Urocortins Research Articles

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 Neuropeptide Levels Are Impaired in the PBMCs of Overweight Children.

The corticotropin-releasing hormone (CRH) and urocortins (UCNs) have been implicated in energy homeostasis and the cellular stress response. However, the expression of these neuropeptides in children remains unclear. Therefore, we determined the impact of obesity on their expression in 40 children who were normal weight, overweight, and had obesity. Peripheral blood mononuclear cells (PBMCs) and plasma were used to assess the expression of neuropeptides. THP1 cells were treated with 25 mM glucose and 200 µM palmitate, and gene expression was measured by real-time polymerase chain reaction (RT-PCR). Transcript levels of neuropeptides were decreased in PBMCs from children with increased body mass index as indicated by a significant decrease in UCN1, UCN3, and CRH mRNA in overweight and obese children. UCN3 mRNA expression was strongly correlated with UCN1, UCN2, and CRH. Exposure of THP1 cells to palmitate or a combination of high glucose and palmitate for 24 h increased CRH, UCN2, and UCN3 mRNA expression with concomitant increased levels of inflammatory and endoplasmic reticulum stress markers, suggesting a crosstalk between these neuropeptides and the cellular stress response. The differential impairment of the transcript levels of CRH and UCNs in PBMCs from overweight and obese children highlights their involvement in obesity-related metabolic and cellular stress.

Urocortins exhibit differential effects on PGE2 and PGF2α output via CRHR2 in human myometrium.

Urocortins (UCNs), belonging to corticotropin-releasing hormone (CRH) family, exert their function via CRH receptor type 1 (CRHR1) and 2 (CRHR2). Our previous studies have demonstrated that CRH acts on CRHR1 to potentiate prostaglandins (PGs) output induced by inflammatory stimuli in myometrial cells. In the present study, we sought to investigate the effects of UCNs on prostaglandin (PG) output via CRHR2 in cultured human uterine smooth muscle cells (HUSMCs) from pregnant women at term. We found that UCN and UCN 3 treatment promoted PGE2 and PGF2α secretion in a dose-dependent manner. In contrast, UCN2 dose-dependently inhibited PGE2 and PGF2α secretion. Their effects were reversed by CRHR2 antagonist and CRHR2 siRNA. Mechanically, we showed that UCN and UCN3 suppressed cAMP production and led to Gi activation while UCN2 stimulated cAMP production and activated Gs signaling. Further, UCN and UCN3 but not UCN2 activated NF-κB and MAPK signaling pathways through Gi signaling. UCN and UCN3 stimulation of PGs secretion were dependent on Gi/adenylyl cyclase (AC)/cAMP, NF-κB and MAPK signaling pathways. UCN2 suppression of PGs output was through Gs/AC/cAMP signaling pathways. Our data suggest that UCN, UCN2 and UCN3 can finely regulate PGs secretion via CRHR2, which facilitates the functional status of the uterus during pregnancy.

Sexually dimorphic metabolic responses mediated by CRF2 receptor during nutritional stress in mice

