Molecular Cloning, Expression, and Characterization of Rat Homolog of Human AP-2{alpha} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}. We further demonstrated that rat AP-2{alpha} promotes efficient NPY transcription activity in response to NGF. Finally, we provide direct evidence that the mice lacking transcription factor AP-2{alpha} exhibit reduced expression of NPY mRNA compared with wild-type mice, further supporting the hypothesis that AP-2{alpha} is an important transcription factor in regulating NPY transcription activity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}. We show that rat AP-2{alpha} 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{alpha}.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation and Characterization of a Rat Homolog of Human Transcription Factor AP-2{alpha}
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. 1Go, 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{alpha} (25) (Fig. 1CGo). 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. 2AGo). To determine the distribution of rat AP-2{alpha} mRNA expression in rat brain, in situ hybridization analysis was performed. The high expression of rat AP-2{alpha} was found in hippocampus, hypothalamus, and cerebral cortex (Fig. 2BGo).



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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{alpha}

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{alpha} to those of human and mouse. Amino acid sequence variation is underlined.

 


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Figure 2. Northern Blot and in Situ Hybridization Analysis of Rat AP-2{alpha} mRNA Expression.

A, Twenty micrograms of total RNA from hypothalamus (lane 1) and hippocampus (lane 2) were immobilized on nitrocellulose and hybridized with rat AP-2{alpha} cDNA. The locations of 18S and 28S ribosomal RNAs are indicated. B, Expression of rat AP-2{alpha} mRNA in adult rat brain coronal sections subjected to the in situ hybridization procedure using 35S-labeled. Darkfield photomicrographs of x-ray film show the hybridization to the 35S-labeled rat AP-2{alpha} antisense (a) and sense (b) probes.

 
Distribution of rat AP-2{alpha} Protein in Rat Brain
To determine more precisely the cellular and subcellular localization of the rat AP-2{alpha} protein, antibodies were raised in rabbits against a glutathione-S-transferase (GST) fusion protein containing the 437 amino acids of the rat AP-2{alpha} 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. 3AGo). This result was further confirmed in NG18–105, PC12 cells, and in adult rat brain by immunocytochemical analysis, revealing that a high level of rat AP-2{alpha} is expressed in NG18–105 and PC12 cell nuclear (Fig. 3BGo) and expressed in rat cerebral cortex, hypothalamus, and hippocampus (Fig. 3CGo). 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. 3DGo).



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Figure 3. Analysis of Rat AP-2{alpha} Protein Expression

A, Western blotting analysis of rat AP-2{alpha} from whole rat brain, hypothalamus, and PC12 cells using rabbit affinity-purified rat AP-2{alpha} antibody. B, Immunofluorescent analysis of PC12 (a and c) and NG108–15 (b and d) cells with rabbit affinity purified rat AP-2{alpha} antibody revealed that rat AP-2 protein expression is confined to nuclei. c and d are negative control. C, Immunohistochemistry analysis of AP-2{alpha} in adult rat brain. Frozen sections of adult rat brain were labeled with rabbit affinity-purified rat AP-2{alpha} antibody, and labeling was detected by fluorescein isothiocyanate-coupled secondary antibody. Immunoreactivity is evident within the hippocampus dentate granule-cell layer region (CA3) (a) and hypothalamus (PVN) (b). D, Double staining analysis of NPY and rat AP-2{alpha} in rat hypothalamic neuronal cultures from PVN with anti-NPY (red) and anti-AP-2 antibodies (green).

 
Analysis of GST-AP-2{alpha} DNA-Binding Activity
To ensure the cloned rat AP-2{alpha} 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{alpha}. After insertion of the NPYPBP cDNA into a pGET-2T vector, a 74 kDa GST-AP-2{alpha} fusion protein was expressed in bacteria and purified by affinity chromatography (Fig. 4Go). The specificity of complexes formed with the NGFRE and the relative affinities of bound rat AP-2{alpha} for the NGFRE were examined using unlabeled probe. DNA-binding analysis of increasing concentrations of GST-AP-2{alpha} in the presence of same probe resulted in an increasing bound activity (Fig. 5Go, lanes 1–6). In contrast, the cold oligonucleotide competed for protein binding at a low concentration (Fig. 5Go, lanes 7 and 8). These results indicate that the specific band represents binding of rat AP-2{alpha} with high affinity for the NGFRE binding site of rat NPY promoter.



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Figure 4. Expression and Purification of Rat GST-AP-2{alpha} Proteins

SDS-PAGE analysis of rat GST-AP-2{alpha}. Lane 1, Uninduced cell proteins; lane 2, isopropyl-ß-D-thiogalactopyranoside-induced cell proteins; lane 3, purified GST protein; lane 4, purified rat GST-AP-2{alpha} fusion protein. Protein size markers indicated in kilodaltons on the left.

 


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Figure 5. DNA Binding Analysis of Rat GST-AP-2{alpha} Proteins

DNA binding analysis of rat GST-AP-2{alpha} fusion proteins. Electrophoretic mobility shift assays were performed with increasing amounts (ng) of purified rat GST-AP-2{alpha} fusion protein (lanes 1–6), and the specificity of binding and the relative affinities of the complexes for rat AP-2{alpha} binding site were examined using cold competition (lanes 7 and 8). Experiments were repeated at least four times.

