(Received for publication, October 1, 1996, and in revised form, November 26, 1996)
From the Departments of Cardiovascular Research and ¶ New Leads Discovery, Ligand Pharmaceuticals, Inc., San Diego, California 92121
We describe the cloning, characterization, and
tissue distribution of the two human peroxisome proliferator activated
receptor isoforms hPPAR2 and hPPAR
1. In cotransfection assays the
two isoforms were activated to approximately the same extent by known PPAR
activators. Human PPAR
binds to DNA as a heterodimer with the retinoid X receptor (RXR). This heterodimer was activated by both
RXR agonists and antagonists and the addition of PPAR
ligands with
retinoids resulted in greater than additive activation. Such
heterodimer-selective modulators may have a role in the treatment of
PPAR
/RXR-modulated diseases like diabetes. Northern blot analysis indicated the presence of PPAR
in skeletal muscle, and a sensitive RNase protection assay confirmed the presence of only PPAR
1 in muscle that was not solely due to fat contamination. However, both
PPAR
1 and PPAR
2 RNA were detected in fat, and the ratio of
PPAR
1 to PPAR
2 RNA varied in different individuals. The presence of tissue-specific distribution of isoforms and the variable ratio of
PPAR
1 to PPAR
2 raised the possibility that isoform expression may
be modulated in disease states like non-insulin-dependent diabetes mellitus. Interestingly, a third protected band was detected with fat RNA indicating the possible existence of a third human PPAR
isoform.
Peroxisome proliferator-activated receptors
(PPARs)1 are members of the intracellular
receptor superfamily. They play a role in lipid metabolism and
metabolic diseases. There are three PPAR subtypes with distinct tissue
distribution in Xenopus, mice, and humans: PPAR, PPAR
(also called NUC1 or PPAR
), and PPAR
(1-10). PPAR
expression
is observed in adipose tissue in rodents. Its expression is induced
early in differentiation of 3T3-L1 preadipocytes into adipocytes, and
its overexpression in fibroblasts induces them to differentiate into
adipocytes (11). Two isoforms of mPPAR
resulting from different
promoters and alternate splicing have been identified (7, 12, 13). A
human isoform, hPPAR
1, has been cloned from a human hematopoietic
cell line and placenta (14, 15), and another from human fat (16) has
been reported. Although a preliminary report on the distribution of
PPAR
in human tissues has been published (16), the distribution of
PPAR
1 versus PPAR
2 has not been reported.
Thiazolidinediones are high affinity ligands and potent activators for
PPAR. They decrease insulin resistance in insulin-responsive tissues
including skeletal muscle (the primary site of insulin-stimulated glucose uptake) in patients with non-insulin-dependent
diabetes mellitus (17). It is assumed that PPAR
is the therapeutic
target for these compounds; yet the presence of PPAR
has not been
conclusively demonstrated in human muscle. The identification of human
PPAR
isoforms and their tissue distribution will help in
understanding their role in metabolic diseases like
non-insulin-dependent diabetes mellitus and obesity.
We undertook to clone and characterize the tissue distribution of human
PPAR1 and PPAR
2 and compare it with that of human PPAR
and
PPAR
. We compared the ability of PPAR
agonists to activate the
two isoforms. A PPAR
antagonist would be a useful tool to dissect
PPAR
action and may also block adipocyte differentiation. Such a
ligand that competitively antagonizes PPAR
activity has not been
reported. An alternative approach would be to block PPAR
/RXR activation with an antagonist of RXR. Surprisingly, an RXR antagonist activated the PPAR
/RXR heterodimer as did an RXR agonist. Greater than additive activation was seen with PPAR
and RXR ligands.
5,8,11,14-Eicosatetraenoic acid and 2-bromopalmitate were
purchased from Sigma, and 15-deoxy-12,14-prostaglandin
J2 was obtained from Cayman Chemicals. BRL 49653, LG100268, and
LG100754 were synthesized at Ligand Pharmaceuticals Inc.
