(Received for publication, March 15, 1995; and in revised form, May 16, 1995)
From the
The peroxisome proliferator-activated receptors (PPAR) and
thyroid hormone receptors (TR) are members of the nuclear receptor
superfamily, which regulate lipid metabolism and tissue
differentiation. In order to bind to DNA and activate transcription,
PPAR requires the formation of heterodimers with the retinoid X
receptor (RXR). In addition to activating transcription through its own
response elements, PPAR is able to selectively down-regulate the
transcriptional activity of TR, but not vitamin D receptor. The
molecular basis of this functional interaction has not been fully
elucidated. By means of site-directed mutagenesis of hPPAR The peroxisome proliferator-activated receptors (PPAR)
Figure 1:
Amino acid sequence comparison of
hPPAR
Figure 2:
Selective inhibition of hTR
Figure 3:
hPPAR
Figure 4:
Leucine residue 433 of hPPAR
The three hPPAR
Figure 5:
Transcriptional capacity of mutant
hPPAR
In
order to test the ability of the mutant PPARs to inhibit ligand-induced
TR transactivation, co-transfection experiments with hTR
Figure 6:
Heterodimerization of hPPAR
Besides sharing structural homologies, and the ability to
heterodimerize with RXR, TR and PPAR are both involved in the
regulation of lipid metabolism. We now show that PPAR is able to
selectively inhibit ligand-induced TR activity by competing for TRAPs,
particularly RXR. In addition, our data suggest that a highly conserved
leucine zipper-like heptad repeat in the ligand-binding domain may be
required for PPAR to dimerize with RXR. However, despite a similar
control mutation located 11 amino acids amino-terminally (L422R), the
possibility of protein misfolding in the mutation L433R cannot be
completely excluded. Nevertheless, immunocytochemical studies of
transfected HepG2 cells show that all the PPAR mutants are expressed
similarly to the wild-type protein (data not shown). Other mechanisms
possibly accounting for the modulatory action of PPAR on TR, such as
the formation of PPAR:TR heterodimers and/or the competition for DNA
binding, were also addressed in the present study. In contrast to what
has been reported for rTR and rPPAR The modulation of TR transactivation by PPAR is highly
specific, since the ligand-dependent VDR activity was not altered by
PPAR. However, since the transcriptional activity of VDR depends also
on RXR on the vitamin D-response element used, PPAR would be expected
to also inhibit the formation of VDR:RXR heterodimers, unless VDR has a
higher affinity for RXR than PPAR. Indeed, our EMSA data suggest that
PPAR also decreases the binding of VDR:RXR heterodimers to DNA, but to
a lesser extent than TR:RXR heterodimers (data not shown). This
together with the observation that the transfection of excess amounts
of RXR does not restore full TR activity in the presence of PPAR argues
in favor of PPAR competing and dimerizing with as yet uncharacterized
non-RXR TRAPs(5) . However, this putative TRAP is expected to
interact with PPAR by means of a similar dimerization region as RXR,
since hPPAR A modulatory effect of PPAR on thyroid hormone signaling in cell
cultures has been recently reported(20) , suggesting a
physiological importance for this mechanism. In humans, the syndrome of
thyroid hormone resistance, which is due to dominant negative mutations
in one hTR In summary, we have demonstrated that PPAR
is able to selectively inhibit the transcriptional activity of TRs by
competing for RXR and possibly also for a specific, but as yet
unidentified non-RXR TRAP. In addition, the results suggest that a
highly conserved leucine zipper-like motif in the ligand-binding domain
of PPAR may be necessary, although not be sufficient, for PPAR to
dimerize with RXR.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
we
mapped its inhibitory action on TR to a leucine zipper-like motif in
the ligand binding domain of PPAR, which is highly conserved among all
subtypes of this receptor and mediates heterodimerization with RXR.
