Michèle Fay

NAD(P)H oxidase Nox-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells

ABSTRACT The mechanisms involved in the cytotoxic action of oxysterols in the pathogenesis of atherosclerosis still remain poorly understood. Among the major oxysterols present in oxidized low-density lipoprotein, we show here that 7-ketocholesterol (7-Kchol) induces oxidative stress and/or apoptotic events in human aortic smooth muscle cells (SMCs). This specific effect of 7-Kchol is mediated by a robust upregulation (threefold from the basal level) of Nox-4, a reactive oxygen species (ROS)-generating NAD(P)H oxidase homologue. This effect was highlighted by silencing Nox-4 expression with a specific small interfering RNA, which significantly reduced the 7-Kchol-induced production of ROS and abolished apoptotic events. Furthermore, the 7-Kchol activating pathway included an early triggering of endoplasmic reticulum stress, as assessed by transient intracellular Ca2+ oscillations, and the induction of the expression of the cell death effector CHOP and of GRP78/Bip chaperone via the activation of IRE-1, all hallmarks of the unfolded protein response (UPR). We also showed that 7-Kchol activated the IRE-1/Jun-NH2-terminal kinase (JNK)/AP-1 signaling pathway to promote Nox-4 expression. Silencing of IRE-1 and JNK inhibition downregulated Nox-4 expression and subsequently prevented the UPR-dependent cell death induced by 7-Kchol. These findings demonstrate that Nox-4 plays a key role in 7-Kchol-induced SMC death, which is consistent with the hypothesis that Nox-4/oxysterols are involved in the pathogenesis of atherosclerosis.

The Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 1/2 Pathway Is Involved in formyl-Methionyl-Leucyl-Phenylalanine-Induced p47phox Phosphorylation in Human Neutrophils

Phosphorylation of p47 phagocyte oxidase, (p47phox), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47phox is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47phox phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47phox. However, PD98059 weakly affected PMA-induced p47phox phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47phox induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47phox phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47phox phosphorylation either directly or indirectly via other kinases. In PMA-induced p47phox phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47phox phosphorylation. However, in fMLP-induced p47phox phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47phox when tested alone, and exerted additive inhibitory effects on p47phox phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47phox. Furthermore, they strongly suggest that p47phox is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.

Phosphorylation of the Respiratory Burst Oxidase Subunit p67 phox during Human Neutrophil Activation REGULATION BY PROTEIN KINASE C-DEPENDENT AND INDEPENDENT PATHWAYS

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen to superoxide anion (O·̄2) at the expense of NADPH. This multicomponent enzyme is dormant in resting cells but is activated on exposure to an appropriate stimulus. The phosphorylation-dependent mechanisms regulating the activation of the respiratory burst oxidase are unclear, particularly the phosphorylation status of the cytosolic component p67 phox . In this study, we found that activation of human neutrophils with formyl-methionyl-leucyl-phenylalanine (fMLP), a chemotactic peptide, or phorbol myristate acetate (PMA), a stimulator of protein kinase C (PKC), resulted in the phosphorylation of p67 phox . Using an anti-p67 phox antibody or an anti-p47 phox antibody, we showed that phosphorylated p67 phox and p47 phox form a complex. Phosphoamino acid analysis of the phosphorylated p67 phox revealed only32P-labeled serine residues. Two-dimensional tryptic peptide mapping analysis showed that p67 phox is phosphorylated at the same peptide whether fMLP or PMA is used as a stimulus. In addition, PKC induced the phosphorylation of recombinant GST-p67 phox in vitro, at the same peptide as that phosphorylated in intact cells. PMA-induced phosphorylation of p67 phox was strongly inhibited by the PKC inhibitor GF109203X. In contrast, fMLP-induced phosphorylation was minimally affected by this PKC inhibitor. Taken together, these results show that p67 phox is phosphorylated in human neutrophils by different pathways, one of which involves protein kinase C.

Priming of Human Neutrophil Respiratory Burst by Granulocyte/Macrophage Colony-stimulating Factor (GM-CSF) Involves Partial Phosphorylation of p47 phox

