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Sabine Gerbal-Chaloin

Publications and source records attributed to Sabine Gerbal-Chaloin.

9 recordsLinked to original sources
PubMed2007

First step of the cell-penetrating peptide mechanism involves Rac1 GTPase-dependent actin-network remodelling.

BACKGROUND INFORMATION: Application of CPPs (cell-penetrating peptides) constitutes a promising strategy for the intracellular delivery of therapeutic molecules. The non-covalent approach based on the amphipathic peptide MPG has been successfully used to improve the delivery of biologically active macromolecules, both in cellulo and in vivo, through a mechanism independent of the endosomal pathway and mediated by the membrane potential. RESULTS: In the present study, we have investigated the first step of the cellular uptake mechanism of MPG and shown that both MPG and MPG-cargo complexes interact with the extracellular matrix through the negatively charged heparan sulfate proteoglycans. We demonstrated that initiation of cellular uptake constitutes a highly dynamic mechanism where the binding of MPG or the MPG-cargo to the extracellular matrix is rapidly followed by a remodelling of the actin network associated with the activation of the GTPase Rac1. We suggest that MPG-induced clustering of the glycosaminoglycan platform constitutes the 'onset' of the cellular uptake mechanism, thereby increasing membrane dynamics and membrane fusion processes. This process favours cell entry of MPG or MPG-DNA complexes, which is further controlled by the ability of MPG to induce a local membrane destabilization. CONCLUSIONS: Although CPPs are taken up through different pathways and mechanisms, the initial step involves electrostatic interactions with the glycosaminoglycan platform, and the dynamics of associated membrane microdomains can be generalized to most non-viral delivery systems.

Actins↗
PubMed2006

Ketoconazole and miconazole are antagonists of the human glucocorticoid receptor: consequences on the expression and function of the constitutive androstane receptor and the pregnane X receptor.

The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) play a major part in the control of drug metabolism and transport. We have previously shown that PXR and CAR expression is controlled by the glucocorticoid receptor (GR) and proposed the existence of a signal transmission cascade GR-(PXR/CAR)-drug metabolizing and transporter systems. In the current study, we investigated the effect of ketoconazole and other azole-derived drugs, miconazole and fluconazole, on the transcriptional activity of the human GR (hGR) in HeLa and HepG2 cells, and in primary human hepatocytes. The data show that ketoconazole inhibits GR transcriptional activity and competes with dexamethasone for hGR binding. In primary human hepatocytes, ketoconazole inhibits the expression of 1) GR-responsive genes tyrosine aminotransferase and both PXR and CAR; 2) CAR and PXR target genes, including cytochromes P450 (P450) CYP2B6, CYP2C9, and CYP3A4; UDP-glucuronosyltransferase 1A1, glutathione S-transferases A1 and A2; and transporter proteins (phase III) solute carrier family 21 form A6 and multidrug resistance protein 2. In parallel experiments, ketoconazole affected neither the expression of GR, the expression of glyceraldehyde-3-phosphate dehydrogenase, nor the inducible expression of CYP1A1 and 1A2. Miconazole behaved like ketoconazole, whereas fluconazole had no effect. We conclude that, in addition to their well known inhibitory effect on P450 enzyme activities, ketoconazole and miconazole are antagonists of hGR. These results provide a novel molecular mechanism by which these compounds may exert adverse and toxic effects on drug metabolism and other functions in human.

Adult↗
PubMed2005

Role of CYP3A4 in the regulation of the aryl hydrocarbon receptor by omeprazole sulphide.

