(Received for publication, May 9, 1994; and in revised form, October 31, 1994)
From the
We have cloned two human peroxisome proliferator- activated
receptor (PPAR) subtypes, hPPAR and hNUC1. hPPAR
is activated
by clofibric acid and other PPAR activators. hNUC1 is not activated by
these compounds acting instead as a repressor of hPPAR
and human
thyroid hormone receptor transcriptional activation. Repression is
specific since hNUC1 does not significantly repress activation by the
progesterone or retinoic acid receptors. We demonstrate co-operative
binding of hNUC1 and hRXR
to a PPAR-responsive element and show
that in the presence of hRXR
, the affinity of hNUC1 for the
peroxisome proliferator is comparable to that of hPPAR
.
Furthermore, repression of hPPAR
can be overcome by transfecting
excess hPPAR
. We propose that hNUC1 represses the activity of
hPPAR
by titrating out a factor required for activation. Our data
further suggests convergence of thyroid hormone- and
peroxisome-mediated fatty acid metabolism pathways. Overcoming hNUC1
repression could be a means of increasing the activity of these
receptors.
Peroxisomes are subcellular organelles found in animals and
plants, and they contain enzymes for respiration, cholesterol, and
lipid metabolism. A variety of chemical agents including hypolipidemic
drugs such as clofibrates cause proliferation of peroxisomes in rodents (1) . Two hypotheses have been put forward to explain the
mechanism of peroxisome proliferation. The first is the ``lipid
overload hypothesis'' whereby an increase in the intracellular
concentration of fatty acids is the main stimulus for peroxisome
proliferation(2, 3) . The second hypothesis postulates
a receptor-mediated mechanism and an as yet unidentified ligand. In
supporting the second postulate, peroxisome proliferator-activated
receptors (PPARs) ()have been cloned from various
species(4, 5, 6, 7, 8) .
We are interested in the effect of various fibrates on human PPAR
subtypes. We have isolated two human PPAR subtypes hPPAR and
hNUC1. While hPPAR
is a transcriptional activator in the presence
of fibrates and ETYA, hNUC1 is not. However, transfected hNUC1
decreased the response from endogenous PPARs. We therefore reasoned
that one function of hNUC1 could be to repress the activity of
hPPAR
. Accordingly, experiments were performed to determine
whether hNUC1 represses hPPAR
and other members of the nuclear
receptor family. We demonstrate that hNUC1 acts as a repressor of
hPPAR
and human thyroid hormone receptor activity.
The activation profile of hPPAR by CFA is shown in Fig. 1A. This receptor is also activated by other known
activators of PPARs, e.g. WY-14,643 and ETYA in HepG2 and CV-1
cells (data not shown). A second human PPAR subtype termed hNUC1 was
cloned from a kidney cDNA library. This receptor has 61% homology to
hPPAR
and the two cysteine residues in the ``D'' box are
separated by three amino acids (E, R, and S, positions 112-114 of
the amino acid sequence). This is a characteristic of
PPARs(5) . All the other nuclear receptors have five amino
acids in the same region. Therefore, we consider hNUC1 a member of the
PPAR family.
Figure 1:
hNUC1, unlike hPPAR is not
activated by PPAR activators. HepG2 cells were transfected with the
pPPRE3-tk-LUC reporter and with pCMVhPPAR
(A) or pBKCMV
(vector) and treated with CFA. Cells were transfected with pCMVhNUC1 or
vector and treated with CFA (B) or ETYA (C) and
luciferase and
-galactosidase assays performed as described under
``Materials and Methods.''
