Moleculer Regulation Of Peroxisome Proliferator Activated Receptor Alpha (PPARA) Gene Expression By Interleukin-G (Il-G) In Human Hepatocarcinoma Hepg2 Cell Line
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Date
2006-06
Authors
Choy Hoong, Chew
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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-1a, IL-1B, 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. Oeletional 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 B1b activity. Electrophoretic mobility shift assays
(EMSA) further revealed that exposure of HepG2 cells to IL-6 dramatically induced the
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 B1 b were observed to comprise of heterodimers of
C/EBP13-C/EBP&, C/EBPu-C/EBP13 and C/EBPu-C/EBP&, while Sp1 and Sp3 were the
minor and major proteins interacting with the Sp1 site, respectively. Co-transfection
experiments not only showed that C/EBP13 and C/EBP8 act as corepressor to PPARu
constitutive expression, but also function to enhance the IL-6-inhibitory action on
PPARu. Therefore, IL-6-inhibitory action on PPARu gene expression is mediated via
C/EBP13-C/EBP8, C/EBPu-C/EBP13 and C/EBPu-C/EBP& heterodimers by direct
interaction with C/EBP site in the PPARu promoter, with C/EBP13-C/EBP& playing a
major role, while C/EBPu-C/EBP13 and C/EBPu-C/EBP& 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 PPARu
expression. These studies therefore, provided a novel mechanism for IL-6 mediated
regulation of PPARu gene expression in human hepatocarcinoma HepG2 cells.
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Keywords
Peroxisome Proliferator Activated Receptor Alpha (PPARA) , Interleukin-G (Il-G)