Urocortin-1 promotes colorectal cancer cell migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway.

PURPOSE: To investigate the effect of urocortin-1 (UCN-1) on growth, migration, and apoptosis in colorectal cancer (CRC) in vivo and vitro and the mechanism by which UCN-1 modulates CRC cells in vitro. METHODS: The correlation between UCN-1 and CRC was evaluated using The Cancer Genome Atlas (TCGA) database and a tissue microarray. The expression of UCN-1 in CRC cells was assessed using quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting. In vitro, the influence of UCN-1 on the proliferation, apoptosis, and migration of HT-29, HCT-116, and RKO cells was explored using the celigo cell counting assay or cell counting kit-8 (CCK8), flow cytometry, and wound healing or Transwell assays, respectively. In vivo, the effect of UCN-1 on CRC growth and progression was evaluated in nude mice. The downstream pathway underlying UCN-1-mediated regulation of CRC was determined using the phospho-kinase profiler array in RKO cells. Lentiviruses were used to knockdown or upregulate UCN-1 expression in cells. RESULTS: Both the TCGA and tissue microarray results showed that UCN-1 was strongly expressed in the tissues of patients with CRC. Furthermore, the tissue microarray results showed that the expression of UCN-1 was higher in male than in female patients, and high expression of UCN-1 was associated with higher risk of lymphatic metastasis and later pathological stage. UCN-1 knockdown caused a reduction in CRC cell proliferation, migration, and colony formation, as well as an increase in apoptosis. In xenograft experiments, tumors generated from RKO cells with UCN-1 knockdown exhibited reduced volumes and weights. A reduction in the expression of Ki-67 in xenograft tumors indicated that UCN-1 knockdown curbed tumor growth. The human phospho-kinase array showed that the p53 signaling pathway participated in UCN-1-mediated CRC development. The suppression in migration and proliferation caused by UCN-1 knockdown was reversed by inhibitors of p53 signal pathway, while the increase in cell apoptosis was suppressed. On the other hand, overexpression of UCN-1 promoted proliferation and migration and inhibited apoptosis in CRC cells. Overexpression of p53 reversed the effect of UCN-1 overexpression on CRC development. CONCLUSION: UCN-1 promotes migration and proliferation and inhibits apoptosis via inhibition of the p53 signaling pathway.

The Lack of TRPA1 Ion Channel Does Not Affect the Chronic Stress-Induced Activation of the Locus Ceruleus.

We have previously proven the involvement of transient receptor potential ankyrin 1 (TRPA1) in stress adaptation. A lack of TRPA1 affects both urocortin 1 (member of the corticotropin-releasing hormone (CRH) family) content of the Edinger-Westphal nucleus. The noradrenergic locus ceruleus (LC) is also an important player in mood control. We aimed at investigating whether the TRPA1 is expressed in the LC, and to test if the response to chronic variable mild stress (CVMS) is affected by a lack of TRPA1. The TRPA1 expression was examined via RNAscope in situ hybridization. We investigated TRPA1 knockout and wildtype mice using the CVMS model of depression. Tyrosine hydroxylase (TH) and FOSB double immunofluorescence were used to test the functional neuromorphological changes in the LC. No TRPA1 expression was detected in the LC. The TH content was not affected by CVMS exposure. The CVMS-induced FOSB immunosignal did not co-localize with the TH neurons. TRPA1 is not expressed in the LC. A lack of functional TRPA1 receptor neither directly nor indirectly affects the TH content of LC neurons under CVMS.

The peripheral corticotropin releasing factor family's role in vasculitis.

