Introduction

Summary Introduction Materials & Methods Results Discussion References

NPY is the most abundant and widely distributed neuropeptide within the central nervous system, where it participates in the control of a large number of physiological functions, including effects on food intake, psychomotor activity, and central endocrine secretion and potent vasoactive effects on the cardiovascular system (1, 2). Two major subtypes of NPY receptors, Y₁ and Y₂, have been defined on the basis of pharmacological criteria; the Y₁ receptor is considered to be a postjunctional receptor, and the Y₂ receptor is considered to be a prejunctional receptor (3). We and others previously reported the molecular cloning of the Y₁ receptor cDNA from rat (4, 5), mouse (6), and human (7, 8) tissues; its primary structure shows that it belongs to the superfamily of G protein-coupled receptors. In peripheral tissues, the Y₁ receptor is found predominantly at the sympathetic postjunctional sites in blood vessels, where it mediates the contractile response to NPY of vascular smooth muscle, both directly and indirectly by potentiating the action of other pressure agents, such as norepinephrine (3). In the central nervous system, the Y₁ receptor has been linked with different physiological processes, including stimulation of feeding behavior (9), stimulation of luteinizing hormone-releasing hormone release (9), a sedative anxiolytic effect (10, 11), and modulation of inflammation and nociception (12, 13).

Recent studies have shown that a marked plasticity in the expression of the Y₁ receptor and its mRNA can be induced under different circumstances. For example, peripheral tissue inflammation evokes up-regulation of Y₁ receptor mRNA in dorsal root ganglia; in the same tissue, peripheral axotomy changes expression of the Y₁ receptor mRNA level (12, 13). The molecular mechanisms responsible for regulation of Y₁ receptor expression are unknown; however, like the mechanisms for ₂ receptors and other G protein-coupled receptor genes (14-16), they may result from alteration of the transcriptional regulatory pathway.

We recently cloned the murine gene of the Y₁ receptor, and we isolated a 1.3-kb genomic fragment of the 5 flanking region that is able to drive the expression of the lacZ reporter gene in the mouse neuroblastoma/rat glioma NG108-15 cell line and in rat corticostriatal neuron primary cultures but not in the Y₁ receptor-deficient rat glial and human embryonic kidney 293 cells (6). Sequence analysis of this region revealed the presence of several potential recognition sequences for known transcription factors, including two decameric sequences corresponding to consensus sites for members of the NF-B/Rel family of transcription factors, three AP-1 sites, three half-palindromic estrogen-responsive elements, and one cAMP-responsive element (Fig. 1A).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Murine Y1R-LUC expression plasmids. A, Putative cis-acting elements residing in the upstream region of the murine Y1 receptor gene. Above each box, relative position of the proximal nucleotide in each motif in relation to the initiator ATG. Below each box, nucleotide sequences. ERE, estrogen-responsive element; CRE, cAMP-responsive element. Underlined, sequences in the Y1-B motif corresponding to the B site. B, Luciferase fusion constructs containing deletion fragments of the murine Y1 receptor 5 flanking region. Top, restriction fragments that were used for deletion mutants.

In the current study, we further characterized the upstream promoter region of the murine Y₁ receptor gene through the use of transient transfection assays with NG108-15 and 293 cells. Analysis of Y₁-R/LUC constructs containing deletions of the Y₁ receptor regulatory region suggested the presence of a 181-bp cell type-specific core promoter spanning nucleotides 399 through 218 from the initiator ATG and, upstream of this region, of two positive and two negative regulatory elements. Furthermore, we present evidence that members of the NF-B/Rel family of transcription factors may participate in regulation of the Y₁ receptor gene expression. The functional role of this family of inducible, ubiquitous transcription factors has been widely characterized in the periphery, where these proteins respond to a variety of signals and control expression of several genes mainly implicated in inflammatory and immune reactions (for reviews, see Refs. 17-19). More recently, several groups have shown that NF-B is also abundant in brain, where it was found as both an inducible and a constitutively activated form (20-27). However, currently, very little is known about the role of NF-B-related factors in regulation of the expression of genes whose products play a functional role in the central nervous system.