A human heart 5-stretch cDNA library (Clontech) was screened with
a mouse PPAR
(7) probe at low stringency (35% formamide, 5 × SSC, 0.1% SDS, 100 µg/ml fish sperm DNA at 37 °C). Several positive clones were isolated and sequenced. Comparison with the mPPAR
sequence indicated that one clone encoded the N terminus and
another the C terminus of hPPAR
, and their sequences overlapped by
485 base pairs. The complete hPPAR
-coding region was reconstructed by a triple ligation using pBKCMV (Stratagene) digested with
EcoRI and KpnI and utilizing the unique ScaI site
in the coding region. This plasmid was then digested with
NcoI, blunt-ended with Klenow enzyme, and redigested with
KpnI. The liberated fragment was subcloned into pBKCMV at
the XbaI site (blunted with Klenow enzyme) and the
KpnI site to give pCMVhPPAR
1.
A third positive clone was isolated and sequenced. This sequence
overlapped that of hPPAR1 by 1268 base pairs but had a unique N
terminus. The technique of crossover polymerase chain reaction was
utilized to create pCMVhPPAR
2. The sequences of the two external primers were 5
-TGAGTCAGCTCGAGATATCAGTGTGAATTACAGC-3
and
5
-GATCCTAGGCGGCCGCTCAGAATAGTGCAACTGG-3
. The internal primers were 5
CATTACGGAGAGAGATCCAC-3
and 5
-ATGGTTGACACAGAGATG-3
. The polymerase
chain reaction product was cloned into the SmaI site of
pBKCMV, and the orientation was determined by restriction mapping.
Human multiple tissue Northern blots were
purchased from Clontech. Hybridization was done according to the
manufacturer's protocol. The probe for hPPAR has been described
(6). pCMVhPPAR
(pCMVhNUC1) (18) was digested with EcoRI
and the 500-base pair fragment was isolated. pCMVhPPAR
1 was digested
with ScaI and KpnI and the 1-kilobase pair
fragment isolated. This probe will recognize hPPAR
1 and hPPAR
2
RNA. All probes were labeled by random priming.
Four human white fat and two skeletal muscle samples were obtained from the National Disease Research Interchange (NDRI, Philadelphia) or the University of California (San Diego) tissue bank. Total RNA was isolated using standard techniques. A sample of human skeletal muscle RNA was purchased from Clontech.
A partial cDNA containing nucleotides 1-252 of hPPAR2 (Fig. 1)
was subcloned into the pCRII vector (Invitrogen). This was linearized
with CelII and labeled antisense riboprobe made with the T7
RNA polymerase and Maxiscript in vitro transcription kit (Ambion). The adipocyte protein 2 (aP2, a kind gift from Dr. Bruce Spiegelman) cDNA was liberated with BamHI and subcloned
into pGEM 3Zf(
) (Promega). The DNA was linearized with
BclI and riboprobe made using SP6 RNA polymerase. RNase
protection assay was done with an Ambion direct protect lysate RNase
protection assay kit. Band intensities were quantitated by a
PhosphorImager (Molecular Dynamics).
Gel Mobility Shift Assays
Human hPPAR2 was translated
in vitro from pCMVhPPAR
2 using the TNT
Coupled Reticulocyte Lysate System (Promega). The baculovirus/Sf21 cell
system was used to express hRXR
(19). Gel retardations were
performed as described (6). The sequence of the oligonucleotides containing PPREs from three genes are
5
-CTAGCGATATCATGACCTTTGTCCTAGGCCTC-3
(acyl-coenzyme A oxidase) (20),
5
-GATCCCCTTTGACCTATTGAACTATTACCTACATTA-3
(bifunctional enzyme)
(21), and 5
-GATCCCCACTGAACCCTTGACCCCTGCCCTGCAGCA-3
(human apoA-1
"A" site) (22). The sequence of the upper strand is shown in all
cases.
