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INTRODUCTION |
Peroxisome proliferator-activated receptors
(PPARs)1 are members of the
nuclear hormone receptor superfamily. Three PPAR isotypes, 
, 
(also called 
, FAAR, and NUC1), and 
, have been cloned from
Xenopus, rodents, and human. PPARs regulate gene expression by binding as heterodimers with retinoid X receptors to peroxisome proliferator response elements in the promoter of genes involved in
lipid metabolism (1-3). The biological roles of PPAR and PPAR
are relatively well understood, not least because specific ligands for
these isotypes have been identified (4). PPAR regulates genes
involved in peroxisomal and mitochondrial -oxidation as well as
lipoprotein metabolism (2, 5). PPAR also suppresses apoptosis in
cultured rat hepatocytes (6) and reduces inflammatory responses (7, 8).
PPAR stimulates adipogenesis, enhances insulin sensitivity, and is
involved in cell cycle control and regulation of tumor growth
(9-13).
In contrast, the functions of PPAR are poorly understood, due partly
to its ubiquitous expression and the lack of a selective ligand. The
objective of this study was to start unraveling PPAR functions using
an experimental model that is easy to manipulate and that expresses
high levels of PPAR compared with PPAR and PPAR. The nervous
system seemed an appropriate target for this, as PPAR is abundantly
expressed in brain from embryogenesis to adulthood, whereas PPAR and
PPAR are barely detectable (14-17). The brain is the organ with the
highest lipid concentration in the body, second only to adipose tissue.
Brain lipids serve primarily in modifying the fluidity, structure, and
functions of the membranes, and both anabolic and catabolic pathways of
lipid metabolism are important in brain development (18, 19). Fatty
acids need to be activated to their acyl-CoA by acyl-CoA synthetases
(ACSs), the activities of which have been found in the brain (20, 21). Moreover, ACS1, ACS2, and ACS3 mRNAs have been analyzed in the postnatal rat brain, and the levels of ACS2 and ACS3 vary during its
development (22). Given the key role of ACSs in fatty acid utilization,
we speculated that they could be potential targets of PPAR in the brain.
We chose cultures of reaggregated neural cells prepared from the
telencephalon of rat embryos, which provide a three-dimensional network
of different neural cell types that progressively acquire a
tissue-specific pattern resembling that in the brain (23, 24). We first
determined the expression pattern of PPARs during maturation of brain
aggregates, providing evidence for PPAR being the prevalent isotype.
Then, using a selective activator for PPAR, we demonstrate that the
ACS2 gene is regulated by PPAR at the transcriptional level.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Reaggregated brain cell primary cultures were
prepared from mechanically dissociated telencephalon of 16-day rat
embryos as described previously (24). Cultures were initiated and grown in serum-free, chemically defined medium consisting of Dulbecco's modified Eagle's medium with high glucose (25 mM) and
supplemented with insulin (0.8 µM), triiodothyronine (30 nM), hydrocortisone-21-phosphate (20 nM),
transferrin (1 µg/ml), biotin (4 µM), vitamin B12 (1 µM), linoleate (10 µM), lipoic acid (1 µM), L-carnitine (10 µM), and
trace elements (listed in Ref. 24). Gentamicin sulfate (25 µg/ml) was
used as antibiotic. Culture media were replenished by exchange of 5 ml
of medium (of a total of 8 ml/flask) every third day until day 14, and
every other day thereafter because of increased metabolic activity in
the cells. The cultures were maintained under constant gyratory
agitation (80 rpm) at 37 °C and in an atmosphere of 10%
CO2/90% humidified air.
Northern Blot Analysis--
PPAR mRNA was probed with the
1.32-kb BamHI fragment of the plasmid pSG5-rPPAR
containing the coding region of the
rPPAR.2 The different ACS
mRNAs were probed with DNA fragments isolated from their respective
rat cDNA containing plasmids (22). ACS1 mRNA was probed with
the 0.96-kb EcoRV fragment of the pRACS15 plasmid. ACS2
mRNA was probed with the 2-kb EcoRI fragment of the
pBACS9 plasmid. ACS3 mRNA was probed with the 2.2-kb
BglII/SacI fragment of the pACS3 plasmid. L27
mRNA, which was used as an internal control, was probed with the
0.22-kb XbaI/EcoRI fragment of the
pKS+/L27 plasmid (25). The above-mentioned gel-purified DNA
fragments of PPAR (100 ng), the three ACSs (100 ng each), and L27
(25 ng) were used as templates to synthesize
[-32P]dCTP-labeled probes using the High Prime kit
(Roche Molecular Biochemicals). Probes were purified on Elutip-d
columns according to the manufacturer's protocol (Schleicher and Schuell).
Brain cell aggregates from each flask were carefully transferred to
round-bottomed 14-ml Falcon tubes, washed with PBS, and frozen rapidly
by plunging the capped tubes into liquid nitrogen after aspirating the
residual amounts of PBS in the samples. Frozen aggregates were stored
at 
80 °C until use. Aggregates were thawed rapidly at 37 °C in
5 ml of TRIzol (Life Technologies, Inc.), and total cellular RNA was
prepared according to the protocol supplied by the manufacturer.
