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A common mRNA modification is 5-methylcytosine (m5C), whose role in gene-transcript processing and cancer remains unclear. Here, we identify serine/arginine-rich splicing factor 2 (SRSF2) as a reader of m5C and impaired SRSF2 m5C binding as a potential contributor to leukemogenesis. Structurally, we identify residues involved in m5C recognition and the impact of the prevalent leukemia-associated mutation SRSF2P95H. We show that SRSF2 binding and m5C colocalize within transcripts. Furthermore, knocking down the m5C writer NSUN2 decreases mRNA m5C, reduces SRSF2 binding, and alters RNA splicing. We also show that the SRSF2P95H mutation impairs the ability of the protein to read m5C-marked mRNA, notably reducing its binding to key leukemia-related transcripts in leukemic cells. In leukemia patients, low NSUN2 expression leads to mRNA m5C hypomethylation and, combined with SRSF2P95H, predicts poor outcomes. Altogether, we highlight an unrecognized mechanistic link between epitranscriptomics and a key oncogenesis driver., SCOPUS: ar.j, info:eu-repo/semantics/published

Aim: Dexamethasone has been shown to induce the formation of epithelial domes by bronchiolar H441 cells. It stimulates the expression of both amiloride inhibitable epithelial sodium channels (ENaC) and dual oxidase-1 (DUOX1). We therefore ask the question whether DUOX1 expression and production of submillimolar amounts of H2O2 is instrumental for the sodium channel upregulation observed in H441 cells. Methods: In vitro cell culture, nystatin-perforated whole-cell patch-clamp technique, immunocytochemistry and RT-PCR methods have been used. Results: Cells forming epithelial domes induced by dexamethasone (0.1 μmol L−1, 24 hours) and by 5-aza-2′-deoxytidine (1 μmol L−1, 48 hours) expressed more DUOX1 protein compared with other cells in the monolayer. Dome formation could be inhibited by exogenous catalase in a concentration-dependent manner and by the NADPH oxidase inhibitor diphenyliodonium, which suggested the involvement of H2O2. While single application of 0.2 mmol L−1 H2O2 induced transient dome formation, lower doses were ineffective and higher doses disrupted the cell monolayer. Hydrogen peroxide (0.1 mmol L−1) activated acutely amiloride-sensitive whole-cell currents from 3.91 ± 0.79 pA pF−1 to 4.76 ± 0.98 pA pF−1 in dome-forming cells and had no effect in cells outside of domes. ENaC but not DUOX1 transcription was potentiated by catalase in the presence of dexamethasone, which suggested negative feedback of H2O2 on ENaC gene expression. Conclusion: Our observations suggest that tonic production of H2O2 by DUOX1 participates in maintaining the level of vectorial sodium transport by lung epithelial cells. Moreover, the system appears to be well tuned as it would allow H2O2-dependent innate immunity without inducing airway/alveolar sodium and fluid hyperabsorption., SCOPUS: ar.j, info:eu-repo/semantics/published

Pulmonary fibrosis is characterized by over-population and excessive activation of fibroblasts and myofibroblasts disrupting normal lung structure and functioning. Rosemary extract rich in carnosic acid (CA) and rosmarinic acid (RA) was reported to cure bleomycin-(BLM)-induced pulmonary fibrosis. We demonstrate that CA decreased human lung fibroblast (HLF) viability with IC50 value of 17.13±1.06 μM, while RA had no cytotoxic effect. In the presence of 50 μM of RA, dose-response for CA shifted to IC50 value of 11.70±1.46 μM, indicating synergic action. TGFβ-transformed HLF, rat lung fibroblasts and L929 cells presented similar sensitivity to CA and CA+RA (20μM+100μM, respectively) treatment. Rat alveolar epithelial cells died only under CA+RA treatment, while A549 cells were not affected. Annexin V staining and DNA quantification suggested that HLF are arrested in G0/G1 cell cycle phase and undergo apoptosis. CA caused sustained activation of phospho-Akt and phospho-p38 expression and inhibition of p21 protein.Addition of RA potentiated these effects, while RA added alone had no action.Only triple combination of inhibitors (MAPK-p38, pan-caspase, PI3K/Akt/autophagy) partially attenuated apoptosis; this suggests that cytotoxicity of CA+RA treatment has a complex mechanism involving several parallel signaling pathways. The in vivo antifibrotic effect of CA and RA was compared with that of Vita-mine-E in BLM-induced fibrosis model in rats. We found comparable reduction in fibrosis score by CA, RA and CA+RA, attenuation of collagen deposition and normalization of oxidative stress markers. In conclusion, antifibrotic effect of CA+RA is due to synergistic pro-apoptotic action on lung fibroblasts and myofibroblasts., SCOPUS: ar.j, info:eu-repo/semantics/published

