Phospholipase D protects ECV304 cells against TNFα-induced apoptosis

AbstractTumor necrosis factor α (TNFα), a pleiotropic cytokine, activates both apoptotic and pro-survival signals depending on the cell model. Using ECV304 cells, which can be made TNFα-sensitive by cycloheximide (CHX) co-treatment, we evaluated the potential roles of ceramide and phospholipase D (PLD) in TNFα-induced apoptosis. TNFα/CHX induced a robust increase in ceramide levels after 16h of treatment when cell death was maximal. PLD activity was increased at early time point (1h) whereas both PLD activity and PLD1 protein were strongly decreased after 24h. TNFα/CHX-induced cell death was significantly lowered by exogenous bacterial PLD and phoshatidic acid, and in cells overexpressing PLD1. Conversely, cells depleted in PLD proteins by small interference RNA (siRNA) treatment exhibited higher susceptibility to apoptosis. These results show that PLD exerts a protective role against TNFα-induced cell death

Phospholipase D, perméabilité endothéliale, et apoptose TNFa dépendante

La phospholipase D (PLD) est une enzyme membranaire qui intervient au cur du métabolisme lipidique en générant un second messager, l\u27acide phosphatidique (PA) impliqué dans de nombreuses fonctions cellulaires. Deux isoformes PLD1 et PLD2 contribuent à sa synthèse chez les mammifères. Nous avons montré que la PLD augmente la perméabilité de monocouches de cellules endothéliales (lignée HUV-EC-C) aux macromolécules en permettant un remodelage du cytosquelette d\u27actine. L\u27isoforme PLD2 jouerait un rôle prépondérant via sa localisation au sein des cavéoles membranaires. Nous avons étudié le rôle de la PLD dans l\u27apoptose des cellules ECV304 induite par le TNFa, une cytokine activant à la fois des voies de survie et des voies pro-apoptotiques. Nous avons aussi étudié le rôle de la PLD dans l\u27apoptose des cellules ECV304 induite par le TNFa une cytokine activant à la fois des voies de survie et des voies pro-apoptotiques. Nous avons montré que la PLD a un rôle protecteur contre la mort cellulaire induite par TNFa en présence de cycloheximide, l\u27isoforme PLD1 étant plus spécifiquement mise en jeu. Divers mécanismes expliquant cet effet protecteur sont proposés

Phospholipase D2 regulates endothelial permeability through cytoskeleton reorganization and occludin downregulation.: PLD2 and endothelial permeability

International audienceEndothelial permeability is controlled by adhesive strengths which connect cells to each other through interendothelial junctions and by contractile forces associated with cytoskeleton reorganization. Phospholipase D (PLD) activation resulting in the generation of phosphatidic acid (PA) is increasingly recognized as a key event in the initiation of various cell responses. In human umbilical vein endothelial cells (HUV-EC), enhancement of intracellular PA by a variety of approaches increased the permeability of endothelial cell monolayers and induced stress fibre formation. Using adenovirus-mediated overexpression and siRNA silencing, we showed that PLD2 but not PLD1 was involved in the enhancement of basal permeability through cytoskeleton reorganization. Furthermore, PLD2 overexpression induced ERK1/2 activation and downregulated the expression of occludin, a major component of tight junctions. A substantial part of PLD2 protein was associated with the low-density caveolin-rich fractions isolated on sucrose gradients. The Raf-1 specific inhibitor GW-5074 drastically reduced hyperpermeability induced by PLD2 overexpression, and inhibited PA-mediated increase of endothelial permeability and ERK1/2 activation. On the whole, the present results demonstrate the selective role of PLD2 isoform in the control of endothelial permeability through a mechanism involving both stress fibre formation and contraction, and occludin downregulation, possibly resulting from PA-mediated activation of Raf-1

Inhibition of de novo ceramide synthesis upregulates phospholipase D and enhances myogenic differentiation.

