From the Core Research for Evolutional Science and Technology,
Japan Science and Technology Corporation and the
Department of Molecular Behavioral Biology, Osaka
Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874 and the
Institute for Molecular and Cellular Regulation, Gunma
University, Showa-machi, Maebashi, Gunma 371-8512, Japan
Received for publication, August 13, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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The promoter function of the rat lipocalin-type
prostaglandin D synthase (L-PGDS) gene was characterized in primary
cultures of leptomeningeal cells prepared from the neonatal rat brain. Luciferase reporter assays with deletion and site-directed mutation of
the promoter region ( Lipocalin-type prostaglandin
(PG)1 D synthase (L-PGDS) was
first identified in the rat brain as an enzyme that catalyzes the conversion to PGD2 from PGH2, the latter being
a common precursor of all prostanoids (1). Later, L-PGDS was found to
be identical to L-PGDS is expressed abundantly in the human heart (14), the male
genital organs (15-17), and the central nervous system (18-20) of
various mammals. In the brain, this enzyme is expressed dominantly in
the leptomeninges, chroid plexus, and oligodendrocytes but poorly in
neurons, astrocytes, and microglial cells (18-20). These distribution
profiles suggest that L-PGDS gene expression is differentially regulated in a tissue- and cell type-specific manner.
García-Fernández et al. (21, 22) found that
L-PGDS gene expression was regulated by the thyroid hormone during
brain development in rats and demonstrated that the thyroid hormone
responsive element was functional in COS-7 cells (23). White et
al. (24) also demonstrated that L-PGDS gene expression was induced
by the thyroid hormone through its responsive element in human TE671
cells derived from a medulloblastoma in the cerebellum. Moreover,
L-PGDS gene expression was induced by dexamethasone in mouse neuronal
GT1-7 cells (25). However, the regulatory mechanisms of L-PGDS gene
expression have never been analyzed in the leptomeningeal cells, which
show the highest expression of this gene (18-20). On the other hand,
the L-PGDS level in the rat CSF was increased by the administration of
a proinflammatory cytokine, interleukin (IL)-1 In this study, we characterized the rat L-PGDS gene promoter in the
primary cultures of leptomeningeal (LM) cells prepared from neonatal
rat brains and identified an AP-2 element for the transcriptional
activation, an E-box element for the repression, and two NF- Cell Culture--
The LM cells were prepared from the external
leptomeninges surrounding the cerebral cortex of neonatal rats as
described previously (33). Rat cell lines, i.e. F2408
(embryo), RNB (astrocyte), and C6 (glia), were obtained from the
Institute for Fermentation (Osaka, Japan). Cells were cultured in
Dulbecco's modified Eagle's medium (Sigma) or Ham's F-10
medium (Invitrogen) supplemented with 10% fetal bovine serum (JRH
Bioscience, Lenexa, KS), 50 units/ml penicillin G (Meiji Seika, Tokyo,
Japan), and 50 µg/ml streptomycin sulfate (Meiji Seika) at 37 °C
under 5% CO2.
RNA Analysis--
Total RNA was prepared by the acid
guanidinium-thiocyanate/phenol/chloroform method (34). For reverse
transcription (RT)-PCR analysis, first-strand cDNAs were
synthesized from total RNA (1 µg) by RNase H
To ensure the transcription level of L-PGDS mRNA, we performed the
competitive RT-PCR. Briefly, 200 ng of total RNA was converted to the
first-strand cDNAs using RNase H
For Northern blot analysis, total RNA (2 µg) was separated
electrophoretically in a 2.2 M formaldehyde-1.2% (w/v)
agarose gel in MOPS buffer (44) and then transferred to a Hybond XL nylon membrane (Amersham Biosciences) in 20× SSC. Probes were labeled with [ Determination of Transcription Initiation Site--
The
transcription initiation site of the rat L-PGDS gene was determined by
the Cap Site Hunting method (46) with rat brain CapSite cDNA
(Nippon Gene, Toyama, Japan). Briefly, first-step PCR was performed by
using LA Taq DNA polymerase (Takara Shuzo) with L-PGDS-E
(5'-CGGAATTCTCTCACCTGTGTTTACTC-3') and 1RDT
(5'-GATGCTAGCTGCGAGTCAAGTC-3'; Nippon Gene) as the primers under the
following amplification conditions: initial denaturation at 95 °C
for 5 min, followed by 94 °C for 15 s, 55 °C for 15 s,
and 72 °C for 1 min for 35 cycles. Nested PCR was carried out by
using L-PGDS-C and 2RDT (5'-CGAGTCAAGTCGACGAAGTGC-3'; Nippon Gene) as
the primers and first-step PCR products as the template under the same
condition as used for the first-step PCR. Resultant PCR products were
cloned into pGEM-T Easy vector. Nucleotide sequences were determined by
the dideoxy termination method with a Thermo Sequenase cycle sequencing
kit (Amersham Biosciences) and a Long Tower Read DNA sequencer
(Amersham Biosciences).
Construction of the Promoter-Luciferase Gene Plasmids--
An
~1.3-kb fragment ( Transient Transfection and Luciferase Assay--
For
transfection, LM cells or those of the three cell lines were seeded at
a density of ~2 × 105 cells/well in 24-well plates
and co-transfected with each construct (0.4 µg) and pRL-CMV (0.1 µg; Promega), the latter carrying the Renilla luciferase
gene under the control of the cytomegalovirus promoter as the
transfection control, by use of Effectene (Qiagen, Hilden, Germany)
according to the method prescribed by the manufacturer. The cells were
cultured for 48 h. In the case of IL-1 Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared as follows: cells were suspended in 500 µl of
ice-cold buffer comprising 10 mM HEPES-KOH, pH 7.5, 10 mM KCl, 0.1 mM EDTA, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride
(PMSF), a mixture of pepstatin, leupeptin, and aprotinin at 2 µg/ml
each, and 0.1% (v/v) Nonidet P-40 and vortexed vigorously. After
centrifugation at 1,000 × g for 1 min at 4 °C, the
nuclear pellet was resuspended in 100 µl of 50 mM
HEPES-KOH, pH 7.5, containing 400 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM PMSF, and a mixture of pepstatin, leupeptin, and
aprotinin at 2 µg/ml each, and incubated for 1 h at 4 °C with
continuous agitation. Nuclear extracts were prepared by centrifugation
at 12,000 × g for 30 min at 4 °C. Protein
concentrations were determined according to the method of Bradford
(47). EMSA was performed as described previously (48).
