Adaptations de la cellule rénale en réponse au Cadmium (rôles du CFTR, de la polyubiquitination sur la Lysine 63 et de l'autophagie)

De nombreux pays sont confrontés aux problèmes causés par la pollution de l air et de l eau par des métaux lourds toxiques. Parmi ces polluants, le cadmium (Cd) est un des plus dangereux car il produit des effets toxiques et cancérogènes, à des doses extrêmement faibles. Chez l Homme, il pénètre par inhalation/ingestion et se concentre principalement dans le rein où il induit une insuffisance rénale mimant le syndrome de Fanconi. A fortes concentrations, il est clairement établi que le Cd induit la production de radicaux libres oxygénés ainsi que l apoptose ou la nécrose, cependant les mécanismes mis-en-jeu restent méconnus. Dans ce travail mené à la fois, in vivo et in vitro, mes objectifs ont été d identifier les cibles moléculaires du Cd ainsi que les stratégies de défense adoptées par les cellules de tubules contournés proximaux (TCP) pour survivre à un tel stress, en utilisant différentes approches cellulaires, moléculaires et biochimiques. Notre première étude réalisée sur des rats intoxiqués avec une faible dose du Cd (0.3 mg/Kg de poids corporel) nous a permis de mettre en évidence l accumulation irréversible du Cd dans les lysosomes avec pour conséquences, la formation des corps d inclusion protéiques spécifiquement au niveau des cellules de TCP suivie de l engagement de ces dernières dans les processus prolifératif et autophagique mais non apoptotique, contrairement à la littérature. Sur la base de ces résultats, nous avons proposé l accumulation des agrégats protéiques et des vésicules autophagiques comme biomarqueurs précoces d intoxication à des doses de Cd relevante de l environnement. Par la suite, grâce à des modèles cellulaires de TCP, nous avons montré que le Cd (i) active via les MAPK le canal CFTR, qui contrôle le niveau des ROS par l efflux des glutathion et entraîne une diminution du volume cellulaire, (ii) induit indépendamment d un stress oxydatif l accumulation massive des protéines poly-ubiquitinées sur la lysine 63, engageant un stress du réticulum et la mort des cellules. De façon intéressante, ces agrégats de protéines poly-ubiquitinées perturbent spécifiquement la dégradation autophagique ; un processus d homéostasie cellulaire important dans la suppression tumorale. Parmi les cibles moléculaires, le Cd induit l ubiquitination du facteur de transcription HIF-1a (hypoxiainducible factor 1) qu il piége dans le cytosol. En conclusion, notre étude représente une étape importante dans la compréhension du mécanisme d action des carcinogènes environnementaux : les capacités du Cd d induire de façon massive une polyubiquitination sur K63 et ainsi de lever la fonction suppresseur de tumeur de l autophagie pourrait en effet fournir le lien manquant entre l exposition à ce métal lourd et les effets toxiques et carcinogènes observés.NICE-BU Sciences (060882101) / SudocSudocFranceTunisiaFRT

CFTR Is Involved in the Fine Tuning of Intracellular Redox Status

International audienceAdaptation to hypoxia is an essential physiological response to decrease in tissue oxygenation. This process is primarily under the control of transcriptional activator hypoxia-inducible factor (HIF1). A better understanding of the intracellular HIF1 stabilization pathway would help in management of various diseases characterized by anemia. Among human pathologies, cystic fibrosis disease is characterized by a chronic anemia that is inadequately compensated by the classical erythroid response mediated by the HIF pathway. Because the kidney expresses CFTR and is a master organ involved in the adaptation to hypoxia, we used renal cells to explore the relationship between CFTR and the HIF1-mediated pathway. To monitor the adaptive response to hypoxia, we engineered a hypoxia-induced fluorescent reporter system to determine whether CFTR modulates hypoxia-induced HIF1 stabilization. We show that CFTR is a regulator of HIF stabilization by controlling the intracellular reactive oxygen species (ROS) level through its ability to transport glutathione (a ROS scavenger) out of the cell. Moreover, we demonstrated in a mouse model that both the pharmacological inhibition and the ΔF508 mutation of CFTR lead to an impairment of the adaptive erythroid response to oxygen deprivation. We conclude that CFTR controls HIF stabilization through control of the level of intracellular ROS that act as signaling agents in the HIF-1 pathway

Subversion of autophagy in adherent invasive Escherichia coli-infected neutrophils induces inflammation and cell death

