(Received for publication, January 27, 1997, and in revised form, May 2, 1997)
From the INSERM U325, Département
d'Athérosclérose, Institut Pasteur, F-59019 Lille, France,
the § INSERM U449, Faculté de Médecine
René Laennec, Université Claude Bernard, F-69373 Lyon,
France, the ¶ Ligand Pharmaceuticals Inc., San Diego, California
92121, the
Department of Medicine and Genetics, University of
Washington, Seattle, Washington 98195, and the ** Department of
Medicine, Beth Israel Hospital, Harvard Medical School, Boston,
Massachusetts 02215
PPAR is a member of the PPAR subfamily of
nuclear receptors. In this work, the structure of the human PPAR
cDNA and gene was determined, and its promoters and tissue-specific
expression were functionally characterized. Similar to the mouse, two
PPAR isoforms, PPAR
1 and PPAR
2, were detected in man. The
relative expression of human PPAR
was studied by a newly developed
and sensitive reverse transcriptase-competitive polymerase chain
reaction method, which allowed us to distinguish between PPAR
1 and
2 mRNA. In all tissues analyzed, PPAR
2 was much less abundant
than PPAR
1. Adipose tissue and large intestine have the highest
levels of PPAR
mRNA; kidney, liver, and small intestine have
intermediate levels; whereas PPAR
is barely detectable in muscle.
This high level expression of PPAR
in colon warrants further study
in view of the well established role of fatty acid and arachidonic acid derivatives in colonic disease. Similarly as mouse PPAR
s, the human
PPAR
s are activated by thiazolidinediones and prostaglandin J and
bind with high affinity to a PPRE. The human PPAR
gene has nine
exons and extends over more than 100 kilobases of genomic DNA.
Alternate transcription start sites and alternate splicing generate the
PPAR
1 and PPAR
2 mRNAs, which differ at their 5
-ends. PPAR
1 is encoded by eight exons, and PPAR
2 is encoded by seven exons. The 5
-untranslated sequence of PPAR
1 is comprised of exons
A1 and A2, whereas that of PPAR
2 plus the additional
PPAR
2-specific N-terminal amino acids are encoded by exon B, located
between exons A2 and A1. The remaining six exons, termed 1 to 6, are
common to the PPAR
1 and
2. Knowledge of the gene structure will
allow screening for PPAR
mutations in humans with metabolic
disorders, whereas knowledge of its expression pattern and factors
regulating its expression could be of major importance in understanding
its biology.
White adipose tissue is composed of adipocytes, which play a central role in lipid homeostasis and the maintenance of energy balance in vertebrates. These cells store energy in the form of triglycerides during periods of nutritional affluence and release it in the form of free fatty acids at times of nutritional deprivation. Excess of white adipose tissue leads to obesity (1-3), whereas its absence is associated with lipodystrophic syndromes (4). In contrast to the development of brown adipose tissue, which mainly takes place before birth, the development of white adipose tissue is the result of a continuous differentiation/development process throughout life (2, 5). During development, cells that are pluripotent become increasingly restricted to specific differentiation pathways. Adipocyte differentiation results from coordinate changes in the expression of several proteins, which are mostly involved in lipid storage and metabolism, that give rise to the characteristic adipocyte phenotype. The changes in expression of these specialized proteins are mainly the result of alterations in the transcription rates of their genes.
Several transcription factors including the nuclear receptor
PPAR (6, 7), the family of CCAATT enhancer binding proteins (C/EBP)1 (8-13) and the basic
helix-loop-helix leucine zipper transcription factor ADD1/SREBP1 (14,
15) orchestrate the adipocyte differentiation process (for reviews, see
Refs. 1, 3, 16-18). In contrast to the wide tissue distribution of the
various C/EBPs, PPAR
has been shown to have an adipose-restricted
pattern of expression in mouse. The currently favored hypothesis is
that C/EBP
and
induce the expression of PPAR
(11), which then
triggers the adipogenic program. Terminal differentiation then requires
the concerted action of both PPAR
, C/EBP
, and ADD-1/SREBP1 (7, 15). Several arguments support the important role of PPAR
in adipocyte differentiation. First, overexpression of PPAR
by itself can induce adipocyte conversion of fibroblasts (6). In addition, PPAR
together with C/EBP
can induce transdifferentiation of myoblasts into adipocytes (19). Second, the description of functional PPREs in the regulatory sequences of several of the genes that are
induced during adipocyte differentiation, such as the genes coding for
adipocyte fatty acid binding protein, aP2 (6), phosphoenolpyruvate carboxykinase (PEPCK) (20), acyl-CoA synthetase (ACS) (21, 22), and
lipoprotein lipase (LPL) (23), is consistent with the crucial role
attributed to PPAR
in adipocyte differentiation. Finally, PPAR
activators, such as fibrates (24, 25) and fatty acids (7, 26-28), or
synthetic PPAR
ligands, such as the thiazolidinediones (7, 28, 29),
induce adipocyte differentiation. In this context, it is interesting to
note that prostanoids, which are potent inducers of adipose
differentiation programs (30-32), may be one of the natural ligands of
PPAR
. In addition to PPAR
, PPAR
, but not PPAR
, has been
shown to have some, albeit weaker, adipogenic activity (33).
