Glucocorticoids differentially regulate the expression of CRFR1 and CRFR2α in MIN6 insulinoma cells and rodent islets

Urocortin 3 (Ucn 3), member of the corticotropin-releasing factor (CRF) family of peptide hormones, is released from β-cells to potentiate insulin secretion. Ucn 3 activates the CRF type-2 receptor (CRFR2) but does not activate the type-1 receptor (CRFR1), which was recently demonstrated on β-cells. While the direct actions of Ucn 3 on insulin secretion suggest the presence of cognate receptors within the islet microenvironment, this has not been established. Here we demonstrate that CRFR2α is expressed by MIN6 insulinoma cells and by primary mouse and human islets, with no detectable expression of CRFR2β. Furthermore, stimulation of MIN6 cells or primary mouse islets in vitro or in vivo with glucocorticoids (GCs) robustly and dose-dependently increases the expression of CRFR2α, while simultaneously inhibiting the expression of CRFR1 and incretin receptors. Luciferase reporters driven by the mouse CRFR1 or CRFR2α promoter in MIN6 cells confirm these differential effects of GCs. In contrast, GCs inhibit CRFR2α promoter activity in HEK293 cells and inhibit the expression of CRFR2β in A7r5 rat aortic smooth muscle cells and differentiated C2C12 myotubes. These findings suggest that the GC-mediated increase of CRFR2α depends on the cellular context of the islet and deviates from the GC-mediated suppression of CRFR1 and incretin receptors. Furthermore, GC-induced increases in CRFR2α expression coincide with increased Ucn 3-dependent activation of cAMP and MAPK pathways. We postulate that differential effect of GCs on the expression of CRFR1 and CRFR2α in the endocrine pancreas represent a mechanism to shift sensitivity from CRFR1 to CRFR2 ligands.     

Cloning and distribution of the bullfrog type 1 and type 2 corticotropin-releasing factor receptors

The molecular cloning of bullfrog cDNAs encoding receptors for corticotropin-releasing factor (CRF) was undertaken as the first step in our investigation of the action of CRF at the receptor level. The specific cDNAs (630 bp) were amplified by reverse-transcribed PCR of a total RNA isolated from hypothalamus of the bullfrog, Rana catesbeiana. With the partial cDNAs as probes, two types of full-length CRF receptor cDNAs (type 1: 2275 bp, type 2: 1600 bp) were obtained from a hypothalamic cDNA library. The deduced amino acid sequences of CRF receptor types 1 (CRFR1) and CRF receptor type 2 (CRFR2) showed 79-94% and 81-92% identity with CRFR1 and CRFR2 of other vertebrates, respectively. The distribution of mRNAs of the two CRF receptors was studied by RT-PCR analysis. CRFR1 mRNA was detected almost exclusively in brain and pituitary, and to a very minor degree in testis, whereas CRFR2 was found in the peripheral tissues as well as in the brain and pituitary.     

Specificity and regulation of extracellularly regulated kinase1/2 phosphorylation through corticotropin-releasing factor (CRF) receptors 1 and 2beta by the CRF/urocortin family of peptides

Corticotropin-releasing factor (CRF) receptor (CRFR)-mediated activation of the ERKs 1/2-p42 and -44) has been reported for CRF, urocortin (Ucn)-I, and sauvagine. Recently two new members of the CRF/Ucn family of peptides have been identified, Ucn-II/stresscopin-related peptide and Ucn-III/stresscopin. Using Chinese hamster ovary cells stably expressing CRFR1 and CRFR2beta, we show that Ucn-I, Ucn-II and Ucn-III activate ERK1/2-p42, 44 via CRFR2beta. CRF and Ucn-I but not Ucn-II or Ucn-III activates ERK1/2-p42, 44 in Chinese hamster ovary cells stably expressing CRFR1. The selectivity of the ligands for CRFR1 and CRFR2beta is shown in a time- and dose-dependent manner. The regulatory mechanisms for ERK1/2-p42, 44 activation by both receptor types are dependent on phosphatidylinositol-3 OH kinase, MAPK kinase 1, and phospholipase C. Raf-1 kinase, tyrosine kinases, and possibly intracellular Ca(2+) provide regulatory roles for Ucn-I activation of ERK1/2-p42, 44 by CRFR1 and CRFR2beta. Studies of the regulation of ERK1/2-p42, 44 by Ucn-I were extended to cell lines that endogenously express CRFR1 (AtT-20 and CATHa cells) and CRFR2 (A7r5 and CATHa cells). Use of the G(i) and G(o) protein inhibitor pertussis toxin showed that ERK1/2-p42, 44 activation by Ucn-I via CRFR1 and CRFR2beta are both G(i) and/or G(o) protein dependent. Based on the data in this study, we present putative signaling pathways by which the CRF/Ucn family of peptides activate ERK1/2-p42, 44 by CRFRs.     