	Materials and Methods

Summary Introduction Materials & Methods Results Discussion References

Cell culture. The mouse neuroblastoma/rat glioma NG108-15 cells were plated onto Falcon Petri dishes coated with 10 µg/ml poly-L-lysine (M_r 70-150 × 10³) and were cultured in the minimum essential medium containing 10% fetal bovine serum and 1× HAT supplement (all from GIBCO, Grand Island, NY). The human embryonic kidney 293 cells were grown in minimum essential medium and 10% fetal bovine serum. Mouse fibroblast NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium containing 10% calf serum (GIBCO). All culture media contained 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Plasmid construction. A SalI/BglII fragment from the original murine Y₁ receptor genomic clone (from nucleotides 1523 to 218 relative to the initiator ATG) was first subcloned into the polylinker of pBluescript SK (Stratagene, La Jolla, CA) (6) (Fig. 1B). The SalI/BglII fragment was isolated by gel electrophoresis; then, the BglII-digested end (all restriction enzymes were from Boehringer-Mannheim Biochemicals, Indianapolis, IN) was filled in with the Klenow fragment of DNA polymerase I (Boehringer-Mannheim) and excess of dNTPs (0.4 mM). The resulting SalI-blunt fragment was ligated into the SalI/ClaI sites of pBluescript SK after the ClaI site of the plasmid was filled to obtain a blunt end. This construct (pBS-Y₁PR) contains 1305 bp of the 5 flanking region of the murine Y₁ receptor gene and includes the first three sites of initiation of transcription (6). The Y₁R-LUC expression plasmids were constructed through subcloning into the polylinker of pGL2-basic (Promega, Madison, WI) with the following restriction fragments from pBS-Y₁PR: a 1305-bp SalI/HindIII fragment (p1305-LUC), a 985-bp HpaI/HindIII fragment (p985-LUC), a 686-bp SmaI/HindIII fragment (p686-LUC), a 618-bp NsiI/HindIII fragment (p618-LUC), a 490-bp SspI/HindIII fragment (p490-LUC), a 306-bp PstI/HindIII fragment (p306-LUC), and a 181-bp EcoRI/HindIII (p181-LUC) fragment (see Figs. 1B and 2B). Constructs containing an upstream sequence between nucleotides 1113 and 960 (p895-LUC, p876-LUC, p808-LUC, and p742-LUC; see Figs. 1B and 2B) were obtained through digestion of pBS-Y₁PR with HpaI and subsequent treatment with Bal31 enzyme (Boehringer-Mannheim). The plasmid DNA was then rendered blunt with the Klenow enzyme, digested a second time with HindIII, and subcloned into the SmaI/HindIII sites of pGL2-basic. The junctions between the insert DNAs and luciferase gene of the fusion constructs were confirmed through sequence analysis (28).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   B-Y1R-LUC expression plasmids. A, Putative regulatory elements residing in the 5 flanking region of the Y1 receptor gene. B, B-Y1R-LUC fusion genes containing the Y1-B motif ligated immediately upstream of different deletion fragments of the murine Y1 receptor promoter. Above each B-Y1R-LUC plasmid, corresponding luciferase fusion constructs containing the deletion fragments of the murine Y1 receptor 5 flanking region. Mutant constructs were obtained as described in Materials and Methods and were confirmed by sequence analysis and restriction mapping. Restriction fragments that were used for deletion mutants are shown in Fig. 1.

Transient transfection experiments. Transfection of reporter plasmids into NG108-15, 293 and NIH 3T3 cells was performed according to the calcium phosphate coprecipitation method (29). In all experiments, pSV--galactosidase control vector (Promega), containing the -galactosidase gene linked to the simian virus 40 early promoter/enhancer, was included as an internal control for the different transfection efficiencies between experiments. When cells reached ~50% confluence, each 35-mm Petri dish received equimolar amounts (2.5 µg) of test plasmid and pSV--galactosidase. Cells were harvested 48 hr after transfection, and the activities of luciferase and -galactosidase were assayed as previously described (30). As controls, the plasmid pGL2-basic, containing the promoterless luciferase gene, and the plasmid pGL2-promoter vector (Promega), containing the luciferase gene driven by the simian virus 40 promoter, were transfected into parallel cultures of each cell line. In all of the cell lines tested, the pGL2-basic was inactive, whereas the pGL2-promoter vector showed high levels of luciferase activity.

