Hepatocyte Nuclear Factor-4
Regulates the Human Apolipoprotein AV Gene: Identification of a Novel Response Element and Involvement in the Control by Peroxisome Proliferator-Activated Receptor-
Coactivator-1
, AMP-Activated Protein Kinase, and Mitogen-Activated Protein Kinase Pathway
Xavier Prieur,
Frank G. Schaap,
Hervé Coste and
Joan C. Rodríguez
GlaxoSmithKline (X.P., H.C., J.C.R.), 91951 Les Ulis cedex, France; and Academic Medical Center Liver Center (F.G.S.), 105 BK Amsterdam, The Netherlands
Address all correspondence and requests for reprints to: Joan C. Rodríguez, GlaxoSmithKline, 25 avenue du Québec, 91951 Les Ulis cedex, France. E-mail: jcrodrig{at}freesurf.fr.
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ABSTRACT
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The recently discovered apolipoprotein AV (apoAV) gene has been reported to be a key player in modulating plasma triglyceride levels. Here we identify the hepatocyte nuclear factor-4
(HNF-4
) as a novel regulator of human apoAV gene. Inhibition of HNF-4
expression by small interfering RNA resulted in down-regulation of apoAV. Deletion, mutagenesis, and binding assays revealed that HNF-4
directly regulates human apoAV promoter through DR1 [a direct repeat separated by one nucleotide (nt)], and via a novel element for HNF-4
consisting of an inverted repeat separated by 8 nt (IR8). In addition, we show that the coactivator peroxisome proliferator-activated receptor-
coactivator-1
was capable of stimulating the HNF-4
-dependent transactivation of apoAV promoter. Furthermore, analyses in human hepatic cells demonstrated that AMP-activated protein kinase (AMPK) and the MAPK signaling pathway regulate human apoAV expression and suggested that this regulation may be mediated, at least in part, by changes in HNF-4
. Intriguingly, EMSAs and mice with a liver-specific disruption of the HNF-4
gene revealed a species-distinct regulation of apoAV by HNF-4
, which resembles that of a subset of HNF-4
target genes. Taken together, our data provide new insights into the binding properties and the modulation of HNF-4
and underscore the role of HNF-4
in regulating triglyceride metabolism.
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INTRODUCTION
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HYPERTRIGLYCERIDEMIA IS AN independent risk factor for coronary heart disease, which remains one of the leading causes of morbidity and mortality in the Western world (1). Recently, the gene of a new member of the apolipoprotein family, named apolipoprotein AV (apoAV), was identified approximately 30 kb downstream from the apoAI/CIII/AIV gene cluster, and it was found to be an important determinant of plasma triglyceride (TG) levels (2, 3, 4). In mice, serum TG concentrations were decreased to one third when a human apoAV transgene was overexpressed (2) or adenoviral vectors expressing mouse apoAV were injected (4). Conversely, plasma TG levels were 4-fold elevated in knockout mice lacking apoAV compared with their wild-type littermates (2). Studies in mice that either overexpressed or completely lacked both apolipoproteins suggested that apoAV and apoCIII independently influence plasma TG concentrations but in an opposing manner (5). In humans, single-nucleotide polymorphisms (SNPs) across the locus of apoAV gene were found to be significantly associated with elevated plasma TG and very low-density lipoprotein (VLDL) particles mass levels in several ethnic groups (2, 6, 7). apoAV is a very hydrophobic protein that circulates in plasma at much lower concentrations than other apolipoproteins and appears to reduce plasma TG by inhibiting VLDL-TG production and stimulating lipoprotein lipase (LPL)-mediated VLDL-TG hydrolysis (2, 3, 8).
Because the amount of apoAV is an important determinant of plasma lipid levels, understanding the factors that control apoAV gene expression is of major importance and may provide new opportunities for therapeutic intervention in atherogenic dyslipidemia. So far, it has been shown that the apoAV gene is regulated by peroxisome proliferator-activated receptor-
(PPAR
; NR1C1), farnesoid X-activated receptor (FXR; NR1H4) and sterol regulatory element-binding protein 1c, which are transcription factors implicated in TG metabolism (9, 10, 11). In addition, apoAV was identified as a positive acute-phase protein in mouse (12). However, although the apoAV gene expression is restricted to the liver and is essential for lipid homeostasis, its potential regulation by hepatocyte nuclear factor-4
(HNF-4
) has not been reported.
HNF-4
(NR2A1) is a highly conserved member of the nuclear receptor superfamily that was initially identified as a transcriptional factor required for liver-specific gene expression (13), although it is also expressed in kidney, intestine, and pancreas (14). HNF-4
has critical roles in hepatocyte differentiation during liver development and in regulating the transcription of genes involved in glucose and lipid metabolism including the apoAI/CIII/AIV gene cluster, apoAII, apoB, apoCII, microsomal triglyceride transfer protein (MTP), liver-fatty acid binding protein, cholesterol 7
-hydroxylase (CYP7A1), glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), carnitine palmitoyltransferase I
(CPT-I
), pregnane X receptor (PXR; NR1l2), and PPAR
(15, 16). Furthermore, naturally occurring mutations in a single allele of the HNF-4
gene cause an inherited form of diabetes known as maturity onset diabetes of the young 1 (MODY1). This syndrome is characterized by impaired glucose-induced insulin secretion, underscoring the importance of HNF-4
in pancreas (17). Homodimers of HNF-4
bind to specific DNA sequence elements consisting of a direct repeat of the hexanucleotide core motif PuGGTCA separated by 0, 1, or 2 nt (DR0, DR1, or DR2) to activate transcription in the absence of exogenously added ligands (18, 19, 20, 21, 22). Some reports have provided convincing evidence that coenzyme A (CoA) derivatives of certain fatty acids could activate HNF-4
(21, 22). Nevertheless, structural models and cofactor recruitment studies have shown that these derivatives are not traditional ligands and suggested that HNF-4
is constitutively bound to endogenous fatty acids that are essential for the structure of the protein (23, 24). Indeed, recent investigations indicate that coactivator rather than ligand binding locks the active conformation of HNF-4
(25). Thus, the interplay with coactivators is a major concern for the understanding of HNF-4
-dependent regulation of genes.
PPAR-
coactivator-1
(PGC-1
) is a versatile coactivator for numerous nuclear receptors and is implicated in diverse biological activities including glucose and lipid metabolism. Recently, PGC-1
has been shown to enhance the HNF-4
-mediated transactivation of PEPCK, G6Pase, L-CPT I, CYP7A1, and FXR genes (26, 27, 28, 29, 30).
Regulation of HNF-4
-dependent transactivation has also been achieved by modulation of HNF-4
gene expression. We have learned from Leff and co-workers (31) that the apoCIII gene can be regulated by the MAPK signaling pathway and that this regulation is mediated by modulation of HNF-4
expression. These findings indicate that hormonal and metabolic signals can control HNF-4
target genes by changes in the amount of the receptor.
Because there are numerous potential phosphorylation sites in HNF-4
, another possibility for the regulation of its activity is posttranslational modification. AMP-activated protein kinase (AMPK) is a central sensor of a cellular signaling system that regulates multiple metabolic enzymes in response to reduced intracellular energy levels. It has been reported that AMPK directly phosphorylates HNF-4
and represses its activity by reducing its ability to form homodimers and bind DNA, and by increasing its degradation rate (32, 33). Consequently, activation of AMPK catalytic activity by 5-amino-4-imidazolecarboxamide ribonucleoside (AICAR) down-regulates the expression of most, although not all, HNF-4
target genes (32).
