Identification of cyclic AMP-phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells

AbstractTo determine whether the expression of different PDE4D variants is unique to the rat or conserved through evolution, we have characterized the different PDE4D mRNAs expressed in human peripheral blood mononuclear cells. RT-PCR was performed using primers based on rat sequences and mRNAs from mononuclear cells. The specifically amplified fragments had a size identical to that predicted for rat PDE4D1, PDE4D2 and PDE4D3. Sequencing confirmed that these fragments are derived from the human PDE4D gene. Their sequence was highly homologous to that reported for the rat variants. cDNAs corresponding to the entire ORF of human PDE4D2 and PDE4D3 were expressed in mammalian cells, causing a large increase in PDE activity. Western blot analysis of human peripheral blood mononuclear cell extracts demonstrated the presence of proteins corresponding to the recombinant PDE4D1 and PDE4D2. The pattern of splicing and different promoter usage of the PDE4D gene is therefore conserved during evolution, which indicates an important physiological role

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

The cAMP-specific Phosphodiesterase PDE4D3 Is Regulated by Phosphatidic Acid Binding CONSEQUENCES FOR cAMP SIGNALING PATHWAY AND CHARACTERIZATION OF A PHOSPHATIDIC ACID BINDING SITE

Hormones and growth factors induce in many cell types the production of phosphatidic acid (PA), which has been proposed to play a role as a second messenger. We have previously shown in an acellular system that PA selectively stimulates certain isoforms of type 4 cAMP-phosphodiesterases (PDE4). Here we studied the effect of endogenous PA on PDE activity of transiently transfected MA10 cells overexpressing the PA-sensitive isoform PDE4D3. Cell treatment with inhibitors of PA degradation, including propranolol, induced an accumulation of endogenous PA accompanied by a stimulation of PDE activity and a significant decrease in both cAMP levels and protein kinase A activity. Furthermore, in FRTL5 cells, which natively express PDE4D3, pretreatment with compounds inducing PA accumulation prevented both cAMP increase and cAMP-responsive element-binding protein phosphorylation triggered by thyroid-stimulating hormone. To determine the mechanism of PDE stimulation by PA, endogenous phospholipids were labeled by preincubating MA10 cells overexpressing PDE4D3 with [(32)P]orthophosphate. Immuno- precipitation experiments showed that PA was specifically bound to PDE4D3, supporting the hypothesis that PDE4D3 activation occurs through direct binding of PA to the protein. PA binding site on PDE4D3 was characterized by engineering deletions of selected regions in the N-terminal regulatory domain of the enzyme. Deletion of amino acid residues 31-59 suppressed both PA-activating effect and PA binding, suggesting that this region rich in basic and hydrophobic residues contains the PA binding site. These observations strongly suggest that endogenous PA can modulate cAMP levels in intact cells, through a direct activation of PDE4D3

The Mechanism of Docosahexaenoic Acid-induced Phospholipase D Activation in Human Lymphocytes Involves Exclusion of the Enzyme from Lipid Rafts

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid that inhibits T lymphocyte activation, has been shown to stimulate phospholipase D (PLD) activity in stimulated human peripheral blood mononuclear cells (PBMC). To elucidate the mechanisms underlying the DHA-induced PLD activation, we first characterized the PLD expression pattern of PBMC. We show that these cells express PLD1 and PLD2 at the protein and mRNA level and are devoid of oleate-dependent PLD activity. DHA enrichment of PBMC increased the DHA content of cell phospholipids, which was directly correlated with the extent of PLD activation. The DHA-induced PLD activation was independent of conventional protein kinase C but inhibited by brefeldin A, which suggests ADP-ribosylation factor (ARF)-dependent mechanism. Furthermore, DHA enrichment dose-dependently stimulated ARF translocation to cell membranes. Whereas 50% of the guanosine 5'-3-O-(thio)triphosphate plus ARF-dependent PLD activity and a substantial part of PLD1 protein were located to the detergent-insoluble membranes, so-called rafts, of non-enriched PBMC, DHA treatment strongly displaced them toward detergent-soluble membranes where ARF is present. Collectively, these results suggest that the exclusion of PLD1 from lipid rafts, due to their partial disorganization by DHA, and its relocalization in the vicinity of ARF, is responsible for its activation. This PLD activation might be responsible for the immunosuppressive effect of DHA because it is known to transmit antiproliferative signals in lymphoid cells

TNF-α- and tumor-induced skeletal muscle atrophy involves sphingolipid metabolism

Additional filesInternational audienceUNLABELLED: ABSTRACT: BACKGROUND: Muscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention. RESULTS: We addressed this question both in vitro using differentiated myotubes treated with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide-synthesis inhibitors that targeted either the de novo pathway (myriocin), or the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy. CONCLUSIONS: Ceramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical target for pharmacological or nutritional interventions aiming at preserving muscle mass in pathological situations

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