脂肪细胞分化过程中影响PAI-1基因转录表达的Dex和C/EBPs顺式调控元件的分析

在研究胰岛素(Ins)、地塞米松(Dex)和甲基异丁基黄嘌呤(Mix)对脂肪细胞分化过程中PAI-1基因表达的影响基础上,为进一步探讨Ins、Dex调控PAI-1基因转录表达的调控机制,应用DNA重组技术,构建含萤光素酶(luciferase)报告基因和PAI-1启动子不同长度片段的嵌合质粒,转染3T3-L1前脂肪细胞并测定报告基因荧光素酶的活性.结果表明,小鼠PAI-1基因起动子-690至-850碱基序列之间有一个Dex的正调控元件.用计算机软件进行分析发现:Dex顺式元件位于PAI-1启动子的-750至-770碱基序列.其组成为:5′ GGTAACCTCTGTTCTCAT 3′.同时还发现在PAI-1启动子的-720至-740碱基序列中,存在一个C/EBPs的结合元件5′CCAAT3′并用凝胶电泳迁移实验对这些元件进行了鉴定.表明Dex正是通过激活转录因子(糖皮质激素受体,GR)和C/EBPα一起与各自的顺式元件结合来促进PAI-1基因的表达.

Analysis of a Novel Dexamethasone Response Element and a Putative C/EBPs cis-Motif: Controlling PAI-1 Gene Expression During Adipocyte Differentiation

It has been reported that there is a significant increase in PAI-1 expression level in obese subjects. To explore the linkage between PAI-1 gene expression and obesity, the restriction enzymes and DNA recombination technologies were used to construct the chimeric plasmids with luciferase and different lengths of PAI-1 promoter. After transfection of the chimeric plasmids into 3T3-L1 preadipocyte and detection of luciferase activity, the results indicated that a positive dexamethasone cis-acting element (bases -690 to -850) may be present in mouse PAI-1 promoter. In addition, computer analysis using Match-Search Software found that a new motif of DexRE (dexamethasone response element) 5′ GGTAACCTCTGTTCTCAT 3′ and a putative C/EBPs binding site (cis-motif) exist respectively in the fragment (nucleotides -751 to -770) of, and a sequence (bases -720 to -740) of, mouse PAI-1 promoter,and GMSA and competition assays identified that the trans-acting factors induced by dexamethasone can specifically bind to those cis-motifs. Meanwhile, the site-directed mutagenesis by PCR was performed to detect the influence of mutant DexRE and C/EBPs cis-motif on PAI-1 gene expression. Similarly, the chimeric plasmids containing luciferase as a reporter gene and a fragment of mouse PAI-1 promoter comprising the mutant cis-motifs were constructed, and then transfected into 3T3-L1 preadipocytes. The measurement revealed that the luciferase activities were markedly lowered by mutant DexRE and mutant C/EBPs cis-motif compared with their wild counterparts, implicating that the DexRE and C/EBPs cis-motif identified may control the expression of PAI-1 gene in 3T3-L1 adipocyte. The study is very helpful to elucidate a molecular mechanism through which the dexamethasone may regulate the expression of PAI-1 gene in 3T3-L1 adipocyte.