The current studies were undertaken to provide a comprehensive analysis of the potential regulation of human apoAV gene expression by HNF-4
. We presently demonstrate that human apoAV is a bona fide HNF-4
target gene. We show that inhibition of HNF-4
expression results in down-regulation of human apoAV and describe that HNF-4
directly activates the human apoAV gene promoter through a DR1 and a novel HNF-4
response element. Furthermore, the coactivator PGC-1
is capable of stimulating this HNF-4
-dependent transactivation of human apoAV promoter. In addition, our data demonstrate that AMPK and the MAPK cascade regulate human apoAV expression and suggest that this regulation may be mediated, at least in part, by changes in HNF-4
function. Furthermore, we presently report that the regulation of apoAV by HNF-4
is somehow species specific, a phenomenon also present in a subset of HNF-4
target genes. Finally, we discuss the implications that the identification of apoAV as an HNF-4
target gene may have in the regulation of TG levels by bile acids (BAs).
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RESULTS
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HNF-4
Enhances the Activity of the Human ApoAV Gene Promoter
ApoAV is an important determinant of plasma TG levels that appears to be expressed exclusively in the liver (2, 3, 10, 11). Inasmuch as the nuclear receptor HNF-4
is essential for maintenance of hepatic gene expression and is a crucial regulator of genes involved in lipid metabolism (16), including particularly the apoAI/CIII/AIV gene cluster (34, 35), we evaluated whether this nuclear receptor modulates human apoAV gene expression. To study the direct effect of HNF-4
on transcription, transient transfection studies were performed in Hep3B cells, which express moderate levels of apoAV (10) and HNF-4
(36). Cotransfection of a human HNF-4
expression plasmid resulted in a significant increase of the activity of the firefly luciferase reporter gene driven by the 617/+18 sequence of the human apoAV promoter (Fig. 1A
). The effect of HNF-4
overexpression was promoter dependent because it was not observed with the promoter-less pGL3-basic vector. To further confirm the induction by HNF-4
, monkey kidney CV-1 cells, a cellular model frequently used to avoid the background of endogenous HNF-4
(19, 37, 38), were also employed. As shown in Fig. 1B
, increasing amounts of the expression plasmid for HNF-4
resulted in a dose-dependent induction of firefly luciferase activity. Activation by exogenously supplied HNF-4
was already appreciable at low doses (Fig. 1B
).
Mapping of the Human ApoAV Promoter Regions Conferring Responsiveness to HNF-4
To localize the promoter region required for HNF-4
activation, a series of progressively larger 5'-deletion human apoAV promoter constructs from nt 2455 to +18 in front of the firefly luciferase reporter gene were transiently transfected into CV-1 cells together with a human HNF-4
expression plasmid. As shown in Fig. 2A
, the sequence upstream to position 437 could be removed without preventing strong activation of the reporter gene by HNF-4
. However, deletion of the fragment spanning 437 to 242 reduced HNF-4
responsiveness. Moreover, further deletion up to nt 82 almost abolished the induction of apoAV promoter activity by HNF-4
, indicating that at least two regions mediate the response.
On the other hand, a T
C SNP at nt 600 relative to the transcription start site (1131 nt upstream from the predicted translation start codon) of the human apoAV gene, called SNP3 in Ref.2 , has been associated with elevated plasma TG levels (2, 6, 7). Analysis of the sequence surrounding this SNP3 revealed no apparent transcription factor binding sites, but assuming a high degree of degeneration one might consider the occurrence of two potential hexamer binding sites separated by a single nt between nt 611 and 599, especially with a C at 600 (GGGGCA A ATCTCA), thereby conforming to the DR1 response element for HNF-4
. Luciferase reporter assays performed in Hep3B (data not shown) and CV-1 cells (Fig. 2A
) revealed that change in common allele T to C had no significant effect on the induction by HNF-4
. Consistent with this observation, the deletion of a fragment containing this SNP, nt 617 to 535, did not affect the response to HNF-4
(Fig. 2A
).
Identification of a DR1 Site and a Novel Element for Transcriptional Activation by HNF-4
in the Human ApoAV Promoter
Analysis of the sequence in the 437/242 fragment revealed two hexamer binding sites separated by a single nt between nt 271 and 259 (Fig. 2B
), which correspond to a canonical DR1 response element for HNF-4
. In contrast, analysis of the sequence in the 242/82 fragment did not reveal any of the previously described HNF-4
response elements DR0, DR1 or DR2 motifs (19, 20, 39). The only repeat of the hexanucleotide core motif PuGGTCA with a low degree of degeneration present within this region corresponds to an inverted repeat separated by 8 nt (IR8) between nt 103 and 84 (Fig. 2C
), which has been previously described to respond to FXR (10). To characterize both DR1 and IR8 sites as HNF-4
response elements, CV-1 cells were cotransfected with a human HNF-4
expression vector and 617/+18 apoAV promoter-luciferase reporter plasmids in which DR1 and/or IR8 elements were mutated (Fig. 2
, B and C). The data shown in Fig. 2D
indicate that both sites confer HNF-4
response to the apoAV promoter and that they contribute similarly, albeit somewhat more IR8 than DR1, to the HNF-4
-mediated induction. The reporter construct bearing mutations in both DR1 and IR8 sites retained a transactivation by HNF-4
of about 25% compared with wild-type. Similar results were obtained with DR1 and IR8 mutant constructs containing the sequence spanning from 305 to +18 of apoAV promoter (data not shown). The basis for this remaining response is unclear because systematic EMSAs performed with numerous fragments within this region showed no binding to HNF-4
(see below), and it might be the result of an indirect action of HNF-4
.
Binding Analysis of HNF-4
to the Human apoAV Promoter
To investigate whether HNF-4
homodimers directly bind to the human apoAV gene promoter, radiolabeled double-stranded oligonucleotides corresponding to fragments of the DNA sequence spanning from nt 617 to +18 were used as probes in EMSA experiments (Fig. 3A
). As expected, the addition of in vitro-translated human HNF-4
protein resulted in the appearance of retarded complexes when using a 275/247 fragment containing the apoAV DR1 site (Fig. 3A
, lane 6) and a 109/80 fragment containing the apoAV IR8 element (lane 12), but not when 399/374, 169/147, 144/117, 52/31 probes (Fig. 3A
, lanes 4, 8, 10, and 14, respectively), or 298/263, 258/221, 220/183, 183/143, 142/104, 240/202, 202/166, 166/131, 141/103 fragments were employed (data not shown). On the other hand, in line with the results from cell transfection assays, no protein-DNA complex band appeared when using a 617/593 fragment containing the SNP3 with a T (Fig. 3A
, lane 2) or a C (data not shown) at 600.

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Fig. 3. Binding Analysis of HNF-4 and HepG2 Nuclear Extracts to the Human apoAV Gene Promoter
A, EMSAs were performed using in vitro-transcribed/translated HNF-4 (2.5 µl) or unprogrammed reticulocyte lysate (), when indicated, and labeled double-stranded oligonucleotides corresponding to the indicated fragments of the human apoAV gene promoter as described in Materials and Methods. CONTROL, PPAR /HNF-4 site of mitHMGS gene (60 ). B, Endogenous HNF-4 binds specifically to the apoAV DR1 and IR8 elements. EMSAs were performed using HepG2 nuclear extracts and labeled double-stranded oligonucleotides corresponding to nt 109 to 80 (wt AvIR8) and 275 to 247 (wt AvDR1) of the human apoAV gene promoter, and an antibody against HNF-4 , when indicated, as described in Materials and Methods. mt3 AvIR8 and mt3 AvDR1 are modified versions of wt AvIR8 and wt AvDR1, respectively, whose mutations are shown in Fig. 2 , B and C. Supershifted immune complexes are indicated by an arrow. CONTROL, HNF-4 -binding element of apoCIII gene (39 ).