Invading bacteria are recognized, captured and killed by a specialized form of autophagy, called xenophagy. Recently, defects in xenophagy in Crohn's disease (CD) have been implicated in the pathogenesis of human chronic inflammatory diseases of uncertain etiology of the gastrointestinal tract. We show here that pathogenic adherent-invasive Escherichia coli (AIEC) isolated from CD patients are able to adhere and invade neutrophils, which represent the first line of defense against bacteria. Of particular interest, AIEC infection of neutrophil-like PLB-985 cells blocked autophagy at the autolysosomal step, which allowed intracellular survival of bacteria and exacerbated interleukin-8 (IL-8) production. Interestingly, this block in autophagy correlated with the induction of autophagic cell death. Likewise, stimulation of autophagy by nutrient starvation or rapamycin treatment reduced intracellular AIEC survival and IL-8 production. Finally, treatment with an inhibitor of autophagy decreased cell death of AIEC-infected neutrophil-like PLB-985 cells. In conclusion, excessive autophagy in AIEC infection triggered cell death of neutrophils

The Carcinogen Cadmium Activates Lysine 63 (K63)-Linked Ubiquitin-Dependent Signaling and Inhibits Selective Autophagy

International audienceSignaling, proliferation, and inflammation are dependent on K63-linked ubiquitination—conjugation of a chain of ubiquitin molecules linked via lysine 63. However, very little information is currently available about how K63-linked ubiquitination is subverted in cancer. The present study provides, for the first time, evidence that cadmium (Cd), a widespread environmental carcinogen, is a potent activator of K63-linked ubiquitination, independently of oxidative damage, activation of ubiquitin ligase, or proteasome impairment. We show that Cd induces the formation of protein aggregates that sequester and inactivate cylindromatosis (CYLD) and selective autophagy, two tumor suppressors that deubiquitinate and degrade K63-ubiquitinated proteins, respectively. The aggregates are constituted of substrates of selective autophagy—SQSTM1, K63-ubiquitinated proteins, and mitochondria. These protein aggregates also cluster double-membrane remnants, which suggests an impairment in autophagosome maturation. However, failure to eliminate these selective cargos is not due to alterations in the general autophagy process, as degradation of long-lived proteins occurs normally. We propose that the simultaneous disruption of CYLD and selective autophagy by Cd feeds a vicious cycle that further amplifies K63-linked ubiquitination and downstream activation of the NF-κB pathway, processes that support cancer progression. These novel findings link together impairment of selective autophagy, K63-linked ubiquitination, and carcinogenesis

Autophagy plays a critical role in the degradation of active RHOA, the control of cell cytokinesis, and genomic stability.

International audienceDegradation of signaling proteins is one of the most powerful tumor-suppressive mechanisms by which a cell can control its own growth. Here, we identify RHOA as the molecular target by which autophagy maintains genomic stability. Specifically, inhibition of autophagosome degradation by the loss of the v-ATPase a3 (TCIRG1) subunit is sufficient to induce aneuploidy. Underlying this phenotype, active RHOA is sequestered via p62 (SQSTM1) within autolysosomes and fails to localize to the plasma membrane or to the spindle midbody. Conversely, inhibition of autophagosome formation by ATG5 shRNA dramatically increases localization of active RHOA at the midbody, followed by diffusion to the flanking zones. As a result, all of the approaches we examined that compromise autophagy (irrespective of the defect: autophagosome formation, sequestration, or degradation) drive cytokinesis failure, multinucleation, and aneuploidy, processes that directly have an impact upon cancer progression. Consistently, we report a positive correlation between autophagy defects and the higher expression of RHOA in human lung carcinoma. We therefore propose that autophagy may act, in part, as a safeguard mechanism that degrades and thereby maintains the appropriate level of active RHOA at the midbody for faithful completion of cytokinesis and genome inheritance

PMNs undergo autophagic death and NETosis on infection with AIEC LF82.