To better understand the physiological role of PPAR in human
physiology, it is crucial that we gain insight into the regulation of
PPAR
gene expression in man. Therefore, we cloned the human PPAR
cDNAs, determined the structure of the human PPAR
gene, and
studied the expression of the PPAR
mRNAs and the regulation of
their promoter. Both PPAR
1 and 2 are produced in human tissues but
PPAR
2 appears to be the minor isoform in man. In addition to adipose
tissue, which contains high levels of PPAR
, we demonstrate high
level expression of human PPAR
in the colon. The structure of the
gene encoding the mouse and human PPAR
s is highly conserved. Furthermore our results demonstrate that 3 and 1 kb of DNA upstream of
the transcription start sites of PPAR
1 and
2, respectively, are
sufficient to control basal and tissue-specific PPAR
gene expression.
Materials and Oligonucleotides
The oligonucleotides used for various experiments in this manuscript are listed in Table I.
|
Isolation of the Human PPAR cDNA and Gene, Restriction
Mapping, Determination of Intron/Exon Boundaries, and DNA
Sequencing
A human adipose tissue gt11 library was screened with a
random primed 32P-labeled 200 bp fragment, covering the
DNA-binding domain of the mouse PPAR
cDNA. After hybridization,
filters were washed in 2 × SSC, 0.1% SDS for 10 min at 20 °C
and twice for 30 min in 1 × SSC, 0.1% SDS at 50 °C and
subsequently exposed to x-ray film (X-OMAT-AR, Kodak). Of several
positive clones, one clone 407 was characterized in detail. The insert
of this clone, starting ±90 bp upstream of the ATG start codon and
extending downstream into the 3
-untranslated region (UTR) sequence,
was subcloned in the EcoRI site of pBluescript
SK
to generate clone 407.2. Sequence analysis of 407.2 confirmed it as being the human homologue of the mouse PPAR
2
cDNA. While this work was in progress, other groups also reported
the isolation of human PPAR
2 cDNA clones (34, 35).
To isolate genomic P1-derived artificial chromosome (PAC) clones
containing the entire human PPAR gene, the primer pair LF-3 and
LF-14 was used to amplify an 86-bp probe with human genomic DNA as
template. This fragment was then used to screen a PAC human genomic
library from human foreskin fibroblasts. Three positive clones, P-8854,
P-8855, and P-8856, were isolated. Restriction digestion and Southern
blotting were performed according to classical protocols as described
by Sambrook et al. (36). Sequencing reactions were
performed, according to the manufacturer instructions, using the T7
sequencing kit (Pharmacia Biotech Inc.).
Determination of the Transcription Initiation Site: Primer
Extension and 5-Rapid Amplification of cDNA Ends (5
-RACE)
The oligonucleotide LF-35 was
32P-labeled with T4-polynucleotide kinase (Amersham Life
Science, Inc) to a specific activity of 107 dpm/50 ng and
purified by gel electrophoresis. For primer extension, 105
dpm of oligonucleotide was added in a final volume of 100 µl to 50 µg of adipose tissue total RNA isolated from different patients. Primer extension analysis was performed following standard protocols utilizing a mixture of 1.25 units of avian mycloblastosis virus reverse
transcriptase (Life Technologies, Inc.) and 100 units of Moloney murine
leukemia virus reverse transcriptase (Life Technologies, Inc.). A
sequencing reaction and molecular mass standards were used to map the
5-end of the extension products.
The Marathon cDNA amplification kit (CLONTECH) was used to obtain a library of adaptor-ligated double-stranded cDNA from human adipose tissue. 1 µg of poly(A)+ RNA was used as a template for the first strand synthesis, with the 52-mer CDS primer and 100 units of the MMLV reverse transcriptase in a total volume of 10 µl. Synthesis was carried out at 42 °C for 1 h. Next, the second strand was synthetized at 16 °C for 90 min in a total volume of 80 µl containing the enzyme mixture (RNase H, Escherichia coli DNA polymerase I, and E. coli DNA ligase), the second strand buffer, the dNTP mix, and the first strand reaction. cDNA ends were then made blunt by adding to the reaction 10 units of T4 DNA polymerase and incubating at 16 °C for 45 min. The double-stranded cDNA was phenol/chloroform extracted, ethanol precipitated, and resuspended in 10 µl of water. Half of this volume was used to ligate the adaptor to the cDNA ends (adaptor sequence CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT) in a total volume of 10 µl using 1 unit of T4 DNA ligase. The ligation reaction was incubated 16 h at 16 °C. The resulting cDNA library was diluted to a final concentration of 0.1 mg/ml.