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HNF-4
Binds Specifically to the Human ApoAV DR1 and IR8 Elements
We next investigated whether HNF-4
was able to bind to apoAV DR1 and IR8 elements in hepatic nuclear extracts. For this purpose, we performed EMSAs with nuclear extracts obtained from human liver-derived HepG2 cells, which express apoAV (10) and HNF-4
(39), in the presence or absence of a specific HNF-4
antibody. As expected, the slower migrating complexes obtained with apoAV IR8 (Fig. 3B
, lane 1) and DR1 (lane 3), or a control probe containing a known HNF-4
-binding element present in the human apoCIII gene promoter (lane 5), were supershifted upon addition of the HNF-4
antibody (lanes 2, 4, and 6, respectively). In contrast, double-stranded oligonucleotides that are equivalent to apoAV IR8 and DR1 probes (mt3 AvIR8 and mt3 AvDR1, respectively) but harbor point mutations in the half-sites (Fig. 2
, B and C) could not form the same slower migrating complexes (Fig. 3B
, lanes 7 and 9). Furthermore, a shift with faster mobility of mt3 AvDR1 probe (lane 9), which had a similar migration to a faint band appeared with the wild-type AvDR1 probe (lane 3), was not supershifted (lane 10). Additionally, because essentially most of the slower DNA-protein complexes were supershifted by the HNF-4
antibody, these results indicated that endogenous HNF-4
in these complexes was the major factor in nuclear extracts that could interact with apoAV DR1 and IR8 elements.
To unequivocally characterize the apoAV DR1 and IR8 as HNF-4
-binding sites, the specificity of these interactions was demonstrated by competition analysis. The formation of the retarded complex of apoAV DR1 was inhibited by the addition of increasing concentrations of either the unlabeled wild-type probe or a cold probe containing the HNF-4
-binding element of the human apoCIII gene promoter (data not shown). In contrast, cold double-stranded oligonucleotides that are equivalent to the apoAV DR1 probe but harbor point mutations (see Table 1
) in the upstream half-site (mt1 AvDR1), in the downstream half-site (mt2 AvDR1) or in both half-sites (mt3 AvDR1 and mt4 AvDR1) could not displace the labeled wild-type element (data not shown). The HNF-4
-IR8 binding was also specific, as demonstrated by competition of an excess of either unlabeled wild-type apoAV IR8 oligonucleotide or a cold probe containing the HNF-4
-binding element of the human apoCIII gene promoter (Fig. 4B
, wt AvIR8 and CONT, respectively). Furthermore, mutation of either half-site (Fig. 4A
, mt1 AvIR8, mt2 AvIR8, mt3 AvIR8) abolished the ability of the probe to compete (Fig. 4B
). Because an HNF-4
binding site with this type of organization has not been reported before, we extended the analysis of this element by introducing a point mutation in the downstream half-site to eliminate the only degeneration present in this hexanucleotide (Fig. 4A
, pft AvIR8). As shown in Fig. 4C
, the binding of HNF-4
to a radiolabeled probe corresponding to this perfect IR8 of the core motif AGGTCA (lane 6) is even more apparent than with the wild-type apoAV IR8 (lane 5). In addition, mutation of nt in the half-site spacing to leave the two hexanucleotide core-binding sites separated by 8 T (Fig. 4A
, pft tttIR8), resulted in a modest reduction, not exclusion, of the binding (Fig. 4C
, lane 7). As a control, no binding is observed with a labeled probe bearing point mutations that increase degeneration of the two hexanucleotides (lane 8). Taken together, these results demonstrate that HNF-4
homodimers are able to directly and specifically interact in vitro with the apoAV IR8 and not with cryptic binding motifs embedded within this site. Next, we examined whether HNF-4
binds human apoAV IR8 in vivo by chromatin immunoprecipitation (ChIP) assay. For technical reasons, this may not be possible on the endogenous apoAV promoter due to the closeness of the DR1 and IR8 sites. Thus, we carried out a DNA immunoprecipitation assay on plasmid p-242/+18hAvLUC, which is an apoAV promoter-luciferase reporter construct that contains IR8 but not DR1. HepG2 cells were transfected, and formaldehyde was added to cross-link the DNA-protein complexes in vivo. After immunoprecipitation, and reversal of cross-linking, the DNA was purified and used as a template for PCR amplification with primers that encompass the region from nt 197 of apoAV promoter to a sequence in the pGL3-basic vector. As shown in Fig. 4D
, this region was precipitated by antibodies directed against HNF-4
. In contrast, no signal was obtained when the immunoprecipitation was carried out with anti-PPAR
antibodies in the same conditions, which is consistent with reports showing that the DR1 element at 271 is required for PPAR
-mediated transactivation of human apoAV promoter (9, 10). Moreover, when ChIP was performed on an equivalent construct harboring mutations within the IR8 site, the signal was dramatically diminished (Fig. 4D
, p-242mutIR8/HNF-4
-Ab).

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Fig. 4. HNF-4 Binds Specifically to the IR8 Element of apoAV Gene Promoter
A, Human apoAV gene promoter sequence surrounding the IR8 element and mutated versions used in experiments B and C. The hexameric consensus sites are in boldface type, and their orientations are indicated by arrows. Mutations in half-sites are shown in lowercase type and within gray squares. B, Competition experiments for binding of in vitro-transcribed/translated HNF-4 to the labeled probe corresponding to nt 109 to 80 (AvIR8) of the human apoAV gene promoter were performed by adding 50-, 250-, and 500-fold molar excess of the indicated unlabeled double-stranded oligonucleotides. CONT, HNF-4 -binding element of apoCIII gene (39 ). C, EMSAs were performed as described in Materials and Methods using in vitro-transcribed/translated HNF-4 (2.5 µl) or unprogrammed reticulocyte lysate (), when indicated, and labeled double-stranded oligonucle-otides corresponding to the sequences in A. D, ChIP analysis of HNF-4 interaction with apoAV IR8 element in vivo. Cross-linked DNA isolated from HepG2 cells transfected with luciferase reporter plasmid p-242/+18hAvLUC (p-242wtIR8), an equivalent construct harboring mutations within the IR8 site (p-242mutIR8), or pGL3-basic vector as a negative control, was immunoprecipitated with the indicated antibodies, and subjected to PCR as described in Materials and Methods. PCR products were electrophoresed on 1% agarose gel. The relative location of the PCR primers is depicted as arrows in diagram below. LUC, Luciferase. Experiments were performed at least two independent times, and representative data are shown.
