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 dement (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 dement) 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.