The 5-end of PPAR
1 was PCR-amplified using 5 µl of the library as
a template with the oligonucleotides AP-1 (binding to the adaptor) and
LF-45 (binding antisense to the 5
-end of the PPAR
1). After an
initial denaturing step at 95 °C for 3 min, 25 cycles were done at
the following conditions: 10 s at 95 °C, 20 s at 60 °C,
and 30 s at 72 °C. The resulting PCR product was reamplified
for 30 additional cycles at the same conditions using the nested
oligonucleotides AP2 (nested to AP1) and LF-2 (nested to LF-45). The
PCR product was analyzed on a 2% agarose gel, treated with
Pfu polymerase (Stratagene) and cloned into the
EcoRV site of pBluescript SK+. A total of 20 white colonies were grown and sequenced from both ends using the
oligonucleotides T3 and T7 (Dye Terminator Cycle sequencing kit,
Applied Biosystems).
For the determination of the 5-end of PPAR
2, the same procedure was
followed except that the oligonucleotide LF-14 (specific for the
PPAR
2 5
-UTR) was used in the first round PCR, and the oligonucleotide LF-35 (nested to LF-14) was used in the second round
PCR with the same cycling conditions.
Tissue Biopsies and Cell Culture
Omental adipose tissue, small and large intestine, kidney, muscle, and liver biopsies were obtained from non-obese adult subjects undergoing elective surgery or endoscopy. All subjects had fasted overnight before surgery (between 8.00 p.m. and 10 a.m.) and received intravenous saline infusion. They had given informed consent, and the project was approved by the ethics committee of the University of Lille. All tissue was immediately frozen in liquid nitrogen until RNA preparation.
Standard cell culture conditions were used to maintain 3T3-L1 (obtained from ATCC), CV-1 (a kind gift from Dr. R. Evans, Salk Institute, La Jolla, CA), and Hep G2 cells (ATCC). BRL-49,653, supplied by Ligand Pharmaceuticals, San Diego, CA (in DMSO) and fatty acids (in ethanol) were added to the medium at the concentrations and times indicated. Control cells received vehicle only. Fatty acids were complexed to serum albumin contained in delipidated and charcoal-treated fetal calf serum by preincubation for 45 min at 37 °C.
mRNA Analysis by RT-Competitive PCR Assay
RNA preparation of total cellular RNA was performed as described
previously (37). The absolute mRNA concentration of the differentially spliced PPAR variants was measured by reverse transcription reaction followed by competitive polymerase chain reaction (RT-competitive PCR) in the presence of known amounts of
competitor DNA yielding amplicons of different size allowing the
separation and the quantification of the PCR products. The competitor
was constructed by deletion of a 74-bp fragment (nucleotides +433 to
+507 by HindIII digestion) of PPAR
1 cloned into
pBluescript KS+, yielding pBSCompPPAR
. Working solution
of the competitor was prepared by in vitro transcription
followed by serial dilution in 10 mM Tris-HCl (pH 8.3), 1 mM EDTA buffer. For RT-competitive PCR, the antisense
primer hybridized to the 3
-end of exon 3 (
AS:5
-GCATTATGAGCATCCCCAC-3
, nt +600 to +620) and the sense primer
to exon 1 (
S:5
-TCTCTCCGTAATGGAAGACC-3
, nt +146 to +165) or to the
B exon (
2S:5
-GCGATTCCTTCACTGATAC-3
, nt +41 to +59). Therefore, the
same competitor served to measure either total PPAR
mRNAs (
1 +
2; with primers
AS and
S) or, specifically, PPAR
2
mRNA (with primers
AS and
2S). The
AS/
S primer pair
gave PCR products of 474 and 400 bp for the PPAR
mRNAs and
competitor, respectively. The primer pair
AS/
2S gave 580 bp for
PPAR
2 mRNA and 506 bp for the competitor. For analysis of the
PCR products, the sense primers
S and
2S were 5
-end labeled with
the fluorescent dye Cy-5 (Eurogentec, Belgium).
