Acides gras polyinsaturés oméga 3 et toxicité hépatique de l'éthanol : rôle du remodelage membranaire

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Identification of the couple GSK3α/c-Myc as a new regulator of hexokinase II in benzo[a]pyrene-induced apoptosis.

International audienceThe early apoptotic events induced by environmental pollutants with carcinogenic properties are poorly understood. Here, we focus on the early cytotoxic effects of benzo[a]pyrene (B[a]P). In F258 rat hepatic epithelial cells, B[a]P induces intrinsic apoptosis via a mitochondrial dysfunction characterized by the release of hexokinase II (HKII) from the mitochondria. Cancer cells often have an anomalous cell energy metabolism; since HKII dysfunction regulates B[a]P-induced apoptosis in F258 cells, but may also alter cell energy metabolism, HKII release from the mitochondria may represent an important B[a]P-related carcinogenic issue. Thus in the present study, we aimed at deciphering the mechanisms underlying HKII dysfunction upon B[a]P exposure. We show that while glycogen synthase kinase 3 beta (GSK3β) regulated the expression of HKII at the transcriptional level, glycogen synthase kinase 3 alpha (GSK3α) was involved in B[a]P-induced apoptosis via a decrease in c-Myc expression. The reduced level of c-Myc caused the relocation of HKII from the mitochondria to the cytosol, thereby being involved in the formation of reactive oxygen species and apoptosis. In conclusion, we show that the couple GSK3α/c-Myc plays a key role in B[a]P-induced early apoptotic cell signaling via HKII dysfunction

A role for lipid rafts in the protection afforded by docosahexaenoic acid against ethanol toxicity in primary rat hepatocytes.

International audience: Previously, we demonstrated that eicosapentaenoic acid enhanced ethanol-induced oxidative stress and cell death in primary rat hepatocytes via an increase in membrane fluidity and lipid raft clustering. In this context, another n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), was tested with a special emphasis on physical and chemical alteration of lipid rafts. Pretreatment of hepatocytes with DHA reduced significantly ethanol-induced oxidative stress and cell death. DHA protection could be related to an alteration of lipid rafts. Indeed, rafts exhibited a marked increase in membrane fluidity and packing defects leading to the exclusion of a raft protein marker, flotillin. Furthermore, DHA strongly inhibited disulfide bridge formation, even in control cells, thus suggesting a disruption of protein-protein interactions inside lipid rafts. This particular spatial organization of lipid rafts due to DHA subsequently prevented the ethanol-induced lipid raft clustering. Such a prevention was then responsible for the inhibition of phospholipase C-γ translocation into rafts, and consequently of both lysosome accumulation and elevation in cellular low-molecular-weight iron content, a prooxidant factor. In total, the present study suggests that DHA supplementation could represent a new preventive approach for patients with alcoholic liver disease based upon modulation of the membrane structures

The cleaved FAS ligand activates the Na+/H+ exchanger NHE1 through Akt/ROCK1 to stimulate cell motility

International audienceTransmembrane CD95L (Fas ligand) can be cleaved to release a promigratory soluble ligand, cl-CD95L, which can contribute to chronic inflammation and cancer cell dissemination. The motility signaling pathway elicited by cl-CD95L remains poorly defined. Here, we show that in the presence of cl-CD95L, CD95 activates the Akt and RhoA signaling pathways, which together orchestrate an allosteric activation of the Na+/H+ exchanger NHE1. Pharmacologic inhibition of Akt or ROCK1 independently blocks the cl-CD95L-induced migration. Confirming these pharmacologic data, disruption of the Akt and ROCK1 phosphorylation sites on NHE1 decreases cell migration in cells exposed to cl-CD95L. Together, these findings demonstrate that NHE1 is a novel molecular actor in the CD95 signaling pathway that drives the cl-CD95L-induced cell migration through both the Akt and RhoA signaling pathways

DNA Damage–Induced Bcl-x(L) Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

The pro-survival protein Bcl-x(L) is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-x(L) deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-x(L) deamidation remains unknown and its functional consequences unclear. We show here that rBcl-x(L) deamidation generates an iso-Asp(52)/iso-Asp(66) species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-x(L) deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-x(L) deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-x(L) deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy

Small molecule anionophores promote transmembrane anion permeation matching CFTR activity

