(Received for publication, August 14, 1995; and in revised form, November 14, 1995)
From the
Peroxisome proliferators cause a rapid and coordinated
transcriptional activation of genes encoding the enzymes of the
peroxisomal -oxidation pathway in rats and mice. Cis-acting
peroxisome proliferator responsive elements (PPREs) have been
identified in the 5`-flanking region of
H
O
-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 proliferator-induced increases in peroxisome volume density
and in the activity of the
-oxidation enzyme system, we have
previously cloned the human ACOX gene, the first and rate-limiting
enzyme of the peroxisomal
-oxidation system. We now present
information on a regulatory element for the peroxisome
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
/RXR
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
and RXR
. The functionality of
this human ACOX promoter was further demonstrated by linking it to a
-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
, whereas only a basal level of promoter activity
is detected in stably transfected cells lacking PPAR
. 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.
Peroxisomes are cellular organelles that are present in
virtually all eukaryotic cells(1) . At present, >50 proteins
have been identified in peroxisomes, and more than half of these play a
role in lipid metabolism(2) . Of particular interest is that
these organelles contain HO
-producing flavin
oxidases together with catalase, which decomposes
H
O
(3) . Peroxisomes in liver
parenchymal cells can be stimulated to proliferate by the
administration of certain nonmutagenic chemicals designated as
peroxisome proliferators(4, 5) . These form a broad
group of compounds of industrial, pharmaceutical, and agricultural
value; they include certain phthalate ester plasticizers, industrial
solvents, herbicides, leukotriene D
antagonists, the
adrenal steroid dehydroepiandrosterone, and amphipathic carboxylates
such as the hypolipidemic drugs clofibrate and
ciprofibrate(2, 5) . When peroxisome proliferators,
with ostensibly dissimilar structures and pharmacokinetic properties,
are administered to rodents and certain nonrodent species including
primates, they cause profound proliferation of peroxisomes in hepatic
parenchymal cells and marked increases in the activities of the enzymes
required for peroxisomal
-oxidation of fatty acids, namely fatty
acyl-CoA oxidase (ACOX), (
)enoyl-CoA
hydratase/3-hydroxyacyl-CoA dehydrogenase bi-(tri)functional enzyme
(PBE), and 3-ketoacyl-CoA thiolase(2, 6) . The
increased activities of these enzymes are related to the rapid and
coordinated transcriptional activation of the nuclear genes encoding
these enzymes(7) .
Peroxisome proliferators owe their
importance, in part, to the fact that they induce hepatocellular
carcinomas in rats and mice on continued
exposure(8, 9) . Since these hepatocarcinogens neither
bind covalently to DNA nor produce somatic mutations directly or after
metabolic activation, it has been postulated that any genetic
alterations responsible for carcinogenesis that may eventually occur
during chronic exposure to these nongenotoxic carcinogens should be
attributable to the tissue-specific pleiotropic responses induced by
such agents(9) . Support for a mechanistic relationship between
peroxisome proliferation and hepatocarcinogencity is provided, in part,
by a close concordance with the magnitude of hepatic peroxisome
proliferation and liver tumor development in rats and
mice(9, 10) . In liver cells with massive increases in
peroxisomal volume density caused by peroxisome proliferators, there is
differential transcriptional regulation of genes encoding catalase and
of HO
- producing peroxisomal ACOX, the first
and rate-limiting enzyme of the
-oxidation
pathway(7, 11, 12) . In these livers catalase
activity is increased
2-fold, whereas the ACOX level increases by
>20-fold, thus leading to excess production of H
O
and possibly other reactive oxygen
intermediates(7, 12) . Corroborative evidence for the
hypothesis that sustained overproduction of intracellular levels of
H
O
in livers of rats and mice with persistent
maximal increase in peroxisome volume density leads to neoplastic
transformation has been derived from a variety of observations,
including the recent finding that African green monkey kidney (CV1)
cells stably overexpressing rat peroxisomal ACOX when exposed to a
fatty acid substrate formed transformed foci, grew efficiently in soft
agar, and developed adenocarcinomas when injected into nude
mice(13, 14) .
The postulated link between
induction of peroxisome proliferation vis à vis ACOX and hepatocarcinogenicity implies that species that do not
respond to peroxisome proliferators, i.e. fail to exhibit
significant degree of hepatic peroxisome proliferative response, are
less likely to develop liver tumors on chronic
exposure(9, 13) . The available data on human
hepatocytes in vivo and in vitro suggest that
compounds that are peroxisome proliferators in rats and mice have
little, if any, effect on human liver cells(15, 16) .
