210 research outputs found

    Immunomodulation by maternal autoantibodies of the fetal serotoninergic 5-HT4 receptor and its consequences in early BALB/c mouse embryonic development

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    <p>Abstract</p> <p>Background</p> <p>The presence of functional 5-HT<sub>4 </sub>receptors in human and its involvement in neonatal lupus erythematosus (NLE) have prompted us to study the receptor expression and role during embryogenesis. Earlier we managed to demonstrate that female BALB/c mice immunized against the second extracellular loop (SEL) of the 5-HT<sub>4 </sub>receptor gave birth to pups with heart block. To explain this phenomenon we investigated the expression of 5-HT<sub>4 </sub>receptors during mouse embryogenesis. At the same time we looked whether the consequence of 5-HT<sub>4 </sub>receptor immunomodulation observed earlier is in relation to receptor expression.</p> <p>We studied the expression of 5-HT<sub>4 </sub>receptor at the mRNA level and its two isoforms 5-HT<sub>4(a) </sub>and 5-HT<sub>4(d) </sub>at the protein level in embryos from BALB/c mice, at 8<sup>th</sup>, 12<sup>th</sup>, 18<sup>th </sup>gestation days (GD) and 1 day post natal (DPN). Simultaneously the receptor activity was inhibited by rising antibodies, in female mice against SEL of the receptor. The mice were mated and embryos were collected at 8<sup>th</sup>, 12<sup>th</sup>, 18<sup>th </sup>GD and 1 DPN.</p> <p>Results</p> <p>5-HT<sub>4 </sub>receptor mRNA increased in brain from 12<sup>th </sup>GD to 1 DPN. Its expression gradually decreased in heart and disappeared at birth. This was consistent with expression of the receptor isoforms 5-HT<sub>4(a) and (d)</sub>. Abnormalities like decreased number of embryos, growth delay, spina bifida and sinus arrhythmia from 12<sup>th </sup>GD were documented in pups of mice showing anti-5-HT<sub>4 </sub>receptor antibodies.</p> <p>Conclusion</p> <p>serotoninergic 5-HT<sub>4 </sub>receptor plays an important role in mouse foetal development. In BALB/c mice there is a direct relation between the expression of receptor and the deleterious effect of maternal anti-5-HT<sub>4 </sub>receptor autoantibodies in early embryogenesis.</p

    Modeling Epac1 interactions with the allosteric inhibitor AM-001 by co-solvent molecular dynamics

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    The exchange proteins activated by cAMP (EPAC) are implicated in a large variety of physiological processes and they are considered as promising targets for a wide range of therapeutic applications. Several recent reports provided evidence for the therapeutic effectiveness of the inhibiting EPAC1 activity cardiac diseases. In that context, we recently characterized a selective EPAC1 antagonist named AM-001. This compound was featured by a non-competitive mechanism of action but the localization of its allosteric site to EPAC1 structure has yet to be investigated. Therefore, we performed cosolvent molecular dynamics with the aim to identify a suitable allosteric binding site. Then, the docking and molecular dynamics were used to determine the binding of the AM-001 to the regions highlighted by cosolvent molecular dynamics for EPAC1. These analyses led us to the identification of a suitable allosteric AM-001 binding pocket at EPAC1. As a model validation, we also evaluated the binding poses of the available AM-001 analogues, with a different biological potency. Finally, the complex EPAC1 with AM-001 bound at the putative allosteric site was further refined by molecular dynamics. The principal component analysis led us to identify the protein motion that resulted in an inactive like conformation upon the allosteric inhibitor binding

    Modeling Epac1 interactions with the allosteric inhibitor AM-001 by co-solvent molecular dynamics

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    The exchange proteins activated by cAMP (EPAC) are implicated in a large variety of physiological processes and they are considered as promising targets for a wide range of therapeutic applications. Several recent reports provided evidence for the therapeutic effectiveness of the inhibiting EPAC1 activity cardiac diseases. In that context, we recently characterized a selective EPAC1 antagonist named AM-001. This compound was featured by a non-competitive mechanism of action but the localization of its allosteric site to EPAC1 structure has yet to be investigated. Therefore, we performed cosolvent molecular dynamics with the aim to identify a suitable allosteric binding site. Then, the docking and molecular dynamics were used to determine the binding of the AM-001 to the regions highlighted by cosolvent molecular dynamics for EPAC1. These analyses led us to the identification of a suitable allosteric AM-001 binding pocket at EPAC1. As a model validation, we also evaluated the binding poses of the available AM-001 analogues, with a different biological potency. Finally, the complex EPAC1 with AM-001 bound at the putative allosteric site was further refined by molecular dynamics. The principal component analysis led us to identify the protein motion that resulted in an inactive like conformation upon the allosteric inhibitor binding

    Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes:A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors

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    Background: Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism including the classical protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). Aim: Investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Methods: Rat hepatocytes were exposed to 400 µmol/L diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by qPCR and Western blot, respectively. The cAMP-elevating agent forskolin, the pan-phosphodiesterase inhibitor IBMX and EPAC inhibitors CE3F4 and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Results: Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition, but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP while both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. Conclusion: cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. Significance Statement Our study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, we propose the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in DILI. Both EPAC1 and EPAC2, but not PKA, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of drug-induced liver injury (DILI) and liver injury involving mitochondrial dysfunction

    Effects of MCF2L2, ADIPOQ and SOX2 genetic polymorphisms on the development of nephropathy in type 1 Diabetes Mellitus

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    <p>Abstract</p> <p>Background</p> <p><it>MCF2L2, ADIPOQ </it>and <it>SOX2 </it>genes are located in chromosome 3q26-27, which is linked to diabetic nephropathy (DN). <it>ADIPOQ </it>and <it>SOX2 </it>genetic polymorphisms are found to be associated with DN. In the present study, we first investigated the association between <it>MCF2L2 </it>and DN, and then evaluated effects of these three genes on the development of DN.</p> <p>Methods</p> <p>A total of 1177 type 1 diabetes patients with and without DN from the GoKinD study were genotyped with TaqMan allelic discrimination. All subjects were of European descent.</p> <p>Results</p> <p>Leu359Ile T/G variant in the <it>MCF2L2 </it>gene was found to be associated with DN in female subjects (P = 0.017, OR = 0.701, 95%CI 0.524-0.938) but not in males. The GG genotype carriers among female patients with DN had tendency decreased creatinine and cystatin levels compared to the carriers with either TT or TG genotypes. This polymorphism <it>MCF2L2-</it>rs7639705 together with SNPs of <it>ADIPOQ</it>-rs266729 and <it>SOX2</it>-rs11915160 had combined effects on decreased risk of DN in females (P = 0.001).</p> <p>Conclusion</p> <p>The present study provides evidence that <it>MCF2L2</it>, <it>ADIPOQ </it>and <it>SOX2 </it>genetic polymorphisms have effects on the resistance of DN in female T1D patients, and suggests that the linkage with DN in chromosome 3q may be explained by the cumulated genetic effects.</p

    Catecholamines facilitate VEGF-dependent angiogenesis via β2-adrenoceptor-induced Epac1 and PKA activation

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    Chronic stress has been associated with the progression of cancer and antagonists for β-adrenoceptors (βAR) are regarded as therapeutic option. As they are also used to treat hemangiomas as well as retinopathy of prematurity, a role of endothelial β2AR in angiogenesis can be envisioned. We therefore investigated the role of β2AR-induced cAMP formation by analyzing the role of the cAMP effector molecules exchange factor directly activated by cAMP 1 (Epac1) and protein kinase A (PKA) in endothelial cells (EC). Epac1-deficient mice showed a reduced amount of pre-retinal neovascularizations in the model of oxygen-induced retinopathy, which is predominantly driven by vascular endothelial growth factor (VEGF). siRNA-mediated knockdown of Epac1 in human umbilical vein EC (HUVEC) decreased angiogenic sprouting by lowering the expression of the endothelial VEGF-receptor-2 (VEGFR-2). Conversely, Epac1 activation by β2AR stimulation or the Epac-selective activator cAMP analog 8-p-CPT-2'-O-Me-cAMP (8-pCPT) increased VEGFR-2 levels and VEGF-dependent sprouting. Similar to Epac1 knockdown, depletion of the monomeric GTPase Rac1 decreased VEGFR-2 expression. As Epac1 stimulation induces Rac1 activation, Epac1 might regulate VEGFR-2 expression through Rac1. In addition, we found that PKA was also involved in the regulation of angiogenesis in EC since the adenylyl cyclase (AC) activator forskolin (Fsk), but not 8-pCPT, increased sprouting in Epac1-depleted HUVEC and this increase was sensitive to a selective synthetic peptide PKA inhibitor. In accordance, β2AR- and AC-activation, but not Epac1 stimulation increased VEGF secretion in HUVEC.Our data indicate that high levels of catecholamines, which occur during chronic stress, prime the endothelium for angiogenesis through a β2AR-mediated increase in endothelial VEGFR-2 expression and VEGF secretion

    Pharmacological Inhibition of Epac1 Averts Ferroptosis Cell Death by Preserving Mitochondrial Integrity

