Molecular Cloning, Expression, and Characterization of Rat Homolog of Human AP-2
That Stimulates Neuropeptide Y Transcription Activity in Response to Nerve Growth Factor
Bing-Sheng Li,
Phillip R. Kramer,
Weiqin Zhao,
Wu Ma,
David A. Stenger and
Lei Zhang
Laboratory of Neurochemistry (B.-S.L., P.R.K.)
Laboratory of Adaptive Systems (W.Z.) NINDS National
Institutes of Health Bethesda, Maryland 20892-4130
Center for Bio/Molecular Science and Engineering, (W.M.,
D.A.S.), Naval Research Laboratory Washington, D.C.
20375
Endocrinology and Behavior Branch (L.Z.) National
Institute of Mental Health National Institutes of Health
Bethesda, Maryland 20892
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ABSTRACT
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Neuropeptide Y (NPY) plays an important role
in the central regulation of neuronal activity, endocrine and sexual
behavior, and food intake. Although transcription activity of the NPY
gene in PC12 cells is regulated by a number of agents such as nerve
growth factor (NGF), the mechanism responsible for the NGF-elicited
increase in the transcription of the NPY gene remains to be explored.
In this study, we isolated and characterized a nuclear protein that is
bound to NGF-response elements (NGFRE) that lie between nucleotide -87
and -33 of the rat NPY promoter gene. This nuclear protein is
identical to the rat homolog of human transcription factor AP-2
. We
further demonstrated that rat AP-2
promotes efficient NPY
transcription activity in response to NGF. Finally, we provide direct
evidence that the mice lacking transcription factor AP-2
exhibit
reduced expression of NPY mRNA compared with wild-type mice, further
supporting the hypothesis that AP-2
is an important transcription
factor in regulating NPY transcription activity.
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INTRODUCTION
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Neuropeptide Y (NPY), a 36-amino acid product of the
precursor prepro-NPY (1, 2), is widely distributed in the brain where
it is involved in various functions, including sexual behavior, feeding
behavior, endocrine, circadian rhythms, memory processing, and
cognition (3, 4, 5). NPY receptor subtypes that belong to the G
protein-coupled receptor superfamily (6) mediate all these actions. In
hypothalamus, NPY has been shown to stimulate food intake and to
decrease thermogenesis and to increase plasma insulin and
corticosterone levels (7, 8). Recently, a number of studies have
indicated an important role for NPY in the regulation of neuronal
activity both under physiological conditions and during pathological
hyperactivity such as that which occurs during seizures (9, 10, 11, 12). Like
many other neuroactive peptides, NPY acts as a cotransmitter at many
sympathetic synapses, producing presynaptic inhibition and inhibiting
Ca2+ currents in the soma of sympathetic neurons
(13, 14, 15, 16).
Transcription activity of the NPY gene is increased in the intermediate
wave of nerve growth factor (NGF)-stimulated gene expression (17).
Previous studies have shown that the rat NPY promoter gene contains
partial consensus sequences for several ubiquitous DNA-binding
proteins, including Sp1 (-57/-51, 5'-CCCCTCC-3'), AP-1 (-72/-65,
5'-TGACTGCC-3') and AP-2 (5'-GCCCGAGG-3') (18, 19). Recently, a NGF
response element (NGFRE) in rat and human NPY promoter genes has been
shown to lie between -87 and -33. At least four nuclear proteins that
interact with this promoter region have been characterized (20, 21).
However, whether these proteins act in conjunction with the NGFRE
binding or by excluding their interaction in NPY promoter remains to be
elucidated. Moreover, the PC12 cells treated with NGF exhibited
increased transcription activity of the NPY gene (20, 21, 22, 42, 43, 44).
Thus, to determine the identity of transcription factors that bind and
interact with the NPY promoter is important for understanding the
regulatory mechanisms that are responsible for the NGF-elicited
increase in transcription activity.