G protein-coupled receptor kinase 2 involvement in desensitization of corticotropin-releasing factor (CRF) receptor type 1 by CRF in murine corticotrophs

Hypothalamic CRF stimulates synthesis and secretion of ACTH via CRF receptor type 1 (CRFR1) in the anterior pituitary gland. After agonist-activated stimulation of receptor signaling, CRFR1 is down-regulated and desensitized. Generally, it is thought that G protein-coupled receptors may be desensitized by G protein-coupled receptor kinases (GRKs). However, the role of GRKs in corticotropic cells has not been determined. In this study we focused on involvement of GRKs in desensitization of CRFR1 by CRF in corticotropic cells. We found that GRK2 (but not GRK3) mRNA and protein were expressed in rat anterior pituitary cells and AtT-20 cells (a line of mouse corticotroph tumor cells). To determine the role of GRK2 in CRF-induced desensitization of CRFR1 in mouse corticotrophs, AtT-20 cells were transfected with a dominant-negative mutant GRK2 construct. CRF desensitized the cAMP-dependent response by CRFR1. Desensitization of CRFR1 by CRF was significantly less in AtT-20 cells transfected with the dominant-negative mutant GRK2 construct compared with desensitization in control (an empty vector-transfected) AtT-20 cells. Furthermore, pretreatment with a protein kinase A inhibitor also partially blocked desensitization of CRFR1 by CRF. These results suggest that GRK2 is involved in CRF-induced desensitization of CRFR1 in AtT-20 cells, and the protein kinase A pathway may also have an important role in desensitization of CRFR1 by CRF seen in corticotropic cells.     

Formal proof that different-size Lyt-2 polypeptides arise from differential splicing and post-transcriptional regulation

We recently isolated the gene and a cDNA clone for the mouse T-cell surface antigen Lyt-2 and showed that Lyt-2 is homologous to the human Leu-2 (T8) antigen and that the gene encoding it is a member of the immunoglobulin gene superfamily. By screening a mouse thymus cDNA library with the Lyt-2 cDNA clone, we isolated two classes of cDNA clones, alpha and alpha', which differ by 31 base pairs. Comparison of the alpha cDNA with genomic sequence data indicates that there are five exons encoding Lyt-2: a fused leader/immunoglobulin variable region-like exon, a spacer region exon, a transmembrane exon, and two cytoplasmic exons. The alpha' cDNA clones lack the first of the two cytoplasmic exons and have a direct splice from the donor splice site of the transmembrane exon to the acceptor of the second cytoplasmic exon. This splice changes the reading frame for the second cytoplasmic exon, causing a stop codon shortly after the splice so that the alpha' cDNA clone codes for a peptide 25 residues shorter than the alpha cDNA-encoded peptide. We have constructed expression vectors with alpha and alpha' cDNAs and have shown that L-cell transfectants of these produce Lyt-2 polypeptides of the predicted sizes and that these associate as homodimers on the cell membranes. We found the two species of mRNA corresponding to alpha and alpha' cDNAs at equal levels in thymus RNA by using S1 nuclease analysis. Although lymph node T cells have only the alpha form of Lyt-2 protein, S1 nuclease analysis shows that lymph nodes have about 20% alpha' mRNA relative to alpha. Thus, Lyt-2 is regulated at RNA processing, translational, and/or post-translational steps.     