First-strand cDNA synthesis was performed from total RNA (0.1 µg)
in the presence of the antisense primer AS (15 pmol) and of
thermostable reverse transcriptase (2.5 units; Tth DNA polymerase, Promega) as described (38). After the reaction, half of the RT volume
was added to the PCR mix (90 µl) containing the primer pair
AS/
S for the assay of PPAR
total mRNA, whereas the other half was added to a PCR mix (10 mM Tris-HCl, pH 8.3, 100 mM KCl, 0.75 M EGTA, 5% glycerol, 0.2 mM dNTP, 5 units of Taq polymerase) containing
the primer pair
AS/
2S for the assay of PPAR
2 mRNA. Four
aliquots (20 µl) of the mixture were then transferred to microtubes
containing a different, but known, amount of competitor. After 120 s at 95 °C, the samples were subjected to 40 PCR cycles (40 s at
95 °C, 50 s at 55 °C, and 50 s at 72 °C). The
fluorescent-labeled PCR products were analyzed by 4% denaturing
polyacrylamide gel electrophoresis using an automated laser
fluorescence DNA sequencer (ALFexpress, Pharmacia, Uppsala, Sweden),
and integration of the area under the curve using the Fragment manager
software (Pharmacia) was performed as described (38).
To validate this technique, human PPAR2 mRNA was synthesized by
in vitro transcription from the expression vector
pSG5hPPAR
(Riboprobe system, Promega) and quantified by competitive
PCR over a wide range of concentrations (0.25-25 attomole (amol) added in the RT reaction). Standard curves obtained when assaying PPAR
total mRNA or PPAR
2 mRNA are shown in Fig. 2C.
The linearity (r = 0.99) and the slopes of the standard
curves (0.98 and 1.11) indicated that the RT-competitive PCR is
quantitative and that all the mRNA molecules are copied into
cDNA during the RT step. The lower limit of the assay was about
0.05 amol of mRNA in the RT reaction, and the interassay variation
of the RT-competitive PCR was 7% with six separated determinations of
the same amount of PPAR
mRNA.
RT-competitive PCR method to measure PPAR
mRNA levels. A, scheme highlighting the features of the
vector pSG5hPPAR
2 and the vector derived from it, pBSCompPPAR
,
which served to synthesize competitor DNA. The primer pairs used in the
RT and PCR reactions as well as the different-sized amplicons obtained are indicated.
B, typical analysis of the fluorescence-labeled PCR products
on an automated fluorescence DNA sequencer using a denaturing 4%
polyacrylamide gel electrophoresis. C, validation of the
RT-competitive PCR assay and standard curves obtained when assaying
PPAR
total mRNA or PPAR
2 mRNA. The linearity
(r = 0.99) and the slopes of the standard curves (0.98 and 1.11) indicated that the RT-competitive PCR is really quantitative
and that all the mRNA molecules are copied into cDNA during the
RT step.
Western Blot Analysis of PPAR
Cells and tissues were homogenized in a lysis buffer of PBS containing 1% Triton X-100 (Sigma). Tissues were homogenized in extraction buffer containing PBS and 1% Nonidet P-40 (Sigma), 0.5% sodium deoxycholate (Sigma), 0.1% SDS (Sigma). Fresh mixture protease inhibitor (ICN) was added (100 mg/ml AEBSF, 5 mg/ml EDTA, 1 mg/ml leupeptin, 1 mg/ml pepstatin). Protein extracts were obtained by centrifugation of the lysate at 4 °C, and concentration was measured with the Bio-Rad DC Protein colorimetric assay system.
Protein (100 µg) was separated by SDS-PAGE, transferred to
nitrocellulose membrane (Amersham Life Science, Inc.), and blocked overnight in blocking buffer (20 mM Tris, 100 mM NaCl, 1% Tween-20, 10% skim milk). Filters were first
incubated for 4 h at room temperature with rabbit IgG
anti-mPPAR (10 mg/ml), raised against an N-terminal PPAR
peptide
(amino acids 20-104), and next developed for 1 h at room
temperature with a goat anti-rabbit IgG (whole molecule) peroxidase
conjugate (Sigma) diluted at 1/500. The complex was visualized with
4-chloro-1-naphtol as reagent.
Analysis of Promoter Activity
To test the activity of the human PPAR promoters several
reporter constructs were made. A 1-kb fragment of PAC clone 8856 was
isolated by PCR using the oligonucleotides LF-35 (binding antisense in
the PPAR
2 5
-UTR) and the oligonucleotide LF-58 (binding sense at
position -1000 of the PPAR
2), was sequenced, and was inserted into
EcoRV site of pBluescript (Stratagene, La Jolla, CA). After
digestion of plasmid pBS
2p1000 with SmaI and KpnI, the insert was cloned into the reporter vector pGL3
(Promega), creating the expression vector pGL3
2p1000. To isolate the
PPAR
1 promoter, an 8-kb EcoRI fragment, which hybridized
with the oligonucleotide LF-2 (corresponding to the 5
-UTR of
1),
was cloned into pBluescript. Partial mapping and sequencing of this
clone revealed the presence of a 3-kb fragment upstream of the
transcription initiation site. To test for promoter activity, a
SacI/XhoI digestion of this clone containing the
3-kb promoter was inserted in the same sites of pGL3, resulting in the
final vector pGL3
1p3000. The pSG5-haPPAR
(39) and pMSV-C/EBP
(10) expression vectors were described elsewhere. Transfections were
carried out in 60-mm plates using standard calcium phosphate
precipitation techniques (for 3T3-L1, CV-1, and COS cells) (22).
