Original Article

Multiple mechanisms, independent of sterol regulatory element binding proteins, regulate low density lipoprotein gene transcription

Correspondence to: Jennifer A. Cuthbert

Transcription of the LDL receptor gene is markedly enhanced in the Jurkat T cell line by stimulation with the combination of the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the protein synthesis inhibitor cycloheximide (CHX). The DNA sequences necessary for this response were identified by analysis of Jurkat T cells permanently transfected with reporter gene expression vectors containing fragments of the LDL receptor promoter extending from 68 bp to 1472 bp 5' of the major transcription start site. The magnitude of the response of this array of promoter fragments to stimulation with PMA and CHX was similar to that previously observed with a ~6.5 kb promoter fragment. However, the various promoter fragments differed with regard to the role of the sterol regulatory element-1 (SRE-1) sequence. Thus, whereas a 142 bp promoter mediated transcription stimulated by PMA and CHX independently of SRE-1, a shorter 115 bp promoter was absolutely dependent on SRE-1. Furthermore, internal deletion of promoter sequences from -142 bp to -113 bp from longer promoter constructs in which the SRE-1 was mutated prevented the induction of transcription by PMA and CHX. Electrophoretic mobility shift assays (EMSAs) demonstrated sequence-specific, stimulus-independent binding by Jurkat nuclear proteins to the novel response element mapped between -142 and -115. Even though the minimal 115 bp or 68 bp promoter fragment required an intact SRE-1 to respond to PMA and CHX, transcriptional induction persisted when nuclear levels of sterol regulatory element binding proteins (SREBPs) were made undetectable by culture in suppressive sterols.

Taken together, these data indicate that non-sterol stimuli such as the combination of PMA and CHX induce LDL receptor gene transcription through at least two distinct promoter elements, neither of which requires the presence of SREBPs. However, the element proximal to the transcription start site is dependent on the SRE-1.—Makar, R. S. J., P. E. Lipsky, and J. A. Cuthbert. Multiple mechanisms, independent of sterol regulatory element binding proteins, regulate low density lipoprotein gene transcription. J. Lipid Res. 2000. 41: 762;–774.

Supplementary key words: phorbol esters, protein synthesis inhibitors, cycloheximide, electromobility shift assays

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