Corticotropin-releasing Factor Family Research Articles

Role of CRH in colitis and colitis-associated cancer: a combinative result of central and peripheral effects?

Corticotropin-releasing factor family peptides (CRF peptides) comprise corticotropin releasing hormone (CRH), urocortin (UCN1), UCN2 and UCN3. CRH is first isolated in the brain and later with UCNs found in many peripheral cells/tissues including the colon. CRH and UCNs function via the two types of receptors, CRF1 and CRF2, with CRH mainly acting on CRF1, UCN1 on both CRF1 &CRF2 and UCN2-3 on CRF2. Compiling evidence shows that CRH participates in inflammation and cancers via both indirect central effects related to stress response and direct peripheral influence. CRH, as a stress-response mediator, plays a significant central role in promoting the development of colitis involving colon motility, immunity and gut flora, while a few anti-colitis results of central CRH are also reported. Moreover, CRH is found to directly influence the motility and immune/inflammatory cells in the colon. Likewise, CRH is believed to be greatly related to tumorigenesis of many kinds of cancers including colon cancer via the central action during chronic stress while the peripheral effects on colitis-associated-colon cancer (CAC) are also proved. We and others observe that CRH/CRF1 plays a significant peripheral role in the development of colitis and CAC in that CRF1 deficiency dramatically suppresses the colon inflammation and CAC. However, up to date, there still exist not many relevant experimental data on this topic, and there seems to be no absolute clearcut between the central and direct peripheral effects of CRH in colitis and colon cancer. Taken together, CRH, as a critical factor in stress and immunity, may participate in colitis and CAC as a centrally active molecule; meanwhile, CRH has direct peripheral effects regulating the development of colitis and CAC, both of which will be summarized in this review.

MRNA expression of CRF family members in urothelial bladder cancer.

The corticotropin-releasing factor (CRF) gene family includes the three urocortins (UCN1, 2 and 3) and the two receptors (CRFR1 and 2), which play a significant role in the physiology of various organs. The expression of the CRF family of genes and its receptors are shown to participate in the pathogenesis of inflammation and even tumorigenesis. However, data regarding the human urinary tract, especially the bladder, are scarce. To the best of our knowledge, no studies are currently available on the CRF system and bladder cancer. The primary goal of the present study was to investigate the mRNA expression of the CRF family members in bladder cancer. The secondary aim was to analyze the differences with the expression of the same mRNAs in normal bladders. From August 2018 to July 2021, 43 recruited patients were divided into three groups. Group A included healthy patients, group B included patients with bladder cancer and group C included patients with a history of cancer from whom samples were taken from the normal bladder mucosa. Detection of mRNA of the CRF family of genes was performed using reverse transcription-quantitative PCR. The mRNA of the three urocortins, CRF and the two receptors were predominantly expressed in all three groups of patients. Statistical analysis using the Kruskal-Wallis test showed that UCN1 was downregulated in patients with bladder cancer and those with possible cancer compared with the healthy group (mean rank group A=24.3 vs. mean rank group B=12.58; P=0.006) and (mean rank group A=24.3 vs. mean rank group C=8.88; P=0.001). The present experiments showed that mRNA of the CRF family of genes was amplified in normal and cancer bladder tissues. Downregulation of the UCN1 gene may be associated with bladder cancer, contributing to the prognosis, diagnosis or therapy of urothelial malignancies.

Abstract 16840: A Novel Long-Acting CRF2 Receptor Agonist COR-1167 Improves Cardiac and Renal Function on Top of Furosemide in a Sheep Model of Pacing Induced Heart Failure

