Inhibition of TNF-α-Induced Neutrophil Apoptosis by Crystals of Calcium Pyrophosphate Dihydrate Is Mediated by the Extracellular Signal-Regulated Kinase and Phosphatidylinositol 3-Kinase/Akt Pathways Up-Stream of Caspase 3

摘要

The role of protein kinases in the inhibition of TNF-α associated apoptosis of human neutrophils by crystals of calcium pyrophosphate dihydrate (CPPD) (25 mg/ml) was investigated. We monitored the activities of the p44 extracellular signal-regulated kinase 1 (ERK1) and p42 ERK2 mitogen-activated protein (MAP) kinases and phosphatidylinositol 3-kinase (PI3-K)-regulated protein kinase B (Akt) in neutrophils incubated with TNF-α and CPPD crystals, separately and in combination, in parallel with the endogenous caspase 3 activity and DNA fragmentation. CPPD crystals were observed to induce a robust and transient activation of ERK1, ERK2, and Akt, whereas TNF-α produced only a modest and delayed activation of Akt. In the presence of TNF-α, Akt activity was enhanced, and CPPD crystal-induced activation of ERK1 and ERK2 was more sustained than with CPPD crystals alone, but TNF-α itself reduced the basal phosphotransferase activities of these MAP kinases. Preincubation with the MAP kinase kinase (MEK1) inhibitors PD98059 (20 ng/ml) and U0126 (250 nM), or the PI3-K inhibitors wortmannin (100 nM) and LY294002 (50 μM) repressed the activation of ERK1, ERK2, and Akt in association with CPPD crystal incubation, in the absence or presence of TNF-α. Furthermore, the inhibition of the Mek1/Mek2→ERK1/ERK2 or PI3-K/Akt pathways reversed CPPD crystal-associated suppression of TNF-α-induced caspase 3 activation and neutrophil apoptosis. Together, these results indicate that CPPD crystals function to induce acute inflammatory responses through ERK1/ERK2 and PI3-K/Akt-mediated stimulation of neutrophil activation and repression of apoptosis.