Andrée Houbion

Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals

Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase are the most important enzymes of the cell antioxidant defense system. However, these molecules are themselves susceptible to oxidation. The aim of this work was to estimate to what extent this system could be inactivated by its own substrates. We tested the effect of hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and hydroxyl and superoxide radicals on GPX, SOD and catalase. For GPX, a 50% inactivation was observed at 10(-1) M (30 min, 37 degrees C) for hydrogen peroxide, 3 x 10(-4) M (15 min, 37 degrees C) for cumene hydroperoxide and 5 x 10(-5) M (11 min, 37 degrees C) for t-butyl hydroperoxide. Unlike the hydroxyl radicals, superoxide anions did not inactivate this enzyme. Catalase was inactivated by hydroxyl radicals and by superoxide anions but organic peroxides had no effect. SOD was inactivated by 50% by hydrogen peroxide at 4 x 10(-4) M (20 min, 37 degrees C), but organic peroxides and hydroxyl radicals were ineffective on this enzyme. Since the three enzymes of the antioxidant system are susceptible to at least one of the oxidative reactive molecules, in the case of high oxidative stresses such an inhibition could take place, leading to an irreversible autocatalytical process in which the production rate of the oxidants will continuously increase, leading to cell death.

CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation

We characterized a new signaling pathway leading to the activation of cAMP‐responsive element‐binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant‐negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a ρ0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNALys (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant‐negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21Waf1/Cip1, a p53‐regulated cyclin‐dependent kinase inhibitor.

Mitochondrial dysfunction induces triglyceride accumulation in 3T3-L1 cells : role of fatty acid beta-oxidation and glucose.

Mitochondrial cytopathy has been associated with modifications of lipid metabolism in various situations, such as the acquisition of an abnormal adipocyte phenotype observed in multiple symmetrical lipomatosis or triglyceride (TG) accumulation in muscles associated with the myoclonic epilepsy with ragged red fibers syndrome. However, the molecular signaling leading to fat metabolism dysregulation in cells with impaired mitochondrial activity is still poorly understood. Here, we found that preadipocytes incubated with inhibitors of mitochondrial respiration such as antimycin A (AA) accumulate TG vesicles but do not acquire specific markers of adipocytes. Although the uptake of TG precursors is not stimulated in 3T3-L1 cells with impaired mitochondrial activity, we found a strong stimulation of glucose uptake in AA-treated cells mediated by calcium and phosphatidylinositol 3-kinase/Akt1/glycogen synthase kinase 3β, a pathway known to trigger the translocation of glucose transporter 4 to the plasma membrane in response to insulin. TG accumulation in AA-treated cells is mediated by a reduced peroxisome proliferator-activated receptor γ activity that downregulates muscle carnitine palmitoyl transferase-1 expression and fatty acid β-oxidation, and by a direct conversion of glucose into TGs accompanied by the activation of carbohydrate-responsive element binding protein, a lipogenic transcription factor. Taken together, these results could explain how mitochondrial impairment leads to the multivesicular phenotype found in some mitochondria-originating diseases associated with a dysfunction in fat metabolism.

A new technique for highly sensitive detection of superoxide dismutase activity by chemiluminescence

A stable enzymatic free radical generation system has been developed which allows a precise production of 02-. and its detection by chemiluminescence between 2 pmol and 8 nmol. This test has been used for assaying superoxide dismutase (SOD) by inhibition of the chemiluminescence (CL) signal. No inhibition was observed with catalase, which excludes the participation of H2O2 in lucigenin CL. N,N-Diethyldithiocarbamate gives 100% inhibition of SOD activity either from a purified enzymatic preparation or from biological samples, which confirms the specificity of the CL assay. SOD assay can be performed either on a purified enzymatic preparation or on biological materials such as cultured cells.

CREB activation induced by mitochondrial dysfunction triggers triglyceride accumulation in 3T3-L1 preadipocytes.