Urocortin-2 (UCN-2), a member of the corticotrophin releasing factor family, has cardiovascular protective effects mediated via activation of CRF2 receptors. Exploratory studies in ovine heart failure (HF) and in HF patients demonstrated that acute intravenous (IV) infusion of UCN-2 (1-4hrs), on top of standard of care, temporarily improved cardiac function. However, the short t 1/2 of recombinant UCN-2 (~15 min) limits its therapeutic utility. A stable CRF2 peptide agonist was developed, named COR-1167, that is potent and selective for CRF2 versus CRF1 receptors and it was tested chronically in vivo. Adult female sheep were anesthetized, and instrumented with 7F His bundle electrodes, arterial, venous and Swan-Ganz catheters, pressure transducers, and flow probes. After recovery, HF was induced by rapid cardiac pacing that was maintained during the study. COR-1167 (5 μg/kg/day SC) in combination with furosemide (0.35 mg/kg/day IV) versus furosemide alone was administered for 5 days and cardiorenal parameters were constantly monitored in conscious sheep. Furosemide had no effect on cardiac function while it increased urine Na + , when compared to pre-treatment measurements. Compared to furosemide, COR-1167 plus furosemide significantly enhanced cardiac and renal function parameters (Table). Mean + SEM for Days 1-5 (CV, biomarker and renal parameters), F (n=5-7), F + COR-1167 (n=6-7), Repeated Measures ANOVA; *BW Day 0 minus Day 5 = BW change, Unpaired t test. COR-1167 cardiorenal benefits occurred within 2 hours of the first dose and were sustained, without changing SBP. It increased natriuresis and decreased net fluid balance (water intake minus urine volume) and BW (reflecting a reduction in congestion). In conclusion, chronic CRF2 receptor activation by COR-1167 significantly improved both cardiac and renal function on top of furosemide in ovine HF and potentially is a unique therapy for HF patients.

Corticotropin-releasing factor signaling and its potential role in the prefrontal cortex-dependent regulation of anxiety.

A large body of literature has highlighted the significance of the corticotropin-releasing factor (CRF) system in the regulation of neuropsychiatric diseases. Anxiety disorders are among the most common neuropsychiatric disorders. An increasing number of studies have demonstrated that the CRF family mediates and regulates the development and maintenance of anxiety. Thus, the CRF family is considered to be a potential target for the treatment of anxiety disorders. The prefrontal cortex (PFC) plays a role in the occurrence and development of anxiety, and both CRF and CRF-R1 are widely expressed in the PFC. This paper begins by reviewing CRF-related signaling pathways and their different roles in anxiety and related processes. Then, the role of the CRF system in other neuropsychiatric diseases is reviewed and the potential role of PFC CRF signaling in the regulation of anxiety disorders is discussed. Although other signaling pathways are potentially involved in the process of anxiety, CRF in the PFC primarily modulates anxiety disorders through the activation of corticotropin-releasing factor type1 receptors (CRF-R1)and the excitation of the cAMP/PKA signaling pathway. Moreover, the main signaling pathways of CRF involved in sex differentiation in the PFC appear to be different. In summary, this review suggests that the CRF system in the PFC plays a critical role in the occurrence of anxiety. Thus, CRF signaling is of great significance as a potential target for the treatment of stress-related disorders in the future.

Unraveling corticotropin-releasing factor family-orchestrated signaling and function in both sexes.

Stress responses to physical, psychological, environmental, or cellular stressors, has two arms: initiation and recovery. Corticotropin-releasing factor (CRF) is primarily responsible for regulating and/or initiating stress responses via, whereas urocortins (UCNs) are involved in the recovery response to stress via feedback inhibition. Stress is a loaded, polysemous word and is experienced in a myriad of ways. Some stressors are good for an individual, in fact essential, whereas other stressors are associated with bad outcomes. Perceived stress, like beauty, lies in the eye of the beholder, and hence the same stressor can result in individual-specific outcomes. In mammals, there are two main biological sexes with reproduction as primary function. Reproduction and nutrition can also be viewed as stressors; based on a body of work from my laboratory, we propose that the functions of all other organs have co-evolved to optimize and facilitate an individual's nutritional and reproductive functions. Hence, sex differences in physiologically relevant outcomes are innate and occur at all levels- molecular, endocrine, immune, and (patho)physiological. CRF and three UCNs are peptide hormones that mediate their physiological effects by binding to two known G protein-coupled receptors (GPCRs), CRF1 and CRF2. Expression and function of CRF family of hormones and their receptors is likely to be sexually dimorphic in all organs. In this chapter, based on the large body of work from others and my laboratory, an overview of the CRF family with special emphasis on sex-specific actions of peripherally expressed CRF2 receptor in health and disease is provided.