RNA (25 µg/well) was electrophoresed in a 2.2 M
formaldehyde-1% agarose gel in MOPS buffer and transferred onto Zeta
Probe GT-membrane (Bio-Rad) by capillary blotting in 10× SSC for about 18 h. The Northern blot membranes were baked for 30 min at
80 °C, prehybridized at 65 °C for 2-6 h in 0.25 M
Na2HPO4 (pH 7.2) containing 7% SDS, and
hybridized at the same temperature with -32P-labeled
probes (2-3 × 106 cpm/ml) for 18 h. After
hybridization, the membranes were sequentially washed at 65 °C for
30 min each in 20 mM Na2HPO4 (pH
7.2) containing 5% SDS and in 20 mM
Na2HPO4 (pH 7.2), 1% SDS and then exposed to
preflashed films at 70 °C for 2-3 days for PPAR and ACSs and
18-24 h for L27. Autoradiographs were quantified in a Elscript 400 AT/SM densitometer (Hirschmann), and the mRNA signals were normalized for the loading error using the L27 mRNA signal as internal control, as described previously (26).
RNase Protection Assay--
[-32P]UTP-labeled
riboprobes were transcribed in vitro with T7 RNA polymerase,
and the RNase protection assay was carried out on 15 µg of total RNA
as described previously (25). The PPAR riboprobe was synthesized
from the 717-bp TaqI fragment of the pKS+/PPAR plasmid and the L27 riboprobe from the
pBS-L27(150) plasmid linearized with EcoRI. Samples were
electrophoresed on 6% polyacrylamide gels and quantified using a Storm
840 phosphorimager (Molecular Dynamics).
Subcloning of the Acyl-CoA Synthetase DNA Fragments--
Short
DNA fragments of the three ACS cDNAs (22) were subcloned into the
pBluescript II KS
vector (pBSII KS) from
Stratagene to use as probes for in situ hybridization. For
ACS1, the 305 bp HindIII fragment corresponding to
nucleotides 1484-1789 in the plasmid pRACS15 was subcloned into the
HindIII site of the vector, and the recombinant plasmid was
named pBSII KS-ACS1. For ACS2, the 368-bp
BamHI/EcoRI fragment corresponding to nucleotides
143-511 in the plasmid pBACS9 was subcloned into the
BamHI/EcoRI site in the bluescript vector, and
the recombinant plasmid was named pBSII KS-ACS2. For
ACS3, the 364-bp SpeI-XbaI fragment corresponding
to nucleotides 682-1047 in the plasmid pACS3 was subcloned into the vector digested with SpeI-XbaI, and the
recombinant plasmid was named pBSII KS-ACS3. The ACS1,
ACS2, and ACS3 recombinant plasmids contained the insert in such an
orientation that the antisense probe was synthesized with T3 RNA
polymerase and the sense probe with T7 RNA polymerase.
In Situ Hybridization--
Digoxigenin-labeled PPAR and
PPAR riboprobes were transcribed in vitro from
linearized, gel-purified plasmids using T7 RNA polymerase as described
previously (17). Antisense probes were synthesized from the plasmids
pKS+/PPAR and pSK+/PPAR, whereas the
sense probes were synthesized from the plasmids pSK+/PPAR and pKS+/PPAR.
Digoxigenin-labeled ACS riboprobes were in vitro transcribed from the plasmids pBSII KS-ACS1, pBSII
KS-ACS2, and pBSII KS-ACS3 linearized with
XbaI for antisense probes of ACS1 and ACS2 and
NotI for ACS3. Sense probes were synthesized from the same plasmids linearized with XhoI for ACS1 and
HindIII for ACS2 and ACS3.
Aggregates washed with PBS were embedded in tissue freezing medium
(Jung, Nussloch), frozen in isopentane cooled with liquid nitrogen as
described previously (24), and stored at 80 °C until use. Cryostat
sections (12 µm thickness) were hybridized for 40 h at 58 °C
with digoxigenin-labeled probes (400 ng/ml) in 5× SSC containing 50%
formamide and 40 µg/ml salmon sperm DNA. Sections were washed, and
the mRNAs were visualized by alkaline phosphatase staining as
described previously (17) and then dehydrated, mounted, and
photographed on an Axiophot microscope (Carl Zeiss).
