Dioxin increases C/EBPβ transcription by activating cAMP/protein kinase A
Dioxin increases C/EBPβ transcription by activating cAMP/protein kinase A
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DOI：
10.1074/jbc.m310190200
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发表时间：
2004-03-05
影响因子：
4.8
通讯作者：
Matsumura, F
中科院分区：
文献类型：
--
作者：
Vogel, CFA;Sciullo, E;Matsumura, F
环境污染物2，3，7，8-四氯二苯并对二恶英(TCDD=二恶英)可增加C/EBPβ的表达。C/EBPbeta基因的调节表达与TCDD的毒性反应有关，如消瘦综合征、糖尿病和抑制脂肪细胞分化。本研究旨在探讨TCDD介导的C/EBPβ转录激活的调控机制。小鼠胚胎成纤维细胞(C(3)H(10)T1/2)和小鼠肝癌细胞(Hepa1c1c7)中C/EBPbeta基因和蛋白水平的升高与C/EBPbeta蛋白结合亲和力的增强有关。用CCAAT/增强子结合蛋白启动子的不同缺失结构进行的转染研究表明，在两个受试细胞系中，TCDD激活C/EBPbeta基因需要位于转录起始点上游60-120bp的一个小区域。利用C/EBPbeta启动子突变结构的进一步分析表明，C/EBPbeta启动子的激活是通过位于C/EBPbeta基因TATA盒附近的不完全cAMP反应元件结合蛋白(CREB)位点介导的。蛋白激酶A(PKA)抑制剂H89完全阻断了TCDD对C/EBPbeta启动子活性的依赖作用，表明TCDD通过cAMP/PKA途径激活CREB结合，这一作用得到了TCDD处理后cAMP水平和PKA活性的支持。凝胶位移分析表明，CREB本身与可能的CREB基序结合，该基序介导TCDD依赖的C/EBPbeta基因转录效应。CREB和PKA表达载体的共转染实验进一步支持了我们的结论，即依赖于TCDD对C/EBPbeta转录的影响是通过依赖于PKA的CREB激活来实现的。
The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD = dioxin) has been shown to increase the expression of C/EBPbeta. The modulated expression of C/EBPbeta has been suggested to be associated with toxic responses of TCDD such as wasting syndrome, diabetes, and inhibition of adipocyte differentiation. This study focused on the regulatory mechanism of TCDD-mediated transcriptional activation of C/EBPbeta. Elevated C/EBPbeta mRNA and protein levels in mouse embryonic fibroblasts (C(3)H(10)T1/2) and in mouse hepatoma cells (Hepa1c1c7) were correlated with increased binding affinity of the C/EBPbeta protein. Transfection studies with different deletion constructs of the CCAAT/enhancer-binding protein promoter indicated that a small region located 60-120 bp upstream of the start site of transcription is required for activation of the C/EBPbeta gene by TCDD in both cell lines tested. Further analysis using mutation constructs of the C/EBPbeta promoter demonstrated that activation of the C/EBPbeta promoter is mediated through incomplete cAMP-response element-binding protein (CREB) sites located close to the TATA box of the C/EBPbeta gene. The protein kinase A (PKA) inhibitor H89 completely blocks the TCDD-dependent effect on C/EBPbeta promoter activity, indicating that TCDD activates CREB binding via a cAMP/PKA pathway, which is supported by the increased cAMP level and PKA activity observed after TCDD treatment. Gel shift analyses demonstrated that CREB itself binds to the putative CREB motif that mediates the TCDD-dependent effect on C/EBPbeta gene transcription. Cotransfection experiments with CREB and PKA expression plasmids further supported our conclusions that the TCDD-dependent effect on C/EBPbeta transcription is mediated via PKA-dependent CREB activation.