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    Exchange proteins directly activated by cAMP (Epac) proteins are implicated in a wide range of cellular functions including oxidative stress and cell survival. Mitochondrial-dependent oxidative stress has been associated with progressive neuronal death underlying the pathology of many neurodegenerative diseases. The role of Epac modulation in neuronal cells in relation to cell survival and death, as well as its potential effect on mitochondrial function, is not well established. In immortalized hippocampal (HT-22) neuronal cells, we examined mitochondria function in the presence of various Epac pharmacological modulators in response to oxidative stress due to ferroptosis. Our study revealed that selective pharmacological modulation of Epac1 or Epac2 isoforms, exerted differential effects in erastin-induced ferroptosis conditions in HT-22 cells. Epac1 inhibition prevented cell death and loss of mitochondrial integrity induced by ferroptosis, while Epac2 inhibition had limited effects. Our data suggest Epac1 as a plausible therapeutic target for preventing ferroptosis cell death associated with neurodegenerative diseases

    Epac1 links prostaglandin E2 to β-catenin-dependent transcription during epithelial-to-mesenchymal transition

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    In epithelial cells, β-catenin is localized at cell-cell junctions where it stabilizes adherens junctions. When these junctions are disrupted, β-catenin can translocate to the nucleus where it functions as a transcriptional cofactor. Recent research has indicated that PGE2 enhances the nuclear function of β-catenin through cyclic AMP. Here, we aim to study the role of the cyclic AMP effector Epac in β-catenin activation by PGE2 in non-small cell lung carcinoma cells.We show that PGE2 induces a down-regulation of E-cadherin, promotes cell migration and enhances β-catenin translocation to the nucleus. This results in β-catenin-dependent gene transcription. We also observed increased expression of Epac1. Inhibition of Epac1 activity using the CE3F4 compound or Epac1 siRNA abolished the effects of PGE2 on β-catenin. Further, we observed that Epac1 and β-catenin associate together. Expression of an Epac1 mutant with a deletion in the nuclear pore localization sequence prevents this association. Furthermore, the scaffold protein Ezrin was shown to be required to link Epac1 to β-catenin.This study indicates a novel role for Epac1 in PGE2-induced EMT and subsequent activation of β-catenin

    Corticotropin-releasing Factor (CRF) and Urocortin Promote the Survival of Cultured Cerebellar GABAergic Neurons Through the Type 1 CRF Receptor

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    Corticotropin releasing factor (CRF) is known to be involved in the stress response and in some degenerative brain disorders. In addition, CRF has a role as a neuromodulator in adult cerebellar circuits. Data from developmental studies suggest a putative role for CRF as a trophic factor during cerebellar development. In this study, we investigated the trophic role for CRF family of peptides by culturing cerebellar neurons in the presence of CRF, urocortin or urocortin II. Primary cell cultures of cerebella from embryonic day 18 mice were established, and cells were treated for either 1, 5 or 9 days with Basal Medium Eagles complete medium alone or complete medium with 1 µM CRF, urocortin, or urocortin II. The number of GABA-positive neurons in each treatment condition was counted at each culture age for monitoring the changes in neuronal survival. Treatment with 1 µM CRF or 1 µM urocortin increased the survival of GABAergic neurons at 6 days in vitro and 10 days in vitro, and this survival promoting effect was abolished by treatment with astressin in the presence of those peptides. Based on these data, we suggest that CRF or urocortin has a trophic role promoting the survival of cerebellar GABAergic neurons in cultures

    Interaction between CRHR1 and BDNF Genes Increases the Risk of Recurrent Major Depressive Disorder in Chinese Population

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    BACKGROUND: An important etiological hypothesis about depression is stress has neurotoxic effects that damage the hippocampal cells. Corticotropin-releasing hormone (CRH) regulates brain-derived neurotrophic factor (BDNF) expression through influencing cAMP and Ca2+ signaling pathways during the course. The aim of this study is to examine the single and combined effects of CRH receptor 1 (CRHR1) and BDNF genes in recurrent major depressive disorder (MDD). METHODOLOGY/PRINCIPAL FINDING: The sample consists of 181 patients with recurrent MDD and 186 healthy controls. Whether genetic variations interaction between CRHR1 and BDNF genes might be associated with increased susceptibility to recurrent MDD was studied by using a gene-based association analysis of single-nucleotide polymorphisms (SNPs). CRHR1 gene (rs1876828, rs242939 and rs242941) and BDNF gene (rs6265) were identified in the samples of patients diagnosed with recurrent MDD and matched controls. Allelic association between CRHR1 rs242939 and recurrent MDD was found in our sample (allelic: p = 0.018, genotypic: p = 0.022) with an Odds Ratio 0.454 (95% CI 0.266-0.775). A global test of these four haplotypes showed a significant difference between recurrent MDD group and control group (chi-2 = 13.117, df = 3, P = 0.016. Furthermore, BDNF and CRHR1 interactions were found in the significant 2-locus, gene-gene interaction models (p = 0.05) using a generalized multifactor dimensionality reduction (GMDR) method. CONCLUSION: Our results suggest that an interaction between CRHR1 and BDNF genes constitutes susceptibility to recurrent MDD
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