Fabienne Desmots

The Reaper-Binding Protein Scythe Modulates Apoptosis and Proliferation during Mammalian Development

ABSTRACT Scythe (BAT3 [HLA-B-associated transcript 3]) is a nuclear protein that has been implicated in apoptosis, as it can modulate Reaper, a central apoptotic regulator in Drosophila melanogaster. While Scythe can markedly affect Reaper-dependent apoptosis in Xenopus laevis cell extracts, the function of Scythe in mammals is unknown. Here, we report that inactivation of Scythe in the mouse results in lethality associated with pronounced developmental defects in the lung, kidney, and brain. In all cases, these developmental defects were associated with dysregulation of apoptosis and cellular proliferation. Scythe− / − cells were also more resistant to apoptosis induced by menadione and thapsigargin. These data show that Scythe is critical for viability and normal development, probably via regulation of programmed cell death and cellular proliferation.

Scythe Regulates Apoptosis-inducing Factor Stability during Endoplasmic Reticulum Stress-induced Apoptosis *□

Scythe (BAT3; HLA-B associated transcript 3, Bag 6) is a protein that has been implicated in apoptosis because it can modulate the Drosophila melanogaster apoptotic regulator, Reaper. Mice lacking Scythe show pronounced defects in organogenesis and in the regulation of apoptosis and proliferation during mammalian development. However, the biochemical pathways important for Scythe function are unknown. We report here multiple levels of interaction between Scythe and the apoptogenic mitochondrial intermembrane protein AIF (apoptosis-inducing factor). Scythe physically interacts with AIF and regulates its stability. AIF stability is markedly reduced in Scythe-/- cells, which are more resistant to endoplasmic reticulum stress induced by thapsigargin. Reintroduction of Scythe or overexpression of AIF in Scythe-/- cells restores their sensitivity to apoptosis. Together, these data implicate Scythe as a regulator of AIF.

Evaluation of the Potential Biological Effects of the 60-GHz Millimeter Waves Upon Human Cells

We investigate potential biological effects of low-power millimeter-wave radiation on human cell viability and intracellular protein homeostasis. A specific exposure system allowing to perform far-field exposures with power densities close to those expected from the future wireless communications in the 60-GHz band has been developed and characterized. Specific absorption rate (SAR) values were determined for the biosamples under test using the FDTD method. It was shown that millimeter-wave radiation at 60.42 GHz and with a maximum incident power density of 1 mW/cm2 does not alter cell viability, gene expression, and protein conformation.

Sequential interplay between BAG6 and HSP70 upon heat shock

Abstract.BAG6/Scythe/Bat3 is a cochaperone of the heat shock protein HSP70 and is involved in various developmental processes, cellular stress and viability. BAG6 interferes with the protein-refolding activity of HSP70 but its precise involvement in proteotoxic stresses remains unknown. We show that BAG6 is required for the accumulation of HSP70 upon heat shock and that conversely, once accumulated, HSP70 leads to the massive and CHIP-independent degradation of BAG6 through the ubiquitin-proteasome system. These reciprocal influences between BAG6 and HSP70 upon heat shock suggest that BAG6 is a central regulator of the cellular content of HSP70. The HSP70-driven degradation of BAG6, following the BAG6-dependent accumulation of HSP70, could allow the protein-refolding activity of HSP70 and limit the extent of its induction.

Roles of heat shock factor 1 and 2 in response to proteasome inhibition: consequence on p53 stability

A single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1−/−, Hsf2−/− or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1−/−- and Hsf2−/−-deficient cells, but through different underlying mechanisms. Contrary to Hsf2−/− cells, Hsf1−/− cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2−/− cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2−/− cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy.

Differential effects of iron overload on GST isoform expression in mouse liver and kidney and correlation between GSTA4 induction and overproduction of free radicles.

We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.

Whole-genome expression analysis in primary human keratinocyte cell cultures exposed to 60 GHz radiation.