Cross-talk between nuclear receptors involved in the control of drug metabolism is being increasingly recognised as a source of drug side effects. Omeprazole is a well known activator of the aryl hydrocarbon receptor (AhR). We investigated the regulation of AhR by omeprazole-sulphide, a degradation metabolite of omeprazole, using CYP1A mRNA induction, reporter gene assay, receptor DNA binding, ligand binding, nuclear translocation, trypsin digests, and drug metabolism analysis in mouse Hepa-1c1c7, human HepG2 cells and primary human hepatocytes. Omeprazole-sulphide is a pure antagonist of AhR in Hepa-1c1c7 and HepG2 hepatoma cell lines. In Hepa-1c1c7 cells, omeprazole-sulphide is a ligand of AhR, inhibits AhR activation to a DNA-binding form, induces a specific pattern of AhR trypsin digestion and inhibits AhR nuclear translocation and subsequent degradation in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, in highly differentiated primary human hepatocytes treated with rifampicin an agonist of the pregnane X receptor (PXR), omeprazole-sulphide behaves as an agonist of AhR. Inhibition of drug metabolizing enzymes by ketoconazole restores the antagonist effect of omeprazole-sulphide. Metabolic LC/MS analysis reveals that omeprazole-sulphide (AhR antagonist) is efficiently converted to omeprazole (AhR activator) by cytochrome P450 CYP3A4, a target gene of PXR, in primary human hepatocytes but not in hepatoma cells in which PXR is not expressed. This report provides the first evidence for a cross-talk between PXR/CYP3A4 and AhR. In addition, it clearly shows that conclusions drawn from experiments carried out in cell lines may lead to erroneous in vivo predictions in man.

Active Transport, Cell Nucleus↗
PubMed2005

Design of a novel class of peptide inhibitors of cyclin-dependent kinase/cyclin activation.

Cyclin dependent kinases (CDKs) are key regulators of the cell cycle progression and therefore constitute excellent targets for the design of anticancer agents. Most of the inhibitors identified to date inhibit kinase activity by interfering with the ATP-binding site of CDKs. We recently proposed that the protein/protein interface and conformational changes required in the molecular mechanism of CDK2-cyclin A activation were potential targets for the design of specific inhibitors of cell cycle progression. To this aim, we have designed and characterized a small peptide, termed C4, derived from amino acids 285-306 in the alpha5 helix of cyclin A. We demonstrate that this peptide does not interfere with complex formation but forms stable complexes with CDK2-cyclin A. The C4 peptide significantly inhibits kinase activity of complexes harboring CDK2 in a competitive fashion with respect to substrates but does not behave as an ATP antagonist. Moreover, when coupled with the protein transduction domain of Tat, the C4 peptide blocks the proliferation of tumor cell lines, thereby constituting a potent lead for the development of specific CDK-cyclin inhibitors.

Amino Acid Sequence↗
PubMed2004

On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids.

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a beta-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.

Amino Acid Sequence↗
PubMed2004

Interleukin 1beta inhibits CAR-induced expression of hepatic genes involved in drug and bilirubin clearance.

During the inflammatory response, intrahepatic cholestasis and decreased drug metabolism are frequently observed. At the hepatic level, the orphan nuclear constitutive androstane receptor (CAR) (NR1I3) controls phase I (cytochrome P450 [CYP] 2B and CYP3A), phase II (UGT1A1), and transporter (SLC21A6, MRP2) genes involved in drug metabolism and bilirubin clearance in response to xenobiotics such as phenobarbital or endobiotics such as bilirubin. We investigated the negative regulation of CAR, a glucocorticoid-responsive gene, via proinflammatory cytokine interleukin 1beta (IL-1beta) and lipopolysaccharides (LPSs) in human hepatocytes. We show that IL-1beta decreases CAR expression and decreases phenobarbital- or bilirubin-mediated induction of CYP2B6, CYP2C9, CYP3A4, UGT1A1, GSTA1, GSTA2, and SLC21A6 messenger RNA. This occurs via nuclear factor kappaB (NF-kappaB) p65 activation, which interferes with the enhancer function of the distal glucocorticoid response element that we have identified recently in the CAR promoter. We demonstrate that: (1) LPSs, IL-1beta, or overexpression of p65RelA inhibit glucocorticoid receptor (GR)-mediated CAR transactivation; (2) these suppressive effects can be blocked both by pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB activation, or by overexpression of SRIkBalpha, a NF-kappaB repressor; and (3) the GR agonist dexamethasone induces histone H4 acetylation at the proximal CAR promoter region, whereas LPSs and IL-1beta inhibit this acetylation as assessed via chromatin immunoprecipitation assay. In conclusion, GR/NF-kappaB interaction affects CAR gene transcription through chromatin remodeling and provide a mechanistic explanation for the long-standing observation that inflammation and sepsis inhibit drug metabolism while inducing intrahepatic cholestasis or hyperbilirubinemia.

