Age-related tyrosine-specific protein phosphorylation defect in human T lymphocytes activated through CD3, CD4, CD8 or the IL-2 receptor

Although transmembrane signaling defect has been recognized as one of the major functional alterations involved in immune senescence, its biochemical nature as well as its precise molecular localization are still unknown. The available data indicate that an early step in the signaling cascade may be affected during the aging process. Because protein tyrosine kinases (PTK) are ubiquitously implicated in the initiation of physiological signals, they appear as prime candidates for age-related changes. The present investigation examined the effect of age on the activity of PTK associated with CD3, CD4, CD8 or the IL-2 receptor (IL-2R) in human T lymphocytes. By comparison with cells derived from young individuals, anti-CD3-activated T lymphocytes from elderly donors were more susceptible to herbimycin A, a PTK inhibitor known to prevent signal transduction by the T cell antigen receptor. This increased sensitivity of cells from senescent organisms to PTK inhibitors is most likely related to a lesser PTK activity since a significant decrease in the tyrosine phosphorylation of particular endogenous substrates was observed as a consequence of either CD3, CD4, CD8 or IL-2R activation. However, no age-related difference in tyrosine phosphorylation could be demonstrated when T cells were activated by pervanadate, a pharmacological activator of PTK. These results suggest that the intrinsic activity of the enzymes is preserved and that the age-associated defect in PTK activation occurs as a consequence of an upstream biochemical alteration. The defect in PTK activation could be the primary cause for the dysfunction of various components of the signaling cascade observed during the course of aging

Age-associated decline in cdk1 activity delays cell cycle progression of human T lymphocytes

Despite the repeatedly observed impaired proliferative response of T lymphocytes from aged donors, the precise molecular basis underlying such a defect is still poorly understood. The aim of this study was to determine whether cyclin-dependent kinase 1 (cdk1), a serine-threonine kinase required for entry into mitosis, is implicated in this age-associated dysregulation of the cell cycle. T lymphocytes derived from young and elderly donors were blocked in S phase by hydroxyurea after a 48-h activation by anti-CD3 Abs. Under these experimental conditions, only the cells that were already located beyond the S phase were able to complete the cell cycle, decreasing their DNA content from 4n to 2n chromosomes. Using this procedure, a delay in the accomplishment of mitosis could be observed in cells from elderly individuals, as evidenced by propidium iodide staining. In this age group, only a minimal cdk1 activity could be immunoprecipitated from cells sorted in G2/M after nocodazole block. The decrease in cdk1 activity observed in T lymphocytes from aged donors could be accounted for by at least three mechanisms: 1) a failure of these cells to express a sufficient amount of cdk1, 2) a reduced level of the associated cyclin B1, and 3) an incomplete dephosphorylation of the kinase on tyrosine. This low cdk1 activity is likely to postpone the progression through the G2/M transition and participates in the dysfunction of the cell cycle during the process of aging

The HIV-1 Nef protein and phagocyte NADPH oxidase activation

Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia (cell line and primary culture) were transduced with lentiviral expression vectors. Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin). These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits. Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway. The following results suggest a key role for Rac in the priming effect of Nef. (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes. Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli

The HIV-1 Nef protein and phagocyte NADPH oxidase activation

Nef, a multifunctional HIV protein, activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway. Given the potential role of this pathway in the activation of the phagocyte NADPH oxidase, we have investigated the effect of the HIV-1 Nef protein on the phagocyte respiratory burst. Microglia (cell line and primary culture) were transduced with lentiviral expression vectors. Expression of Nef did not activate the NADPH oxidase by itself but led to a massive enhancement of the responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin). These effects were not caused by up-regulation of phagocyte NADPH oxidase subunits. Nef mutants lacking motifs involved in the interaction with Vav and PAK failed to reproduce the effects of wild type Nef, suggesting a role for the Vav/Rac/PAK signaling pathway. The following results suggest a key role for Rac in the priming effect of Nef. (i) Inactivation of Rac by Clostridium difficile toxin B abolished the Nef effect. (ii) The fraction of activated Rac1 was increased in Nef-transduced cells, and (iii) the dominant positive Rac1(V12) mutant mimicked the effect of Nef. These results are to our knowledge the first analysis of the effect of Rac activation on the NADPH oxidase in intact phagocytes. Rac activation is not sufficient to stimulate the phagocyte NADPH oxidase; however, it markedly enhances the NADPH oxidase response to other stimuli

NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation

NOX5 is a ROS-generating NADPH oxidase which contains an N-terminal EF-hand region and can be activated by cytosolic Ca(2+) elevations. However the C-terminal region of NOX5 also contains putative phosphorylation sites. In this study we used HEK cells stably expressing NOX5 to analyze the size and subcellular localization of the NOX5 protein, its mechanisms of activation, and the characteristics of the ROS released. We demonstrate that NOX5 can be activated both by the protein kinase C activating phorbol esther PMA and by the Ca(2+) ionophore ionomycin. The PMA- but not the ionomycin-dependent activation can be inhibited by protein kinase C inhibitors. NOX5 activity is inhibited by submicromolar concentrations of diphenyl iodonium (DPI), but not by apocynin. Western blot analysis showed a lower ( approximately 70 kDa) than expected (82 kDa) molecular mass. Two arguments suggest that NOX5 is at least partially expressed on the plasma membrane: (i) the membrane-impermeant superoxide was readily detected by extracellular probes, and (ii) immunofluorescent labeling of NOX5 detected a fraction of the NOX5 protein at the plasma membrane. In summary, we demonstrate that NOX5 can be found intracellularly and at the cell surface. We also describe that it can be activated through protein kinase C, in addition to its Ca(2+) activation

Three common polymorphisms in the CYBA gene form a haplotype associated with decreased ROS generation

NOX enzymes are reactive oxygen species (ROS)-generating NADPH oxidases. Several members of the NOX family depend on the p22(phox) subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein-Barr (EBV)-transformed B-lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2-dependent ROS generation. Seven single-nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A-G). Haplogroup C (c.214T>C, c.521T>C, and c.(*)24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22(phox)-deficient B-lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.(*)24G>A lies within the 3'UTR. Using a luciferase/3'UTR construct, we showed that the (*)24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation

Evaluation of NADPH oxidases as drug targets in a mouse model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by progressive loss of motor neurons, gliosis, neuroinflammation and oxidative stress. The aim of this study was to evaluate the involvement of NADPH oxidases (NOX) in the oxidative damage and progression of ALS neuropathology. We examined the pattern of NOX expression in spinal cords of patients and mouse models of ALS and analyzed the impact of genetic deletion of the NOX1 and 2 isoforms as well as pharmacological NOX inhibition in the SOD1(G93A) ALS mouse model. A substantial (10-60 times) increase of NOX2 expression was detected in three etiologically different ALS mouse models while up-regulation of some other NOX isoforms was model-specific. In human spinal cord samples, high NOX2 expression was detected in microglia. In contrast to previous publications, survival of SOD1(G93A) mice was not modified upon breeding with constitutive NOX1 and NOX2 deficient mice. As genetic deficiency of a single NOX isoform is not necessarily predictive of a pharmacological intervention, we treated SOD1(G93A) mice with broad-spectrum NOX inhibitors perphenazine and thioridazine. Both compounds reached in vivo CNS concentrations compatible with NOX inhibition and thioridazine significantly decreased superoxide levels in the spinal cord of SOD1(G93A) mice in vivo. Yet, neither perphenazine nor thioridazine prolonged survival. Thioridazine, but not perphenazine, dampened the increase of microglia markers in SOD1(G93A) mice. Thioridazine induced an immediate and temporary enhancement of motor performance (rotarod) but its precise mode of action needs further investigation. Additional studies using specific NOX inhibitors will provide further evidence on the relevance of NOX as drug targets for ALS and other neurodegenerative disorders

NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action

International audienceBACKGROUND Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood. EXPERIMENTAL APPROACH Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins. KEY RESULTS Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration-response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 mM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47 phox translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47 phox and disrupted the binding of the proline rich region of p22 phox to the tandem SH3 domain of p47 phox and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively. CONCLUSIONS AND IMPLICATIONS These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome

NOX4 activity is determined by mRNA levels and reveals a unique pattern of ROS generation

NOX4 is an enigmatic member of the NOX (NADPH oxidase) family of ROS (reactive oxygen species)-generating NADPH oxidases. NOX4 has a wide tissue distribution, but the physiological function and activation mechanisms are largely unknown, and its pharmacology is poorly understood. We have generated cell lines expressing NOX4 upon tetracycline induction. Tetracycline induced a rapid increase in NOX4 mRNA (1 h) followed closely (2 h) by a release of ROS. Upon tetracycline withdrawal, NOX4 mRNA levels and ROS release decreased rapidly (<24 h). In membrane preparations, NOX4 activity was selective for NADPH over NADH and did not require the addition of cytosol. The pharmacological profile of NOX4 was distinct from other NOX isoforms: DPI (diphenyleneiodonium chloride) and thioridazine inhibited the enzyme efficiently, whereas apocynin and gliotoxin did not (IC50>100 μM). The pattern of NOX4-dependent ROS generation was unique: (i) ROS release upon NOX4 induction was spontaneous without need for a stimulus, and (ii) the type of ROS released from NOX4-expressing cells was H2O2, whereas superoxide (O2−) was almost undetectable. Probes that allow detection of intracellular O2− generation yielded differential results: DHE (dihydroethidium) fluorescence and ACP (1-acetoxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine) ESR measurements did not detect any NOX4 signal, whereas a robust signal was observed with NBT. Thus NOX4 probably generates O2− within an intracellular compartment that is accessible to NBT (Nitro Blue Tetrazolium), but not to DHE or ACP. In conclusion, NOX4 has a distinct pharmacology and pattern of ROS generation. The close correlation between NOX4 mRNA and ROS generation might hint towards a function as an inducible NOX isoform