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.     

Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.     

Urocortin 1 inhibits rat leydig cell function

Corticotropin-releasing factor (CRF) has previously been reported in rat testes in which it inhibits Leydig cells activity. However, recent studies in our laboratory have suggested that some of the effects originally attributed to CRF were instead due to the related peptide Urocortin 1 (Ucn 1) and that this latter hormone, not CRF, was detectable in Leydig cells. We show here that Ucn 1 [a mixed CRF receptor (CRFR) type 1 and CRFR2 agonist] and the CRFR1-selective peptide Stressin 1, but not Ucn 2 or Ucn 3 (both considered selective CRFR2 ligands), significantly blunt the testosterone response to human chorionic gonadotropin. The effect of Ucn 1 is observed regardless of whether this peptide is injected iv or directly into the testes, and it is reversed by the mixed CRFR1/R2 antagonist Astressin B. Blockade of GnRH receptors with the antagonist Azalin B does not interfere with the influence of Ucn 1, thereby demonstrating that pituitary luteinizing hormone does not appear to be involved in this model. Collectively these results suggest that Ucn 1, not CRF, is present in the rat testes and interferes with Leydig cell activity. However, whereas we previously reported that alcohol up-regulated gonadal Ucn 1 gene expression, CRF receptor antagonists were unable to reverse the inhibitory effect exerted by alcohol on human chorionic gonadotropin-induced testosterone release. The functional role played by testicular Ucn 1 in stress models characterized by blunted androgen levels therefore needs to be further investigated.     

CRFR1 is expressed on pancreatic β cells,promotes β cell proliferation,and potentiates insulin secretion in a glucose-dependent manner

Corticotropin-releasing factor (CRF), originally characterized as the principal neuroregulator of the hypothalamus-pituitary-adrenal axis, has broad central and peripheral distribution and actions. We demonstrate the presence of CRF receptor type 1 (CRFR1) on primary β cells and show that activation of pancreatic CRFR1 promotes insulin secretion, thus contributing to the restoration of normoglycemic equilibrium. Stimulation of pancreatic CRFR1 initiates a cAMP response that promotes insulin secretion in vitro and in vivo and leads to the phosphorylation of cAMP response element binding and the induction of the expression of several immediate-early genes. Thus, the insulinotropic actions of pancreatic CRFR1 oppose the activation of CRFR1 on anterior pituitary corticotropes, leading to the release of glucocorticoids that functionally antagonize the actions of insulin. Stimulation of the MIN6 insulinoma line and primary rat islets with CRF also activates the MAPK signaling cascade leading to rapid phosphorylation of Erk1/2 in response to CRFR1-selective ligands, which induce proliferation in primary rat neonatal β cells. Importantly, CRFR1 stimulates insulin secretion only during conditions of intermediate to high ambient glucose, and the CRFR1-dependent phosphorylation of Erk1/2 is greater with elevated glucose concentrations. This response is reminiscent of the actions of the incretins, which potentiate insulin secretion only during elevated glucose conditions. The presence of CRFR1 on β cells adds another layer of complexity to the intricate network of paracrine and autocrine factors and their cognate receptors whose coordinated efforts can dictate islet hormone output and regulate β cell proliferation.     

A method for soluble overexpression of the alpha7 nicotinic acetylcholine receptor extracellular domain

We describe the construction of a soluble protein carrying the N-terminal extracellular domain (ECD) of the alpha7 subunit of the nicotinic acetylcholine receptor. The approach was to fuse the alpha7 ECD at the C and N termini of several monomeric and pentameric soluble carrier proteins and to investigate the soluble expression of the product in Escherichia coli. An initial screening of six carrier proteins resulted in the selection of a fusion protein comprising, from the N to the C terminus, the maltose binding protein, a 17-aa linker containing an enterokinase binding site, and the alpha7 ECD. This protein is soluble upon expression in bacteria and is purified by affinity chromatography. It binds the competitive nicotinic antagonist alpha-bungarotoxin with 2.5 microM affinity and displays a CD spectrum corresponding to a folded protein. The method might be suitable to produce large quantities of protein for crystallization and immunochemical experiments.     

