Abstract
Peroxisome proliferators cause a rapid and coordinated transcriptional activation of genes encoding the enzymes of the peroxisomal beta-oxidation pathway in rats and mice. Cis-acting peroxisome proliferator responsive elements (PPREs) have been identified in the 5'-flanking region of H202-producing rat acyl-CoA oxidase (ACOX) gene and in other genes inducible by peroxisome proliferators. To gain more insight into the purported nonresponsiveness of human liver cells to peroxisome volume density and in the activity of the beta-oxidation enzyme system, we have previously cloned the human ACOX gene, the first and rate-limiting enzyme of the peroxisomal beta-oxidation system. We now present information on a regulatory element for the peroxidase proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. The PPRE, consists of AGGTCA C TGGTCA, which is a direct repeat of hexamer half-sites interspaced by a single nucleotide (DR1 motif). It is located at -1918 to -1906 base pairs upstream of the transcription initiation site of this human ACOX gene. This PPRE specifically binds to baculovirus-expressed recombinant rat PPAR alpha/RXR alpha heterodimers. In transient transfection experiments, the maximum induction of luciferase expression by ciprofibrate and/or 9-cis-retinoic acid is dependent upon cotransfection of expression plasmids for PPAR alpha and RXR alpha. The functionally of this human ACOX promoter was further demonstrated by linking it to a beta-galactosidase reporter gene or to a rat urate oxidase cDNA and establishing stably transfected African green monkey kidney (CV1) cell lines expressing reporter protein. The human ACOX promoter has been found to be responsive to peroxisome proliferators in CV1 cells stably expressing PPAR alpha, whereas only a basal level of promoter activity is detected in stably transfected cells lacking PPAR alpha. The presence of a PPRE in the promoter of this human peroxisomal ACOX gene and its responsiveness to peroxisome proliferators suggests that factors other than the PPRE in the 5'-flanking sequence of the human ACOX gene may account for differences, if any, in the pleiotropic responses of humans to peroxisome proliferators.
Highlights
Peroxisomes are cellular organelles that are present in virtually all eukaryotic cells [1]
Since peroxisome proliferator-activated receptor (PPAR) isoform(s) as well as enzymes of peroxisomal -oxidation system are present in the human liver [43] and cannot account for the nonresponsiveness of human liver cells to peroxisome proliferator-induced early pleiotropic responses, it is essential to determine whether peroxisome proliferator responsive elements (PPREs) are present and functional in human acyl-CoA oxidase (ACOX) gene and other human genes involved in lipid metabolism
We show that the 5Ј region of the human peroxisomal ACOX gene contains a PPRE and that it appears essential for the response of this gene to peroxisome proliferators
Summary
Peroxisomes are cellular organelles that are present in virtually all eukaryotic cells [1]. Analysis of the Human ACOX Promoter in H4IIEC3 Cells— Treatment of H4IIEC3 rat hepatoma cells with ciprofibrate or other peroxisome proliferators results in the transcriptional activation of the genes encoding the -oxidation enzymes [7, 38].
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