In this study, we report the isolation and characterization of a
nuclear protein that is bound to rat NPY promoter NGFRE. This nuclear
protein is identical to a rat homolog of human transcription factor
AP-2
. We show that rat AP-2
promotes efficient NPY promoter
transcription activity in response to NGF. Finally, we demonstrate that
expression of NPY mRNA is significantly reduced in mice lacking
transcription factor AP-2
.
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RESULTS
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Isolation and Characterization of a Rat Homolog of Human
Transcription Factor AP-2
Screening of a rat brain cDNA library was performed with a
40-bp oligonucleotide probe corresponding to the NGFRE that occurs in
rat neuropeptide Y promoter and that is shown to be homologous to human
NPY promoter by Southwestern screen method (23, 24) (Fig. 1
, A and B). We have isolated five
positive clones from 4 x 106 clones
screened. The longest of the cDNAs contains a predicted open reading
frame of 437 amino acid residues with a predicted molecular mass of 48
kDa and is 98% conserved relative to human and mouse AP-2
(25)
(Fig. 1C
). When the cDNA of this clone was used as a probe in Northern
blot analysis, an approximately 3.2-kb mRNA was identified in rat brain
(Fig. 2A
). To determine the distribution
of rat AP-2
mRNA expression in rat brain, in situ
hybridization analysis was performed. The high expression of rat
AP-2
was found in hippocampus, hypothalamus, and cerebral cortex
(Fig. 2B
).

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Figure 1. Oligonuclotied Probe Corresponding to NGFRE in Rat
NPY Promoter and Comparison of the Amino Acid Sequence Rat AP-2
A, The sequence of the rat NPY promoter spanning -80 to -40. The
AP-1-like, Sp1-like CT-box and the putative AP-2 binding site are shown
by underline. B, Homology between part of the sequence
corresponds to NGFREs in rat and human NPY promoters. C, Comparison of
the amino acid sequence of rat AP-2 to those of human and
mouse. Amino acid sequence variation is underlined.
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Distribution of rat AP-2
Protein in Rat Brain
To determine more precisely the cellular and subcellular
localization of the rat AP-2
protein, antibodies were raised in
rabbits against a glutathione-S-transferase (GST) fusion
protein containing the 437 amino acids of the rat AP-2
protein.
Affinity-purified polyclonal antisera were used to probe immunoblots of
nuclear protein from rat brain and PC12 cells by Western blotting; a
single unique protein of 48 kDa was detected (Fig. 3A
). This result was further confirmed in
NG18105, PC12 cells, and in adult rat brain by immunocytochemical
analysis, revealing that a high level of rat AP-2
is expressed in
NG18105 and PC12 cell nuclear (Fig. 3B
) and expressed in rat cerebral
cortex, hypothalamus, and hippocampus (Fig. 3C
). To examine whether NPY
colocalized with rat AP-2 in hypothalamic cells,
double-immunostaining analysis was performed in rat hypothalamic
neuronal cultures from hypothalamic paraventricular nuclei (PVN). The
result showed that both AP-2 and NPY are expressed in hypothalamic
cells (Fig. 3D
).
Analysis of GST-AP-2
DNA-Binding Activity
To ensure the cloned rat AP-2
could bind to the NGFRE in
the rat NPY promoter gene, we performed gel-shift assays using
oligonucleotides containing NGFRE together with purified rat
GST-AP-2
. After insertion of the NPYPBP cDNA into a pGET-2T vector,
a 74 kDa GST-AP-2
fusion protein was expressed in bacteria and
purified by affinity chromatography (Fig. 4
). The specificity of complexes formed
with the NGFRE and the relative affinities of bound rat AP-2
for the
NGFRE were examined using unlabeled probe. DNA-binding analysis of
increasing concentrations of GST-AP-2
in the presence of same probe
resulted in an increasing bound activity (Fig. 5
, lanes 16). In contrast, the cold
oligonucleotide competed for protein binding at a low concentration
(Fig. 5
, lanes 7 and 8). These results indicate that the specific band
represents binding of rat AP-2
with high affinity for the NGFRE
binding site of rat NPY promoter.
