Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms
| Autor: | D Eidlen, William P. Arend, M F Smith, Arthur Gutierrez-Hartmann, C.J. Guthridge |
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| Rok vydání: | 1997 |
| Předmět: |
MAPK/ERK pathway
Lipopolysaccharides Recombinant Fusion Proteins Sialoglycoproteins MAP Kinase Kinase Kinase 1 Biology Protein Serine-Threonine Kinases Transfection Cell Line Mice Proto-Oncogene Proteins Animals Kinase activity Protein kinase A Promoter Regions Genetic Molecular Biology Regulation of gene expression Mitogen-Activated Protein Kinase 1 Expression vector Kinase Macrophages Cell Biology Protein-Tyrosine Kinases Molecular biology Proto-Oncogene Proteins c-raf Interleukin 1 Receptor Antagonist Protein Gene Expression Regulation Calcium-Calmodulin-Dependent Protein Kinases lipids (amino acids peptides and proteins) Signal transduction Signal Transduction Research Article |
| Zdroj: | Molecular and cellular biology. 17(3) |
| ISSN: | 0270-7306 |
| Popis: | Lipopolysaccharide (LPS) treatment of monocytic cells has been shown to activate the Raf-1/mitogen-activated protein kinase (MAPK) signaling pathway and to increase secretory interleukin-1 receptor antagonist (sIL-1Ra) gene expression. The significance of the activation of the Raf-1/MAPK signaling pathway to LPS regulation of sIL-1Ra gene expression, however, has not been determined. This study addresses the role of the Raf-1/MAPK signaling pathway in regulation of sIL-1Ra gene expression by LPS. Cotransfection of the murine macrophage cell line RAW 264.7 with a 294-bp sIL-1Ra promoter/luciferase construct (pRA-294-luc) and a constitutively active Raf-1 kinase expression vector (pRSV-Raf-BXB) resulted in induction of sIL-1Ra promoter activity, indicating that Raf-1, like LPS, can regulate sIL-1Ra promoter activity. An in vitro MAPK analysis indicated that both LPS treatment and pRSV-Raf-BXB transfection of RAW 264.7 cells increases p42 MAPK activity. An in vitro Raf-1 kinase assay, however, failed to detect LPS-induced Raf-1 kinase activity in RAW 264.7 cells, suggesting that in RAW 264.7 cells, Raf-1 kinase is not an activating component of the LPS signaling pathway regulating MAPK activity or sIL-1Ra promoter activity. This observation was supported by results from transfection studies which demonstrated that expression of a dominant-inhibitory Raf-1 mutant in RAW 264.7 cells does not inhibit LPS-induced MAPK activity or sIL-1Ra promoter activity, indicating that LPS-induced sIL-1Ra promoter activation occurs independent of the Raf-1/MAPK signaling pathway. In additional studies, cotransfection of RAW 264.7 cells with pRA-294-luc and increasing amounts of pRSV-Raf-BXB caused a dose-dependent inhibition of LPS-induced sIL-1Ra promoter activity, indicating that the role of the Raf-1 pathway in the regulation of sIL-1Ra promoter activity by LPS is as an antagonizer. Interestingly, LPS treatment of RAW 264.7 cells, cotransfected with pRA-294-luc and pRSV-Raf-BXB, also inhibited pRSV-Raf-BXB-induced sIL-1Ra promoter activity, suggesting that inductions of sIL-1Ra promoter activity by LPS and Raf-1 actually occur by mutually antagonistic mechanisms. In support of this conclusion, sIL-1Ra promoter mapping studies indicated that LPS and Raf-1 responses localized to different regions of the sIL-1Ra promoter. Further studies demonstrated that mutual antagonism between the LPS and Raf-1 kinase pathways is not promoter specific, as the same phenomenon is observed in assays using a c-fos enhancer/thymidine kinase promoter/luciferase construct (pc-fos-TK81-luc). Additionally, mutual antagonism with regard to sIL-1Ra promoter activity also was observed between the LPS and MEK kinase pathways, indicating that mutual antagonism can occur in more than one MAPK activation pathway. |
| Databáze: | OpenAIRE |
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