Transfections in CV-1 cells were
performed as described (6, 23). The reporter plasmid pPPREA3-tk-Luc
containing three copies of the PPRE identified in the acyl CoA oxidase
(AOX) gene has been described (24). The -galactosidase
expression plasmid pCH110 was used to normalize difference in
transfection efficiencies. The normalized response is the luciferase
activity of the extract divided by the
-galactosidase activity of
the same. Compounds were dissolved in Me2SO (vehicle). Each
data point is the mean of triplicate transfections, and the error bars
represent the standard error of the mean. Each experiment was repeated
at least two times. A representative experiment is shown in each
case.
A human heart cDNA library was screened with a probe
corresponding to the mouse PPAR (7). Three overlapping clones were identified, purified, and sequenced. The nucleotide sequence is shown
in Fig. 1. The longest open reading frame starting from the nucleotide at position 91 coded for a polypeptide of 505 amino acids. There was an in-frame stop codon upstream of this methionine suggesting the translation initiation occurred from this codon. The
second and third methionine codons were at positions 29 and 31 in the
amino acid sequence. The first and third methionine codons in hPPAR
2
were in a context appropriate for translation initiation,
i.e. the Kozak sequence (25), and were conserved between
mice and man. The second methionine codon was unique to human PPAR
2,
and the corresponding amino acid in mice was isoleucine.
Amino acid sequence comparison indicated 97% identity overall between
hPPAR2 and mPPAR
2. The DNA binding domains were 83% conserved
between hPPAR
2 and hPPAR
or hPPAR
. Further, three amino acids
were present between the two cysteines in the D-box (amino acids
177-179), a characteristic feature of all PPARs known to date. Based
on these observations we believe this human isoform is hPPAR
2.
To determine if there are multiple translation start sites for
hPPAR2, as in mPPAR
2, coupled in vitro
transcription/translation reactions were performed in the presence of
[35S]methionine and pCMVhPPAR
2 as template. Two bands
were observed by PAGE (Fig. 2). The upper band (57 kDa)
corresponded to translation initiation from the methionine at position
1. The lower band (53 kDa) probably corresponded to translation
initiation from the methionine at position 31. We cannot rigorously
discount translation initiation from the methionine at position 29. However, since this methionine was not within a good Kozak sequence and
was also absent in mPPAR
2, we think it is unlikely. Indeed, in
vitro transcription/translation of pCMXmPPAR
1 (10) and
pCMVhPPAR
1 also gives rise to bands that comigrate with the lower
band observed with pCMVPPAR
2. Hence, in analogy with mPPAR
1, we
called the smaller polypeptide hPPAR
1.
PPARs bind to PPREs as heterodimers with RXRs (24). Mobility shift
assays using in vitro-translated hPPAR2 (Fig.
3) and recombinant baculovirus-expressed RXR
were
performed. PPAR
2 alone did not form a complex with oligonucleotides
containing PPRE sequences identified in the promoters from the
enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional
enzyme), acyl-CoA oxidase, and the A site of the apoA-1 gene
(lanes 1-3). Similarly, no complex was observed with RXR
and the PPRE-containing oligonucleotides (lanes 7-9).
However, with PPAR
2 and RXR
, retarded complexes are clearly
observed (lanes 4-6). Similar results were obtained with
hPPAR
1 (data not shown). We concluded that hPPAR
1 and hPPAR
2
bound to PPREs as heterodimers with RXR. Complexes were also observed
between hPPAR
1 or hPPAR
2 and RXR
and RXR
(data not
shown).
The transcriptional response of hPPAR2 to PPAR
activators was
determined in a cotransfection assay (Fig.
4A) and compared with hPPAR
1 (Fig.
4B). PPAR
1 and PPAR
2 are activated by BRL 49653 with
an EC50 of approximately 100 nM and by
15-deoxy-
12,14-prostaglandin J2 (an endogenous PPAR
ligand) (26, 27) with an EC50 around 3 µM.