Nuclear extracts and electrophoretic mobility shift assays. Nuclear extracts from rat brain areas were prepared essentially as described by Kang et al. (31). Nuclear extracts from cell lines and from A.E7 cells were prepared according to a small-scale protocol (25) with minor modifications. Briefly, 5-10 × 10⁶ cells were scraped into cold phosphate-buffered saline, washed once in phosphate-buffered saline, and pelleted for 10 sec in an Eppendorff centrifuge. Cells were then resuspended in 400 µl of cold buffer A (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride), allowed to swell on ice for 10 min, and vortexed for 10 sec. Samples were centrifuged for 10 sec, and the pellets were resuspended in 50 µl of cold buffer C (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM phenylmethylsulfonyl fluoride) and incubated on ice for 20 min for high salt extraction. Cellular debris were removed by centrifugation for 2 min at 4°, and the supernatant was stored at 70°. Protein concentration was assessed by BioRad (Hercules, CA) Bradford assay according to the manufacturer's instructions. DNA binding reaction were initiated by the combination of 2 µg of nuclear extracts with 20,000 cpm (0.1 ng) of -³²P-labeled oligonucleotide probes in 1× lipage buffer (10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 10% glycerol) containing 0.5 µg of poly(dI/dC) in a total volume of 10 µl. In competition experiments, indicated amounts of unlabeled competitor oligonucleotides were added with ³²P-labeled probes. Reactions were carried out for 20 min at room temperature, and protein/DNA complexes were resolved on nondenaturing 4% polyacrylamide gels in 1× Tris/glycine/EDTA buffer (1× = 50 mM Tris, 380 mM glycine, and 2.7 mM EDTA). Gels were then dried and subjected to autoradiography at room temperature.

Synthetic DNA oligonucleotides. Oligonucleotide sequences and their respective complementary strands were synthesized with a DNA synthesizer (Applied Biosystems, Norwalk, CT) and purified through denaturing gel electrophoresis. Oligonucleotides were annealed to complementary strands by heating to 68° and cooling slowly to room temperature. For gel shift analysis, double-stranded oligonucleotides were end-labeled with [-³²P]ATP (> 7000 Ci/mmol; ICN Pharmaceuticals, Costa Mesa) and T4 polynucleotide kinase (Boehringer-Mannheim) to obtain a specific activity of >10⁸ cpm/µg. The sequences of the oligonucleotides were as follows: Y₁-B, 5-GATCATGGGATTTCATTGGGATTTCACTT-3 (sense); mY₁-B, 5-GATCCATctcATTTCATTctcATTTCACTT-3 (sense); Ig-B, 5-CAGAGGGGACTTTCCGAGAGGC-3; and octamer (octamer binding site from the IL-2 gene enhancer region), 5-TATGTGTAATATGTAAAACATTTTGACACC-3. Sequences corresponding to the B site are underlined.

Statistical analysis. Statistical analysis was performed by using the Mann-Whitney U test.

	Results

Summary Introduction Materials & Methods Results Discussion References

Analysis of the effect of progressive deletion within the Y₁ receptor promoter on heterologous gene expression. The ability of several deletion mutants of the 5 flanking region of the murine Y₁ receptor gene to drive the expression of the luciferase reporter gene was analyzed in transient transfection experiments. As shown in Fig. 3, the 1.3-kb genomic fragment, spanning nucleotides 1523 through 218 relative to the initiator ATG (p1305-LUC), drives the luciferase activity in NG108-15 cells, whereas a very low enzyme activity was determined in 293 cells. Sequential deletion from nucleotides 1523 through 1026 (p985-LUC, p895-LUC, p876-LUC, and p808-LUC) had no significant effect on luciferase activity (Figs. 3 and 4). Further deletion to nucleotide 960 (p742-LUC) resulted in an ~2-fold increase in luciferase activity in NG108-15 cells but not in 293 cells, suggesting the presence of a negative regulatory element between nucleotides 1026 and 960 that is operative in NG108-15 cells. The promoter activity remained unchanged by removal of the region between 960 and 904 (p686-LUC), whereas further reduction of the upstream sequence to nucleotide 836 (p618-LUC) increased the luciferase activity by ~2-fold in NG108-15 cells but not in 293 cells, suggesting that the sequence between nucleotides 904 and 836 contains a negative regulatory element that contributes to lower Y₁ receptor gene expression in this cell type (Fig. 3). Further deletion to nucleotide 708 (p490-LUC) decreased the luciferase activity to the level driven by the undeleted Y₁ receptor promoter (p1305-LUC) in NG108-15 cells, suggesting the presence of a positive cis-acting element between nucleotides 836 and 708. Extension of 5 deletion to nucleotide 524 (p306-LUC) did not affect the luciferase activity significantly, whereas further deletion to nucleotide 399 (p181-LUC) reduced the reporter gene expression by 2.8-fold in NG108-15 cells, suggesting the presence of a positive cis-acting element between nucleotides 524 and 399. The remaining 181 bp-genomic fragment, spanning nucleotides 399 and 218 of the Y₁ receptor gene, drives the luciferase activity in NG108-15 cells at a level significantly above the pGL2-basic (see legend to Fig. 3), indicating that it represents the minimal promoter region still capable of directing expression of the Y₁ receptor gene in the neuroblastoma/glioma cell line. It should also be pointed out that the same sequence is unable to drive luciferase activity in 293 cells. Furthermore, all of the Y₁ receptor deletion mutants/fusion constructs drove negligible luciferase activity when transiently transfected into the mouse fibroblast NIH 3T3 cell line (data not shown).

