Sterol-independent, sterol response element-dependent, regulation of low density lipoprotein receptor gene expression

Abstract

Stimulation with phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin increased native low density lipoprotein (LDL) receptor gene expression in the human leukemic T cell line Jurkat when cells were cultured in the absence of sterols and also increased nuclear accumulation of sterol regulatory element binding protein (SREBP)-1. PMA and ionomycin likewise increased LDL receptor mRNA levels when cells were cultured in the presence of suppressive concentrations of sterols, when neither SREBP-1 nor SREBP-2 was detectable in the nucleus. These findings indicated that mitogen-induced up-regulation of the LDL receptor gene could be independent of sterolregulated transcription factors. The involvement of sterol regulatory element (SRE)-1 was analyzed by transfection of LDL receptor promoter constructs. Promoter fragments of either the 5 1472 or 142 base pairs induced reporter gene expression after mitogenic stimulation when cells were cultured in the absence or presence of sterols. Mutation of the SRE-1 sequence in either construct abolished sterol-mediated regulation of transcription. However, mutation of the SRE-1 sequence in the 1472 base pair promoter fragment did not alter mitogenic induction of transcription, whereas mutation of SRE-1 in the 142 base pair promoter fragment completely prevented up-regulation of transcription. Taken together, these results demonstrate that the LDL receptor promoter contains at least one 5 SRE-independent as well as an SRE-dependent response element. Furthermore, the data suggest that the SRE-dependent response may not involve the action of either SREBP-1 or -2. Thus, mitogen-induced transcription of the LDL receptor promoter is regulated by diverse sterol-independent mechanisms.-

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

“…The construction of the vectors 1472wt-CAT, 142wt-CAT, 1472mut-CAT, and 142mut-CAT was detailed previously (17). These vectors contain either 1472 bp or 142 bp from the LDL receptor promoter and each contains 36 bp from exon 1.…”

“…Those vectors designated wt-CAT contain a wild-type SRE-1. In those designated mut-CAT, 9 of 10 bp in the central core of SRE-1 were mutated (17).…”

“…CAT activity was measured as detailed previously (17). Briefly, whole cell lysates were prepared by three cycles of freeze-thawing and their protein content was determined using Bradford reagent (Bio-Rad).…”

“…Levels of mRNA were quantified as described previously (15,17). Briefly, total RNA (10-40 g) isolated from Jurkat T cells was hybridized with single-stranded cDNA probes for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), LDL receptor, and CAT.…”

“…Nuclear extracts for immunoblot analysis of SREBP-1 and -2 were made from Jurkat cells processed as described (23,24) with minor modifications as detailed (17). Briefly, after harvesting in the presence of protease inhibitors (50 g/ml calpain inhibitor I, 10 g/ml leupeptin, 2.2 g/ml aprotinin, 5 g/ml pepstatin, and 0.5 mm phenylmethylsulfonyl fluoride) and incubation in hypotonic buffer (10 mm HEPES-KOH at pH 7.9, 1.5 mm MgCl 2 , 10 mm KCl, 1 mm EDTA, 1 mm EGTA, 0.5 mm DTT), cells were sheared, centrifuged, and the nuclear pellet was resuspended in high salt buffer (20 mm HEPES-KOH at pH 7.9, 25% v/v glycerol, 400 mm NaCl, 1.5 mm MgCl 2 , 1 mm EDTA, 1 mm EGTA, 0.5 mm DTT) containing all protease inhibitors.…”