Replacement of a single leucine by arginine at position 433 of
hPPAR
(L433R) abolished heterodimerization of PPAR with RXR and
consequently its trans-activating capacity. However, a similar mutation
of a leucine residue to arginine at position 422 showed no alteration
of heterodimerization, DNA binding, or transcriptional activation. The
dimerization deficient mutant L433R was no longer able to inhibit TR
action, demonstrating that the selective inhibitory effect of PPAR
results from the competition for RXR as well as possibly for other
TR-auxiliary proteins. In contrast, abolition of DNA binding by a
mutation in the P-box of PPAR (C122S) did not eliminate the inhibition
of TR trans-activation, indicating that competition for DNA binding is
not involved. Additionally, no evidence for the formation of PPAR:TR
heterodimers was found in co-immunoprecipitation experiments. In
summary, we have demonstrated that PPAR selectively inhibits the
transcriptional activity of TRs by competition for RXR and possibly
non-RXR TR-auxiliary proteins. In contrast, this functional interaction
is independent of the formation of PPAR:TR heterodimers or competition
for DNA binding.
(
)are a novel subfamily of the steroid/thyroid hormone
nuclear receptor proteins involved in the ligand-inducible regulation
of lipid metabolism, adipose tissue differentiation, and possibly
hepatocarcinogenesis in rodents(1) . Their closest relatives in
the superfamily are the type II nuclear hormone receptors, such as the
retinoic acid, vitamin D, and thyroid hormone receptors. In particular,
the P-box of the first zinc-finger responsible for DNA-binding
specificity is fully conserved between hPPAR
and the L-triiodothyronine (T
)-receptors (TR)
1 and
1 (Fig. 1A), reflecting the preferential binding
of both receptors to differently spaced AGGTCA half-sites(2) .
Additionally, a high degree of sequence conservation is found between a
putative leucine zipper motif in the ligand-binding domain of PPAR
and TR(3) . This motif is very highly conserved among various
species and PPAR subtypes (Fig. 1B). Besides the
structural homology, TRs and PPARs require heterodimerization with the
retinoid X receptor (RXR) for optimal DNA binding and both receptors
are co-expressed in brain, liver, and adipocytes, where they are
involved in the regulation of lipid metabolism(1) . We and
others have recently reported that the PPAR
is able to modulate TR
activity either positively or negatively, depending on the
T
-response element (TRE)(4, 5) . Although
it has been suggested that rTR
is able to form heterodimers with
rPPAR
in solution, it has not been demonstrated whether this
interaction quantitatively accounts for the observed transcriptional
changes. Here we examined the mechanism by which hPPAR
structurally and functionally interacts with TR and RXR. We show that
hPPAR
is an efficient competitor for RXR and most likely for other
TR-auxiliary proteins (TRAPs), thereby specifically inhibiting TR
activity by disrupting the formation of TR:RXR heterodimers. A series
of point mutations in hPPAR
allowed the mapping of a region that
is indispensable for this cross-talk to a carboxyl-terminal leucine
zipper-like motif, which is highly conserved among all subtypes
(PPAR
,
,
, and
) of this receptor.
with hTR
1 and other PPARs. A, the first zinc
fingers of the DNA-binding domain of hPPAR
and hTR
1 were
aligned, showing a 100% sequence conservation of the P-box (I indicates identical amino acids). The position of the point
mutation in hPPAR
-C122S changing a cysteine to a serine residue is
indicated. B, the heptad repeat motif (boxed) of the
leucine-zipper in the dimerization domain of hPPAR
, corresponding
to the ninth heptad repeat in the TR, was aligned with hTR
1 and
all currently available PPAR sequences. The heptad repeats are formed
by hydrophobic amino acids with leucine or other hydrophobic amino
acids (e.g. Ile, Val, Ala, Met, or Phe) at positions 1 and 8
and hydrophobic or charged amino acids with hydrophobic side chains (e.g. Arg and Gln) in the fifth
position(3, 22, 23) . This motif is well
conserved between hPPAR
and hTR
1 and even more so within the
PPAR subtypes even across different species. The two hPPAR
mutations L433R and L422R changing leucine to arginine residues are
indicated (prefixes: h = human, r =
rat, m = mouse, cg = hamster, and x = Xenopus).
Construction of Plasmids and Site-directed
Mutagenesis
The pSG5-hPPAR plasmid was kindly provided by Dr. F.
Gonzalez(6) . The pBL2-BFE-CAT containing the peroxisome
proliferator-response element (PPRE) from the bifunctional enzyme (BFE)
promoter (position -2950 to -2925) as well as the pSG5-VDR
vector for the human vitamin D receptor and the pBL2-DR3-CAT with a DR3
vitamin D response element were generously provided by Dr. C.