Renal and lung epithelial cells are exposed to some significant concentrations of H2O2. In urine it may reach 100 M, while in the epithelial lining fluid in the lung it is estimated to be in micromolar to tens-micromolar range. Hydrogen peroxide has a stimulatory action on the epithelial sodium channel (ENaC) single-channel activity. It also increases stability of the channel at the membrane and slows down the transcription of the ENaC subunits. The expression and the activity of the channel may be inhibited in some other, likely higher, oxidative states of the cell. This review discusses the role and the origin of H2O2 in the lung and kidney. Concentration-dependent effects of hydrogen peroxide on ENaC and the mechanisms of its action have been summarized. This review also describes outlooks for future investigations linking oxidative stress, epithelial sodium transport, and lung and kidney function., SCOPUS: re.j, info:eu-repo/semantics/published

Aristolochic acids (AA) are nephrotoxic and profibrotic agents, leading to chronic kidney disease. As some controversial studies have reported a nephroprotective effect of exogenous recombinant human bone morphogenetic protein (rhBMP)-7 in several models of renal fibrosis, we investigated the putative effect of rhBMP-7 to prevent progressive tubulointerstitial damage after AA intoxication in vitro and in vivo. In vitro, the toxicity of AA on renal tubular cells was demonstrated by an increase in vimentin as well as a decrease in β-catenin expressions, reflecting a dedifferentiation process. Increased fibronectin and interleukin-6 levels were measured in the supernatants. Enhanced α-SMA mRNA levels associated to decreased E-cadherin mRNA levels were also measured. Incubation with rhBMP-7 only prevented the increase in vimentin and the decrease in β-catenin expressions. In vivo, in a rat model of AA nephropathy, severe tubulointerstitial lesions induced by AA after 10 and 35days (collagen IV deposition and tubular atrophy), were not prevented by the rhBMP-7 treatment. Similarly, rhBMP-7 did not ameliorate the significant increase in urinary concentrations of transforming growth factor-β. In summary, our in vitro data demonstrated a poor beneficial effect of rhBMP-7 to reverse cell toxicity while, in vivo, there was no beneficial effect of rhBMP-7. Therefore, further investigations are needed to confirm the exact role of BMP-7 in progressive chronic kidney disease., SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