Luciferase and
-galactosidase assays were carried out exactly as
described previously (22).
Electrophoretic Mobility Shift Assays (EMSA) and Oligonucleotide Sequences
haPPAR (39), hPPAR
2, and mRXR
(40) proteins were
synthesized in vitro in rabbit reticulocyte lysate
(Promega). Molecular weights and quality of the in vitro
translated proteins were verified by SDS-PAGE. PPAR (2 µl) and/or RXR
(2 µl) were incubated for 15 min on ice in a total volume of 20 µl
with 1-ng probe, 2.5 µg of poly(dI-dC) and 1 µg of herring sperm
DNA in binding buffer (10 mM Tris-HCl pH 7.9, 40 mM KCl, 10% glycerol, 0.05% Nonidet P-40, and 1 mM dithiothreitol). For competition experiments, increasing amounts (from 10- to 200-fold molar excess) of cold
oligonucleotide (AII-J-PPRE, 5
-GATCCTTCAACCTTTACCCTGGTAGA-3
(41);
acyl-CoA oxidase (ACO)-PPRE, 5
-GATCCCGAACGTGACCTTTGTCCTGGTCCC-3
(42); or LPL-PPRE, 5
-GATCCGTCTGCCCTTTCCCCCTCTTCA-3
) (23) were
included just before adding T4-PNK end-labeled AII-J-PPRE
oligonucleotide. DNA-protein complexes were separated by
electrophoresis on a 4% polyacrylamide gel in 0.25 × TBE buffer
at 4 °C (43).
A cDNA probe
containing a 200-bp (KpnI-BglII) fragment
encoding the DNA binding domain of the mouse PPAR (44) was used to
screen a human adipose tissue cDNA library. Several independent human PPAR
cDNA clones, representing both the PPAR
1 and
PPAR
2 subtypes, were isolated and sequenced (Fig.
1A). The human PPAR
protein shows a 99%
similarity and a 95% identity on the amino acid level with mouse
PPAR
(Fig. 1A). Interestingly, the initiation codon for
human PPAR
1 is different from the mouse PPAR
1 (Fig. 1B). Therefore, human PPAR
1 is 2 amino acid residues
longer than its mouse homologue.
Expression of PPAR
To analyze the
expression pattern of the two PPAR isoforms, we developed a
sensitive RT-competitive PCR assay in which relative amounts of
PPAR
1 and
2 mRNA could be measured from minute quantities of
RNA (0.1 µg total RNA). This method relies on the co-amplification in
the same tube of known amounts of competitor DNA (Fig.
2A) with PPAR
cDNA, obtained after
reverse transcription from total tissue RNA. The competitor and the
target use the same fluorescently labeled PCR primers but yield
amplicons with a different size (Fig. 2, A and
B), allowing their separation and quantification on an
automated sequencing gel at the end of the reaction (Fig. 2C). All tissue preparations were carefully dissected, and
the RNA was shown to be free of contamination with adipose tissue as
evidenced by the absence of human leptin mRNA by RT-competitive PCR
assay (38) (data not shown). PPAR
1 mRNA was the predominant PPAR
isoform in all human tissues analyzed (Fig. 3).
PPAR
2 was detected in both liver and adipose tissue where it
accounted for 15% of all PPAR
mRNA. Interestingly, in addition
to the high level of expression of PPAR
mRNA expected in adipose
tissue, we found a very high level of PPAR
1 in large intestine. In
contrast to adipose tissue, large intestine contained no PPAR
2
mRNA. Kidney, liver, and small intestine contained intermediate
levels of PPAR
mRNA, whereas PPAR
mRNA was barely
detectable in skeletal muscle (Fig. 3).
Next, the expression of the human PPAR protein was analyzed in human
adipose tissue. A PPAR
specific antibody, raised against a peptide
corresponding to amino acids 20-104 of mPPAR
, was used. This
antibody is highly specific for PPAR
and does not cross-react with
PPAR
and
in Western blot experiments (Fig. 4,
A and B). Using this antibody in a Western blot
of protein extracts from human adipose tissue, we detected a band
(potentially representing a doublet) with an approximate molecular mass
of 60 kDa, consistent with the predicted mass of PPAR
1 and 2 and
with the protein product generated by in vitro
transcription/translation in the presence of
[35S]methionine (Fig. 4, B and
C).