Carlberg(7, 8) . The pSV2-hTR1 and
pMTV-TRElap-CAT plasmids are described
elsewhere(9, 10) . Mutant hPPAR
(pSG5-hPPAR
-L433R and -L422R) were created by the polymerase chain
reaction-mediated splice donor site overlap extension method and by
subsequently replacing the HindIII/XbaI fragment of
pSG5-hPPAR
with the mutated polymerase chain reaction
product(10) . The clones were verified by dideoxy sequencing to
rule out spurious mutations. hPPAR
-L433R and -L422R have a T to G
point mutation in codons 433 and 422 at nucleotide positions 1514 and
1400, respectively, changing a leucine to arginine.
pSG5-hPPAR
-C122S has a point mutation, replacing a C by a G at
nucleotide position 581, changing codon 122 from cysteine to serine.
This mutant polymerase chain reaction fragment was used to replace the
wild-type AvaI/AvaI fragment in pSG5-hPPAR
.
Preparation of Antibodies
Rabbit polyclonal
anti-TR1 antibodies were raised against a synthetic peptide
corresponding to the unique hTR
1 amino acid sequence 61-81
as described by Falcone et al.(11) . The peptide was
coupled to the maleimide-activated keyhole limpet hemocyanin (Pierce)
for immunization. Fifty µg of protein was injected with Specol
(Central Veterinary Institute, Lelystad, the Netherlands). The
specificity of the antibody was confirmed by immunoprecipitation of
[
S]methionine-labeled in vitro translated hTR
1, hTR
2, and hTR
1 as already
described(12) . To prepare an anti-PPAR antibody the cDNA
encoding the 101 first amino acids of the mouse PPAR
were cloned
into the pQE-9 bacterial expression vector. The expressed polypeptide
was purified on a Ni-NTA-agarose column under native conditions
according to the manufacturer's instructions (Quiagen, Hilden,
Germany), and injected subcutaneously into KOBU rabbits. After the
primary injection (200 µg of polypeptide with Freund's
adjuvant), the rabbits were boosted 4 times (200 µg/boost). The
serum was collected 10 days after the final boost.
In Vitro Transcription/Translation of
Receptors
[S]Methionine-labeled and
unlabeled receptors were synthesized using the rabbit reticulocyte
lysate transcription/translation kit TnT/T7 (Promega, Madison, WI)
according to the manufacturer's instructions. The labeled
receptors were analyzed for appropriate size by electrophoresis on a
12.5% sodium dodecyl sulfate-polyacrylamide gel and quantitated by the
trichloroacetic acid precipitation method as described(10) .
Co-immunoprecipitation of Receptors in
Solution
In vitro translated
[S]methionine-labeled and unlabeled receptors
were brought to a final volume of 20 µl with EMSA binding buffer
and incubated with the appropriate rabbit polyclonal antibodies or
preimmune sera overnight at 4 °C. Complexes were precipitated with
50 µl of protein A-agarose (slurry 50%) (Boehringer Mannheim,
Germany) previously washed with phosphate-buffered saline containing
0.3% Tween 20, 0.5 mM methionine, and 1% bovine serum albumin.
Samples were incubated for 1 h at 4 °C with regular shaking. After
microcentrifugation the pellet was washed 4 times with 1 ml of
phosphate-buffered saline containing 0.3% Tween 20 and 0.5 mM methionine. 50 µl of SDS-PAGE denaturing sample buffer was
added to the final pellet and boiled for 5 min at 94 °C. The
supernatant was subjected to electrophoresis on a 12.5% polyacrylamide
gel.
Electrophoretic Gel Mobility Shift Assay
(EMSA)
Single stranded oligonucleotides were synthesized by
Microsynth (Balgach, Switzerland) and annealed with the complementary
strands. The following sequences were used(7, 10) :
TRE-LAP, 5`-AAGGGGATCCAGCTTGACCTGACGTCAGGTCAAGTC-3`; and PPRE (BFE),
5`-AGGGCTTTGACCTATTGAACTATTACCTAC-3`. The ends were filled in using Taq polymerase (Promega, Madison, WI) in the presence of
[-
P]dCTP (Amersham, United Kingdom). In
vitro translated receptors were incubated with 20,000 cpm of the
labeled double stranded oligonucleotides in the presence of 2 µg of
poly[d(I-C)] and EMSA binding buffer (50 mM KCl, 20
mM Hepes, 20% glycerol, 0.05% Nonidet P-40, 10 mM
-mercaptoethanol, pH 7.5) added to a final volume of 25
µl. Incubation was performed at room temperature for 20 min and the
mixture was then loaded on a 5% polyacrylamide gel. The electrophoresis
was performed at 4 °C and 300 V for 80 min.