I. DESCRIPTION DE PROJET DE RECHERCHELe canal sodium épithélial bloquable par l’amiloride (ENaC) est une protéine intégrale de la membrane apicale des épithéliums impliqués dans l’absorption du sodium. Deux fonctions majeures sont directement liées au fonctionnement d’ENaC. D’une part, la régulation de la balance sodée par le rein et donc de la pression artérielle et d’autre part, la clairance du fluide alvéolaire pulmonaire.Le transport vectoriel de sel et d’eau à travers ces épithéliums à jonctions serrées repose sur un transport actif de sodium entraînant un flux osmotique d’eau. Ce transport de sodium s’effectue en deux étapes: l’entrée apicale, par diffusion, facilitée via ENaC, et la sortie basolatérale, active, par les pompes Na+/K+ ATPases.Ces dernières années, un intérêt grandissant est porté sur les acides gras polyinsaturés à longues chaînes de type oméga 3 (PUFAs) et leurs implications dans divers processus physiologiques. Entre autres effets, les PUFAs modulent différents types de canaux ioniques (canaux Na+ dépendant du voltage, Ca++ L-type, K+).Les études in vivo impliquant un effet à long terme des PUFAs décrivent des mécanismes inhibiteurs. Cependant, lors d’une étude précédente, axée sur la composition lipidique des membranes de cellules rénales en culture et l’influence de l’ajout d’acides gras saturés et insaturés sur le transport du sodium, nous avons constaté que les acides gras polyinsaturés à longues chaînes de type oméga 3 augmentaient la réabsorption du sodium. Ces résultats pourraient être intéressants, car les canaux sodiques de l’épithélium alvéolaire sont en contact direct avec le surfactant, dont la composition lipidique varie en fonction de l’apport alimentaire en PUFAs. Chez les prématurés humains, le syndrome de détresse respiratoire est une des causes les plus fréquentes de mortalité. Dans un certain nombre de cas, on peut restaurer une fonction pulmonaire satisfaisante par l’administration de surfactant.Dan, Doctorat en Sciences biomédicales et pharmaceutiques, info:eu-repo/semantics/nonPublished

During inhalational anesthesia, halogenated gases are in direct contact with the alveolar epithelium, in which they may affect transepithelial ion and fluid transport. The effects of halogenated gases in vivo on epithelial Na+ and K+ channels, which participate in alveolar liquid clearance, remain unclear. In the present study, the effects of halothane (1, 2, and 4% atm) on ion-channel function in cultured human alveolar cells were investigated using the patch-clamp technique. After exposure to 4% halothane, amiloride-sensitive whole-cell inward currents increased by 84+/-22%, whereas tetraethylammonium-sensitive outward currents decreased by 63+/-7%. These effects, which occurred within 30 s, remained for 30-min periods of exposure to the gas, were concentration-dependent, and were reversible upon washout. Pretreatment with amiloride prevented 90+/-7% of the increase in inward currents without change in outward currents, consistent with an activation of amiloride-sensitive epithelial sodium channels. Tetraethylammonium obliterated 90+/-9% of the effect of halothane on outward currents, without change in inward currents, indicating inhibition of Ca2+-activated K+ channels. These channels were identified in excised patches to be small-conductance Ca2+-activated K+ channels. These effects of halothane were not modified after the inhibition of cytosolic phospholipase A2 by aristolochic acid. Exposure of the cells to either trypsin or to low Na+ completely prevented the increase in amiloride-sensitive currents induced by halothane, suggesting a release of Na+ channels self-inhibition. Thus, halothane modifies differentially and independently Na+ and K+ permeabilities in human alveolar cells., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The epithelial sodium channel is found in apical membranes of a variety of native epithelial tissues, where it regulates sodium and fluid balance. In vivo, a number of hormones and other endogenous factors, including polyunsaturated fatty acids (PUFAs), regulate these channels. We tested the effects of essential n-3 and n-6 PUFAs on amiloride-sensitive sodium transport in A6 epithelial cells. Eicosapentaenoic acid [EPA; C20:5(n-3)] transiently stimulated amiloride-sensitive open-circuit current (I(Na)) from 4.0 +/- 0.3 to 7.7 +/- 0.3 microA/cm2 within 30 min (P < 0.001). No activation was seen in the presence of 10 microM amiloride. In cell-attached but not in cell-excised patches, EPA acutely increased the open probability of sodium channels from 0.45 +/- 0.08 to 0.63 +/- 0.10 (P = 0.02, paired t-test). n-6 PUFAs, including linoleic acid (C18:2), eicosatetraynoic acid (C20:4), and docosapentanoic acid (C22:5) had no effect, whereas n-3 docosahexanoic acid (C22:6) activated amiloride-sensitive I(Na) in a manner similar to EPA. Activation of I(Na) by EPA was prevented by H-89, a PKA inhibitor. Similarly, PKA activity was stimulated by EPA. Nonspecific stimulation of phosphodiesterase activity by CoCl2 completely prevented the effect of EPA on sodium transport. We conclude that n-3 PUFAs activate epithelial sodium channels downstream of cAMP in a cAMP-dependent pathway also involving PKA., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The activity of epithelial Na(+) selective channels is modulated by various factors, with growing evidence that membrane lipids also participate in the regulation. In the present study, Triton X-100 extracts of whole cells and of apical membrane-enriched preparations from cultured A6 renal epithelial cells were floated on continuous-sucrose-density gradients. Na(+) channel protein, probed by immunostaining of Western blots, was detected in the high-density fractions of the gradients (between 18 and 30% sucrose), which contain the detergent-soluble material but also in the lighter, detergent-resistant 16% sucrose fraction. Single amiloride-sensitive Na(+) channel activity, recorded after incorporation of reconstituted proteoliposomes into lipid bilayers, was exclusively localized in the 16% sucrose fraction. In accordance with other studies, high- and low-density fractions of sucrose gradients likely represent membrane domains with different lipid contents. However, exposure of the cells to cholesterol-depleting or sphingomyelin-depleting agents did not affect transepithelial Na(+) current, single-Na(+) channel activity, or the expression of Na(+) channel protein. This is the first reconstitution study of native epithelial Na(+) channels, which suggests that functional channels are compartmentalized in discrete domains within the plane of the apical cell membrane., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