Immunohistochemistry--
Monoclonal antibodies against cell
type-specific proteins were used for immunohistochemical analysis of
the aggregates. Antibodies against glial fibrillary acidic protein
(GFAP), microtubule-associated protein 2 (MAP2) (Roche Molecular
Biochemicals), and myelin basic protein (Boehringer-Ingelheim) were
used to identify astrocytes, neurons, and oligodendrocytes,
respectively. Cryosections (12 µM) were fixed for 1 h in 4% paraformaldehyde-PBS at room temperature, washed in PBS (three
times for 5 min each), and incubated overnight at 4 °C with the
primary antibody diluted 1:100 in dilution buffer (PBS containing 1%
bovine serum albumin and 0.3% Triton X-100). After washing away the
primary antibody, sections were first incubated at room temperature for
1 h with an anti-mouse IgG (Sigma) bridging antibody diluted 1:100
in dilution buffer, and then incubated at room temperature for 1 h
with the alkaline phosphatase/anti-alkaline phosphatase complex diluted
1:100. Sections were then washed with PBS, stained for alkaline
phosphatase for 20 min, dehydrated, mounted, and photographed on an
Axiophot microscope.
Reporter Gene Assay in HeLa Cells--
Cells were cultured at
37 °C, 5% CO2 in Dulbecco's modified Eagle's medium
supplemented with antibiotics and 5% fetal calf serum. For transient
transfection by the calcium phosphate method, 2.5 × 105 cells/well were plated on six-well plates. After
24 h, DNA mixture (200 µl) containing 0.1 µg of PPAR
expression plasmid,2 0.5 µg of the internal control
plasmid CMV-gal (CLONTECH), 2 µg of the
reporter plasmid Cyp2XPalCAT (27), and 5 µg of sonicated salmon sperm
DNA was added to each well, and cells were transfected for 12 h.
The medium was then replaced with Dulbecco's modified Eagle's medium
supplemented with 5% charcoal-treated fetal calf serum, activators
were added, and cells were incubated at 37 °C for 24 h. Cells
were then scraped off the dishes, and total cell extracts were prepared
by three cycles of freezing in liquid nitrogen and thawing at 37 °C.
-Galactosidase and chloramphenicol acetyltransferase (CAT)
activities were measured in these extracts by standard methods (28).
Relative CAT activity was calculated as the fold increase in CAT
activity over the basal activity obtained with the empty vector in
untreated cells.
Treatment with Activators and Marker Enzyme Assays--
Stock
solutions of bezafibrate (Sigma) and L-165041 compound (a gift from
Parke-Davis) were prepared in ethanol and dimethyl sulfoxide
(Me2SO), respectively. The effect of activators on marker enzyme activities were measured on reaggregated cultures, equivalent to
one-fourth of the original cultures, treated on day 34 or 35 with
either 0.05% ethanol (control-bezafibrate), 0.05% Me2SO
(control-L-165041), 10 µM bezafibrate, or 5 and 10 µM L-165041 for 24 h. After treatment, the samples
were homogenized in ground glass homogenizers. The marker enzymes used
were glutamine synthetase for astrocytes, glutamic acid decarboxylase
for GABAergic neurons, and choline acetyltransferase for cholinergic
neurons, and they were measured using radiometric methods as described
previously (24). Determination of protein amounts by the Lowry method
was done on replicate cultures representing one-fourth of the original cultures.
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RESULTS |
PPAR mRNAs in Reaggregated Brain Cell Cultures--
As PPARs
have not been studied so far in the developing postnatal brain, we
first characterized the aggregates for the basal level of PPAR
mRNAs. We examined these cultures at four ages representing different stages of maturation of neurons and glia (henceforth referred
to as differentiation). At day 7 (d7), the cells were undifferentiated;
at d21, differentiation of neuron and glia subtypes was ongoing, d35
cultures reached the steady state of differentiation, and d49
represented late cultures with the highest level of neuron-specific parameters, although some demyelination may have already occurred (29,
30).
The basal levels of the PPAR isotype mRNAs were different in these
aggregates. PPAR mRNA was barely detectable by Northern blot
analysis but was detected by RNase protection assay (Fig. 1A). In contrast, PPAR
mRNA was abundant and could be detected easily by Northern blot
analysis of total RNA (Fig. 1B). Total cellular RNA from
adult rat liver and brain was used as a positive control for PPAR
and PPAR, respectively. PPAR mRNA was not detected in the
aggregates even by reverse transcription-PCR (data not shown), and
therefore, this isotype was not analyzed in subsequent experiments.
There was also a different age-dependent pattern of
expression of both PPAR isotypes. On the one hand, the PPAR mRNA
basal level was low in undifferentiated cultures (d2 and d7), increased
3-5-fold during differentiation (d14, d21, and d28), and remained high
in differentiated (d35) and late (d42 and d49) cultures (Fig.
1C). On the other hand, the level of PPAR mRNA
remained low at the four developmental stages examined (Fig. 1,
A and C, inset). Thus, of the three PPARs, only
the two isotypes and were present in the aggregates, and the
levels of PPAR increased with the onset of cell differentiation.
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Fig. 1.
PPARs in reaggregated brain cell
cultures. A, basal level of PPAR mRNA was
determined by RNase protection assay of total RNA from reaggregated
brain cell cultures of 7, 21, 35, and 49 days. Adult rat liver was used
as a positive control. B, Northern blot analysis of PPAR
mRNA in aggregates of 7, 21, 35, and 49 days in which total RNA
from adult rat brain was used as a positive control and L27 mRNA as
an internal loading control. C, Northern blots of RNA from
cultures 2, 7, 14, 21, 28, 35, 42, and 49 days old were probed for
PPAR and L27 mRNAs. Data are mean ± S.D.