Cell Culture and Transfection Studies
HepG2 cells
were plated 24 h before transfection in modified Eagle's medium
containing 10% (v/v) hormone-depleted fetal calf serum(13) ,
penicillin (100 units/ml), streptomycin (100 µg/ml), and
amphotericin B (0.25 µg/ml) in 6-well plates at a density of 0.5
10
cells/well. The medium was changed 4 h before
transfection. Using the calcium-phosphate method (CellPhect kit,
Pharmacia Biotech Inc., Piscataway, NJ) the cells were transfected with
the appropriate plasmids. Eighteen hours later the plates were washed
once with phosphate-buffered saline, and fresh medium was added
together with 500 nM of either L-triiodothyronine,
the peroxisome proliferator, and arachidonic acid analogue ETYA or
1,25(OH)
-vitamin D (VD). After another 24 h the cells were
harvested, lysed, and the chloramphenicol acetyltransferase activity
determined in the extract as described(14) . Chloramphenicol
acetyltransferase activity was normalized for the protein concentration
as measured by the Coomassie Blue method. Experiments were performed in
triplicate and repeated two to four times.
hPPAR
We have
previously reported an inhibitory effect of hPPAR Selectively Inhibits hTRs
on hTRs in HeLa
cells(5) . In contrast to the dominant negative effect of
mutant hTR
1 from kindreds with thyroid hormone resistance, this
effect was observed on all types of TREs, i.e. palindromic,
inverted palindromic, and direct repeat arrangements of the half-sites.
The present experiments were performed in the human hepatocarcinoma
cell line HepG2 representing a tissue of high expression of both TR and
PPAR(15, 16) . Fig. 2A shows the dose
dependent inhibition by PPAR of ligand-dependent transcription of
hTR
1 when assessed on a TRE-LAP. Similar results were obtained on
reporters containing a DR+4 or TRE-PAL (data not shown). However,
this inhibitory effect of hPPAR
was specific for hTR, since no
alteration of vitamin D-dependent transactivation was observed on a
DR+3 element in the presence of increasing doses of hPPAR
(Fig. 2B). Similarly, no change in the transcriptional
activity of hTR
1 on a TRE-LAP was present when increasing amounts
of VDR were transfected (Fig. 2C).
1, but not
hVDR, by hPPAR
. A, transfection of increasing amounts of
hPPAR
inhibits the T
-induced transcriptional capacity
of hTR
1. HepG2 cells were transfected with 1 µg of
pMTV-TRElap-CAT, 0.2 µg of pSG5-hRXR
and pSV2-hTR
1 each,
as well as 0, 0.2, 0.6, or 1.8 µg of pSG5-hPPAR
as indicated.
The amount of pSG5 promoter sequences was kept constant by adding empty
pSG5 vector where necessary. Cells were treated with hormone, lysed,
and chloramphenicol acetyltransferase (CAT) activity
determined as described under ``Materials and Methods.'' B, transfection of increasing amounts of hPPAR
does not
alter the VD-induced transcriptional capacity of hVDR. HepG2 cells were
transfected with 1 µg of pBL2-DR3-CAT, 0.2 µg of pSG5-hRXR
and pSG5-hVDR each, as well as 0, 0.2, 0.6, or 1.8 µg of
pSG5-hPPAR
as indicated. C, transfection of increasing
amounts of hVDR does not alter the T
-induced
transcriptional capacity of hTR
1. HepG2 cells were transfected
with 1 µg of pMTV-TRElap-CAT, 0.2 µg of pSG5-hRXR
and
pSV2-hTR
1 each, as well as 0, 0.2, 0.6, or 1.8 µg of pSG5-hVDR
as indicated.
hPPAR
Co-immunoprecipitation experiments of
[ Inhibits the Formation of TR:RXR Heterodimers
without Forming PPAR:TR Heterodimers in
Solution
S]methionine-labeled hPPAR
with unlabeled
hTR
1 in the presence of an anti-hTR
1 antibody did not show
evidence for the formation of relevant quantities of PPAR:TR
heterodimers in solution (Fig. 3A, lane 3). However,
the precipitation of
S-labeled hRXR
after incubation
with cold hTR
1 demonstrates the ability of the anti-hTR
1
antibody to precipitate hTR
1 also in its heterodimerized state.
