Involvement of ASMase and ceramide in JNK activation by LPS.

(A) LPS stimulates ASMase; quiescent BAC-1.2F5 cells were either left untreated or pretreated with D609 (10 μmol/L, 60 minutes) to block PC-PLC before stimulation with 1.5 μg/mL LPS for different times before solubilization. ASMase activity was determined in whole cell extracts as described in “Materials and methods.” Results are expressed as the percentage increase with respect to the control values. The plot represents the mean of 2 independent experiments, and vertical bars represent the range of the samples. (B) BIM pretreatment, but not down-regulation of DAG-dependent PKC, affects the activation of ASMase by LPS. Quiescent BAC-1.2F5 cells were left untreated (black bars) or were treated with BIM (10 μmol/L, 60 minutes; gray bars); alternatively, down-regulation of DAG-dependent PKC was performed as described in Figure 3B (white bars). Cells were stimulated with LPS for 15 minutes before lysis and determination of ASMase activity in whole cell extracts. Results are expressed as the percentage increase with respect to the control values. (C) Exogenous ceramide stimulates JNK in macrophages; BAC-1.2F5 cells were left untreated or were pretreated with D609 (10 μmol/L, 60 minutes) to block PC-PLC or with BIM (10 μmol/L, 60 minutes) to inhibit PKC. Cells were then stimulated with C2 ceramide (C2, 250 μmol/L) or with the inactive analog dihydro-C2 ceramide (DH-C2, 250 μmol/L), as indicated. The presence of phosphorylated JNK was detected by immunoblotting with the corresponding antibodies. An anti-JNK1 blot is shown as a loading control.