(n = 3-8). Inset, PPAR and L27 mRNA
signals obtained by RNase protection assay. The data are a mean of two
sets of samples.
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Cell Type-specific Expression of PPAR Isotypes in Reaggregated
Brain Cell Cultures--
In order to identify the cells expressing
PPARs in the aggregates, we used in situ hybridization to
detect PPAR transcripts in cells (Fig.
2A), as well as
immunohistochemical staining for cell type-specific markers to identify
these cells (Fig. 2B). The marker antigens used for
immunohistochemistry were MAP2 for neurons, GFAP for astrocytes, and
myelin basic protein for oligodendrocytes. Because MAP2 and GFAP are
cytoskeletal proteins, immunostaining for these antigens is seen in the
cell bodies and processes, and the in situ hybridization
signal of PPAR mRNAs is cytoplasmic. Furthermore, we used thionine
staining in parallel sections to visualize the nuclei in the aggregates
(Fig. 2, A, q-t, and B, i and k). At
d7, a faint signal of PPAR mRNA was detected in most cells of
the aggregates (Fig. 2A, compare a with
q). At d21, the signal was slightly stronger and restricted
to cells with small nuclei (Fig. 2A, compare b
with r), which were identified primarily as astrocytes by
immunohistochemistry (Fig. 2B, compare c with
d, h, and l). The pattern was similar at d35
(Fig. 2A, c). At d49, the same cell type was positive for
PPAR but with a decreased signal intensity (Fig. 2A, d).
The sense probe for PPAR did not stain the sections (Fig. 2A,
e-h), indicating that the signal detected with the antisense
probe was specific.
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Fig. 2.
Cell type-specific expression of
PPAR and PPAR
mRNAs in the reaggregated brain cell cultures.
A, in situ hybridization for PPAR
(a-h) and PPAR (i-p) on aggregates of 7, 21, 35, and 49 days (as indicated above columns). Panels a-d
are sections of the aggregates hybridized with the antisense probe for
PPAR mRNA (PPARAS), whereas panels e-h
are those hybridized with the sense probe (PPARS).
Panels i-l are sections hybridized with the antisense probe
for PPAR mRNA (PPARAS), and panels m-p
are those for the sense probe (PPARS). Panels in the
bottom row (q-t) are sections of aggregates
stained with thionine to visualize the general morphology of the
aggregates. Bar, 100 µm. B, immunohistochemical
analysis of 7- and 21-day-old aggregates (second and
fourth columns from left, respectively). Shown is
immunostaining for GFAP (b and d), MAP2
(f and h) and myelin basic protein (j
and l) to identify astrocytes, neurons, and
oligodendrocytes, respectively. For panel h, aggregates were
fixed overnight in Carnoy, dehydrated, and embedded in paraffin.
Sections of 12 µm thickness were rehydrated and stained for MAP2 as
described for cryosections. In situ hybridization with the
antisense probe for PPAR (a and c) and PPAR
(e and g) in aggregates of 7 and 21 days
(first and third columns from left,
respectively). Panels i and k are sections of
aggregates stained with thionine. Bar, 100 µm.
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The in situ hybridization signal for PPAR was higher than
that for PPAR in all developmental stages of the aggregates (Fig. 2A, compare i-l with a-d). At d7,
the PPAR signal was of medium intensity and was present in most
cells of the aggregates (Fig. 2A, i). At d21, the signal was
stronger and exhibited a cell type-specific expression pattern. A
signal of very high intensity was observed in cells with large nuclei
(Fig. 2A, compare j with r), which formed a broad layer in the aggregates. Immunostaining with MAP2 revealed that these cells were neurons (Fig. 2B, compare
g with h). A PPAR signal of medium and high
intensity was also observed in the same region of the aggregates in
cells with smaller nuclei, which were neurons, astrocytes, and
oligodendrocytes (Fig. 2B, compare g with
d, h, and l). At d35 and d49, the pattern of
expression of PPAR mRNA was similar to that of d21, but the
signal intensity was lower (Fig. 2A, k and l).
The peripheral layer of glial cells were positive for PPAR at all
developmental stages except d7 (Fig. 2B, d and
l). Again, the sense probe barely stained the sections (Fig.
2A, m-p), confirming the specificity of the signal obtained
for PPAR. These results showed that PPAR mRNA was ubiquitous in the aggregates and high in some neurons, whereas PPAR mRNA was mainly detected in the astrocytes.
Acyl-CoA Synthetases in the Aggregates--
We used a three-step
approach to establish whether ACSs were target genes of PPAR in this
model of the developing rat brain. First, we determined the basal
levels of the three ACSs in the aggregates. We then examined their cell
type-specific expression pattern, and finally studied whether PPAR
activators regulate their expression.