Similarly, when using an anti-PPAR antiserum together with
[
S]methionine-labeled hTR
1 and cold
hPPAR
, no PPAR:TR heterodimers were detected (Fig. 4A,
lane 9). In order to test for inhibitory mechanisms other than the
formation of TR:PPAR heterodimers which we were unable to detect, we
performed EMSA on a radiolabeled TRE-LAP (Fig. 3B). The
addition of a 1-4-fold excess of hPPAR
did not inhibit the
formation and DNA binding of hTR
1 homodimers, compatible with the
lack of formation of TR:PPAR heterodimers (Fig. 3B, lanes
2-5). However, hPPAR
was able to substantially reduce
the formation of TR:RXR heterodimers already at equimolar receptor
concentrations, suggesting that hPPAR
competes with high affinity
for this TRAP (lane 7). At excess amounts of hPPAR
, the
TR:RXR complex was almost completely abolished, and the reappearance of
TR:TR homodimers could be observed (lanes 8 and 9).
This suggests that PPAR competes for RXR, rather than for DNA binding.
However, for reasons that have not been further investigated, the
intensity of the homodimeric band in lane 9 does not equal
that observed in the absence of PPAR and RXR in lane 2. It is
nevertheless likely that the still visible DNA-bound TR:RXR complex as
well as possibly unbound TR:RXR heterodimers sufficiently lower the
free TR
1 concentration to reduce the cooperative formation of
homodimers. The dimeric nature of the TR
1 complexes on this
element was established previously by our and other
groups(5, 17, 18) . The composition of the
homo- and heterodimeric complexes of hTR
1 on TRE-LAP was confirmed
by quantitatively supershifting both bands with the anti-TR
1
antiserum (data not shown).
competes with hTR
1 for
hRXR
without the formation of PPAR:TR heterodimers. A,
co-immunoprecipitation of hTR
1 and hPPAR
with an
anti-TR
1 antibody. While this antibody precipitates TR:RXR
heterodimers (lane 2), no evidence for the formation of
heterodimers between TR and PPAR wild-type (lane 3) or mutants (lanes 4-6) is present. 6 fmol of in vitro translated [
S]methionine-labeled wild-type (WT) or mutant hPPAR
(hPPAR
) were
incubated with 4 fmol of hTR
1 and 2 µl of anti-hTR
1
antibody (lanes 2-6) or preimmune serum (lane
1). After precipitation with protein A-agarose, samples were run
on SDS-polyacrylamide gel electrophoresis. B, effect of
hPPAR
wild-type on TR:TR homo- and TR:RXR heterodimer binding on
TRE-LAP as analyzed by EMSA. While no effect of increasing amounts of in vitro translated hPPAR
on the TR:TR homodimer was
present (lanes 2-5) the formation of TR:RXR heterodimers
was strongly reduced (lanes 6-9). At a 2- and 4-fold
excess of PPAR, a homodimeric TR:TR band reappeared.
[
P]TRE-LAP was incubated either with
unprogrammed reticulocyte lysate (lane 1) or in vitro translated hTR
1 (lanes 2-8) as described under
``Materials and Methods.'' Numbers represent molar
ratio of receptors.
