MOLECULAR REGULATION OF PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR ALPHA (PPARa) GENE EXPRESSION BY INTERLEUKIN-6 (IL-6) IN HUMAN HEPATOCARCINOMA HEPG2 CELL LINE
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Date
2006-06
Authors
HOONG, CHEW CHOY
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Abstract
PPARa is a ligand-activated transcription factor, which plays a pivotal role in
coordinating the expression of multiple genes during acute phase response (APR). The
regulation of PPARa by cytokines and lipopolysaccharide (LPS) is therefore, of
potentially crucial importance in the development and initiation of APR However, the
precise mechanisms by which cytokines and LPS modulate the expression of PPARa
in human liver cells are poorly understood. The aim of the work presented here was to
investigate the regulation of PPARa gene expression by the cytokines IL-1 a, IL-1 [3, IL-
6, TNFa and LPS and to elucidate the molecular mechanisms responsible in mediating
the cytokine effect on PPARa gene expression in human hepatocarcinoma HepG2
cells. By utilising RT -PCR and Real-Time RT -PCR, cytokines and LPS were shown to
down-regulate the PPARa mRNA expression. Concomitantly, the corresponding
PPARa protein was significantly reduced after cytokines and LPS treatment. IL-6 was
found to produce the most potent dose- and time-dependent reduction in PPARa
mRNA levels. Transient transfection experiments carried out using the four promoters
(A, B, C and D) of human PPARa in HepG2 cells showed that IL-6 suppressed the
transcriptional activities of all four promoters. Deletional analysis revealed that the
promoter region B1b (-164/+34) contained the minimal IL-6 responsive elements. By
carrying out site-directed mutagenesis experiments, the C/EBP site within this region
was observed to play a crucial role, while Sp1 site played a minor role in the IL-6-
mediated suppression of promoter 81 b activity. Electrophoretic mobility shift assays
(EMSA) further revealed that exposure of HepG2 cells to IL-6 dramatically induced the
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binding of proteins to regions in the C/EBP site. By contrast, IL-6 treatment did not
cause any major changes in the binding activity in the Sp1 binding site. Through
competition EMSA and antibody supershift assays, the interacting proteins with the
C/EBP site of promoter B1b were observed to comprise of heterodimers of
C/EBPJ3•C/EBP8, C/EBPa•C/EBPJ3 and C/EBPa•C/EBPi>, while Sp1 and Sp3 were the
minor and major proteins interacting with the Sp1 site, respectively. Co-transfection
experiments not only showed that C/EBPJ3 and C/EBP8 act as corepressor to PPARa
constitutive expression, but also function to enhance the IL-6-inhibitory action on
PPARa. Therefore, IL-6-inhibitory action on PPARa gene expression is mediated via
C/EBPJ3•C/EBP8, C/EBPa•C/EBPJ3 and C/EBPa•C/EBPi> heterodimers by direct
interaction with C/EBP site in the PPARa promoter, with C/EBPJ3•C/EBP8 playing a
major role, while C/EBPa•C/EBPJ3 and C/EBPa•C/EBPi> play a minor role.
Furthermore, a cooperative interaction between C/EBP and Sp1 family proteins is
necessary to synergistically exert a full inhibitory response of IL-6 on PPARa
expression. These studies therefore, provided a novel mechanism for IL-6 mediated
regulation of PPARa gene expression in human hepatocarcinoma HepG2 cells
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Keywords
MOLECULAR REGULATION OF PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR ALPHA