All three ACS mRNAs were easily detectable by Northern blot
analysis of total RNA (Fig. 3). ACS1
mRNA was present at a low level in the four developmental stages
examined. At d21, a small but significant increase of 1.8-fold over the
level at d7 was observed (t test, p 
0.05). The basal level of ACS2 mRNA was very low at d7, but it
increased 5-7-fold in d21 and d35 cultures and remained steady in the
late cultures. In undifferentiated cultures, the basal level of ACS3
mRNA was the highest among the three ACSs, and this increased
further 2-3-fold until d49. The increase over the basal level at d7
was statistically significant for ACS2 and ACS3 mRNAs (t
test, p 0.01).
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Fig. 3.
Acyl-CoA synthetases in the reaggregated
brain cell cultures. Northern blot analysis of ACS mRNAs in
cultures 7, 21, 35, and 49 days old (as indicated above the
lanes). The total RNA was blotted in duplicate. One blot was probed in
the order ACS1, L27, and ACS3, and the other was probed in the order
PPAR, L27, and ACS2. Lane B in each panel corresponds to
total RNA from adult rat brain. Data are mean ± S.D.
(n = 3-5).
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The comparison of the temporal expression pattern of ACS mRNAs with
that of PPARs revealed similarities between ACS1 and PPAR mRNAs
on one hand and ACS2 and PPAR mRNAs on the other hand (Figs. 1
and 3).
Identification of the Cell Types Expressing acyl-CoA Synthetases in
the Aggregates--
We examined by in situ hybridization
the cell type-specific pattern of expression of ACS mRNAs and
compared it with the immunohistochemical analysis shown in Fig.
2B. ACS1 mRNA was barely detectable in d7 cultures (Fig.
4A, a). At d21, a signal of
medium intensity was observed in small cells in most regions of the
aggregates, which were predominantly astrocytes (Fig. 4A, b
and Fig. 2B, d). The intensity of this signal decreased at
d35 and d49 but was still primarily astrocytic (Fig. 4A, c
and d). The glial cells at the periphery of the aggregates
did not stain for ACS1 mRNA. ACS2 mRNA signal was higher than
that of ACS1 mRNA in the aggregates and was observed in most cells
at d7 (Fig. 4A, e). At d21, the signal was of high intensity
in neurons with large nuclei, which form a broad layer in the
aggregates. In the same region, a signal of medium intensity was
observed in other neurons, astrocytes, and oligodendrocytes (Fig.
4A, f). In aggregates of d35 and d49, the ACS2 mRNA
signal decreased in intensity (Fig. 4A, g and h) but was present in the same cell types as observed at d21. ACS3 mRNA was found in most cells of the aggregates at days 7 and 21 (Fig. 4A, i and j), with a few cells showing a
very strong signal. At d35 and d49, the ACS3 mRNA signal decreased
in intensity and was observed in neurons, astrocytes, and
oligodendrocytes (Fig. 4A, k and l). Large cells,
which stained intensely at d7 and d21, were not apparent in these
differentiated cultures. The peripheral layer of glial cells was
positive for ACS2 and ACS3 mRNAs in cultures of all developmental
stages except d7. No signal was observed with the sense probes for
ACS1, ACS2, and ACS3 mRNAs (data not shown).
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Fig. 4.
In situ hybridization for acyl-CoA
synthetases in reaggregated brain cell cultures. A,
sections of aggregates of 7, 21, 35, and 49 days (as indicated above
columns) were hybridized with the antisense probes for ACS1
(a-d), ACS2 (e-h), and ACS3 (i-l).
Bar, 100 µm. B, in situ hybridization with the
antisense probes for ACS1 (a), ACS2 (b), PPAR
(c), and ACS3 (d) in aggregates of 21 days.
Bar, 100 µm.
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The in situ hybridization analysis of the three ACSs in d21
aggregates was compared with that of PPAR and is shown at a higher magnification in Fig. 4B. The expression pattern of ACS2
mRNA was similar to that of PPAR (Fig. 4B, b and
c). This finding, taken together with their temporal pattern
of expression, suggested that ACS2 might be a target gene of
PPAR.
Activation of Rat PPARs in a Cell-based Reporter Gene
Assay--
To test whether ACS2 was a PPAR target gene, a PPAR
activator was required that preferentially activates rPPAR. In a
recent study from our laboratory, several compounds were screened with the coactivator-dependent receptor ligand assay. Fatty
acids, eicosanoids, and hypolipidemic drugs were identified as ligands for Xenopus PPARs (27). Most of the ligands identified for
xPPAR by this assay exhibited similar or greater activity toward
xPPAR. As bezafibrate, which was strong on xPPAR, was the least
active on xPPAR, we tested this hypolipidemic drug on rPPARs in a
reporter gene assay in HeLa cells. Transfection of the PPAR
expression vector resulted in an apparent ligand independent activation
of the reporter gene (Fig.