mediates
the interaction with hRXR
. A, interaction of wild-type
and mutant hPPAR
with hRXR
assessed by co-immunoprecipitation
with an anti-PPAR antibody. While the hPPAR
wild-type (lanes 1 and 2), -C122S (lanes 5 and 6), and
L422R (lanes 7 and 8) were able to form heterodimers
with RXR, the L433R mutant completely lost its ability to interact with
RXR (lanes 3 and 4). Lane 9 confirms the
absence of PPAR:TR heterodimers with an anti-PPAR antibody when
[
S]methionine-labeled hTR
1
(hTR
1
) was used instead of hRXR
. 4 fmol of
[
S]methionine-labeled hRXR
(hRXR
) are incubated with 6 fmol of in vitro translated wild-type or mutated PPARs (lanes 1-8)
and 2 ml anti-PPAR antiserum (lanes 2, 4, 6, 8, and 9). When no antiserum was added, volume is replaced by
preimmune serum (lanes 1, 3, 5, and 7). Immune
complexes were precipitated by protein A-agarose and electrophoresed. B, binding of wild-type and mutant hPPAR
to the PPRE from
BFE in EMSA. In the absence of RXR, neither the wild-type nor mutant
PPARs were able to bind to DNA (lanes 3-6). Upon
addition of RXR, PPAR wild-type and the control mutant L422R were
capable of interacting with the BFE element (lanes 7 and 9), whereas the heptad repeat (L433R, lane 8) and
P-box (C122S, lane 10) mutants showed no detectable binding.
In all incubation mixtures, the total volume of reticulocyte lysate was
kept constant and completed with unprogrammed reticulocyte lysate where
necessary.
The Inhibitory Effect of PPAR on TR Activity Requires Its
Heterodimerization with RXR
In order to test directly whether
hPPAR competes for RXR, rather than for DNA binding, three mutant
hPPAR
were created as illustrated in Fig. 1. Mutation C122S
is located in the P-box at the base of the first zinc-finger (Fig. 1A). The amino acid sequence alignment of the
ligand-binding domains of hTR
1 and hPPAR
revealed the
presence of a highly conserved leucine zipper-like heptad repeat,
corresponding to the ninth heptad repeat in hTR
1 (Fig. 1B)(3, 19) . In order to test
its function in the context of hPPAR
, the distal leucine was
mutated to an arginine (L433R). To exclude a nonspecific effect of this
mutation on the secondary or tertiary protein structure of PPAR, a
control mutation was created 11 amino acids amino-terminally (L422R).
mutants were characterized with respect to
their ability to interact with RXR in solution, to bind to DNA, and to
transactivate through a PPRE and to modulate TR activity.
Co-immunoprecipitation experiments with the anti-PPAR antibody detected
the presence of PPAR:RXR heterodimers in solution when
hPPAR
-wild-type, or the -C122S and -L422R mutants where used. In
contrast, the Leu to Arg mutation at position 433 abolished the
heterodimerization with hRXR
(Fig. 4A). In
addition, like the wild-type hPPAR
, none of the mutants was able
to heterodimerize with hTR
1 (Fig. 3A). When
analyzed in an EMSA using the PPRE from the BFE promoter, no binding to
DNA was observed when wild-type or mutant hPPAR
was used alone (Fig. 4B, lanes 3-6). However, only the wild-type
and hPPAR
-L422R mutant bound to DNA when RXR was present (Fig. 4B, lanes 7 and 9). The hPPAR
-C122S
was expected to be unable to bind to DNA due to its mutation in the
DNA-binding domain (lane 10), while the hPPAR
-L433R was
incapable of interacting with DNA due to its defect in heterodimerizing
with RXR (lane 8). Taken together, these results suggest that
the distal leucine residue 433 of the minimal leucine zipper motif in
the ligand-binding domain of PPAR is required for the
heterodimerization with RXR, while the cysteine residue 122 in the
P-box is necessary for binding to DNA without affecting the
heterodimerization with RXR. The control mutation hPPAR
-L422R
close to this putative leucine zipper heptad repeat altered neither the
formation of heterodimers nor DNA binding. The panel of mutant
hPPAR
was tested in a transient transfection system for the
capacity to transactivate as well as to modulate TR activity. When
tested on the PPRE from the BFE promoter, only wild-type hPPAR
and
L422R showed ligand-induced transcriptional activity, as anticipated
from the EMSA experiments (Fig. 5). The inhibitory effect of
hPPAR
-L433R and -C122S in the presence of ETYA on the basal
activity of the reporter gene was not further analyzed here.
on the PPRE from bifunctional enzyme. HepG2 cells
transiently transfected with the pBL2-BFE-CAT reporter gene and treated
with the peroxisome proliferator ETYA showed no evidence for
functionally active endogenous PPAR activity(-). Upon
transfection of pSG5-hPPAR
(PPAR-WT) or the control mutant
pSG5-hPPAR
-L422R (L422R), a severalfold increase in
chloramphenicol acetyltransferase (CAT) activity was observed.