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Inhibition of HNF-4
Expression Results in Down-Regulation of Human ApoAV
To further provide evidence for the involvement of HNF-4
in the regulation of human apoAV expression, we used RNA interference technology to knock down endogenous HNF-4
. Transfection of HepG2 with increasing concentrations of small interfering RNA (siRNA) targeting HNF-4
expression resulted in a dose-dependent decrease of apoAV mRNA levels (Fig. 5A
). This effect appeared to be specific because the expression of apoCIII, a well-known HNF-4
target gene, was also diminished, whereas cyclophilin, used as internal control, was unaffected by siRNA transfection (Fig. 5A
and data not shown). Moreover, immunoblotting experiments revealed that this HNF-4
siRNA-mediated down-regulation of apoAV occurred also at the protein level (Fig. 5
, B and C) and confirmed that this effect is specific because the nonsilencing control siRNA did not significantly affect endogenous HNF-4
and apoAV protein expression (Fig. 5B
). In addition, actin protein levels, which were measured as a control, were not modified after siRNA transfection, thereby demonstrating the specificity of this effect (Fig. 5B
). Taken together, these results establish the essential role of HNF-4
in the regulation of human apoAV expression.
HNF-4
Is Involved in the Stimulation of ApoAV Gene Promoter by the Nuclear Receptor Coactivator PGC-1
It has been demonstrated that PGC-1
stimulates the promoter activity of multiple hepatic genes involved in gluconeogenesis (26, 27), fatty acid oxidation (28), BA synthesis (29), and TG metabolism (30) through coactivation of HNF-4
. To determine whether PGC-1
modulates apoAV promoter activity and to evaluate the potential involvement of HNF-4
, transient transfection assays were performed with the firefly luciferase reporter gene expression vector driven by sequences of the human apoAV promoter. In Hep3B, cotransfection of a human PGC-1
expression plasmid significantly enhanced the 617/+18 apoAV promoter activity (Fig. 6A
). Cotransfection of HNF-4
and PGC-1
together resulted in a further activation, although only slightly greater than with HNF-4
alone (Fig. 6A
). The effect of PGC-1
overexpression was promoter dependent because it was not observed with the promoter-less pGL3-basic vector. To identify the regulatory elements mediating the effect of PGC-1
, a reporter construct bearing mutations in the DR1 and IR8 elements (Fig. 2
, B and C) was tested. Although some remaining response was observed, the mutations markedly diminished the response to PGC-1
and HNF-4
(Fig. 6A
). In addition, a truncated construct representing the 82/+18 apoAV promoter was completely unresponsive to HNF-4
and PGC-1
. These results indicate that PGC-1
moderately stimulates human apoAV promoter activity, preferentially via DR1 and IR8 elements. PGC-1
can also coactivate PPAR
(40) and FXR (30). Because apoAV DR1 and IR8 have been previously described as PPAR
and FXR response elements, respectively (10), it is also possible that PGC-1
is acting on the human apoAV promoter through these transcription factors. To prove that PGC-1
-dependent stimuli on apoAV promoter activity can be achieved by coactivation of HNF-4
, we performed cotransfection studies in the CV-1 line, a cellular model frequently used by others in PGC-1
studies (30, 40) to avoid the background of endogenous HNF-4
, PPAR
and FXR. As shown in Fig. 6B
, cotransfection of PGC-1
alone resulted in a weak activation of apoAV promoter, an effect also observed in other promoters (Fig. 2
in Ref.30 and Fig. 1
in Ref.40). However, cotransfection of PGC-1
and HNF-4
together caused a dramatic activation of this promoter that was markedly higher than with either factor alone. These results reveal a clear synergistic effect between PGC-1
and HNF-4
on the activity of the human apoAV promoter.
The MAPK Pathway Regulates the Expression of Human ApoAV
Leff and colleagues (31) have demonstrated that apoCIII expression can be regulated by signals acting through the MAPK signaling pathway and that this regulation is mediated, at least in part, by changes in the amount of HNF-4
. To determine whether this could be also true for apoAV, HepG2 cells were incubated for 24 h in medium containing PD98059 (41), an inhibitor of the upstream activator of Erk1/2, which are members of the MAPK family, or vehicle. As determined by quantitative real-time RT-PCR, treatment with PD98059 caused a significant increase of apoAV mRNA levels (Fig. 7A
). These effects were specific because the expression of apoCIII was also increased by PD98059, in accordance with previous studies (31), whereas cyclophilin, used as internal control, remained unaffected by the treatments (Fig. 7A
and data not shown). Furthermore, in contrast to apoAV and apoCIII, low-density lipoprotein receptor (LDLr) mRNA levels were slightly reduced by PD98059 after 24 h of treatment under standard serum conditions, in agreement with published results (42). ApoAI mRNA levels were affected similarly to apoAV and apoCIII (Fig. 7A
).

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Fig. 7. Regulation of Human apoAV Expression by the MAPK Pathway and AMPK
A, HepG2 cells were treated for 24 h with vehicle (Control) or the Erk1/2 kinase inhibitor PD98059 (20 µM). Total RNA was extracted for analysis by real time RT-PCR as described in Materials and Methods. Specific mRNA levels normalized to cyclophilin content are expressed relative to untreated cells set as 1 for each gene (mean ± SD). Significant differences compared with the untreated control are as follows: *, P < 0.05; **, P < 0.001. B, Western analyses of HNF-4 , apoAV, and actin expression in whole cell lysates from HepG2 treated for 24 h as in A. C, HepG2 cells were treated as in A with increasing concentrations of AMPK activator AICAR. D, Western analyses performed as in B with increasing concentrations of AICAR. The contents of phosphorylated acetyl-CoA carboxylase were also monitored as positive control. E, AMPK and the MAPK pathway modulate the binding of HepG2 nuclear extracts to the DR1 and IR8 elements of human apoAV promoter. HepG2 cells were treated for 24 h with vehicle, AICAR (2.5 mM), or PD98059 (20 µM). Nuclear extracts were prepared and EMSAs were performed using labeled double-stranded oligonucleotides corresponding to nt 109 to 80 (AvIR8) and 275 to 247 (AvDR1) of the human apoAV gene promoter as described in Materials and Methods. CONTROL, HNF-4 -binding element of apoCIII gene (39 ). Experiments were performed twice with identical results.
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Next, Western blot analysis performed on whole cell lysates from treated HepG2 revealed that the quantity of apoAV and HNF-4
proteins was notably increased by PD98059, whereas the levels of actin were indistinguishable between treatments (Fig. 7B
). Taken together, these observations demonstrate that the MAPK/Erk kinase signaling pathway controls human apoAV expression and suggest that this regulation may be mediated, at least in part, by changes in the amount of HNF-4
.
Activation of AMPK Down-Regulates Expression of Human ApoAV
Activation of AMPK diminishes HNF-4
protein levels and inhibits its ability to form homodimers and bind to DNA, and consequently down-regulates the expression of several HNF-4
target genes (32, 33). To investigate whether activation of AMPK catalytic activity down-regulates human apoAV expression, HepG2 cells were incubated for 24 h in the presence of increasing concentrations of AICAR, which is converted in cells to an analog of AMP that activates the kinase (43). As shown in Fig. 7C
, AICAR significantly decreased apoAV mRNA levels. As a positive control, apoCIII mRNA levels were measured, and consistent with the literature (32), a clear down-regulation was observed. ApoAI mRNA levels were also decreased. However, no changes were detected in cyclophilin levels, which was used as internal control, and in contrast to apolipoproteins, LDLr mRNA levels were robustly induced by AICAR treatment (Fig. 7C
).
Immunoblotting assays with whole cell lysates revealed that increasing concentrations of AICAR decrease HNF-4
protein levels in HepG2 (Fig. 7D
), likewise as described in murine hepatocytes (32). The results, depicted in Fig. 7D
, show that apoAV protein levels declined with increasing concentration of AICAR, whereas actin levels were not modified and while the phosphorylation of acetyl-CoA carboxylase increased. Given the key role of HNF-4
in apoAV expression, these data clearly indicate that the activation of AMPK catalytic activity down-regulates human apoAV expression and suggest that this negative regulation occurs, at least in part, via HNF-4
.