5A), which has been observed
previously in HeLa cells (31). In cells treated with 10 and 100 µM bezafibrate, PPAR-mediated transcription of the
reporter gene was enhanced. In contrast, expression of PPAR did not
affect the basal activity of the reporter, and treatment with the 100 µM bezafibrate activated the reporter gene only weakly.
Thus, at a concentration of 10 µM, bezafibrate can be
used as a specific activator of rPPAR.
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Fig. 5.
Activation of rat PPARs in cell-based
reporter gene assay. HeLa cells were cotransfected with reporter
plasmid Cyp2XPalCAT and either the empty expression vector (pSG5),
rPPAR (pSG5-rPPAR), or rPPAR (pSG5-rPPAR). After
transfection, cells were treated with bezafibrate (Bz)
(A) or L-165041 (B) for 24 h. Relative CAT
activities for the two activators are plotted for the empty expression
vector and PPAR and . Data are mean ± S.D.
(n = 3-5).
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L-165041 compound has been recently identified as a human PPAR
agonist (32). In order to characterize its activity profile on rPPARs,
we tested this compound in the reporter gene assay in HeLa cells. In
cells treated with 5 and 10 µM L-165041, there was only a
small increase in PPAR-mediated transcription of the reporter gene,
indicating a weak effect of the compound on this isotype (Fig.
5B). After a similar treatment,
PPAR-dependent activity of the reporter gene increased
dramatically above its basal level (Fig. 5B). This strong
activation contrasts with the absence of background activity of
rPPAR and provides strong evidence for the high L-165041
responsiveness of this isotype. We therefore used this compound as a
PPAR activator on the aggregates.
Effect of Bezafibrate and L-165041 on the Aggregates--
We next
tested whether the two activators had any strong and general effect on
the aggregates. These cultures are an established model for
neurotoxicological studies (33), and the major subtypes of neurons
present in the differentiated aggregates are cholinergic, glutamatergic, and GABAergic neurons. In the aggregates, the lactate dehydrogenase release assay underestimates the cytotoxic effects of
test compounds, and therefore, estimation of cell type marker enzyme
activities is a more accurate indicator of the effect of chemical
treatments. Routine biochemical analysis for the cellular effects of
compounds involves measuring glutamine synthetase, choline
acetyltransferase, and glutamic acid decarboxylase, representing glial
cells, excitatory neurons, and inhibitory neurons, respectively, whereas the total protein content is used as an indicator of general cytotoxicity. Treatment of the differentiated aggregates with bezafibrate or L-165041 for 24 h did not cause any visible
alteration in their size or glucose metabolism. Similarly, there was
little effect on the marker enzyme activities and on the total protein content of the aggregates (Table I).
Therefore, we conclude that treatment with the two activators did not
modify the neuron and glial cell parameters and did not have general
cytotoxic effects on the aggregates. This also suggested that the
marker enzymes were probably not regulated by either PPAR.
Acyl-CoA Synthetase 2 Is a Target Gene of PPAR in Reaggregated
Brain Cell Cultures--
Specific activators or ligands of PPAR and
PPAR have been successfully used to identify their target genes.
Therefore, it was reasonable to expect an effect of L-165041 on ACS2
expression if it was a target gene of PPAR. Treatment of
differentiated aggregates for 24 h with bezafibrate had no effect
on either the ACS mRNAs or PPAR mRNA (Fig.
6A). However, a similar
treatment of the aggregates with 5 and 10 µM L-165041
increased the ACS2 mRNA levels significantly (2.4- and 3-fold) but
did not affect the ACS1 and ACS3 mRNAs (Fig. 6B). We
conclude that ACS2 is a target gene of PPAR in the postnatal rat
brain because its temporal and cell type-specific expression pattern
correlated with that of PPAR, and its mRNA is induced after
treatment with a selective activator of this PPAR isotype. The 2-fold
increase in PPAR mRNA level itself after L-165041 treatment
(Fig. 6B) was statistically significant (t test,
p 0.05).
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Fig. 6.
L-165041 treatment increases ACS2 and
PPAR mRNA levels. Reaggregated brain
cell cultures were treated on day 35 for 24 h with 0.05% ethanol
(control) or 10 µM bezafibrate (A) or with
0.05% Me2SO (control) or 5 or 10 µM L-165041
(B). Total RNA was Northern blotted in duplicate. One blot
was hybridized in the order ACS1, L27, and ACS3, and the other was
probed in the order PPAR, L27, and ACS2. Data are mean ± S.D.
(n = 3).
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L-165041-mediated Induction of ACS2 mRNA Occurs by
Transcriptional Activation and Requires Protein Synthesis--
Next,
we tested whether the L-165041 effect on ACS2 mRNA expression was
at the transcriptional or posttranscriptional level by treating
aggregates for 6 and 30 h with 10 µM L-165041 in the presence of 0.1 µM of the transcriptional inhibitor
actinomycin D (Fig. 7, ActD).
