Biomedical subjects

Jacques Piette

Publications and source records attributed to Jacques Piette.

At least 19 recordsLinked to original sources

PubMed2006

Extending the nuclear roles of IkappaB kinase subunits.

The transcription factor NF-kappaB plays a key role in a wide variety of cellular processes such as innate and adaptive immunity, cellular proliferation, apoptosis and development. In unstimulated cells, NF-kappaB is sequestered in the cytoplasm through its tight association with inhibitory proteins called IkappaBs, comprising notably IkappaBalpha. A key step in NF-kappaB activation is the phosphorylation of IkappaBalpha by the so-called IkappaB kinase (IKK) complex, which targets the inhibitory protein for proteasomal degradation and allows the freed NF-kappaB to enter the nucleus where it can transactivate its target genes. The IKK complex is composed of two catalytic subunits called IKKalpha and IKKbeta, and a regulatory subunit called NEMO/IKKgamma. Despite their key role in mediating IkappaBalpha phosphorylation in the cytoplasm, recent works have provided evidence that IKK subunits also translocate into the nucleus to regulate NF-kappaB-dependent and -independent gene expression, paving the way of a novel and exciting field of research. In this review, we will describe the current knowledge in that research area.

Animals↗

PubMed2006

NF-kappaB activation by reactive oxygen species: fifteen years later.

The transcription factor NF-kappaB plays a major role in coordinating innate and adaptative immunity, cellular proliferation, apoptosis and development. Since the discovery in 1991 that NF-kappaB may be activated by H(2)O(2), several laboratories have put a considerable effort into dissecting the molecular mechanisms underlying this activation. Whereas early studies revealed an atypical mechanism of activation, leading to IkappaBalpha Y42 phosphorylation independently of IkappaB kinase (IKK), recent findings suggest that H(2)O(2) activates NF-kappaB mainly through the classical IKK-dependent pathway. The molecular mechanisms leading to IKK activation are, however, cell-type specific and will be presented here. In this review, we also describe the effect of other ROS (HOCl and (1)O(2)) and reactive nitrogen species on NF-kappaB activation. Finally, we critically review the recent data highlighting the role of ROS in NF-kappaB activation by proinflammatory cytokines (TNF-alpha and IL-1beta) and lipopolysaccharide (LPS), two major components of innate immunity.

Animals↗

PubMed2006

TNFalpha- and IKKbeta-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKKgamma and NF-kappaB activation.

Pro-inflammatory cytokines trigger signalling cascades leading to NF-kappaB (nuclear factor-kappaB)-dependent gene expression through IKK [IkappaB (inhibitory kappaB) kinase]-dependent phosphorylation and subsequent degradation of the IkappaB proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNFalpha (tumour necrosis factor alpha) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-kappaB activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKKbeta upon TNFalpha stimulation and that this modification negatively regulates TANK binding to NEMO (NF-kappaB essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNFalpha-mediated induction of a subset of NF-kappaB target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNFalpha by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKKbeta-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-kappaB activation.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Physical and chemical properties of pyropheophorbide-a methyl ester in ethanol, phosphate buffer and aqueous dispersion of small unilamellar dimyristoyl-L-alpha-phosphatidylcholine vesicles.

The aggregation process of pyropheophorbide-a methyl ester (PPME), a second-generation photosensitizer, was investigated in various solvents. Absorption and fluorescence spectra showed that the photosensitizer was under a monomeric form in ethanol as well as in dimyristoyl-L-alpha-phosphatidylcholine liposomes while it was strongly aggregated in phosphate buffer. A quantitative determination of reactive oxygen species production by PPME in these solvents has been undertaken by electron spin resonance associated with spin trapping technique and absorption spectroscopy. In phosphate buffer, both electron spin resonance and absorption measurements led to the conclusion that singlet oxygen production was not detectable while hydroxyl radical production was very weak. In liposomes and ethanol, singlet oxygen and hydroxyl radical production increased highly; the singlet oxygen quantum yield was determined to be 0.2 in ethanol and 0.13 in liposomes. The hydroxyl radical production origin was also investigated. Singlet oxygen was formed from PPME triplet state deactivation in the presence of oxygen. Indeed, the triplet state formation quantum yield of PPME was found to be about 0.23 in ethanol, 0.15 in liposomes (too small to be measured in PBS).