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Deletion analysis of the murine Y1 receptor gene promoter. The promoter activity of the Y1R-LUC expression plasmids was determined in (open bars) NG108-15 and (hatched bars) 293 cells. Left, tested reporter plasmids. The luciferase activity was normalized to -galactosidase activity obtained by cotransfecting cells with the control plasmid pSV--galactosidase (in cpm). Values are mean ± standard error from eight or more transfection experiments, each performed in triplicate, with plasmid DNAs from at least two different preparations. Cultures transfected with the promoterless plasmid pGL2-basic had a mean luciferase activity of 0.16 ± 0.018 and 0.04 ± 0.003 cpm × 106 in NG108-15 and 293 cells, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Enhancer activity of the Y1-B sequence in NG108-15 cells. Histograms show the mean ± standard error of at least eight transfection experiments, each performed in triplicate. Each reporter plasmid tested (left) was independently prepared at least two times. The luciferase activity was normalized to -galactosidase activity from a cotransfected internal control plasmid pSV--galactosidase (in cpm). The luciferase activity of pB-741-LUC, pB-686-LUC, and pB-181-LUC was significantly more than that of p742-LUC, p686-LUC, and p181-LUC, respectively (*, p < 0.05).

Binding of Y₁-B sequences to members of the family of NF-B transcription factors. Detailed sequence analysis of the Y₁ receptor promoter region reveals the presence of putative binding sites for known transactivating factors that may play a role in the regulation of the tissue-specific expression of this gene (Fig. 1A). Particularly, we focused our interest on the sequence located in position 1302 through 1282 in the Y₁ receptor gene regulatory region because it contains two decameric sequences corresponding to consensus sites for members of the NF-B/Rel family of transcription factors (6). We performed experiments to verify whether the aforementioned sequences are indeed binding sites for transcriptional control proteins belonging to this family. A well-studied model of gene regulation mediated by B/Rel proteins is represented by A.E7 cells, a CD4⁺ murine T cell clone in which B-mediated gene expression has been extensively analyzed. Kang et al. (31) demonstrated that these untransformed cells constitutively express at least two nuclear complexes belonging to the NF-B/Rel family: the p50-p65 (relA) heterodimer and the p50 homodimer. In this well-characterized model, we initially studied binding properties of the putative B sequences from the Y₁ receptor gene.

View larger version (56K): [in this window] [in a new window]   Fig. 5.   Analysis of the binding properties of the Y1-B sequence on nuclear extracts from a T cell clone endowed with constitutive NF-B/Rel activities and on the p50 subunit. A, Oligonucleotide sequence comprising the two B sequences from the Y1 receptor gene (Y1-B) binds three specific nuclear complexes in extracts from a murine T cell clone, A.E7. Electrophoretic mobility shift assay obtained through incubation of 2 µg of A.E7 nuclear extracts with 32P-labeled Y1-B probe in the absence (0 ng) or in the presence of 1, 16, or 32 ng of unlabeled Y1-B or Ig-B as competitors. B, Affinity purified, bacterially produced p50 subunit of the NF-B/Rel family can bind the Y1-B oligonucleotide probe. bp50, bacterially purified p50.