AMPK and the MAPK Cascade Modulate the Binding of HepG2 Nuclear Extracts to the DR1 and IR8 Elements of Human ApoAV Promoter
We next examined whether the MAPK/Erk kinase pathway and the activation of AMPK could modify the binding of HepG2 nuclear extracts to the HNF-4
response elements of human apoAV. For this purpose, we carried out EMSAs using nuclear extracts prepared from HepG2 treated with AICAR, PD98059, or vehicle and labeled oligonucleotides corresponding to the apoAV DR1 and IR8 elements described in this report (Fig. 7E
, AvDR1 and AvIR8, respectively). As expected, we found that AICAR treatment caused a dramatic reduction in DNA binding of the nuclear extracts to the IR8 (Fig. 7E
, compare lane 1 with lane 4) and DR1 (Fig. 7E
, compare lane 2 with lane 5), whereas PD98059 robustly increased binding to both HNF-4
sites of apoAV (Fig. 7E
, compare lanes 7 and 8 with lanes 10 and 11, respectively). These results further support the hypothesis that HNF-4
is mediating the effects of AMPK and the MAPK/Erk kinase pathway on human apoAV expression.
Species-Distinct Regulation of ApoAV by HNF-4
Next, we sought to extend the analysis to mouse, an animal model commonly used for apoAV studies (2, 3, 4, 5, 8, 11, 12). Sequence comparisons revealed that the human apoAV DR1 element that we describe here as an HNF-4
site is apparently not conserved in the mouse apoAV gene promoter. Indeed, the alignment of the sequences, depicted in Fig. 8A
, reveals 3-nt differences that render higher degeneration of the two hexameric PuGGTCA core-binding sites in murine sequences compared with the human DR1. In addition, it has been reported that the occurrence of A as the spacer nucleotide between the hexamers is crucial for the binding of HNF-4
(20, 38). To determine whether these nucleotide differences could impair the binding of HNF-4
, EMSA experiments were performed. As shown in Fig. 8B
, HNF-4
binds to a human, but not to a mouse, probe containing the DR1 sequence. These results prompted us to explore the possibility of a species-distinct regulation of the apoAV gene by HNF-4
.
To obtain direct evidence for different regulation of murine apoAV by HNF-4
, we measured the levels of apoAV in H4LivKO mice, which are conditionally HNF-4
-null mice lacking hepatic HNF-4
expression (16). Strikingly, immunoblotting assays showed that the absence of HNF-4
in the liver of standard chow-fed adult mice had no effect on liver or serum protein concentrations of apoAV, further confirming the species-distinct regulation of apoAV by HNF-4
(Fig. 8C
). As a control, we verified that serum of H4LivKO mice was virtually devoid of apoAII, a canonical HNF-4
target gene (16).
 |
DISCUSSION
|
---|
Overexpression of apoAV results in a dramatic reduction of plasma TG, whereas the lack of apoAV transcripts causes a marked hypertriglyceridemia (2, 3, 4, 5, 8). Therefore, the potential regulation of apoAV expression by hormonal and metabolic signals may play a significant role in controlling plasma TG levels. The present study reveals that a liver-enriched nuclear receptor involved in the regulation of lipid-related genes, namely HNF-4
, controls the human apoAV gene. We show that depletion of the endogenous HNF-4
by siRNA-mediated RNA interference causes down-regulation of apoAV mRNA levels. Our results demonstrate unequivocally that HNF-4
directly regulates the apoAV gene promoter via two elements, a DR1 and an IR8, a hitherto unknown element for HNF-4
. We show for the first time that HNF-4
binds to an IR8 both in vitro and in vivo, and by using deletion and site-directed mutagenesis we demonstrate that this IR8 is required for full transactivation by HNF-4
. Thus, we conclude that this novel IR8 element is a bona fide HNF-4
response element.
Numerous studies have reported that a T
C polymorphism in the 5'-region of the human apoAV gene, called SNP3, is associated with elevated plasma TG levels (2, 6, 7). Assuming a high degree of degeneration, one could consider the occurrence of a DR1 element spanning nt 611 to 599, particularly if a C is present (GGGGCA A ATCTCA). We had previously reported that the basal activity or the PPAR
or FXR-mediated induction of the apoAV promoter was not affected by this change of T to C (10). Similarly, our gel shift analyses (Fig. 3A
and data not shown) indicate that HNF-4
is not binding to this fragment. Moreover, luciferase reporter assays revealed that deletion of the sequence surrounding this SNP or the change of the common allele T to C has no significant effect on the induction by HNF-4
of the apoAV promoter activity (Fig. 2A
), further arguing against the functional character of this polymorphism.
Two reports on MODY1 showed that, in addition to abnormal pancreatic ß-cell function, mutations in a single allele of the HNF-4
gene, which result in truncation of the protein, were associated with mild reductions in serum levels of TG, apoAII, and apoCIII (44, 45). Despite the key role of HNF-4
in hepatic expression, no liver defect was identified in individuals with these HNF-4
mutations. Notably, the serum levels of some HNF-4
-regulated genes, including transferrin,
-1 antitrypsin, and apoAI were not decreased in subjects with HNF-4
mutations (44, 45). The reasons for these findings remain unknown, but compensatory mechanisms related to the diabetic condition have been suggested (45). Our RNA interference data show that apoAV expression is less sensitive than apoCIII to a reduction of HNF-4
levels (Fig. 5A
), thereby suggesting that a dramatic reduction of apoAV levels in MODY1 patients is unlikely.
In the present study, we also explored which effects may produce the modulation of HNF-4
activity on the expression of human apoAV. A previous report has shown that inhibition of the Erk1/2 signaling in HepG2 augmented HNF-4
levels, which in turn resulted in an increase of apoCIII expression (31). Here we show that this also may be valid for apoAV (Fig. 7
). Furthermore, it has been reported that AMPK represses the activity and increases the degradation of HNF-4
by direct phosphorylation, which consequently results in down-regulation of the expression of many HNF-4
target genes (32, 33). As expected, the activation of AMPK catalytic activity by AICAR led to the down-regulation of apoAV and apoCIII (Fig. 7
). Interestingly, in contrast to rodent hepatocytes (32), we observed that AICAR decreases apoAI expression in human HepG2. Moreover, these effects on HNF-4
function were confirmed in gel shift analysis showing that the binding of HepG2 nuclear extracts to the DR1 and IR8 elements of human apoAV (mainly due to HNF-4
, as shown in Fig. 3B
) was diminished by activation of AMPK and increased by inhibition of the Erk1/2 signaling (Fig. 7E
). Notwithstanding the above, we do not exclude the possibility that besides HNF-4
there might be other yet undiscovered factors contributing to the effects of MAPK and AMPK. At present, the physiological role of the MAPK/Erk kinase cascade in the regulation of apoAV expression remains unknown. We have observed that IL-1ß negatively regulates apoAV expression in human hepatocytes and HepG2 (data not shown). Because IL-1ß activates Erk1/2 signaling (46) and reduces HNF-4
protein levels in HepG2 (Ref.47 and data not shown), it is tempting to speculate that the response of human apoAV to this inflammatory cytokine may be mediated, at least in part, by changes in the HNF-4
-dependent regulation. However, further work is required to confirm or rebut this hypothesis. AMPK acts as a fuel sensor that is activated by states of low energy charge (i.e. reduced ATP/AMP ratios). When activated, AMPK inhibits ATP-consuming anabolic pathways and stimulates ATP-producing catabolic signals. In the liver, AMPK activation results in reduced lipid and glucose synthesis by repressing gene expression of fatty acid synthase, PEPCK, and G6Pase (48). Because all the effects of AMPK activation that have been described caused reduction of gene expression, it has been proposed that one of the primary functions of AMPK might be to reduce the general rate of transcription, a process that utilizes large amounts of energy (48). The inhibition of HNF-4
activity would indeed reduce the transcription of a relatively large set of genes, which is consistent with this general role of the kinase in reducing the rate of energy-utilizing processes. However, the apoAV down-regulation by AMPK appears counterproductive because it would result in a reduction in the VLDL-TG hydrolysis. Further studies are needed to elucidate the precise physiological significance of the repression of human apoAV by activated AMPK.