BackgroundChronic stress is a major contributor in the development of metabolic syndrome and associated diseases, such as diabetes. High-fat diet (HFD) and sex are known modifiers of metabolic parameters. Peptide hormones corticotropin-releasing factor (CRF) and urocortins (UCN) mediate stress responses via activation and feedback to the hypothalamic-pituitary-adrenal (HPA) axis. UCN3 is a marker of pancreatic β-cell differentiation, and UCN2 is known to ameliorate glucose levels in mice rendered diabetic with HFD. CRF receptor 2 (CRF2) is the only known cognate receptor for UCN2/3. Here, we ascertained the role of CRF2 in glucose clearance, insulin sensitivity, and other parameters associated with metabolic syndrome in a mouse model of nutritional stress.MethodsWild-type (WT) and Crhr2−/− (null) mice of both sexes were fed either normal chow diet or HFD. After 8 weeks, blood glucose levels in response to glucose and insulin challenge were determined. Change in body and fat mass, plasma insulin, and lipid profile were assessed. Histological evaluation of liver sections was performed.ResultsHere, we show that genotype (Crhr2), sex, and diet were all independent variables in the regulation of blood glucose levels, body and fat mass gain/redistribution, and insulin resistance. Surprisingly, CRF2-deficient mice (Crhr2−/−) male mice showed similarly impaired glucose clearance on HFD and chow. HFD-fed female Crhr2−/− mice redistributed their fat depots that were distinct from wild-type females and male mice on either diet. Blood cholesterol and low-density lipoprotein (LDL) levels were elevated significantly in male Crhr2−/− mice; female Crhr2−/− mice were protected. Male, but not female Crhr2−/− mice developed peripheral insulin resistance. HFD, but not chow-fed wild-type male mice developed hepatic macrovesicular steatosis. In contrast, livers of Crhr2−/− male mice showed microvesicular steatosis on either diet, whereas livers of female mice on this 8-week HFD regimen did not develop steatosis.ConclusionsCRF2 receptor dysregulation is a sexually dimorphic risk factor in development of pre-diabetic and metabolic symptoms.

The CRF Family of Neuropeptides and their Receptors - Mediators of the Central Stress Response.

Background: Dysregulated stress neurocircuits, caused by genetic and/or environmental changes, underlie the development of many neuropsychiatric disorders. Corticotropin-releasing factor (CRF) is the major physiological activator of the hypothalamic-pituitary-adrenal (HPA) axis and conse-quently a primary regulator of the mammalian stress response. Together with its three family members, urocortins (UCNs) 1, 2, and 3, CRF integrates the neuroendocrine, autonomic, metabolic and behavioral responses to stress by activating its cognate receptors CRFR1 and CRFR2.Objective: Here we review the past and current state of the CRF/CRFR field, ranging from pharmacologi-cal studies to genetic mouse models and virus-mediated manipulations.Results: Although it is well established that CRF/CRFR1 signaling mediates aversive responses, includ-ing anxiety and depression-like behaviors, a number of recent studies have challenged this viewpoint by revealing anxiolytic and appetitive properties of specific CRF/CRFR1 circuits. In contrast, the UCN/CRFR2 system is less well understood and may possibly also exert divergent functions on physiol-ogy and behavior depending on the brain region, underlying circuit, and/or experienced stress conditions.Conclusion: A plethora of available genetic tools, including conventional and conditional mouse mutants targeting CRF system components, has greatly advanced our understanding about the endogenous mecha-nisms underlying HPA system regulation and CRF/UCN-related neuronal circuits involved in stress-related behaviors. Yet, the detailed pathways and molecular mechanisms by which the CRF/UCN-system translates negative or positive stimuli into the final, integrated biological response are not completely un-derstood. The utilization of future complementary methodologies, such as cell-type specific Cre-driver lines, viral and optogenetic tools will help to further dissect the function of genetically defined CRF/UCN neurocircuits in the context of adaptive and maladaptive stress responses.

UCN enhances TGF-beta-mediated mitoinhibition of VSMCs via counteracting TGF-beta-induced cPLA2 expression and activation

Urocortins (UCNs) and transforming growth factor-beta (TGF-beta) have been demonstrated to participate in various cardiovascular diseases, many of which involve vascular smooth muscle cells (VSMCs) proliferation. Cytosolic phospholipase A2 (cPLA2)-mediated arachidonic acid (AA) release is an important cause of VSMCs proliferation. The work was to investigate the regulation of VSMCs proliferation by UCN/TGF-beta and whether cPLA2 was a link between their signaling pathways. VSMCs proliferation was measured by colorimetric assay and immunofluorescence microscopy. Using cell flow cytometry, the changes in the cell cycle phases were investigated. Lentiviral Vector Particle was performed to overexpress cPLA2 gene. Both UCN and TGF-beta inhibited VSMCs proliferation and an additive effect was observed when the cells were treated with UCN plus TGF-beta. TGF-beta increased the percentage of cells in G1-phase while UCN increased the cell percentage in G2-phase with a concomitant decrease in S-phase. Furthermore, cPLA2 expression was increased by TGF-beta but decreased by UCN and UCN attenuated TGF-beta-induced cPLA2 expression. In primary VSMCs, TGF-beta induced cPLA2 phosphorylation, and this effect was also attenuated by UCN. Similar to UCN, the cPLA2 inhibitor, pyrrophenone (PYR), also played a role in enhancing TGF-beta-mediated mitoinhibition. Inversely, overexpression of cPLA2 eliminated the effect of UCN on the mitoinhibition. The pretreatment with UCN counteracted TGF-beta-mediated cPLA2 expression and activation, thereby contributing to TGF-beta-mediated mitoinhibition of VSMCs.

Urocortin 2 But Not Urocortin 3 Promotes the Synaptic Formation in Hipppocampal Neurons via Induction of NGF Production by Astrocytes.

CRH family peptides play differential role during various physiological and pathophysiological responses, such as stress. Urocortins (UCNs) have been implicated to play complementary or contrasting actions for the effects of CRH during stress. It has been shown that activation of CRH receptor type 1 (CRHR1) results in decreased synapse formation in hippocampus. We therefore explored the effect of UCN2 and UCN3, the exclusive CRHR2 agonists, on synaptic formation in hippocampus. In hippocampal slices cultures, UCN2 but not UCN3 treatment increased the levels of presynaptic protein synapsinI and postsynaptic protein postsynaptic density 95 (PSD95), which was reversed by CRHR2 antagonist astressin 2B. In isolated hippocampal neurons, however, UCN2 decreased the numbers of synapsinI- and PSD95-labeled terminals/clusters via CRHR2. Treatment of hippocampal neurons with the media of UCN2-treated astrocytes led to an increase in synapsinI- and PSD95-labeled terminals. In neuron-astrocyte cocultures, UCN2 also enhanced the numbers and level of synapsinI- and PSD95-labeled terminals. These effects did not occur if glial cells were transfected with CRHR2 small interfering RNA. UCN2 but not UCN3 treatment induced nerve growth factor (NGF) production in astrocytes via CRHR2. The effects of the media of UCN2-treated glial cells on synapse formation in hippocampal neurons were prevented by administration of NGF receptor antagonists. Our data indicate that UCN2 promotes synapse formation in hippocampus via induction of NGF secretion from astrocytes. CRHR2 in glial cells mediates the stimulatory effects of CRH. Glia-neuron communication is critical for neuronal circuits remodeling and synaptic plasticity in response to neurohormones or neuromodulators.

Reduced expression of CRHR2 and Sp-1 in myocardium of ovariectomized rats is improved by exercise training

Exercise training has been looked on as a non-pharmacologic approach to treating ovariectomy (OVX)-induced dysfunctions. In this study, we investigated whether chronic exercise impacts on expression of urocortins (UCNs) and corticotropin-releasing hormone receptor type 2 (CRHR2) in myocardium of OVX rats. Bilateral OVX or sham-operation was performed under anesthesia. Both groups were then divided into two subgroups, with or without treadmill training for 8 weeks. It was found that OVX as well as exercise did not affect the mRNA levels of UCN, UCN2 and UCN3 in myocardium. OVX caused down-regulation of CRHR2 in myocardium. Exercise training reversed the OVX-induced reduction of CRHR2, but had no influence on CRHR2 level in sham rats. OVX resulted in a decrease in estrogen receptor α (ERα) expression in myocardium, which was restored by exercise. Moreover, exercise training also reversed OVX-induced down-regulation of specific protein-1 (Sp-1) expression in myocardium. CRHR2 expression level correlated with Sp-1 and ERα level in myocardium. These results indicate that exercise training can restore the CRHR2 level in myocardium of OVX rats, which is associated with ERα and Sp-1 expression.

Expression of corticotropin-releasing factor and urocortins in the normal and Schistosoma mansoni-infected mouse ileum.

Corticotropin-releasing factor (CRF) and urocortins (UCNs) are important ligands in the CRF signaling pathways, which are most known for their role in the hypothalamic-pituitary-adrenal stress axis. However, peripheral CRF signaling also has profound effects on gastrointestinal functions. Although the murine animal model is highly relevant for the exploration of this complexly balanced pathway via genetic manipulation, little is known about the expression of CRF and UCNs in the mouse intestine. This study aims to investigate the cellular localization of CRF and UCNs in the ileum and to explore whether and how this cellular expression is altered in conditions of intestinal Schistosoma mansoni-induced inflammation. The results show a distinct expression pattern for the different CRF receptor ligands in the ileum. CRF was located in nerve fibers and stromal cells. All UCNs were expressed in polymorphonuclear leukocytes. Furthermore, UCN2 and UCN3 were found in the musculature. During acute schistosomiasis, UCN1 showed an increased immunoreactivity in blood vessels and UCN3 was de novo expressed mainly in submucous neurons. Typical features of S. mansoni-inflamed ileum, such as nerve fiber sprouting, muscle layer thickening and granuloma formation thus all have an impact on the CRF signaling pathways. In conclusion, we outline for the first time the expression of CRF signaling ligands in the mouse ileum; our results point to important changes of this signaling system in S. mansoni-induced intestinal inflammation, which warrants further functional investigation with specific focus on CRF2, given the exclusive binding of UCN2 and UCN3 to this receptor.

Secretion of urocortin I by human glioblastoma cell lines, possibly via the constitutive pathway

Corticotropin-releasing factor (CRF) and its family of peptides, i.e., urocortins (UCNs), play a critical role in systemic and peripheral stress-response systems and are widely expressed not only in normal tissues but also in various types of cancer cells. Given limited understanding of the mechanism of UCN I secretion, we investigated the UCN I secretory pathway in human neural stem cells (HNSCs) and in two glioblastoma cell lines, e.g., A172 and U-138 MG. Immunoreactivities for CRF receptors were detected in A172 glioblastoma cells, but not in HNSCs or U-138 glioblastoma cells, while UCN I immunoreactivity was detected in A172 and U-138 MG glioblastoma cell lines by both light field and electron microscopy. Interestingly, electron microscopy revealed UCN I immunoreactivtiy in vesicle-like structures in the plasma membrane of the glioblastoma cells. Tracking of a hybrid fluorescent protein containing a UCN I signal peptide expressed in A172 human glioblastoma cells revealed that fluorescence in secretory granules could be decreased by cycloheximide (100μg/ml), indicating that the forward transport of secretory granules containing fluorescent protein was not altered by the inhibition of protein synthesis by cycloheximide. Retrograde transport and the fusion of fluorescent granules in A172 human glioblastoma cells was induced by brefeldin A (10μg/ml), indicating that UCN I secretory granules may be transported via the constitutive pathway. Based on these results, it appears that UCN I is secreted from human glioblastoma cells by exocytosis through constitutive secretory granules, indicating that transcription of UCN I mRNA may be correlated to secretion of UCN I protein.

Urocortin 3 activates AMPK and AKT pathways and enhances glucose disposal in rat skeletal muscle.

Insulin resistance (IR) in skeletal muscle is an important component of both type 2 diabetes and the syndrome of sarcopaenic obesity, for which there are no effective therapies. Urocortins (UCNs) are not only well established as neuropeptides but also have their roles in metabolism in peripheral tissues. We have shown recently that global overexpression of UCN3 resulted in muscular hypertrophy and resistance to the adverse metabolic effects of a high-fat diet. Herein, we aimed to establish whether short-term local UCN3 expression could enhance glucose disposal and insulin signalling in skeletal muscle. UCN3 was found to be expressed in right tibialis cranialis and extensor digitorum longus muscles of rats by in vivo electrotransfer and the effects studied vs the contralateral muscles after 1 week. No increase in muscle mass was detected, but test muscles showed 19% larger muscle fibre diameter (P=0.030), associated with increased IGF1 and IGF1 receptor mRNA and increased SER256 phosphorylation of forkhead transcription factor. Glucose clearance into the test muscles after an intraperitoneal glucose load was increased by 23% (P=0.018) per unit mass, associated with increased GLUT1 (34% increase; P=0.026) and GLUT4 (48% increase; P=0.0009) proteins, and significantly increased phosphorylation of insulin receptor substrate-1, AKT, AKT substrate of 160 kDa, glycogen synthase kinase-3β, AMP-activated protein kinase and its substrate acetyl coA carboxylase. Thus, UCN3 expression enhances glucose disposal and signalling in muscle by an autocrine/paracrine mechanism that is separate from its pro-hypertrophic effects, implying that such a manipulation may have promised for the treatment of IR syndromes including sarcopaenic obesity.

