin-releasing factor (CRF) is a key mediator of the behavioral, autonomic, and endocrine responses to stress. CRF binds two receptors and a CRF-binding protein (CRF-BP), which may inactivate or modulate the actions of CRF at its receptors. The amygdala is an important anatomical substrate for CRF and contains CRF, its receptors, and CRF-BP. Few studies have examined the effects of acute stress on the regulation of amygdala CRF-BP with other CRF system genes. Therefore, we examined the time course of the effects of acute restraint stress on central (CeA) and basolateral (BLA) amygdala CRF system genes. Consistent with our previous study, acute stress increased BLA CRF-BP mRNA shortly after stress offset. Surprisingly, BLA CRF-BP mRNA remained elevated up to 21 h after the stressor. This effect was selective in the BLA as stress did not alter CeA CRF-BP mRNA, and there were no changes in CRF or CRF receptor mRNAs in either amygdala nucleus. These results suggest that alterations in BLA CRF-BP gene expression are a primary response of the BLA/CeA CRF system to acute stress. Because CRF-BP can modulate CRF action, changes in amygdala CRF-BP levels after stress exposure may affect the ability of an organism to adapt to future stressors.

Regulation of Corticotropin‐Releasing Factor‐Binding Protein Expression in Amygdalar Neuronal Cultures

Corticotropin-releasing factor-binding protein (CRF-BP) is known to regulate the bioavailability of CRF and may also play a role in stress behaviours. CRF-BP has been localized in the pituitary as well as central nervous system (CNS) limbic and cortical areas, including the amygdala. The signal transduction pathways which regulate amygdalar CRF-BP are not well understood. In this report, we have examined the effect of protein kinase A and C activators, CRF, dexamethasone and interleukin-6 (IL6) on CRF-BP mRNA and protein expression in dissociated fetal amygdalar cultures. CRF-BP mRNA levels were determined by Northern analysis following 12 h treatment with the following agents: forskolin (1-30 microM), CRF (1-1000 nM), phorbol-12-myristate-13-acetate (TPA; 1-50 nM), dexamethasone (1-100 nM) and IL6 (10-500 pM). Significant increases in CRF-BP mRNA were observed in response to forskolin (30 mM), CRF (100, 1000 nM), IL6 (100, 500 pM), TPA (50 nM) and dexamethasone (100 nM; P<0.05 for all; n=3-6 for all). We extended our observations of CRF-BP expression to the protein level by performing semiquantitative Western analysis of total cellular protein after treatment with the same agents. Twenty-four hour treatment with 30 microM forskolin, 1000 nM CRF, 50 nM TPA, 100 pM IL6 or 100 nM dexamethasone significantly increased CRF-BP expression (P<0.05, n=3 for each treatment). The primary cultures were then transfected with a rat CRF-BP-reporter construct containing 3500 base pairs of CRF-BP 5' flanking DNA. Treatment with all five agents produced statistically significant increases above control (P<0.05; n=3 for each). The results suggest that CRF-BP in the amygdala is stimulated by numerous pathways which may play a significant role in promoting behavioural changes.

Corticotropin releasing factor binding protein (CRF-BP) is expressed in neuronal and astrocytic cells

Corticotropin releasing factor (CRF) binding protein (CRF-BP) was measured in media and cell lysates of primary rat astrocytes, microglia and neurons with the use of a ligand immunoradiometric assay (LIRMA). A low basal level of CRF-BP was detected in the media and cell lysates from primary neuronal and astrocyte cells after 48 h in culture. No basal expression of CRF-BP was detected in cell lysates or media from primary microglial cultures. The CRF-BP expressed in cultured astrocytes and neurons had the same pharmacological characteristics as the human recombinant molecule. After forskolin, IBMX or forskolin/IBMX treatment, a robust increase in secreted CRF-BP levels in the media from astrocytes and neurons, but not microglia, was observed. An increase in CRF-BP-like immunoreactivity in cell lysates was also observed after IBMX/forskolin treatment. In situ hybridization analysis revealed that CRF-BP mRNA was increased in primary cultured astrocytes after IBMX/forskolin stimulation suggesting that regulation was at the level of gene transcription. ‘Axon sparing’ lesions produced with 0.12 M quinolinic acid in PBS injected intracerebrally (unilaterally into dorsal hippocampus) resulted in loss CRF-BP expressed in neurons. These data provide evidence for the differential localization and regulation of CRF-BP in different cell types in brain and suggest that CRF-BP expression may be locally increased in disease states associated with astrocytosis and gliosis.

Corticotropin-releasing factor-binding protein is produced by human placenta and intrauterine tissues.

CRF circulates in high concentration in pregnant woman. It is produced by the placenta and the other intrauterine tissues (maternal decidua, amnion, and chorion). Recently, a CRF-binding protein (CRF-BP) has been identified and cloned. It binds the circulating CRF, reducing its biological action during pregnancy. Liver is the major source of CRF-BP. The aim of the present study was to evaluate whether human placenta and intrauterine tissues produce CRF-BP. The localization of mRNA and immune CRF-BP by in situ hybridization and immunohistochemistry, respectively, was performed. Antisense and sense riboprobes synthesized from a fragment of human CRF-BP cRNA and a specific rabbit anti-hCRF-BP serum was used. The syncytial layer of placental villi at term intensely expressed CRF-BP mRNA and immunoreactivity, whereas rare positively hybridized cells were observed within the cytotrophoblasts and mesenchymal cells. Large decidual cells, amniotic epithelial cells, and chorionic cytotrophoblast stained positively for CRF-BP mRNA and protein. Control sections collected from the same tissues failed to show any positive localization of sense strand cRNA probe and antiserum preadsorbed with immunogen. Finally, the addition of recombinant CRF-BP to human cultured placental cells significantly decreased CRF-induced ACTH release, with a dose-dependent effect. The present data show that local production of CRF-BP occurs in human trophoblast and intrauterine tissues and may represent one of the major mechanisms used by targets tissues to control CRF activity during pregnancy.

The central distribution of a corticotropin-releasing factor (CRF)-binding protein predicts multiple sites and modes of interaction with CRF.

In recent studies to clone and characterize genes coding for the corticotropin-releasing factor-binding protein (CRF-BP), analysis of the tissue distribution of the CRF-BP gene indicated a high level of expression in the rat brain. We have now characterized by immunohistochemical and hybridization histochemical means the cellular localization of CRF-BP protein and mRNA expression, respectively. Results from both approaches converged to indicate that CRF-BP is expressed predominantly in the cerebral cortex, including all major archi-, paleo-, and neocortical fields. Other prominent sites of mRNA and protein expression include subcortical limbic system structures (amygdala, bed nucleus of the stria terminalis), sensory relays associated with the auditory, olfactory, vestibular, and trigeminal systems, severe raphe nuclei, and a number of cell groups in the brainstem reticular core. Expression in the hypothalamus appears largely limited to the ventral premammillary and dorsomedial nuclei; only isolated CRF-BP-stained cells are apparent in neurosecretory cell groups. Dual immunostaining for CRF and CRF-BP revealed a partial colocalization in some of these regions. In addition, prominent CRF-BP-stained terminal fields have been identified in association with CRF-expressing cell groups in circumscribed hypothalamic and limbic structures. In the anterior pituitary, CRF-BP mRNA and immunoreactivity were colocalized with corticotropin-immunoreactivity in a majority of corticotropes. Thus, CRF-BP could serve to modify the actions of CRF by intra- and intercellular mechanisms, in CRF-related pathways in the central nervous system and pituitary.