We used antisera recognizing rat GST-AP-2
to confirm the composition
of the specific complexes observed in the electrophoretic mobility
shift assays. As shown for the NGFRE site, the specific band is
entirely supershifted by a rat AP-2
antiserum (Fig. 6A
), indicating that the rat AP-2
protein specifically binds this element in the rat NPY promoter. To
determine whether another protein also can bind to this element, we
used rat AP-2
antiserum to investigate proteins binding to NGFRE in
nuclear extracts from rat brain. We observed a partial supershift (Fig. 6B
), indicating that at least more than two proteins can bind to NGF
response element. This result was consistent with previous studies (20, 21).

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Figure 6. Supershift Analysis of DNA Binding Activity
A, Binding to the NGFRE site was not quantitatively altered in rat
GST-AP-2 fusion proteins. In the presence of a rat AP-2
antiserum, the band, which was show to be specific by cold competition,
is entirely supershifted. B, As shown for nuclear extracts, which are
partly supershifted with increasing amounts of rat brain nuclear
extracts and PC12 cells.
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Regulation of NPY Transcription Activity by AP-2
in Response to
NGF
To determine whether rat AP-2
is up-regulated by NGF, we
investigated the AP-2
mRNA and protein expression by Northern and
Western blot analysis in PC12 cells treated with or without NGF for
48 h. We showed that AP-2
mRNA and protein expression were
increased in NGF treatment cells compared with nontreatment cells (Fig. 7
, A and B), suggesting that stimulation
of NPY transcription activity by NGF may have occurred through
regulation of AP-2
expression.
The results of the above experiments (Fig. 6B
) showed that rat AP-2
protein specifically binds to the element (-80/-40) in the rat NPY
promoter. However, rat AP-2
antiserum only caused a partial
supershift in nuclear extracts from rat brain, thus suggesting that
more than two proteins can bind to this NGF response element
(-80/-40). To determine the specificity of AP-2 binding site, we
performed the electrophoretic mobility shift assays using deletion or
mutation probes as shown in Fig. 7C
and purified AP-2 protein. We found
that AP-2 protein specifically bound to probe (-67/-51), but not
probe (-80/-65), probe (-57/-40) and mutation probe (-67/-51
M) (Fig. 7B
). We also demonstrated that the specific band
of AP-2 protein bound to probe (-67/-51) is entirely supershifted by
a rat AP-2
antiserum.
To verify the specificity of AP-2 function on the NPY promoter,
PC12 cells were transiently transfected with luciferase reporter gene
containing the AP-2
specific binding sequences (-67/-51) and
subjected to stimulation with NGF for different times, or, as a
control, cells were not exposed to any reagents. As shown in Fig. 8
, the increases in luciferase activity
were obtained from PC12 cells treated with NGF after 24, 48, and
72 h compared to those in nontreated cells, suggesting that
NGF-inducible transcription of the NPY gene involved these elements
(-67/-51) and AP-2
protein.

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Figure 8. Transcriptional Activation Analysis of Rat AP-2
in Response to NGF
PC12 cells were transiently transfected with a luciferase reporter
carrying the oligonucleotides (5X, -67/-51) in the presence or
absence of NGF. Cells were subsequently incubated with or without NGF
(50 ng/ml) for different times. Extracts were prepared, and
luciferase assays were carried out. Luciferase activity is expressed as
fold increase relative to unstimulated cells transfected with vector.
All transfections and luciferase assays were performed at least three
times, with triplicates in each experiment. Error bars
indicate the SDs from the means. Results were standardized,
where values for uninduced resting cells were set to 1.
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Reduced NPY mRNA Expression in Mice Lacking Transcription Factor
AP-2
To further investigate whether the AP-2
transcription
factor can regulate NPY transcription activity in vivo, we
tested the effect of AP-2
on NPY mRNA expression by in
situ hybridization and RT-PCR analysis in mice lacking
transcription factor AP-2
. AP-2
deficient mice were generated by
targeted disruption of exon 5 of murine AP-2
gene, and these
homozygous AP-2
(AP-2-/-) mutant mice died perinatally (26, 27).
