Abstract
BackgroundPeroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and commonly play an important role in the regulation of lipid homeostasis. To identify human PPARs-responsive genes, we established tetracycline-regulated human hepatoblastoma cell lines that can be induced to express each human PPAR and investigated the gene expression profiles of these cells.ResultsThe expression of each introduced PPAR gene was investigated using the various concentrations of doxycycline in the culture media. We found that the expression of each PPAR subtype was tightly controlled by the concentration of doxycycline in these established cell lines. DNA microarray analyses using these cell lines were performed with or without adding each subtype ligand and provided much important information on the PPAR target genes involved in lipid metabolism, transport, storage and other activities. Interestingly, it was noted that while ligand-activated PPARδ induced target gene expression, unliganded PPARδ repressed these genes. The real-time RT-PCR was used to verify the altered expression of selected genes by PPARs and we found that these genes were induced to express in the same pattern as detected in the microarray analyses. Furthermore, we analysed the 5'-flanking region of the human adipose differentiation-related protein (adrp) gene that responded to all subtypes of PPARs. From the detailed analyses by reporter assays, the EMSAs, and ChIP assays, we determined the functional PPRE of the human adrp gene.ConclusionThe results suggest that these cell lines are important tools used to identify the human PPARs-responsive genes.
Highlights
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and commonly play an important role in the regulation of lipid homeostasis
We demonstrated that human PPARs are important regulators of lipid homeostasis in these cell lines using DNA microarray and real-time RT-PCR technologies
The tet-off regulatable HY-Toff cells were transfected with the pBabepuro and pBI-EGFP vector harbouring the cDNA for human PPARα, PPARδ, PPARγ1 or PPARγ2
Summary
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and commonly play an important role in the regulation of lipid homeostasis. To identify human PPARs-responsive genes, we established tetracycline-regulated human hepatoblastoma cell lines that can be induced to express each human PPAR and investigated the gene expression profiles of these cells. One of the approaches for investigating target genes of PPARs is to construct stable cell lines that can be induced to express PPARs. The tet-off system is a well-established system for inducible gene expression. The tet-off system is a well-established system for inducible gene expression In this system, transcription is turned on or off in response to doxycycline (Dox; a tetracycline derivative) in a strictly dose-dependent manner. We established the human hepatoblastoma cell lines (HepG2 cells) which were strictly induced to express the hepatitis C non-structural proteins by removing Dox from the media, and we characterized the changes in mRNA expression profile using DNA microarray analyses [22]
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