Several mitochondrial pathologies are characterized by lipid redistribution and microvesicular cell phenotypes resulting from triglyceride accumulation in lipid-metabolizing tissues. However, the molecular mechanisms underlying abnormal fat distribution induced by mitochondrial dysfunction remain poorly understood. In this study, we show that inhibition of respiratory complex III by antimycin A as well as inhibition of mitochondrial protein synthesis trigger the accumulation of triglyceride vesicles in 3T3-L1 fibroblasts. We also show that treatment with antimycin A triggers CREB activation in these cells. To better delineate how mitochondrial dysfunction induces triglyceride accumulation in preadipocytes, we developed a low-density DNA microarray containing 89 probes, which allows gene expression analysis for major effectors and/or markers of adipogenesis. We thus determined gene expression profiles in 3T3-L1 cells incubated with antimycin A and compared the patterns obtained with differentially expressed genes during the course of in vitro adipogenesis induced by a standard pro-adipogenic cocktail. After an 8-day treatment, a set of 39 genes was found to be differentially expressed in cells treated with antimycin A, among them CCAAT/enhancer-binding protein α (C/EBPα), C/EBP homologous protein-10 (CHOP-10), mitochondrial glycerol-3-phosphate dehydrogenase (GPDmit), and stearoyl-CoA desaturase 1 (SCD1). We also demonstrate that overexpression of two dominant negative mutants of the cAMP-response element-binding protein CREB (K-CREB and M1-CREB) and siRNA transfection, which disrupt the factor activity and expression, respectively, inhibit antimycin-A-induced triglyceride accumulation. Furthermore, CREB knockdown with siRNA also downregulates the expression of several genes that contain cAMP-response element (CRE) sites in their promoter, among them one that is potentially involved in synthesis of triglycerides such as SCD1. These results highlight a new role for CREB in the control of triglyceride metabolism during the adaptative response of preadipocytes to mitochondrial dysfunction.

Relationship between the critical level of oxidative stresses and the glutathione peroxidase activity

The production and removal of the various oxygen-derived free radicals is a dynamic and complex process which normally results in a steady state of very low concentrations of these reactive molecules in the cell. The mathematical modelling of this process showed that any lowering of the glutathione peroxidase activity will increase the steady state level of the hydroperoxides and will decrease the level of organic peroxides necessary to destabilize the system. In this paper, we experimentally tested this relationship by the estimation of the level of peroxidative stresses which lead to cell degeneration in the presence of more or less active selenium-dependent glutathione peroxidase (GPX). The enzyme was inhibited by mercaptosuccinate (MS) and the cells were submitted to various extents of oxidative stress using tert-butylhydroperoxide (TBHP). Critical levels of this peroxidative molecule could be determined by the determination of the concentration leading to 50% cell death. A relationship between this critical level of TBHP and the GPX activity was established. The critical level strongly decreased with the inhibition of GPX and was found to be zero when 44% of the GPX activity is inhibited. Presented in this way, the results clearly show the pattern of the inverse relationship between the susceptibility of the cell to oxidative stress and the GPX activity.

Subcellular localization and modification with ageing of glutathione, glutathione peroxidase and glutathione reductase activities in human fibroblasts

Differential centrifugation and isopycnic equilibration in density gradients were used to localize glutathione (GSH), glutathione peroxidase and glutathione reductase in the subcellular organelles of WI-38 fibroblasts. GSH was present in all the subcellular fractions, whereas the glutathione peroxidase and reductase activities were restrained to the cytoplasm and the mitochondrial fractions. After equilibration in density gradients, the results showed the presence of GSH, glutathione peroxidase and glutathione reductase in both the cytoplasm and mitochondria. GSH was also located in plasma membranes and probably in peroxisomes, endoplasmic reticulum and lysosomal membranes. Evolution of GSH in ageing fibroblasts showed a sudden increase of its concentration just before cell death. The glutathione peroxidase activity already decreases in the early passages, while the decrease of the glutathione reductase activity was constant and reached a drastic low level at the end of the culture. In conclusion, GSH is probably involved in the cell degeneration associated with ageing but because of its multiple functions and its ubiquitous localization, it is difficult to assert to which extent this metabolite is implicated in the ageing process.