They are also activated by 5,8,11,14-eicosatetraenoic acid and
2-bromopalmitate. The response of hPPAR
2 to these four activators is
very similar to that of hPPAR
1. We conclude that both hPPAR
1 and
hPPAR
2 are similarly activated by known PPAR
activators.
Since PPAR2 binds to PPREs as a heterodimer with RXR, we next
determined the transcriptional response of the PPAR
2/RXR heterodimer to an RXR ligand. LG100268 (28) is a highly selective RXR ligand (Kd ~3 nM). Both BRL 49653 and
LG100268 transcriptionally activated the PPAR
2/RXR heterodimer (Fig.
5A), and the transcriptional response
observed with both ligands was greater than that observed individually.
RXR agonists activated a reporter containing the hydratase
(bifunctional enzyme) PPRE. They also induced expression of the
hydratase gene in vivo, and increased induction is seen with
a combination of RXR and PPAR
agonists.2
Recently LG100754, another high affinity RXR ligand
(Kd ~12 nM), has been described
as an RXR/RXR homodimer antagonist on a CRBPII-tk-Luc reporter (29). To
determine if the response of PPAR2/RXR to BRL 49653 will be
antagonized by LG100754 binding to RXR, a cotransfection assay with
PPAR
2/RXR was performed (Fig. 5A). Surprisingly,
LG100754, like LG100268, is an agonist of hPPAR
2/RXR, and
activation by the combination of BRL 49653 and LG100754 is greater than
the individual compounds. It is also an agonist of hPPAR
1/RXR (data
not shown) and hPPAR
/RXR (30).
Since LG100754 is a high affinity RXR ligand and also activates the
hPPAR/RXR heterodimer, we determined whether LG100754 also activates
RXR homodimers using the same reporter used for the PPAR assays
(pPPREA3-tk-Luc). Since the consensus PPRE and RXR response element are
of the DR-1 type (24), it would be interesting to compare the effect of
RXR modulators on the two response elements. LG100268 strongly
activated the RXR/RXR homodimer on PPREA3-tk-Luc (Fig. 5B).
LG100754 was a very weak activator of the RXR homodimer. Interestingly,
it antagonized the activation of RXR/RXR by LG100268 (Fig.
5B). Hence, LG100754 acted as a PPAR
/RXR heterodimer
agonist but as an RXR homodimer antagonist on the same response
element, a PPRE. This was the first demonstration of an RXR ligand
having such dimer-selective effects on the same reporter. This
dimer-selective activity is probably not due to LG100754 binding with
high affinity to PPAR
since LG100754 displaces labeled BRL 49653 from PPAR
only at very high concentrations in a
DNA-dependent ligand binding assay using PPAR
/RXR
heterodimers (40) (data not shown).
We next determined the tissue distribution of human PPAR RNA (using
a probe common to PPAR
1 and PPAR
2) and compared it with that of
hPPAR
and hPPAR
by Northern blotting (Fig.
6A). Human PPAR
was found predominantly in
skeletal muscle, liver, heart, and kidney, a distribution similar to
that reported for mPPAR
. PPAR
RNA was more ubiquitously expressed
with maximal expression in placenta and skeletal muscle. One band
approximately 2 kilobases in length was observed with the PPAR
probe. Human PPAR
is expressed in the insulin-responsive tissues
(skeletal muscle, heart, and liver) and is consistent with the
distribution in mice (31).
Since the probe used in the Northern blot experiments could not
distinguish between PPAR1 and PPAR
2 RNA, we developed an RNase
protection assay to distinguish the two isoforms. The majority of
insulin-stimulated glucose uptake occurs in skeletal muscle, therefore,
we determined the expression of PPAR
2 versus PPAR
1 in
muscle. Since mPPAR
2 expression is restricted to fat (32), and the
commercial blot used in the Northern analysis did not have a sample of
fat RNA, we included human fat RNA in the study (Fig. 6B).
