NLK 通过Smad4负调控 TGFβ信号通路

摘要

Objective To study the effect of NLK on TGFβsignaling pathway and explore the molecular mechanism .Methods Protein stability assay was used to determine the influence of NLK on the degradation of Smad 4 .In vivo ubiquitination assay was applied to detect the effect of NLK on the ubiquitination of Smad4 .Luciferase reporter gene assay was used to detect the effect of NLK on CAGA‐luc and 3TP‐luc ,the responsive reporter of TGFβ signaling pathway .Real time PCR was applied to examine the effect of NLK on the expression of p21 and PAI‐1 ,the target genes of TGFβsignaling pathway .Results In HEK293T cell ,over ex‐pression of NLK promotes the degradation and ubiquitination of Smad4 .In HEK293T cells ,Ectopic expression of NLK inhibits the activity of CAGA luc and 3TP luc stimulated by TGFβ.In HepG2 cells ,over expression of NLK inhibits the expression of p21 and PAI 1 ,the target genes of TGFβsignaling pathway .Conclusion NLK promotes the ubiquitination and degradation of Smad4 ,conse‐quently inhibits TGFβsignaling pathway .%目的：探讨NLK对转化生长因子β（TGFβ）信号通路的影响及其分子机制。方法采用蛋白质稳定性实验检测NLK对Smad4蛋白质降解的影响；体内泛素化实验检测过表达NLK对Smad4泛素化修饰的影响；荧光素酶报告基因实验检测NLK对TGFβ信号通路报告基因载体CAGA‐luc和3TP‐luc的影响；实时定量PCR技术检测NLK对TGFβ信号通路靶基因p21和PAI‐1的影响。结果在 HEK293T细胞内，过表达NLK促进Smad4的降解和泛素化修饰；在HEK293T细胞内过表达NLK抑制细胞因子 TGFβ对CAGA‐luc和3TP‐luc的激活作用；在 HepG2细胞内过表达 NLK 抑制 TGFβ信号通路靶基因p21和PAI‐1的表达。结论 NLK促进Smad4的泛素化修饰和降解，进而抑制TGFβ信号通路。