 
We used antisera recognizing rat GST-AP-2{alpha} 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{alpha} antiserum (Fig. 6AGo), indicating that the rat AP-2{alpha} 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{alpha} antiserum to investigate proteins binding to NGFRE in nuclear extracts from rat brain. We observed a partial supershift (Fig. 6BGo), 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{alpha} fusion proteins. In the presence of a rat AP-2{alpha} 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.

 
Regulation of NPY Transcription Activity by AP-2{alpha} in Response to NGF
To determine whether rat AP-2{alpha} is up-regulated by NGF, we investigated the AP-2{alpha} 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{alpha} mRNA and protein expression were increased in NGF treatment cells compared with nontreatment cells (Fig. 7Go, A and B), suggesting that stimulation of NPY transcription activity by NGF may have occurred through regulation of AP-2{alpha} expression.



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Figure 7. Analysis of Mutations of AP-2 Probe and Transcription Activation in PC12 Cells

A, Left panel: Total RNA was collected from PC12 cells treated with or without NGF. The blot was probed with rat AP-2{alpha} cDNA. The positions of 28S and 18S ribosomal RNAs are indicated. Right panel: the difference in mean density of AP-2 mRNA between control and NGF treatment groups (n = 3); ***, P < 0.001. B, Western blot analysis of rat AP-2{alpha} using anti-AP-2{alpha} antibody from nuclear extracts of PC12 cells treated with or without NGF. C, The sequences of deletion (a) and mutation (b) of the rat NPY promoter. D, Gel shift (lanes 1–5) and supershift (lanes 6 and 7) analysis of purified AP-2{alpha} binding activity in the deletion and mutation probes.

 
The results of the above experiments (Fig. 6BGo) showed that rat AP-2{alpha} protein specifically binds to the element (-80/-40) in the rat NPY promoter. However, rat AP-2{alpha} 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. 7CGo 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. 7BGo). We also demonstrated that the specific band of AP-2 protein bound to probe (-67/-51) is entirely supershifted by a rat AP-2{alpha} 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{alpha} 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. 8Go, 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{alpha} protein.



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Figure 8. Transcriptional Activation Analysis of Rat AP-2{alpha} 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.

 
Reduced NPY mRNA Expression in Mice Lacking Transcription Factor AP-2{alpha}
To further investigate whether the AP-2{alpha} transcription factor can regulate NPY transcription activity in vivo, we tested the effect of AP-2{alpha} on NPY mRNA expression by in situ hybridization and RT-PCR analysis in mice lacking transcription factor AP-2{alpha}. AP-2{alpha} deficient mice were generated by targeted disruption of exon 5 of murine AP-2{alpha} gene, and these homozygous AP-2{alpha} (AP-2-/-) mutant mice died perinatally (26, 27). We found that expression of NPY mRNA in AP-2{alpha} deficient mice was significantly reduced in hypothalamus regions (Fig. 9Go, B and C), and also in whole brain (Fig. 9Go, D and E), indicating that AP-2{alpha} 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}. Rat AP-2{alpha} is expressed in the NG108–15 and PC12 cells and also in hypothalamus and hippocampus. Our transient transfection studies further suggest that rat AP-2{alpha} promotes NPY transcription activity in response to NGF. Moreover, analysis of AP-2{alpha} null mice embryonic brain reveals a decrease in NPY mRNA expression. Taken all together, these findings providing strong evidence that rat AP-2{alpha} is an important transcription factor in promoting NPY gene transcription.

The finding that rat AP-2{alpha} 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{alpha} is expressed with a pattern that coincides with that of NPY gene expression and it would be consistent for AP-2{alpha} via NPY to play a role in the regulation of neuron and endocrine functions in hypothalamus.

The transcription factor AP-2{alpha} 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{alpha} with high affinity for this binding site of rat NPY promoter. Thus, the identification of induced AP-2{alpha} 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{alpha} 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{alpha} 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
PC12 and NG108–15 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 {lambda} 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{alpha} 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 Denhardt’s 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 Denhardt’s solution, 10% dextran sulfate] with 2.5 x 106 cpm/ml [35S] UTP-labeled 800-bp fragment of rat AP-2{alpha} 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 2–3 weeks, and developed in (Eastman Kodak Co.) D-19.

Preparation of Fusion Protein and Antiserum
DNA fragment encoding rat AP-2{alpha} 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 Freund’s complete adjuvant and injected intraperitoneally and subcutaneously into New Zealand White rabbits. Booster injections of another 1 mg of the fusion protein in Freud’s incomplete adjuvant were given subcutaneously every 3 weeks. Rabbits were bled 10–14 days after each booster. The crude antiserum was prepared with rat GST-AP-2{alpha} 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. 1AGo), probe (-80/-65), probe (-67/-51), probe (-57/-40), and probe (-67/-51M) (Fig. 7AGo). 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 (515–540 nm, fluorescein; 590–610 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{alpha} 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{alpha} 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{alpha} antibody. Western blots were performed using the Amersham Pharmacia Biotech Enhanced Chemiluminescence (ECL) kit following the manufacturer’s 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'.


    ACKNOWLEDGMENTS
 
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.


    FOOTNOTES
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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