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首页> 外文期刊>Molecular and Cellular Biology >Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms.
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Lipopolysaccharide and Raf-1 kinase regulate secretory interleukin-1 receptor antagonist gene expression by mutually antagonistic mechanisms.

机译：脂多糖和Raf-1激酶通过相互拮抗机制调节分泌性白介素-1受体拮抗剂基因的表达。

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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.
机译：脂多糖（LPS）处理单核细胞已显示可激活Raf-1 /丝裂原激活的蛋白激酶（MAPK）信号通路并增加分泌性白介素1受体拮抗剂（sIL-1Ra）基因表达。但是，尚未确定Raf-1 / MAPK信号通路的激活对sIL-1Ra基因表达的LPS调节的重要性。这项研究解决了Raf-1 / MAPK信号通路在LPS调控sIL-1Ra基因表达中的作用。鼠巨噬细胞系RAW 264.7与294-bp sIL-1Ra启动子/荧光素酶构建体（pRA-294-luc）和组成型活性Raf-1激酶表达载体（pRSV-Raf-BXB）的共转染导致sIL的诱导-1Ra启动子活性，表明Raf-1与LPS一样可以调节sIL-1Ra启动子活性。体外MAPK分析表明，LPS处理和RAW 264.7细胞的pRSV-Raf-BXB转染均可增加p42 MAPK活性。但是，体外Raf-1激酶测定未能检测到LP 26诱导的RAW 264.7细胞中Raf-1激酶活性，这表明在RAW 264.7细胞中，Raf-1激酶不是调节MAPK的LPS信号通路的激活成分。活性或sIL-1Ra启动子活性。转染研究的结果支持了这一观察结果，该结果表明在RAW 264.7细胞中显性抑制性Raf-1突变体的表达不抑制LPS诱导的MAPK活性或sIL-1Ra启动子活性，表明LPS诱导的sIL-1Ra启动子激活发生独立于Raf-1 / MAPK信号通路。在其他研究中，RAW 264.7细胞与pRA-294-luc共转染并增加pRSV-Raf-BXB的量引起LPS诱导的sIL-1Ra启动子活性的剂量依赖性抑制，表明Raf-1途径的作用LPS调节sIL-1Ra启动子活性的作用是作为拮抗剂。有趣的是，LPS处理与pRA-294-luc和pRSV-Raf-BXB共转染的RAW 264.7细胞，也抑制了pRSV-Raf-BXB诱导的sIL-1Ra启动子活性，提示LPS和sIL-1Ra启动子活性的诱导。 Raf-1实际上是通过相互对立的机制发生的。支持该结论的sIL-1Ra启动子作图研究表明，LPS和Raf-1响应位于sIL-1Ra启动子的不同区域。进一步的研究表明，LPS和Raf-1激酶途径之间的相互拮抗不是启动子特异性的，因为在使用c-fos增强子/胸苷激酶启动子/荧光素酶构建体（pc-fos-TK81-luc）的分析中观察到了相同的现象。另外，在LPS和MEK激酶途径之间也观察到了关于sIL-1Ra启动子活性的相互拮抗作用，表明相互拮抗作用可以在一种以上的MAPK激活途径中发生。

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