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J Biol Chem, Vol. 274, Issue 14, 9342-9350, April 2, 1999
, and
From the Department of Microbiology and Immunology, Uniformed
Services University of the Health Sciences, Bethesda, Maryland 20814 and The involvement of ceramide in
lipopolysaccharide-mediated activation of mouse macrophages was
studied. Lipopolysaccharide, cell-permeable ceramide analogs, and
bacterial sphingomyelinase led to phosphorylation of the extracellular
signal-regulated kinases, c-Jun NH2-terminal kinases,
and p38 kinase and induced AP-1 DNA binding in C3H/OuJ
(Lpsn) but not in C3H/HeJ
(Lpsd) macrophages. Lipopolysaccharide and
ceramide mimetics showed distinct kinetics of mitogen-activated protein
kinase phosphorylation and AP-1 induction and activated AP-1 complexes
with different subunit compositions. Lipopolysaccharide-activated
AP-1 consisted of c-Fos, Jun-B, Jun-D, and c-Jun, while
C2-ceramide induced Jun-D and c-Jun only.
Lipopolysaccharide and, less potently, C2-ceramide or
sphingomyelinase, stimulated AP-1-dependent reporter gene
transcription in RAW 264.7 cells. Unlike lipopolysaccharide,
C2-ceramide failed to activate NF- William S. Middleton Veterans Memorial Medical
Center, Madison, Wisconsin 53705
B and did not
induce production of tumor necrosis factor or interleukin-6. The
lipopolysaccharide antagonist, Rhodobacter sphae-roides diphosphoryl lipid A, inhibited
lipopolysaccharide activation of NF-B and AP-1 but did not
block C2-ceramide-induced AP-1. Pretreatment of C3H/OuJ
macrophages with C2-ceramide greatly diminished AP-1
induction following subsequent C2-ceramide stimulation. However, lipopolysaccharide-induced transcription factor activation and
cytokine release were not influenced. In contrast, lipopolysaccharide pretreatment inhibited both lipopolysaccharide- and
C2-ceramide-mediated responses. Thus, ceramide partially
mimics lipopolysaccharide in activating the mitogen-activated protein
kinases and AP-1 but not in mediating NF-B induction or cytokine
production, suggesting a limited role in lipopolysaccharide signaling.
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