We found that expression of NPY mRNA in AP-2
deficient mice was
significantly reduced in hypothalamus regions (Fig. 9
, B and C), and also in whole brain
(Fig. 9
, D and E), indicating that AP-2
is a key transcription
factor in the regulation of NPY transcription activity.

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Figure 9. In Situ Hybridization Analysis of
NPY mRNA Expression in Mice Lacking AP-2
A, Schematic diagram of the E15.5 mouse brain. CX, Cortex; TH,
thalamus; HT, hypothalamus; MC, mesencephalon; RC, rhombencephalon; SC
c, spinal cord cervical; SC t, spinal cord thoracic; SCl s, spinal cord
lumbosacral. B and C, In situ hybridization analysis of
expression of NPY mRNA in E15.5 wild-type and AP-2 knockout mice
embryos revealed that NPY mRNA expression was confined to the nervous
system and this expression was significantly reduced in mice lacking
AP-2 gene. D, Quantification of NPY mRNA expression density in AP-2+/+
and AP-2 -/- mice. Data represent mean ± SD of three
experiments shown in panels B and C. ***, P < 0.001. E,
RT-PCR analysis of NPY mRNA expression from AP-2+/+ and AP-2-/-
embryonic brain. RT-PCR for ß-actin mRNA to document equal amounts of
cDNA. The PCR products for NPY and ß-actin are 498 bp and 240 bp,
respectively. M indicates a 1-kb ladder DNA marker.
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DISCUSSION
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In this report, we describe the molecular cloning and
characterization of a nuclear protein that binds to the NGF response
element of the rat NPY promoter and stimulates the transcription
activity in response to NGF. We have identified this protein as a rat
homolog of human transcription factor AP-2
. Rat AP-2
is expressed
in the NG10815 and PC12 cells and also in hypothalamus and
hippocampus. Our transient transfection studies further suggest that
rat AP-2
promotes NPY transcription activity in response to NGF.
Moreover, analysis of AP-2
null mice embryonic brain reveals a
decrease in NPY mRNA expression. Taken all together, these findings
providing strong evidence that rat AP-2
is an important
transcription factor in promoting NPY gene transcription.
The finding that rat AP-2
is expressed in hypothalamus is of
particular interest, as this region contains extremely high levels of
NPY mRNA, and NPY- induced endocrine function has been identified
(2). Thus, AP-2
is expressed with a pattern that coincides with that
of NPY gene expression and it would be consistent for AP-2
via NPY
to play a role in the regulation of neuron and endocrine functions in
hypothalamus.
The transcription factor AP-2
is a retinoic acid
(RA)-responsive gene that is highly expressed in neural crest cells and
their major derivatives, which are undergoing complex morphogenic
changes during vertebrate embryogenesis (28, 29, 30, 31). A functional AP-2
gene is vital for normal mammalian embryogenesis. Mice that contain a
homozygous disruption of the AP-2 gene die perinatally and exhibit
severe developmental defects (26, 27). In situ hybridization
and immunohistochemistry studies showed that AP-2 also is highly
expressed in the mature rat central nervous system. Nevertheless,
consensus AP-2 sites are frequently noted in gene promoter regions,
suggesting that this factor or related proteins may play a wider role.
The AP-2 protein binds as a dimer to sequences with the consensus
5'-GCCCCAGGC-3'. The AP-2-like sequences lie between -67 and -51 from
the rat NPY promoter. In the present study, we clearly observed that
the specific band represents binding of rat AP-2
with high affinity
for this binding site of rat NPY promoter. Thus, the identification of
induced AP-2
transcription activities leading to NPY mRNA expression
further corroborates a crucial function for this transcription factor
in neuron and endocrine function.
The experiments in mutations of AP-2 probe and then in the
transfection indicate that NGF-inducible transcription of the NPY may
occur through AP-2
directly binding to NPY promoter. The
question arises as to how the NGF signals are translated into
regulation of AP-2 binding activity, or indirectly by interacting with
other protein(s), which leads to up-regulation of NPY gene
transcription. One possible mechanism is that NGF-induced signal
cascade culminates in the phosphorylation of AP-2
or associated
proteins and activation of NPY transcription. It has been demonstrated
previously that specific activators of protein kinase C (PKC) mimic and
potentate the effect of NGF on NPY gene activity (19). That NGF-induced
activation is mainly mediated by PKC is suggested by the ability of
calphostin C to block both NGF and phorbol-12-myristate
13-acetate-induced rises in chloramphenicol acetyltransferase
activity. NGF can lead to the activation of numerous genes, including
the immediate-early genes (IEGs) (32) and the late response gene (33).
The IEGs, such as c-fos, encode transcription factors
that may participate in regulation of transcription of
late response genes (reviewed by Refs. 32, 34). Although the
mechanism of NGF-induced AP-2 expression and NPY transcription activity
remains to be elucidated, our AP-2 knockout mice studies provide
evidence that AP-2 is a key transcription factor in regulation of NPY
transcription activity.
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MATERIALS AND METHODS
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Cell Culture
PC12 and NG10815 cells were from the American Type Culture Collection (ATCC, Manassas, VA). Cells were
maintained by serial passage in DMEM supplemented with 10% FCS, 50
U/ml penicillin, and 50 µg/ml streptomycin at 37 C in a 10%
CO2 incubator as described (23, 35). Preparation
of rat hypothalamic neuronal cultures was described previously by
Kusano et al. (41).
Southwestern Screening
A rat brain
gt11 library was screened with the
multimerized -80 to -40 fragment of the rat NPY promoter as a probe
as described (35, 36, 37). Five positive clones were obtained. Among these
positive clones, clone N12 was selected, and its cDNA insert was
subcloned as a EcoRI fragment into EcoRI site of
Bluescript II SK- and sequenced by fluorescent DNA sequencing (PE Applied Biosystems, Norwalk, CT).
Northern Blot Analysis
Random primed 32P-labeled DNA probe,
specific activity (2 x 109 cpm per µg
DNA) from rat AP-2
cDNA was used to probe total RNA extracted from
tissues extracted with guanidine isothiocyanate/phenol/chloroform.
Total RNA (20 µg) was separated on 1.2% agarose-formaldehyde
gels, and capillary blotted onto Hibond-N filter (Amersham Pharmacia Biotech, Piscataway, NJ) (37). The amounts of RNA on
the blot were roughly equivalent, as judged by ethidium bromide
staining of ribosomal RNA. Filters were prehybridized in 50% (vol/vol)
formaldehyde, 5 x SSC containing 0.5% SDS, 5 x Denhardts
solution, 100 µg/ml boiled salmon-sperm DNA, 10 µg/ml poly (U) and
10 µg/ml poly(C) at 45 C for 6 h. After 36 h of
hybridization in the same conditions using 107
cpm per ml hybridization probe, the filters were washed briefly in
2 x SSC at room temperature, and then twice with 2 x SSC
with 0.5% SDS at 68 C. The filters were autoradiographed overnight on
X-omat film (Eastman Kodak Co., Rochester, NY)
In Situ Hybridization
Coronal sections (20 µm) from fresh frozen adult Sprague
Dawley rat brain and E15.5 wild-type and null mutant of AP-2 mice
(Jackson ImmunoResearch Laboratories, Inc., West Grove,
PA) were cut at -20 C on a cryostat (Leitz,
Germany). The sections were thawed onto slides, pretreated with
poly-L-lysine (100 µg/ml), fixed in 4% paraformaldehyde
for 15 min, and rinsed once in PBS (pH 7.5) and twice in distilled
water. The sections were subsequently treated with 0.25% acetic
anhydride in 0.1 M triethanolamine (pH 8.0) and acetic
anhydride (0.25 and vol/vol). After additional rinses in 2 x SSC,
PBS, and 0.1 M Tris-glycine buffer (pH 7.0), sections were
dehydrated in graded ethanols up to 95% and allowed to air dry. For
hybridization, each section was covered with 10 µl of buffer [50%
formamide, 20 mM Tris-HCl (pH 7.6), 1
mMM EDTA (pH 8.0), 0.3 M NaCl, 0.1
M dithiothreitol, 0.5 mg/ml yeast tRNA, 0.1 mg/ml poly (A)
RNA (Sigma, St. Louis, MO), 1 x Denhardts
solution, 10% dextran sulfate] with 2.5 x
106 cpm/ml [35S]
UTP-labeled 800-bp fragment of rat AP-2
cRNA probe or 2.5 x
106 cpm/ml [35S]
UTP-labeled 500-bp fragment of NPY cRNA probe. Sections were then
incubated overnight at 60 C. After hybridization, the sections were
washed once in 1 x SSC at 48 C for 40 min, treated with RNase (10
µg/ml) in 0.5 M NaCl, 20 mM Tris-HCl (pH
7.5), 2 mM EDTA at 37 C for 30 min, and washed twice with
0.5 x SSC and twice with 0.1 x SSC for 10 min each at 60 C.
