Jean-Pascal Piret

Is HIF-1α a pro- or an anti-apoptotic protein? ☆

Abstract Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor specifically activated by hypoxia. It induces the expression of different genes whose products play an adaptive role for hypoxic cells and tissues. Besides these protective responses, HIF-1 and/or hypoxia have also been shown to be either anti-apoptotic or pro-apoptotic, according to the cell type and experimental conditions. More severe or prolonged hypoxia rather induces apoptosis that is, at least in part, initiated by the direct association of HIF-1α and p53 and p53-induced gene expression. On the other hand, HIF-1α dimerized with ARNT, as an active transcription factor, can protect cells from apoptosis induced by several conditions. This review is aimed to describe the different mechanisms that account for these opposite effects of HIF-1α.

CoCl2, a Chemical Inducer of Hypoxia‐Inducible Factor‐1, and Hypoxia Reduce Apoptotic Cell Death in Hepatoma Cell Line HepG2

Abstract: HIF‐1 (hypoxia‐inducible factor‐1) is the major transcription factor that is specifically activated during hypoxia. This transcription factor is composed of two subunits: HIF‐1α and ARNT (aryl hydrocarbon receptor nuclear translocator). ARNT is constitutively expressed, whereas HIF‐1α is targeted to proteasome degradation by ubiquitination during normoxia. In hypoxia, HIF‐1α is stabilized and translocates to the nucleus, where it binds to ARNT. The active HIF‐1 induces expression of various genes whose products play an adaptive role to the new conditions induced by hypoxia. Besides the role played by HIF‐1 in the adaptation to hypoxia, recent data describe a possible role for HIF‐1 in the modulation of apoptosis. According to some authors, hypoxia induces apoptosis. However, it has also been reported that hypoxia could protect cells against apoptotic cell death induced by various agents such as serum deprivation and incubation in the presence of chemotherapy agents. These contradictory data suggest that HIF‐1 could display either a proapoptotic or an antiapoptotic role according to the conditions. In order to study how HIF‐1 can modulate apoptosis, we studied whether hypoxia or cobalt chloride, a chemical inducer of HIF‐1, could influence apoptosis induced by tert‐butyl hydroperoxide (t‐BHP), serum deprivation, or both in hepatoma cell line HepG2. HepG2 cells were incubated 8 hours under normoxia or hypoxia in the presence of t‐BHP with or without CoCl2. CoCl2 reduced the apoptotic death of HepG2 cells induced by t‐BHP and serum deprivation, as measured by DNA fragmentation. This effect was confirmed by measurement of the caspase activity. Moreover, hypoxia also prevented t‐BHP‐ or serum deprivation‐induced DNA fragmentation and caspase activation‐however, to a lower extent than CoCl2. These different data suggest a possible antiapoptotic role of HIF‐1. More experiments are needed to define if HIF‐1 actually plays an active role in cell death protection and to determine the exact mechanism underlying this effect.

HIF-1 and AP-1 Cooperate to Increase Gene Expression in Hypoxia: Role of MAP Kinases

HIF‐1 is the main transcription factor responsible for increased gene expression in hypoxia: VEGF, erythropoietin, GLUT‐1, and glycolytic enzymes are such target genes and all participate in the adaptative response of cells to hypoxia. AP‐1 activation by hypoxia has also been demonstrated in several cell lines and it cooperates with HIF‐1 for increasing VEGF gene transcription in hypoxia. Both HIF‐1 and AP‐1 activation by hypoxia seems to involve members of the MAP kinase family. Here, we summarize the data indicating that ERK and JNK are needed for activation of HIF‐1 and AP‐1, respectively.

Copper(II) oxide nanoparticles penetrate into HepG2 cells, exert cytotoxicity via oxidative stress and induce pro-inflammatory response

The potential toxic effects of two types of copper(II) oxide (CuO) nanoparticles (NPs) with different specific surface areas, different shapes (rod or spheric), different sizes as raw materials and similar hydrodynamic diameter in suspension were studied on human hepatocarcinoma HepG2 cells. Both CuO NPs were shown to be able to enter into HepG2 cells and induce cellular toxicity by generating reactive oxygen species. CuO NPs increased the abundance of several transcripts coding for pro-inflammatory interleukins and chemokines. Transcriptomic data, siRNA knockdown and DNA binding activities suggested that Nrf2, NF-κB and AP-1 were implicated in the response of HepG2 cells to CuO NPs. CuO NP incubation also induced activation of MAPK pathways, ERKs and JNK/SAPK, playing a major role in the activation of AP-1. In addition, cytotoxicity, inflammatory and antioxidative responses and activation of intracellular transduction pathways induced by rod-shaped CuO NPs were more important than spherical CuO NPs. Measurement of Cu(2+) released in cell culture medium suggested that Cu(2+) cations released from CuO NPs were involved only to a small extent in the toxicity induced by these NPs on HepG2 cells.