Differential expression of corticotropin releasing factor and urocortins in the Schistosoma mansoni‐infected mouse ileum (214.2)

Corticotropin releasing factor (CRF) and urocortins (UCNs) are important ligands in the CRF signaling pathways, most well‐known from the hypothalamic‐pituitary‐adrenal stress axis. However, peripheral CRF signaling has profound effects on gastrointestinal functions. Despite the potential of the murine animal model in elucidating this complexly balanced pathway through genetic manipulation, little is known regarding the expression of CRF and UCNs in the intestinal wall of the mouse. This study therefore investigates the cellular localization of CRF and UCNs in the ileum and examines how this cellular expression is altered in intestinal schistosomiasis. The results show a diverse expression of CRF and UCNs in the enteric nervous system, granulocytes and the ileal musculature. Of special interest was the de novo expression of UCN3 in a majority of submucous vasodilator/secretomotor neurons, possibly strengthening CRF2‐mediated effects during inflammation. In conclusion, we outline for the first time the ileal expression of CRF signaling ligands in the mouse, and point to important changes of this signaling system in intestinal inflammation.Grant Funding Source: Supported by a grant of the Flemish Organization for Scientific Research (FWO G.0179.08)

Alcohol‐Preferring Rats Show Decreased Corticotropin‐Releasing Hormone‐2 Receptor Expression and Differences in HPA Activation Compared to Alcohol‐Nonpreferring Rats

Corticotropin-releasing hormone (CRH) and urocortins (UCNs) bind to corticotropin-releasing hormone type 2 receptor (CRF2 receptor ), a Gs protein-coupled receptor that plays an important role in modulation of anxiety and stress responses. The Crhr2 gene maps to a quantitative trait locus (QTL) for alcohol preference on chromosome 4 previously identified in inbred alcohol-preferring (iP) and-nonpreferring (iNP) F2 rats. Real-time polymerase chain reaction was utilized to screen for differences in Crhr2 mRNA expression in the central nervous system (CNS) of male iP and iNP rats. DNA sequence analysis was then performed to screen for polymorphism in Crhr2 in order to identify genetic variation, and luciferase reporter assays were then applied to test their functional significance. Next, binding assays were used to determine whether this polymorphism affected CRF2 receptor binding affinity as well as CRF2 receptor density in the CNS. Finally, social interaction and corticosterone levels were measured in the P and NP rats before and after 30-minute restraint stress. Crhr2 mRNA expression studies found lower levels of Crhr2 mRNA in iP rats compared to iNP rats. In addition, DNA sequencing identified polymorphisms in the promoter region, coding region, and 3'-untranslated region between the iP and iNP rats. A 7 bp insertion in the Crhr2 promoter of iP rats altered expression in vitro as measured by reporter assays, and we found that CRF2 receptor density was lower in the amygdala of iP as compared to iNP rats. Male P rats displayed decreased social interaction and significantly higher corticosterone levels directly following 30-minute restraint when compared to male NP rats. This study identified Crhr2 as a candidate gene of interest underlying the chromosome 4 QTL for alcohol consumption that was previously identified in the P and NP model. Crhr2 promoter polymorphism is associated with reduced mRNA expression in certain brain regions, particularly the amygdala, and lowered the density of CRF2 receptor in the amygdala of iP compared to iNP rats. Together, these differences between the animals may contribute to the drinking disparity as well as the anxiety differences of the P and NP rats.