Two bands (78 and 252 nucleotides long) were observed in all adipose
tissue samples arising from protection of the probe by PPAR
1 and
PPAR
2 RNA, respectively, as shown. In contrast to the findings in
mice (31), PPAR
1 was expressed at higher levels in all human fat
samples studied. Quantitation of the band intensities indicated that
the ratio of PPAR
1 to -
2 varied in the human samples (from 2 (lane 5) to 10 (lane 7)).
With RNA from human skeletal muscle, we observed the protected fragment
due to hPPAR1 but not from PPAR
2 in all three samples. This was
not observed with yeast RNA, which was used as a negative control.
To test whether PPAR1 RNA observed in muscle was solely due to fat
contaminating the muscle samples, we performed RNase protection assays
with the muscle RNA and a mouse aP2 probe and compared that with a
sample of fat RNA. aP2 (adipocyte protein 2) gene expression is
fat-specific (33). Very little specific protection of the probe was
seen with 10 µg of muscle RNA (Fig. 6C, lanes 5-7) while an intense band is seen with only 2 µg of fat RNA
(lane 4). The autoradiogram was deliberately overexposed
(see lane 3) to reveal any protected bands in lanes
5-7. The smear observed in lanes 5-7 was probably due
to nonspecific hybridization between human RNA and the mouse probe and
is also seen with yeast RNA (lane 8). We concluded that
PPAR
was expressed in human skeletal muscle and PPAR
1 was the
predominant isoform in this tissue. In contrast, both PPAR
1 and
PPAR
2 were expressed in human fat and at much higher levels compared
with muscle.
Interestingly, a third protected fragment (170 nucleotides long) was
also observed (denoted by an asterisk) in all four fat samples but not in the muscle samples (Fig. 6B). This could
be simply due to RNase digestion in regions of imperfect hybridization. However, the ratio of the intensity of this fragment compared with
PPAR2 varied in the different fat samples, hence, it is unlikely
that this was due to breakdown of the larger protected fragment. We
therefore hypothesized a third isoform of PPAR
in humans that may
arise due to alternate splicing and promoter usage.
We have cloned the cDNA for a second isoform of the human
PPAR, hPPAR
2. Sequence comparison with mPPAR
2 revealed 97%
amino acid identity. Human PPAR
2 bound to PPREs as a heterodimer
with RXR and was activated by the PPAR
ligands BRL 49653 and
15-deoxy-
12,14-prostaglandin J2. Based on these
observations we believe that hPPAR
2 was a genuine member of the PPAR
subfamily. The amino acid sequence shown in Fig. 1 was identical to the
hPPAR
2 amino acid sequence predicted from the cDNA isolated from
a human adipose library (16). However, our clone contained an
additional 90 nucleotides of 5
-untranslated sequence including the
upstream in-frame translation stop codon.
PPAR2, like PPAR
1, bound to PPREs as a heterodimer with RXRs as
do all the PPARs known to date. Human PPAR
1 and hPPAR
2 have
similar activation profiles in reponse to BRL 49653 and
15-deoxy-
12,14-prostaglandin J2. Interestingly, there
was only 63% identity in the N-terminal 30 amino acids between human
and mouse PPAR
2, far less than in the rest of the polypeptide
(98%). This suggests that the N terminus coded by a different exon
(13) has diverged more rapidly than the rest of the protein during
evolution. The function of these amino acids is unclear.
The hPPAR2/RXR and hPPAR
1/RXR heterodimers were activated by the
RXR modulators LG100268 and LG100754. They increased the transcriptional response seen with the PPAR
agonist BRL 49653. This
is consistent with our previous studies showing that RXR modulators
increase the responsiveness of the PPAR
/RXR heterodimer (6, 24).