Absorption↗

PubMed2005

Varicella-zoster virus IE63 protein phosphorylation by roscovitine-sensitive cyclin-dependent kinases modulates its cellular localization and activity.

During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested.

Animals↗

PubMed2005

Distinct transduction mechanisms of cyclooxygenase 2 gene activation in tumour cells after photodynamic therapy.

Photodynamic therapy (PDT) is a minimally invasive treatment for cancer and several noncancerous proliferating cell diseases. PDT relies on the uptake of a photosensitizing compound by the pathologic tissue followed by a selective irradiation with visible light, which leads to oxidative stress-mediated cell death. However, some studies showed that PDT induces the release of proangiogenic factors, such as vascular endothelial growth factor, and/or cyclooxygenase-2 (COX-2), thereby promoting cancer cell regrowth following PDT. In this work, we focused on the molecular mechanisms regulating COX-2 expression after low-dose PDT in two cancer cell lines, namely HeLa and T24. We report that PDT induces COX-2 expression in these cells and this expression is mainly due to nuclear factor kappa B (NF-kappaB)-dependent transcription of cox-2 gene without any post-transcriptional regulation. However, the transduction mechanism leading to NF-kappaB activation and subsequent cox-2 gene transcription differs in both cell types. In T24, NF-kappaB activation occurs through a protein kinase C (PKC)alpha- and phosphoinositide-3-kinase (PI3K)-dependent I kappa B kinase (IKK) complex activation, whereas in HeLa cells, NF-kappaB activation is mediated by PKC- and PI3K-independent IKK complex activation.

Cyclooxygenase 2↗

PubMed2005

Perturbation of actin dynamics induces NF-kappaB activation in myelomonocytic cells through an NADPH oxidase-dependent pathway.

Although several reports showed the effect of compounds disrupting microtubules on NF-kappaB (nuclear factor kappaB) activation, nothing is known about agents perturbing actin dynamics. In the present study, we have shown that actin cytoskeleton disruption induced by actin-depolymerizing agents such as cytochalasin D and latrunculin B and actin-polymerizing compounds such as jasplakinolide induced NF-kappaB activation in myelomonocytic cells. The transduction pathway involved the IkappaB (inhibitory kappaB) kinase complex and a degradation of IkappaBalpha. We have shown that NF-kappaB activation in response to the perturbation of actin dynamics required reactive oxygen species, as demonstrated by the effect of antioxidants. Actin cytoskeleton disruption by cytochalasin D induced O2- release from human monocytes, through the activation of the NADPH oxidase, as confirmed by the phosphorylation and by the membrane translocation of p47phox. NF-kappaB activation after actin cytoskeleton disruption could be physiologically relevant during monocyte activation and/or recruitment into injured tissues, where cellular attachment, migration and phagocytosis result in cyclic shifts in cytoskeletal organization and disorganization.

Actins↗

PubMed2005

Sodium nitroprusside-induced osteoblast apoptosis is mediated by long chain ceramide and is decreased by raloxifene.

Release of high levels of nitric oxide (NO) is associated with osteoblastic cell death. The mechanisms of NO-induced cytotoxicity are not well documented and it is presently not known if estrogenic compounds prevent this effect. We studied the role of ceramides in cell death induced by the NO donor sodium nitroprusside (SNP) and we tested the possibility that 17beta-estradiol, the anti-estrogen ICI 182.780 and two selective estrogen receptor modulators raloxifene and tamoxifen modify osteoblastic cell apoptosis. SNP dose-dependently decreased MC3T3-E1 osteoblast viability, increased NO production in the culture media and enhanced the release of intracellular ceramides C22 and C24. Cell death induced by SNP was partially inhibited when MC3T3-E1 cells were pretreated with raloxifene and tamoxifen but was not modified when the cells were pretreated with 17beta-estradiol or ICI 182.780. Cell death induced by SNP resulted from apoptosis as demonstrated by Annexin-V and propidium iodide labeling and a reduction of SNP-induced MC3T3-E1 apoptosis was confirmed in the presence of raloxifene and tamoxifen. SNP induction of C22 and C24 production was inhibited by a pretreatment with raloxifene but not with 17beta-estradiol. Moreover, the synthetic ceramide C24 (0.75 and 1microM) decreased MC3T3-E1 cell viability and osteoblast cell death induced by C24 was partially decreased by raloxifene and to a lesser extent by 17beta-estradiol. These data demonstrate that SNP-induced cell death is mediated by the long chain ceramides C22 and C24 and that raloxifene protected osteoblast from apoptosis induced by SNP, an effect that might be relevant to its pharmacological properties on bone remodeling.