International audienceIn L6 skeletal myoblasts induced to differentiate by Arg8-vasopressin treatment, a short-lived lowering of ceramide levels was observed, followed by a long-lasting elevation that was prevented by inhibitors of the de novo synthesis pathway, fumonisin B1 and myriocin. Both inhibitors increased the expression of myogenic differentiation markers and cell fusion rate, whereas short-chain ceramides inhibited these responses. Similar drug effects were observed on primary mouse satellite cell differentiation. Furthermore, bacterial sphingomyelinase overexpression suppressed myogenin nuclear accumulation in L6 cells. These data suggested that endogenous ceramide mediates a negative feedback mechanism limiting myogenic differentiation, and that inhibitors of ceramide synthesis promoted myogenesis by removing this control. Phospholipase D (PLD), a recognized target of ceramide, is required for myogenesis, as shown by the negative effects of PLD1 isoform depletion obtained by siRNA treatment. Fumonisin induced an increase in PLD activity of L6 cells, whereas C6-ceramide decreased it. The expression of PLD1 mRNA transcripts was selectively decreased by C6-ceramide, and increased by ceramide synthesis inhibitors. An early step of myogenic response is the PLD1-dependent formation of actin stress fiber-like structures. C6-ceramide addition or overexpression of sphingomyelinase impaired actin fiber formation. Ceramide might thus regulate myogenesis through downregulation of PLD1 expression and activity

In vitro impact of pegvisomant on growth hormone-secreting pituitary adenoma cells

International audiencePegvisomant (PEG), an antagonist of growth hormone (GH)-receptor (GHR), normalizes insulin-like growth factor 1 (IGF1) oversecretion in most acromegalic patients unresponsive to somatostatin analogs (SSAs) and/or uncontrolled by transsphenoidal surgery. The residual GH-secreting tumor is therefore exposed to the action of circulating PEG. However, the biological effect of PEG at the pituitary level remains unknown. To assess the impact of PEG in vitro on the hormonal secretion (GH and prolactin (PRL)), proliferation and cellular viability of eight human GH-secreting tumors in primary cultures and of the rat somatolactotroph cell line GH4C1. We found that the mRNA expression levels of GHR were characterized in 31 human GH-secreting adenomas (0.086 copy/copy β-Gus) and the GHR was identified by immunocytochemistry staining. In 5/8 adenomas, a dose-dependent inhibition of GH secretion was observed under PEG with a maximum of 38.2 ± 17% at 1 μg/mL (P < 0.0001 vs control). A dose-dependent inhibition of PRL secretion occurred in three mixed GH/PRL adenomas under PEG with a maximum of 52.8 ± 11.5% at 10 μg/mL (P < 0.0001 vs control). No impact on proliferation of either human primary tumors or GH4C1 cell line was observed. We conclude that PEG inhibits the secretion of GH and PRL in primary cultures of human GH(/PRL)-secreting pituitary adenomas without effect on cell viability or cell proliferation

Phospholipase D Regulates Myogenic Differentiation through the Activation of Both mTORC1 and mTORC2 Complexes*

How phospholipase D (PLD) is involved in myogenesis remains unclear. At the onset of myogenic differentiation of L6 cells induced by the PLD agonist vasopressin in the absence of serum, mTORC1 complex was rapidly activated, as reflected by phosphorylation of S6 kinase1 (S6K1). Both the long (p85) and short (p70) S6K1 isoforms were phosphorylated in a PLD1-dependent way. Short rapamycin treatment specifically inhibiting mTORC1 suppressed p70 but not p85 phosphorylation, suggesting that p85 might be directly activated by phosphatidic acid. Vasopressin stimulation also induced phosphorylation of Akt on Ser-473 through PLD1-dependent activation of mTORC2 complex. In this model of myogenesis, mTORC2 had a positive role mostly unrelated to Akt activation, whereas mTORC1 had a negative role, associated with S6K1-induced Rictor phosphorylation. The PLD requirement for differentiation can thus be attributed to its ability to trigger via mTORC2 activation the phosphorylation of an effector that could be PKCα. Moreover, PLD is involved in a counter-regulation loop expected to limit the response. This study thus brings new insights in the intricate way PLD and mTOR cooperate to control myogenesis