In the absence of actinomycin D, the expected L-165041-dependent induction was observed after a 30-h
treatment. However, treatment with actinomycin D prevented induction by
L-165041, ruling out an effect of the compound on the stability of the
ACS2 mRNA. If L-165041 stabilized the ACS2 mRNA, then higher
levels of this mRNA would have been observed in presence of the
compound in actinomycin D-treated samples. Therefore, regulation is at the transcriptional level, and it was of interest to test whether it
requires de novo protein synthesis. This possibility was
examined in aggregates incubated in the presence of a 5 µM concentration of the protein synthesis inhibitor
cycloheximide (Fig. 7, CHX). In the presence of
cycloheximide, induction of the ACS2 gene by L-165041 was blocked,
providing evidence that de novo protein synthesis was
required for ACS2 mRNA stimulation. The requirement for ongoing
protein synthesis suggests either that the PPAR protein or another
key factor is turned over rapidly or that L-165041 induces the de
novo synthesis of a missing regulatory protein.
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Fig. 7.
L-165041-mediated increase in ACS2 mRNA
occurs at the transcriptional level and requires protein
synthesis. Reaggregated brain cell cultures were treated on day 34 with 0.05% Me2SO (control) or 10 µM L-165041
for 6 and 30 h. Cycloheximide (CHX) (5 µM) and actinomycin D (ActD) (0.1 µM) were used to inhibit protein synthesis and
transcription, respectively. Total RNA was Northern blotted and probed
for ACS2 and L27 mRNAs. Treatments with cycloheximide or
actinomycin D for 30 h had an effect on the internal control
itself, and therefore the nonnormalized data for these samples are
plotted. Data are mean ± S.D. (n = 3).
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DISCUSSION |
This is the first report on PPAR expression in reaggregated rat
brain cell cultures, which is an in vitro experimental model representative of the developing postnatal brain in vivo.
The finding that PPAR was the prevalent isotype in these cultures allowed us to test whether ACS1, ACS2, and ACS3 were target genes of
this PPAR. Using L-165041 as a selective activator of rPPAR in the
aggregates, we have identified ACS2 as the first PPAR target gene.
The Aggregates as an in Vitro Experimental Model of the Developing
Postnatal Brain--
Morphological and biochemical studies have shown
that the reaggregated cultures of mechanically dissociated fetal rat
telencephalon are able to mimic several morphogenetic events occurring
in vivo, including cell migration, synaptogenesis, and
myelination. These three-dimensional cell cultures provide maximum
intercellular contacts and interactions, which facilitates the
maturation-dependent expression and deposition of
extracellular matrix components (34). The distinct stages of cell
proliferation and differentiation observed are comparable to the tissue
in vivo. The aggregates also exhibit cell type-specific and
development-dependent expression of neuronal and glial
cytoskeletal proteins and reproduce the developmental pattern of
Na+-K+-ATPase gene expression observed in
vivo (35, 36). Therefore, the observations made in this model can
be considered to reflect the in vivo situation. Because
these aggregates were analyzed between day 2 and day 49, this study
covers a period of time corresponding to the development of the brain
from embryonic day 18 to about postnatal week 6.
PPARs in the Aggregates--
The differential expression of PPARs
in the aggregates, taken together with their cell type-specific
expression pattern, suggests that the neurons contain a low level of
PPAR and a high level of PPAR, whereas the glia contain a low
level of both PPARs. This is in concert with the expression of PPAR
and in monolayer cultures of neonatal neurons (37). However, in
astrocyte-enriched monolayer cultures, it was observed that PPAR
mRNA levels were higher than those of PPAR mRNA.
Furthermore, in both neuron- and astrocyte-enriched monolayer cultures,
a very low level of PPAR was present (37). It has also been reported
that PPAR was abundant in monolayer cultures enriched for
oligodendrocytes but present only at low levels in primary astrocyte
cultures (38). Our in situ hybridization results indicate
that in the aggregates, PPAR is present at a low level in both these
cell types. These differences in PPAR expression between monolayer and
aggregate cultures most likely reflect the effects of these two modes
of culture on gene expression.
The general differential expression of PPAR mRNAs in d49 aggregates
(representative of the 6-week-old rat brain) is consistent with that
observed in the adult rodent (14, 17, 39) and human (40) brain in
vivo. Furthermore, the cell type-specific expression of PPAR in
these aggregates is also consistent with that found in the rat brain
(16, 17). Thus, PPAR expression in the aggregates has more
similarities with that in vivo than in monolayer cultures.
This confirms that reaggregated cultures provide a better model to
study the biological role of this PPAR isotype.
Acyl CoA Synthetases in the Aggregates--
The differential
expression of ACSs in the aggregates, with ACS2 and ACS3 being abundant
and ACS1 at a very low level, is consistent with that observed in the
developing postnatal rodent brain (22, 41). Because the cell
type-specific expression of ACS1, ACS2, and ACS3 mRNAs has not been
studied in vivo, we analyzed the distribution of these
mRNAs in the aggregates and found that ACS1 mRNA is localized
mainly in the astrocytes and ACS2 mRNA in astrocytes and neurons.