Co-expression Study with Hes-1--
The expression vector for
human Hes-1 was kindly provided by Dr. Michael Caudy (Cornell
University) and contained the human Hes-1 coding region fused to a
FLAG-tagged sequence (49). Rat LM cells were co-transfected with the
human Hes-1 expression vector and the Chromatin Immunoprecipitation (ChIP) Assay--
LM cells were
cross-linked with 1% formaldehyde at 37 °C for 10 min and
subsequently washed with ice-cold phosphate-buffered saline containing
0.5 mM PMSF and a protease inhibitor mixture of pepstatin,
leupeptin, and aprotinin at 2 µg/ml each. The cells were then lysed
in 200 µl of buffer containing 1% (w/v) SDS, 10 mM EDTA,
50 mM Tris-Cl, pH 8.0, and 0.5 mM PMSF and a
mixture of pepstatin, leupeptin, and aprotinin at 2 µg/ml each and
then sonicated. After centrifugation to remove the cell debris, the supernatant was diluted with 20 mM Tris-Cl, pH 8.0, buffer
containing 150 mM NaCl, 2 mM EDTA, 1% (v/v)
Triton X-100, 0.01% (w/v) SDS, and 0.5 mM PMSF and a
mixture of pepstatin, leupeptin, and aprotinin at 2 µg/ml each and
incubated at 4 °C for 1 h with 60 µl of 50% slurry of
protein G-Sepharose (Amersham Biosciences), 20 µg of shared salmon
sperm DNA (Sigma), and 60 µg of bovine serum albumin (Sigma). After
the protein G-Sepharose had been removed by centrifugation, the
resulting supernatant was incubated with the specific antibody and then
50 µl of protein G-Sepharose was added, and the incubation was
continued at 4 °C for 1 h. The Sepharose beads were washed five
times with washing buffer containing 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 0.05% (v/v) Triton X-100 and twice with
10 mM Tris-Cl, pH 8.0, and 1 mM EDTA. The
immunoprecipitated DNA-protein complexes were eluted with 500 µl of
1% (w/v) SDS and 0.1 M NaHCO3. DNA-protein
cross-links were reversed at 65 °C for 5 h, after which the DNA
was purified with a Qiaquick PCR purification kit (Qiagen) and then
used for PCR amplification with the following gene-specific primer
sets: S1-F (5'-CTGTATAAAGCAGGAGAGTGC-3') and S1-R
(5'-GGCTGATTTGGGAGTTTTCTCC-3'), S2-F (5'-TGTGAGAAGCAGGTCTTAGCC-3') and
S2-R (5'-CATACCTCAGTGAAGAACGGG-3'), and H1-F
(5'-CTCCAGTGGGCAGTCCTTGGG-3') and H1-R (5'-GGGCTGCACGGAGACCTGGGC-3') as
the sense and antisense primers for NF- Western Blot Analysis--
Nuclear extracts were prepared as
described above and separated by SDS-PAGE and transferred onto
polyvinylidene difluoride membranes (Immobilon P; Millipore Corp.,
Bedford, MA). The membranes were incubated with specific primary
antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
washed, and then incubated with second antibodies conjugated to
horseradish peroxidase (Santa Cruz Biotechnology, Inc.). Immunoreactive
proteins were detected by using the ECL Western blotting detection
system according to the manufacturer's instructions (Amersham Biosciences).
Cell Type-specific Expression of the Rat L-PGDS Gene--
The
expression of the L-PGDS gene in the rat LM cells and rat cell lines
was examined by RT-PCR. The rat L-PGDS mRNA was expressed abundantly in the LM cells (Fig.
1A, lane 1) and
negligibly in F2408 (lane 2), RNB (lane 3), and
C6 (lane 4) cell lines, whereas the expression of the
mRNA for G3PDH as the internal control was almost identical in each
cell (Fig. 1A). Transient transfection experiments with the
Transcription Initiation Sites of the Rat L-PGDS
Gene--
Transcription initiation sites were determined by the
CapSite Hunting method. The sequence analysis of 18 clones demonstrated that nine of them had a nucleotide C of 77 base pairs upstream (defined
as +1) of the A of the translation initiation codon, ATG, and that the
remaining nine had a C of 75 base pairs upstream (Fig.
2), indicating that rat L-PGDS gene
expression was initiated predominantly from both of these transcription
initiation sites in the brain. Surrounding both transcription
initiation sites, a TATA-like box (ATAAATA) was found at a position
from Promoter Activity of the Rat L-PGDS Gene--
To determine the
promoter activity and the important cis-elements involved in
the regulation of L-PGDS gene expression, we introduced deletion and
site-directed mutation into the promoter region and then conducted
reporter analyses using rat LM cells. When the region from
A deletion from
In contrast, the deletion of the region from +40 to +77 in exon 1 caused the reporter activity to increase significantly by ~160%
compared with that of the Analysis of Factor Binding to Regulatory Elements by EMSA--
To
demonstrate the nuclear factor(s) specific for binding to the putative
cis-element(s) identified by the promoter luciferase assay,
we conducted an EMSA using 32P-labeled double-stranded
oligonucleotides containing the putative cis-element,
shown in Table I, and nuclear
extracts prepared from the rat LM cells. As shown in Fig.
4A, when the double-stranded rL3 oligonucleotide carrying the AP-2 element was incubated with the
nuclear extracts, two major DNA-protein complexes were found (Fig.
4A, lane 1). The formation of these complexes was
inhibited by the addition of a 10- or 50-fold excess amount of the
unlabeled rL3 oligonucleotide (lanes 2 and 3) or
the authentic AP-2 wild-type (WT) DNA probe (lane
6). In contrast, a 100-fold excess amount of the nonspecific pUC19
DNA (lane 4) or the inactive AP-2 mutant (mu) DNA probe
(lane 5) did not prevent the complex formation. These
results demonstrate the specific binding of nuclear factors to the AP-2
element.
Furthermore, 32P-labeled rL4 oligonucleotide containing the
E-box bound a nuclear factor (Fig. 4B, lane 1).