Animals↗

PubMed2005

Interleukin-6 receptor shedding is enhanced by interleukin-1beta and tumor necrosis factor alpha and is partially mediated by tumor necrosis factor alpha-converting enzyme in osteoblast-like cells.

OBJECTIVE: Interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) activation of gp130 represents an alternative pathway for osteoclast development in inflammatory conditions. The goal of the present study was to investigate changes in sIL-6R levels in response to the inflammatory cytokines IL-1beta and tumor necrosis factor alpha (TNFalpha) and to determine the role of TNFalpha-converting enzyme (TACE) in this process. METHODS: Levels of sIL-6R in the culture media of MG63 and SAOS-2 osteoblast-like cell lines after exposure to various agents were determined by immunoassay. TACE protein levels were measured by Western immunoblotting. Cells were transfected with small interfering RNA (siRNA) or with an expression plasmid for IL-6R and TACE to determine the potential involvement of TACE in IL-6R shedding. RESULTS: IL-1beta and TNFalpha increased the levels of sIL-6R in the culture media of MG63 osteoblast-like cells. This effect was not influenced by cycloheximide or 5,6-dichlorobenzimidazole riboside but was markedly inhibited by the calcium chelator EGTA and by the TACE and matrix metalloproteinase inhibitor hydroxamate (Ru36156). IL-1beta and TNFalpha had no influence on the alternatively spliced form of IL-6R RNA. Levels of sIL-6R were reduced when MG63 cells were transiently transfected with TACE siRNA. Transfection of SAOS-2 cells with expression plasmids for IL-6R and TACE produced a dose-dependent increase in sIL-6R levels. CONCLUSION: IL-1beta- and TNFalpha-mediated induction of IL-6R shedding in osteoblast-like cells is at least partly dependent on TACE activation.

ADAM Proteins↗

PubMed2005

Discovery of new rheumatoid arthritis biomarkers using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry ProteinChip approach.

OBJECTIVE: To identify serum protein biomarkers specific for rheumatoid arthritis (RA), using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) technology. METHODS: A total of 103 serum samples from patients and healthy controls were analyzed. Thirty-four of the patients had a diagnosis of RA, based on the American College of Rheumatology criteria. The inflammation control group comprised 20 patients with psoriatic arthritis (PsA), 9 with asthma, and 10 with Crohn's disease. The noninflammation control group comprised 14 patients with knee osteoarthritis and 16 healthy control subjects. Serum protein profiles were obtained by SELDI-TOF-MS and compared in order to identify new biomarkers specific for RA. Data were analyzed by a machine learning algorithm called decision tree boosting, according to different preprocessing steps. RESULTS: The most discriminative mass/charge (m/z) values serving as potential biomarkers for RA were identified on arrays for both patients with RA versus controls and patients with RA versus patients with PsA. From among several candidates, the following peaks were highlighted: m/z values of 2,924 (RA versus controls on H4 arrays), 10,832 and 11,632 (RA versus controls on CM10 arrays), 4,824 (RA versus PsA on H4 arrays), and 4,666 (RA versus PsA on CM10 arrays). Positive results of proteomic analysis were associated with positive results of the anti-cyclic citrullinated peptide test. Our observations suggested that the 10,832 peak could represent myeloid-related protein 8. CONCLUSION: SELDI-TOF-MS technology allows rapid analysis of many serum samples, and use of decision tree boosting analysis as the main statistical method allowed us to propose a pattern of protein peaks specific for RA.

Adult↗

PubMed2005

Varicella-zoster virus IE63 protein represses the basal transcription machinery by disorganizing the pre-initiation complex.

