Abstract
In the absence of survival factors, blood monocytes undergo spontaneous apoptosis, which involves the activation of caspase-3. Although nitric oxide can block caspase-3 activation and promote cell survival, it can also induce apoptosis. We hypothesized that nitrosothiols that promote protein S-nitrosylation would reduce caspase-3 activation and cell survival, whereas nitric oxide donors (such as 1-propamine 3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA) NONOate and diethylamine (DEA) NONOate) that do not target thiol residues would not. Using human monocytes as a model, we observed that nitrosothiol donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine suppressed caspase-9 and caspase-3 activity and DNA fragmentation. In contrast, PAPA or DEA NONOate did not promote monocyte survival events and appeared to inhibit monocyte survival induced by macrophage colony-stimulating factor. The caspase-3-selective inhibitor DEVD-fluoromethyl ketone reversed DNA fragmentation events, and the caspase-9 inhibitor LEHD-fluoromethyl ketone reversed caspase-3 activity in monocytes treated with PAPA or DEA NONOate in the presence of macrophage colony-stimulating factor. These results were not caused by differences in glutathione levels or the kinetics of nitric oxide release. Moreover, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine directly blocked the activity of recombinant caspase-3, which was reversed by the reducing agent dithiothreitol, whereas PAPA or DEA NONOate did not block the enzymatic activity of caspase-3. These data support the hypothesis that nitrosylation of protein thiol residues by nitric oxide is critical for promoting the survival of human monocytes.
Highlights
In the absence of specific survival factors, human monocytes spontaneously undergo apoptosis in 24 – 48 h [1, 2]
We investigated whether PAPA NONOate or DEA NONOate would reverse the suppression of caspase-3 and DNA fragmentation promoted by macrophage colony-stimulating factor (M-CSF) in human monocytes
Incubating human monocytes with the nitrosothiol donors GSNO and SNAP suppressed the activation of native caspase-3, reduced the enzymatic activity of active recombinant caspase-3, and reduced DNA fragmentation in monocytes incubated with or without M-CSF
Summary
Materials—RPMI 1640 medium and Dulbecco’s modified Eagle’s medium were purchased from BioWhittaker, Inc. (Walkersville, MD). Recombinant human M-CSF was purchased from R&D Systems (Minneapolis, MN). The anti-phosphotyrosine antibody used is a 30:30:1 ratio of clonal antibodies PY72, PY20, and 4G10 obtained from Dr Bart Sefton (Transduction Laboratories, San Diego, CA) and Upstate Biotechnology, Inc. The anticaspase-3 antibody was purchased from Upstate Biotechnology, Inc. The caspase inhibitors DEVD-fluoromethyl ketone (FMK) and LEHDFMK and the fluorogenic substrates DEVD-aminotrifluoromethylcoumarin (AFC) and LEHD-AFC were obtained from Enzyme Systems Products (Livermore, CA). The active recombinant human caspase-3 enzyme was purchased from BioVision (Mountain View, CA). For DNA fragmentation analysis, nitric oxide measurement, and caspase 3-like activity measurement, monocytes (5 ϫ 106/sample) were treated in the indicated conditions immediately after monocyte isolation. In signaling experiments for tyrosine-phosphorylated proteins, monocytes were resuspended at 10 ϫ 106 cells/ml of RPMI 1640 medium, 10% fetal bovine serum, 10 g/ml polymyxin B, and 20 ng/ml recombinant human M-CSF. The DNA fragments were analyzed on a digital gel documentation system (Gel-Doc 1000, Bio-Rad)
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