The formation of this DNA-protein complex was decreased in a
concentration-dependent manner by the addition of a 10- or
50-fold excess amount of the unlabeled rL4 oligonucleotide (lanes
2 and 3) but was not inhibited by a 100-fold excess
amount of the nonspecific pUC19 DNA (lane 4) or the inactive
E-box (mu) mutant oligonucleotide (lane 5). Thus, the
nuclear factor prepared from the rat LM cells bound to the E-box in a
nucleotide sequence-specific manner.
Function of the E-box in the Regulation of L-PGDS Gene
Expression--
The E-box is well known as the cis-element
for the Hes protein that is involved in the Notch-Hes signaling
pathway. Thus, we examined the expression of the genes involved in the
Notch-Hes signaling pathway by RT-PCR and found that Jagged,
a ligand for Notch receptors, Notch1 and Notch3,
and Hes-1 were expressed in the rat LM cells (Fig.
5A).
Co-expression of the
We then conducted EMSA by incubating the rL4 oligonucleotide containing
the E-box with nuclear extracts prepared from rat LM cells transiently
expressing the human Hes-1 protein. Evidence of formation of a
DNA-protein complex was observed (Fig. 5D, lane 2), but no complex was found in the absence of the nuclear
extracts (lane 1). This complex failed to form in the
presence of 10- or 50-fold excess amount of the unlabeled rL4
oligonucleotide (lanes 3 and 4). The formation of
the complex was not inhibited by a 100-fold excess amount of
nonspecific pUC18 DNA (lane 5). In the supershift assay,
this DNA-protein complex migrated more slowly when anti-FLAG monoclonal
antibody had been added (lane 6). In the non-transfected LM
cells, the corresponding shifted band (*) was observed in both in the
presence or absence of the FLAG antibody (lanes 7 and
8). In contrast, no corresponding supershifted band (**) was
observed even when FLAG antibody was added (lane 8).
Furthermore, we confirmed the in vivo binding of the
recombinant Hes-1 protein to the E-box of the rat L-PGDS gene by using the ChIP assay. The Hes-1 protein bound to the E-box. The amount of
DNA-protein complex was increased in a concentration-dependent manner by the transfection with the Hes-1 vector (Fig. 5E).
In the negative control experiment, when S1-F and S1-R as primers for
NF- Enhancement of Transcription by IL-1
Through the data base search, we found two NF-
Furthermore, we confirmed that nuclear factors bound to each of the
NF- We earlier reported that the L-PGDS gene was expressed
predominantly in the leptomeninges in the rat brain (18, 19). Indeed, its expression in the rat LM cells was higher than that in other cell
lines such as astrocytes and other glial cells (Fig. 1). These results
indicate that rat L-PGDS gene expression is strictly regulated in a
cell type-specific manner. In this study, we examined the promoter
function of the rat L-PGDS gene in the LM cells. Fig.
8 shows a schematic representation of our
proposed regulatory mechanism governing the transcription of the rat
L-PGDS gene in the LM cells.
1250 to +77) showed that an AP-2 element at
109 was required for activation and an E-box at +57, for repression. Binding of nuclear factors to each of these cis-elements
was demonstrated by an electrophoretic mobility shift assay. Several
components of the Notch-Hes signaling pathway, Jagged,
Notch1, Notch3, and Hes-1, were
expressed in the leptomeningeal cells. Human Hes-1 co-expressed in the
leptomeningeal cells bound to the E-box of the rat L-PGDS gene, and
repressed the promoter activity of the rat L-PGDS gene in a
dose-dependent manner. The L-PGDS gene expression was
up-regulated slowly by interleukin-1
to the maximum level at 24 h. The reporter assay with deletion and mutation revealed that two
NF-
B elements at
1106 and
291 were essential for this up-regulation. Binding of two NF-
B subunits, p65 and c-Rel, to these
two NF-
B elements occurred after the interleukin-1
treatment. Therefore, the L-PGDS gene is the first gene identified as the target
for the Notch-Hes signal through the E-box among a variety of genes
involved in the prostanoid biosynthesis, classified to the lipocalin
family, and expressed in the leptomeninges. Moreover, the L-PGDS gene
is a unique gene that is activated slowly by the NF-
B system.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-trace (2, 3), which had been identified earlier (4) as the major protein in human cerebrospinal fluid (CSF).
PGD2 is a major prostanoid in the brain, known as the most
potent endogenous somnogenic substance thus far identified (5), and is
involved in various physiological events such as regulation of sleep
and pain responses (5-8). Because human L-PGDS-overexpressing
transgenic mice (9) and L-PGDS-gene knock-out mice (10, 11) showed abnormality in the regulation of non-rapid eye movement (NREM) sleep
and pain responses, PGD2 produced by L-PGDS is thought to play important roles in the regulation of NREM sleep and pain sensation
in the central nervous system. Alternatively L-PGDS binds small
lipophilic molecules such as retinal and retinoic acid
(Kd = 70-80 nM) (12) and biliverdin and
bilirubin (Kd = 33-37 nM) (13) with
affinities higher than those of other members of the lipocalin family.
Therefore, L-PGDS is a unique bifunctional protein acting as a
PGD2-producing enzyme and as a lipophilic molecule-binding
protein (6-8).
(26). IL-1
and tumor necrosis factor-
activate the NF-
B system (27) to elicit somnogenic activities (28, 29). The sleep-inducing effects of IL-1
and tumor necrosis factor-
were proposed to be mediated by L-PGDS,
because these effects were inhibited by administration of inhibitors of
PG synthesis to animals (29). However, the transcriptional regulation
of the L-PGDS gene by the NF-
B system has never been clarified.
B
elements for slow induction by IL-1
. Among various genes involved in
prostanoid biosynthesis, classified as members of the lipocalin family
and expressed in the leptomeninges, the L-PGDS gene is the first one
identified to be a target of Hes-1, a mammalian homolog of
Drosophila Hairy and Enhancer of
split of the Notch-Hes signal pathway. The L-PGDS gene is
thus the second identified gene encoding an enzyme suppressed by the
Notch-Hes signal, as the human acid
-glucosidase gene was the first
one (30, 31). Moreover, the L-PGDS gene is a novel example of a small
number of genes slowly up-regulated through activation of NF-
B
subunits by IL-1
, similar to the Mn-superoxide dismutase gene (32)
but different from many of the IL-1
-activated genes identified up to
the present.