Although additional studies are nonetheless required to establish
unequivocally the nonresponsiveness of human hepatocytes, the apparent
disparity between the effects of peroxisome proliferators in rodent and
human hepatocytes may be due to a number of modulating or confounding
factors. These include the dose administered, pharmacokinetics,
bioavailability, affinity of the agents at the target site,
distribution of peroxisome proliferator-activated receptor (PPAR)
isoforms in the target (responsive) cells, and the nature of the
cis-acting peroxisome proliferator-responsive element (PPRE) in the
5`-flanking region of target genes(2) . Transactivation of
peroxisome proliferator-responsive genes is mediated through
ligand-activated receptors, collectively referred to as PPARs; these
receptors are members of the steroid/thyroid hormone receptor
superfamily(17, 18) . Three PPAR isoforms (PPAR,
PPAR
, and PPAR
) have been isolated both from the mouse and Xenopus(19, 20, 21, 22) .
These PPARs have been shown to be activated by a wide array of
peroxisome proliferators, as well as, natural and synthetic fatty
acids(23) . The discovery of PPAR isoforms has facilitated the
identification of PPRE in the upstream regions of the rat ACOX
gene(17, 24) . This PPRE is composed of two direct
AGG(A/T)CA repeats separated by a single nucleotide(24) .
Similar PPREs have been identified in other genes known to be activated
by peroxisome proliferators, such as the rat PBE
gene(25, 26) , the rabbit CYP4A6 fatty acid
-hydroxylase gene(27) , the rat CYP4A1
-hydroxylase
gene(28) , the rat malic enzyme gene(29) , and the rat
mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase
gene(30) . The PPREs characterized to date consist of direct
repeats (DR) of AGGTCA related sequences, the consensus half-site
recognition sequence for the TR/RAR/RXR receptor
family(31, 32) . Like TR, RAR, and VDR, which strongly
heterodimerize with RXR to enhance binding to their respective response
elements, PPAR also heterodimerizes with RXR to enhance the
transactivation of the PPREs of rat ACOX and PBE
genes(23, 33) . The relative spacing and orientation
of the TGACCT/AGGTCA half-site motifs determine the selective binding
of PPAR/RXR to these response elements (34) . The DNA binding
of the PPAR
/RXR
heterodimers occurs preferentially when the
spacing between the direct repeats was one base pair (DR1) as
demonstrated with the PPRE of rat ACOX
gene(23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) .
Thus, the convergence of the peroxisome proliferator/retinoid signaling
pathways resulting in PPAR/RXR heterodimers to interact with PPREs
appears to be a crucial mechanism in the transcriptional activation of
ACOX of the rat peroxisomal
-oxidation enzyme system and by
inference in hepatocarcinogenesis by peroxisome proliferators.
To
gain more insight into the purported nonresponsiveness of human liver
cells to peroxisome proliferator-induced early pleiotropic responses,
such as peroxisome proliferation and increase in the activity of
-oxidation enzyme system, we have undertaken studies to examine
the transcriptional regulation of the human ACOX gene. Analysis of the
regulation of the human ACOX gene is an important step in our attempts
to understand the implications of peroxisome proliferator-induced
pleiotropic responses to human health. We previously cloned the human
ACOX gene (36) and its corresponding cDNA (37) and now
demonstrate the presence of a PPRE in the promoter of this gene. The
human ACOX promoter is constitutively active in CV1 cells. This
regulatory element of human ACOX gene is a DR1, which interacts
specifically with PPAR
/RXR
and is responsive to peroxisome
proliferators in CV1 cells stably expressing PPAR
. These
observations indicate that factors other than the PPRE in the
5`-flanking region of the ACOX gene may account for possible
differences in the peroxisome proliferator-induced responses between
human and rodent.
Figure 1:
Ciprofibrate-mediated induction of the
HACOX promoter requires an upstream cis-acting site. A,
diagram of the promoter constructs linked to the luciferase reporter
gene, pGL2-basic. A restriction map of the 7.8-kb SacI
fragment of the HACOX gene (pSKACOX), which includes the transcription
start site as well as the first and second exons is shown at the top. The luciferase reporter constructs are numbered pHACOXLUC1-7 (a-g). Their
position with respect to pSKACOX is indicated. pHACOXLUC7 represents
the 294-bp minimal promoter generated by polymerase chain reaction as
described under ``Experimental Procedures.'' B, the
HACOX promoter requires the sequence of the 200-bp fragment between
-2015 and -1812 for responsiveness to ciprofibrate. All of
the constructs were transfected into H4IIEC3 cells in the presence of
MeSO alone (black bar) or 0.5 mM ciprofibrate (gray bar). -Fold induction is the ratio of
the value obtained from ciprofibrate-treated cells to those obtained
from untreated cells. Values are the means of three experiments
normalized to a
-galactosidase transfection
control.