Strikingly, we observed that liver and plasma concentrations of apoAV in adult mice were unaffected by liver-specific disruption of the HNF-4
gene (Fig. 8C
), whereas expression of other HNF-4
target genes including CYP7A1, MTP, apoAII, apoB, and apoCIII genes was found markedly decreased (Ref.16 and Fig. 8C
). We also show that nucleotide differences between human and mouse in the most upstream HNF-4
element of apoAV promoter abolish binding by HNF-4
(Fig. 8B
), which could be a sign of a species-distinct regulation of the apoAV gene by HNF-4
. However, the sequence of the IR8 in the human apoAV promoter is perfectly conserved in murine counterpart (data not shown), which is difficult at present to reconcile with the former results in HNF-4
-null livers. One possibility might be that in these modified mice another activator substitutes for HNF-4
in apoAV IR8 site. In addition, given that the expression of some mouse genes containing a functional HNF-4
site in their proximal promoters is down-regulated by disruption of HNF-4
in fetal hepatocytes but not in the adult liver (15, 16, 49, 50), it is tempting to speculate that the apoAV IR8 element might be used by HNF-4
in the fetal liver but not the mouse adult liver. In fact, an HNF-4
binding site was characterized in the mouse PXR gene and found to be required for activation of the PXR promoter in fetal hepatocytes (49). However, the role of this element in the adult liver is uncertain because the expression of PXR is decreased by deletion of the HNF-4
gene in fetal hepatocytes but not in the fully differentiated liver (15, 16, 49). By analogy to apoAV, the case of apoAI is particularly intriguing because the expression of the human gene is clearly regulated by HNF-4
and the promoter contains two HNF-4
functional elements (34, 35, 36, 37). In contrast, 3-nt differences abolish activation by HNF-4
through the most upstream element of the rat and mouse apoAI gene promoters (51). Still, the activity of rodent apoAI is increased by HNF-4
via a proximal element (50, 51) at nt 132/120. This finding is in direct contradiction with the demonstration that mRNA levels for apoAI were unaffected by disruption of the HNF-4
in the adult mice liver (16), but it is consistent with the abolishment of apoAI expression in fetal HNF-4
-null livers (15). Thus, it will be of considerable interest for future liver development studies to determine whether there is also an HNF-4
-dependent regulation of mouse apoAV expression specific to the fetal stage, and to clarify the physiological significance of the IR8 element in apoAV gene.
It is well established that, in the liver, PGC-1
plays a key role in the nutritional regulation of gluconeogenesis, fatty acid oxidation, ketogenesis, and BA synthesis (26, 27, 28, 29, 30). Spiegelman and co-workers (26) have reported that PGC-1
coactivates HNF-4
by direct interaction in a ligand-independent manner. These authors have shown that HNF-4
is absolutely required for PGC-1
-mediated induction of G6Pase and PEPCK, whereas PGC-1
can still activate, albeit at much lesser extent, the expression of the CPT-I
in the absence of HNF-4
(27). In fact, PGC-1
can stimulate CPT-I
through both HNF-4
and thyroid hormone receptor-ß (28, 52). In reporter assays, transfection of either PGC-1
or HNF-4
activated G6Pase promoter very poorly, whereas cotransfection of both PGC-1
and HNF-4
caused a dramatic activation (27). In similar assays, a synergism between PGC-1
and HNF-4 was clear in the promoters of CPT-I
(28), CYP7A1 (29) and FXR genes (30), respectively, but to a much lesser extent than in G6Pase promoter. Our studies show that there is also a clear synergism between PGC-1
and HNF-4
in the activation of apoAV promoter, even stronger than those reported for other genes (i.e. FXR), but less dramatic than in G6Pase promoter. Whether other transcription factor besides HNF-4
could participate in recruiting PGC-1
to the apoAV promoter is under investigation. Additionally, because the hepatic expression of HNF-4
itself is induced after fasting and PGC-1
abundance is increased in fasting and diabetes (26, 30), it will be of great interest to examine the levels of human apoAV expression in these states.
The identification of apoAV as an HNF-4
target gene has serious implications in the regulation of TG levels by BAs. Several investigators have recently reported that in the liver BAs can regulate the transcription of genes involved in lipoprotein and TG metabolism via a pathway involving FXR and the small heterodimer partner (SHP) (53, 54, 55, 56). BAs down-regulate HNF-4
target genes by reducing the expression of HNF-4
itself (53). In addition, BAs reduce MTP and apoB mRNA levels because of increased expression of the FXR target gene SHP, which in turn suppresses HNF-4
activity (53). Notably, BAs suppress hepatic expression of apoCIII, an inhibitor of LPL-mediated lipolysis, via an active repression by FXR and displacement of HNF-4
from the DNA site (54). On the other hand, BAs induce the expression of the HNF-4
target apoCII, an obligate cofactor for LPL, through two IR1 FXR response elements (55). Thus, one may speculate that BAs would repress HNF-4
target genes if no positive BA response elements were present. We have previously reported that the apoAV IR8 element is a positive FXR response element (10). However, we observed that apoAV mRNA levels were not modified by FXR agonists in HepG2, thus suggesting that the regulation of apoAV expression by BAs was not straightforward (10). It is possible that the role of IR8 as a positive FXR element is to counteract a negative BA-triggered signal through HNF-4
, thereby avoiding the down-regulation of apoAV, an activator of LPL-mediated hydrolysis of VLDL-TG.
Finally, while performing this study, a genome-scale location analysis using ChIP assays and DNA microarrays containing 700-bp promoter regions of 13,000 human genes identified the apoAV gene promoter bound by HNF-4
in human hepatocytes (57), further confirming our results.
In conclusion, here we demonstrate that human apoAV is de facto an HNF-4
target gene. Our data provide new insights into the binding properties of HNF-4
, recognize HNF-4
as a highly regulated factor that is involved in multiple hormonal and nutritional signaling pathways, and underscore the physiological role of HNF-4
in TG metabolism.
 |
MATERIALS AND METHODS
|
---|
Plasmids
Constructs p-2455/+18hAvLUC, p-617/+18hAvLUC, p-535/+18hAvLUC, p-437/+18hAvLUC, p-242/+18hAvLUC and p-82/+18hAvLUC containing the corresponding sequences of the 5' flanking region of human apoAV gene cloned in front of the promoter-less firefly (Photinus pyralis) luciferase gene have been previously described (10). Site-directed mutagenesis of the construct p-617/+18hAvLUC was performed using the QuikChange Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the recommendations of the manufacturer. For mutation of the DR1 element, the oligonucleotides JCR169 and JCR170 were used to generate a preliminary construct, and then a second site-directed mutagenesis was accomplished with the primers JCR188 and JCR189 (see Table 1
). The IR8 site was mutated using the oligonucleotides JCR167 and JCR168. Plasmids expressing human cDNAs for HNF-4
and PGC-1
, respectively, were provided by J. M. Maglich (GlaxoSmithKline, Research Triangle Park, NC). The backbone of those plasmids was the mammalian expression vector pSG5 with a modified polylinker. Plasmid DNA was prepared using the QIAGEN (Valencia, CA) endotoxin-free maxipreparation method, and quantitated spectrophotometrically. The integrities of all plasmids were verified by DNA sequencing.