<p>Differentiated PLB-985 cells or PMN were infected (MOI 50) with K12 or AIEC LF82 bacteria for 1 h and gentamycin was added for the following 5 h, cells were then analysed. (<b>A</b>) Control or K12- or AIEC LF82-infected PLB-985 cells, treated or not with 3-methyladenine (3-MA, 5 mM, autophagy inhibitor) or Zvad (5 µM, a pancaspase inhibitor), were incubated with propidium iodide (PI) (5 µg/ml) and cell death was analysed by flow cytometric analysis as described in the materials and methods section. The average ± S.D. is shown for three independent experiments, *p<0.003. Inset: immunoblot analysis of LC3-II testifying to the induction of autophagy in infected cells. (<b>B,</b> left panel) PMNs were infected with AIEC LF82 and cell death was assayed as described in A. Maximal cell death (+) was obtained after treatment with etoposide phosphate (100 µg/ml). To test whether cell death was due to apoptosis, a caspase-3 activity test was performed as described in the materials and methods section (right panel). Maximal cell apoptosis (+) was obtained by treatment with staurosporine (10 µM). (<b>C</b>) Cleavage of PARP and caspase-3 in non-infected or K12- and AIEC LF82-infected differentiated PLB-985 cells were analysed by immunoblot analyses. Etoposide phosphate (100 µg/ml) was used as a positive control. LC3-II was detected in LF82-infected cells and compared to uninfected or K12-infected cells (1+5 h, MOI 50). Actin was used as a loading control. (<b>D</b>) Survival of AIEC LF82 and K12 in differentiated PLB-985 cells was analysed with the gentamycin assay (panel one). To compare the ultrastructural morphology of vesicles and bacteria in PLB-985 cells transmission electron microscopy was performed. Ultrastructural analysis of K12-infected differentiated PLB-985 cells shows bacterial sequestration and degradation in phagocytosis vacuoles (panel two). In contrast, accumulation of autophagic vesicles and bacteria (arrowheads) inside the vacuoles was observed in PLB-985 cells (panel three) and PMNs (panel four). Scale bar = 1 µm. (<b>E</b>) Differentiated PLB-985 were seeded on glass coverslips coated with poly D lysine (5 µg/cm²) and allowed to settle for 1 h. Cells were then infected with AIEC-LF82 (MOI 50) for 4 h (1 h+3 h) and 6 h (1 h+5 h), fixed with 3% paraformaldehyde and stained with Hoechst 33342 (0.5 µg/ml) to visualize DNA. Cells were examined with an epifluorescence Axiophot microscope (Zeiss). Early apoptosis and necrosis were assayed by measuring Annexin-V-fluos (Roche) and propidium iodide (PI). Differentiated non-infected PLB-985 cells or cells infected with AIEC LF82 (MOI 50) for 1 h+5 h were incubated with Annexin-V-fluos and PI and fluorescence was detected on a FACS Calibure.</p

Inhibition of autophagic flux by AIEC LF82 infection.

<p>Human neutrophils or neutrophil-like PLB-985 cells were infected with AIEC LF82 at a MOI of 50 for 1 h than gentamicin (100 µg/ml) was added for 3 h. Cells were and processed for immunoblotting (<b>A,</b> left panel, <b>B</b>), quantitative RT-PCR (<b>A</b> rigth panel), immunofluorescence (<b>C</b>, <b>D</b>) and ultrastructural TEM analysis (<b>E</b>). (<b>A</b>) Time-dependent accumulation (1–8 h) of LC3-II and p62 in infected human neutrophils or neutrophil-like PLB-985 cells compared to uninfected cells analyzed at the end time point. Longer exposure detects the LC3-I band. We checked that AIEC infection did not affect p62 mRNA levels by qRT-PCR analysis (<b>A</b> right panel). Data are means ± SEM of three experiments. ** p<0.001. (<b>B</b>) Autophagic flux was analysed by immunoblot analysis in differentiated PLB-985 cells infected for 3 h with AIEC LF82 bacteria (MOI 50) in the absence or in the presence of E64d/PEPS. Actin was used as a loading control. Control unstimulated cells were analysed at the end time point. (<b>C</b>) Representative confocal images of control (0) or infected cells (LF82) (3 h post infection) showed the colocalization of bacteria with LC3-II and LAMP-1 proteins as indicated by yellow punctiform staining. Insets highlight individual staining of bacteria (DNA staining, blue), LC3-II (Alexa 488, green) and LAMP-1 (Alexa 594, red). (<b>D</b>) Representative confocal micrographs of control (0) and LF82 infected cells (LF82) showing the co-localization of bacteria (DNA staining, blue) within autophagic (LC3-II positive, Alexa 594, red) but not acidic compartments (LysoTracker negative, green). (<b>E</b>) Representative TEM images showing bacteria within endosomes (asterisk), autophagosomes (arrowheads) or free in the cytosol (arrow) in LF82-infected cells (3 h post infection). Bar = 2 µm.</p