Cardioprotection of 17β-estradiol against hypoxia/reoxygenation in cardiomyocytes is partly through up-regulation of CRH receptor type 2

Estrogens have been suggested to exert cardioprotection through maintaining endogenous cardioprotective mechanisms. In the present study, we investigated whether estrogens protect cardiomyocytes against hypoxia/reoxygenation (H/R) via modulating urocortins (UCNs) and their receptor corticotrophin-releasing hormone receptor type 2 (CRHR2). We found that 17β-estradiol (E2) enhanced UCN cardioprotection against H/R and increased CRHR2 expression in neonatal rat cardiomyocytes. E2 protected cardiomyocytes against H/R, which was impaired by CRHR2 antagonist or knockdown of CRHR2. Estrogen receptor α (ERα) antagonist treatment or ERα knockdown could abolish E2-induced CRHR2 up-regulation. Moreover, knockdown of Sp1 also attenuated E2-induced CRHR2 up-regulation. Ovariectomy resulted in down-regulation of CRHR2 and Sp-1 in myocardium of mice, which was restored by E2 or ERα agonist treatment. These results suggest that estrogens act on ERα to up-regulate CRHR2 expression in cardiomyocytes, thereby enhancing cardioprotection of UCNs against H/R.

Mechanisms of cardiovascular actions of urocortins in the hypothalamic arcuate nucleus of the rat

The presence of urocortins (UCNs) and corticotropin-releasing factor (CRF) receptors has been reported in the hypothalamic arcuate nucleus (ARCN). We have previously reported that UCNs are involved in central cardiovascular regulation. Based on this information, we hypothesized that the ARCN may be one of the sites where UCNs exert their central cardiovascular actions. Experiments were done in artificially ventilated, adult male Wistar rats anesthetized with urethane. Unilateral microinjections (30 nl) of UCN1 (0.12-2 mM) elicited decreases in mean arterial pressure (MAP) and heart rate (HR). Maximum cardiovascular responses were elicited by a 1 mM concentration of UCN1. Microinjections of UCN2 and UCN3 (1 mM each) into the ARCN elicited similar decreases in MAP and HR. UCN1 was used as a prototype for the other experiments described below. HR responses elicited by UCN1 were significantly attenuated by bilateral vagotomy. Prior microinjections of NBI-27914 (CRF-1 receptor antagonist) and astressin (CRF-1 receptor and CRF-2 receptor antagonist) (1 mM each) into the ARCN significantly attenuated the cardiovascular responses elicited by UCN1 microinjections at the same site. Microinjections of UCN1 into the ARCN decreased efferent renal sympathetic nerve activity. It was concluded that microinjections of UCN1, UCN2, and UCN3 into the ARCN elicited decreases in MAP and HR. Decreases in MAP, HR, and renal sympathetic nerve activity elicited by UCN1 microinjections into the ARCN were mediated via CRF receptors. Bradycardic responses to UCN1 were mediated via the activation of vagus nerves, and decreases in MAP may be mediated via decreases in sympathetic nerve activity.

A New Potential Approach to Inotropic Therapy in the Treatment of Heart Failure

Urocortins (UCNs), peptides that belong to the corticotrophin-releasing hormone family, represent a novel group of inotropic agents that have a multifaceted effect on the body with significant effects on the cardiovascular, hemodynamic, neurohormonal, and renal systems. UCNs can potentially improve the overall picture of heart failure by targeting not only the cardiovascular and hemodynamic systems like many current inotropic agents but also other systemic tissues that contribute significantly to the mortality and morbidity of heart failure. The 3 types of UCNs (1, 2, and 3) have been shown in preclinical studies to be effective in improving cardiovascular, neurohormonal, and renal function. UCN 2 has been shown in clinical studies to induce significant cardiovascular benefit with limited systemic effects. UCNs, specifically UCNs 2 and 3, show great potential as additional treatment in the management of systolic heart failure.

