RESUMO
Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.
Assuntos
Sinalização do Cálcio , Proteínas de Neoplasias , Pancreatite Crônica , Molécula 1 de Interação Estromal , Cálcio/metabolismo , Sinalização do Cálcio/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Pancreatite Crônica/genética , Pancreatite Crônica/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
Arrhythmogenic cardiomyopathy (ACM) is a genetic disease associated with sudden cardiac death and cardiac fibro-fatty replacement. Over the last years, several works have demonstrated that different epigenetic enzymes can affect not only gene expression changes in cardiac diseases but also cellular metabolism. Specifically, the histone acetyltransferase GCN5 is known to facilitate adipogenesis and modulate cardiac metabolism in heart failure. Our group previously demonstrated that human primary cardiac stromal cells (CStCs) contribute to adipogenesis in the ACM pathology. Thus, this study aims to evaluate the role of GCN5 in ACM intracellular lipid accumulation. To do so, CStCs were obtained from right ventricle biopsies of ACM patients and from samples of healthy cadaveric donors (CTR). GCN5 expression was increased both in ex vivo and in vitro ACM samples compared to CTR. When GCN5 expression was silenced or pharmacologically inhibited by the administration of MB-3, we observed a reduction in lipid accumulation and a mitigation of reactive oxygen species (ROS) production in ACM CStCs. In agreement, transcriptome analysis revealed that the presence of MB-3 modified the expression of pathways related to cellular redox balance. Altogether, our findings suggest that GCN5 inhibition reduces fat accumulation in ACM CStCs, partially by modulating intracellular redox balance pathways.
Assuntos
Displasia Arritmogênica Ventricular Direita , Adipogenia/fisiologia , Displasia Arritmogênica Ventricular Direita/genética , Displasia Arritmogênica Ventricular Direita/metabolismo , Displasia Arritmogênica Ventricular Direita/patologia , Morte Súbita Cardíaca/patologia , Humanos , Lipídeos , Células Estromais/metabolismoRESUMO
In cystic fibrosis (CF), impaired airway surface hydration (ASL) and mucociliary clearance that promote chronic bacterial colonization, persistent inflammation, and progressive structural damage to the airway wall architecture are typically explained by ion transport abnormalities related to the mutation of the gene coding for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. However, the progressive and unrelenting inflammation of the CF airway begins early in life, becomes persistent, and is excessive relative to the bacterial burden. Intrinsic abnormalities of the inflammatory response in cystic fibrosis have been suggested but the mechanisms involved remain poorly understood. This review aims to give an overview of the recent advances in the understanding of the defective resolution of inflammation in CF including the abnormal production of specialized pro-resolving lipid mediators (lipoxin and resolvin) and their impact on the pathogenesis of the CF airway disease.
Assuntos
Fibrose Cística/fisiopatologia , Ácidos Docosa-Hexaenoicos/metabolismo , Inflamação/fisiopatologia , Lipoxinas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , MutaçãoRESUMO
Cystic Fibrosis (CF) disease is caused by mutations in the CFTR gene (CF transmembrane conductance regulator). F508 deletion is the most represented mutation, and F508del-CFTR is absent of plasma membrane and accumulates into the endoplasmic reticulum (ER) compartment. Using specific Ca2+ genetics cameleon probes, we showed in the human bronchial CF epithelial cell line CFBE that ER Ca2+ concentration was strongly increased compared to non-CF (16HBE) cells, and normalized by the F508del-CFTR corrector agent, VX-809. We also showed that ER F508del-CFTR retention increases SERCA (Sarcoplasmic/Reticulum Ca2+ ATPase) pump activity whereas PMCA (Plasma Membrane Ca2+ ATPase) activities were reduced in these CF cells compared to corrected CF cells (VX-809) and non-CF cells. We are showing for the first time CFTR/SERCA and CFTR/PMCA interactions that are modulated in CF cells and could explain part of Ca2+ homeostasis deregulation due to mislocalization of F508del-CFTR. Using ER or mitochondria genetics Ca2+ probes, we are showing that ER Ca2+ content, mitochondrial Ca2+ uptake, SERCA and PMCA pump, activities are strongly affected by the localization of F508del-CFTR protein.
