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Isolation and Characterization of the 5'-Flanking Regulatory Region of the Human Natriuretic Peptide Receptor C Gene¹

Lead Generation Research Laboratory, Tanabe Seiyaku Co., Yodogawa-ku, Osaka 532, Japan

Address all correspondence and requests for reprints to: Dr. Kenji Omori, Lead Generation Research Laboratory, Tanabe Seiyaku Co., 16–89 Kashima-3-chome, Yodogawa-ku, Osaka 532, Japan. E-mail: k-omori{at}tanabe.co.jp

	Abstract

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Phenotypic modulation of vascular smooth muscle cells (SMCs) plays a central role in the pathogenesis of atherosclerosis. Natriuretic peptide receptor-C (NPR-C) is highly expressed in vascular SMCs in the experimental arteriosclerotic neointimal area as well as in cultured SMCs, suggesting that increased expression of the NPR-C gene is related to the phenotypic alteration of vascular SMCs. To elucidate the molecular mechanisms and to identify the essential DNA sequences in NPR-C gene expression, a genomic clone containing over 8 kilobases of the 5'-flanking region of the human NPR-C gene has been isolated. Sequence analysis revealed that a number of putative regulatory elements including unusual tandem repeated AP-2-like sequences were observed in the 5'-flanking region. Primer extension and ribonuclease protection analyses revealed that transcription of the human NPR-C gene starts from two major regions. Promoter analysis using deletion constructs in human cells, highly producing NPR-C transcripts, showing that the region (from -33 to +13 relative to the transcription start point) had a potential promoter activity suggested that the region from -33 to +13, containing a pyrimidine-rich stretch composed of four CTTTTT-repeated sequences, is sufficient for the proximal promoter activity. Moreover, three distinct DNA sequences surrounding the transcription start site (P1, from -60 to -33; P2, from +14 to +40; P3, from +41 to +66) were revealed to be functional as a cis-acting positive enhancer, and a nuclear protein(s) from the human cells was demonstrated to specifically bind to the sequences, respectively. However, promoter analysis has shown that the P2 and P3 sequences could not activate the human NPR-C promoter in a synergistic manner. On the basis of deoxyribonuclease I footprinting analysis showing that a DNA element from +48 to +60 within the P3 sequence is preferentially protected, the P3 sequence appears to contain a potential regulatory element involved in NPR-C gene expression. The present study demonstrated the structure of the 5'-regulatory region of the human NPR-C gene and multiple cis-acting positive sequences closely located around the transcription start points with an important role in regulation of human NPR-C gene expression.

	Introduction

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NATRIURETIC peptides (NP) are known to play important roles in cardiovascular homeostasis. Three isoforms termed atrial NP (ANP), brain NP, and C-type NP (1, 2) constitute a natriuretic peptide family. ANP and brain NP have been considered to be responsible for systemic blood pressure control and body fluid homeostasis (3, 4). In contrast, C-type NP has been recently demonstrated to be involved in the suppression of cell proliferation and in phenotypic development in osteoblasts (5). These biological functions of NPs were mediated with production of intracellular cGMP through the guanylyl cyclase (GC)-coupled receptors, termed GC-A and GC-B. NP receptor-C (NPR-C) has a very short putative intracytoplasmic extension, with no guanylyl cyclase activity (6, 7, 8). This receptor is supposed to regulate the biological effects of NPs by eliminating the peptides from the blood circulation. On the other hand, NPR-C has been reported to control adenylyl cyclase activity via G_i protein, phospholipid hydrosis, thymidine kinase activity, and mitogen-activated protein kinase activity (9, 10, 11, 12, 13) without any cGMP response. Recent studies have shown that NPR-C is involved in inhibition of catecholamine secretion in chromaffin cells and the pheochromocytoma (PC12) cell line (14, 15). In vascular smooth muscle cells (SMCs), expression of the NPR-C gene has been revealed to be modulated by ANP and endothelium-derived relaxing factor, both of which are key mediators for protection against cardiovascular disorders, and by ß₂-adrenergic stimulants (16, 17). Additionally, NPR-C gene expression in vivo has been shown to be down-regulated in the aorta of hypertensive rats (18), in the lung of rats adapted to hypoxia (19), in the kidney of rats with renal failure (20) and streptozotocin-induced diabetes mellitus (21), and during the development of cardiac hypertrophy in aortic banded rats (22). Although NPR-C has been demonstrated to localize in tracheal and glomerular epithelium (23, 24), no information is yet available on the mechanisms underlying the down-regulation of NPR-C expression.