Figure 2:
Constructs used in the generation of the
stable cell lines CVPHACOX, CVHACOX and CVHACOXUOX. a, this
construct designated pHACOX, contains the promoter fragment
corresponding to the pHACOXLUC1 plasmid linked to the
-galactosidase cDNA, which was used in the generation of both the
CVPHACOX and the CVHACOX cell lines. b, this construct,
designated pHACOXUOX, contains the same ACOX promoter fragment
mentioned above linked to the full-length rat UOX cDNA. c,
this represents the PPAR
cDNA under the transcriptional control of
the cytomegalovirus promoter. A stably expressing PPAR
cell line,
termed PPAR
-CV1, was used in the generation of CVPHACOX cell
line.
These double-stranded oligonucleotides were end-labeled using
[P]dCTP, dATP, dTTP, and dGTP with Klenow DNA
polymerase. The unincorporated nucleotides were removed by a Sephadex
G-50 spin column. The purified probes were diluted to
20,000
cpm/µl for binding assays. Purified receptor proteins were
incubated with 5 µl of the radiolabeled double-stranded
oligonucleotide probe at room temperature for 20 min as described
previously(26) . The respective double-stranded
oligonucleotides were also used for cloning into a pTKLUC reporter
plasmid to assess their activity following transfection into CV1
cells(26) . Transfections typically contained 1 µg of a
reporter gene construct, 0.2 µg of rPPAR
expression plasmid,
0.2 µg of rRXR
expression plasmid, and 0.5 µg of a
bacterial
-galactosidase expression vector pCMV
(Invitrogen)
as an internal control. Cells were incubated in the presence of 1.5
mM ciprofibrate or 0.5% dimethyl sulfoxide, as required.
Following a 40-h incubation, cells were processed to assess luciferase
activity; the activity obtained for individual transfections was
expressed relative to the
-galactosidase activity obtained for the
same preparation of lysate(26) .
The plasmid pHACOXLUC1, which includes
the native minimal promoter linked in tandem to the 3.7-kb 5`-most SacI-BamHI fragment (Fig. 1A, a) revealed a comparatively higher level of induction (
4.0-fold) with ciprofibrate than that observed using the minimal
promoter alone (Fig. 1B; a and g).
The highest inducible luciferase activity, 5-fold over the minimal, was
obtained with pHACOXLUC5 (-2165 to +53). This construct
encompasses the minimal, the 1.6-kb BamHI fragment, and 200 bp
further upstream (Fig. 1A). To delineate this region
further, a 200-bp fragment immediately upstream of the second BamHI site (-2015 to -1812) was linked to the
minimal (-241 to +53) (Fig. 1A, f).
The level of activity obtained for this construct, pHACOXLUC6, was
3.5-fold over that obtained using the minimal alone (Fig. 1B, f, and g). Since this
200-bp region between -2015 and -1812 is able to confer
inducibility on the otherwise minimally responsive minimal human ACOX
promoter, this region most probably contains a positively acting PPRE
that functions independent of the other regions of this promoter. Also
the other two constructs, pHACOXLUC1 and pHACOXLUC5 (Fig. 1, a and e), which include this 200-bp fragment, have a
higher level of induction than pHACOXLUCs 2, 3 and 4 that lack this
region. The level of inducible luciferase activity that was obtained
for the HACOXLUC constructs was similar to that observed for the
inducible rat ACOX in our system (data not shown).
Figure 3:
PPAR and RXR
transactivate
reporter expression cooperatively for the HACOX promoter. CV1 cells
were transfected with constructs pHACOXLUCs 6 or 7 reporters, which are
the two constructs from Fig. 1, together with PPAR
and
RXR
expression vectors. pSG5 plasmid DNA was used as a control.
Treatments included no ligand (Me
SO alone), ciprofibrate
alone, 9-cis-retinoic acid and both ciprofibrate and
9-cis-retinoic acid. The luciferase expression, depicted as
-fold induction, is calculated in relative light
units.