Urocortin stimulates ERK1/2 phosphorylation and proliferation but reduces ATP production of MCF7 breast cancer cells

Urocortins are members of the stress-related corticotropin-releasing factor family. Small amounts of them are present in the circulation and they are produced locally in various tissues of higher vertebrates. Aside from regulating circulation, or food uptake they also influence, via auto- and paracrine mechanisms, cell proliferation. In the present study we investigated in MCF7 human breast cancer cells the effect of urocortin onto mitogenic signaling via ERK1/2. Our results revealed that already 10 nM urocortin could stimulate the phosphorylation of these kinases and cell proliferation of MCF7 cells while ATP production was reduced when kept in the presence of the peptide up to two days. We examined the expression and contribution of the specific receptors of urocortin to the activation of ERK1/2 and to cell proliferation, the intracellular distribution of phosphorylated ERK1/2, and the involvement of additional proteins like PKA, PKB/Akt, MEK, p53, Rb and E2F-1 behind the observed phenomena.

Role of CRF and the hypothalamic-pituitary-adrenal axis in stroke: revisiting temporal considerations and targeting a new generation of therapeutics.

Ischaemic neurovascular stroke represents a leading cause of death in the developed world. Preclinical and human epidemiological evidence implicates the corticotropin-releasing factor (CRF) family of neuropeptides as mediators of acute neurovascular injury pathology. Preclinical investigations of the role of CRF, CRF receptors and CRF-dependent activation of the hypothalamic-pituitary-adrenal (HPA) axis have pointed toward a tissue-specific and temporal relationship between activation of these pathways and physiological outcomes. Based on the literature, the major phases of ischaemic stroke aetiology may be separated into an acute phase in which CRF and anti-inflammatory stress signalling are beneficial and a chronic phase in which these contribute to neural degeneration, toxicity and apoptotic signalling. Significant gaps in knowledge remain regarding the pathway, temporality and systemic impact of CRF signalling and stress biology in neurovascular injury progression. Heterogeneity among experimental designs poses a challenge to defining the apparent reciprocal relationship between neurological injury and stress metabolism. Despite these challenges, it is our opinion that the elucidated temporality may be best matched with an antibody against CRF with a half-life of days to weeks as opposed to minutes to hours as with small-molecule CRF receptor antagonists. This state-of-the-art review will take a multipronged approach to explore the expected potential benefit of a CRF antibody by modulating CRF and corticotropin-releasing factor receptor 1 signalling, glucocorticoids and autonomic nervous system activity. Additionally, this review compares the modulation of CRF and HPA axis activity in neuropsychiatric diseases and their counterpart outcomes post-stroke and assess lessons learned from antibody therapies in neurodegenerative diseases.

Urocortins as biomarkers in cardiovascular disease.

The urocortins (Ucns) belong to the corticotropin-releasing factor (CRF) family of peptides and have multiple effects within the central nervous and the cardiovascular systems. With growing evidence indicating significant cardioprotective properties and cardiovascular actions of these peptides, the question arises as to whether the plasma profiles of the Ucns are altered in pathologic settings. While reports have shown conflicting results and findings have not been corroborated in multiple independent cohorts, it seems likely that plasma Ucn concentrations are elevated in multiple cardiovascular conditions. The degree of increase and accurate determination of circulating values of the Ucns requires further validation.

Expression Patterns of the Neuropeptide Urocortin 3 and Its Receptor CRFR2 in the Mouse Central Auditory System.

Sensory systems have to be malleable to context-dependent modulations occurring over different time scales, in order to serve their evolutionary function of informing about the external world while also eliciting survival-promoting behaviors. Stress is a major context-dependent signal that can have fast and delayed effects on sensory systems, especially on the auditory system. Urocortin 3 (UCN3) is a member of the corticotropin-releasing factor family. As a neuropeptide, UCN3 regulates synaptic activity much faster than the classic steroid hormones of the hypothalamic-pituitary-adrenal axis. Moreover, due to the lack of synaptic re-uptake mechanisms, UCN3 can have more long-lasting and far-reaching effects. To date, a modest number of studies have reported the presence of UCN3 or its receptor CRFR2 in the auditory system, particularly in the cochlea and the superior olivary complex, and have highlighted the importance of this stress neuropeptide for protecting auditory function. However, a comprehensive map of all neurons synthesizing UCN3 or CRFR2 within the auditory pathway is lacking. Here, we utilize two reporter mouse lines to elucidate the expression patterns of UCN3 and CRFR2 in the auditory system. Additional immunolabelling enables further characterization of the neurons that synthesize UCN3 or CRFR2. Surprisingly, our results indicate that within the auditory system, UCN3 is expressed predominantly in principal cells, whereas CRFR2 expression is strongest in non-principal, presumably multisensory, cell types. Based on the presence or absence of overlap between UCN3 and CRFR2 labeling, our data suggest unusual modes of neuromodulation by UCN3, involving volume transmission and autocrine signaling.