The slides were then dehydrated with ethanol and exposed to beta Max
x-ray film (Amersham Pharmacia Biotech) for the indicated
times. In addition, sections were dipped in NTB-2 nuclear emulsion,
exposed for 23 weeks, and developed in (Eastman Kodak Co.) D-19.
Preparation of Fusion Protein and Antiserum
DNA fragment encoding rat AP-2
was generated by PCR. All
sequences were confirmed by DNA sequencing. DNA fragment was subcloned
into pGEX-4T-2 vector (Amersham Pharmacia Biotech) to
produce GST-tagged fusion protein, which was purified using
glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). The purified fusion protein (1 mg) was emulsified in
Freunds complete adjuvant and injected intraperitoneally and
subcutaneously into New Zealand White rabbits. Booster injections of
another 1 mg of the fusion protein in Freuds incomplete adjuvant were
given subcutaneously every 3 weeks. Rabbits were bled 1014 days after
each booster. The crude antiserum was prepared with rat GST-AP-2
protein and further purified by Mono Q-sepharose FPLC.
Nuclear Extract Preparation and Electrophoretic Mobility Shift
Assay
Nuclear extracts from rat brain or culture cells were
prepared as described (38), or by a mininuclear extract procedure (39).
Gel shift assays were essentially performed as described previously
(40). To assay protein binding, 32P-labeled
synthetic double-stranded oligonucleotides encompassing the
NGF-response element of the rat NPY promoter gene were used: probe
(-80/-40) (Fig. 1A
), probe (-80/-65), probe (-67/-51), probe
(-57/-40), and probe (-67/-51M) (Fig. 7A
). Binding reaction (25
µl) contained 12 mM Tris-KCl (pH 7.9), 60 mM
KCl, 1 mM dithiothreitol, 12% glycerol, 1 µg poly
(dI-dC)poly(dI-dC), 20,000 cpm of 32P-labeled
probe, 5 µg nuclear extracts, and GST-rat AP-2 protein, as indicated.
The binding reactions were incubated at 30 C for 30 min and then loaded
4% polyacrylamide gel, run at 150 V for 2 h in a solution
containing 6.7 mM Tris-glycine (pH 7.9), 3.3 mM
sodium acetate and 1 mM EDTA, and the bands of DNA-protein
complexes were quantitated by a Fuji X Bioimage Analyzer (BAS2000)
(Fuji Photo Film Co., Ltd., Stamford, CT). In
competition assay, unlabeled probes were incubated with the nuclear
extracts or GST fusion protein for 5 min before the addition of labeled
probe. For immunomobility shift assays immune or preimmune
rabbit polyclonal antiserum was diluted with PBS as indicated and
incubated with rat GST-AP-2 or nuclear extract for 10 min after the
addition of labeled probe DNA for 15 min on ice.