Figure 4:
Transient and stable transfection with the
-galactosidase expression vector (p
HACOX plasmid) in
PPAR
-CV1 cells. A, transient expression of
-galactosiadse under the influence of human ACOX promoter. B and C represent CVPHACOX cells that were generated by
stable transfection with p
HACOX. This cell line, CVPHACOX, had
approximately 98% of the cells expressing
-galactosidase as
visualized by the histochemical staining (B, stably
transfected control; C, stably transfected treated with
ciprofibrate).
The pHACOX and pHACOXUOX
plasmids were also transfected individually into regular CV1 cells (i.e. those not expressing PPAR
), and stable
transfectants were generated using G418 as a selection marker. The cell
line expressing
-galactosidase was termed CVHACOX, and that of rat
UOX was designated CVHACOXUOX. No distinct differences in the
-galactosidase staining intensities were noted between control and
ciprofibrate-treated CVHACOX cells (data not shown). Further evidence
that the human ACOX promoter is constitutively active in CV1 cells is
derived from the demonstration that this promoter is capable of driving
the expression of UOX, which can be visualized as crystalloid cores in
the stably transfected CV1 cells. In these stably transfected CV1
cells, UOX containing crystalloid core-like structures are detected
within single-membrane limited structures, indicating that human ACOX
promoter is active (Fig. 5, A and B). The
identity of these UOX containing recombinant organelles as peroxisomes
was confirmed by the demonstration of catalase (data not shown). As
expected, UOX, which is a liver-specific protein, is not detectable in
untransfected CV1 cells, which are derived from monkey kidney (Fig. 5C).
Figure 5: Stable transfection using the pHACOXUOX plasmid, which contains the full-length rat urate oxidase cDNA under the transcriptional control of the HACOX promoter. Stable transfectants express the typical crystalloid-core like structure characteristic of UOX. A, peroxisomes indicated by arrowheads in CV1 cells stably expressing UOX under the transcriptional control of HACOX display dense cores. B, higher magnification of these in the peroxisomes of stably transfected cells show crystalloid cores characteristic of UOX. C, regular CV1 cells that lack the core-like structures as these cells normally do not express UOX.
Figure 6:
Dose-response of the CVPHACOX cell line.
Ciprofibrate, Wy-14,643, dioctyl phthalate (DEHP), and elaidic acid
were used for this experiment. Concentrations of the drugs ranged from
0.01 to 3 mM. -galactosidase activity was assessed as
described under ``Experimental Procedures.'' Data points
represent the mean of two individual experiments, each containing
quadruplicate wells for the doses tested. Percent
-galactosidase
activity represents -fold induction over the control (untreated)
cells.
Figure 7:
Time-course of response of the CVPHACOX
cell line. DEHP (1.5 mM), ciprofibrate (1.5 mM),
Wy-14,643 (1.5 mM), and elaidic acid (1 mM) were
added to the medium, and cells were harvested at 0, 2, 4, 6, 8, 10, 12,
18, 24, 36, and 48 h following treatment to assess the reporter
activity. Data points represent the mean of two individual experiments,
each containing quadruplicate wells for the doses tested. Percent
-galactosidase activity represents -fold induction over the
control (untreated) cells.
Figure 8: Sequence of the human ACOX promoter. The -2015 to -1725 region of the human ACOX promoter is shown. The numbering of bp is with respect to the transcription start site = +1. The potential PPRE (boldfaced and underlined) is very similar to the cognate response elements recognized by the nuclear hormone receptor superfamily and is composed of AGGTCACTGGCTA (-1918/-1906). Two putative CACCC boxes (italicized and underlined) are presumed to be important for hormone-mediated responsiveness of several genes having response elements further upstream of their transcription start sites such as the one seen with human ACOX.
Figure 9:
Specific binding of RXR and PPAR
with the PPRE of the HACOX promoter. A
P-labeled DNA
fragment containing the human ACOX PPRE (WT) was incubated with
PPAR
and RXR
purified from insect Sf9 cells. Lanes
1-5 have the same amount of PPAR (50 ng). Lanes
2-5 proceeding from left to right have a
10-50 ng increase in RXR
. In the reverse direction, lanes 9-5 have the same amount of RXR (50 ng), while
increasing PPAR from 10 to 50 ng in lanes 8-5. Lanes
10-12 used same amount of receptor proteins as lane
5. Lane 10 represents nonspecific competition using a
25-fold molar excess of an unrelated, unlabeled oligonucleotide. Lanes 11 and 12 represent specific competition using
a 25- and 100-fold molar excess of unlabled human ACOX PPRE
DNA.
