©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Human Peroxisome Proliferator-activated Receptor (PPAR) Subtype NUC1 Represses the Activation of hPPAR and Thyroid Hormone Receptors (*)

(Received for publication, May 9, 1994; and in revised form, October 31, 1994)

Lily Jow Ranjan Mukherjee (§)

From the Department of Molecular Biology, Ligand Pharmaceuticals Inc., San Diego, California 92121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have cloned two human peroxisome proliferator- activated receptor (PPAR) subtypes, hPPARalpha and hNUC1. hPPARalpha is activated by clofibric acid and other PPAR activators. hNUC1 is not activated by these compounds acting instead as a repressor of hPPARalpha 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 hRXRalpha to a PPAR-responsive element and show that in the presence of hRXRalpha, the affinity of hNUC1 for the peroxisome proliferator is comparable to that of hPPARalpha. Furthermore, repression of hPPARalpha can be overcome by transfecting excess hPPARalpha. We propose that hNUC1 represses the activity of hPPARalpha 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.


INTRODUCTION

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) (^1)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 hPPARalpha and hNUC1. While hPPARalpha 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 hPPARalpha. Accordingly, experiments were performed to determine whether hNUC1 represses hPPARalpha and other members of the nuclear receptor family. We demonstrate that hNUC1 acts as a repressor of hPPARalpha and human thyroid hormone receptor activity.


MATERIALS AND METHODS

Reagents

ETYA, ATRA, LT3, and CFA were purchased from Sigma, and WY-14,643 from Chemsyn Science Laboratories, Lenexa, KS. Stock solutions of these compounds were made in ethanol or methanol.

Isolation of Human PPARalpha cDNA

A human homolog of rat PPARalpha was isolated from a human liver 5`-stretch gt10 cDNA library (Clontech). The library was screened at medium stringency (40% formamide, 5 times SSC at 37 °C), with a rPPAR nick-translated DNA fragment specific to the A/B and DNA binding domain (from the EcoRI to the BglII site, nucleotides 450-909) (6) . Positive clones were isolated and subcloned into the Bluescript KS vector (Stratagene) for sequencing. The sequence is identical to that published by Sher et al.(7) except for two amino acid differences, alanine at position 268 and glycine at position 296(28) .

Isolation of hNUC1 cDNA

A second human PPAR subtype hNUC1 was isolated from a human kidney cDNA library by similarly screening with a probe specific to the rat PPAR DNA binding domain (from the PvuII to the BglII site, nucleotides 618-909) (6) as described above. A recombinant clone was isolated, subcloned into pGEM-5Zf (Promega), and sequenced. The sequence of this receptor is identical to that of the hNUC1 sequence (8) except for alanine at position 292.

Receptor Expression and Reporter Constructs

For expression in mammalian cells, the hPPARalpha cDNA was cloned into the NotI site of pBKCMV (Stratagene) to give pCMVhPPARalpha. The hNUC1 cDNA was directionally cloned into the SalI-SacII site of pBKCMV to give pCMVhNUC1. The reporter plasmid pPPREA3-tk-LUC containing three copies of the ``A'' site identified in the acyl-CoA oxidase gene regulatory sequence (9) has been described(10) . This PPRE conforms to the DR1 configuration(26) . The reporter plasmid AOX-LUC (10) contains nucleotides -602 to +20 of the rat AOX promoter. The plasmids pRShRARalpha, pSVhPRB, and pRShTRbeta and the reporters MTV-TREp2 and PRE2-tk-LUC have been described(11, 12, 13, 14) . The human TRalpha1 cDNA (15) was liberated from pME21 by digestion with EcoRI and blunt-ended by digestion with mung bean nuclease. pRS plasmid (16) was digested with BamHI, dephosphorylated, and repaired with Klenow enzyme. The TRalpha1 cDNA was was then joined to the vector by blunt end ligation. The reporter TRE(DR4)2-tk-LUC was made by inserting two copies of an oligonucleotide containing the TRE (DR4) sequence into the pBL-tk-LUC reporter(27) . The sequence of the DR4 oligonucleotide is 5`-GATCTAGGTCACAGGAGGTCACG-3`.