Corticotropin releasing factor family peptides (CRF peptides) include 4 members, corticotropin releasing hormone (CRH), Urocortin (UCN1), UCN2 and UCN3. CRF peptides function via the two distinct receptors, CRF1 and CRF2. Among them, CRH/CRF1 has been recognized to influence immunity/inflammation peripherally. Both pro- and anti-inflammatory effects of CRH are reported. Likewise, UCNs, peripherally in cardiovascular system have been documented to have both potent protective and harmful effects, with UCN1 acting on both CRF1 & CRF2 and UCN2 & UCN3 on CRF2. We and others also observe protective and detrimental effects of CRF peptides/receptors on vasculature, with the latter of predominantly higher incidence, i.e., they play an important role in the development of vasculitis while in some cases they are found to counteract vascular inflammation. The pro-vasculitis effects of CRH & UCNs include increasing vascular endothelial permeability, interrupting endothelial adherens & tight junctions leading to hyperpermeability, stimulating immune/inflammatory cells to release inflammatory factors, and promoting angiogenesis by VEGF release while the anti-vasculitis effects may be just the opposite, depending on many factors such as different CRF receptor types, species and systemic conditions. Furthermore, CRF peptides' pro-vasculitis effects are found to be likely related to cPLA2 and S1P receptor signal pathway. This minireview will focus on summarizing the peripheral effects of CRF peptides on vasculature participating in the processes of vasculitis.

The expression of corticotropin-releasing hormone family peptides in premalignant and malignant vulvar lesions.

OBJECTIVES: To examine the relation of corticotropin-releasing hormone (CRH) family peptides with inflammatory processes and oncogenesis, emphasizing in vulvar inflammatory, premalignant and malignant lesions, as well as to investigate the possibility of lesion cells immunoescaping, utilizing FAS/FAS-L complex. METHODS: Immunohistochemical expression of CRH, urocortin (UCN), FasL and their receptors CRHR1, CRHR2 and Fas was studied in vulvar tissue sections obtained from patients with histologically confirmed diagnosis of lichen, vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell carcinoma (VSCC). The patient cohort was selected from a tertiary teaching Hospital in Greece, between 2005 and 2015. For each of the disease categories, immunohistochemical staining was evaluated and the results were statistically compared. RESULTS: A progressive increase of the cytoplasmic immunohistochemical expression of CRH and UCN, from precancerous lesions to VSCC was observed. A similar increase was detected for Fas and FasL expression. Nuclear localization of UCN was demonstrated in both premalignant and VSCC lesions, with staining being significantly intensified in carcinomas, particularly in the less differentiated tumor areas or in the areas at invasive tumor front. CONCLUSIONS: Stress response system and CRH family peptides seem to have a role in inflammation maintenance and progression of vulvar premalignant lesions to malignancy. It seems that stress peptides may locally modulate the stroma through Fas/FasL upregulation, possibly contributing to vulvar cancer development.

CRH-R2 signalling modulates feeding and circadian gene expression in hypothalamic mHypoA-2/30 neurons.

The hypothalamic type 2 corticotropin releasing hormone receptor (CRH-R2) plays critical roles in homeostatic regulation, particularly in fine tuning stress recovery. During acute stress, the CRH-R2 ligands CRH and urocortins promote adaptive responses and feeding inhibition. However, in rodent models of chronic stress, over-exposure of hypothalamic CRH-R2 to its cognate agonists is associated with urocortin 2 (Ucn2) resistance; attenuated cAMP-response element binding protein (CREB) phosphorylation and increased food intake. The molecular mechanisms involved in these altered CRH-R2 signalling responses are not well described. In the present study, we used the adult mouse hypothalamus-derived cell line mHypoA-2/30 to investigate CRH-R2 signalling characteristics focusing on gene expression of molecules involved in feeding and circadian regulation given the role of clock genes in metabolic control. We identified functional CRH-R2 receptors expressed in mHypoA-2/30 cells that differentially regulate CREB and AMP-activated protein kinase (AMPK) phosphorylation and downstream expression of the appetite-regulatory genes proopiomelanocortin (Pomc) and neuropeptide Y (Npy) in accordance with an anorexigenic effect. We studied for the first time the effects of Ucn2 on clock genes in native and in a circadian bioluminescence reporter expressing mHypoA-2/30 cells, detecting enhancing effects of Ucn2 on mRNA levels and rhythm amplitude of the circadian regulator Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), which could facilitate anorexic responses in the activity circadian phase. These data uncover novel aspects of CRH-R2 hypothalamic signalling that might be important in regulation of circadian feeding during stress responses.

Chronic UCN2 treatment desensitizes CRHR2 and improves insulin sensitivity.