Cultured vascular SMCs possess a phenotype of growing rapidly and showing pathological characteristics (synthetic phenotype). Replication of SMCs representing the synthetic phenotype is found in atheromatous intimal lesions, indicating that abnormal proliferation of SMCs in the vascular wall is a key process of atheromatous lesion formation (25, 26, 27). Although cultured vascular SMCs showing the synthetic phenotype express NPR-C (28) and various molecules including embryonic myosin heavy chain (SMemb) (29), cyclooxygenase-2 (30), and 95-kDa gelatinase (MMP-9) (31), these molecules are not identified in the medial SMCs showing a contractile phenotype. Recent studies demonstrated that expression of these genes was induced in the SMCs in the intimal area of the experimental model in vivo. In this study, we have shown that NPR-C is highly expressed in SMCs in the neointimal area of the rat aorta after balloon injury, suggesting that the NPR-C gene is a useful marker molecule representing the synthetic phenotype in vascular SMCs. Additionally, NPR-C has been shown to decrease during phenotypic differentiation of osteoblastic cells (32), also suggesting that the expression level of the NPR-C gene was dependent on the developmental stage of the osteoblasts.

Despite the significant progress that has been made in describing NPR-C expression, no information is yet available on the mechanisms underlying the basic regulation of NPR-C gene transcription. For further understanding of the regulation of NPR-C gene expression, detailed information of the structural and functional properties of its transcriptional regulatory region will be required. Although we have previously demonstrated the structure of the 5'-flanking regulatory region of the mouse NPR-C gene and functional features using serial 5'-deletions of the promoter region (33), little is known regarding the molecular mechanisms that regulate proximal transcription of the NPR-C gene. In the present work, we isolated the 5'-flanking regulatory region of the human (h) NPR-C gene, determined the transcription start points, and defined the DNA sequences that are essential for its expression to elucidate the basic transcriptional regulation of the hNPR-C gene. Three distinct cis-acting positive regulatory sequences surrounding the transcription start sites have been determined according to functional promoter analysis using a series of hNPR-C promoter-reporter constructs and human epithelial-like cells highly expressing NPR-C messenger RNA (mRNA). Further investigations using the nuclear proteins were carried out to examine the molecular mechanisms of the cis-acting sequences. Moreover, another finding in this study was that NPR-C mRNA was highly expressed in the human megakaryocyte-like cell line and the human epithelial-like cell line, suggesting that these cell lines were not only an abundant source of the hNPR-C transcripts, but also a useful tool to investigate the regulation of NPR-C expression in platelet and epithelium and its pathophysiological roles.

	Materials and Methods

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Materials
Restriction endonucleases and DNA-modifying enzymes were obtained from Takara Shuzo (Kyoto, Japan). [-³²P]Deoxy-CTP, [-³²P]UTP, [-³²P]ATP, [¹²⁵I]human ANP, and Hybond-N plus nylon filters were obtained from Amersham (Arlington Heights, IL). The ANP analog, des[Gln¹⁸,Ser¹⁹,Gly²⁰,Leu²¹,Gly²²]ANP-(4–23)-NH₂ [C-ANF-(4–23)], was purchased from Sigma Chemical Co. (St. Louis, MO). The expression vector pGVB, containing a firefly luciferase gene, was obtained from Toyo Inki (Tokyo, Japan). Control plasmid pRL-cytomegalovirus (pRL-CMV), containing a Renilla luciferase gene, was obtained from Promega (Madison, WI). DMEM, Iscove’s Modified Dulbecco’s Medium, FBS, and horse serum were obtained from Life Technologies (Grand Island, NY).

