Georges Némoz

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 [32P]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.

Phosphodiesterase inhibitory profile of some related xanthine derivatives pharmacologically active on the peripheral microcirculation

The cyclic nucleotide phosphodiesterase (PDE) inhibitory profile of four related xanthine derivatives: pentoxifylline (BL 191), propentofylline (HWA 285), torbafylline (HWA 448) and albifylline (HWA 138), pharmacologically active on the peripheral and/or cerebral microcirculation was established using the four main PDE isoforms present in rat heart cytosol. HPLC on a Mono Q ion-exchange column resolved four separate cyclic nucleotide PDE activities: a calmodulin-activated fraction (PDE I), a cGMP-stimulated fraction (PDE II), a cAMP-specific rolipram-sensitive fraction (PDE IV) and a cGMP-inhibited fraction (PDE III). Among the four compounds studies, only torbafylline and pentoxifylline inhibited more efficiently the calcium plus calmodulin-stimulated than the basal activity of PDE I. The four xanthine derivatives inhibited more potently the cGMP-stimulated than the basal activity of the cGMP-stimulatable PDE II, propentofylline being the most inhibitory (IC50: 20 microM). Except for propentofylline, which exhibited a marked selectivity toward the rolipram-sensitive PDE versus the cGMP-inhibited PDE III, the other xanthines modestly (IC50 in the 10(-4) M range) inhibited both cAMP-specific isoforms with similar potency. Propentofylline proved to be the best inhibitor whatever the considered isoform whereas torbafylline exhibited the weakest inhibitory potency with, however, some selectivity for PDE I.

Protective effect of docosahexaenoic acid against hydrogen peroxide-induced oxidative stress in human lymphocytes.

Oxidatively stressed lymphocytes exhibit decreased proliferative response to mitogenic stimulation. Although several sensitive targets involved in lymphocyte suppression have already been identified, little is known about the influence of oxidative stress on cyclic nucleotide phosphodiesterases (PDE) (EC 3.1.4.17), thought to play a major role in the control of cyclic AMP (cAMP) level, a well-recognized negative effector of lymphoproliferation. Although the polyunsaturated fatty acid content of membrane phospholipids is thought to be directly related to the extent of oxidant-induced lipid peroxidation, some n-3 fatty acids also seem to have antioxidant effects, depending on the concentration used and the overall redox status of the cells in question. Results of the present study showed that human peripheral blood mononuclear cells (PBMC) as well as rat thymocytes were relatively resistant to a short-term exposure (10 min) to hydrogen peroxide (H2O2). Indeed, H2O2-induced lipid peroxidation, estimated by malondialdehyde (MDA) production, was only 2-fold increased by H2O2 concentrations lower than 2 mM, whereas a larger increase (10-fold) could be observed in PBMC at the highest dose (5 mM). Previous enrichment of PBMC with 5 microM docosahexaenoic acid (22:6n-3), brought to the cells as a fatty acid-albumin complex (ratio 1), significantly reduced MDA production induced by low doses of H2O2, the protective effect no longer being observed at the highest doses. In contrast, eicosapentaenoic acid (20:5n-3) did not have any protective effect. Cytosolic PDE activities of both human PBMC and rat thymocytes were significantly inhibited (40-50%) after H2O2 treatment of the cells, whereas particulate PDE activities were not modified. Different responses of PDE activities to H2O2 treatment were observed when PBMC were first enriched with 22:6n-3 prior to H2O2 addition. In 22:6n-3-treated cells, the H2O2-induced inhibition of both cAMP- and cGMP-PDE cytosolic activities was abolished, whereas the particulate activities were increased by the highest H2O2 concentration used (5 mM). At the same time, the glutathione peroxidase (glutathione: oxidoreductase, EC 1.11.1.9) (GSH-Px) activity of PBMC and thymocytes was only marginally inhibited by H2O2 addition (20%), and pretreatment of the cells with 22:6n-3 did not modify the slight inhibitory effect of H2O2. Collectively, these results suggest that lymphocytes are relatively resistant to H2O2-induced lipid peroxidation due to their high GSH-Px content, and that low doses of 22:6n-3 are able to prevent some of the H2O2-induced alterations such as lipid peroxidation and PDE inhibition. Docosahexaenoic acid might thus offer some protection against oxidant-induced lymphocyte suppression.

A New Role for Sterol Regulatory Element Binding Protein 1 Transcription Factors in the Regulation of Muscle Mass and Muscle Cell Differentiation

ABSTRACT The role of the transcription factors sterol regulatory element binding protein 1a (SREBP-1a) and SREBP-1c in the regulation of cholesterol and fatty acid metabolism has been well studied; however, little is known about their specific function in muscle. In the present study, analysis of recent microarray data from muscle cells overexpressing SREBP1 suggested that they may play a role in the regulation of myogenesis. We then demonstrated that SREBP-1a and -1c inhibit myoblast-to-myotube differentiation and also induce in vivo and in vitro muscle atrophy. Furthermore, we have identified the transcriptional repressors BHLHB2 and BHLHB3 as mediators of these effects of SREBP-1a and -1c in muscle. Both repressors are SREBP-1 target genes, and they affect the expression of numerous genes involved in the myogenic program. Our findings identify a new role for SREBP-1 transcription factors in muscle, thus linking the control of muscle mass to metabolic pathways.

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

BackgroundMuscle 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.ResultsWe 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.ConclusionsCeramide 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.

Comparison of cyclic nucleotide phosphodiesterase isoforms from rat heart and bovine aorta: Separation and inhibition by selective reference phosphodiesterase inhibitors

The resolution as well as the biochemical properties of the multiple molecular forms of cyclic nucleotide phosphodiesterase, in a given tissue, may be strongly dependent upon experimental conditions of preparation (extraction of crude enzyme from tissues and fractionation procedures). In the present study, we compare the different molecular forms of cardiac (rat heart ventricle) and vascular (bovine aorta) phosphodiesterase isolated from crude extracts prepared either in sucrose medium or in hypotonic medium (in the presence of protease inhibitors and ion chelators) using two different fractionation procedures: isoelectric focusing on flat gel bed and DEAE-Trisacryl anion exchange chromatography. Both the calmodulin-dependent and the cAMP-specific forms exhibited close IEF and chromatographic patterns and showed similar sensitivities towards reference inhibitors regardless of the tissue of origin. In marked contrast, the cGMP-specific isoform notably differed from one to another tissue with respect to its biochemical properties (only the cardiac tissue being capable of stimulation by cGMP) and sensitivities to xenobiotics. Thus the possibility exists that pharmacological agents may modulate phosphodiesterase activity differently in cardiac and vascular target tissues.

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

In 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.

Disruption of Lipid Rafts Stimulates Phospholipase D Activity in Human Lymphocytes: Implication in the Regulation of Immune Function

Recent evidence suggests that phospholipase D (PLD) can be regulated through its association/dissociation to lipid rafts. We show here that modifying lipid rafts either by cholesterol depletion using methyl-β-cyclodextrin and filipin or by conversion of sphingomyelin to ceramide with exogenous bacterial sphingomyelinase (bSMase) markedly activated the PLD of human PBMC. bSMase was the most potent PLD activator, giving maximal 6- to 7-fold increase in PLD activity. Triton X-100-treated lysates prepared from control PBMC and from bSMase-treated cells were fractionated by centrifugation on sucrose density gradient. We observed that bSMase treatment of the cells induced a larger ceramide increase in raft than in nonraft membranes and displaced both the Src kinase Lck and PLD1 out of the raft fractions. In addition, the three raft-modifying agents markedly inhibited the lymphoproliferative response to mitogenic lectin. To examine further the potential role of PLD activation in the control of lymphocyte responses, we transiently overexpressed either of the PLD1 and PLD2 isoforms in Jurkat cells and analyzed the phorbol ester plus ionomycin-induced expression of IL-2 mRNA, which is one of the early responses of lymphocyte to activation. We observed a 43% decrease of IL-2 mRNA level in Jurkat cells overexpressing PLD1 as compared with mock- or PLD2-transfected cells, which indicates that elevated PLD1, but not PLD2, activity impairs lymphocyte activation. Altogether, the present results support the hypothesis that PLD1 is activated by exclusion from lipid rafts and that this activation conveys antiproliferative signals in lymphoid cells.

Concanavalin A stimulates the rolipram-sensitive isoforms of cyclic nucleotide phosphodiesterase in rat thymic lymphocytes

Pretreatment of rat thymic lymphocytes with Concanavalin A induced a very early (30 min) and substantial increase (+90%) of the soluble cAMP phosphodiesterase activity. The crude cytosolic phosphodiesterase activity of rat thymocytes could reproducively be resolved by Mono-Q ion exchange high performance liquid chromatography into four separate phosphodiesterase peaks: a cGMP-stimulated, two cAMP-specific Rolipram-sensitive and a cGMP-inhibited cardiotrope-sensitive peaks. Concanavalin A stimulated very specifically the activity of the two predominant cAMP-specific Rolipram sensitive peaks whereas it only slightly modified the cGMP-stimulated and the cGMP-inhibited forms. The present results strongly suggest that the Rolipram-sensitive cAMP PDE activity may play a key role in the control and regulation of mitogen-induced thymocyte proliferation.

Phosphatidic acid stimulates the rolipram-sensitive cyclic nucleotide phosphodiesterase from rat thymocytes

The role of phospholipid metabolites in the modulation of cyclic AMP degradation during the early response of rat thymic lymphocytes to mitogenic stimulation was investigated by measuring their in vitro effect on the activity of five different cyclic nucleotide phosphodiesterase forms separated from thymocyte cytosol by means of an HPLC technique. Arachidonic acid was found to markedly inhibit four of the enzyme forms, with IC50 ranging from 14 to 60 microM, while its hydroperoxy and hydroxy derivatives proved inefficient. Dioctanoylglycerol, a biologically active diacylglycerol, was weakly inhibitory while phosphatidic acid, the diacylglycerol phosphorylated derivative, markedly stimulated the two cyclic-AMP-specific type-IV forms identified in thymocyte cytosol, by 50 and 70%. In intact cells labelled with tritiated arachidonate, the mitogenic lectin concanavalin A induced a rapid 4-5-fold increase in radiolabelled phosphatidic acid which peaked at 1 min, and remained elevated for at least 30 min. These observations suggest that phosphatidic acid formed during the mitogenic stimulation of T-cells might be responsible for an early activation of cyclic AMP degradation with, as a consequence, a lowering of cyclic AMP level, which is reported to be necessary for the occurrence of the first steps of mitogenesis.