Stress and glucocorticoids regulated corticotropin releasing factor in rat prefrontal cortex

Corticotropin releasing factor (CRF) is considered as the central driving force in the stress response and plays a key role in the pathogenesis of depression. CRF neurons have been identified to locate in most regions of the prefrontal cortex (PFC), a brain region that is highly associated with the control of emotion and cognition. However, little is known on the regulation of CRF in this region. In this study, we aimed to identify the regulatory effect of acute restraint stress and glucocorticoid on PFC CRF and characterize the possible function of CRF in the PFC. We found that acute restraint stress increased and glucocorticoid decreased PFC CRF mRNA expression. The expression of glucocorticoid receptor (GR) was found to colocalize with CRF neurons in the PFC. In addition, recruitment of GR by the CRF promoter was observed in vivo. Specific attention was paid to the effect of CRF on CRF receptor 1 (CRFR1) expression in primary PFC cultures. The results showed that CRF increased CRFR1 expression through the MEK-ERK1/2 pathway. In summary, this study may contribute to the better understanding of CRF functions in the PFC.     

A new role for corticotropin-releasing factor receptor-2: suppression of vascularization

The corticotropin-releasing factor (CRF) family of receptors and ligands are known to have potent effects on vasculature function. Our recent data has described a novel role of corticotropin-releasing factor receptor-2 (CRFR2) as a tonic inhibitor of neovascularization. CRFR2 is found in both endothelial and smooth muscle cells (SMC) in the vasculature, where its function has been elusive. From investigation into the role of CRFR2 as a determinant of tissue vascularization, it was discovered that mice deficient for CRFR2 become hypervascularized postnatally and express increased levels of vascular endothelial growth factor (VEGF). Mechanistically, CRFR2 activation in vitro was found to result in reduced VEGF release from SMCs, an inhibition of SMC proliferation, and an inhibition of capillary tube formation in collagen gels. Treatment of a subcutaneously injected gel matrix with a CRFR2 agonist inhibited growth factor-induced vascularization in vivo. Western blots for the cell-cycle retinoblastoma protein (Rb)--essential for cell-cycle progression, showed decreased levels of phosphorylated Rb following CRFR2 agonist treatment in SMCs--supporting a role for CRFR2 in regulation of SMC proliferation. These results suggest that CRFR2 is a critical component of a novel pathway necessary for tonic inhibition of adult neovascularization, and that CRFR2 may be a potential target for therapeutic modulation of angiogenesis.     

Synthesis and characterization of a photoactivatable analog of corticotropin-releasing factor for specific receptor labeling.

A novel photoactivatable analog of ovine corticotropin-releasing factor (ovine photoCRF) has been synthesized and characterized. A diazirine group, the 4-(1-azi-2,2,2-trifluoroethyl)benzoyl residue, was covalently bound to the amino terminus of ovine CRF (oCRF), which was N-terminally extended by a tyrosyl residue for radioactive labeling with 125I. Under mild conditions, photolysis yielded highly reactive carbenes, responsible for the formation of covalent bonds to the CRF receptor. Ovine photoCRF was shown to bind to the high-affinity site of the CRF receptor with a similar Kd value as oCRF. When radioactively iodinated ovine photoCRF (ovine 125I-photoCRF) was covalently linked to rat CRF receptor, type 1 (rCRFR1), permanently transfected into human embryonic kidney (HEK) 293 cells, a highly glycosylated 75-kDa protein was identified with SDS/PAGE. The specificity of ovine 125I-photoCRF was demonstrated by the finding that this analog could be displaced from the receptor by oCRF, but not other unrelated peptides such as vasoactive intestinal peptide. The observed size of the 75-kDa cross-link was in agreement with the molecular weight reported earlier for native CRFR1 from rat brain. Deglycosylation of the 75-kDa cross-link with peptide:N-glycosidase (PNGase) yielded a 46-kDa protein, in agreement with the molecular weight estimated from cDNA coding for rat CRFR1. The developed CRF analog, photoCRF, is expected to facilitate future biochemical and physiological analysis of CRF receptors and--by analogous strategies--of other peptide receptors.     