Co-transfection Assay

HepG2 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (HyClone), 2 mML-glutamine, and 55 µg/ml gentamicin (BioWhittaker). Cells were plated at 1.7 times 10^5 cells/well for HepG2 in 12-well cell culture dishes (Costar). The medium was replaced with fresh medium 20 h later. After 4 h, DNA was added by the calcium phosphate coprecipitation technique (17) . Typically, 0.1 µg of expression plasmid, 0.5 µg of the beta-galactosidase expression plasmid pCH110 (internal control), and 0.5 µg of reporter plasmid were added to each well. Where indicated, 0-0.5 µg of hNUC1 plasmid (repressor) was added. Repressor plasmid dosage was kept constant by the addition of appropriate amounts of the empty expression vector pBKCMV. The total amount of DNA was kept at 2 µg by the addition of pGEM DNA. After 14 h the cells were washed with 1 times phosphate-buffered saline and fresh medium added (Dulbecco's modified Eagle's medium with 10% charcoal-stripped fetal bovine serum (HyClone) plus the above supplements). Ligands or PPAR activators were added to the final concentrations indicated. Control cells were treated with vehicle. After another 24 h the cells were harvested, and the luciferase and beta-galactosidase activities were quantified on a Dynatech ML 1000 luminometer and a Beckman Biomek 1000 workstation, respectively. The normalized response is the luciferase activity of the extract divided by the beta-galactosidase activity of the same. Each data point is the mean of triplicate transfections, and the error bars represent the standard deviation from the mean. Each experiment has been repeated at least three times and a representative experiment is shown in each case.

Gel Retardation Assays

hPPARalpha and hNUC1 were made by coupled in vitro transcription/translation using 1 µg of pCMVhPPARalpha or pCMVhNUC1 plasmid DNA and the T3-coupled reticulocyte lysate system (Promega). The baculovirus/Sf21 cell system was used to express hRXRalpha(18) . Gel retardation assays were performed by incubating 1 µl of in vitro translated hPPARalpha or hNUC1 and 2 µg of hRXRalpha in buffer containing 10 mM Hepes (pH 7.8), 50 mM KCl, 1 mM dithiothreitol, 2.5 mM MgCl(2), 0.4 mg/ml poly(dI-dC), and 20% glycerol at 4 °C for 5 min. About 12 fmol of P-end-labeled probe (approximately 100,000 cpm) were then added and incubated at 25 °C for another 5 min. Protein-DNA complexes were resolved by electrophoresis on 5% polyacrylamide gels in 0.5 times TBE. For the competition assays, annealed, unlabeled oligonucleotides were mixed with the labeled probe just before adding to the binding reactions. Oligonucleotides containing the PPRE sequence from the acyl-CoA oxidase (AOX) gene used as probe have the sequence 5`-CTAGCGATATCATGACCTTTGTCCTAGGCCTC-3` (upper strand) and 5`-CTAGGAGGCCTAGGACAAAGGTCATGATATCG-3` (lower strand). Oligonucleotides with sequences unrelated to the PPRE were used to determine specificity of binding. Their sequences are 5`-CGGGTTAAAAACCGATGTCACATCGGCCGTTCGAA-3` (upper strand) and 5`-TTTCGAACGGCCGATGTGACATCGGTTTTTAACCC-3` (lower strand).


RESULTS

The activation profile of hPPARalpha 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 hPPARalpha 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 hPPARalpha is not activated by PPAR activators. HepG2 cells were transfected with the pPPRE3-tk-LUC reporter and with pCMVhPPARalpha (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 beta-galactosidase assays performed as described under ``Materials and Methods.''



The hNUC1 receptor, unlike hPPARalpha 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 hPPARalpha activity by hNUC1, we co-transfected increasing amounts of hNUC1 plasmid with a constant amount of hPPARalpha expressing plasmid. We saw a strong dose dependent repression of hPPARalpha 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 PPARalpha and on hPPARalpha in the presence of ETYA, WY-14,643, and other fibrates (data not shown).