Assuntos
Cálcio/metabolismo , Fibrose Cística/patologia , Células Epiteliais/enzimologia , Homeostase , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trifosfato de Adenosina/farmacologia , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Brônquios/patologia , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Homeostase/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of centromere transcripts (cenRNAs). The latter are ensured by the downregulation of RNA polymerase II (RNAPII) activity, and cenRNA turnover by the nuclear exosome. Using S. cerevisiae, we now add protein kinase Rio1 to this scheme. Yeast cenRNAs are produced either as short (median lengths of 231 nt) or long (4458 nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII accessibility and promotes cenRNA degradation by the 5'-3'exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs. These exist as short transcripts (225 nt) at levels that are minimally two orders of magnitude higher than the cenRNAs. In yeast depleted of Rio1, cen- and pericenRNAs accumulate, CEN nucleosomes and kinetochores misform, causing chromosome instability. The latter phenotypes are also observed with human cells lacking orthologue RioK1, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved.
Assuntos
Cinetocoros , Proteínas de Saccharomyces cerevisiae , Humanos , Cinetocoros/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/genética , Centrômero/metabolismo , Nucleossomos/metabolismo , Exorribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Specialized pro-resolving lipid mediators (SPMs) as lipoxins (LX), resolvins (Rv), protectins (PD) and maresins (MaR) promote the resolution of inflammation. We and others previously reported reduced levels of LXA4 in bronchoalveolar lavages from cystic fibrosis (CF) patients. Here, we investigated the role of CF airway epithelium in SPMs biosynthesis, and we evaluated its sex specificity. Human nasal epithelial cells (hNEC) were obtained from women and men with or without CF. Lipids were quantified by mass spectrometry in the culture medium of hNEC grown at air-liquid interface and the expression level and localization of the main enzymes of SPMs biosynthesis were assessed. The 5-HETE, LXA4, LXB4, RvD2, RvD5, PD1 and RvE3 levels were significantly lower in samples derived from CF patients compared with non-CF subjects. Within CF samples, the 12-HETE, 15-HETE, RvD3, RvD4, 17-HODHE and PD1 were significantly lower in samples derived from females. While the mean expression levels of 15-LO, 5-LO and 12-LO do not significantly differ either between CF and non-CF or between female and male samples, the SPMs content correlates with the level of expression of several enzymes involved in SPMs metabolism. In addition, the 5-LO localization significantly differed from cytoplasmic in non-CF to nucleic (or nuclear envelope) in CF hNEC. Our studies provided evidence for lower abilities of airway epithelial cells derived from CF patients and more markedly, females to produce SPMs. These data are consistent with a contribution of CF airway epithelium in the abnormal resolution of inflammation and with worse pulmonary outcomes in women.
Assuntos
Fibrose Cística , Lipoxinas , Epitélio/metabolismo , Feminino , Humanos , Inflamação , Lipoxinas/metabolismo , Pulmão/metabolismo , MasculinoRESUMO
Ciguatera fish poisoning is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The most distressing symptoms are cutaneous sensory disturbances, including cold dysesthesia and itch. CTXs are neurotoxins known to activate voltage-gated sodium channels, but no specific treatment exists. Peptidergic neurons have been critically involved in ciguatera fish poisoning sensory disturbances. Protease-activated receptor-2 (PAR2) is an itch- and pain-related G proteinâcoupled receptor whose activation leads to a calcium-dependent neuropeptide release. In this study, we studied the role of voltage-gated sodium channels, PAR2, and the PAR2 agonist cathepsin S in the cytosolic calcium increase and subsequent release of the neuropeptide substance P elicited by Pacific CTX-2 (P-CTX-2) in rat sensory neurons and human epidermal keratinocytes. In sensory neurons, the P-CTX-2âevoked calcium response was driven by voltage-gated sodium channels and PAR2-dependent mechanisms. In keratinocytes, P-CTX-2 also induced voltage-gated sodium channels and PAR2-dependent marked calcium response. In the cocultured cells, P-CTX-2 significantly increased cathepsin S activity, and cathepsin S and PAR2 antagonists almost abolished P-CTX-2âelicited substance P release. Keratinocytes synergistically favored the induced substance P release. Our results demonstrate that the sensory effects of CTXs involve the cathepsin S-PAR2 pathway and are potentiated by their direct action on nonexcitable keratinocytes through the same pathway.