Corticotropin-releasing factor receptor-2-deficient mice display abnormal homeostatic responses to challenges of increased dietary fat and cold

Corticotropin-releasing factor (CRF) and its family of ligands are key regulators of energy balance. These ligands function via activation of their two receptors, CRFR1 and CRFR2. CRFR1 has been shown to be the dominant receptor in activation of the hypothalamic-pituitary-adrenal axis in response to stress as well as a key mediator of anxiety in the limbic system. To specifically examine the role of CRFR2 in energy balance, mice deficient for CRFR2 were exposed to physiological perturbations of homeostasis, including high-fat diet, repeated cold stress, and glucose and insulin challenges, and their responses measured. While on a high-fat diet, CRFR2-mutant mice consumed substantially more food and maintaining the same weight but had significantly lower body fat and lower plasma lipids than their wild-type littermates. These mice were also less inclined to develop diet-induced insulin resistance and more sensitive to changes in plasma glucose, indicating increased insulin sensitivity. Following repeated cold stress, mutant mice had significantly lower body fat and a transient reduction in feed efficiency, despite similar body weights, suggesting a possible preference for fat as an energy substrate. Elevated levels of uncoupling protein-1 in brown adipose tissue as well as smaller white and brown adipocytes from CRFR2-mutant mice were indications of possible increased sympathetic tone. These results demonstrate that CRFR2 plays a critical role in regulation of energy expenditure and is important for responses to homeostatic challenges.     

Urocortin-II and urocortin-III are cardioprotective against ischemia reperfusion injury: an essential endogenous cardioprotective role for corticotropin releasing factor receptor type 2 in the murine heart

Corticotropin-releasing factor (CRF) receptor type 2beta (CRFR2beta) is expressed in the heart. Urocortin (Ucn)-I activation of CRFR2beta is cardioprotective against ischemic reperfusion (I/R) injury by stimulation of the ERKs1/2 p42, 44. However, by binding CRF receptor type 1, Ucn-I can also activate the hypothalamic stress axis. Ucn-II/stresscopin related peptide and Ucn-III/stresscopin are two new members of the CRF/Ucn-I gene family and are selective for CRFR2beta. We propose that CRFR2beta selective Ucn-II or Ucn-III will protect cardiomyocytes and the ex vivo Langendorff perfused rat heart from I/R injury by activation of ERK1/2-p42, 44. Ucn-II is expressed in mouse cardiomyocytes, and Ucn-II or Ucn-III can bind to CRFR2beta, resulting in ERK1/2-p42, p-44 phosphorylation and cAMP stimulation. Phosphorylation of ERK1/2-p42, p-44 is regulated by the Ras/Raf-1 kinase pathway, independent of adenylate cyclase and, therefore, cAMP activation. Ucn-II and Ucn-III protect cardiomyocytes from I/R injury and reduce the percentage of infarct size:risk ratio in Langendorff perfused rat hearts exposed to regional I/R (P<0.001). The CRFR2 selective antagonist astressin2-B and an ERK1/2-p42, 44 inhibitor abolish the cardioprotective actions of Ucn-II and Ucn-III in reperfusion. Cardiomyocytes isolated from CRFR2-null mice are less resistant to I/R injury, compared with wild-type cardiomyocytes. We propose the use of CRFR2 selective agonists, Ucn-II and Ucn-III, to treat ischemic heart disease because of their potent cardioprotective effects in the murine heart and their minimal impact on the hypothalamic stress axis. We emphasize an important endogenous cardioprotective role for CRFR2beta in the murine heart.