View larger version (79K): [in this window] [in a new window]   Fig. 6.   Gel shift analysis of nuclear complexes specifically interacting with the Y1-B probe in NG108-15 cell extracts. A, Comparison of the migration properties of complexes intercepted by (lane 1) Y1-B and (lane 2) Ig-B oligonucleotide probes in NG108-15 cell extracts. B, Competition analysis of the complexes bound by the Y1-B oligonucleotide probe. Competition was performed by adding the indicated amounts (in ng) of the unlabeled oligonucleotide sequences Y1-B (lanes 2-4) or Ig-B (lanes 5-7) or the unrelated oligonucleotide sequence for octamer binding proteins (lanes 9-12). , No competitor (lanes 1 and 8). Apparent discrepancies in the migration properties of complexes in A and B are due to differences in gel running length in the two different sets of experiments.

View larger version (45K): [in this window] [in a new window]   Fig. 7.   The Y1-B oligonucleotide sequence binds a single B-related complex in nuclear extracts from several rat brain regions. Protein extracts were from hippocampus (lane 1, hipp.), striatum (lane 2), cerebellum (lane 3, cereb.), cortex (lanes 4 and 6), and olfactory bulb (lane 5, olf. bulb). Lane 6, binding competition was performed with 16 ng of the unlabeled oligonucleotide Ig-B and nuclear extracts from cortex.

The Y₁-B oligonucleotide sequence from the Y₁ receptor gene acts as enhancer element in NG108-15 cells. Although the deletion from nucleotides 1523 to 1203 (p985-LUC) in the Y₁ receptor sequence did not affect luciferase activity in our in vitro model (Fig. 4), based on the essential role of the NF-B/Rel family of transcription factors for the expression of several genes (for reviews, see Refs. 17-19), we surmised that the Y₁-B sequence may participate in the regulation of the transcriptional activity of this gene. To test this possibility, we constructed a series of expression plasmids (pB-Y₁R-LUC) in which an oligonucleotide corresponding to the Y₁-B sequence was placed immediately upstream of deletion fragments of the Y₁ receptor 5 flanking region that did not contain the endogenous sites (Fig. 2B). Results indicated that the Y₁-B sequence, when placed upstream of nucleotides 959, 904, and 399 from the initiator ATG (pB-741-LUC, pB-686-LUC, and pB-181-LUC, respectively), enhances by >2-fold the luciferase activity in NG108-15 cells (Fig. 4). Specificity of the effect was demonstrated by the fact that mutation of selected nucleotides within the motifs, which abolished binding activity in gel shift assay (not shown), completely abolished enhancer activity (Fig. 8). Interestingly, the Y₁-B sequence seemed to be functional in NG108-15 cells but not in 293 cells (Fig. 8). Furthermore, the Y₁-B motif failed to increase luciferase activity when placed upstream of nucleotides 1203 (pB-985-LUC), 1111 (pB-893-LUC), 1091 (pB-873-LUC), and 1029 (pB-811-LUC) suggesting that the 70-bp sequence spanning nucleotides 1029 and 959 of the Y₁ receptor promoter contains a negative regulatory element that inhibits Y₁-B enhancer activity in NG108-15 cells (Fig. 4). The B-related factors were found as both inducible and constitutively activated complexes in the central nervous system (20-27). The B nuclear activity interacting with the Y₁ receptor gene is constitutive. We verified whether specific extracellular signals might further activate the enhancer activity of the Y₁-B sequence. To investigate this possibility, we treated NG108-15 cells transfected with pB-686-LUC or pB-181-LUC plasmids with various agents that are known to modulate activity of these transcriptional regulators in either peripheral or central nervous system-derived cells (17, 22, 25-27). In particular, we tested cytokines, such as IL-1 (30 units/ml), IL-2 (2 nM), and tumor necrosis factor- (100 ng/ml); lipopolysaccharide (25 µg/ml); concanavalin A (25 µg/ml); 12-O-tetradecanoylphorbol-13-acetate (0.1 µM); hydrogen peroxide (50 and 100 µM), KCl (30 mM); and glutamate (100 µM). However, none of these agents were able to further augment the transcriptional activity of pB-686-LUC or pB-181-LUC expression plasmids (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Mutational analysis of the Y1-B sequence. A three-nucleotide mutation was made in each B motif from the Y1-B oligonucleotide sequence as described in Materials and Methods. The pmB-686-LUC fusion plasmid was prepared by ligating the mutated Y1-B motif (mY1-B) immediately upstream of the 686-bp (SmaI/HindIII) deletion fragment of the Y1 receptor promoter. Promoter activity of the p1305-LUC, p686-LUC, pB-686-LUC, and pmB-686-LUC was determined in (open bars) NG108-15 and (hatched bars) 293 cells. The enhancer activity of the mutated Y1-B motif was depicted in NG108-15 cells. *, p < 0.05 versus p1305-LUC. **, p < 0.05 versus p686-LUC.

