| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2005: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
Lipopolysaccharide (LPS) and interferon gamma (IFNγ) have been show to cooperatively regulate the expression of many macrophage genes such as chemokines. Although the transcriptional regulation of these inflammatory genes has been regulated by transcription factors NF-κB and STAT1, the role of protein kinase induced by LPS and IFNγ in the transcriptional regulation has not been fully understood. In the present study, we performed a series of experiments to determine the functional role of p38 MAP kinase in the transcriptional regulation of the chemokine CXCL9 gene in macrophage cell line RAW264.7 cells, and obtained the following results.
1) To determine whether p38 MAP kinase regulates LPS-induced NF-κB-dependent transcriptional activity, we constructed an expression vector in which NF-κB RelA transactivation domain fused to the GAL4 DNA-binding domain, co-transfected with a luciferase reporter construct containing GAL4 DNA-binding sequences into RAW264.7 cells, and assessed for the lu
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ciferase activity. The results demonstrated that stimulation with LPS enhanced the RelA-dependent transcriptional activity and the enhanced activity was suppressed by SB203580, an inhibitor of p38MAPK.
2) Chromatin immunoprecipitation analysis demonstrated that co-treatment of RAW264.7 cells with IFNγ and LPS cooperatively recruited STAT1, RelA and RNA polymerase II at the promoter region of the CXCL9 gene and that this cooperative recruitment was suppressed by SB203580.
3) Phosphorylation on Ser727 of STAT1 was additively enhanced by co-treatment with LPS and IFNγ, though the magnitude of induction was not synergistic as seen in the transcriptional activity of the CXCL9 gene induced by LPS and IFNγ.
4) Anisomycin, a p38 MAPK activator which enhances Ser727 phosphorylation but has no effect on NF-κB activation, only marginally potentiated IFNγinduced CXCL9 gene expression, suggesting that phosphorylation on Ser727 alone is not sufficient for the synergistic induction of CXCL9 gene.
These results suggest p38MAPK regulates the STAT1- and RelA-mediated enhanceosome formation at the promoter/enhance region of the CXCL9 gene in IFNγ and LPS-stimulated macrophages, which formation then leads to the synergistic transcriptional activity. Less
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