Urocortin 2 (UCN2) acts as a ligand for the G protein-coupled receptor corticotropin-releasing hormone receptor 2 (CRHR2). UCN2 has been reported to improve or worsen insulin sensitivity and glucose tolerance in vivo. Here we show that acute dosing of UCN2 induces systemic insulin resistance in male mice and skeletal muscle. Inversely, chronic elevation of UCN2 by injection with adenovirus encoding UCN2 resolves metabolic complications, improving glucose tolerance. CRHR2 recruits Gs in response to low concentrations of UCN2, as well as Gi and ß-Arrestin at high concentrations of UCN2. Pre-treating cells and skeletal muscle ex vivo with UCN2 leads to internalization of CRHR2, dampened ligand-dependent increases in cAMP, and blunted reductions in insulin signaling. These results provide mechanistic insights into how UCN2 regulates insulin sensitivity and glucose metabolism in skeletal muscle and in vivo. Importantly, a working model was derived from these results that unifies the contradictory metabolic effects of UCN2.

Urocortin 2 - a protective effect in hypertension?

Urocortin 2, an endogenous selective ligand for the corticotropin-releasing hormone receptor type 2, has been suggested to exert cardioprotective effects. We analyzed the possible relationship between the level of Ucn2 and specific indicators of cardiovascular risk factors in patients with untreated hypertension and in healthy subjects. Sixty seven subjects were recruited: 38 with newly diagnosed treatment-naive hypertension (with no pharmacological treatment - HT group) and 29 healthy subjects without hypertension (nHT group). We evaluated ambulatory blood pressure monitoring, Ucn2 levels and metabolic indices. Multivariable regression analyses were performed to assess the effects of gender, age, and Ucn2 levels on metabolic indices or blood pressure (BP) level. Log of Ucn2 levels were higher in healthy subjects than in hypertensive patients (2.44±0.7 versus 2.09±0.66, p<.05) and correlated inversely with 24-hour diastolic blood pressure, and both night-time systolic and diastolic blood pressure regardless of age and gender (R2=0.06; R2=0.06; R2=0.052; respectively). Furthermore, Ucn2 levels inversely correlated with cholesterol and low-density cholesterol (LDL) concentrations in healthy subjects only. Ucn2 was independently related to total cholesterol (but not to LDL) regardless of age, gender and the presence of hypertension (R2=0.18). However, we did not find any relationship between urocortin 2, body mass index or waist-hip ratio as well as parameters of glucose metabolism. Our data indicates that higher levels of urocortin 2 are related to more favorable lipid profiles and lower blood pressure.

Urocortin stimulates the ERK1/2 signaling pathway and the proliferation of HeLa cells via CRF receptor 1.

Corticotropin-releasing factor (CRF) stimulates adrenocorticotropic hormone (ACTH) secretion from the pituitary gland and is an essential regulator of the hypothalamic-pituitary-adrenocortical axis. Isoforms of CRF receptor are known to mediate the effects of urocortin stress ligands on the regulation of stress responses, anxiety, and feeding behavior; however, urocortin stress ligands also influence cell proliferation. In view of the tumor-promoting capacity of prolonged stress, here we investigated (a) the effect of urocortin on cell proliferative signaling via extracellular signal-regulated kinase 1/2, (b) the expression and cellular distribution of the specific CRF receptor isoforms, and (c) the intracellular localization of phosphorylated ERK1/2 in HeLa cells. Stimulation of cell proliferation was observed in the presence of 10 nm urocortin. Our data also suggest that MAP kinase MEK, the transcription factors E2F-1 and p53, and PKB/Akt are involved in this process. These findings may have therapeutic relevance for the targeted treatment of various malignancies.

Structure insights into selective coupling of G protein subtypes by a class B G protein-coupled receptor.

