From the Unit of Biochemistry, School of Pharmacy, University of Barcelona, 08028 Barcelona, Spain
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ABSTRACT |
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The expression of several genes involved in
intra- and extracellular lipid metabolism, notably those involved in
peroxisomal and mitochondrial -oxidation, is mediated by
ligand-activated receptors, collectively referred to as peroxisome
proliferator-activated receptors (PPARs). To gain more insight into the
control of expression of carnitine palmitoyltransferase (CPT) genes,
which are regulated by fatty acids, we have examined the
transcriptional regulation of the human MCPT I gene. We have cloned by
polymerase chain reaction the 5'-flanking region of this gene and
demonstrated its transcriptional activity by transfection experiments
with the CAT gene as a reporter. We have also shown that this is a
target gene for the action of PPARs, and we have localized a PPAR
responsive element upstream of the first exon. These results show that
PPAR regulates the entry of fatty acids into the mitochondria, which is
a crucial step in their metabolism, especially in tissues like heart,
skeletal muscle and brown adipose tissue in which fatty acids are a
major source of energy.
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INTRODUCTION |
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The incorporation of activated long-chain fatty acids into the
mitochondria to be catabolized through -oxidation is produced by the
mitochondrial carnitine palmitoyltransferase
(CPT)1 enzyme system. CPT I,
the outer membrane component of this system, is the main control point
in the
-oxidation pathway. CPT I is thus a suitable site for
pharmacological control of fatty acid oxidation in conditions such as
diabetes or heart diseases.
Two isoforms of CPT I have been described, which have been designated LCPT I and MCPT I since these isoforms are mainly expressed in liver and muscle respectively. The MCPT I gene is expressed not only in skeletal muscle but also in heart and brown and white adipose tissue (1-4). This expression pattern may be of great significance since fatty acids are a major source of energy for heart, skeletal muscle, and brown adipose tissue.
The CPT I gene expression is regulated by fatty acids and peroxisome
proliferators (5, 6). To gain more insight into the control of CPT I
gene expression by fatty acids, we have examined the transcriptional
regulation of CPT I genes. The expression of several genes involved in
intra- and extracellular lipid metabolism, notably those involved in
peroxisomal and mitochondrial -oxidation, is mediated by
ligand-activated receptors collectively referred to as peroxisome
proliferator-activated receptors (PPARs); these receptors are members
of the nuclear receptor superfamily. PPARs are activated by a wide
array of peroxisome proliferators and also by natural and synthetic
fatty acids (7, 8), antidiabetic drugs (9, 10), prostaglandin
J2 (10), and leukotriene B4 (11).
We have amplified by polymerase chain reaction (PCR) the 5' region of the human heart and brown adipose tissue CPT I gene and demonstrate, first, the transcriptional activity of this fragment and, second, the presence of a PPRE in the 5'-flanking region of this gene. In CV1 cells, the activation of the CPT I gene by PPAR was dependent on the addition of exogenous ligands.
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EXPERIMENTAL PROCEDURES |
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Plasmids--
pCPTCAT, containing an 882-base pair fragment of
the human MCPT I gene, was constructed by the application of the PCR
using a pair of oligonucleotide primers, CPTF
(5'-CCTGGCTGCAGCTTAGAATAA) and CPTR (5'-GGAGTTGATCCCAGACAGG TAGAC),
corresponding to coordinates 909 to
889 and +126 to +92,
respectively, of the human MCPT I gene (12) and human genomic DNA as a
template. The PstI-AvrII-digested PCR product was
cloned into the PstI-XbaI sites of
chloramphenicol acetyltransferase (CAT) vector pCAT-BASIC reporter gene
(Promega). To confirm the sequence, the PCR-amplified fragment was
automatically sequenced using the fluorescent terminator kit
(Perkin-Elmer).
Cell Culture and Transfections--
CV1 cells were cultured in
minimal essential media supplemented with 10% fetal calf serum. Cells
were typically cotransfected with 10 µg of the reporter MCPT I-CAT
gene construct and, when indicated, with 1 µg of effector plasmids
expressing full-length cDNAs for mouse PPAR, PPAR
2, or
PPAR
. 4 µg of plasmid pRSV
GAL (Rous sarcoma virus promoter
-galactosidase) was included as internal control in cotransfections.
Transfection experiments were carried out by the calcium-phosphate
method as described (14, 15). After removal of the
calcium-phosphate-DNA precipitate, cells were re-fed with medium
supplemented with 10% delipidated calf serum. Experiments with ligand
included either vehicle (dimethyl sulfoxide or ethyl alcohol) or ligand
(10 µM PGJ2 (15-deoxy-
12,14-prostaglandin J2),
30 µM LY-171883, or 30 µM linoleic acid). All ligands used were from Sigma. Cells were harvested 48 h after re-feeding.
-Galactosidase and CAT Assays--
Extracts of harvested
cells were prepared by liquid nitrogen freeze/thaw disruption (three
times) after resuspension in 100 µl of 0.25 M Tris-HCl,
pH 7.5.
-Galactosidase activity was determined (15) in a 10-20-µl
volume of extract to normalize for transfection efficiency. All samples
assayed for CAT activity were first incubated at 65 °C for 5 min.
CAT assays were performed (14) for 60 min. Radioactivity of samples was
measured on an LKB-1217 liquid scintillation counter.
Transcription/Translation in Vitro--
cDNAs for the
receptors (mouse PPAR, PPAR
2, PPAR
, and human RXR
) were
transcribed and translated by using a commercially available kit
according to the instructions of the manufacturer (Promega).