Urocortin Role in Ischemia Cardioprotection and the Adverse Cardiac Remodeling.

Despite the considerable progress in strategies of myocardial protection, ischemic heart diseases (IHD) and consequent heart failure (HF) remain the main cause of mortality worldwide. Several procedures are used routinely to guarantee the prompt and successful reestablishment of blood flow to preserve the myocardial viability of infarcted hearts from ischemia injuries. However, ischemic heart reperfusion/revascularization triggers additional damages that occur when oxygen-rich blood re-enters the vulnerable myocardial tissue, which is a phenomenon known as ischemia and reperfusion (I/R) syndrome. Complications of I/R injuries provoke the adverse cardiac remodeling, involving inflammation, mishandling of Ca2+ homeostasis, apoptotic genes activation, cardiac myocytes loss, etc., which often progress toward HF. Therefore, there is an urgent need to develop new cardioprotective therapies for IHD and HF. Compelling evidence from animal studies and pilot clinical trials in HF patients suggest that urocortin (Ucn) isoforms, which are peptides associated with stress and belonging to the corticotropin releasing factor family, have promising potential to improve cardiovascular functions by targeting many signaling pathways at different molecular levels. This review highlights the current knowledge on the role of urocortin isoforms in cardioprotection, focusing on its acute and long-term effects.

Ghrelin, via corticotropin-releasing factor receptors, reduces glucose uptake and increases lipid content in mouse myoblasts cells.

Ghrelin and the corticotropin‐releasing factor (CRF) family are known regulators of cellular metabolism and energy balance. We previously demonstrated that myoblast glucose metabolism is regulated by ghrelin and that this effect is mediated by CRF receptor type 2 (CRF‐R2). Here we explored the effect of des‐acyl ghrelin, the major circulating isoform of ghrelin, on cellular metabolism in mouse myoblast C2C12 cells, and examined whether CRF family receptors mediate its metabolic effects in muscle cells. C2C12 cells were exposed to des‐acyl ghrelin with or without the CRF‐R1‐ and CRF‐R2‐specific antagonists antalarmin or antisauvagine‐30, respectively. Des‐acyl ghrelin reduced glucose uptake and expression of the glucose transporter GLUT4, but induced retinol‐binding protein 4 (RBP4) expression. Antalarmin and antisauvagine‐30 inhibited the induction of glucose uptake by des‐acyl ghrelin and its effect on GLUT4 and RBP4 expression. Moreover, treating C2C12 cells with des‐acyl ghrelin resulted in cAMP activation in response to the CRF‐R1‐specific ligand stressin, and the CRF‐R2‐specific ligand Ucn3. Furthermore, des‐acyl ghrelin reduced the expression of uncoupling proteins UCP2 and UCP3. Adding antalarmin or antisauvagine‐30 to the medium reversed this effect. Finally, des‐acyl ghrelin elevated lipid content and acetyl‐CoA carboxylase expression in C2C12 cells. Our results suggest that during food deprivation, des‐acyl ghrelin signals the muscle cells that glucose levels are low and that they should switch to fatty acids for their metabolic fuel.

Corticotropin-Releasing Factor: An Ancient Peptide Family Related to the Secretin Peptide Superfamily.