The ability to couple with multiple G protein subtypes, such as Gs, Gi/o, or Gq/11, by a given G protein-coupled receptor (GPCR) is critical for many physiological processes. Over the past few years, the cryo-EM structures for all 15 members of the medically important class B GPCRs, all in complex with Gs protein, have been determined. However, no structure of class B GPCRs with Gq/11 has been solved to date, limiting our understanding of the precise mechanisms of G protein coupling selectivity. Here we report the structures of corticotropin releasing factor receptor 2 (CRF2R) bound to Urocortin 1 (UCN1), coupled with different classes of heterotrimeric G proteins, G11 and Go. We compare these structures with the structure of CRF2R in complex with Gs to uncover the structural differences that determine the selective coupling of G protein subtypes by CRF2R. These results provide important insights into the structural basis for the ability of CRF2R to couple with multiple G protein subtypes.

Fluoxetine treatment supports predictive validity of the three hit model of depression in male PACAP heterozygous mice and underpins the impact of early life adversity on therapeutic efficacy.

According to the three hit concept of depression, interaction of genetic predisposition altered epigenetic programming and environmental stress factors contribute to the disease. Earlier we demonstrated the construct and face validity of our three hit concept-based mouse model. In the present work, we aimed to examine the predictive validity of our model, the third willnerian criterion. Fluoxetine treatment was applied in chronic variable mild stress (CVMS)-exposed (environmental hit) CD1 mice carrying one mutated allele of pituitary adenylate cyclase-activating polypeptide gene (genetic hit) that were previously exposed to maternal deprivation (epigenetic hit) vs. controls. Fluoxetine reduced the anxiety level in CVMS-exposed mice in marble burying test, and decreased the depression level in tail suspension test if mice were not deprived maternally. History of maternal deprivation caused fundamental functional-morphological changes in response to CVMS and fluoxetine treatment in the corticotropin-releasing hormone-producing cells of the bed nucleus of the stria terminalis and central amygdala, in tyrosine-hydroxylase content of ventral tegmental area, in urocortin 1-expressing cells of the centrally projecting Edinger-Westphal nucleus, and serotonergic cells of the dorsal raphe nucleus. The epigenetic background of alterations was approved by altered acetylation of histone H3. Our findings further support the validity of both the three hit concept and that of our animal model. Reversal of behavioral and functional-morphological anomalies by fluoxetine treatment supports the predictive validity of the model. This study highlights that early life stress does not only interact with the genetic and environmental factors, but has strong influence also on therapeutic efficacy.

Urocortin 2 Gene Transfer for Systolic and Diastolic Dysfunction Due to Chronically Increased Left Ventricular Pressure.

We used transverse aortic constriction (TAC) in mice to test the hypothesis that urocortin 2 (Ucn2) gene transfer would increase left ventricular (LV) systolic and diastolic function in the pressure-stressed LV. Three groups were studied: (1) control mice (no TAC); (2) mice that received saline 6 weeks after TAC; and (3) mice that received Ucn2 gene transfer 6 weeks after TAC, using adeno-associated virus 8 encoding murine Ucn2 (AAV8.mUcn2; 2 × 1013 genome copies (gc)/kg, i.v. per mouse). Echocardiography was performed 6 and 12 weeks after TAC. In terminal studies 12 weeks after TAC, rates of LV pressure development and decay and Tau were measured, and LV cardiac myocytes (CMs) were isolated and cytosolic Ca2+ transients and sarcomere shortening rates recorded. Reverse transcription polymerase chain reaction and immunoblotting were used to measure key proteins in LV samples. A CM cell line (HL-1) was used to explore mechanisms. Concentric LV hypertrophy was evident on echocardiography 6 weeks after TAC. Twelve weeks after TAC, LV ejection fraction (EF) was higher in mice that received Ucn2 gene transfer (TAC-saline: 65% ± 3%; TAC-Ucn2: 75% ± 2%; p = 0.01), as was LV peak +dP/dt (1.9-fold increase; p = 0.001) and LV peak -dP/dt (1.7-fold increase; p = 0.017). Tau was more rapid (23% reduction, p = 0.02), indicating improved diastolic function. The peak rates of sarcomere shortening (p = 0.002) and lengthening (p = 0.002) were higher in CMs from TAC-Ucn2 mice, and Tau was reduced (p = 0.001). LV (Ser-16) phosphorylation of phospholamban (PLB) was increased in TAC-Ucn2 mice (p = 0.025), and also was increased in HL-1 cells treated with angiotensin II to induce hypertrophy and incubated with Ucn2 peptide (p = 0.001). Ucn2 gene transfer in TAC-induced heart failure with preserved ejection fraction increased cardiac function in the intact LV and provided corresponding benefits in CMs isolated from study animals, including increased myofilament Ca2+ sensitivity during contraction. The mechanism includes enhanced CM Ca2+ handling associated with increased (Ser-16)-PLB.