Neutrophil superoxide production can be potentiated by prior exposure to “priming” agents such as granulocyte/macrophage colony stimulating factor (GM-CSF). Because the mechanism underlying GM-CSF-dependent priming is not understood, we investigated the effects of GM-CSF on the phosphorylation of the cytosolic NADPH oxidase components p47 phox and p67 phox . Preincubation of neutrophils with GM-CSF alone increased the phosphorylation of p47 phox but not that of p67 phox . Addition of formyl-methionyl-leucyl-phenylalanine (fMLP) to GM-CSF-pretreated neutrophils resulted in more intense phosphorylation of p47 phox than with GM-CSF alone and fMLP alone. GM-CSF-induced p47 phox phosphorylation was time- and concentration-dependent and ran parallel to the priming effect of GM-CSF on superoxide production. Two-dimensional tryptic peptide mapping of p47 phox showed that GM-CSF induced phosphorylation of one major peptide. fMLP alone induced phosphorylation of several peptides, an effect enhanced by GM-CSF pretreatment. In contrast to fMLP and phorbol 12-myristate 13-acetate, GM-CSF-induced phosphorylation of p47 phox was not inhibited by the protein kinase C inhibitor GF109203X. The protein-tyrosine kinase inhibitor genistein and the phosphatidylinositol 3-kinase inhibitor wortmannin inhibited the phosphorylation of p47 phox induced by GM-CSF and by fMLP but not that induced by phorbol 12-myristate 13-acetate. GM-CSF alone did not induce p47 phox or p67 phox translocation to the membrane, but neutrophils treated consecutively with GM-CSF and fMLP showed an increase (compared with fMLP alone) in membrane translocation of p47 phox and p67 phox . Taken together, these results show that the priming action of GM-CSF on the neutrophil respiratory burst involves partial phosphorylation of p47 phox on specific serines and suggest the involvement of a priming pathway regulated by protein-tyrosine kinase and phosphatidylinositol 3-kinase.

NADPH oxidase 1 modulates WNT and NOTCH1 signaling to control the fate of proliferative progenitor cells in the colon.

ABSTRACT The homeostatic self-renewal of the colonic epithelium requires coordinated regulation of the canonical Wnt/β-catenin and Notch signaling pathways to control proliferation and lineage commitment of multipotent stem cells. However, the molecular mechanisms by which the Wnt/β-catenin and Notch1 pathways interplay in controlling cell proliferation and fate in the colon are poorly understood. Here we show that NADPH oxidase 1 (NOX1), a reactive oxygen species (ROS)-producing oxidase that is highly expressed in colonic epithelial cells, is a pivotal determinant of cell proliferation and fate that integrates Wnt/β-catenin and Notch1 signals. NOX1-deficient mice reveal a massive conversion of progenitor cells into postmitotic goblet cells at the cost of colonocytes due to the concerted repression of phosphatidylinositol 3-kinase (PI3K)/AKT/Wnt/β-catenin and Notch1 signaling. This conversion correlates with the following: (i) the redox-dependent activation of the dual phosphatase PTEN, causing the inactivation of the Wnt pathway effector β-catenin, and (ii) the downregulation of Notch1 signaling that provokes derepression of mouse atonal homolog 1 (Math1) expression. We conclude that NOX1 controls the balance between goblet and absorptive cell types in the colon by coordinately modulating PI3K/AKT/Wnt/β-catenin and Notch1 signaling. This finding provides the molecular basis for the role of NOX1 in cell proliferation and postmitotic differentiation.

Differential regulation of IL 6, IL 1 A, IL 1β and TNFα production in LPS-stimulated human monocytes: Role of cyclic AMP

Abstract Interleukin 6 (IL 6), IL 1α, IL β and tumor necrosis factor (TNF)α are four cytokines induced in monocytes by lipopolysaccharide (LPS); however, it is unclear whether the mechanisms which control their production are similar. In this study, we report the effects of prostaglandin E2 (PGE2), and two other cAMP-elevating agents, dibutyryl cAMP and 3-isobutyl-1-methylxanthine, on the in vitro LPS-induced production of IL 6, IL 1α, IL 1β and TNFα by human monocytes. The production of these four cytokines was found to be selectively regulated in monocytes, by increases in intracellular cAMP levels. In effect, such agents enhanced, in a dose-dependent manner, both extracellular and cell-associated IL 6 production by LPS-stimulated monocytes. In contrast, it was confirmed, using the same samples, that these cAMP-elevating agents inhibit both extracellular and cell-associated TNFα production in a dose-dependent manner. IL 1α and IL 1β production, measured by means of specific immunoreactive assays, were not significantly modified. Kinetic analysis showed that the potentiating effect of cAMP on IL 6 production, along with its inhibiting effect on TNFα production, could be seen as early as 1 hr after LPS stimulation. These results demonstrate that IL 6, TNFα, IL 1α and IL 1β production can be differently modulated by an agent, PGE2, which is produced simultaneously by LPS-stimulated monocytes. Such differential autocrine modulation may play an important role in the regulation of the production of cytokines participating in immune and inflammatory responses.