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ABSTRACT
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SuperScript II reverse transcriptase (Invitrogen) and oligo(dT)-adapter primer (Takara Shuzo, Kyoto, Japan) at 42 °C for 60 min after denaturation of the RNA at 72 °C for 3 min. PCR was conducted with
RT products used as the template, LA Taq DNA
polymerase (Takara Shuzo), and gene-specific primer sets under the
following conditions: initial denaturation at 96 °C for 5 min,
followed by 96 °C for 20 s, 55 °C for 20 s, and
72 °C for 1 min for 30-40 cycles. The gene-specific primer sets
used were chemically synthesized with the reference to the deposited
sequence information. L-PGDS-T (5'-GACAAGTTTCTGGGGCGCTGGT-3')
and L-PGDS-C (5'-GCTGTAGAGGGTGGCCATGCGG-3') for L-PGDS (35), G3PDH-5'
(5'-ACCACAGTCCATGCCATCAC-3') and G3PDH-3' (5'-TCCACCACCCTGTTGCTGTA-3')
for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (36),
Jagged-F (5'-ATGCGGTCCCCACGGACGCGC-3') and Jagged-R (5'-GCCTTTTCAATTATGCTATCAGG-3') for Jagged (37), Jagged2-F
(5'-CCCGTGCTGGGTAGCAACTCC-3) and Jagged2-R
(5'-GCCCTGCCAGCCATAGCTGCACC-3') for Jagged2 (38), HES1-F
(5'-GTCATCAAAGCCTATCATGGAG-3') and HES1-R
(5'-GTGCGCCTGCCCGGGGTAGGTC-3') for Hes-1 (36), HES2-F
(5'-ATGAGACTGCCTAGAGGAGTAGGGG-3') and HES2-R
(5'-CTACCATGGCCTCCAAAGTCCAAGGC-3') for Hes-2 (39), HES3-F (5'-ATGGAGAAGAAGCGTCGTGCCCGC-3') and HES3-R
(5'-TCACCAGGGCCGCCACACGCGAAACC-3') for Hes-3 (40), HES5-F
(5'-ATGGCCCCAAGTACCGTGGCGG-3') and HES5-R (5'-TCACCAGGGCCGCCAGAGGCCG-3') for Hes-5 (41), Notch1-F1
(5'-ATGCCGCGGCTCCTGGCGCCC-3') and Notch1-F2
(5'-CGTGCTATGTTGTGGACCATGGC-3') as the forward primers and Notch1-R
(5'-CACACTCGTGGGTGGTGTCCCCCG-3') as the reverse primer for
Notch1 (42), Notch2-F1 (5'-GTCCTCAGGCTTCCCTGGAGGAC-3') and Notch2-F2 (5'-GGCAATCCTTTGCTTGCATCCC-3') as the forward primers and
Notch2-R (5'-GGGGTGAGAGGTCGAGTATTGGC-3') as the reverse primer for
Notch2 (43), and Notch3-F1 (5'-CCCCCTTGTCTGGATGGAAGCCC-3') and Notch3-F2 (5'-CCCGGGAGGCTGCTTGCCTGTGC-3') as the forward primers and Notch3-R (5'-GCCAGGAAGACAAGCACAGTC-3') as the reverse primer for Notch3 (GenBankTM accession
number AF164486). Notch mRNAs were detected by nested PCR. In the
first-step PCR, the following primer sets were used: Notch1-F1 for
Notch1, Notch2-F1 for Notch2, or Notch3-F1 for
Notch3 as the forward primer and M13 M4 primer (Takara
Shuzo) as the reverse primer. In the nested PCR, Notch1-F2 and Notch1-R
for Notch1, Notch2-F2 and Notch2-R for Notch2, or
Notch3-F2 and Notch3-R for Notch3 were used as the forward
and reverse primers, respectively. The resultant PCR products were
analyzed by agarose gel electrophoresis, purified, and cloned into
pGEM-T Easy vector (Promega, Madison, WI) to verify the nucleotide sequences.
SuperScript II
reverse transcriptase in the presence of serial diluted RNA standard
(competitor) with random 9-mer primer (Takara Shuzo). PCR amplification
was performed using sets of specific primers of L-PGDS-T and L-PGDS-C
for L-PGDS or G3PDH-5' and G3PDH-3' for G3PDH, ExTaq DNA polymerase
(Takara Shuzo), and the RT products. After the initial denaturation at
95 °C for 5 min, PCR was carried out for 15 s at 94 °C,
15 s at 55 °C, and 20 s at 74 °C for 28 cycles. The
amplified products were analyzed by electrophoresis on 2% agarose gel
followed by staining with ethidium bromide. The stained products were
scanned and analyzed by ChemiImager system (Alpha Innotech, San
Leandro, CA). The expression level of L-PGDS gene was expressed
relative to that of the G3PDH gene.
-32P]dCTP by the random priming method
(45). Hybridization was performed at 42 °C overnight in a solution
consisting of 50% (v/v) formamide, 5× Denhardt's solution, 6× SSC,
0.5% (w/v) SDS, and 100 µg/ml shared salmon sperm DNA (44). The
blots were washed twice in 2× SSC/0.1% (w/v) SDS at room temperature
for 10 min and twice in 0.2× SSC/0.1% (w/v) SDS at 55 °C for 15 min. Blots were analyzed with a fluorescent imaging analyzer (FLA2000;
Fuji Photo Film, Tokyo, Japan).