Role of Posterodorsal Medial Amygdala Urocortin-3 in Pubertal Timing in Female Mice.

Post-traumatic stress disorder impedes pubertal development and disrupts pulsatile LH secretion in humans and rodents. The posterodorsal sub-nucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, pubertal timing, as well as emotional processing and anxiety. Psychosocial stress exposure alters neuronal activity within the MePD increasing the expression of Urocortin3 (Ucn3) and its receptor corticotropin-releasing factor type-2 receptor (CRFR2) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. We test the hypothesis that psychosocial stress, processed by the MePD, is relayed to the hypothalamic GnRH pulse generator to delay puberty in female mice. We exposed C57Bl6/J female mice to the predator odor, 2,4,5-Trimethylthiazole (TMT), during pubertal transition and examined the effect on pubertal timing, pre-pubertal LH pulses and anxiety-like behaviour. Subsequently, we virally infected Ucn3-cre-tdTomato female mice with stimulatory DREADDs targeting MePD Ucn3 neurons and determined the effect on pubertal timing and pre-pubertal LH pulse frequency. Exposure to TMT during pubertal development delayed puberty, suppressed pre-pubertal LH pulsatility and enhanced anxiety-like behaviour, while activation of MePD Ucn3 neurons reduced LH pulse frequency and delayed puberty. Early psychosocial stress exposure decreases GnRH pulse generator frequency delaying puberty while inducing anxiety-behaviour in female mice, an effect potentially involving Ucn3 neurons in the MePD.

Pharmacological Evaluation of a Pegylated Urocortin-1 Peptide in Experimental Autoimmune Disease Models.

Urocortin-1 (UCN1) is a member of the corticotropin releasing hormone (CRH) family of peptides that acts through CRH-receptor 1 (CRHR1) and CRH-receptor 2 (CRHR2). UCN1 can induce the adrenocorticotropin hormone and downstream glucocorticoids through CRHR1 and promote beneficial metabolic effects through CRHR2. UCN1 has a short half-life and has been shown to improve experimental autoimmune disease. A pegylated UCN1 peptide (PEG-hUCN1) was generated to extend half-life and was tested in multiple experimental autoimmune disease models and in healthy mice to determine effects on corticosterone induction, autoimmune disease, and glucocorticoid induced adverse effects. Cardiovascular effects were also assessed by telemetry. PEG-hUCN1 demonstrated a dose dependent 4-6-fold elevation of serum corticosterone and significantly improved autoimmune disease comparable to prednisolone in several experimental models. In healthy mice, PEG-hUCN1 showed less adverse effects compared with corticosterone treatment. PEG-hUCN1 peptide induced an initial 30% reduction in blood pressure that was followed by a gradual and sustained 30% increase in blood pressure at the highest dose. Additionally, an adeno-associated viral 8 (AAV8) UCN1 was used to assess adverse effects of chronic elevation of UCN1 in wild type and CRHR2 knockout mice. Chronic UCN1 expression by an AAV8 approach in wild type and CRHR2 knockout mice demonstrated an important role of CRHR2 in countering the adverse metabolic effects of elevated corticosterone from UCN1. Our findings demonstrate that PEG-hUCN1 shows profound effects in treating autoimmune disease with an improved safety profile relative to corticosterone and that CRHR2 activity is important in metabolic regulation. SIGNIFICANCE STATEMENT: This study reports the generation and characterization of a pegylated UCN1 peptide and the role of CRHR2 in UCN1-induced metabolic effects. The potency/selectivity, pharmacokinetic properties, pharmacodynamic effects, and efficacy in four autoimmune models and safety profiles are presented. This pegylated UCN1 shows potential for treating autoimmune diseases with reduced adverse effects compared to corticosterone treatment. Continuous exposure to UCN1 through an AAV8 approach demonstrates some glucocorticoid mediated adverse metabolic effects that are exacerbated in the absence of the CRHR2 receptor.