LG100754 was interesting because it is an agonist of PPAR
/RXR but an
RXR/RXR antagonist. Binding of LG100754 to RXR may lead to distinct
conformational changes of the receptor dimer such that PPAR
/RXR is
read as an activator by the transcriptional machinery, but the RXR/RXR
homodimer is transcriptionally silent. Such compounds like LG100268,
LG100754, and BRL 49653 may therefore modulate distinct but overlapping
sets of target genes and might have a role in the treatment of
PPAR/RXR-modulated diseases like diabetes.
The tissue distribution of hPPAR is important for the therapeutic
activity of drugs targeting the PPAR
/RXR heterodimer. Thiazolidinediones act as insulin sensitizers in skeletal muscle and
are high affinity PPAR
ligands (34). Structure activity relationship
indicates a good correlation between in vivo potency and
in vitro activity of thiazolidinediones (35), implicating PPAR
as the therapeutic target for these compounds. However, earlier
data indicated PPAR
is expressed at high levels, specifically in
adipose tissue in rodents (11, 32), and is essentially undetectable in
muscle where approximately 80% of insulin-stimulated glucose uptake
occurs (36). It was not clear how PPAR
expressed almost exclusively
in adipose tissue could have the effect of insulin sensitization in
skeletal muscle and raised the possibility that insulin sensitization
by thiazolidinediones in skeletal muscle was not mediated by
PPAR
-dependent mechanisms.
Our data demonstrated that PPAR was expressed in human skeletal
muscle, fat, and heart, tissues where the majority of
insulin-stimulated glucose uptake occurs. Further, while both PPAR
1
and PPAR
2 are expressed in human fat, the dominant isoform in human
muscle is PPAR
1. These findings are consistent with recently
published data in mice (31) and suggest that PPAR
1 might be the
relevant target for thiazolidinediones in human skeletal muscle. The
close conservation of sequence, subtype, and tissue distribution of PPAR
between mice and humans is consistent with the observation that
thiazolidinediones act as insulin sensitizers in both species (17, 37).
However, we do not yet know the distribution of PPAR
1
versus PPAR
2 protein in these tissues.
The ratio of the intensities of the PPAR1 and -
2 isoforms varied
in the four individual fat samples, hinting that isoform expression may
be modulated. Further, only PPAR
1 was detected in muscle, not
PPAR
2, pointing to differential expression of PPAR
isoforms in
tissues. Therefore, an analysis of PPAR
isoform distribution in
skeletal muscle and fat in normal, obese, and diabetic individuals
might yield valuable information and is currently underway.
We used a commercially available Northern blot to determine PPAR
distribution. Although we did not observe hybridization to placenta and
lung RNA on this blot we note that PPAR
expression was observed in
these tissues (15). We cannot explain this other than as a variation
between individuals. For an accurate determination it was important to
assay expression levels in several individuals as was done in our
RNase-protection assays.
Thiazolidinediones appear to act as insulin sensitizers in
vivo through activation of PPAR/RXR. Our data indicate the
presence of PPAR
in insulin-responsive tissues in humans. One may
speculate that thiazolidinediones bind to and activate PPAR
altering
the expression of key genes in target tissues rendering them more responsive to insulin, increasing glucose uptake, lowering hepatic glucose output, and lowering hyperglycemia. Since RXR modulators are
also able to activate the PPAR
/RXR heterodimer, they could activate
a set of thiazolidinedione-responsive genes and may therefore either
alone or in combination with thiazolidinediones have utility in the
treatment of non-insulin-dependent diabetes mellitus.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U79012[GenBank].
We thank Rich Heyman and Patricia Hoener for critical comments on the manuscript, Lawrence Hamann for synthesizing BRL 49653, Dr. Jerrold Olefsky for providing a human skeletal muscle biopsy sample, Regis Saladin for isolating human fat RNA, Johan Auwerx for useful suggestions, Kay Klausing for the DNA-dependent ligand displacement assay, and Sharon Dana, Thuan Le, Hung Lam, and members of the Departments of Cardiovascular Research, Retinoid Research, and New Leads Discovery for help.