Using transient transfection assays, regulation properties of varicella-zoster virus (VZV)-encoded IE63 protein were analyzed on several VZV immediate early (ORF4), early (ORF28) and late (ORF67) promoters. IE63 was shown to repress the basal activity of most of the promoters tested in epithelial (Vero) and neuronal (ND7) cells to various extents. Trans-repressing activities were also observed on heterologous viral and cellular promoters. Since a construct carrying only a TATA box sequence and a series of wild-type or mutated interleukin (IL)-8 promoters was also repressed by IE63, the role of upstream regulatory elements was ruled out. Importantly, the basal activity of a TATA-less promoter was not affected by IE63. Using a series of IE63 deletion constructs, amino acids 151-213 were shown to be essential to the trans-repressing activity in Vero cells, while in ND7 cells the essential region extended to a much larger carboxy-terminal part of the protein. We also demonstrate that IE63 is capable of disrupting the transcriptional pre-initiation complex and of interacting with several general transcription factors. The central and carboxy-terminal domains of IE63 are important for these effects. Altogether, these results demonstrate that IE63 protein is a transcriptional repressor whose activity is directed towards general transcription factors.

Animals↗

PubMed2004

Downregulation of ICAM-1 and VCAM-1 expression in endothelial cells treated by photodynamic therapy.

Photodynamic therapy (PDT) is a treatment for cancer and several noncancerous proliferating cell diseases that depends on the uptake of a photosensitizing compound followed by selective irradiation with visible light. In the presence of oxygen, irradiation leads to the production of reactive oxygen species (ROS). A large production of ROS induces the death of cancer cells by apoptosis or necrosis. A small ROS production can activate various cellular pathways. Here, we show that PDT by pyropheophorbide-a methyl ester (PPME) induces the activation of nuclear factor kappa B (NF-kappaB) in HMEC-1 cells. NF-kappaB is active since it binds to the NF-kappaB sites of both ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) promoters and induces the transcription of several NF-kappaB target genes such as those of IL-6, ICAM-1, VCAM-1. In contrast, expression of ICAM-1 and VCAM-1 at the protein level was not observed, although we measured an IL-6 secretion. Using specific chemical inhibitors, we showed that the lack of ICAM-1 and VCAM-1 expression is the consequence of their degradation by lysosomal proteases. The proteasome and calpain pathways were not involved. All these observations were consistent with the fact that no adhesion of granulocytes was observed in these conditions.

Cell Adhesion↗

PubMed2004

GSK3-mediated BCL-3 phosphorylation modulates its degradation and its oncogenicity.

The oncoprotein BCL-3 is a nuclear transcription factor that activates NF-kappaB target genes through formation of heterocomplexes with p50 or p52. BCL-3 is phosphorylated in vivo, but specific BCL-3 kinases have not been identified so far. In this report, we show that BCL-3 is a substrate for the protein kinase GSK3 and that GSK3-mediated BCL-3 phosphorylation, which is inhibited by Akt activation, targets its degradation through the proteasome pathway. This phosphorylation modulates its association with HDAC1, -3, and -6 and attenuates its oncogenicity by selectively controlling the expression of a subset of newly identified target genes such as SLPI and Cxcl1. Our results therefore suggest that constitutive BCL-3 phosphorylation by GSK3 regulates BCL-3 turnover and transcriptional activity.

Animals↗

PubMed2004

p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress.

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.

CDC2 Protein Kinase↗

PubMed2004

Enhanced osteoclast development in collagen-induced arthritis in interferon-gamma receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis.

Collagen-induced arthritis (CIA) in mice is accompanied by splenomegaly due to the selective expansion of immature CD11b+ myeloblasts. Both disease manifestations are more pronounced in interferon-gamma receptor knock-out (IFN-gammaR KO) mice. We have taken advantage of this difference to test the hypothesis that the expanding CD11b+ splenic cell population constitutes a source from which osteoclast precursors are recruited to the joint synovia. We found larger numbers of osteoclasts and more severe bone destruction in joints of IFN-gammaR KO mice than in joints of wild-type mice. Osteoclast-like multinucleated cells appeared in splenocyte cultures established in the presence of macrophage colony-stimulating factor (M-CSF) and stimulated with the osteoclast-differentiating factor receptor activator of NF-kappaB ligand (RANKL) or with tumour necrosis factor-alpha (TNF-alpha). Significantly larger numbers of such cells could be generated from splenocytes of IFN-gammaR KO mice than from those of wild-type mice. This was not accompanied, as might have been expected, by increased concentrations of the intracellular adaptor protein TRAF6, known to be involved in signalling of RANKL- and TNF-alpha-induced osteoclast formation. Splenocyte cultures of IFN-gammaR KO mice also produced more TNF-alpha and more RANKL than those of wild-type mice. Finally, splenocytes isolated from immunised IFN-gammaR KO mice contained comparatively low levels of pro-interleukin-1beta (pro-IL-1beta) and pro-caspase-1, indicating more extensive conversion of pro-IL-1beta into secreted active IL-1beta. These observations provide evidence that all conditions are fulfilled for the expanding CD11b+ splenocytes to act as a source of osteoclasts and to be indirectly responsible for bone destruction in CIA. They also provide a plausible explanation for the higher susceptibility of IFN-gammaR KO mice to CIA.