The hNUC1 receptor, unlike hPPAR is not activated
in HepG2 or CV-1 cells by CFA or ETYA (Fig. 1, B and C). The slight activation seen in the absence of transfected
receptor is presumably due to the endogenous PPARs in the cell line
utilized. Transfected hNUC1 did however decrease the response from the
endogenous PPARs. This suggested that hNUC1 may act as a repressor of
hPPAR function. Therefore, to demonstrate repression of hPPAR
activity by hNUC1, we co-transfected increasing amounts of hNUC1
plasmid with a constant amount of hPPAR
expressing plasmid. We saw
a strong dose dependent repression of hPPAR
activity by hNUC1 (Fig. 2A). Complete repression was observed with 0.1
µg of cotransfected hNUC1 plasmid. Repression by hNUC1 was also
observed on rat PPAR
and on hPPAR
in the presence of ETYA,
WY-14,643, and other fibrates (data not shown).
Figure 2:
hNUC1 inhibits activation of hPPAR.
HepG2 cells were transfected with 0.1 µg of pCMVhPPAR
and
increasing amounts of pCMVhNUC1 (indicated in micrograms). The
reporters were pPPRE3-tk-LUC (A) or AOX-LUC (B). CFA
or WY-14,643 was added to a final concentration of 1 and 0.1
mM, respectively, where indicated. Control cells received an
equal volume of ethanol (vehicle).
To determine whether
hNUC1 also represses activation of hPPAR on the natural acyl-CoA
oxidase gene promoter, we performed co-transfection assays with the
AOX-LUC reporter (Fig. 2B). We observe activation of
hPPAR
from the AOX promoter in the presence of WY-14,643.
Co-transfected hNUC1 completely blocks this activation. No activation
was observed by hNUC1 itself in the presence or absence of WY-14,643.
We conclude that hNUC1 can repress hPPAR
activation through the
AOX promoter.
We next determined the specificity of hNUC1
repression. We tested the effect of hNUC1 on other members of the
steroid receptor family (Fig. 3, A-D). Activation
of hTR by LT3 through a palindromic TRE was repressed by 65% by
hNUC1 (Fig. 3A). Repression increased to 75% in the
presence of CFA. However, many TR inducible genes contain TREs that
involve direct repeats of the 5`-AGGTCA-3` motif. We therefore tested
whether hNUC1 could also repress activation of hTR
through a DR4
motif(26) . We observe repression of TR activation through a
DR4 element by hNUC1 in the absence and presence of CFA (Fig. 3B). Repression was also observed with hTR
,
although to a lesser degree (data not shown).
Figure 3:
hNUC1 represses activation of TR but not
of PR or RAR. HepG2 cells were transfected with 0.1 µg of
pRShTR (A and B), pSVhPRB (C), and
pRShRAR
(D). The reporters were MTV-TREp2-LUC (A), TRE(DR4)2-tk-LUC (B), PRE2-tk-LUC (C),
and MTV-TREp2-LUC (D). pCMVhNUC1 was co-transfected where
indicated (0.1 or 0.5 µg). The respective ligands for the
transfected receptors were L-T3 (100 nM), progesterone (PROG, 1 µM) and ATRA (1 µM), the
final concentration indicated within parentheses. CFA was added where
indicated to a final concentration of 1
mM.
To determine whether
hNUC1 could repress activation of other members of the steroid receptor
family, we performed similar assays with the PR and RAR (Fig. 3, C and D). With 0.1 µg of
transfected hNUC1 plasmid no repression of PR activity was observed.
Similarly with RAR
no significant repression by hNUC1 in the
absence of CFA was observed. With CFA, 50% repression of RAR
activity was observed (Fig. 3D). At higher levels of
transfected hNUC1 (0.5 µg) we observe a modest stimulation of
activity (Fig. 3, C and D). CFA reduces this
induction. However, this stimulation was not observed with hPPAR
and 0.5 µg of co-transfected hNUC1 (Fig. 2A). This
result indicates that even at high levels of hNUC1, no repression of PR
or RAR
is observed. Further, hNUC1 did not repress the activity of
the estrogen receptor through an estrogen-responsive promoter (data not
shown). We conclude that hNUC1 is not a general transcriptional
repressor. Among the receptors tested, it strongly repressed activation
of hPPAR
and hTR. Repression occurred in the absence of clofibric
acid, but was enhanced in its presence.