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Specific hydrolysis of GTP catalyzed by membranes prepared from A6 epithelial cells grown on porous supports was measured. Aldosterone treatment of the cells for 4 h increased Na+ transport and stimulated GTP hydrolysis by apical membranes in vitro more than twofold over basal levels. This stimulation was attributed to an increase in maximum velocity with little change in Michaelis-Menten constant values. Na+ transport rate and GTP hydrolysis were linearly correlated after aldosterone. This relationship was maintained when aldosterone's response was blunted by various inhibitors. Spironolactone decreased both the hormone-stimulated guanosinetriphosphatase (GTPase) and the Na+ transport rate. Pertussis toxin, which exerted minimal effects on basal rates, reduced the increase of Na+ current normally observed after aldosterone and the hormone stimulation of GTPase activity. The expression of classical G(i)/G(o)-type G proteins was not increased after hormone treatment. When A6 cells were grown on nonporous plastic dishes, aldosterone neither stimulated GTPase activity nor increased amiloride- blockable 22Na+ fluxes. We propose that activation of one or more G proteins in the apical membrane of A6 cells is directly involved in the natriferic action of aldosterone., SCOPUS: ar.j, info:eu-repo/semantics/published

Aldosterone treatment of A6 cultured renal epithelial cells methylates the apical membrane, and we examined the aldosterone-induced carboxymethylation of the apical membrane of these cells to determine the targeted polypeptides. Methionine-deprived A6 cells were incubated with aldosterone and [3H]methionine. Homogenates and apical membranes were solubilized and analyzed by SDS-polyacrylamide gel electrophoresis. Label incorporation in a 90-kDa polypeptide was more intense (4-fold) in membranes after aldosterone compared to control. For in vitro methylation, membranes were isolated, incubated with S-adenosyl-L-[methyl-3H]methionine, and analyzed for 3H-methyl uptake. Label incorporation was low in control membranes but markedly stimulated (4-fold) in membrane preparations from aldosterone-treated cells. Guanosine 5′-O-(3-thiotriphosphate) increased in vitro methylation of a 90-kDa polypeptide 5-fold in control membranes but after aldosterone, where methylation was already stimulated, little change was observed. We conclude that aldosterone induces methylation of an apical membrane 90-kDa polypeptide, possibly a subunit of the epithelial Na+ channel, in a GTP-dependent manner, and this may be one of the final steps in a cascade of reactions leading to the natriferic action of this hormone., SCOPUS: ar.j, info:eu-repo/semantics/published