The main purpose of this study is to investigate potential responses of skin cells to millimeter wave (MMW) radiation increasingly used in the wireless technologies. Primary human skin cells were exposed for 1, 6, or 24 h to 60.4 GHz with an average incident power density of 1.8 mW/cm(2) and an average specific absorption rate of 42.4 W/kg. A large-scale analysis was performed to determine whether these exposures could affect the gene expression. Gene expression microarrays containing over 41,000 unique human transcript probe sets were used, and data obtained for sham and exposed cells were compared. No significant difference in gene expression was observed when gene expression values were subjected to a stringent statistical analysis such as the Benjamini-Hochberg procedure. However, when a t-test was employed to analyze microarray data, 130 transcripts were found to be potentially modulated after exposure. To further quantitatively analyze these preselected transcripts, real-time PCR was performed on 24 genes with the best combination of high fold change and low P-value. Five of them, namely CRIP2, PLXND1, PTX3, SERPINF1, and TRPV2, were confirmed as differentially expressed after 6 h of exposure. To the best of our knowledge, this is the first large-scale study reporting on potential gene expression modification associated with MMW radiation used in wireless communication applications.

Study of narrow band millimeter-wave potential interactions with endoplasmic reticulum stress sensor genes.

The main purpose of this article is to study potential biological effects of low-power millimeter waves (MMWs) on endoplasmic reticulum (ER), an organelle sensitive to a wide variety of environmental insults and involved in a number of pathologies. We considered exposure frequencies around 60 GHz in the context of their near-future applications in wireless communication systems. Radiations within this frequency range are strongly absorbed by oxygen molecules, and biological species have never been exposed to such radiations in natural environmental conditions. A set of five discrete frequencies has been selected; three of them coincide with oxygen spectral lines (59.16, 60.43, and 61.15 GHz) and two frequencies correspond to the spectral line overlap regions (59.87 and 60.83 GHz). Moreover, we used a microwave spectroscopy approach to select eight frequencies corresponding to the spectral lines of various molecular groups within 59-61 GHz frequency range. The human glial cell line, U-251 MG, was exposed or sham-exposed for 24 h with a peak incident power density of 0.14 mW/cm(2). The average specific absorption rate (SAR) within the cell monolayer ranges from 2.64 +/- 0.08 to 3.3 +/- 0.1 W/kg depending on the location of the exposed well. We analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) the level of expression of two endogenous ER-stress biomarkers, namely, the chaperones BiP/GRP78 and ORP150/GRP170. It was found that exposure to low-power MMW does not significantly modify the mRNA levels of these stress-sensitive genes suggesting that ER homeostasis is not altered by low-power MMW at the considered frequencies.

Activation of C-Jun N-terminal kinase is required for glutathione transferase A4 induction during oxidative stress, not during cell proliferation, in mouse hepatocytes

Expression of the mouse glutathione transferase Alpha 4 (mGSTA4) has been studied during hepatocyte isolation and in cultured hepatocytes. Transient mGSTA4 induction during liver disruption correlated to strong oxidative stress and induction of the Jun N‐terminal kinase (JNK) pathway. Similarly, tumor necrosis factor α induced both JNK phosphorylation and mGSTA4 expression while specific JNK inhibitor JNKI1 prevented these two events and JNK activator anisomycin strongly induced mGSTA4 expression. We also found that endogenous JNK and mGSTA4 co‐immunoprecipitate. A second mGSTA4 induction occurred 2 days after cell seeding concomitantly to DNA replication and was prevented by treatment with mitogen‐activated protein kinase (MEK) inhibitor U0126. Our data demonstrate that mGSTA4 is strongly increased during oxidative stress possibly via JNK pathway and during proliferation via MEK/extracellular signal‐regulated kinase pathway, and suggest that mGSTA4 might be an endogenous regulator of JNK activity by direct binding.