Corticotropin-releasing factor (CRF) is the hypothalamic releasing peptide that regulates the hypothalamic-pituitary-adrenal/inter-renal (HPA/I) axis in vertebrates. Over the last 25 years, there has been considerable discussion on its paralogs genes, urotensin-I/urocortin-1, and urocortins-2 and-3 and their subsequent role in the vertebrate stress response. Phylogenetically, the CRF family of peptides also belong to the diverse assemblage of Secretin- and Calcitonin-based peptides as evidenced by comparative-based studies of both their ligand and G-protein-coupled receptor (GPCR) structures. Despite this, the common origin of this large assemblage of peptides has not been ascertained. An unusual peptide, teneurin-C-terminal associated peptide (TCAP), reported in 2004, comprises the distal extracellular tip of the teneurin transmembrane proteins. Further studies indicated that this teneurin region binds to the latrophilin family of GPCRs. Initially thought to be a member of the Secretin GPCR family, evidence indicates that the latrophilins are a member of the Adhesion family of GPCRs and are related to the common ancestor of both Adhesion and Secretin GPCR families. In this study, we posit that TCAP may be a distantly related ancestor of the CRF-Calcitonin-Secretin peptide family and evolved near the base of metazoan phylogeny.

Corticotropin-Releasing Factor Family: A Stress Hormone-Receptor System's Emerging Role in Mediating Sex-Specific Signaling.

No organ in the body is impervious to the effects of stress, and a coordinated response from all organs is essential to deal with stressors. A dysregulated stress response that fails to bring systems back to homeostasis leads to compromised function and ultimately a diseased state. The components of the corticotropin-releasing factor (CRF) family, an ancient and evolutionarily conserved stress hormone-receptor system, helps both initiate stress responses and bring systems back to homeostasis once the stressors are removed. The mammalian CRF family comprises of four known agonists, CRF and urocortins (UCN1–3), and two known G protein-coupled receptors (GPCRs), CRF1 and CRF2. Evolutionarily, precursors of CRF- and urocortin-like peptides and their receptors were involved in osmoregulation/diuretic functions, in addition to nutrient sensing. Both CRF and UCN1 peptide hormones as well as their receptors appeared after a duplication event nearly 400 million years ago. All four agonists and both CRF receptors show sex-specific changes in expression and/or function, and single nucleotide polymorphisms are associated with a plethora of human diseases. CRF receptors harbor N-terminal cleavable peptide sequences, conferring biased ligand properties. CRF receptors have the ability to heteromerize with each other as well as with other GPCRs. Taken together, CRF receptors and their agonists due to their versatile functional adaptability mediate nuanced responses and are uniquely positioned to orchestrate sex-specific signaling and function in several tissues.

Urocortin 1: A putative excitatory neurotransmitter in the enteric nervous system.

Urocortin 1 (Ucn1), a stress-related peptide, is a member of the corticotropin-releasing factor (CRF) family and acts as a CRF1 receptor agonist. Ucn1 and CRF1 receptor immunoreactivity are present in the enteric nervous system (ENS), and Ucn1 elicits contraction of colonic muscle strips. Considering these findings, we have hypothesized that Ucn1 acts as an excitatory neurotransmitter in the ENS. The present study was conducted to determine whether exogenously applied Ucn1 causes contractions, whether it participates in neurally mediated contraction, and whether it is released from the ENS of the rat colon. Isometric tension of the rat colonic muscle strips (middle to distal colon) in a longitudinal direction was measured. The effects of Ucn1 on phasic contractions were examined in the absence and presence of antalarmin (CRF1 receptor antagonist), tetrodotoxin (TTX), and atropine. The effects of antalarmin on electrical field stimulation (EFS)-induced contractions were examined in the absence and presence of atropine. Ucn1 peptide in the bath solution was measured after EFS using an EIA kit. Ucn1 caused a significant and dose-dependent increase in phasic contractions. These effects were completely inhibited by antalarmin, TTX, and atropine. EFS-induced contractions were inhibited by antalarmin. Atropine markedly reduced EFS-induced contractions, and antalarmin did not decrease these contractions further. EFS elicited a significant increase in the concentration of Ucn1 in the bath solution, and this increase was completely inhibited by TTX. These results suggest that Ucn1 acts as an excitatory neurotransmitter in the ENS enhancing the cholinergic neurotransmission.

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.

Involvement of Centrally Projecting Edinger-Westphal Nucleus Neuropeptides in Actions of Addictive Drugs.