Cell Transfection and Reporter Assays
Human hepatoblastoma Hep3B and monkey kidney CV-1 cell lines were cultured in Eagles basal medium supplemented with nonessential amino acids, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, and 10% (vol/vol) fetal calf serum (medium A). On d 0, cells were seeded on 24-well plates at a density of 3 x 105 or 5 x 104 cells/well for Hep3B or CV-1, respectively. On d 1, cells were transfected with FuGENE 6 reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturers instructions. Typically, each well of a 24-well plate received 100 ng of firefly luciferase reporter plasmid and, when indicated, 0200 ng of plasmids expressing human HNF-4
or PGC-1
. Effector plasmid dosage was kept constant by the addition of appropriate amounts of the empty expression vector pSG5. A total of 100 ng/well of a sea pansy (Renilla reniformis) luciferase plasmid pRL-TK (Promega, Madison, WI) was included in all transfections as an internal control for transfection efficiency. On d 3, cell lysates were prepared by shaking the cells in 200 µl of 1x Promega Lysis Buffer for 15 min at room temperature. Firefly and Renilla luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) and a Lumistar luminometer (BMG Lab Technologies, Offenburg, Germany). Firefly luciferase activity values were divided by Renilla luciferase activity values to obtain normalized luciferase activities. To facilitate comparisons within a given experiment, activity data were presented either as relative luciferase activities or as fold induction over the normalized activity of the reporter plasmid in the absence of nuclear receptor cotransfection. All transfection experiments were performed at least three times, and with each experimental point done in triplicate. The data are expressed as the means ± SD. Statistic significance analyses were done with Students t test.
RNA Interference
Twenty-one-nucleotide RNA oligonucleotides targeting the human HNF-4
mRNA (5'-AACCACAUGUACUCCUGCAGA-3') were chemically synthesized and supplied in the 2'-deprotected, annealed, and desalted form by Dharmacon (Lafayette, CO). Nonsilencing control siRNA was provided by QIAGEN (see Table 1
). The sequence of the specific siRNA duplex was designed according to the manufacturers recommendations and subjected to a basic local assignment and search tool search against the human genome sequence to ensure that non-HNF-4
genes were not targeted. On d 0, human hepatoblastoma HepG2 cells were seeded on 24-well plates at a density of 3 x 105 cells/well in medium A. On d 1, cells were transfected with siRNAs by using TansIT-TKO reagent (Mirus, Madison, WI) following the manufacturers instructions. Twenty-four hours after transfection, cells were refed with fresh medium A. On d 3, the cells were washed twice with PBS and harvested for isolation of RNA or Western analysis.
Treatment of HepG2
On d 0, human hepatoblastoma HepG2 cells were plated on 24-well plates at 3.5 x 105 in medium A. On d 1, the cells were refed with the corresponding fresh media and vehicle (Me2SO), 20 µM PD98059 (Sigma, St. Louis, MO) or AICAR (Toronto Research Chemicals, Toronto, Ontario, Canada; A611700) as indicated. On d 2, the cells were washed twice with PBS and harvested for isolation of RNA or Western analysis.
Real-Time PCR Quantification of mRNAs
Total RNA was prepared from primary HepG2 cells, and real-time RT-PCRs were performed as previously described with minor modifications (10). The reactions contained, in a final volume of 20 µl, 4 µl of diluted (1:10) cDNA, a 300 nM concentration of the forward and reverse primers, and 2x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Real-time PCRs were carried out in 384-well plates by using the ABI PRISM 7900 sequence detection system (Applied Biosystems). Levels of HNF-4
, apoCIII, apoAV, apoAI, and LDLr were normalized to cyclophilin to compensate for variations in input RNA amounts (cyclophilin levels were unaffected by the treatments). All points were performed in triplicate during two independent experiments, and the RT-PCRs were carried out in duplicate for each sample. The relative amounts of mRNAs were calculated using the comparative threshold method.
Nuclear Extracts
On d 0, HepG2 cells were seeded on 100-mm diameter dishes at a density of 1.5 x 107 cells/dish in medium A. On d 1, the cells were treated with vehicle (Me2SO), 20 µM PD98059, or 2.5 mM AICAR in fresh medium A. On d 2, the cells were washed twice with PBS and harvested in 1.5 ml of ice-cold PBS. The extraction of DNA-binding proteins was performed essentially as described (58). Briefly, cells were pelleted at 4 C and resuspended in 400 µl of hypotonic buffer [10 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 10 µl/ml Protease Inhibitor Cocktail from Sigma]. The cells were allowed to swell on ice for 10 min, and then vortexed for 10 sec and centrifuged at 4 C for 1 min. The pellet was resuspended in 100 µl of extraction buffer [20 mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, 10 µl/ml Protease Inhibitor Cocktail from Sigma] and incubated on ice for 20 min. Cellular debris was removed by centrifugation for 2 min at 4 C and 2.5 µl of the supernatant fraction (containing 4 µg/µl nuclear proteins) were used for EMSAs.
In Vitro Transcription/Translation and EMSAs
Human HNF-4
protein was synthesized in vitro from the expression plasmid in rabbit reticulocyte lysate by using TNT Quick Coupled transcription/translation system (Promega) according to the instructions of the manufacturer. To obtain an unprogrammed lysate as a negative control for EMSA, a reaction was performed with the empty vector pSG5. Double-stranded oligonucleotides were radiolabeled by fill in with the Klenow fragment of DNA polymerase I and used as probes. For competition experiments, increasing fold molar excesses of unlabeled probes were included during a 15-min preincubation on ice. Protein-DNA binding assays were performed as described (10). Supershift reactions were carried out by including 2 µl of HNF-4
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; SC-6556X). AvDR1 is a double-stranded oligonucleotide corresponding to nt 275 to 247 of human apoAV promoter. The probe AvIR8 contains the sequence spanning nt 109 to 80 of the human apoAV promoter. The mutations present in the modified versions of AvDR1 (mt1 AvDR1, mt2 AvDR1, mt3 AvDR1 and mt4 AvDR1) and AvIR8 (mt1 AvIR8, mt2 AvIR8, mt3 AvIR8, pft AvIR8 and pft tttIR8) are shown in Table 1
and Fig. 4A
. The control probe used in EMSA with HNF-4
and several fragments of apoAV promoter contains the PPAR
/HNF-4
site of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mitHMGS) gene promoter (59, 60). The probe used as a control for assays with nuclear extracts and competition experiments corresponds to the nt 90 to 68 of human apoCIII gene promoter containing a known HNF-4
-binding element (39). Samples were electrophoresed at 4 C on a 4.5% polyacrylamide gel in 0.5x TBE buffer [45 mM Tris, 45 mM boric acid, 1 mM EDTA (pH 8.0)]. Gels were dried and analyzed using a PhosphorImager STORM 860 and ImageQuant software (Amersham Biosciences, Buckinghamshire, UK).