Animals↗

PubMed2004

Expression of 25 kDa thiamine triphosphatase in rodent tissues using quantitative PCR and characterization of its mRNA.

Thiamine triphosphate (ThTP) is found in most organisms, but its biological role remains unclear. In mammalian tissues, cellular ThTP concentrations remain low, probably because of hydrolysis by a specific 25 kDa thiamine triphosphatase (ThTPase). The aim of the present study was to use quantitative PCR, for comparing the 25 kDa ThTPase mRNA expression in various mouse tissues with its enzyme activities. ThTPase mRNA was expressed at only a few copies per cell. The highest amount of mRNA was found in testis, followed by lung and muscle, while the highest enzyme activities were found in liver and kidney. The poor correlation between mRNA levels and enzyme activities might result either from tissue-specific post-transcriptional regulation of mRNA processing and/or translation or from the regulation of enzyme activities by post-translational mechanisms. Purified recombinant human ThTPase was phosphorylated by casein kinase II, but this phosphorylation did not modify the enzyme activity. However, the characterization of the 3'-untranslated mRNA region revealed a unique, highly conserved, 200-nucleotide sequence that might be involved in translational control. In situ hybridization studies in testis suggest a predominant localization of ThTPase mRNA in poorly differentiated spermatogenic cells. This is the first study demonstrating a cell-specific 25 kDa ThTPase mRNA expression, suggesting that this enzyme might be related to the degree of differentiation or the metabolic state of the cell.

3' Untranslated Regions↗

PubMed2004

Ascorbate potentiates the cytotoxicity of menadione leading to an oxidative stress that kills cancer cells by a non-apoptotic caspase-3 independent form of cell death.

Hepatocarcinoma cells (TLT) were incubated in the presence of ascorbate and menadione, either alone or in combination. Cell death was only observed when such compounds were added simultaneously, most probably due to hydrogen peroxide (H2O2) generated by ascorbate-driven menadione redox cycling. TLT cells were particularly sensitive to such an oxidative stress due to its poor antioxidant status. DNA strand breaks were induced by this association but this process did not correspond to oligosomal DNA fragmentation (a hallmark of cell death by apoptosis). Neither caspase-3-like DEVDase activity, nor processing of procaspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) were observed in the presence of ascorbate and menadione. Cell death induced by such an association was actively dependent on protein phosphorylation since it was totally prevented by preincubating cells with sodium orthovanadate, a tyrosine phosphatase inhibitor. Finally, while H2O2, when administered as a bolus, strongly enhances a constitutive basal NF-kappaB activity in TLT cells, their incubation in the presence of ascorbate and menadione results in a total abolition of such a constitutive activity.

Animals↗

PubMed2003

Cell death and growth arrest in response to photodynamic therapy with membrane-bound photosensitizers.

Photodynamic therapy (PDT) is a treatment for cancer and for certain benign conditions that is based on the use of a photosensitizer and light to produce reactive oxygen species in cells. Many of the photosensitizers currently used in PDT localize in different cell compartments such as mitochondria, lysosomes, endoplasmic reticulum and generate cell death by triggering necrosis and/or apoptosis. Efficient cell death is observed when light, oxygen and the photosensitizer are not limiting ("high dose PDT"). When one of these components is limiting ("low dose PDT"), most of the cells do not immediately undergo apoptosis or necrosis but are growth arrested with several transduction pathways activated. This commentary will review the mechanism of apoptosis and growth arrest mediated by two important PDT agents, i.e. pyropheophorbide and hypericin.

Animals↗