PPAR
To analyze
whether PPAR could bind to a PPRE, classically composed of direct
repeats spaced by one intervening nucleotide (DR-1), EMSA was performed
using in vitro transcribed/translated PPAR
2 protein. An
oligonucleotide containing a high affinity PPRE, previously identified
in the apoA-II promoter J site, was used in EMSA (29). This
oligonucleotide was capable of binding both human and hamster
PPAR
/mRXR
heterodimers in EMSA (Fig. 5,
lanes 5 and 6). Homodimers of either hPPAR
or
mRXR
, however, were incapable of binding to this oligonucleotide.
When increasing concentrations of unlabeled apoA-II J site were added
as competitor, binding of the hPPAR
/mRXR
heterodimer to the
labeled PPRE was almost completely inhibited (Fig. 5, lanes
7-9). In addition, oligonucleotides corresponding to the PPRE
elements of the ACO or LPL genes competed, albeit less efficiently
(Fig. 5, compare lanes 7 with 10 and
13).
We next verified that the human PPAR2 cDNA was capable of
activating gene transcription through a PPRE. Therefore, 3T3-L1 preadipocytes were cotransfected with the PPAR
2 expression vector pSG5hPPAR
2 and a PPRE-driven luciferase reporter gene. The
luciferase gene was under the control of a multimerized ACO-PPRE site
and the TK promoter (Fig. 6). hPPAR
2 was capable of
activating this PPRE-based reporter 2-fold, an effect which was
substantially enhanced when hPPAR
2 was cotransfected
together with RXR
. Upon the addition of the PPAR
ligand
BRL-14653, luciferase expression was increased 6-fold when the
transfection was done with hPPAR
2 alone or at least 10-fold when the
cells were co-transfected with both hPPAR
2 and mRXR
. Similar
results were obtained when prostaglandin J2 was used as a PPAR
ligand (data not shown).
Characterization of the Transcription Initiation Site of the Human PPAR
To unambiguously identify the 5-end of the
cDNA, several approaches were undertaken. First, primer extension
experiments were performed, utilizing different human adipose tissue
RNA samples, and results were independently confirmed by using 5
-RACE.
Several primer extension products were seen for the PPAR
1 mRNA
using primer LF2 (Fig. 7, A and
C). The relative positions of the transcription initiation
sites as determined by the 5
-RACE were in agreement with the results
for primer extension.
One major extension product of 62 bp was observed consistently with
the primer LF-35 for PPAR2. A second extension product of 96 bp was
found using the same primer (Fig. 7, B and C).
The results of 5
-RACE were consistent with the primer extension (Fig. 7B). The transcription initiation sites identified
correlated well with the transcription initiation sites observed for
the mouse PPAR
2 mRNA (45). A striking feature of the human
PPAR
2 5
-UTR is its high degree of sequence conservation with the
mouse 5
-UTR (see Fig. 9). It awaits further study to determine the exact implications of this conservation.
Structural Organization of the Human PPAR
To clone
the human PPAR gene and to determine its promoter sequence, we
screened a PAC human genomic library derived from human foreskin
fibroblasts. Three positive clones (P-8854, P-8855, and P-8856), each
spanning >100 kb of genomic sequence, were isolated. All three clones
were next shown to hybridize with the oligos LF-14 (corresponding to
exon B) and LF-36 (exon 6), which indicates that they span most of the
PPAR
coding region. More importantly, clone P-8856 also hybridized
to oligo LF-2 and, hence, contains the transcription initiation site
for PPAR
1 and 2. This clone was further characterized by Southern
blotting and partial sequence analysis, which allowed the construction
of a physical map of the human PPAR
locus (Fig. 8).
The human PPAR
gene spans more than 100 kb. The PPAR
1 and
PPAR
2 mRNAs are encoded by 8 and 7 exons, respectively. The
5
-untranslated region of the PPAR
1 mRNA is encoded by two
exons, which we, in analogy to the nomenclature used for the mouse
gene, named exon A1 and A2. The coding region of PPAR
1 is contained
in the next six exons (exons 1 to 6). Exons 1 to 6 also encode the
majority of PPAR
2 mRNA. The additional 28 amino acids of
PPAR
2 as well as the 5
-UTR are encoded by the B exon, which is
located between exons A2 and A1.