Impact of chondroitin sulfate treatment on immunoregulatory neuropeptides levels in OA patients synovitis

Purpose: Osteoarthritis (OA) is the most prevalent joint disease, characterized by synovial inflammation, cartilage destruction and subchondral bone sclerosis. Synovitis has been described to be an important process in OA progression, where several immune mediators, such as immunoregulatory neuropeptides, play an important role. The vasoactive intestinal peptide (VIP) and the corticotropin-releasing factor (CRF)/urocortins (UCNs) family are neuropeptides expressed in central and peripheral tissues, including immune cells, that modulate the expression of inflammatory and extracellular matrix remodelling mediators. VIP has previously shown a plethora of beneficial effects, both in inflammatory disorder animal models and in human OA, being a potential therapeutic agent. Besides, it has shown that high VIP levels are correlated with a decrease of the OA severity progress. In addition, CRF and Urocortin1 (UCN1) are secreted to the OA joint microenvironment exerting an autocrine and paracrine immuno/inflammatory regulation. Chondroitin sulfate (CS) is a Symptomatic Slow Action Drug for Osteoarthritis (SYSADOA) described to improve the symptoms of OA disease. The NIH-funded GAIT study demonstrated that CS is an effective drug in reducing OA patients’ synovitis. The aim of this study was to compare the effect of CS vs. paracetamol on synovitis in OA patients, and to evaluate their impact on VIP, CRF and UCN1 concentrations. Methods: 71 OA patients with synovitis (synovial hypertrophy+effusion≥4mm) were treated with CS (Condrosan®, CS Bio-ActiveTM, Bioibérica S.A., Barcelona, Spain) (800mg/day) or paracetamol (4g/day) for 6 months. Patients were followed-up until month 9 to evaluate the CS carry-over effect. In each visit (0, 6 and 9 months) synovitis was evaluated by sonography and plasma and synovial fluid were collected for the quantification of VIP, CRF and UCN1 by EIA (Enzyme Immunoassay). Once the clinical data have been collected, CS treated patients were subdivided into two groups, as responding and non-responding to treatment. A responding patient was defined by a sinovitis reduction at 6 months greater than 20% from basal value. Time-course analysis study was performed by a Wilcoxon two-related samples test. The comparation between responding and non-responding patients was analyzed by a Mann-Whitney U test. P values less than or equal to 0.05 were considered significant. Results: The analysis of the results obtained showed that CS but not paracetamol reduces effectively the synovitis observed in OA patients. The improvement remained after 3 months treatment cessation. Synovial fluid presents a progressive decrease neuropeptides levels along the CS treatment, reaching significancy VIP reduction at 9 months. This tendency is more pronounced when only the responding patients are analyzed. In contrast no tendency was observed with the paracetamol group, showing a lonely significant decrease of UCN1 at 6 months. The neuropeptides levels in plasma did not show significant differences neither in the chondroitin treated patients nor in the responding group. However, the paracetamol grup present a progressive increment of CRF/UCN levels being significant the UCN1 increase at 9 months. When comparing the basal levels of the neuropeptides between responding and non-responding CS groups, we observed that patients with high level of neuropeptides before CS treatment, demonstrated a positive response to the treatment. Conclusions: These results indicate that CS but not paracetamol effectively reduces synovitis, even after leaving the CS treatment. This improvement lead to a decreased VIP level in synovial fluid, probably because of a fall of the mediators that regulates its expression. Neuropeptides levels could represent an important biomarker to distinguish which patients are responder to CS, since high level of them in synovial fluid at the onset of treatment are correlated with a positive response to CS treatment.