In contrast, the hPPAR
mutants deficient in heterodimerization
(L433R) or DNA binding (C122S) showed no transcriptional activity.
Cells were transfected with 1 µg of reporter plasmid, 0.2 µg of
pSG5-hRXR
, and 0.2 µg of wild-type (WT) or mutant
pSG5-hPPAR
as indicated. The amount of pSG5 promoter sequences was
kept constant by adding empty pSG5 vector where necessary. Cells were
treated with ETYA, lysed, and chloramphenicol acetyltransferase
activity determined as described under ``Materials and
Methods.''
1 and
mutant hPPAR
were performed as shown in Fig. 6, A-C. While the leucine zipper mutant hPPAR
-L433R lost its
ability to down-regulate TR activity (Fig. 6A), the DNA
binding-deficient mutant hPPAR
-C122S (Fig. 6B) and
the control mutant hPPAR
-L422R (Fig. 6C) remained
efficient inhibitors of TR-dependent transcription. These data
demonstrate that the leucine residue at position 433 of hPPAR
is
essential for mediating cross-talk with hTR by a mechanism involving
heterodimerization with RXR.
with
hRXR
, but no DNA binding, is required for the inhibition of
hTR
1 activity. HepG2 cells were transiently transfected with
constant amounts of pSV2-hTR
1 (0.2 µg), pSG5-hRXR
(0.2
µg), and the pMTV-TRElap-CAT reporter (1 µg) as described under
``Materials and Methods.'' The marked inhibition of hTR
1
activity observed with hPPAR
wild-type (Fig. 2) is
completely abolished when the pSG5-hPPAR
-L433R mutant is used at
increasing doses (0.2, 0.6, and 1.8 µg) as shown in panel
A. However, the same mutation located 11 amino acids upstream
(pSG5-hPPAR
-L422R) as well as the DBD mutant pSG5-hPPAR
-C122S
were still able to inhibit the T
-induced transactivation by
hTR
1, as shown in panels C and B, respectively. CAT, chloramphenicol
acetyltransferase.
(4) , we found no
evidence for the formation of significant amounts of
hPPAR
:hTR
1 heterodimers. While the formation of PPAR:TR
heterodimers may exist, it is not likely to be quantitatively important
as is already evident from the initial report. The present results
obtained with the P-box mutant C122S show that competition for DNA
binding is also not a major mechanism for the inhibition of TR action
by PPAR. Nevertheless, the hPPAR
-C122S was a somewhat less
efficient inhibitor of thyroid hormone action, although its
dimerization with RXR was preserved and binding to a TRE-LAP has been
excluded by gel shift assays. This characteristic of the mutant has not
been analyzed further, but possible explanations are differences in
expression levels, post-translational modifications, or heterodimer
stability.
-L433R eliminates the inhibitory effect of PPAR on TR.
1 allele, also clearly demonstrates the ability of a
nuclear factor to modulate thyroid hormone action in a dominant
negative manner in vivo and in vitro(21) . In
addition, TRs and PPARs are co-expressed in many tissues, such as liver
and brain, and both receptors regulate similar metabolic steps in lipid
metabolism, such as the ones controlled by malic enzyme and
bifunctional enzyme. The modulatory effect of PPAR on TR depends on the
PPAR protein levels as well as its affinity for RXR, the former being
controlled by glucocorticoids and the latter possibly by
phosphorylation as demonstrated for other nuclear
receptors(15) .
, L-triiodothyronine; TR, T
receptor; RXR, retinoid
X receptor; TRE, T
-response element; TRAP, TR-auxiliary
proteins; PPRE, peroxisome proliferator-activator response element;
BFE, bifunctional enzyme; VDR, vitamin D receptor; VD,
1,25(OH)
-vitamin D; EMSA, electrophoretic gel mobility
shift assay; ETYA, 5,8,11,14-eicosatetraynoic acid.
We are grateful to Drs. F. Gonzalez (National Cancer
Institute, Bethesda, MD) and C. Carlberg (University Hospital Geneva)
for providing the PPAR and VDR expression and reporter plasmids,
respectively. We also thank Prof. B. Desvergne (Institut de Biologie
Animale, University of Lausanne) for helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.