NPR-C immunohistochemistry
The antiserum against the NPR-C protein used was obtained as follows. The multiple antigen peptide-complex containing the sequence NH₂-RKKYRITIERRNHQEESNIGKHRELREDSIRSHFSVA-COOH derived from the cytoplasmic domain of rat NPR-C protein was synthesized using the 9-fluorenylmethyloxycarbonyl synthesis strategy. Polyclonal antibody toward the peptide was obtained by injecting rabbits with the peptide in Freund’s complete adjuvant. Male Sprague-Dawley rats (12 weeks old) were used in this experiment. Aortic balloon injury was performed on anesthetized rats as previously reported (31). After 11 or 21 days, tissues were rapidly fixed with paraformaldehyde and 4-µm thick sections were prepared on a cryostat. Immunostaining was performed with a primary rabbit anti-NPR-C antibody (diluted 1:1000) with or without preabsorption (10 µM antigen) and the avidin-biotin-peroxidase complex kit (Vector Laboratories, Burlingame, CA) as described by the manufacturer.

Cell cultures and binding assay of NPR-C
HeLa cells were obtained from Dainippon Pharmaceutical Co. (Osaka, Japan) and cultured in DMEM supplemented with 10% FCS. The human megakaryocyte-like cell line (Dami cells) was obtained from American Type Culture Collection (Rockville, MD) and cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 10% horse serum. Cultured human coronary artery SMCs were purchased from Clonetics Corp. (Walkerville, MD) and cultured using medium supplemented by the manufacturer. These cells were passaged in a controlled atmosphere of 5% CO₂-95% air at 37 C. Dami cells in 12-well dishes were washed twice with 1 ml PBS. Binding of 1 nM [¹²⁵I]human ANP was allowed to proceed for 30 min at 4 C to equilibrium in the presence of increasing concentrations of unlabeled human ANP and C-ANF-(4–23) (1 pM to 1 µM). After 30 min, cells were washed twice with PBS and solubilized with 500 µl 0.5 N NaOH. Aliquots were assayed for radioactivity in a Cobra -counter (Packard, Downers Grove, IL).

RNA analysis by Northern blotting hybridization
Total RNA was isolated from the Dami and HeLa cells using ISOGEN (Nippon gene, Toyama, Japan), and polyadenylated [poly(A)⁺] RNA was purified by oligo(deoxythymidine)-cellulose column chromatography using a mRNA separator kit (Clontech, Palo Alto, CA). Poly(A)⁺ RNA (5 µg) was fractionated in a 1% agarose gel containing 0.66 M formaldehyde and 0.02 M morpholinopropanesulfonic acid (pH 7.0). Fractionated RNA was transferred onto a nylon filter by capillary blotting and then cross-linked by UV irradiation. A ³²P-labeled complementary DNA (cDNA) fragment encoding the hNPR-C was used for Northern blotting hybridization as a probe.

Preparation of a probe DNA encoding the hNPR-C cDNA
To obtain a probe for human genomic DNA library screening, we performed RT-PCR using two sets of hNPR-C primers and human placental poly(A)⁺ RNA as a template (6). Total RNA was isolated from human placenta by standard protocol using the guanidinium thiocyanate procedure, and poly(A)⁺ RNA was purified by oligo(deoxythymidine)-cellulose column chromatography. First strand complementary DNA (cDNA) synthesis was performed using a first strand cDNA synthesis kit (Amersham) and the specific primer CR4 (5'-GGGAATTCATGTCTTTCAACCCGTCTATCTCC-3') corresponding to the 3'-sequence of the hNPR-C cDNA. After cDNA synthesis, the 5'-PCR primer C21 (5'-TTGGTACCGCCAGGAGAGAGAGGCGCTGCCGC-3') and the 3'-PCR primer CR7 (5'-GTGACTGTCTGGAGGGACGAGTATGC-3') were used for amplification by PCR. Denaturing, annealing, and polymerase reaction were performed 30 times at 94 C for 1 min, 50 C for 2 min, and 72 C for 3 min, respectively. The amplified 0.8-kilobase (kb) DNA fragment was separated on PAGE, digested by KpnI and SacI, and subcloned into the corresponding sites of pUC18. The nucleotide sequence was determined by the dideoxy chain termination method, using an Applied Biosystems model 373A sequencer and the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer/Cetus, Norwalk, CT). The fragment obtained by RT-PCR was confirmed to encode the N-terminal portion of the hNPR-C.