Figure 2: hNUC1 inhibits activation of hPPARalpha. HepG2 cells were transfected with 0.1 µg of pCMVhPPARalpha 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 hPPARalpha on the natural acyl-CoA oxidase gene promoter, we performed co-transfection assays with the AOX-LUC reporter (Fig. 2B). We observe activation of hPPARalpha 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 hPPARalpha 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 hTRbeta 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 hTRbeta 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 hTRalpha, 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 pRShTRbeta (A and B), pSVhPRB (C), and pRShRARalpha (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 RARalpha (Fig. 3, C and D). With 0.1 µg of transfected hNUC1 plasmid no repression of PR activity was observed. Similarly with RARalpha no significant repression by hNUC1 in the absence of CFA was observed. With CFA, 50% repression of RARalpha 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 hPPARalpha 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 RARalpha 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 hPPARalpha 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 hPPARalpha is by binding to the PPRE. To demonstrate binding of hNUC1 to a PPRE, we performed gel retardation assays. With hNUC1 or hRXRalpha alone, very weak retarded complexes are seen (Fig. 4A, lanes 1 and 4). Addition of RXRalpha enhances binding of hNUC1 (lane 2) demonstrating cooperative binding of hNUC1 and hRXRalpha 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 hRXRalpha and between hPPARalpha and hRXRalpha 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 hPPARalpha and hNUC1 were expressed at roughly equal levels as judged by the retarded band intensities. Therefore, to account for the almost complete inhibition of hPPARalpha induced response at 1:1 ratio of hNUC1 to hPPARalpha plasmid DNA, the affinity of the hNUC1-RXR complex for the PPRE must be significantly higher than that of hPPARalpha. 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 hPPARalpha-RXR-PPRE complex. The relative affinity of hNUC1-hRXRalpha for the PPRE is comparable to that of hPPAR-hRXRalpha (compare lanes 1-6 with lanes 7-12). Given that the expression levels of hNUC1 and hPPARalpha in transfected cells and their affinities for the PPRE are similar, competition for PPRE alone cannot wholly account for the strong repression of hPPARalpha activity by hNUC1. We therefore investigated whether hNUC1 could be titrating a limiting factor required for hPPARalpha activity.


Figure 4: (A) hNUC1 and hRXRalpha bind co-operatively to the PPRE. DNA binding assays were performed with in vitro translated hNUC1 and recombinant baculovirus expressed hRXRalpha (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) hPPARalpha-hRXRalpha and hNUC1-hRXRalpha bind to the PPRE with similar affinities. Recombinant baculovirus expressed hRXRalpha with in vitro translated hPPARalpha (hPPARalpha) 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 hPPARalpha-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 (hPPARalpha) would overcome the repression. We observe that increasing the ratio of hPPARalpha to hNUC1 overcame the repression by hNUC1. The activation observed with 0.25 and 0.5 µg of hPPARalpha 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 hPPARalpha by titrating a limiting factor required for hPPARalpha activation. We cannot rigorously rule out the possibility of competitive DNA binding by hNUC1. This is however unlikely since hNUC1 and hPPARalpha 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 hPPARalpha by sequestering a limiting transcription factor. HepG2 cells were transfected with pPPREA3-tk-LUC as reporter and different amounts of pCMVhPPARalpha 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.




DISCUSSION

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, xPPARbeta has the closest homology to hNUC1. It is possible that hNUC1 is the human homolog of xPPARbeta.

Although hNUC1 is a member of the PPAR family, we have shown that hNUC1 is not transcriptionally activated by compounds that normally activate hPPARalpha 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 hPPARalpha 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-alpha2(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 hPPARalpha. COUP-TF has been shown to inhibit PPAR activation by a similar mechanism(22) . We have demonstrated cooperative binding of hNUC1 and hRXRalpha to a PPRE. In the absence of a CFA inducible transcription activation function of hNUC1, this mechanism could explain the repression of hPPARalpha activity by hNUC1. However, since the affinity of the hNUC1-RXR complex for the PPRE is comparable to that of hPPARalpha-RXR, this cannot wholly explain the strong repression observed with hNUC1. Finally we show that repression by hNUC1 can be reversed by excess transfected hPPARalpha. This suggests competition for a limiting factor required for transcriptional activity of hPPARalpha.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Ligand Pharmaceuticals Inc., Dept. of Molecular Biology, 9393 Towne Centre Dr., San Diego, CA 92121. Tel.: 619-535-3900; Fax: 619-535-3906.

(^1)
The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; PR, progesterone receptor; RAR, retinoid acid receptor; RXR, retinoid X receptor; TR, thyroid hormone receptor; PPRE, peroxisome proliferator-responsive element; TREp, thyroid hormone-responsive element (palindromic); ETYA, 5,8,11,14-eicosatetraynoic acid; ATRA, all-trans-retinoic acid; CFA, clofibric acid; GR, glucocorticoid receptor.


ACKNOWLEDGEMENTS

We acknowledge the contribution of Dan Noonan in the cloning of hNUC1. We thank Donald McDonnell, Jon Rosen, and Jeff Miner for very helpful discussions and critically reading the manuscript, Rich Heyman and Dave Clemm for the hRXRalpha protein extract, and our colleagues in the Molecular Biology, Cell Biology, and the New Leads Discovery Department for their help. We thank Glaxo Research and Development Ltd. UK for helpful discussions and support.

Note Added in Proof-While this manuscript was under review, repression of mouse PPARalpha 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).


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