1250 to +77) consisting of the rat L-PGDS gene
promoter was amplified by PCR with the gene-specific primer set with
XhoI (sense) or HindIII (antisense) sites at
their respective 5'-end. PCR fragments doubly digested with
XhoI and Hind III were cloned into the upstream
region of the luciferase reporter gene of the pGL3-Enhancer vector
(Promega). The luciferase-reporter construct carrying the promoter
region from
1250 to +77 was designated as
1250/+77. A deletion
series was constructed in the same manner. Site-directed mutations were
introduced into the putative cis-elements by PCR using
mutated primers. All constructs were subjected to nucleotide sequencing
to verify their correct sequence and orientation.
stimulation, the cells
were transfected in the same way as described above. After 48 h of
transfection, the medium was replaced with fresh medium with or without
IL-1
(1 ng/ml; R&D Systems, Minneapolis, MN), and the cells were
then incubated further for 24 h. The luciferase activities were
measured by using a dual luciferase reporter assay kit (Promega). The
reporter activity was calculated relative to that of pGL3-Enhancer
vector and was defined as 1. All data were obtained from at least three
independent experiments, and each experiment was performed in
duplicate. The relative promoter activities were depicted as the
mean ± S.D.
1250/+77 construct as described
above. After 48 h of transfection, the cells were harvested, and
the luciferase activities were then measured.
B site 1, site 2, and E-box,
respectively, of the rat L-PGDS promoter. PCR amplification was
performed as follows: initial denaturation at 95 °C for 3 min,
followed by 94 °C for 25 s, 55 °C for 25 s, and
72 °C for 30 s for 34-40 cycles. The resultant PCR products
were analyzed on 2% (w/v) agarose gels.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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1250/+77 construct in the rat LM cells and each of the three rat cell
lines demonstrated that efficient reporter activity was detected only
in the LM cells (Fig. 1B). This result is in agreement with
the expression of the L-PGDS mRNA demonstrated by the RT-PCR
analysis and shows that the promoter region from
1250 to +77 contains
the critical element(s) responsible for the cell type-specific
expression of the rat L-PGDS gene. Therefore, we used this promoter
region to study the transcriptional regulation mechanism involving the
activation and/or suppression of L-PGDS gene expression in the LM
cells.
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Fig. 1.
Cell type-specific expression of the rat
L-PGDS gene. A, total RNA was prepared from the rat LM cells
or each rat cell line, and the L-PGDS mRNA was detected by RT-PCR
analysis as described under "Experimental Procedures."
M, molecular size marker. Lane 1, LM cells;
lane 2, F2408 cells; lane 3, RNB cells; and
lane 4, C6 cells. G3PDH mRNA was also amplified as the
internal control. Sizes of expected PCR products were 342 and 452 base
pairs for L-PGDS and G3PDH, respectively. B, promoter
activity of the L-PGDS gene in rat cells. The 1250/+77 construct was
used to transfect the rat LM cells (lane 1) and rat cell
lines F2408 (lane 2), RNB (lane 3), and C6
(lane 4). The luciferase activity in the rat LM cells was
defined as 100%.
28 to
22, but no typical CCAAT box was observed. Through a
TRANSFAC search of the promoter region, several putative
cis-elements such as NF-
B elements at
1106 and
291,
an Sp-1 element at
926, an AP-2 element at
109, and an E-box at +57
were found. To elucidate the evolutional conservation of the promoter
sequences between the human and rat L-PGDS genes, we aligned the
5'-flanking regions of both sequences (Fig. 2). The overall homology
(61.9%) between them was observed in a region of ~400 bp around the
transcriptional initiation site. One of the transcription initiation
sites of the rat L-PGDS gene showed a one-nucleotide difference from
that of the human L-PGDS gene (24). Furthermore, several
cis-elements were conserved between them, including the
TATA-like box (
28 to
22 for rat and human), the AP-2 element (
109
to
101 and
98 to
90 for rat and human, respectively), and the E-
or N-box (+57 to +62 and
334 to
329 for rat and human,
respectively). These results suggest that the human and rat L-PGDS
genes are regulated in a similar manner.
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Fig. 2.
Conservation of the promoter regions of the
rat and human L-PGDS genes. Dashes show the gaps to
make maximum identity. Conserved nucleotide residues are
dotted, and putative cis-elements for
transcription factors are underlined and indicated.
TATA-like box is boxed and indicated. Transcription
initiation sites of the rat L-PGDS gene are indicated, and
numbers refer to the number of 5'-end of transcripts in the
5'-oligo capping analysis. The first transcription initiation site is
defined as +1. The transcription initiation site identified previously
at +39 (50) is circled. The translational initiation codon,
ATG, is shown in bold.
1250 to
+77 was used for the luciferase-reporter assay, efficient reporter
activity was detected, indicating that this region contained
cis-element(s) responsible for the regulation of gene
expression (Fig. 1B). By deletion of the region from
1250 to
281, which contained two NF-
B elements at
1106 and
291, and
the Sp-1 element at
926, the promoter activity was decreased by
~30% compared with that of the
1250/+77 construct (Fig.
3), indicating that this region contained
weak activation activity for the expression.
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Fig. 3.
Deletion and site-directed mutation analyses
of the promoter region of the rat L-PGDS gene. A, each
of two putative cis-elements, AP-2 and E-box, was subjected
to site-directed mutagenesis with mutations indicated by the nucleotide
changes. B, scheme of the rat L-PGDS promoter-luciferase
constructs used in the transient transfection experiments and the
relative luciferase activity. The putative cis-acting and
TATA-like elements are indicated at the top. Site-directed
mutagenesis was introduced by PCR using primers with mutations.
Relative reporter activities of the deletion and mutated constructs are
shown by black and gray columns, respectively.
Empty vector means pGL3-Enhancer vector. Data are the means ± S.D. from at least three independent experiments.
280 to
100 resulted in a drastic decrease in the
promoter activity by nearly 80% as compared with that of the
1250/+77 construct. This region contained the AP-2 element at
109.
By introduction of the site-directed mutagenesis into the AP-2 element,
the luciferase reporter activity was decreased to almost the same level
as when the AP-2 element had been deleted. These results indicate that
the AP-2 element is an important cis-element for the
transcriptional activation of the rat L-PGDS gene. Furthermore, when the
1250/
75 construct was used for transfection, measurable reporter activity was no longer detected, indicating that the region
from
74 to +1, containing the TATA-like box at
28 was essential for
the basal expression of the L-PGDS gene in the LM cells.
1250/+77 construct, indicating that a
negative regulator binds to this region from +40 to +77. This region
contained the E-box at +57 involved in the Notch-Hes signaling pathway.
Introduction of a mutation into the E-box increased the reporter
activity by ~130% compared with that of the wild-type
1250/+77
construct. These results clearly indicate that L-PGDS gene expression
was suppressed through the E-box in the LM cells.