One mechanism by which hNUC1
could repress activation by hPPAR is by binding to the PPRE. To
demonstrate binding of hNUC1 to a PPRE, we performed gel retardation
assays. With hNUC1 or hRXR
alone, very weak retarded complexes are
seen (Fig. 4A, lanes 1 and 4).
Addition of RXR
enhances binding of hNUC1 (lane 2)
demonstrating cooperative binding of hNUC1 and hRXR
to the PPRE.
The specific complex is not observed in control reactions using Sf21
and unprogrammed reticulocyte lysate (lanes 3, 5, and 6), nor with a probe with an unrelated sequence (lane
7). Co-operative binding to the PPRE was also observed between
hNUC1 and hRXR
and between hPPAR
and hRXR
in gel
retardation assays using whole cell extracts from COS cells transfected
with the respective expression plasmids (data not shown). These
experiments also suggested that transfected hPPAR
and hNUC1 were
expressed at roughly equal levels as judged by the retarded band
intensities. Therefore, to account for the almost complete inhibition
of hPPAR
induced response at 1:1 ratio of hNUC1 to hPPAR
plasmid DNA, the affinity of the hNUC1-RXR complex for the PPRE must be
significantly higher than that of hPPAR
. Fig. 4B shows the result of a competition experiment using increasing
amounts of unlabeled PPRE or probes with unrelated sequences. The
hNUC1-RXR-PPRE complex is specific since the unrelated oligonucleotides (lanes 14-18) compete very poorly compared to the
specific PPRE containing oligonucleotides (lanes 8-12).
It also has a higher mobility than the hPPAR
-RXR-PPRE complex. The
relative affinity of hNUC1-hRXR
for the PPRE is comparable to that
of hPPAR-hRXR
(compare lanes 1-6 with lanes
7-12). Given that the expression levels of hNUC1 and
hPPAR
in transfected cells and their affinities for the PPRE are
similar, competition for PPRE alone cannot wholly account for the
strong repression of hPPAR
activity by hNUC1. We therefore
investigated whether hNUC1 could be titrating a limiting factor
required for hPPAR
activity.
Figure 4:
(A) hNUC1 and hRXR bind
co-operatively to the PPRE. DNA binding assays were performed with in vitro translated hNUC1 and recombinant baculovirus
expressed hRXR
(RXR) as described under ``Materials
and Methods.'' As controls, Sf21 cell extracts (Sf21) and
unprogrammed lysate (unprog. lys.) were used. Labeled
oligonucleotides containing a PPRE sequence (lanes 1-6)
or an unrelated sequence (lane 7) was used as probes. (B) hPPAR
-hRXR
and hNUC1-hRXR
bind to the PPRE
with similar affinities. Recombinant baculovirus expressed hRXR
with in vitro translated hPPAR
(hPPAR
) or
hNUC1 (hNUC1) was used in binding reactions with labeled PPRE.
Unlabeled oligonucleotides containing a PPRE sequence (lanes
2-6 and lanes 8-12) or an unrelated sequence (lanes 14-18) at various fold molar excess as indicated
were premixed with the labeled probe and added to the reactions. The
hPPAR
-specific complex is denoted by the solid arrowhead and the hNUC1-specific complex by the open
arrowhead.
To investigate this possibility,
we performed an experiment where we systematically altered the ratio of
activator to repressor (Fig. 5). If hNUC1 was indeed titrating
out a limiting factor, one would predict from simple equilibrium
considerations that increasing the amount of activator (hPPAR)
would overcome the repression. We observe that increasing the ratio of
hPPAR
to hNUC1 overcame the repression by hNUC1. The activation
observed with 0.25 and 0.5 µg of hPPAR
in the presence of 0.05
µg of hNUC is the same as that observed in the absence of hNUC.