ChIP Assay
DNA immunoprecipitation assay was performed according to the ChIP protocols of Upstate Biotechnology (Lake Placid, NY) with minor modifications. Briefly, on d 0 HepG2 cells were seeded on 10-cm culture dishes at a density of 8.5 x 106 cells/dish in medium A. On d 1, cells were transfected with 7 µg of wild-type luciferase reporter plasmid p-242/+18hAvLUC (described in Ref.10), an equivalent construct harboring mutations within IR8 site (see site-directed mutagenesis described in Plasmids), or pGL3-basic vector. Sixteen hours after transfection, the cells were washed twice with PBS and protein-DNA complexes were fixed by 1% formaldehyde for 10 min at 37 C in PBS. Cells were washed twice with PBS with protease inhibitors (Protease Inhibitor Cocktail from Sigma) and then collected in ice-cold PBS with protease inhibitors. Cell pellets were obtained by centrifugation at 1000 x g in PBS for 4 min, resuspended in 1 ml of hypotonic buffer [50 mM Tris-HCl (pH 8.0), 85 mM KCl, 0.5% Nonidet P-40, 10 µl/ml Protease Inhibitor Cocktail from Sigma], and incubated on ice for 10 min. Nuclei were isolated by centrifugation at 2000 x g for 10 min, then resuspended in 1 ml of sodium dodecyl sulfate (SDS)-containing sonication buffer [0.01% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1)] with protease inhibitors and incubated on ice for 10 min. Lysates were sonicated for four cycles of 10 sec each. Debris was removed from the samples by centrifugation at 14,000 x g for 10 min. Supernatants were collected and diluted 5-fold in ChIP dilution buffer [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl] with protease inhibitors, divided into aliquots of 1 ml and precleared with salmon sperm DNA preabsorbed protein A-Agarose slurry (Upstate Biotechnology) at 4 C for 1 h with rotation. An aliquot was removed as a control (input) and stored at 4 C until decross-linking. Immunoprecipitations were performed overnight with rotation at 4 C with either 4 µg of anti-HNF-4
(Santa Cruz; SC-6556) or 4 µg of anti-PPAR
(Abcam, Cambridge, UK; ab-2779) antibodies for each aliquot. Immunoprecipitated complexes were collected with the addition of protein A-Agarose slurry followed by sequential washes for 5 min each time in low-salt wash buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl], high-salt wash buffer (same as the low-salt buffer but with 500 mM NaCl), LiCl buffer [0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)], and Tris-EDTA buffer. Precipitates were eluted with elution buffer (1% SDS, 0.1 M NaHCO3) for 30 min with rotation. The cross-linking of protein-DNA complexes in precipitates and inputs was reversed by incubation with 5 M NaCl at 65 C for 4 h. DNA-associated proteins were digested with proteinase K (QIAGEN) for 1 h at 45 C. The DNA was extracted with a QIAquick PCR purification kit (QIAGEN) and one tenth of purified sample was subjected to PCR amplification with a forward primer corresponding to a sequence upstream of IR8 in human apoAV promoter and a reverse primer corresponding to a sequence in the pGL3-basic vector (Table 1
). PCR products (one fifth for input) were resolved by 1% agarose-ethidium bromide gel electrophoresis and visualized by UV.
Western Blot Analysis
Whole cell lysates were prepared by shaking the cells from in 100 µl/well lysis buffer (PBS, 1% Triton, 50 mM NaF, 5 mM sodium pyrophosphate, 10 µl/ml Protease Inhibitor Cocktail from Sigma) for 30 min at 4 C. The lysates were centrifuged at 103 x g at 4 C for 5 min. Equal amounts of protein (40 µg) from supernatant were run on 10% polyacrylamide 3[N-morpholino]propanesulfonic acid NuPAGE Nobex gels (Invitrogen Life Technologies, Carlsbad, CA) and transferred onto nitrocellulose membranes in NuPAGE transfer buffer (Invitrogen Life Technologies). Membranes were preincubated for 1 h at room temperature in blocking buffer, 5% nonfat dry milk in TBST [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% (vol/vol) Tween 20]. Subsequently, the blots were incubated overnight at 4 C with rabbit antihuman apoAV (8), rabbit antiactin (Sigma; A5060), or goat anti-HNF-4
(Santa Cruz; SC-6556) antibodies in blocking buffer. For phosphorylated acetyl-CoA carboxylase, lysates were run on 7.5% polyacrylamide gels, and blot was incubated with rabbit antiphospho-acetyl-CoA carboxylase (Ser79) antibody (Upstate Biotechnology). After washing five times in TBST for 5 min, blots were incubated with donkey horseradish-peroxidase-conjugated antirabbit IgG antibody (Amersham Biosciences) or mouse horseradish-peroxidase-conjugated antigoat IgG antibody (Pierce Biotechnology, Rockford, IL) for 1 h at room temperature. Finally, blots were washed as above and immunoreactive bands were detected by ECL (Amersham Biosciences). ApoAV contents were quantitated and normalized to actin using a densitometer and ImageQuant software (Amersham Biosciences). Specimens of conditionally HNF-4
-null mice (16) and controls (n = 5/group) were generously provided by Dr. Y. Inoue (National Institutes of Health, Bethesda, MD). Livers were homogenized in PBS containing a protease inhibitor cocktail (Roche), and equal amounts of proteins were pooled. Two hundred micrograms of protein from liver homogenates and 25 µl of pooled sera were separated on denaturing polyacrylamide gels. After transfer to polyvinylidene difluoride membrane (Millipore, Billerica, MA), proteins were visualized as described above by using anti-HNF-4
(SC-6556), antirat apoAV (3) and antimouse apoAII (Biodesign, Saco, ME) antibodies.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. J. Kirilovsky for encouragement of this work; Dr. Y. Inoue (National Institutes of Health, Bethesda, MD) for kindly providing specimens of conditionally HNF-4
-null mice and controls; Drs. P. Grondin and N. Ancellin for help on AMPK studies; Dr. P. Delerive for assistance in the design of siRNAs; and Drs. T. Huby and J. Chapman (Unité 551, Institut National de la Santé et de la Recherche Médicale, France) for scientific discussions and critical reading of the manuscript.
 |
FOOTNOTES
|
---|
First Published Online July 28, 2005
Abbreviations: AMPK, AMP-activated protein kinase; AICAR, 5-amino-4-imidazolecarboxamide ribonucleoside; apo, apolipoprotein; apoAV, apolipoprotein AV; BA, bile acid; CoA, coenzyme A; CPT-I
, carnitine palmitoyltransferase I
; CYP7A1, cholesterol 7
-hydroxylase; DR, direct repeat; FXR, farnesoid X-activated receptor; G6Pase, glucose-6-phosphatase; HNF-4
, hepatocyte nuclear factor-4
; IR, inverted repeat; LDLr, low-density lipoprotein receptor; LPL, lipoprotein lipase; mitHMGS, mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase; MODY1, maturity onset diabetes of the young 1; MTP, microsomal triglyceride transfer protein; nt, nucleotides; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1
, peroxisome proliferator-activated receptor-
coactivator-1
; PPAR
, peroxisome proliferator-activated receptor
; PXR, pregnane X receptor; SDS, sodium dodecyl sulfate; SHP, small heterodimer partner; siRNA, small interfering RNA; SNP, single nucleotide polymorphism; TG, triglycerides; VLDL, very low-density lipoprotein.
Received for publication January 18, 2005.
Accepted for publication July 18, 2005.
 |
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