Tumor necrosis factor-α eliminates binding of NF-Y and an octamer-binding protein to the lipoprotein lipase promoter in 3T3-L1 adipocytes

Abstract

TNFα has been shown to reduce lipoprotein lipase (LPL) activity in adipose tissue. Regulation of LPL by TNFα occurs at the level of LPL gene transcription and posttranscriptionally. To elucidate further the transcriptional mechanism of TNFα inhibition of LPL gene transcription, transfection analysis was used to locate the site(s) of the LPL promoter that imparts the TNFα response. Transient transfections using LPL promoter deletions fused to luciferase in differentiated 3T3-L1 cells with and without TNFα treatment indicated that a DNA region downstream of -180 bp confers the TNFα effect. Electrophoretic mobility shift assays using two 32P-labeled LPL probes spanning the region between -180 and +44 bp revealed the loss of several LPL DNA-protein interactions after TNFα treatment, including the binding of NF-Y to the CCAAT box and a protein to the octamer consensus sequence. Protein binding to the OCT-1 consensus sequence is unaffected until after 4 h of TNFα treatment. In addition, the amount of mRNA for OCT-1 is not altered with TNFα treatment. These results indicate that TNFα regulates at least two DNA-binding proteins on the proximal promoter, thereby inhibiting LPL gene transcription.