Casein kinase 2 inhibition decreases hypoxia-inducible factor-1 activity under hypoxia through elevated p53 protein level

HIF-1 (hypoxia-inducible factor-1) is the main transcription factor involved in the adaptation of cells to hypoxia. In addition to regulation of HIF-1α protein level, HIF-1 activity is also enhanced by several pathways involving asparagine hydroxylation and phosphorylation. Here, we investigated the relationship between casein kinase 2 (CK2), p53 and HIF-1. An increase in p53 protein level and transcriptional activity was observed when CK2 was inhibited by different inhibitors under normoxia and hypoxia. This increase was in parallel with a decrease in HIF-1 activity without changes in HIF-1α protein level, indicating a regulation of its transcriptional activity. Similar results were obtained using CK2α siRNA. Ectopic overexpression of p53 also led to an inhibition of HIF-1 activity. Conversely, CK2 inhibition had no effect in p53-null cells indicating that the inhibitory effect of CK2 inhibitors requires the presence of p53. p53 activity was not required because overexpression of a p53 mutated in its DNA-binding domain exerted the same effect as wild-type p53 and because the effect of CK2 inhibitors was still observed when p53 activity was inhibited by pifithrin-α. Since CK2 activity is increased in hypoxic conditions, this process provides one more mechanism to ensure enhanced HIF-1 activity under such conditions.

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.

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.

Validation of the calibrated thrombin generation test (cTGT) as the reference assay to evaluate the procoagulant activity of nanomaterials

Abstract We validated a preclinical toxicological screening assay and provided guidelines to evaluate the potential impact of nanoparticles (NPs) on blood coagulation. Five NPs with various physicochemical properties were studied using several existing methods of clotting times and thrombin generation assays in human normal pool plasma. In both recalcification clotting time (RCT) and calibrated thrombin generation test (cTGT), the NPs exhibited procoagulant activity (SiO2 ≥ SiC ≥ TiC > CuO > CB) but cTGT was more sensitive and relevant than RCT. Thus, the cTGT appears as a reference assay to investigate the nanoparticle (NP) procoagulant activity in human plasma. It should be used as the reference toxicity test for evaluating the effects of nanomaterials on coagulation cascade. In addition, we also showed that the use of the Pluronic F-108 dispersant and/or the sonication for the NP suspension preparation may mask their procoagulant activity and thus should be avoided.

Role of p38MAPK and oxidative stress in copper-induced senescence

In the present work, we indicate that copper is involved in the senescence of human diploid fibroblasts and we describe mechanisms to explain it. Using different techniques, we show for the first time an accumulation of copper in cells during replicative senescence. This accumulation seems to be co-localized with lipofuscin. Second, we observed that an incubation of cells with copper sulfate induced oxidative stress, antioxidant response and premature senescence. Antioxidant molecules reduced the appearance of premature senescence. Third, we found that Nrf2 transcription factor was activated and regulated the expression of genes involved in antioxidant response while p38MAPK regulated the appearance of premature senescence.

How does the deposited dose of oxide nanomaterials evolve in an in vitro assay

In this work, the evolution of some key physicochemical parameters of oxide engineered nanomaterial (ENM) dispersions was studied during an in vitro biological assessment. Commercial oxide ENMs, SiO2 and TiO2, were dispersed in aqueous solutions (20 μg/mL) to A549 cells and N-hTERT keratinocytes and were assessed at several incubation times: 6, 24, 48, and 72 hours. The ENMs deposited dose and its particle size distribution (PSD) were followed each time. Centrifuge Liquid Sedimentation (CLS) measured the PSD and the ENMs deposited dose from a particle entity perspective, while Particle-Induced X-ray Emission (PIXE) measured the ENMs deposited dose from an elemental mass perspective. No significant variations in PSD were observed for SiO2 ENMs during incubation in A549 cells and TiO2 ENMs in both cell lines, while a continuous evolution of the PSD is observed for SiO2 in N-hTERT keratinocytes. The deposited dose for TiO2 ENMs remained stable and similar in both cell lines due to a smaller specific surface area and a higher quantity of primary particles present during incubation. It is concluded that the observed differences in the deposited dose are related to an interaction between the proteins present in the media and the ENMs specific surface.