Marie Paule Merville

Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.

ABSTRACT We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-κB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1β (IL-1β) led to ROI production and NF-κB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-κB activation in these cells. IL-1β stimulation of epithelial cells did not generate any ROIs and NF-κB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-κB activation. In monocytic cells, IL-1β treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-κB activation by IL-1β in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-κB activation by IL-1β: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.

Cell type-specific role for reactive oxygen species in nuclear factor-kappaB activation by interleukin-1.

The role of reactive oxygen intermediates (ROIs) in nuclear factor-kappaB (NF-kappaB) activation remains a matter of controversy. We have studied whether ROIs played any role in NF-kappaB induction by interleukin-1beta (IL-1beta) in different cell types. Our studies indicated three different pathways. IL-1beta stimulation of lymphoid cells generates ROIs, which are required for IkappaB-alpha degradation and NF-kappaB activation. The source of these ROIs is the 5-lipoxygenase (5-LOX) enzyme. In monocytic cells, ROIs are also produced in response to IL-1beta and necessary for NF-kappaB induction, but their source appears to be the NADPH oxidase complex. Finally, epithelial cells do not generate ROIs after IL-1beta stimulation, but do rapidly activate NF-kappaB. Interestingly, transfection of epithelial cells with the 5-LOX and 5-LOX activating protein expression vectors restored ROI production and ROI-dependent NF-kappaB activation in response to IL-1beta. Our data thus indicate that ROIs are cell type-specific second messengers for NF-kappaB induction by IL-1beta.

NF-κB activation in response to toxical and therapeutical agents: role in inflammation and cancer treatment

The NF-kappaB transcription factor is ubiquitously expressed and controls the expression of a large number of genes. Experimental data clearly indicate that NF-kappaB is a major regulator of the inflammatory reaction by controlling the expression of pro-inflammatory molecules in response to cytokines, oxidative stress and infectious agents. We demonstrated that NF-kappaB activation by IL-1beta follows three distinct cell-specific pathways. Moreover, our studies indicated that in one model of inflammatory diseases, horse recurrent airway obstruction (RAO), the extent of NF-kappaB basal activity correlates with pulmonary dysfunction. Another role of NF-kappaB activity protects cancer cells against apoptosis and could participate in the resistance to cancer treatment. However, we did not observe any increased cytotoxicity after treatment with anticancer drugs or TNF-alpha of cells expressing a NF-kappaB inhibitor. Therefore, we can conclude that the inhibition of apoptosis by NF-kappaB is likely to be cell type and stimulus-dependent. Further studies are required to determine whether NF-kappaB could be a target for anticancer treatments.

Role of the protein kinase C λ/ι isoform in nuclear factor-κB activation by interleukin-1β or tumor necrosis factor-α: cell type specificities

Abstract It has previously been reported that distinct signaling pathways can lead to nuclear factor (NF)-κB activation following stimulation of different cell types with inflammatory cytokines. As the role of atypical protein kinase C (PKC) isoforms in NF-κB activation remains a matter of controversy, we investigated whether this role might be cell type-dependent. Immunoblots detected atypical PKC expression in all the analyzed cell lines. The PKC inhibitor calphostin C inhibited NF-κB activation by tumor necrosis factor (TNF)-α or interleukin (IL)-1β in Jurkat or NIH3T3 cells but not in MCF7 A/Z cells. Cell transfections with a PKC λ/ι dominant negative mutant abolished TNF-α-induced NF-κB-dependent transcription in NIH3T3 and Jurkat cells but not in MCF7 A/Z cells. Similarly, the same mutant blocked NF-κB-dependent transactivation after IL-1β stimulation of NIH3T3 cells, but was ineffective after IL-1β treatment of MCF7 A/Z cells. In MCF7 A/Z cells, however, the PKC λ/ι dominant negative mutant could abolish transactivation of an AP-1-dependent reporter plasmid after stimulation with TNF-α but not with IL-1β. These data thus confirm that transduction pathways for NF-κB activation after cell stimulation with TNF-α or IL-1β are cell-type specific and that atypical PKC isoforms participate in this pathway in NIH3T3 and Jurkat cells.