	Discussion

Summary Introduction Materials & Methods Results Discussion References

The Y₁ receptor subtype plays important roles in mediating NPY-induced control of several functions, including cardiovascular system activity, neuroendocrine secretion, food intake, and nociception (1, 2). In situ hybridization studies have shown that Y₁ receptor mRNA is indeed highly expressed in several regions of the rat forebrain, in the hypothalamus, and in dorsal root ganglia (4, 13, 32, 33). We previously isolated the murine gene encoding the Y₁ receptor and demonstrated that the 5 flanking region of this gene contains a functional promoter that is active in neuronal cells (primary cultured neurons and NG108-15 cells) but not in glial or 293 cells (6).

In the current study, we investigated which regions of the Y₁ receptor promoter contain potential negative or positive cis-acting elements participating in the regulation of gene expression. For this purpose, luciferase constructs comprising the 1.3-kb 5 flanking regions of the Y₁ receptor or various 5 deletions of it were transfected into a cell line that expresses the Y₁ receptor endogenously, NG108-15 cells, and in the Y₁ receptor-deficient 293 cell line. Deletional analysis has shown the presence of several potential areas of transcriptional regulation. Our data suggest the presence of at least two positive acting regulatory elements lying between nucleotides 836 and 708 and between nucleotides 524 and 399. Although the sequence of the fragment contained between 836 and 708 has no clear homology to any known regulatory element, it is noteworthy that an AP-1 binding site resides between nucleotides 473 and 479. Further mutational and deletional analysis will be required to define whether this site functions as a cis-acting element. In preliminary experiments, we were able to show that the Y₁ receptor promoter/luciferase reporter gene can be positively modulated by treatment with phorbol esters but that deletion of the sequence containing the AP-1 site (473 to 479) fails to suppress this type of responsiveness (data not shown).

A second type of regulation that may exist for the Y₁ receptor gene expression involves silencer domains. Our work suggests the presence of at least two negative regulatory elements contained between nucleotides 1026 and 960 and between nucleotides 904 and 836. To assess the cell type specificity of these sequences, 293 and NIH 3T3 cells were transfected with the corresponding luciferase constructs. All deletion mutants were ineffective in modulating transcription of the heterologous luciferase gene in these cell lines, indicating that the negative regulatory elements of the Y₁ receptor gene are not responsible for repression in these cell lines.

Reporter gene assay also suggested that the core promoter (399 to 218) of the Y₁ receptor gene exhibited substantial cell type specificity. Ball et al. (34) recently reported that the human Y₁ receptor gene is under the control of three promoters that are activated in a tissue-specific manner. It is noteworthy that the core promoter of the murine Y₁ receptor gene displays a high sequence homology with the corresponding region of the human promoter directing the expression of the most abundant Y₁ receptor transcript.

Detailed analysis of the sequence of the murine Y₁ receptor 5 flanking region reveals the presence of many putative binding sites for known transcription factors (6) (Fig. 1A). We focused our attention on the murine Y₁ receptor promoter region that contains two decameric sequences located in tandem in position 1302 to 1282 bp, relative to the ATG. These sequences correspond to consensus sites for members of the B-Rel family of transcription factors (17). In the current study, we showed that this sequence can indeed bind B-related nuclear complexes in a specific manner and acts as an enhancer element in transiently transfected NG108-15 cells.

NF-B/Rel proteins are constitutive and inducible transcription factors that are present in most cell types. Each B complex corresponds to homodimers and heterodimers whose subunits belong to a superfamily that comprises at least five DNA binding proteins: p50, p52 (p50B), p65 (RelA), c-rel, and RelB (17-19). The inducible form of NF-B contains an additional inhibitory subunit called IB and can be activated in response to stimuli that mostly represent pathogenic conditions, including viruses, bacterial lipopolysaccharide, inflammatory cytokines, and oxidants. It was previously suggested that only a limited number of lymphoid cells contain constitutively active NF-B-related factors (35, 36). More recent evidence, however, indicates that in the central nervous system, members of the B family of transcription factors are constitutively active and are present in the nucleus of cultured neurons as well as in neurons in vivo (21, 24, 25).

