NF-κB结合位点在LPS诱导人TNF-α基因转录中的调节作用

Regulation of hTNF-α Gene Transcription by NF-κB Binding Sites

为探讨人TNF α基因启动子区域NF κB结合位点对LPS诱导性基因表达的调节作用 ,将构建的含人TNF α基因启动子区域及其不同缺失片段的pGL2萤光素酶报道基因重组体 ,体外转染HL 60细胞 ,观察LPS刺激对TNF基因启动子指导萤光素酶表达的影响 .发现TNF α基因启动子区域的 3个NF κB结合位点的存在是该基因最大组成性表达和LPS诱导性表达所必需 ;将这 3个位点缺失可完全阻断报道基因的LPS诱导性表达 ,并使其组成性表达明显受到抑制 (P <0 .0 0 1) .缺失κB1和κB2位点 ,使基因组成性表达与LPS诱导性表达均减少约 50 % (P <0 .0 1) ,但诱导倍数则与非突变体相近 ;反之 ,用κB3反义寡核苷酸封闭κB3位点 ,保留κB1和κB2功能 ,使LPS诱导性基因表达抑制 70 % (P <0 .0 0 1) ,诱导倍数明显降低 .保留原有κB3位点 ,再将κB3位点取代κB1和κB2位点 ,可使基因组成性和诱导性表达几乎完全恢复 ;将κB3位点向TSS移近 (- 98→ - 52 ) ,取代Sp1位点 ,虽然κB1和κB2位点仍然缺失 ,但仅一个κB3位点即可使基因组成性和LPS诱导性表达恢复到 80 % .用反义寡核苷酸封闭这个κB3位点 ,不但可使TNF α启动子完全丧失对LPS的反应性 ,还可完全阻断所控基因的组成性表达 .结果表明 ,人TNF α启动子区域 3个NF κB位点均参与该?

The regulatory effect of NF κB binding sites located in TNF α gene promoter on LPS induced gene expression was explored. Human TNF α gene promoter contained three κB binding sites and its several mutants ligated to luciferase reporter plasmid pGL2 were transiently introduced into HL 60 cells , and the LPS inducibility of TNF α gene promoter and its mutants directed reporter gene expression was observed. It was found that three κB binding sites in TNF promoter had been shown to be required for maximal levels of constitutive and LPS induced gene expression. Deletion of all three κB binding sites from TNF α promoter resulted in a complete inhibition of LPS induced luciferase expression and an obvious reduction in its constitutive expression( P <0.001). The deletion of κB1 and κB2 binding sites decreased the constitutive and LPS induced expression of reporter gene by 50%( P <0 01),but the inducible factor remained unchanged. On the contrary, when κB3 was blocked by antisense oligonucleotide, LPS induced gene expression was repressed by 70%( P <0.001) and the inducibility also significantly declined, although κB1 and κB2 binding sites were present.Replacement of κB1 and κB2 binding sites with a κB3 site, keeping the original κB3 site in TNF α gene promoter, almost entirely recovered the gene expression. κB3 site removed closer to TSS(-98→-52)and substitution of it for Sp1, only this one κB3 binding site could compensate for the functional deficiency of mutagenic TNF promoter by 80% despite of lack of κB1 and κB2 sites. Blocking this κB3 binding site by antisense oligonucleotide gave rise not only to the unresponsiveness of the TNF α gene promoter to LPS, but also to the complete suppression of its constitutive expression. The results indicated that all three κB binding sites were involved in the LPS inducibility of the TNF α promoter and κB3 site might play the most important role among them. Moreover, Sp1 might be one of the factors responsible for the constitutive gene expression.