Figure 10:
Mutation of HACOX PPRE influences
receptor binding and peroxisome proliferator responsiveness. A, electropheretic mobility shift assay using P-labeled WT (lane 1), M1 (lane 2), M2 (lane 3), M3 (lane 4), M4 (lane 5) HACOX
PPRE probes. B, transfection of CV1 cells was carried out with
rat PPAR
and rat RXR
expression vector and TK reporter
constructs containing WT (lane 1), M1 (lane 2), M2 (lane 3), M3 (lane 4), M4 (lane 5) HACOX
PPREs, respectively. Lane 6 is the pTKLUC control plasmid.
After incubation with DNA for 16 h, the cells were washed and either
solvent (Me
SO, open bars) or 1.5 mM of
ciprofibrate was added to the fresh medium for an additional incubation
of 48 h. The bar graphs represent the mean values plus
standard deviation obtained from three independent transfections and
show the activity normalized to that obtained with WT PPRE. The
pCMV
(Clontech) plasmid was used as an internal
control.
Elucidation of the mechanism(s) by which peroxisome
proliferators modulate gene expression necessitates, in part, the
identification of promoter elements and transcription factors that are
responsible for mediating the biological responses to these
nongenotoxic agents. Induction of peroxisomal ACOX is the most widely
used marker to assess the magnitude of peroxisome proliferator-induced
pleiotropic responses. Different species appear to exhibit varying
degrees of increase in ACOX activity and peroxisome volume density in
response to peroxisome proliferators(13, 16) .
Evidence indicates that human hepatocytes show either minimal or no
increases in peroxisome volume density when exposed to peroxisome
proliferators(15, 16) . Since PPAR isoform(s) as well
as enzymes of peroxisomal -oxidation system are present in the
human liver (43) and thus cannot account for the
nonresponsiveness of human liver cells to peroxisome
proliferator-induced early pleiotropic responses, it is essential to
determine whether PPREs are present and functional in human ACOX gene
and other human genes involved in lipid metabolism. In this paper, we
show that the 5` region of the human peroxisomal ACOX gene contains a
PPRE and that it appears essential for the response of this gene to
peroxisome proliferators. The PPRE of human ACOX gene is a direct
repeat of the consensus AGGTCA half-site interspaced by one nucleotide,
the so-called DR1. Several genes, which are transactivated by PPAR
and RXR
heterodimers and are involved in the function of
peroxisomes, mitochondria, and cytosol are comprised of DR1 motifs.
Thus, the presence of a functionally active PPRE in human ACOX gene as
demonstrated in this report, and the existence of a functional PPAR in
human liver(43) , suggests that malfunctioning of these two
components of the transcriptional machinery cannot account for the
reported nonresponsiveness, or weak responsiveness, of human liver to
peroxisome proliferators.
Recent evidence demonstrates that
peroxisome proliferators, like other lipophilic molecules such as
steroids, retinoic acid, thyroid hormone, and vitamin D function by interacting with ligand-activable transcription
factors that comprise the steroid/nuclear receptor
superfamily(17, 18, 21) . VDR, TR, and RAR
form heterodimers with RXR on bipartite hormone-response elements
composed of two direct repeats of the consensus sequence 5`-AGGTCA-3`
separated by 3-5 bp(31, 32) . The hormone
response elements for VDR, TR, and RAR vary from one another only in
the number of base pairs (3-5 bp) separating the hexameric direct
repeats (DR3-DR5). To date, almost all of the PPREs identified in
rat and rabbit peroxisomal and nonperoxisomal enzyme genes that are
inducible by peroxisome proliferators have a DR1 motif that binds
PPAR/RXR heterodimers with greater affinity. The PPAR/RXR heterodimers
also exhibit greater potential for transactivation than either PPAR or
RXR alone. As demonstrated in the present study, the PPRE of human ACOX
gene is also a DR1 and appears essentially similar to the PPRE of rat
ACOX gene in its ability to bind PPAR/RXR heterodimers (Fig. 9).
