Sébastien Vankoningsloo

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.

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.

Eco-, geno- and human toxicology of bio-active nanoparticles for biomedical applications

Gene delivery has become an increasingly important strategy for treating a variety of human diseases, including infections, genetic disorders and tumours. To avoid the difficulties of using viral carriers, more and more non-viral gene delivery nanoparticles are developed. Among these new approaches polyethylene imine (PEI) is currently considered as one of the most effective polymer based method solution and considered as the gold standard. The toxicity of nanoparticles is a major concern when used for medical application. In this study we chose two nanoparticles for an in depth toxicological and ecotoxicological evaluation, one well characterized, PEI, and another novel polymer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA). In the present study we have assessed the toxicity of these cation nanoparticles as such and of the polyplexes - nanoparticles covered with DNA. As these nanoparticles are also frequently used in high volumes in various industries and as such may enter in the environment, we also made an initial assessment of ecotoxicological effects assessment. The following nanoparticles related aspects have been studied during the project: development and characterization, ecotoxicity, general toxicity and specific toxicity. To this end a battery of different tests was used. The conclusion of these tests is that toxicity is varying between different nanoparticles and between different DNA covering ratios. In general, in the different systems tested, the PEI polymer is more toxic than the PDMAEMA polymer. The same difference is seen for the polyplexes and the higher the charge ratio, the more toxic are the polyplexes. Our study also clearly shows the need for a broad spectrum of toxicity assays for a comprehensive risk assessment. Our study has performed such a comprehensive analysis of two biomedical nanoparticles.

Differential toxicity of copper (II) oxide nanoparticles of similar hydrodynamic diameter on human differentiated intestinal Caco-2 cell monolayers is correlated in part to copper release and shape

Abstract The potential toxic effects of copper oxide (CuO) nanoparticles (NPs) were studied on differentiated Caco-2 cell monolayers, a classical in vitro model of human small intestine epithelium. Two types of CuO NPs, with different specific surface area, different sizes as raw material but the same hydrodynamic diameter in suspension, differentially disturbed the monolayer integrity, were cytotoxic and triggered an increase of the abundance of several transcripts coding for pro-inflammatory cytokines and chemokines. Specific surface area was not a major variable explaining the increased toxicity when intestinal epithelium is exposed to rod-shaped CuO NPs, compared with spherical CuO NPs. The results suggest that release of Cu(II) cations and shape of these CuO NPs are likely to be implicated in the toxicity of these CuO NPs.

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.

Cytotoxicity of multi-walled carbon nanotubes in three skin cellular models: Effects of sonication, dispersive agents and corneous layer of reconstructed epidermis

Abstract The effects of multi-walled carbon nanotubes were investigated in SZ95 sebocytes, IHK keratinocytes and reconstructed human epidermises. Carbon nanotubes were subjected to dispersion protocols leading to different agglomeration states. Toxicological methods were chosen and adapted in order to ensure compatibility with nanotubes. Results show that: (i) Water-suspended nanotubes, as micrometric agglomerates, were not harmful to skin cells, except minor effects in keratinocytes, (ii) mild sonication slightly decreased nanotube agglomeration but increased cytotoxicity on keratinocytes, (iii) addition of hydroxypropylcellulose or Pluronic F108, which improved nanotube dispersion, masked the harmful effects of sonicated nanotubes. Altogether, these results indicate that carbon nanotubes induced cytotoxicity in human keratinocytes after a short exposure (24–48 h), particularly when they were sonicated before cell incubations. However, the cytotoxic effects of raw and sonicated nanotubes could be prevented in presence of dispersive agents. No cytotoxic effects were observed in SZ95 sebocytes or in stratified epidermises reconstructed in vitro.

Identification of p53-dependent genes potentially involved in UVB-mediated premature senescence of human skin fibroblasts using siRNA technology

Premature senescence of skin human diploid fibroblasts is induced after a series of 10 sublethal exposures to UVB at 2.5 kJ/m(2) with appearance of several biomarkers of cellular senescence like senescence-associated beta-galactosidase activity (SA beta-gal) and cell cycle arrest. Herein it is shown that the induction of UVB-induced premature senescence is associated with a transient increase of protein abundance and DNA-binding activity of p53. Silencing p53 expression with small interfering RNA (siRNA) affected the basal level of SA beta-gal and proliferative potential, but did not prevent UVB-induced increase of SA beta-gal and decrease of DNA synthesis. We used a senescence-specific low-density DNA array and p53 siRNA to study the mRNA abundance of 240 senescence-related genes and identified several potential p53-dependent genes differentially expressed after the repeated exposures to UVB.

Gene expression silencing with ‘specific’ small interfering RNA goes beyond specificity – a study of key parameters to take into account in the onset of small interfering RNA off‐target effects

RNA‐mediated gene silencing (RNA interference) is a powerful way to knock down gene expression and has revolutionized the fields of cellular and molecular biology. Indeed, the transfection of cultured cells with small interfering RNAs (siRNAs) is currently considered to be the best and easiest approach to loss‐of‐function experiments. However, several recent studies underscore the off‐target and potential cytotoxic effects of siRNAs, which can lead to the silencing of unintended mRNAs. In this study, we used a low‐density microarray to assess gene expression modifications in response to five different siRNAs in various cell types and transfection conditions. We found major differences in off‐target signature according to: (a) siRNA sequence; (b) cell type; (c) duration of transfection; and (d) post‐transfection time before analysis. These results contribute to a better understanding of important parameters that could impact on siRNA side effects in knockdown experiments.

Pro-inflammatory effects of different MWCNTs dispersions in p16INK4A-deficient telomerase-expressing human keratinocytes but not in human SV-40 immortalized sebocytes

Abstract We tested whether multi-walled carbon nanotubes (MWCNTs) induce oxidative stress and a pro-inflammatory response in human N-hTERT telomerase-immortalized keratinocytes, in human SZ95 SV-40 immortalized sebocytes and in in vitro reconstructed epidermises. MWCNTS were tested in various dispersion states, from raw and agglomerated particles to isolated entities obtained by sonication in the presence of dispersive agents (hydroxypropylcellulose and Pluronic F108). It was observed that: (a) Contrary to individualized MWCNTs, agglomerated particles prepared by suspension into pure water increased the intracellular levels of reactive oxygen species as well as the expression and secretion of interleukin-8 in N-hTERT cells; (b) the inflammatory signature of MWCNTs in N-hTERT cells, drawn by transcriptomic analysis with low-density microfluidic cards, included various other cytokines such as interleukin-6 or C-C motif ligand 3; (c) the pro-inflammatory effects of MWCNTs, as assessed by interleukin-8 transcript level and protein release, were not observed in SZ95 cells; and (d) the secretion of interleukins-1α and -8 from in vitro reconstructed epidermal tissues, used as specific markers for skin irritation and sensitization, was unaffected in presence of MWCNTs, confirming that the cornified layer is an efficient barrier against MWCNTs.