Lipopolysaccharide response is linked to the GTP binding protein, Gi2, in the promonocytic cell line U937

S. Daniel-Issakani, Allen M. Spiegel, B. Strulovici

Research output: Contribution to journalArticle

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Abstract

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 1β (IL-1β) 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-1β 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.

Original languageEnglish (US)
Pages (from-to)20240-20247
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number34
StatePublished - 1989
Externally publishedYes

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GTP-Binding Proteins
Lipopolysaccharides
Pertussis Toxin
Cells
Cell Line
U937 Cells
Phosphorylation
Phorbol Esters
Interleukin-1
Protein Kinase C
Biochemistry
Macrophages
Isoelectric Point
Molecular mass
Tetradecanoylphorbol Acetate
Electrophoresis
Gene expression
Sodium Dodecyl Sulfate
Methionine
Adenosine Diphosphate

ASJC Scopus subject areas

  • Biochemistry

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Lipopolysaccharide response is linked to the GTP binding protein, Gi2, in the promonocytic cell line U937. / Daniel-Issakani, S.; Spiegel, Allen M.; Strulovici, B.

In: Journal of Biological Chemistry, Vol. 264, No. 34, 1989, p. 20240-20247.

Research output: Contribution to journalArticle

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abstract = "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 1β (IL-1β) 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-1β 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.",
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N2 - 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 1β (IL-1β) 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-1β 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.

AB - 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 1β (IL-1β) 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-1β 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.

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