Anion selective ionophores, anionophores, are small molecules capable of facilitating the transmembrane transport of anions. Inspired in the structure of natural product prodigiosin, four novel anionophores 1a-d, including a 1,2,3-triazole group, were prepared. These compounds proved highly efficient anion exchangers in model phospholipid liposomes. The changes in the hydrogen bond cleft modified the anion transport selectivity exhibited by these compounds compared to prodigiosin and suppressed the characteristic high toxicity of the natural product. Their activity as anionophores in living cells was studied and chloride efflux and iodine influx from living cells mediated by these derivatives was demonstrated. These compounds were shown to permeabilize cellular membranes to halides with efficiencies close to the natural anion channel CFTR at doses that do not compromise cellular viability. Remarkably, optimal transport efficiency was measured in the presence of pH gradients mimicking those found in the airway epithelia of Cystic Fibrosis patients. These results support the viability of developing small molecule anionophores as anion channel protein surrogates with potential applications in the treatment of conditions such as Cystic Fibrosis derived from the malfunction of natural anion transport mechanisms.European Union’s Horizon 2020 research and innovation programme under grant agreement No. 667079, La Marató de TV3 Foundation (20132730), Consejería de Educación de la Junta de Castilla y León (Projects BU340U13 and BU092U16

2-Aminophenoxazine-3-one and 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one cause cellular apoptosis by reducing higher intracellular pH in cancer cells

We examined intracellular pH (pHi) of ten cancer cell lines derived from different organs and two normal cell lines including human embryonic lung fibroblast cells (HEL) and human umbilical vein endothelial cells (HUVEC) in vitro, and found that pHi of most of these cancer cells was evidently higher (pH 7.5 to 7.7) than that of normal cells (7.32 and 7.44 for HEL and HUVEC, respectively) and that of primary leukemic cells and erythrocytes hitherto reported (≤7.2). Higher pHi in these cancer cells could be related to the Warburg effect in cancer cells with enhanced glycolytic metabolism. Since reversal of the Warburg effect may perturb intracellular homeostasis in cancer cells, we looked for compounds that cause extensive reduction of pHi, a major regulator of the glycolytic pathway and its associated metabolic pathway. We found that phenoxazine compounds, 2-aminophenoxazine-3-one (Phx-3) and 2-amino-4,4α-dihydro-4α,7-dimethyl-3H-phenoxazine-3-one (Phx-1) caused a rapid and drastic dose-dependent decrease of pHi in ten different cancer cells within 30 min, though the extent of the decrease of pHi was significantly larger for Phx-3 (ΔpHi = 0.6 pH units or more for 100 µM Phx-3) than for Phx-1 (ΔpHi = 0.1 pH units or more for 100 µM Phx-1). This rapid and drastic decrease of pHi in a variety of cancer cells caused by Phx-3 and Phx-1 possibly perturbed their intracellular homeostasis, and extensively affected the subsequent cell death, because these phenoxazines exerted dose-dependent proapoptotic and cytotoxic effects on these cells during 72 h incubation, confirming a causal relationship between ΔpHi and cytotoxic effects due to Phx-3 and Phx-1. Phx-3 and Phx-1 also reduced pHi of normal cells including HEL and HUVEC, although they exerted less proapoptotic and cytotoxic effects on these cells than on cancer cells. Drugs such as Phx-3 and Phx-1 that reduce pHi and thereby induce cellular apoptosis might serve as benevolent anticancer drugs

On the Role of the Difference in Surface Tensions Involved in the Allosteric Regulation of NHE-1 Induced by Low to Mild Osmotic Pressure, Membrane Tension and Lipid Asymmetry

The sodium-proton exchanger 1 (NHE-1) is a membrane transporter that exchanges Na+ for H+ ion across the membrane of eukaryotic cells. It is cooperatively activated by intracellular protons, and this allosteric regulation is modulated by the biophysical properties of the plasma membrane and related lipid environment. Consequently, NHE-1 is a mechanosensitive transporter that responds to osmotic pressure, and changes in membrane composition. The purpose of this study was to develop the relationship between membrane surface tension, and the allosteric balance of a mechanosensitive transporter such as NHE-1. In eukaryotes, the asymmetric composition of membrane leaflets results in a difference in surface tensions that is involved in the creation of a reservoir of intracellular vesicles and membrane buds contributing to buffer mechanical constraints. Therefore, we took this phenomenon into account in this study and developed a set of relations between the mean surface tension, membrane asymmetry, fluid phase endocytosis and the allosteric equilibrium constant of the transporter. We then used the experimental data published on the effects of osmotic pressure and membrane modification on the NHE-1 allosteric constant to fit these equations. We show here that NHE-1 mechanosensitivity is more based on its high sensitivity towards the asymmetry between the bilayer leaflets compared to mean global membrane tension. This compliance to membrane asymmetry is physiologically relevant as with their slower transport rates than ion channels, transporters cannot respond as high pressure-high conductance fast-gating emergency valves