Interleukin-8-induced Priming of Neutrophil Oxidative Burst Requires Sequential Recruitment of NADPH Oxidase Components into Lipid Rafts

The superoxide-producing phagocyte NADPH oxidase consists of a membrane-bound flavocytochrome b558, the cytosol factors p47phox, p67phox, p40phox, and the small GTPase Rac2, which translocate to the membrane to assemble the active complex following neutrophil activation. Interleukin-8 (IL-8) does not activate NADPH oxidase, but potentiates the oxidative burst induced by stimuli such as formyl-methionyl-leucyl-phenylalanine (fMLP) via a priming mechanism. The effect of IL-8 on the components of NADPH oxidase during the priming process has never been investigated in human neutrophils. Here we showed that within 3 min, IL-8 treatment enhanced the Btk- and ERK1/2-dependent phosphorylation of p47phox, as well as the recruitment of flavocytochrome b558, p47phox, and Rac2 into cholesterol-enriched detergent-resistant microdomains (or lipid rafts). Conversely, IL-8 treatment lasting 15 min failed to recruit flavocytochrome b558, p47phox, or Rac2, but did enhance the Btk- and p38 MAPK-dependent phosphorylation and the translocation of p67phox into detergent-resistant microdomains. Moreover, methyl-β-cyclodextrin, which disrupts lipid rafts, inhibited IL-8-induced priming in response to fMLP. Our findings indicate that IL-8-induced priming of the oxidative burst in response to fMLP involves a sequential assembly of the NADPH oxidase components in the lipid rafts of neutrophils.

Effects of quinolones on tumor necrosis factor production by human monocytes

Previous studies have shown that in lipopolysaccharide (LPS)-stimulated human monocytes, interleukin 1 (IL-1) production is altered by quinoline derivative antibiotics (quinolones), in a way which depends both on the dose and on the agents used. Given that IL-1 and tumor necrosis factor alpha (TNF) are produced in response to LPS and have some overlapping and synergistic activities, we sought to determine if TNF production was altered under the above-mentioned conditions. We investigated the effects of three quinolones: ciprofloxacin (Cip), pefloxacin (Pef) and ofloxacin (Ofl). These quinolones were found to decrease extracellular TNF production in a dose-dependent manner at concentrations higher than 25 micrograms/ml as previously described by our laboratory with regard to IL-1 production. Moreover, the order of the extracellular decrease in TNF and IL-1 induced by each drug was similar. However, in contrast to IL-1 activity, the quinolones studied also reduced cell-associated TNF. The kinetics of TNF production suggested that the quinolones affected TNF production at a very early step, probably during TNF synthesis rather than during its secretion into the extracellular medium. Furthermore, the quinolone-induced accumulation of intracellular cAMP could explain the extracellular decrease in both IL-1 and TNF production.

Intracellular Pool of IL-10 Receptors in Specific Granules of Human Neutrophils: Differential Mobilization by Proinflammatory Mediators

IL-10 has a wide range of effects tending to control inflammatory responses. We used flow cytometry to study IL-10 binding at the polymorphonuclear neutrophil (PMN) surface and its modulation by various proinflammatory agents. Little IL-10 bound to the surface of resting PMN. However, binding was strongly increased after stimulation with LPS and proinflammatory cytokines such as TNF and GM-CSF. IL-1 and IL-8 did not significantly modify IL-10 binding. Cycloheximide had no effect on TNF-induced IL-10 binding, strongly suggesting the release of a pre-existing pool of IL-10R rather than de novo receptor synthesis by PMN. This was confirmed by the inhibitory effect of pentoxifylline, an inhibitor of degranulation. The existence of an intracellular pool of IL-10R was shown by flow cytometry, immunocytochemical staining, and Western blotting with several anti-human IL-10R Abs. In subcellular fractions of resting PMN, IL-10R was mainly located in the specific granule fraction, and was absent from azurophil granules and cytosol. We also tested the mobilization of specific granules by measuring the release of lactoferrin, their reference marker. The differential effects of the proinflammatory agents on IL-10 binding matched their effects on lactoferrin release and may therefore be related to differential mobilization of specific granules by these agents. Furthermore, the kinetics of TNF-induced up-regulation of IL-10 binding to PMN ran parallel to the kinetics of the inhibitory effect of IL-10 on the oxidative burst, suggesting a key role of IL-10R mobilization from specific granules to the membranes in optimal regulation of inflammatory responses.

Differentiation of PLB-985 myeloid cells into mature neutrophils, shown by degranulation of terminally differentiated compartments in response to N-formyl peptide and priming of superoxide anion production by granulocyte–macrophage colony-stimulating factor

Summary.  Mature blood neutrophils have a short lifespan in vitro and are not easily transfectable. We obtained terminally mature neutrophils after differentiation of immature transfectable PLB‐985 myeloid cells by treatment with dimethylformamide (0·5%), Nutridoma SP (1%) and fetal calf serum (0·5%). Maturation was shown by functional degranulation, in response to bacterial N‐formyl peptide (fMLP), of specific granules and secretory vesicle contents; the latter emerge during the last step of normal neutrophil differentiation into bone marrow. These differentiated cells also produced quantities of superoxide anion similar to those produced by blood neutrophils, in response to physiological stimuli (fMLP); in addition, the fMLP‐induced respiratory burst was primed by the proinflammatory cytokine granulocyte–macrophage colony‐stimulating factor. Thus, in our experimental conditions, PLB‐985 cells transformed into fully differentiated neutrophils capable of fine regulation by inflammatory agents. This cell model will help in the understanding of the molecular mechanisms underlying neutrophil functions.