The centrally-projecting Edinger–Westphal nucleus (EWcp) is a brain region distinct from the preganglionic Edinger–Westphal nucleus (EWpg). In contrast to the EWpg, the EWcp does not send projections to the ciliary ganglion and appears not to regulate oculomotor function. Instead, evidence is accumulating that the EWcp is extremely sensitive to alcohol and several other drugs of abuse. Studies using surgical, genetic knockout, and shRNA approaches further implicate the EWcp in the regulation of alcohol sensitivity and self-administration. The EWcp is also known as the site of preferential expression of urocortin 1, a peptide of the corticotropin-releasing factor family. However, neuroanatomical data indicate that the EWcp is not a monotypic brain region and consists of several distinct subpopulations of neurons. It is most likely that these subpopulations of the EWcp are differentially involved in the regulation of actions of addictive drugs. This review summarizes and analyzes the current literature of the EWcp’s involvement in actions of drugs of abuse in male and female subjects in light of the accumulating evidence of complexities of this brain region.

Evaluation of Serum Urocortin 2 Levels in Patients with Hypertension.

Urocortin 2 (UCN2), is an endogenous stress-related peptide belonging to the corticotropin-releasing factor (CRF) family, has a major role in the pathogenesis of congestive heart failure, ischemic heart disease, and hypertension. To investigate the role of UCN2 levels in patients with hypertension (HTN). Serum UCN2 levels measured by ELISA were compared between patients with HTN (n = 86) and nonHTN (n = 53). Eighty-six patients with HTN [median age 66 (45-76); 38 men] and 53 patients with non-HTN [median age 62 (40-80); 39 men] were included into this study. Serum UCN2 (5.17ng/ml; IQR, 1.26-11.68ng/ml vs 0.79ng/ml; IQR, 0.07-4.10ng/ml, p < 0.0005) levels were found significantly elevated in patients with HTN compared to nonHTN control group.Concentrations of serum UCN2 were positively correlated with left ventricle mass index to body surface area (LV mass index to BSA, r = 0.20, p = 0.03), LV mass index to height2.7 (r = 0.28, p = 0.002) and body mass index (r = 0.24, p = 0.008). Additionally, logistic regression analysis was performed to UCN2, uric acid, creatinine, glomerular filtration rate, age, body mass index, coronary artery disease and diabetes mellitus which are the potential confounders of hypertension. According to logistic regression analysis serum UCN2 values were found out as an independent predictor of HTN. UCN2 levels, correlated with LV mass index were increased in HTN patients compared to nonHTN patients. These data provide evidence that there could be a relationship between high concentrations of UCN2 and HTN. UCN2 may appear as a promising choice of HTN treatment in the future.

Significant alteration of liver metabolites by AAV8.Urocortin 2 gene transfer in mice with insulin resistance.

IntroductionUrocortin 2 (Ucn2) is a 38-amino acid peptide of the corticotropin-releasing factor family. Intravenous (IV) delivery of an adeno-associated virus vector serotype 8 encoding Ucn2 (AAV8.Ucn2) increases insulin sensitivity and glucose disposal in mice with insulin resistance.ObjectiveTo determine the effects of Ucn2 on liver metabolome.MethodsSix-week-old C57BL6 mice were divided into normal chow (CHOW)-fed and high fat diet (HFD)-fed groups. The animals received saline, AAV8 encoding no gene (AAV8.Empt) or AAV8.Ucn2 (2x1013 genome copy/kg, IV injection). Livers were isolated from CHOW-fed and HFD-fed mice and analyzed by untargeted metabolomics. Group differences were statistically analyzed.ResultsIn CHOW-fed mice, AAV8.Ucn2 gene transfer (vs. saline) altered the metabolites in glycolysis, pentose phosphate, glycogen synthesis, glycogenolysis, and choline-folate-methionine signaling pathways. In addition, AAV8.Ucn2 gene transfer increased amino acids and peptides, which were associated with reduced protein synthesis. In insulin resistant (HFD-induced) mice, HFD (vs CHOW) altered 448 (112 increased and 336 decreased) metabolites and AAV8.Ucn2 altered 239 metabolites (124 increased and 115 reduced) in multiple pathways. There are 61 metabolites in 5 super pathways showed interactions between diet and AAV8.Ucn2 treatment. Among them, AAV8.Ucn2 gene transfer reversed HFD effects on 13 metabolites. Finally, plasma Ucn2 effects were determined using a 3-group comparison of HFD-fed mice that received AAV8.Ucn2, AAV.Empt or saline, where 18 metabolites that altered by HFD (15 increased and 3 decreased), but restored levels to that seen in CHOW-fed mice by increased plasma Ucn2.ConclusionsMetabolomics study revealed that AAV8.Ucn2 gene transfer, through increased plasma Ucn2, provided counter-HFD effects in restoring hepatic metabolites to normal levels, which could be the underlying mechanisms for Ucn2 effects on increasing glucose disposal and reducing insulin assistance.