Our mobility shift analysis indicates that rat RXR
/PPAR
heterdimers are capable of binding to the PPRE of human ACOX
cooperatively, indicating that they (i.e. their counterparts
in human) possibly heterodimerize in order to transactivate the human
ACOX PPRE (Fig. 9). Also, our mobility shift analysis with the
mutant PPREs has demonstrated that the second and third nucleotides of
both the half-site motifs are crucial for binding and transcriptional
activity (Fig. 10). It has been previously reported that the
highest stimulation for the rat ACOX reporter plasmid was observed by
cotransfection with PPAR
and RXR
in the presence of their
respective ligands(23) . Transfection experiments have clearly
indicated that both ciprofibrate and 9-cis-retinoic acid
contribute toward gene activation for rat ACOX(23) . This
synergistic induction has been reported for other systems as
well(18, 28, 29) . Our results also indicate
that ciprofibrate and 9-cis-retinoic acid act synergistically
in transactivating the human ACOX promoter constructs that include the
PPRE if cotransfected with PPAR
and RXR
. Recently, it has
been demonstrated that the DR1 motif alone is not sufficient to
constitute a PPRE in the case of gene encoding rabbit CYP4A6, and that
sequences immediately 5` of the DR1 are required for the
PPAR
/RXR
heterodimer binding to the PPRE of CYP4A6
gene(44) . Apparently, the minimal sequence that preserves
strong binding with PPAR
/RXR
heterodimers for the CYP4A6 gene
includes six nucleotides upstream of the DR1 motif(44) . The
role of sequences 5` and 3` of the human ACOX PPRE in promoting the
binding of the PPAR/RXR heterodimers to DR1 and in the transcriptional
efficiency of this gene remains unclear.
Transient transfection
analysis of the pHACOX promoter fragment into the CV1 cell line
stably expressing PPAR
reveals a number of blue cells staining
positively for
-galactosidase activity. Transient expression
assays have provided extensive information on the peroxisome
proliferator-induced regulations on receptor-mediated transactivations,
but the development of a stably transfected mammalian cell line will
facilitate studies on the ligand/human PPRE/PPAR-mediated
transactivation of a reporter gene. Toward this goal, we have developed
a CV1 cell line, in which the human ACOX promoter is driving the
-galactosidase, and CMV promoter is driving the rat PPAR
.
This cell line, designated CVPHACOX, reveals a 700% increase in
activity of the reporter gene using Wy-14,643 when compared with
untreated controls. In contrast, both the CVHACOX and CVHACOXUOX cell
lines, both of which lack stably transfected PPAR
gene,
demonstrate a very minimal increase in the activity of
-galactosidase and UOX, respectively, upon treatment with
Wy-14,643. The CVPHACOX cell line stably expressing PPAR
, has
provided new data regarding the inducibility of the human ACOX promoter
with structurally different peroxisome proliferators, namely
ciprofibrate, Wy-14,643, DEHP, and a fatty acid. Although published
studies on the responsiveness of human liver cells to peroxisome
proliferators have provided negative or inconclusive
results(15, 16) , our transfection analysis data both
from the transient luciferase, as well as stable
-galactosidase
and UOX experiments, indicate that the human ACOX promoter is indeed
constitutively active and inducible. Thus the presence of a
functionally active PPRE in human ACOX as shown here, and a functional
PPAR in human liver(43) , suggest that the reported
nonresponsiveness of human liver cells to peroxisome proliferators may
be due to other factors such as the bioavailability of the ligand,
interference with PPAR/RXR heterodimerization and its binding to PPRE
and possibly a myriad of complex interactions involved in the
transcriptional activation/repression of genes in vivo. It is
of interest to note that mice, in which the PPAR
gene has been
disrupted by homologous recombination, failed to display the peroxisome
proliferator-induced early pleiotropic responses, implying that
PPAR
plays a crucial role in mediating the effects of peroxisome
proliferators(45) . Nonetheless, many other DR1 binding
proteins are present in liver that may antagonize PPAR signaling; these
include COUP-TF1, ARP-1, and
HNF-4(27, 44, 46, 47) . Also, since
direct binding of peroxisome proliferators to PPAR has yet to be
demonstrated, it remains uncertain whether peroxisome proliferators
activate PPAR
directly. Additional stringently controlled studies
are nevertheless desirable to unequivocally establish the
nonresponsiveness of human hepatocytes to peroxisome proliferators. The
transcriptional controls of genes involved in lipid metabolism appear
extremely complex, and the net effect of peroxisome proliferators may
depend upon the heterodimerization and binding or displacement of
nuclear factors that may have positive or negative transcriptional
influence. It is worth noting that hNUC1, a PPAR isoform isolated from
an osteogenic sarcoma (48) , acts as repressor of human
PPAR
-mediated transcriptional activation of rat ACOX
gene(49) . The tissue distribution of hNUC1 is not known, and
it remains to be ascertained whether hNUC is a dominant isoform in
human liver when compared with rat liver and that the relative lack of
peroxisome proliferation in human liver is due to the inability of
peroxisome proliferators to overcome hNUC1 repression.