The proapoptotic C16-ceramide-dependent pathway requires the death-promoting factor Btf in colon adenocarcinoma cells.

Ceramides are central molecules in sphingolipid metabolism. They are involved in the regulation of cancer-cell growth, differentiation, senescence and apoptosis. To better understand how these secondary messengers induce their biological effects, adenocarcinoma cells (HCT116) were treated with exogenous long-chain ceramides (C16-ceramide) in order to mimic endogenous sphingolipids. This treatment induced a decrease of cell viability partly due to apoptosis as shown by PARP cleavage and a decrease of pro-caspase 3. Two-dimensional differential in-gel electrophoresis (2D-DIGE) revealed the differential expression of 51 proteins in response to C16-ceramide. These proteins are notably involved in cell proliferation, apoptosis, protein transport and transcriptional regulation. Among them, the cell death-promoting factor Btf was found to be implicated in the apoptotic signal triggered by ceramide. In adenocarcinoma cells, Btf regulates apoptosis related proteins such as Mdm2, p53, BAX and pBcl-2 and thus plays an important role in the ceramide mediated cell death. These findings bring new insight into the proapoptotic ceramide-dependent signaling pathway.

New biomarkers for primary mitral regurgitation

BackgroundMitral regurgitation is a frequent valvular heart disease affecting around 2.5xa0% of the population with prevalence directly related to aging. Degeneration of mitral valve is broadly considered as a passive ongoing pathophysiological process and little is known about its physiological deregulation. The purpose of this study was to highlight new biomarkers of mitral regurgitation in order to decipher the underlying pathological mechanism as well as to allow the diagnosis and the monitoring of the disease.ResultsModulation of various blood proteins expression was examined in patients suffering from different grades of mitral regurgitation (mild, moderate and severe) compared to healthy controls. To this end, several routine clinical assays and the multi analyte profile technology targeting 184 proteins were used. High-density lipoprotein, apolipoprotein-A1, haptoglobin and haptoglobin-α2 chain levels significantly decreased proportionally to the degree of mitral regurgitation when compared to controls. High-density lipoprotein and apolipoprotein-A1 levels were associated with effective regurgitant orifice area and regurgitant volume. Apolipoprotein-A1 was an independent predictor of severe mitral regurgitation. Moreover, with ordinal logistic regression, apolipoprotein-A1 remained the only independent factor associated with mitral regurgitation. In addition, myxomatous mitral valves were studied by immunocytochemistry. We observed an increase of LC3, the marker of autophagy, in myxomatous mitral valves compared with healthy mitral valves.ConclusionThese potential biomarkers of mitral regurgitation highlighted different cellular processes that could be modified in myxomatous degenerescence: reverse cholesterol transport, antioxidant properties and autophagy.

Le facteur transcriptionnel NF-κB : rôle au cours de l'oncogenèse et de la réponse au traitement anticancéreux.

Le facteur transcriptionnel NF-κB joue un role central dans la reponse immunitaire et dans les reactions inflammatoires. Diverses observations indiquent, en outre, que NF-κB est probablement implique dans le developpement et la progression de cancers humains. Un role oncogenique direct des proteines Rel/NF-κB na pas pu etre mis en evidence a ce jour, a lexception de loncoproteine virale v-Rel. Il est cependant concevable quune activation, ou une inhibition, du facteur NF-κB puisse participer a la progression de la maladie cancereuse. En effet, NF-κB inhibe lapoptose provoquee par le TNF-α et est implique dans la reponse cellulaire aux dommages induits sur lADN par les therapeutiques anticancereuses. Cela conduit a suggerer que des agents inhibiteurs de lactivite NF-κB pourraient accroitre lefficacite de la chimiotherapie anticancereuse.