Urocortin Induces Phosphorylation of Distinct Residues of Signal Transducer and Activator of Transcription 3 (STAT3) via Different Signaling Pathways.

BackgroundUrocortin (Ucn) is a member of the hypothalamic corticotrophin-releasing factor family and has been shown to reduce cell death in the heart caused by ischemia/reperfusion (I/R) injury. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor known to function as a pro-survival and anti-apoptotic factor, whose activation depends on a variety of cytokines, including IL-6. A recent study demonstrated that urocortin induced IL-6 release from cardiomyocytes in a CRF-R2-dependent manner, suggesting a possible link between CRF-R2 stimulation and STAT3 activation.Material/MethodsExperimental work was carried out in HL-1 cardiac myocytes exposed to serum starvation for 16–24 h.ResultsUcn stimulation led to IL-6 expression and release from mouse atrial HL-1 cardiomyocytes. Ucn treatment led to rapid phosphorylation of JAK2, which was blocked by the protein synthesis inhibitor cycloheximide or the JAK inhibitor AG490. Urocortin treatment induced STAT3 phosphorylation at Y705 and S727 through transactivation of JAK2 in an IL-6-dependent manner, but had no effect on STAT1 activity. Kinase inhibition experiments revealed that urocortin induces STAT3 S727 phosphorylation through ERK1/2 and Y705 phosphorylation through Src tyrosine kinase. In line with this finding, urocortin failed to induce phosphorylation of Y705 residue in SYF cells bearing null mutation of Src, while phosphorylation of S727 residue was unchanged.ConclusionsHere, we have shown that Ucn induces activation of STAT3 through diverging signaling pathways. Full understanding of these signaling pathways will help fully exploit the cardioprotective properties of endogenous and exogenous Ucn.

Urocortin 3 Gene Transfer Increases Function of the Failing Murine Heart.

Peptide infusions of peptides the corticotropin releasing factor family, including urocortin 2, stresscopin, and urocortin 3 (UCn3), have favorable acute effects in clinical heart failure (HF), but their short half-lives make them unsuitable for chronic therapy. This study asked whether UCn3 gene transfer, which provides sustained elevation of plasma UCn3 levels, increases the function of the failing heart. HF was induced by transmural left ventricular (LV) cryoinjury in mice. LV function was assessed 3 weeks later by echocardiography. Those with ejection fractions (EF) <40% received intravenous saline or intravenous adeno-associated virus type-8 encoding murine UCn3 (AAV8.mUCn3; 1.9 × 1013 genome copies/kg). Five weeks after randomization, repeat echocardiography, assessment of LV function (+dP/dt, -dP/dt), and quantification of Ca2+ transients and sarcomere shortening in isolated cardiac myocytes were conducted, and assessment of LV Ca2+ handling and stress proteins was performed. Three weeks after myocardial infarction, prior to treatment, EFs were reduced (mean 31%, from 63% in sham-operated animals). Mice randomized to receive UCn3 gene transfer showed increased plasma UCn3 (from 0.1 ± 0.01 ng/mL in the saline group to 5.6 ± 1.1 ng/mL; n = 12 each group; p < 0.0001). Compared to mice that received saline, UCn3 gene transfer was associated with higher values for EF (p = 0.0006); LV +dP/dt (p < 0.0001), and LV -dP/dt (p < 0.0001). Cardiac myocytes from mice that received UCn3 gene transfer showed higher peak Ca2+ transients (p = 0.0005), lower time constant of cytosolic Ca2+ decline (tau, p < 0.0001), and higher rates of sarcomere shortening (+dL/dt, p = 0.03) and lengthening (-dL/dt, p = 0.04). LV samples from mice that received UCn3 gene transfer contained higher levels of SERCA2a (p = 0.0004 vs. HF) and increased amounts of phosphorylated troponin I (p = 0.04 vs. HF). UCn3 gene transfer is associated with improved Ca2+ handling and LV function in mice with HF and reduced EF.