El silenciamiento de GPAT2 en células MDA-MB-231 afecta el remodelado del ácido araquidónico

La síntesis de novo de glicerolípidos en células de mamífero comienza con la acilación del glicerol-3-fosfato. Este paso esta catalizado por la glicerol-3-fosfato aciltransferasa (GPAT); hasta el momento se han clonado cuatro genes que codifican GPATs. La GPAT2, se clonó por homología de secuencia con GPAT1, sin embrago hemos demostrado que presenta características que la distinguen notablemente de las otras GPATs: se comporta como un antígeno cáncer/ testículo (expresión selectiva en testículo y en tumores de distintas localizaciones), presenta una expresión transitoria en un estadío particular de la meiosis y promueve el fenotipo tumoral incrementando la proliferación y supervivencia celular. Si bien los mecanismos mediante los cuales GPAT2 ejerce su rol funcional, tanto en condiciones fisiológicas como patológicas, no se han dilucidado, nuestros resultados previos indican que juega un rol relevante en el metabolismo del ácido araquidónico.Facultad de Ciencias Médica

El silenciamiento de GPAT2 en células MDA-MB-231 afecta el remodelado del ácido araquidónico

La síntesis de novo de glicerolípidos en células de mamífero comienza con la acilación del glicerol-3-fosfato. Este paso esta catalizado por la glicerol-3-fosfato aciltransferasa (GPAT); hasta el momento se han clonado cuatro genes que codifican GPATs. La GPAT2, se clonó por homología de secuencia con GPAT1, sin embrago hemos demostrado que presenta características que la distinguen notablemente de las otras GPATs: se comporta como un antígeno cáncer/ testículo (expresión selectiva en testículo y en tumores de distintas localizaciones), presenta una expresión transitoria en un estadío particular de la meiosis y promueve el fenotipo tumoral incrementando la proliferación y supervivencia celular. Si bien los mecanismos mediante los cuales GPAT2 ejerce su rol funcional, tanto en condiciones fisiológicas como patológicas, no se han dilucidado, nuestros resultados previos indican que juega un rol relevante en el metabolismo del ácido araquidónico.Facultad de Ciencias Médica

El silenciamiento de GPAT2 en células MDA-MB-231 afecta el remodelado del ácido araquidónico

La síntesis de novo de glicerolípidos en células de mamífero comienza con la acilación del glicerol-3-fosfato. Este paso esta catalizado por la glicerol-3-fosfato aciltransferasa (GPAT); hasta el momento se han clonado cuatro genes que codifican GPATs. La GPAT2, se clonó por homología de secuencia con GPAT1, sin embrago hemos demostrado que presenta características que la distinguen notablemente de las otras GPATs: se comporta como un antígeno cáncer/ testículo (expresión selectiva en testículo y en tumores de distintas localizaciones), presenta una expresión transitoria en un estadío particular de la meiosis y promueve el fenotipo tumoral incrementando la proliferación y supervivencia celular. Si bien los mecanismos mediante los cuales GPAT2 ejerce su rol funcional, tanto en condiciones fisiológicas como patológicas, no se han dilucidado, nuestros resultados previos indican que juega un rol relevante en el metabolismo del ácido araquidónico.Facultad de Ciencias Médica

Asmase Regulates autophagy and lysosomal membrane permeabilization and its inhibition prevents early stage nonalcoholic steatohepatitis

Background & Aims: Acid sphingomyelinase (ASMase) is activated in nonalcoholic steatohepatitis (NASH). However, ASMase's contribution to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined ASMase's role in high fat diet (HFD)-induced NASH. Methods: Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase-/- mice fed HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed HFD. Results: ASMase deficiency determined resistance to HFD or methionine and choline deficient diet-mediated hepatic steatosis. ASMase-/- mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress and steatosis, indicating selectivity in the resistance of ASMase-/- mice to ER stress. Autophagic flux determined in the presence of rapamycin and/or chloroquine was lower in primary mouse hepatocytes (PMH) from ASMase-/- mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldinA caused ER stress in PMH from ASMase+/+ mice but not ASMase-/- mice. ASMase-/- PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by the oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD- induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH. Conclusions: These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH

Agonist-induced calcium entry correlates with STIM1 translocation

The mechanisms of agonist-induced calcium entry (ACE) following depletion of intracellular calcium stores have not been fully established. We report here that calcium-independent phospholipase A (iPLA2) is required for robust Ca2+ entry in HaCaT keratinocytes following ATP or UTP stimulation. Lysophosphatidic acid (LPA), an unrelated agonist, evoked Ca2+ release without inducing robust Ca2+ entry. Both LPA and UTP induced the redistribution of STIM1 into puncta which localized to regions near or at the plasma membrane, as well as within the cytoplasm. Plasma membrane-associated STIM1 remained high for up to 10 min after UTP stimulation, whereas it had returned almost to baseline by that time point in LPA-stimulated cells. This correlated with faster reloading of the endoplasmic reticulum Ca2+ stores in LPA treated cells. Thus by differentially regulating store-refilling after agonist-mediated depletion, LPA and UTP may exert distinct effects on the duration of STIM1 localization at the plasma membrane, and thus, on the magnitude and duration of ACE

Novel role of cPLA2α in membrane and actin dynamics

Actin-directed processes such as membrane ruffling and cell migration are regulated by specific signal transduction pathways that become activated by growth factor receptors. The same signaling pathways that lead to modifications in actin dynamics also activate cPLA2α. Moreover, arachidonic acid, the product of cPLA2α activity, is involved in regulation of actin dynamics. Therefore, it was investigated whether cPLA2α plays a role in actin dynamics, more specifically during growth factor-induced membrane ruffling and cell migration. Upon stimulation of ruffling and cell migration by growth factors, endogenous cPLA2α and its active phosphorylated form were shown to relocate at protrusions of the cell membrane involved in actin and membrane dynamics. Inhibition of cPLA2α activity with specific inhibitors blocked growth factor-induced membrane and actin dynamics, suggesting an important role for cPLA2α in these processes

Platelet activating factor stimulates arachidonic acid release in differentiated keratinocytes via arachidonyl non-selective phospholipase A2

Platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is known to be present in excess in psoriatic skin, but its exact role is uncertain. In the present study we demonstrate for the first time the role of group VI PLA2 in PAF-induced arachidonic acid release in highly differentiated human keratinocytes. The group IVα PLA2 also participates in the release, while secretory PLA2s play a minor role. Two anti-inflammatory synthetic fatty acids, tetradecylthioacetic acid and tetradecylselenoacetic acid, are shown to interfere with signalling events upstream of group IVα PLA2 activation. In summary, our major novel finding is the involvement of the arachidonyl non-selective group VI PLA2 in PAF-induced inflammatory responses

Depletion of Murine Intestinal Microbiota: Effects on Gut Mucosa and Epithelial Gene Expression

Background Inappropriate cross talk between mammals and their gut microbiota may trigger intestinal inflammation and drive extra-intestinal immune-mediated diseases. Epithelial cells constitute the interface between gut microbiota and host tissue, and may regulate host responses to commensal enteric bacteria. Gnotobiotic animals represent a powerful approach to study bacterial-host interaction but are not readily accessible to the wide scientific community. We aimed at refining a protocol that in a robust manner would deplete the cultivable intestinal microbiota of conventionally raised mice and that would prove to have significant biologic validity. Methodology/Principal Findings Previously published protocols for depleting mice of their intestinal microbiota by administering broad-spectrum antibiotics in drinking water were difficult to reproduce. We show that twice daily delivery of antibiotics by gavage depleted mice of their cultivable fecal microbiota and reduced the fecal bacterial DNA load by 400 fold while ensuring the animals' health. Mice subjected to the protocol for 17 days displayed enlarged ceca, reduced Peyer's patches and small spleens. Antibiotic treatment significantly reduced the expression of antimicrobial factors to a level similar to that of germ-free mice and altered the expression of 517 genes in total in the colonic epithelium. Genes involved in cell cycle were significantly altered concomitant with reduced epithelial proliferative activity in situ assessed by Ki-67 expression, suggesting that commensal microbiota drives cellular proliferation in colonic epithelium. Conclusion We present a robust protocol for depleting conventionally raised mice of their cultivatable intestinal microbiota with antibiotics by gavage and show that the biological effect of this depletion phenocopies physiological characteristics of germ-free mice

Intracellular Phospholipase A1 and Acyltransferase, Which Are Involved in Caenorhabditis elegans Stem Cell Divisions, Determine the sn-1 Fatty Acyl Chain of Phosphatidylinositol

Phosphatidylinositol (PI) is unique in the abundance of stearic acid at the sn-1 position. This fatty acid is thought to be incorporated through fatty acid remodeling. Here, we identified a phospholipase and acyltransferases involved in the fatty acid remodeling at the sn-1 position of PI and provide a link between the sn-1 fatty acid of PI and asymmetric cell division