Oligonucleotide used for EMSA
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Fig. 4.
EMSA for the rat L-PGDS promoter. Each
of the [ -32P]-labeled double-stranded
oligonucleotides, rL3 containing the AP-2 element (A) and
rL4 containing the E-box (B), was incubated for 20 min at
room temperature with the nuclear extracts prepared from the rat LM
cells. The incubation mixture was analyzed by electrophoresis on 5%
polyacrylamide gels as described under "Experimental Procedures."
The shifted complexes are indicated by arrows. Unlabeled
oligonucleotides were added as the competitor.
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Fig. 5.
Transcriptional suppression of the rat L-PGDS
gene by Hes-1. A, expression of the genes involved in
the Notch-Hes signaling pathway in the LM cells. Expression was
analyzed by RT-PCR with the gene-specific primer sets shown in Table I.
M indicates the 100-base pair ladder marker. The predicted
sizes of the PCR products for Jagged, Jagged2,
Notch1, Notch2, Notch3,
Hes-1, Hes-2, Hes-3, and
Hes-5 were 563, 523, 512, 560, 502, 373, 474, 528, and 501 base pairs, respectively. Amplified signals are indicated by
arrows. B, recombinant Hes-1 protein in the
nuclear fraction of the LM cells transfected with the indicated amount
of Hes-1 expression vector. Recombinant Hes-1 protein fused with FLAG
tag in the nucleus was detected with anti-FLAG monoclonal antibody.
C, suppression of L-PGDS gene expression by Hes-1. Rat LM
cells were co-transfected with the 1250/+77 construct and 0, 0.1, 1, or 5 µg of the human Hes-1 expression vector. The luciferase
activities were measured 48 h after transfection. When no
expression vector was used, the reporter activity was defined as 100%.
Firefly and Renilla luciferase activities are indicated by
closed and open columns, respectively.
D, double-stranded rL4 oligonucleotide containing the E-box
labeled with [
-32P]ATP was incubated with nuclear
extracts prepared from the rat LM cells transfected with the human
Hes-1 expression vector, and the mixtures were then subjected to EMSA.
Unlabeled rL4 oligonucleotide was added as the competitor. The shifted
band is indicated by an asterisk. Supershift assay was
conducted by using anti-FLAG monoclonal antibody, and the supershifted
signal is indicated by a double asterisk. E,
in vivo binding of the Hes-1 protein to the E-box of the rat
L-PGDS gene. The LM cells transfected with the Hes-1 expression vector
were treated with formaldehyde to cross-link DNA and proteins. The
DNA-protein complexes were immunoprecipitated by the anti-FLAG
antibody, and the extracted DNA was used as the template for PCR
amplification (IP). A small aliquot before
immunoprecipitation was used for PCR amplification as the input control
(Input). N.C. means the negative control for this
assay using a primer set of S1-F and S1-R for the NF-
B site 1, whose
expected amplified region does not contain the E-box consensus
sequence. Results are representative of two independent
experiments.
1250/+77 construct with the human Hes-1 protein
was performed in the rat LM cells. The recombinant human Hes-1 protein
fused with a FLAG tag was detected with anti-FLAG antibody in the
nuclear extracts prepared from the transfected LM cells (Fig.
5B). The recombinant human Hes-1 protein level in the
nucleus of the transfected LM cells was dependent on the amount of the
expression vector used for the transfection (Fig. 5B).
Furthermore, the promoter activity was decreased in a
concentration-dependent manner by the Hes-1 expression
vector (Fig. 5C). On the other hand, Renilla
luciferase activities as the internal control for transfection were
almost the same in all experiments, even when the Hes-1 protein was
overexpressed in the LM cells (Fig. 5C).
B site 1 of L-PGDS gene were used for the subsequent PCR amplification, the amplification of its region containing no E-box consensus sequence was not detected (N.C. in Fig.
5E). Therefore, these results, taken together, indicate that
the Hes-1 protein binds specifically to the promoter region of the
L-PGDS gene in vivo, as well as in vitro, and
thereby suppresses the L-PGDS gene expression through the E-box in the
rat LM cells. This is the first proof that the L-PGDS gene, which is
involved in prostanoid biosynthesis, classified as a member of the
lipocalin gene family, and expressed in the leptomeninges, is regulated
transcriptionally by the Notch-Hes signal pathway.
--
We examined the
change of L-PGDS mRNA level in the rat LM cells treated with or
without IL-1
(1 ng/ml) by the competitive RT-PCR assay. When the LM
cells were treated with IL-1
, L-PGDS mRNA level was increased
slowly with a peak at 24 h after the start of treatment
(~2.5-fold over the control) (Fig.
6A). We also confirmed its
induction by Northern blot analysis. L-PGDS gene expression was
enhanced to a level of ~2.5-fold over the control after 24 h of
IL-1
treatment (Fig. 6B, upper panel), whereas
G3PDH gene expression was not altered by the IL-1
treatment (Fig.
6B, lower panel). In the nuclear fraction of the
LM cells, NF-
B subunits, p65 and c-Rel, but not p50, were detected
by Western blot analysis, and their nuclear accumulations was enhanced
by the treatment with IL-1
(Fig. 6C).
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Fig. 6.
Identification of the
cis-elements responsible for the effect of
IL-1 treatment. A, rat LM
cells were cultured in the presence or absence of IL-1
(1 ng/ml) for
the indicated time and then collected for the isolation of RNA and the
subsequent competitive RT-PCR assay. Open and closed
bars indicate no-treatment (control) and IL-1
treatment. The
data represent the mean ± S.D. from three independent assays.
B, induction of L-PGDS gene expression by IL-1
. The cells
were cultured for 24 h with or without 1 ng/ml IL-1
. Total RNA
(2 µg) was utilized for the Northern blot analysis with rat L-PGDS
cDNA (upper panel) (35) or G3PDH cDNA (lower
panel) (36) as the probes. C, induction of the NF-
B
proteins by the treatment of LM cells with IL-1
. NF-
B proteins,
p50, p65, or c-Rel were detected with each specific antibody in the
nuclear fraction prepared from LM cells incubated with (+) or without
(
) IL-1
(1 ng/ml) for 24 h. D, a site-directed
mutation was introduced into each of the two NF-
B elements, and the
nucleotide changes are indicated. E, each region-deleted or
mutated NF-
B element was connected to the luciferase reporter gene,
and LM cells were transfected with each construct along with the
Renilla luciferase expression plasmid. After 48 h,
IL-1
(1 ng/ml) was added (closed columns) or not
(open columns), and the cells were then cultured for 24 h. Data are presented as the means ± S.D. from at least three
independent experiments.
B elements in the
promoter region of the rat L-PGDS gene and termed them NF-
B site 1 at
1106 and site 2 at
291 (see Fig. 3B and Fig.
6D). Progressive deletion analysis of the L-PGDS promoter
region of the
1250/+77 construct demonstrated that two regions
containing the NF-
B element from
1250 to
950 and from
450 to
280 were required for the IL-1
-mediated induction of L-PGDS gene
expression (Fig. 6E). By introducing mutation into each of
these two NF-
B sites, we found the luciferase reporter activity in
the presence of IL-1
to have decreased to almost the same extent as
when the region containing the NF-
B site had been deleted (Fig.
6D).
B elements by conducting EMSA using nuclear extracts prepared
from the rat LM cells treated with IL-1
and specific oligonucleotides, rL1 and rL2 containing NF-
B site 1 and 2, respectively. Fig. 7A shows
that nuclear factor-DNA complexes (
and
) were observed in both
NF-
B sites (lanes 2 and 6). In addition, a
weak signal (
) was often observed only in the site 2 (lane
6). Each signal was absent when an excess amount of the
corresponding DNA probes was added (lanes 3, 4,
7, and 8). To confirm the binding of NF-
B
subunits to each of the two NF-
B sites, we carried out the ChIP
assay using specific antibodies for p50, p65, and c-Rel of NF-
B
subunits. No binding of NF-
B subunits was observed at either NF-
B
site when the LM cells were not treated with IL-1
(Fig.
7B). When the LM cells were treated with IL-1
, however, p65 and c-Rel predominantly bound to both NF-
B site 1 and site 2. These results indicate that L-PGDS gene expression was induced by
IL-1
through these two NF-
B elements in the LM cells and that
this induction occurred through the binding of p65 and c-Rel to both
NF-
B site 1 and site 2.
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Fig. 7.
Binding of nuclear factors to the two
NF- B elements. A,
double-stranded rL1 or rL2 oligonucleotide containing NF-
B site 1 or
site 2 labeled with [
-32P]ATP was incubated with
nuclear extracts prepared from the rat LM cells that had been treated
with 1 ng/ml IL-1
for 24 h, and the mixtures were then
subjected to EMSA. Unlabeled rL1 or rL2 oligonucleotide was added as
the competitor. Shifted bands are indicated by arrows.
B, ChIP assay to detect the in vivo binding
between NF-
B proteins and L-PGDS promoter. The LM cells were treated
(+) or not (
) with IL-1
for 24 h and then fixed with
formaldehyde. Chromatin prepared from the formaldehyde-fixed cells was
sonicated and then immunoprecipitated with anti-p50, anti-p65, or
anti-c-Rel antibodies. Cross-linkage of the co-precipitated DNA-protein
complexes was reversed, and DNA was used as the template for PCR
amplification with specific primers for NF-
B site 1 or site 2 of the
L-PGDS promoter (IP). The aliquot prior to
immunoprecipitation was also amplified as the input control
(Input). Results are representative of two or three
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Schematic representation of the proposed
mechanism for transcriptional regulation of the rat L-PGDS gene in LM
cells.
In a previous study, the transcription initiation site for the rat
enzyme was determined at +39 by primer extension analysis (50).
However, recent efforts at expressed sequence tags sequencing gave several rat expressed sequence tags clones showing high homology to L-PGDS and carrying the upstream region of the transcription initiation site decided previously. We determined the novel
transcription initiation sites of the rat L-PGDS gene by the 5'-oligo
capping method to be located 77 or 75 base pairs upstream from the
translational initiation ATG codon (Fig. 2). The upstream transcription
initiation site showed only a 1-base difference from that for the human
L-PGDS gene (24) or the mouse
gene.2 Furthermore,
the transcription of the mouse gene was initiated from one other
position2 that was identical
to the one decided previously at +39 in the rat (50). Therefore, the
initiation site identified previously at +39 in the rat gene is
considered to also be one of the transcriptional initiation sites of
the rat gene. A TATA-like sequence
(28ATAAATA
22) was found around the
transcription initiation site in the rat gene. The deletion of this
TATA-like element abolished completely the promoter activity,
indicating that the TATA-like element was essential for the basal
expression of the rat L-PGDS gene. The sequences around the TATA-like
element and transcription initiation sites of human and rat genes
showed a high degree of conservation, indicating that the
transcriptional regulation of these L-PGDS genes is similar among
various animal species.
We analyzed the 5'-flanking region of the rat L-PGDS gene in the LM
cells. The AP-2 element at 109, GCCGCCCGC, whose consensus sequence
is GCCNNNGCC (51), is a cis-element that binds AP-2 proteins, which are involved in the regulation of numerous cellular events and are responsible for transmitting the signal from cAMP or
phorbol ester (52, 53). Deletion of the AP-2 element caused a
significant decrease in the promoter activity, but the basal promoter
activity still remained intact (see Figs. 3 and 6). EMSA demonstrated
that a nuclear factor bound to the AP-2 element (Fig. 4B).
These findings indicate that AP-2 activates rat L-PGDS gene expression
but is not associated with the basal promoter activity, unlike the case
of the human hematopoietic PGDS gene, where it is associated with both
(48).
The rat L-PGDS promoter carries the thyroid hormone responsive element
at 596, and it was shown to function in COS-7 cells (23). On the
contrary, this element was not functional in the LM cells under our
experimental conditions (Fig. 3). In hyperthyroidism, L-PGDS gene
expression was decreased in the interlaminar meninges but not in the
external leptomeninges surrounding the cerebellum, although its
expression was strong in both membranes in normal rats (22). Thus
L-PGDS gene expression in the leptomeninges is not affected by thyroid
hormone and is regulated by a cell type-specific mechanism.