Acetylation↗
PubMed2003

Pathophysiological factors affecting CAR gene expression.

The body defends itself against potentially harmful compounds, such as drugs and toxic endogenous compounds and their metabolites, by inducing the expression of enzymes and transporters involved in their metabolism and elimination. The orphan nuclear receptor CAR (NR1I3 controls phase I (CYP2B, CYP2C, CYP3A), phase II (UGT1A1), and transporter (SLC21A6, MRP2) genes involved in drug metabolism and bilirubin clearance. Constitutive androstane receptor (CAR) is activated by xenobiotics, such as phenobarbital, but also by toxic endogenous compounds such as bilirubin metabolite(s). To better understand the inter- and intravariability in drug detoxification, we studied the molecular mechanisms involved in CAR gene expression in human hepatocytes. We clearly identified CAR as a glucocorticoid receptor (GR) target gene, and we proposed the hypothesis of a signal transduction where the activation of GR plays a critical function in CAR-mediated cellular response. According to our model, chemicals or pathophysiological factors that affect GR function should decrease CAR function. To test this hypothesis, we recently investigated the effect of microtubule disrupting agents (MIAs) or proinflammatory cytokines. These compounds are well-known inhibitors of GR transactivation property. MIAs activate c-Jun N-terminal kinase (JNK), which phosphorylates and inactivates GR, whereas proinflammatory cytokines, such as IL-6 or IL1beta, induce AP-1 or NF-kB activation, respectively, leading to GR inhibition. As expected, we observed that these molecules inhibit both CAR gene expression and phenobarbital-mediated CYP gene expression in human hepatocytes.

Animals↗
PubMed2001

Transcriptional regulation of CYP2C9 gene. Role of glucocorticoid receptor and constitutive androstane receptor.

Although cytochrome P450 2C9 (CYP2C9) is a major CYP expressed in the adult human liver, its mechanism of regulation is poorly known. In previous work, we have shown that CYP2C9 is inducible in primary human hepatocytes by xenobiotics including dexamethasone, rifampicin, and phenobarbital. The aim of this work was to investigate the molecular mechanism(s) controlling the inducible expression of CYP2C9. Deletional analysis of CYP2C9 regulatory region (+21 to -2088) in the presence of various hormone nuclear receptors suggested the presence of two functional response elements, a glucocorticoid receptor-responsive element (-1648/-1684) and a constitutive androstane receptor-responsive element (CAR, -1783/-1856). Each of these were characterized by co-transfection experiments, directed mutagenesis, gel shift assays, and response to specific antagonists RU486 and androstanol. By these experiments we located a glucocorticoid-responsive element imperfect palindrome at -1662/-1676, and a DR4 motif at -1803/-1818 recognized and transactivated by human glucocorticoid receptor and by hCAR and pregnane X receptor, respectively. Identification of these functional elements provides rational mechanistic basis for CYP2C9 induction by dexamethasone (submicromolar concentrations), and by phenobarbital and rifampicin, respectively. CYP2C9 appears therefore to be a primary glucocorticoid-responsive gene, which in addition, may be induced by xenobiotics through CAR/pregnane X receptor activation.

Aryl Hydrocarbon Hydroxylases↗