Regulation of interleukin-1 and tumor necrosis factor-alpha induced granulocyte-macrophage colony-stimulating factor gene expression: potential involvement of arachidonic acid metabolism.
Signal transduction pathways evoked by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) to stimulate expression of other cytokines in mesenchymal cells are not clearly understood. Stimulation of the murine bone marrow stromal cell line +/(+)-1.LDA 11 with IL-1 (500 U/ml) in combination with TNF-alpha (500 U/ml) (IL-1 plus TNF-alpha) induced expression of c-jun mRNA as well as granulocyte-macrophage colony stimulating factor (GM-CSF) mRNA. We investigated the possibility that arachidonic acid metabolites, acting through protein kinase C (PKC) and perhaps also through the PKC-responsive transcription factor c-jun/AP-1, may be responsible for regulating GM-CSF transcription in these stromal cells. Expression of GM-CSF mRNA was preceded by IL-1 plus TNF-alpha induced arachidonate release (assayed using the 3H-derivative). Pretreatment of cells with the phospholipase A2 inhibitor quinacrine (20 microM) inhibited accumulation of both c-jun and GM-CSF mRNA but had no influence on expression of other genes induced by IL-1 and TNF-alpha, including leukemia inhibitory factor (LIF). In addition, quinacrine partially blocked IL-1 plus TNF-alpha induced 3H-arachidonic acid release from prelabeled stromal cells. Furthermore, exogenous arachidonate (10 to 50 microM) induced expression of c-jun. To investigate the role of arachidonate in GM-CSF transcription, we used a reporter vector consisting of the murine GM-CSF promoter linked to firefly luciferase. Transfection efficiency was monitored by assessing expression of a constitutively active gene, RSV-beta galactosidase. In this system, quinacrine significantly inhibited IL-1 plus TNF-alpha induced GM-CSF transcription assayed with the reporter construct. Exogenous arachidonic acid alone (10 microM) increased activity of GM-CSF reporter vector 1.5-fold over control. These results are consistent with the hypothesis that arachidonate metabolites are involved in the signaling pathway that leads to IL-1 plus TNF-alpha induced GM-CSF gene expression. Thus, transcriptional activation of GM-CSF gene is mediated, in part, by the arachidonate cascade.
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Division of Hematology/Oncology, Indiana University School of Medicine, Walter Oncology Center, Indianapolis.
Boswell
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D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism* / Q000502:physiology; D015152:Blotting, Northern; D001853:Bone Marrow / Q000187:drug effects / Q000378:metabolism; D001854:Bone Marrow Cells; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000235:genetics* / Q000502:physiology*; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D051379:Mice; D011493:Protein Kinase C / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011796:Quinacrine / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection; D014316:Tritium; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D001095:Arachidonic Acids; D007375:Interleukin-1; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D014316:Tritium; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011493:Protein Kinase C; D011796:Quinacrine