The Notch-Hes signaling contributed to the regulation of rat L-PGDS
gene expression in the LM cells. Hes is a member of the basic
helix-loop-helix protein family and acts as a transcriptional repressor
by binding to the E- or N-box of target genes (39, 54). The Notch-Hes
signaling pathway is important for the determination of the cell fate
or the embryonic development by inhibiting cellular differentiation in
a variety of cells (55, 56). The rat L-PGDS gene expression was
repressed through the E-box, and the binding of the Hes-1 protein to
the E-box was proved in vitro and in vivo by EMSA
and ChIP assay. Further evidence for the role of Hes-1 as a
transcriptional repressor was provided by the observation that
co-expression of the human Hes-1 protein decreased the reporter activity of the 1250/+77 construct in the LM cells (Fig.
5C). In the human L-PGDS promoter, an N-box (CACCAG) was
found at
334, and the lack of the region containing the N-box
increased significantly the reporter activity, indicating that this
N-box might be functional for transcriptional repression and probably
that its regulation is the same as in the case of the rat gene (24).
However, such action was not discussed with respect to the human gene
(24). Major component proteins involved in the Notch-Hes signaling
pathway such as Jagged, Notch1,
Notch3, and Hes-1 were expressed in the rat LM
cells (Fig. 5A). Thus it is possible that the Notch-Hes signal is functionally involved in the regulation of L-PGDS gene expression through the binding of the Hes-1 to the E-box in the rat LM
cells. Recently, presenilin-1 was found to digest the intracellular domain of the Notch receptor to activate the Notch-Hes signal (57).
Furthermore, presenilin-1 is involved in the development of the central
nervous system. Its mRNA is expressed in the leptomeninges in the
mouse brain, and, moreover, in presenilin-1-deficient mice, the
leptomeninges appeared to be thicker (58). Thus, the L-PGDS gene,
highly expressed in the leptomeninges, might have potential roles
mediated by the signal of the Notch-Hes pathway during development. Our
present data provide the first proof that the Notch-Hes signal regulates transcriptionally a gene involved in prostanoid biosynthesis, classified as a member of the lipocalin gene family, and expressed dominantly in the leptomeninges. Roles of L-PGDS under the control of
the Notch-Hes signal need to be further elucidated.
The region from 1250 to
280 contained three putative
cis-elements, two NF-
B elements at
1106 and
291 and
the Sp-1 element at
926; however, deletion of this region did not
result in a significant decrease in the promoter activity. On the other
hand, when the cells were treated with the proinflammatory cytokine IL-1
, L-PGDS gene expression was enhanced ~2.5-fold, but it was slowly induced to its maximal level, which was attained after 24 h
of treatment. Such a slow induction by proinflammatory cytokines resembles the case of Mn-superoxide dismutase gene, in which there was
a peak at 24 h after the start of IL-1
treatment (59). These
two NF-
B sites (GGGGGTTTCA at
1106 and GGGCTTTGCT at
291) of the
L-PGDS gene are well in line with the NF-
B consensus sequence, GGGNNTYYCC (60). Site-directed mutation analysis and EMSA demonstrated that these two NF-
B sequences were required for IL-1
-mediated promoter activity of the L-PGDS gene. Moreover, the ChIP assay with
antibodies against NF-
B subunits demonstrated that each of the two
NF-
B sites was recognized and bound in vivo by the NF-
B p65 and c-Rel as the homodimeric or heterodimeric complex. These results indicate the activation mechanism of the L-PGDS gene by
proinflammatory cytokine IL-1
acting through these two NF-
B
elements in the LM cells. Moreover, IL-1
induces the L-PGDS level in
the CSF (26), in which case its induction may occur through enhancement
of L-PGDS gene expression via activation of NF-
B subunits.
Cyclooxygenase-2, an upstream enzyme of PGDS, is responsible for the
inflammation signal and is also transcriptionally activated by NF-
B
(61, 62). However, IL-1
-mediated induction of the cyclooxygenase-2
gene is rapid and completely different from the slow induction of the
L-PGDS gene. The molecular mechanism and physiological roles of such
slow induction of L-PGDS gene expression are currently under investigation.
L-PGDS gene is involved in the regulation of NREM sleep (9), and
IL-1 is one of the somnogenic substances and is involved in NREM
sleep induction through the activation of NF-
B (27-29). Furthermore, IL-1
receptor was detected in high concentration in the
leptomeninges (63, 64). Our present findings, together with previous
results, indicate that the IL-1
-mediated NREM sleep may, at least in
part, occur by enhancement of L-PGDS gene expression in the leptomeninges.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Michael Caudy (Cornell University) for kindly providing the human Hes-1 expression vector and Dr. Osamu Hayaishi (Osaka Bioscience Institute) for continuous encouragement and support of this study. We also thank Shuko Sakae for secretarial assistance.
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FOOTNOTES |
---|
* This work was supported in part by a grant from the program for Core Research for Evolutional Science and Technology of Japan Science Technology Corporation (to Y. U.), Grant-in-aid for Scientific Research 12558078 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y. U.), grants from the Ground Research Announcement for Space Utilization Promotion from the Japan Space Forum (to Y. U.), the Pilot Applied Research Project for the Industrial Use of Space from the National Space Development Agency of Japan (NASDA) and Japan Space Utilization Promotion Center (JSUP) (to Y. U.), the Takeda Science Foundation (to K. F., Y. F., H. K., and Y. U.), the Special Coordination Fund for Promoting Science and Technology (to Y. U.), and Osaka City.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: Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 1-98 Kasugadenaka 3-chome, Konohana-ku, Osaka 554-8558, Japan.
¶ Present address: Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., 17-85, Jusohonmachi 2-chome, Yodogawa-ku, Osaka 532-8686, Japan.
** To whom correspondence should be addressed. Tel.: 81-6-6872-4851; Fax: 81-6-6872-2841; E-mail: uradey@obi.or.jp.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M208288200
2 K. Fujimori and Y. Urade, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: PG, prostaglandin; L-PGDS, lipocalin-type PGD synthase; CSF, cerebrospinal fluid; NREM, non-rapid eye movement sleep; IL, interleukin; LM, primary cultures of leptomeningeal; RT, reverse transcription; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; WT, wild-type; mu, mutant; MOPS, 4-morpholinepropanesulfonic acid.
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