Mitochondrial biogenesis in mtDNA-depleted cells involves a Ca2+-dependent pathway and a reduced mitochondrial protein import.

Alterations in mitochondrial activity resulting from defects in mitochondrial DNA (mtDNA) can modulate the biogenesis of mitochondria by mechanisms that are still poorly understood. In order to study mitochondrial biogenesis in cells with impaired mitochondrial activity, we used rho‐L929 and rho0143 B cells (partially and totally depleted of mtDNA, respectively), that maintain and even up‐regulate mitochondrial population, to characterize the activity of major transcriptional regulators (Sp1, YY1, MEF2, PPARgamma, NRF‐1, NRF‐2, CREB and PGC‐1α) known to control the expression of numerous nuclear genes encoding mitochondrial proteins. Among these regulators, cyclic AMP‐responsive element binding protein (CREB) activity was the only one to be increased in mtDNA‐depleted cells. CREB activation mediated by a calcium‐dependent pathway in these cells also regulates the expression of cytochrome c and the abundance of mitochondrial population as both are decreased in mtDNA‐depleted cells that over‐express CREB dominant negative mutants. Mitochondrial biogenesis in mtDNA‐depleted cells is also dependent on intracellular calcium as its chelation reduces mitochondrial mass. Despite a slight increase in mitochondrial mass in mtDNA‐depleted cells, the mitochondrial protein import activity was reduced as shown by a decrease in the import of radiolabeled matrix‐targeted recombinant proteins into isolated mitochondria and by the reduced mitochondrial localization of ectopically expressed HA‐apoaequorin targeted to the mitochondria. Decrease in ATP content, in mitochondrial membrane potential as well as reduction in mitochondrial Tim44 abundance could explain the lower mitochondrial protein import in mtDNA‐depleted cells. Taken together, these results suggest that mitochondrial biogenesis is stimulated in mtDNA‐depleted cells and involves a calcium‐CREB signalling pathway but is associated with a reduced mitochondrial import for matrix proteins.

Aging as a multi-step process characterized by a lowering of entropy production leading the cell to a sequence of defined stages. II. Testing some predictions on aging human fibroblasts in culture.

The concepts of irreversible thermodynamics have been used in order to develop a theory of aging considered as a multi-step process leading the cell through a sequence of defined stages characterized by a lower level of entropy production and finally to a critical level of errors involving cell death (Toussaint et al., 1991). One of the predictions of this model is that external stresses which can be considered as fluctuations would accelerate the evolution of the cell from one state to the other according to the intensity of the stress. Seven morphotypes have been observed in the serially cultivated human fibroblasts, cells passing progressively from one morphotype to the other. In this paper, we experimentally tested the effect of two different molecules, tert-butylhydroperoxide and ethanol, in order to determine their influence on the shift from one morphotype to the other. When applied for a single period of time on cultivated cells, both molecules effectively showed a modification in the pattern of the different morphotypes which was dependent on the stress intensity: a decreased proportion of the early morphotypes and an increased proportion of the late and post-mitotic morphotypes were observed within three days after the stresses. Similar results were obtained when successive stresses were performed at every subculture. The results also indicated that all stages are not equally stable with morphotypes III and IV being the most stable. The positive effect on the increased shift of these cells from one morphotype to the other by two different stresses firms one of the prediction of the thermodynamic model which states that cellular aging can be considered as a multi-step process which can be speeded up by various external modifications.

mtCLIC is up-regulated and maintains a mitochondrial membrane potential in mtDNA-depleted L929 cells

To explain why mitochondrial DNA (mtDNA)‐depleted or rho0 cells still keep a mitochondrial membrane potential (Δψm) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1‐ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase‐polymerase chain reaction on L929 mtDNA‐depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up‐regulated in mtDNA‐depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor α. Interestingly, up‐ or down‐regulation of mtCLIC protein expression changes Δψm whereas the chloride channel inhibitor NPPB reduces the Δψm in mtDNA‐depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA‐depleted cells incorporate, in a NPPB‐sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA‐depleted cells, a feature required to prevent apoptosis and to drive continous protein import into mitochondria.