The length of the introns was determined by long-range PCR
(CLONTECH Tth polymerase mix) using the
oligonucleotide pairs LF-3/LF-18, LF-20/LF-21, LF-22/LF-23,
LF-24/LF-25, LF-26/LF-27, and LF-28/LF-29 and the PAC clone P-8856 as a
template. The intron-exon boundaries were sequenced using genomic DNA
as template. The 5 donor and 3
acceptor splice sites were found to be
conforming to the consensus splice donor and acceptor sequences
(Table II). The DNA binding domain of the receptors is
encoded by exons 2 and 3, each encoding a separate zinc finger. The
entire ligand binding domain is encoded by exons 5 and 6, which are
separated by 16.3 kb of intron sequence.
|
We next subcloned the region 5 to the transcription
initiation sites of PPAR
1 and
2 and sequenced the proximal
promoters (Figs. 7 and 9). No canonical TATA box was found in the
PPAR
1 promoter region close to the transcription initiation site
(Fig. 7). The sequence immediately upstream of the transcription
initation site is extremely GC-rich, including several consensus Sp1
binding sites. Also a CCAAC box was found in the proximal promoter.
Whether any of these factors are important for the regulation of
PPAR
1 gene expression awaits further study. The PPAR
2 promoter
contains a TATA-like element at position
68, relative to the
transcription initiation site. Furthermore, sequence analysis
identified a potential CAAT-like consensus C/EBP protein binding site
at
56 (CCAATT) and a perfect AP-1 site at +10 (TGACTCA) (Figs. 7 and
9).
In experiments to evaluate the tissue specificity of these promoters,
DNA fragment extending from about 3 kb to +110 bp and
1 kb to +122
bp relative to the transcription initiation sites of PPAR
1 and
PPAR
2 were inserted into the pGL3-basic luciferase vector (Promega)
to generate the constructs pGL3-
1p3000 and pGL3-
2p1000 (Fig.
10). These vectors were then transfected into mouse
3T3-L1 and Hep G2 cells. Transfection efficiency of the various cell lines was monitored by evaluation of the activity of control vectors. Relative to the promoterless parent vector, the human PPAR
1 promoter fragment stimulated luciferase expression up to 3.5-fold in 3T3-L1 cells, maintained under non-differentiating conditions. In Hep G2
cells, luciferase expression was 9-fold higher with the pGL3-
1p3000 vector relative to the pGL3-basic vector (Fig. 10). Similar results were obtained with COS cells (data not shown). The expression of the
pGL3-
2p1000 construct containing the PPAR
2 promoter was not
different from the pGL3-basic promoterless vector in Hep G2 cells. In
undifferentiated 3T3-L1 cells, the PPAR
2 promoter induced luciferase
expression 2-fold relative to the promoterless control.
Two important findings recently underlined the importance of the
PPAR transcription factor. First, PPAR
has been identified as one
of the key factors controlling adipocyte differentiation and function
in rodent systems (6, 7). Second, the recent identification of
prostaglandin J2 derivatives and antidiabetic thiazolidinediones as
natural and synthetic PPAR
ligands, respectively (28, 29, 46-48).
Thiazolidinediones are a new group of anti-diabetic drugs which improve
insulin-resistance (for review, see Refs. 49 and 50). The
identification of thiazolidinediones as PPAR
ligands together with
the central role that adipose tissue plays in the pathogenesis of
important metabolic disorders, such as obesity and
non-insulin-dependent diabetes mellitus (NIDDM), have generated a major interest to determine the role of this PPAR subtype
in normal and abnormal adipocyte function in humans.
The PPAR gene spans about 100 kb and is composed of 9 exons, which
give rise to PPAR
1 and PPAR
2 mRNAs by differential promoter usage and differential splicing. The gene structure as well as the
sequence of the encoded protein are well conserved between human and
mice (45) (99% similarity and 95% identity). Relative to the mouse,
hamster, and Xenopus PPAR
(6, 39, 51), the human protein
contains two additional amino acids. This is in agreement with the
previous reports on the human PPAR
cDNA (34, 35, 52). The
availability of the structure of the human PPAR
gene and protein
will now allow for genetic studies, evaluating its role in disorders
such as insulin resistance, NIDDM, and diseases characterized by
altered adipose tissue function such as obesity or lipodystrophic
syndromes.
To determine tissue-specific patterns of expression of the human
PPAR gene, we developed an RT-competitive PCR assay. Unlike results
of previous reports, which used commercially available blots or single
RNA samples (34, 35), we used multiple independent samples to base our
conclusions on. As was observed in rodents (6, 7), we found PPAR
to
be strongly expressed in adipose tissue. In addition to adipose tissue,
the large intestine had high levels of PPAR
expression. Several
other tissues, such as liver, kidney, and small intestine contained
lower but nevertheless considerable levels of PPAR
RNA. Skeletal
muscle, in contrast, contained only trace amounts of PPAR
mRNA.