Assuntos
Ciguatera/patologia , Ciguatoxinas/toxicidade , Epiderme/patologia , Queratinócitos/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Cálcio/metabolismo , Catepsinas/metabolismo , Ciguatera/complicações , Técnicas de Cocultura , Citosol/metabolismo , Modelos Animais de Doenças , Epiderme/inervação , Humanos , Microscopia Intravital , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Parestesia/etiologia , Parestesia/patologia , Cultura Primária de Células , Prurido/etiologia , Prurido/patologia , Ratos , Receptor PAR-2/agonistas , Receptor PAR-2/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Análise de Célula Única , Substância P/metabolismoRESUMO
Calumenin is a secretory pathway protein regulating different endoplasmic reticulum (ER) proteins such as the sarco-endoplasmic reticulum calcium ATPase (SERCA) pumps. Combined with its diverse cellular distribution, its calcium-binding ability, and its interaction with proteins involved in calcium signaling, it is easy to speculate on future description of important roles of calumenin in calcium homeostasis in many cell types, as it was initially observed in muscle cells. In this chapter, we describe basic techniques to modulate calumenin expression and detect its impact on ER calcium content using classic transfection and Western blot techniques, as well as ER calcium measurement using microplate reader.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Sinalização do Cálcio , Linhagem Celular , Inativação Gênica , Humanos , Ligação Proteica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Regulação para CimaAssuntos
Displasia Arritmogênica Ventricular Direita , Mitocôndrias Cardíacas , Humanos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Animais , Displasia Arritmogênica Ventricular Direita/fisiopatologia , Displasia Arritmogênica Ventricular Direita/metabolismo , Displasia Arritmogênica Ventricular Direita/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologiaRESUMO
PAR2 activation in basal keratinocytes stimulates inflammation via the Ca2+-dependent production of mediators such as IL-1ß, TNF-α, and TSLP. In this study, we investigated PAR2 calcium signaling and the consequent production of inflammatory mediators in differentiated human primary keratinocytes (DhPKs). Stimulation with the PAR2-activating peptide SLIGKV promoted Ca2+ store depletion in both undifferentiated human primary keratinocytes and DhPKs. SLIGKV-evoked Ca2+ store depletion did not trigger the store-operated Ca2+ entry (i.e., SOCE) through ORAI1 in DhPKs compared with undifferentiated human primary keratinocytes. The inhibition of phospholipase C and the concomitant inhibition of TRPV1 and inositol triphosphate receptor in DhPKs abrogated the SLIGKV-evoked Ca2+ store depletion; NF-κB activity; and the production of inflammatory mediators such as IL-1ß, TNF-α, and TSLP. Taken together, these results indicate a key role for both InsP3R and TRPV1 in Ca2+ internal stores in the PAR2-evoked Ca2+ release and consequent skin inflammation in DhPKs. These findings may provide clues to understanding the pathological role of DhPKs in skin disorders in which PAR2 is known to be involved, such as atopic dermatitis, Netherton syndrome, and psoriasis.
Assuntos
Mediadores da Inflamação/imunologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Queratinócitos/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Canais de Cátion TRPV/metabolismo , Sinalização do Cálcio/imunologia , Diferenciação Celular , Dermatite/imunologia , Humanos , Mediadores da Inflamação/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/imunologia , Queratinócitos/efeitos dos fármacos , Proteína ORAI1/genética , Proteína ORAI1/imunologia , Proteína ORAI1/metabolismo , Oligopeptídeos/farmacologia , Cultura Primária de Células , RNA Interferente Pequeno/metabolismo , Receptor PAR-2 , Receptores Acoplados a Proteínas G/imunologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/imunologiaRESUMO
Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca2+) homeostasis. In this study, we discovered that the Ca2+ binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca2+ calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca2+ concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR.