Inhibition of TNF-#alpha#-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 jkinases in the inhibition of TNF-#alpha# 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 (KRK1) and p42 mitogen-activated protein (MAP) kinases and phosphaticdylinositol 3-kinase (P13-K)-regulated protein kinase B (Akt) in neutrophils incubated with TNF-#alpha# and CPPD crystals,separately and in combinationm,in parallel with the endogenous caspase 3 activity and DNA fragmentaion.CPPD crystals were observed to induce a robust and transient activation of ERK1,ERK2,and Akt,whereas TNF-#alpha# produced only a modest and delayed activation of Akt.In the pressence of TNF-#alpha#,Akt activity was enhanced,and CPPD crystal-induced activation of ERK1 and ERK2 was more sustained than with CPPD crystals alone,but TNF-#alpha# itself reduced thebaseal phosphotrnsferase activities of these MAP kinases.Preincubation with the MAP kinase kinase (MEK) inhibitors PD98059 (20 ng/ml) and U0126 (250 nM),or the P13-K inhibitors wortmannin (100 nM) and LY294002 (50 #mu#M) repressed tha ctivation of ERK1,ERK2,and Akt in association with CPPD crystal incubation,in the absence or presence fo TNF-#alpha#,Furthermore,the inhibition of the Mek1/Mek2->ERK1/ERK2 or P13-K/Akt- pathways reversed CPPD crystal-associated suppression of TNF-#alpha#-induced caspase 3 activation and neutrophil apoptosis.Together,these results indicate that CPPD crystals function to induce acute inflammatory responses through ERK1/KER2 and P13-K/Akt-mediated stimulation of neutrophl activation and repression of apoptosis.