Premium
LRAT gene promoter exhibits regulated activity, but is not responsive to retinoic acid in HEK and HepG2 cells.
Author(s) -
Zolfaghari Reza,
Ross A. Catharine
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a353-c
Subject(s) - retinoic acid , microbiology and biotechnology , retinoid x receptor , transfection , transcription (linguistics) , response element , luciferase , caat box , gene knockdown , hek 293 cells , biology , nuclear receptor , promoter , tata box , transactivation , transcription factor , retinoic acid receptor , gene , gene expression , biochemistry , linguistics , philosophy
Lecithin:retinol acyltransferase (LRAT) catalyzes the formation of retinyl esters and is essential for normal transport and intracellular storage of vitamin A (VA). In this study, we confirmed that liver LRAT mRNA was up‐regulated by dietary VA and exogenous retinoic acid (RA). To identify the promoter for the LRAT gene, we cloned a 3.2 kb DNA segment from rat genomic DNA, spanning 2.5 kb in the 5′ upstream to 800 bp downstream of the transcription start site (TSS) and prepared LRAT promoter‐luciferase (Luc) constructs used to transfect human kidney (HEK293T) and hepatoma (HepG2) cells. None of the promoter constructs responded to all‐trans‐RA or after cotransfection with RA receptors in either cells, but they showed significantly more activity when the cells were cotransfected with PPAR‐a and RXR‐a in the presence of 9‐cis RA and WY‐14643, a PPAR‐a ligand. The proximal region up to −268 bp from the TSS, which is conserved in mouse and rat, conferred the highest Luc activity in both cells, and a PPAR‐a/RXR‐a responsive region is present within −268 to −111 from TSS. Within the region up to −110 bp from the TSS there are basic transcription elements (TATA box, CAAT box, and SP1 site) and an element for binding tlx, a nuclear receptor, which is apparently required for eye development in mice. The elimination of this region resulted in complete knockdown of Luc activity whereas cotransfection of this region with rat tlx cDNA construct resulted in an increase in activity > 20%. Results from this study showed that the proximal 5′‐flanking region of the LRAT gene may be sufficient to drive the gene for expression of LRAT and further suggest that LRAT may belong to a category of retinoid‐regulated genes which are regulated indirectly, rather than through a direct effect of RA on RA response elements in the promoter region of the LRAT gene. (Support: CA90214)