Electrophoretic Mobility Shift Analysis--
2 µl of mPPAR,
mPPAR
, and mPPAR
with or without hRXR
(2 µl) synthesized
in vitro were preincubated on ice for 10 min in 10 mM Tris-HCl, pH 8.0, 40 mM KCl, 0.05% (v/v)
Nonidet P-40, 6% glycerol, 1 mM dithiothreitol, and 2 µg
of poly(dI-dC). The total amount of reticulocyte lysate was kept
constant in each reaction (4 µl) by the addition of unprogrammed
lysate. For competition experiments, a 25-100-fold molar excess of
MCPT I PPRE or MCPT I MPPRE double-stranded probes, relative to the
labeled probe, was included during preincubation. MCPT I PPRE is the
fragment corresponding to coordinates
774 to
755 of the MCPT I
gene, which was used to prepare pTKCATCPT. MCPT I MPPRE is the fragment corresponding to coordinates
782 to
748 of the MCPT I gene, but the
nucleotides corresponding to the PPAR binding sequence have been
mutated (CACATCGGTGACCctcgagggatccTTGGCTATTT, nucleotides described in
lowercase correspond to those that have been changed from the wild type
sequence). Next, 2 ng of MCPT I PPRE, 32P-labeled by
fill-in with Klenow polymerase, was added, and the incubation was
continued for 15 min at room temperature. The final volume for all
reactions was 20 µl. Samples were electrophoresed at 4 °C on a
4.5% polyacrylamide gel in 0.5× TBE buffer (45 mM Tris,
45 mM boric acid, 1 mM EDTA, pH 8.0).
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RESULTS AND DISCUSSION |
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The Human MCPT I Gene 5'-Flanking Region Contains a Consensus
PPRE--
PPAR ,
, and
bind to the MCPT I PPRE as
heterodimers with RXR. To elucidate the control of CPT I gene
expression by fatty acids, we have examined the transcriptional
regulation of CPT I genes. A BLAST search performed using the NCBI
BLAST WWW Server revealed that the sequence for the human muscle type
CPT I gene was included in the sequence of a BAC clone containing a
part of the q arm of chromosome 22 (GenBankTM accession
number U62317). The analysis of the 5'-flanking region of this gene by
the TFSEARCH routine, performed using the Kyoto Center's GenomeNet WWW
Server, shows the presence of a putative PPAR binding sequence upstream
of exon 1A. The comparison of this sequence with the consensus sequence
required for the binding of the PPAR-RXR heterodimer, as proposed by
Palmer et al. (16), shows the coincidence of 17 out of 20 bases (Fig. 1). We performed gel mobility
shift assays to analyze whether PPAR-RXR heterodimers bind to the
putative PPAR binding sequence of the human muscle type CPT I gene. As
can be seen in Fig. 2 neither PPARs nor
RXR alone binds significantly to this sequence. However, incubation of
this probe with a mixture of PPAR (
,
, or
) and RXR
resulted in a prominent complex. An oligonucleotide containing a
mutated PPRE was not able to compete with the wild-type probe for the formation of the complex. The binding of the three subtypes of PPAR to
the MCPT I PPRE is as strong as the binding to the mitochondrial HMG-CoA synthase PPRE, which allows the formation of the strongest complexes for all PPAR subtypes (17) (data not shown).
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CAT Constructs Containing the 5'-Flanking Region of the MCPT I Are
Activated by PPAR--
To investigate the effect of the observed
binding of PPAR to the human MCPT I gene promoter on its
transcriptional activity, we made constructs in which the 5'-flanking
region of this gene was linked to a promoter-less bacterial CAT gene.
These plasmids were introduced into cultured CV1 cells by the
calcium-phosphate method, with or without an expression vector for
PPARs, together with a plasmid that contains the -galactosidase
coding region driven by the SV40 promoter as a control of the
efficiency of the transfection. Following transfection, cells were
incubated in the presence or absence of a PPAR activator, and after
48 h, the cells were harvested and CAT activity measured.
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The Human MCPT I PPRE Confers PPAR Responsiveness to Thymidine
Kinase Gene Promoter--
Next a pair of oligonucleotides containing
the human MCPT I PPRE were inserted into pBLCAT2, a plasmid containing
the CAT gene under the control of the thymidine kinase gene promoter. As can be seen in Fig. 4, this sequence
conferred PPAR responsiveness to the otherwise unresponsive thymidine
kinase gene promoter. The results demonstrate that this human MCPT I
element is able to confer PPAR and
responsiveness both on its
natural context and on a normally unresponsive promoter.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. Ronald M. Evans,
Stephen Green, and Bruce M. Spiegelman for supplying the expression
vectors for RXR and PPAR
, PPAR
, and PPAR
, respectively. We
are also grateful to Robin Rycroft of the Language Service, University
of Barcelona, for valuable assistance in the preparation of the English
manuscript.
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FOOTNOTES |
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* This research was supported by Grant PB94-0840 from Dirección General de Investigación Científica y Técnica.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: IGBMC, CNRS INSERM, Université Louis
Pasteur, C.U. de Strasbourg 67404, France.
§ To whom correspondence should be addressed: Unitat de Bioquímica, Facultat de Farmàcia, Avda. Diagonal, 643, 08028 Barcelona, Spain. Tel.: 34-3-402 45 23; Fax: 34-3-402 18 96.
1
The abbreviations used are: CPT, carnitine
palmitoyltransferase; CAT, chloramphenicol acetyltransferase; PPAR,
peroxisome proliferator-activated receptor; PPRE, peroxisome
proliferator-responsive element; RXR, retinoid X receptor; PCR,
polymerase chain reaction; hRXR, human 9-cis-retinoic
acid receptor
; TK, thymidine kinase; NIDDM,
non-insulin-dependent diabetes mellitus; HMG-CoA,
3-hydroxy-3-methylglutaryl-CoA.
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REFERENCES |
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