Immunohistochemistry
The adult rat brain cryosections or cultured cells were
fixed with 4% paraformaldehyde in PBS for 30 min. The cryosections
were washed with PBS, and then incubated with PBS-0.3% Triton and
antibody to rat AP-2 at a dilution of 1:500. After overnight
incubation, the cryosections were washed in PBS before incubation
with fluorescent isothiocyanate-conjugated goat antirabbit IgG at a
dilution of 1:500 for 1 h at room temperature, rinsed in PBS, and
counterstained with Evans blue before examination under an Axiophot
fluorescence microscope (Carl Zeiss, Thornwood, NY).
Control experiments included incubation in the presence of a second
antibody only in the presence of an irrelevant first antibody. Double
staining was incubated with monoclonal anti-AP-2 antibody (dilution of
1:100, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and
polyclonal anti-NPY antibody (1:1000, Sigma). Fluorescent
images were obtained using an LSM-410 laser scanning confocal
microscope (Carl Zeiss) . Fluorescence was done with dual
excitation (488 and 568 nm) and emission (515540 nm, fluorescein;
590610 nm, Texas Red) filter sets. Images were processed and merged
using Adobe PhotoShop software (Adobe Systems, Inc., San Jose, CA) and
printed using FUJIX Pictography 3000 (Fuji Photo Film Co., Ltd.).
Transient Transfection and Luciferase Assay
PC12 cells were plated into six-well plates at a density
of 200,000 cells per well and grown overnight. The luciferase reporter
plasmid with the rat AP-2
binding site in the -67/-51(5X) NPY
promoter gene was constructed. PC12 cells were transiently transfected
with 10 µg of Luciferase reporter plasmids by LipofectAMINE PLUS
reagent method (Life Technologies, Inc., Gaithersburg,
MD). After cells were subsequently incubated with or without NGF (50
ng/ml) for different times, the cells were harvested and lysates
were prepared for luciferase activity assay. The amount of luciferase
activity in the lysates was assayed by integrating total light emission
over 20 sec, according to the Promega Corp. (Madison, WI)
protocol. Each transfection was normalized to concomitant
ß-galactosidase expression from a control-transfected. Each
sample was performed in triplicate in a single experiment and repeated
in four different experiments. Error bars indicate the SDs
deviations from the means. Results were standardized, where values for
uninduced resting cells were set to 1.
Western Blotting
Nuclear extracts from rat brain and PC12 cells were prepared as
described (38). Protein expression rat AP-2
was determined by
Western blotting. An equal amount of total protein was resolved on 10%
SDS-polyacrylamide gel and bolted onto polyvinylidene fluoride (PVDF)
membrane for immunoblotting analysis with antirat AP-2
antibody.
Western blots were performed using the Amersham Pharmacia Biotech Enhanced Chemiluminescence (ECL) kit following the
manufacturers instructions (Amersham Pharmacia Biotech).
Preparation of RNA and RT-PCR
Embryonic brain from AP-2+/+ and AP-2 -/- were dissected and
total RNA was extracted using Rneasy Total RNA kit
(QIAGEN, Chatsworth, CA). First-strand cDNA synthesis was
accomplished using SuperscriptII reverse transcriptase (Life Technologies, Inc.). An aliquot of 50 ng of cDNA was used as
template for the PCR reactions. After an initial denaturation step at
94 C for 2 min, the PCR conditions were as follows: 95 C for 30 sec, 55
C for 1 min, 72 C for 1 min for 30 cycles, followed by incubating
for 10 min at 72 C. PCR products were analyzed on a 1.5% TAE
gel. The primers were as follows: NPY,
5'-GCGCCCAGAGCAGAGCACCCGC-3' and
5'-GACAACAAGGGAAATGGGTCGG-3'; actin,
5'-TAAAGACC-TCTATGCCAACACAGT-3'.
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ACKNOWLEDGMENTS
|
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We thank Dr. Harish C. Pant for supporting this work. We thank
Dr. R. Wayne Albers for critically reading this manuscript. We also
thank Mr. Renhong-Wei for his help with antiserum preparation.
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FOOTNOTES
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Address requests for reprints to: Lei Zhang, M.D., Building 36, Room 2C02, Behavioral and Endocrinology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892.
Received for publication December 14, 1998.
Revision received August 4, 1999.
Accepted for publication February 25, 2000.
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