Lipopolysaccharide response is linked to the GTP binding protein, Gi2, in the promonocytic cell line U937.
Phorbol esters induce the differentiation of the human promonocytic cell line U937 to a monocyte/macrophage. This process is associated with the induction of interleukin 1 beta (IL-1 beta) gene expression (Strulovici, B., Daniel-Issakani, S., Oto, E., Nestor, J., Jr., Chan, H., and Ping-Tsou, A. (1989) Biochemistry 28, 3569-3576). Here we describe the induction by phorbol esters of lipopolysaccharide (LPS) responsiveness in U937 cells. Preincubation with phorbol myristate acetate (TPA, 5 x 10(-8) M) for at least 4-6 h and up to 12 h followed by 3 h of LPS treatment induced a 4-fold enhancement in the accumulation of IL-1 beta transcripts compared to treatment with TPA alone. This "priming" effect was specific for protein kinase C agonists and required de novo protein synthesis. Exposure of [35S]methionine-labeled U937 cells to phorbol esters induced the de novo synthesis of a protein which migrated with a 40-kDa molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had an isoelectric point of 5.7 (p 40/5.7), and was recognized by a specific antibody to the pertussis toxin (PT)-sensitive Gi2. The time course for the appearance of Gi2 correlated with that for the induction of LPS responsiveness by TPA. Moreover, the LPS response was PT-sensitive. In cells treated with LPS for 5 min, Gi2 showed diminished ADP-ribosylation by PT. Treatment of U937 cells with LPS for 30 min induced phosphorylation of Gi2 and enhanced PT labeling. In a cell-free assay, phosphorylation of Gi2 by protein kinase C type III, rendered it a better PT substrate. The present findings thus suggest: 1) that TPA induces LPS responsiveness in U937 cells via de novo synthesis of Gi2; 2) that the LPS response (enhanced IL-1 production) is linked to a pertussis toxin-sensitive G protein which we identified as Gi2; and 3) that LPS leads to phosphorylation of Gi2.
Daniel-Issakani
S
S
Department of Biochemistry, Syntex Research, Palo Alto, California 94304.
Spiegel
A M
AM
Strulovici
B
B
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016403:Lymphoma, Large B-Cell, Diffuse; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D037342:Pertussis Toxin; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007375:Interleukin-1; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D010710:Phosphates; D010566:Virulence Factors, Bordetella; D004247:DNA; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate