Identification Of The TNF-α Response Element In Human Acyl-coenzyme A: Cholesterol Acyltransferase-1 Gene P1 Promoter

Acyl-coenzyme A: cholesterol acyltransferase (ACAT) is an intracellular enzyme involved in cellular cholesterol homeostasis and in atherosclerotic foam cell formation. The ACAT-1 that is present in most tissues is believed to be critical for maintaining cholesterol homeostasis and involved in the developing atherosclerotic plaque. Tumor necrosis factor-α (TNF-α), a cytokine that exerts many pro-atherosclerotic effects in vivo, causes up-regulation of ACAT-1 gene expression in THP-1, human monocytic cell line. Luciferase activity assays by using different constructs containing human ACAT-1 gene 1M promoter with 5'-/T-dclctions show tluil the maximal transcriptional activity is located within the 47bp core region from -125 to -79.To examine the molecular nature of this observation, we firstly develop the DEAE-Dextran-mediated luciferase reporter gene transfection system. Optimizing condition of transfection using GFP(Green Fluorescent Protein) as reporter gene. RT-PCR assays indicated that the endogenous PI promoter in THP-1 cells could be stimulated by treatment of TNF-α, The results of co-transfection with mutant 1KB and mutant p65 expression plasmid and treatment with NF-кB inhibitor TPCK, MG-132 showed that the up-regulation effect of TNF-a was abrogated, the same results was observed in luciferase assay using constructs containing human ACAT-1 gene PI promoter core region. Indicating that NF-кB signal pathway might involve in this action. Sequence analysis by computer and site-directed mutagenesis results revealed NF-кB elements are located in this core region. Gel retardation results show that the nuclear extracts from THP-1 cells treated with TNF-a can form specific bands withthe wild-type P1 promoter NF-KB element. And these specific hands can he supershifted by anti- NF-KB antibody.On the whole, our work identified the functional NF-KB element responding to TNF-α effect. TNF-a causes large activation of NF-KB in TRIM, then the helerodimer p65-p50 nuclear-translocated and bound to the NF-KB site in the ACAT-1 PI promoter, at last caused up-regulation of P1 promoter. We provides a molecular mechanism to account for the effect of TNF-a in causing transcriptional activation of human ACAT-1 gene in human monocytic cell. Further study on the cis-elements and Iram-factors is well underway.