Characterization of Calcineurin in Human Neutrophils

Abstract

We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca 2؉ , inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H 2 O 2 ), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H 2 O 2 . A similar inhibitory effect of H 2 O 2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-B in human neutrophils is inhibited by cell pretreatment with H 2 O 2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharideinduced NF-B/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H 2 O 2 -dependent NF-B inactivation in neutrophils occurs in concert with inhibition of calcineurin.

Oxidative Stress Induces Neuronal Death by Recruiting a Protease and Phosphatase-gated Mechanism

“…This phosphatase has been previously described to regulate dephosphorylation of pCREB in hippocampal neurons (43), and calcineurin has been implicated in neuronal apoptosis (19,20) and in neurodegenerative diseases (18,60). However, activation of this phosphatase during oxidative stress is not in line with other reports that superoxide-induced oxidative stress results in an inhibition of calcineurin activity (61)(62)(63) can impinge on different signaling pathways (51); in addition, cell and tissue type may be important in determining these differences.…”

Oxidative Stress Triggers STAT3 Tyrosine Phosphorylation and Nuclear Translocation in Human Lymphocytes

“…Very recently, we have described that H 2 O 2 and other peroxides are able to inhibit calcineurin activity and to inactivate nuclear factor B DNA binding in human neutrophils (36). In contrast, Schreck et al (2) have provided evidence that peroxides activate nuclear factor B in lymphocytes through an undetermined mechanism involving the synthesis of ROI.…”

“…Next, the membranes were washed twice with TBST, and incubation with a 1:5000 dilution of goat anti-rabbit secondary antibody coupled to horseradish peroxidase was performed for 1 h at room temperature. After washing three times with TBST, immunoreactive bands were visualized by using the enhanced chemiluminescence assay as described previously (36).…”

Stimulators of AMP‐activated protein kinase inhibit the respiratory burst in human neutrophils

“…Proteins extracts in the cell supernatant were separated by SDS-PAGE on 7.5% (w/v) polyacrylamide gels and transferred to PVDF membranes using a semi-dry device (Bio-Rad, Richmond, USA) [27]. The membranes were probed with specific antibodies against phospho-AMPK (Cell Signaling Technology) (1:1000 dilution) in phosphate buffer saline (PBS) containing BSA (1%) and Tween 20 (0.2%), at 4°C overnight, and after washing with PBS the membrane was incubated with HRP-conjugated goat anti-rabbit IgG, followed by enhanced chemiluminescence [27,28]. For the detection of p38MAPK, ERK1/2 and JNK1/2 phosphorylation was carried out as in [29].…”

“…Antibody binding was detected as indicated above. For immunoprecipitation experiments, incubation with anti-p47 phox specific antibodies and subsequent procedure were carried out as indicated [28].…”