Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms
Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms
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DOI：
10.1128/mcb.17.3.1118
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发表时间：
1997-03-01
影响因子：
5.3
通讯作者：
Smith, MF
中科院分区：
文献类型：
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作者：
Guthridge, CJ;Eidlen, D;Smith, MF
单核细胞经脂多糖处理后，Raf-1/MAPK信号通路被激活，分泌型白介素1-L受体拮抗剂(sIL-1Ra)基因表达增加，但Raf-1/MAPK信号通路激活对内毒素调节sIL-1Ra基因表达的意义尚不明确。将含有sIL-1Ra启动子/荧光素酶基因片段的pRA-294-Luc与具有内在活性的Raf-激酶表达载体(pRSV-Raf-BXB)共转染小鼠巨噬细胞RAW 264.7，可诱导RAW 264.7细胞中sIL-1ra启动子的活性，表明Raf-L与脂多糖一样，能调节sIL-1ra启动子的活性。体外MAPK分析表明，脂多糖处理和pRSV-Raf-BXB转染人RAW 264.7细胞均可提高p42MAPK活性，而体外Raf-L激酶实验未能检测到脂多糖诱导的RAW 264.7细胞中Raf-L激酶的活性在RAW 264.7细胞中，Raf-L激酶不是调节MAPK活性或sIL-1ra启动子活性的脂多糖信号通路中的激活成分，这一观察结果得到了转基因研究的支持，这些结果表明在RAW 264.7细胞中显性抑制Raf-L突变体的表达并不抑制内毒素诱导的MAPK活性或sIL-1ra启动子活性，表明内毒素诱导的sIL-1ra启动子激活不依赖于Raf-1/MAPK信号通路。在进一步的研究中，RAW 264.7细胞与PrA-294-Luc共转染组和pRSV-Raf-BXB组均呈剂量依赖性抑制sIL-1ra启动子活性，提示Raf-L通路在脂多糖调节sIL-1ra启动子活性中的作用是拮抗剂。有趣的是，脂多糖处理RAW 264.7细胞后，pRSV-Raf-BXB和pRSV-Raf-BXB共转染组也抑制了pRSV-Raf-BXB诱导的sIL-1ra启动子活性，提示脂多糖和Raf-L诱导sIL-1ra启动子活性实际上是通过相互拮抗的机制发生的。SIL-1Ra启动子定位研究表明，脂多糖和Raf-L反应定位于sIL-1Ra启动子的不同区域，进一步的研究表明，脂多糖和Raf-L激酶通路之间的相互拮抗作用不是启动子专一性的，同样的现象也在使用c-fos增强子/胸苷激酶启动子/荧光素酶构建物(PC-fos-TK81-Luc)的检测中观察到。此外，在sIL-1ra启动子活性方面，还观察到了内毒素和MEK激酶途径之间的相互拮抗，这表明MAPK激活途径中可能存在相互拮抗。
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-l 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-l kinase expression vector (pRSV-Raf-BXB) resulted in induction of sIL-1Ra promoter activity, indicating that Raf-l, 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-l kinase assay, however, failed to detect LPS-induced Raf-l kinase activity in RAW 264.7 cells, suggesting that in RAW 264.7 cells, Raf-l 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-l 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-l 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-l actually occur by mutually antagonistic mechanisms, In support of this conclusion, sIL-1Ra promoter mapping studies indicated that LPS and Raf-l responses localized to different regions of the sIL-1Ra promoter, Further studies demonstrated that mutual antagonism between the LPS and Raf-l 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.