Regulation of Estradiol and Progesterone Production by CRH-R1 and -R2 Is through Divergent Signaling Pathways in Cultured Human Placental Trophoblasts

CRH and its related peptides urocortins (UCN) have been identified in placenta and implicated to play pivotal roles in the regulation of pregnancy and parturition in humans. The objectives of present study were to investigate the effects of endogenous CRH and its related peptides in the regulation of steroid production in placenta. Placental trophoblasts were isolated from term placenta tissues and cultured for 72 h. Estradiol (E(2)) and progesterone (P(4)) contents in culture media were determined by radioimmunoassay. Treatment of cultured trophoblasts with CRH or UCNI antibody showed decreased E(2), whereas increased P(4) production. Treatment of cells with CRH receptor type 1 antagonist antalarmin or CRH receptor type 2 (CRH-R2) antagonist astressin-2b also decreased E(2) but increased P(4) production. Knockdown of CRH receptor type 1 or CRH-R2 cells showed a decrease in E(2) production and an increase in P(4) production. In CRH-R2 knockdown cells, CRH stimulated GTP-bound Gαs protein and phosphorylated phospholipase C-β3. Adenylyl cyclase and protein kinase A inhibitors blocked CRH-induced increased E(2) production but not decreased P(4) production. PLC inhibitor U73122 and protein kinase C inhibitor chelerythrine blocked the effects of CRH on E(2) and P(4) production in CRH-R2 knockdown cells. UCNIII, the specific CRH-R2 agonist, stimulated GTP-bound Gαi protein and phosphorylated phospholipase C-β3 expression. Both U73122 and chelerythrine blocked UCNIII-induced increased E(2) production and decreased P(4) production. We suggest that CRH and its related peptides might be involved in changes in the progesterone to estrogen ratio during human pregnancy.

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release – The impacts on the hypothalamic–pituitary–adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2.Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release.CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective.We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic–pituitary–adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.

Colitis-Associated Adipose Tissue Responses to Corticotropin-Releasing Hormone Family of Neuropeptides

Background and Aims: Numerous studies have indicated that adipose tissue is an active endocrine organ and several adipose-associated molecules have been implicated as potential mediators of intestinal inflammation. Results from our group and others indicate that corticotrophin releasing hormone (CRH or CRF), and its peptide family members urocortins (UCNs) participate in the pathophysiology of intestinal inflammation and inflammatory bowel disease (IBD). Recent evidence also indicates that CRH receptors 1 and 2 are expressed by human adipocytes. Whether these ligands and receptors are expressed in the mesenteric fat and are modulated during colitis has never been studied. Here we compared levels of expression of the CRH peptide family at in the mesenteric fat depots of mice before and after experimental TNBS-induced colitis. We also exposed isolated human mesenteric preadipocytes to CRH and measured changes in cytokine production at the mRNA and protein levels. Methods: C57BL6 mice (n=10 per group) were injected intracolonically with either vehicle (40% ethanol, control) or TNBS (5 mg/20 grams) for 48 hr. Mesenteric adipose tissue was harvested from the mice and mRNA was isolated and converted to cDNA for real time RT PCR. Human mesenteric pre-adipocytes grown in culture were stimulated with 100nM CRH for 8 hours. Supernatants were collected for protein array and ELISA while mRNA was isolated for conversion to cDNA and use in real time RT PCR experiments. Data were analyzed using a two tailedMannWhitney test. Results: CRHmRNAwas significantly increased inmesenteric fat of TNBS-exposed mice compared to controls (by 112.9 fold, p<0.01). UCNs 2 and 3 were also significantly increased in these tissues (by 30.7 and 9.2 fold, p< 0.0016 and p< 0.0052, respectively). Protein arrays on CRH stimulated human mesenteric preadipocytes showed increased secretion of TIMP-2 and Rantes (CCL5 gene product) and decreased MIF, MCP-3 and PAI-1 levels. Changes in released MIF were also confirmed by ELISA in the medium of preadipocytes (p< 0.01). Conclusions: Increased expression of CRH and urocortins in adipose tissue during colitis together with the ability of CRH to induce changes in cytokine release from isolated adipocytes suggest that the CRH family of peptides may mediate inflammatory responses in mesenteric fat and affect the development of intestinal inflammation. These findings may represent a novel signaling pathway in colitis and could have important implications for inflammatory bowel disease treatment targets. Research Support: NIH NIDDK T32 DK07180-36 Gastroenterology Training Grant and P01 DK 33506