The urocortin peptides: biological relevance and laboratory aspects of UCN3 and its receptor.

The urocortins are polypeptides belonging to the corticotropin-releasing hormone family, known to modulate stress responses in mammals. Stress, whether induced physically or psychologically, is an underlying cause or consequence of numerous clinical syndromes. Identifying biological markers associated with the homeostatic regulation of stress could provide a clinical laboratory approach for the management of stress-related disorders. The neuropeptide, urocortin 3 (UCN3), and the corticotropin-releasing hormone receptor 2 (CRHR2) constitute a regulatory axis known to mediate stress homeostasis. Dysregulation of this peptide/receptor axis is believed to play a role in several clinical conditions including post-traumatic stress, sleep apnea, cardiovascular disease, and other health problems related to stress. Understanding the physiology and measurement of the UCN3/CRHR2 axis is important for establishing a viable clinical laboratory diagnostic. In this article, we focus on evidence supporting the role of UCN3 and its receptor in stress-related clinical syndromes. We also provide insight into the measurements of UCN3 in blood and urine. These potential biomarkers provide new opportunities for clinical research and applications of laboratory medicine diagnostics in stress management.

Peptidergic neurons of the Edinger-Westphal nucleus express TRPA1 ion channel that is downregulated both upon chronic variable mild stress in male mice and in humans who died by suicide.

BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1), a cation channel, is expressed predominantly in primary sensory neurons, but its central distribution and role in mood control are not well understood. We investigated whether TRPA1 is expressed in the urocortin 1 (UCN1)-immunoreactive centrally projecting Edinger-Westphal nucleus (EWcp), and we hypothesized that chronic variable mild stress (CVMS) would reduce its expression in mice. We anticipated that TRPA1 mRNA would be present in the human EWcp, and that it would be downregulated in people who died by suicide. METHODS: We exposed Trpa1 knockout and wild-type mice to CVMS or no-stress control conditions. We then performed behavioural tests for depression and anxiety, and we evaluated physical and endocrinological parameters of stress. We assessed EWcp Trpa1 and Ucn1 mRNA expression, as well as UCN1 peptide content, using RNA-scope in situ hybridization and immunofluorescence. We tested human EWcp samples for TRPA1 using reverse transcription polymerase chain reaction. RESULTS: Trpa1 mRNA was colocalized with EWcp/UCN1 neurons. Non-stressed Trpa1 knockout mice expressed higher levels of Ucn1 mRNA, had less body weight gain and showed greater immobility in the forced swim test than wild-type mice. CVMS downregulated EWcp/Trpa1 expression and increased immobility in the forced swim test only in wild-type mice. We confirmed that TRPA1 mRNA expression was downregulated in the human EWcp in people who died by suicide. LIMITATIONS: Developmental compensations and the global lack of TRPA1 may have influenced our findings. Because experimental data came from male brains only, we have no evidence for whether findings would be similar in female brains. Because a TRPA1-specific antibody is lacking, we have provided mRNA data only. Limited access to high-quality human tissues restricted sample size. CONCLUSION: TRPA1 in EWcp/UCN1 neurons might contribute to the regulation of depression-like behaviour and stress adaptation response in mice. In humans, TRPA1 might contribute to mood control via EWcp/UCN1 neurons.

Urocortin-1 Is Chondroprotective in Response to Acute Cartilage Injury via Modulation of Piezo1.