In adipose tissue and liver, about 15% of all PPAR mRNA was of
the PPAR
2 type, whereas in the remaining tissues no PPAR
2 mRNA was detected. These observations have several important
implications. First, our data question the relative
importance of PPAR
2. Indeed, our results in humans as well as the
data by Xue et al. (53) in rodent adipocytes show
consistently lower levels of PPAR
2 mRNA and protein relative
to the PPAR
1 subtype. These observations are in line with the
previous observations that the N-terminal domain of PPAR
was
dispensable, both regarding transcriptional activation and capacity to
induce adipocyte differentiation in vitro (7). However, the
N-terminal domain is highly conserved between different species,
suggesting it might have an important function in vivo.
Second, PPAR
expression is much more widespread than previously
realized, which implies that PPAR
controls gene expression in
several tissues in addition to adipose tissue. Especially striking is
the high level of PPAR
expression in the human large intestine.
These reports are consistent with the reported high level expression of
PPAR
in colonic mucosa in mouse (54). It is interesting to note that
fatty acids, potential PPAR activators, have been shown to play an
important role in modulating the function of the large intestine. For
instance diets enriched in saturated lipids have been shown to
predispose to the development of colon cancer (55). Furthermore, it has
been shown that diets enriched in
-3 fatty acids, powerful PPAR
activators, have a beneficial response on inflammatory diseases of the
gastrointestinal tract such as colitis ulcerosa and Crohn's disease
(56, 57). Since the high level expression of PPAR
suggest that it
might play an important role in normal and abnormal colonic function,
further studies aimed at exploring this are definitely needed.
Finally, the low levels of PPAR
expression in skeletal
muscle cells also deserve some reflection. Muscle is responsible for
clearance of the majority of glucose in the body and abnormal muscle
glucose uptake is one of the prime features of insulin resistance and NIDDM. The low levels of PPAR
in muscle argue, therefore, that the
beneficial effects of thiazolidinedione antidiabetic agents are not
likely to be due to a direct effect of these agents on PPAR
present
in the muscle. In fact, even though the liver has considerably higher
levels of PPAR
relative to muscle, thiazolidinediones do not seem to
affect PPAR responsive genes in liver tissue at the concentrations
commonly used to lower glucose levels (23). This observation together
with the observed tissue distribution of PPAR
suggests that the
glucose lowering effects of the thiazolidinedione PPAR
ligands are
primarily a result of their activity on adipose tissue, which then, via
a secreted signal, might influence muscle glucose uptake.
To identify the molecular circuitry underlying tissue-specific
expression of PPAR, we cloned and performed an initial characterization of the human PPAR promoters. As shown, 3000 bp of the PPAR
1 and
1000 bp of the PPAR
2 promoter account for substantial levels of
basal promoter activity. Further functional studies are underway to
determine elements necessary for tissue-specific and regulated expression of the PPAR
gene. In this context, it will be interesting to determine the effects of transcription factors known to induce adipocyte differentiation on PPAR
expression in this tissue and to
define the hierachical role that PPAR
plays in this process. PPAR
is not the only transcription factor involved in adipocyte differentiation. In addition to PPAR
, the basic helix-loop-helix leucine zipper factor ADD-1/SREBP1 and transcription factors of the
C/EBP family also play a role in determining adipocyte differentiation. It is interesting to note that, as in the mouse PPAR
2 promoter (45),
a potential consensus C/EBP response element could be identified in the
human PPAR
2 promoter by homology searches. This observation fits
well with the previous observation that forced expression of C/EBP
could induce PPAR
expression and further studies on this subject are
underway (11, 12).
In conclusion, we report the characterization of the human PPAR gene
structure and furthermore define the structure of the PPAR
1 and
2
promoter. In addition, our data show that human PPAR
has a similar
structure and similar transactivation function as the rodent PPARs. The
expression patterns of PPAR
1 and
2 show that in man, PPAR
1 is
the predominant form. Our results furthermore demonstrate that, in
addition to adipose tissue, human colon expresses high levels of
PPAR
. It is expected that the gene structure will facilitate our
analysis of eventual PPAR
mutations in humans, whereas knowledge of
expression patterns and sequence elements, as well as factors
regulating PPAR
gene expression, could be of major importance in
understanding PPAR biology.
The technical help of Delphine Cayet, Veerle Beelen, and Odile Vidal and the support and/or discussion with Drs. Rich Heyman and James Paterniti from Ligand Pharmaceuticals are kindly acknowledged. We acknowledge the gift of materials from Drs. Ronald Evans and Alex Nadzan. Access to an automatic sequencer by Drs. Philippe Froguel and Jorg Hager in the initial phases of this project are greatly appreciated.