蛋白激酶D1在烟曲霉介导的NF-κB信号通路激活及转录活性中的作用  

Functional role of protein kinase D1 in Aspergillums fumigates-induced activation of nuclear factor-κB signal pathway and transcription

作　　者：牛晓璐[1,2] 许万福[1,2] 李松玉[1,2] 柯志勇[1,2] 曾方银[3] 罗深秋[1,2] 邓凡[1,2] 

机构地区：[1]南方医科大学基础医学院细胞生物学教研室,广东广州510515 [2]南方医科大学骨科研究所,广东广州510515 [3]南方医科大学南方医院检验科,广东广州510515

出　　处：《南方医科大学学报》2013年第3期351-355,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(30973014;81272852);广东省自然科学基金(10151051501000040);广东省自然科学基金博士启动基金(9451051501003704)~~

摘　　要：目的研究蛋白激酶D1(PKD1)在烟曲霉介导的NF-κB信号通路激活及转录活性中的作用,为进一步探讨PKD1在烟曲霉感染引起的肺曲霉病中的作用及其机制奠定基础。方法首先将GFP、GFP-PKD1表达质粒分别转染人肺腺癌细胞A549和HEK293细胞中,分别将灭活的烟曲霉分生孢子(1×105CFU/ml)于不同时间处理上述两组细胞,Western blotting证实PKD1的过表达,并检测PKD1的磷酸化活性;其次在A549细胞中分别转染GFP-PKD1、siRNA-PKD1并以灭活的烟曲霉分生孢子处理细胞30 min,分别检测下游NF-κB通路相关信号分子的磷酸化活性;最后,将NF-κB-luc荧光素酶报告基因及内参照报告质粒海肾荧光素酶(pRL-SV40)共转染入表达GFP、GFP-PKD1的A549细胞中,两组细胞在有无烟曲霉分生孢子的作用下处理24 h,收集细胞裂解液,进行双色荧光素酶活性检测。结果 Western blotting证实PKD1的过表达时间依赖性地增强烟曲霉刺激的A549细胞和HEK293细胞中PKD1的磷酸化活性及NF-κB通路中IКB和p65(pS276)的磷酸化活性,反之,敲低PKD1的表达则抑制烟曲霉介导的NF-κB通路中IКB和p65(pS276)的磷酸化活性。过表达PKD1明显增加NF-κB-luc荧光素酶的转录激活活性。结论 PKD1可能在烟曲霉刺激的NF-κB通路的激活及转录活性中起重要作用。Objective To explore the functional role of protein kinase D1 （PKD1） in the activation of nuclear factor-κB （NF-κB） signal pathway and NF-κB transcription mediated by Aspergillums fumigates. Methods A549 cells and HEK293 cells were transfected with green fluorescence protein （GFP） or GFP-PKD1 followed by treatment with 1× 10^5 CFU/ml Aspergillus fum igatus conidia for different time lengths. The phosphorylation levels of PKD1, IκB and p65 （pS276） in the transfected cells were measured by Western blotting. A549 cells were transfected with GFP-PKD1 or siRNA-PKD1, and the phosphorylation of IκB and p65 （pS276） was examined. Finally, NF-κB-luc and renilla luciferase reporter pRL-SV40 were cotransfected into GFP- or GFP-PKDl-transfected A549 cells before exposure of the cells to Aspergillus fumigatus conidia for 24 h, and NF-κB transcriptional activity in the cells was determined using dual-luciferase reporter assay. Results Overexpression of PKD1 significantly increased Aspergillus fumigatus conidia-sfimulated phosphorylation of PKD1, IκB and p65 （pS276）, whereas PKD1 knockdown by siRNA-PKD1 suppressed IκB and p65 （pS276） phosphorylation. Dual luciferase assay demonstrated that PKD1 overexpression markedly enhanced Aspergillus fumigatus-induced NF-κB transcription in A549 cells. Conclusion PKD1 may contribute to the activation of NF-κB signal pathway and NF-κB transcription induced by Aspergillums fumigates.

关 键 词：蛋白激酶D1 烟曲霉分生孢子 NF-ΚB 转录激活 磷酸化 

分 类 号：R378[医药卫生—病原生物学]