These expression patterns of ACS1 and ACS2 correlate well with PPAR
and PPAR expression, respectively. ACS3 mRNA was present in most
cells of the aggregates, a pattern of expression quite different from
that of PPAR or PPAR. The similarity in the ACS2 and PPAR
expression patterns identified the former as the product of a potential
target gene of the latter. We could confirm this in the differentiated
aggregates, which have a high level of PPAR, using an activator that
exhibited a preference for this PPAR isotype.
Activators of PPAR--
One of the key issues in analyzing
PPAR function is that of the availability of PPAR isotype selective
ligands. All compounds that have been reported to activate PPAR so
far in various types of assays are also active on PPAR (7, 14, 27,
42-44). Species differences in PPAR ligand affinity are another factor
that needs to be taken into account. For example, bezafibrate was
identified as a ligand for xPPAR (27), but in reporter gene assay in
HeLa cells, we found that bezafibrate was an activator of rPPAR but not of rPPAR. In the same type of assay, L-165041 preferentially activated rPPAR, which enabled us to identify ACS2 as the first target gene of this isotype.
ACS2 as a PPAR Target Gene--
In this study, we provide
evidence that ACS2 is a target gene of PPAR, indeed the first one
identified. The L-165041-mediated induction of ACS2 mRNA occurs by
transcriptional activation of the gene and is not a consequence of
stabilization of the mRNA itself. In the presence of a ligand, a
direct target gene of PPAR would be activated via a PPRE present in
its promoter, even in the absence of protein synthesis, whereas an
indirect response gene would require protein synthesis for its
activation. It is not known whether the promoter of the ACS2 gene
contains a PPRE. However, it is noteworthy that a functional PPRE has
been identified in the promoter of the ACS1 gene (45). The requirement
of de novo protein synthesis to achieve L-165041-mediated
induction of ACS2 mRNA suggests that the ACS2 gene might be an
indirect target of PPAR. However, at this stage we cannot exclude
that a short half-life of the PPAR protein itself is the limiting parameter. This possibility will be tested when a PPAR selective antibody becomes available.
Possible Biological Functions of ACS2 in the Aggregates--
ACS2
mRNA levels increase significantly in the differentiating
aggregates, which coincides with the maturation of neurons, onset of
myelination, and high metabolic activity (29). Because no specific
roles have yet been attributed to ACS2 in the brain, we present below
several functions in the modulation of which a regulated expression of
ACS2 might be beneficial. Maturation of neurons (that is, their
cytodifferentiation and the formation of neuronal connectivity)
involves extensive lipid biosynthesis and turnover. Biochemical
analysis of ACS2 has revealed that its preferred substrates are
arachidonic, eicosapentaenoic, and docosahexaenoic acids (46), which
are major components of the neural membranes. Another characteristic of
neuron maturation is the production of neurotransmitters and their
vesicular transport, storage, and release. Recently, ACS1 has been
localized in Glut 4-containing vesicles prepared from rat adipocytes
(47), and palmitoyl-CoA appears to be required for vesicular transport
in vitro (48). With respect to signal transduction pathways
that play key roles in the brain, there are several examples of the
involvement of acyl-CoA esters (reviewed in Ref. 49), such as in the
stimulation of Ca2+ release from intracellular compartments
(50-52) and stimulation of ion channels (53, 54). Finally, acylation
is a common posttranslational modification of myelin proteins (reviewed
in Ref. 55), the bound fatty acids of which are turned over very
rapidly (3). ACS activity has been reported in myelin (21), but the
type involved not yet determined. It will be the aim of future work to
investigate whether and how ACS2 is involved in the processes described above.
Conclusions--
In reaggregated brain cell cultures, PPARs are
expressed differentially, with a cell type-specific pattern. PPAR is
the prevalent isotype, and it is ubiquitous in the aggregates. PPAR
is present at a low level and is mainly astrocytic, whereas PPAR is
absent. Among the three ACSs analyzed in this study, the temporal and cell type-specific expression pattern of ACS2 correlates very well with
that of PPAR. The identification of this ACS as a PPAR target
gene demonstrates a role of this receptor in lipid metabolism in the
brain. The diversity of the cellular processes in which acyl-CoA esters
are known to be involved may partly explain why PPAR appears early
during development and is ubiquitous in adult tissues. A systematic
analysis to correlate PPAR directly with the cellular processes
regulated by acyl-CoA esters will help to identify other target genes
of this receptor.
Regulates Acyl-CoA
Synthetase 2 in Reaggregated Rat Brain Cell Cultures*
,
TOP
ABSTRACT
INTRODUCTION
Supported by a Marie Heim Vögtlin fellowship of the Swiss
National Science Foundation, the Société Académique
Vaudoise, and the Novartis Foundation.
To whom correspondence should be addressed. Tel.:
41-21-692-4110; Fax: 41-21-692-4115; E-mail:
Walter.Wahli@iba.unil.ch.