Therefore, at sufficiently high ratios of activator to repressor (5-
and 10-fold), repression by hNUC was completely overcome. This data is
consistent with the hypothesis that hNUC represses hPPAR
by
titrating a limiting factor required for hPPAR
activation. We
cannot rigorously rule out the possibility of competitive DNA binding
by hNUC1. This is however unlikely since hNUC1 and hPPAR
have
similar affinities for the PPRE in presence of excess RXR (Fig. 4). This factor is probably not utilized by all receptors
since hNUC1 does not have a pronounced repressive effect on the PR and
RAR.
Figure 5:
hNUC1 represses hPPAR by sequestering
a limiting transcription factor. HepG2 cells were transfected with
pPPREA3-tk-LUC as reporter and different amounts of pCMVhPPAR
plasmid (indicated in micrograms) in the absence(-) or presence
(+) of 0.05 µg of pCMVNUC1. CFA was added to a final
concentration of 1 mM.
We have cloned a subtype of the human PPAR family, hNUC1. The
sequence of our clone is similar to the previously published sequence (8) except for alanine at position 292 of the amino acid
sequence instead of proline. Among the Xenopus PPAR subtypes,
xPPAR has the closest homology to hNUC1. It is possible that hNUC1
is the human homolog of xPPAR
.
Although hNUC1 is a member of
the PPAR family, we have shown that hNUC1 is not transcriptionally
activated by compounds that normally activate hPPAR through the
PPRE identified in the acyl-coenzyme A oxidase gene. This has also been
observed by Schmidt et al.(8) with hNUC1 and certain
fatty acids. We further demonstrate that hNUC1 is a dominant negative
repressor of hPPAR
and hTR.
Several mechanisms of repression
can be suggested. First, the mechanism of repression by hNUC1 could be
similar to the repression of thyroid hormone action by the non-hormone
binding rat erbA-2(19) . However, Schmidt et al.(8) have demonstrated that a chimera of the N terminus
including the DNA binding domain of the GR fused to the ligand binding
domain of hNUC1 (GR-NUC) was activated by WY-14,643. This suggests that
hNUC1 can bind the as yet unidentified ``ligand'' for PPAR.
Interestingly, a similar estrogen receptor-NUC chimera was not
activated by WY-14,643. This suggests that there is probably no
WY-14,643 inducible transcriptional activation function in the ligand
binding domain of hNUC1 and further that the activation observed with
the GR-NUC chimera was from the strong activation function in the N
terminus of the GR(20, 21) . Secondly, hNUC1 could be
binding to the PPRE thereby antagonizing activation of hPPAR
.
COUP-TF has been shown to inhibit PPAR activation by a similar
mechanism(22) . We have demonstrated cooperative binding of
hNUC1 and hRXR
to a PPRE. In the absence of a CFA inducible
transcription activation function of hNUC1, this mechanism could
explain the repression of hPPAR
activity by hNUC1. However, since
the affinity of the hNUC1-RXR complex for the PPRE is comparable to
that of hPPAR
-RXR, this cannot wholly explain the strong
repression observed with hNUC1. Finally we show that repression by
hNUC1 can be reversed by excess transfected hPPAR
. This suggests
competition for a limiting factor required for transcriptional activity
of hPPAR
.
How hNUC1 represses TR activity is not clear at present. One possibility is the formation of transcriptionally inactive heterodimers as in the case of helix loop helix proteins(23) . Heterodimerization between rPPAR and TR has recently been demonstrated(24) . The role of CFA on hNUC1-mediated repression is also unclear at present.
This is the first demonstration of repression by one PPAR subtype on another and on TR. Our data and that of others (25) suggest a convergence of the thyroid hormone- and peroxisome- mediated fatty acid metabolism pathways. Overcoming repression by hNUC1 may be a way to increase activity of PPARs and thyroid hormone receptors.
Note
Added in Proof-While this manuscript was under review,
repression of mouse PPAR by mouse NUC1 was demonstrated (Kleiwer,
S. A., Forman, B. M., Blumberg, B., Ong, E. S., Borgmeyer, U.,
Mangelsdorf, D. J., Umesono, K., and Evans, R. M.(1994) Proc. Natl.
Acad. Sci. U. S. A.91, 7355-7359).