Post-traumatic OA (PTOA) is often triggered by injurious, high-impact loading events which result in rapid, excessive chondrocyte cell death and a phenotypic shift in residual cells toward a more catabolic state. As such, the identification of a disease-modifying OA drug (DMOAD) that can protect chondrocytes from death following impact injury, and thereby prevent cartilage degradation and progression to PTOA, would offer a novel intervention. We have previously shown that urocortin-1 (Ucn) is an essential endogenous pro-survival factor that protects chondrocytes from OA-associated pro-apoptotic stimuli. Here, using a drop tower PTOA-induction model, we demonstrate the extent of Ucn's chondroprotective role in cartilage explants exposed to excessive impact load. Using pathway-specific agonists and antagonists, we show that Ucn acts to block load-induced intracellular calcium accumulation through blockade of the non-selective cation channel Piezo1 rather than TRPV4. This protective effect is mediated primarily through the Ucn receptor CRF-R1 rather than CRF-R2. Crucially, we demonstrate that the chondroprotective effect of Ucn is maintained whether it is applied pre-impact or post-impact, highlighting the potential of Ucn as a novel DMOAD for the prevention of injurious impact overload-induced PTOA.

Urocortin 2 promotes hypertrophy and enhances skeletal muscle function through cAMP and insulin/IGF-1 signaling pathways.

OBJECTIVE: Although it is well established that urocortin 2 (Ucn2), a peptide member of the corticotrophin releasing factor (CRF) family, and its specific corticotrophin-releasing factor 2 receptor (CRF2R) are highly expressed in skeletal muscle, the role of this peptide in the regulation of skeletal muscle mass and protein metabolism remains elusive. METHODS: To elucidate the mechanisms how Ucn2 directly controls protein metabolism in skeletal muscles of normal mice, we carried out genetic tools, physiological and molecular analyses of muscles in vivo and in vitro. RESULTS: Here, we demonstrated that Ucn2 overexpression activated cAMP signaling and promoted an expressive muscle hypertrophy associated with higher rates of protein synthesis and activation of Akt/mTOR and ERK1/2 signaling pathways. Furthermore, Ucn2 induced a decrease in mRNA levels of atrogin-1 and in autophagic flux inferred by an increase in the protein content of LC3-I, LC3-II and p62. Accordingly, Ucn2 reduced both the transcriptional activity of FoxO in vivo and the overall protein degradation in vitro through an inhibition of lysosomal proteolytic activity. In addition, we demonstrated that Ucn2 induced a fast-to-slow fiber type shift and improved fatigue muscle resistance, an effect that was completely blocked in muscles co-transfected with mitogen-activated protein kinase phosphatase 1 (MKP-1), but not with dominant-negative Akt mutant (Aktmt). CONCLUSIONS: These data suggest that Ucn2 triggers an anabolic and anti-catabolic response in skeletal muscle of normal mice probably through the activation of cAMP cascade and participation of Akt and ERK1/2 signaling. These findings open new perspectives in the development of therapeutic strategies to cope with the loss of muscle mass.

Urocortin I Protects against Myocardial Ischemia/Reperfusion Injury by Sustaining Respiratory Function and Cardiolipin Content via Mitochondrial ATP-Sensitive Potassium Channel Opening.