Our data demonstrate that the Y₁-B sequence binds with high affinity members of the B/Rel family of transcription factors in nuclear extracts from rat brain areas, from the NG108-15 neuronal cell line, and from the murine T cell clone A.E7. Interestingly, different binding properties were observed in nuclear extracts from different sources. In nuclear extracts from rat brain regions, a single B-related complex was detected. In nuclear extracts prepared from the cell line NG108-15, as well as in extracts from the murine T cell clone A.E7, three complexes with different migration properties interacted specifically with the Y₁-B sequence, but only two of them seemed to be B-related nuclear activities in competition experiments. The molecular nature of the third complex, specifically interacting with the Y₁-B sequence, remains to be elucidated. Furthermore, detailed mutation studies are necessary to better define the binding requirements of each complex to the Y₁-B sequence containing the two B sites. Also, it will be interesting to clarify the significance of the differences in binding activities in rat brain extracts compared with extracts from NG108-15 cells and from the murine T cell clone A.E7. It is noteworthy that the single complex identified by the Y₁-B oligonucleotide probe in rat brain extract is reminiscent of the binding specificity of a B-binding site recently identified and characterized in the regulatory region of the amyloid precursor protein, which in rat brain extracts recognizes specific complexes that are either identical or very similar to p50 homodimers (25).

In transient transfection assays, we also demonstrated that the Y₁-B sequence behaves as an enhancer element when placed upstream of deletion fragments of the Y₁ receptor regulatory region. Surprisingly, the Y₁-B site does not enhances the transcriptional activity of the fusion gene constructs when placed 70 bases upstream of nucleotide 959 relative to the initiator ATG. These results suggest that the 70-bp region lying between 1029 and 959 bases of the 5 flanking region of the Y₁ receptor gene might contain a negative regulatory element that is able to suppress the enhancer activity of Y₁-B sequence in NG108-15 cells.

The NF-B activity interacting with the Y₁ receptor gene promoter seems to be a constitutive activity, but it is well known that these transcriptional activators can be present as both activated and inducible forms in neurons (20, 22, 25-27). A wide variety of stimuli can modulate NF-B/Rel activities, depending on the cell type (17-19). However, the treatment of NG108-15 cells with several agents, including inflammatory cytokines, oxidants, bacterial lipopolysaccharide, and neurotransmitters, failed to stimulate the transcriptional activity of the Y₁-B sequence. It is possible that in NG108-15 cells, the intracellular signals that activate NF-B proteins are coupled to specific membrane receptors that we were unable to identify, or that in this tumoral cell line, the Y₁-B binding activity is maximally up-regulated. To answer these questions, we are analyzing the modulation of the Y₁-B binding activity in primary cultures of neuronal cells.

The results that we report are, to our knowledge, the first demonstration that a B-related motif contained in the regulatory region of a neuropeptide receptor gene binds B-related nuclear proteins and might be activated by this family of transcription factors. The functional significance of these data remains to be elucidated. Kaltschmidt et al. (22) suggested that in neurons, the B-related factors might participate in the normal physiology and development of the nervous system. Sequence analysis revealed the presence of putative B-related sequences in the regulatory region of other neuropeptide receptor genes, such as the vasoactive intestinal peptide receptor (37) and the -opioid receptor (38). It is possible that this family of transcription factors participates in the control of neurotransmission by transcriptionally regulating the expression of neuropeptide and neurotransmitter receptor genes.

In most cell types, the B-related proteins mediate an immediate-early response to stimuli that represent stress conditions. Vascular responsiveness to NPY was shown to be increased in conditions that were occurring physiologically during prolonged stress or in disease states, such as hypertension (39). In addition, NPY seems to play a critical role in the transmission of stress-related information to the hypothalamic/hypophysial system and in the activation of neuroendocrine responses essential for the survival of the organism (40). An interesting possibility is that the Y₁ receptor for NPY may represent one of the B site-containing genes that is modulated in the mammalian nervous system by B-related factors in response to stimuli that require an immediate defensive response.