Objective: Our experiments were aimed at probing whether urocortin I postconditioning was beneficial for maintaining the mitochondrial respiratory function and inhibiting the surging of reactive oxygen species. In addition, our experiments also intended to reveal the relationships between urocortin I postconditioning and mitochondrial ATP-sensitive potassium channel. Methods: Langendorff and MPA perfusion systems were used to establish myocardial ischemia-reperfusion injury model and cardiomyocytes hypoxia-reoxygenation injury model in rats, respectively. Isolated hearts and cardiomyocytes were randomly divided into normal group, ischemia-reperfusion/hypoxia-reoxygenation group, urocortin I postconditioning group, and 5-hydroxysolanoic acid (5-HD)+urocortin I group. At the end of balance (T1) and reperfusion (T2), cardiac functions, mitochondrial state3 respiratory, respiratory control ratio, mitochondrial respiratory enzyme activity, and mitochondrial cardiolipin content were measured. Our experiments also observed the ultrastructure of myocardium. The changes of cardiomyocyte mitochondrial permeability transition pore, mitochondrial membrane potential, reactive oxygen species, expression of apoptosis protein, and cardiomyocytes activity were detected at the end of reoxygenation. Results: The cardiac functions, mitochondrial respiratory function, and enzyme activity of the normal group were better than other three groups at T2, and urocortin I postconditioning group was better than the IR group and 5-HD+urocortin I group. LVEDP, +dp/dtmax, mitochondrial respiratory function, and enzyme activity of IR group were worse than 5-HD+urocortin I group. Cardiolipin content of the normal group was higher than the other three groups at T2, urocortin I postconditioning group was higher than the IR group and 5-HD+urocortin I group, and 5-HD+urocortin I group was still higher than the IR group. The ultrastructure of the normal group maintained the most integrated than the other groups, IR group suffered the most serious damage, and ultrastructure of the urocortin I postconditioning group was better than the IR group and 5-HD+urocortin I group. At the end of reoxygenation, activity of mitochondrial permeability transition pore and generation of reactive oxygen species of normal group were lower than the other groups, HR group and 5-HD+urocortin I group were higher than the urocortin I postconditioning group, and 5-HD+urocortin I group was still higher than the urocortin I postconditioning group. Normal group had the highest level of mitochondrial membrane potential at the end of reoxygenation, and the urocortin I postconditioning group was higher than the HR group and 5-HD+urocortin I group. The normal group had the lowest expression level of Bax and the highest expression level of Bcl-2 at the end of reoxygenation. Urocortin I postconditioning group had lower Bax expression but higher Bcl-2 expression than the HR and 5-HD+urocortin I group. Accordingly, the normal group had the highest activity of cardiomyocytes, and the urocortin I postconditioning group was higher than the HR group and 5-HD+urocortin I group. Conclusions: Urocortin I postconditioning can protect the activity of cardiomyocytes after hypoxia-reoxygenation injury, improve the mitochondrial respiratory function, and enhance the contractility of isolated heart after myocardial ischemia-reperfusion injury. The alleviation of myocardial injury relates to the opening of mitochondrial ATP-sensitive potassium channel.

Opposing roles of the entero-pancreatic hormone urocortin-3 in glucose metabolism in rats.

AIM/HYPOTHESIS: Urocortin-3 (UCN3) is a glucoregulatory peptide produced in the gut and pancreatic islets. The aim of this study was to clarify the acute effects of UCN3 on glucose regulation following an oral glucose challenge and to investigate the mechanisms involved. METHODS: We studied the effect of UCN3 on blood glucose, gastric emptying, glucose absorption and secretion of gut and pancreatic hormones in male rats. To supplement these physiological studies, we mapped the expression of UCN3 and the UCN3-sensitive receptor, type 2 corticotropin-releasing factor receptor (CRHR2), by means of fluorescence in situ hybridisation and by gene expression analysis. RESULTS: In rats, s.c. administration of UCN3 strongly inhibited gastric emptying and glucose absorption after oral administration of glucose. Direct inhibition of gastrointestinal motility may be responsible because UCN3's cognate receptor, CRHR2, was detected in gastric submucosal plexus and in interstitial cells of Cajal. Despite inhibited glucose absorption, post-challenge blood glucose levels matched those of rats given vehicle in the low-dose UCN3 group, because UCN3 concomitantly inhibited insulin secretion. Higher UCN3 doses did not further inhibit gastric emptying, but the insulin inhibition progressed resulting in elevated post-challenge glucose and lipolysis. Incretin hormones and somatostatin (SST) secretion from isolated perfused rat small intestine was unaffected by UCN3 infusion; however, UCN3 infusion stimulated secretion of somatostatin from delta cells in the isolated perfused rat pancreas which, unlike alpha cells and beta cells, expressed Crhr2. Conversely, acute antagonism of CRHR2 signalling increased insulin secretion by reducing SST signalling. Consistent with these observations, acute drug-induced inhibition of CRHR2 signalling improved glucose tolerance in rats to a similar degree as administration of glucagon-like peptide-1. UCN3 also powerfully inhibited glucagon secretion from isolated perfused rat pancreas (perfused with 3.5 mmol/l glucose) in a SST-dependent manner, suggesting that UCN3 may be involved in glucose-induced inhibition of glucagon secretion. CONCLUSIONS/INTERPRETATION: Our combined data indicate that UCN3 is an important glucoregulatory hormone that acts through regulation of gastrointestinal and pancreatic functions.