Library screening and DNA sequencing
The genomic DNA encoding the NPR-C was isolated from a human leukocyte genomic DNA library in EMBL3 (Clontech) using Escherichia coli K803 as a host and the 0.8-kb KpnI-SacI cDNA fragment as a probe. Plaques were lifted using Plaque Screen (DuPont-New England Nuclear, Boston, MA) and fixed by UV light exposure (Stratalinker, Stratagene, La Jolla, CA). The filters were prehybridized for 2 h at 55 C in 6 x SSC (standard saline citrate), 0.15 M NaCl, 15 mM sodium citrate (pH 7.0), 0.1% SDS, 5 x Denhardt’s solution (1 x Denhardt’s and 0.1% each of BSA, polyvinylpyrrolidone, and Ficoll), and 100 µg/ml sonicated heat-denatured salmon sperm DNA, and then hybridized with the denatured radiolabeled probe in the same solution for 16 h at 55 C. Labeling of a probe was performed using [-³²P]deoxy-CTP and a random primer labeling kit (Takara Shuzo). After hybridization, the filters were washed twice with 1 x SSC-0.1% SDS at 65 C for 1 h each time and exposed to x-ray film for 48 h at -80 C. Four positive clones were isolated from 1 x 10⁶ phage plaques and purified by following plaque hybridization. The 2.2-kb XhoI fragment was subcloned into the SalI-digested pUC19, producing pUC/hNPCR1, and determined for the nucleotide sequence described above.

Southern blot analysis
Human genomic DNA was isolated from peripheral blood cells using Genomix (Talent SRL, Trieste, Italy). A probe DNA encoding the 5'-flanking region upstream of the initiation codon ATG of the NPR-C gene was produced as follows. The oligonucleotides 5'-GGGAGCTCTCTAGAACCATCCCTTTCCCCCAG-3' and 5'-GGAGATCTCGTGCCGCAAGAAAGAGCTTGCCC-3', derived from the sequence of the 5'-flanking region of the gene, were used as a primer, and the cloned human genomic fragment was used as a template. The amplified 1.0-kb fragment was digested by SacI and BglII, and subcloned into the corresponding sites of pGVB (Pica Gene from Toyo Inki), generating phNPCR1. The genomic DNA was completely digested with restriction enzymes, XhoI, HindIII, and KpnI, and fractionated by agarose gel electrophoresis (10 µg/lane). The DNA fragments were transfered onto a filter, and then hybridized with a ³²P-labeled 1.0-kb DNA fragment, as described above. The filter was washed three times in 0.1 x SSC-0.1% SDS at 65 C for 30 min each time and subjected to autoradiography.

Determination of transcription start points
To determine the transcription start sites within the hNPR-C promoter, primer extension was carried out with poly(A)⁺ RNA from Dami cells as a template. The ³²P-labeled synthetic primers (primer A, 5'-AAGTGCTTGTAATTGACGGTG-3'; primer B, 5'-TTCCTCTGTCCCTGCGCCCC-3'), which are complementary to regions upstream of the ATG codon, respectively, were hybridized with 10 µg poly(A)⁺ RNA in 40 mM bis-Tris (pH 6.4), 400 mM NaCl, 1 mM EDTA, and 50% formamide at 25 C for 18 h. Extension reaction was carried out in 50 mM Tris-HCl (pH 8.3), 100 mM KCl, 10 mM MgCl₂, 1 mM each of deoxy-NTP, 20 U RNasin, and 25 U Moloney murine leukemia virus reverse transcriptase (Perkin-Elmer). Extended products were fractionated on an 8-M urea-8% PAGE. Dideoxy sequencing products primed by the corresponding primers were run in parallel for size comparison.

Ribonuclease (RNase) protection assay was performed with an RPA II kit from Ambion (Houston, TX). Bluescript SK^- (Stratagene) was ligated with a BamHI-HindIII fragment (473 bp) that covered the 5'-flanking region of the hNPR-C gene, cut with BamHI completely, and transcribed in vitro with T7 RNA polymerase (Pharmacia) to yield an ³²P-labeled riboprobe (Fig. 7C). Five micrograms of poly(A)⁺ RNA from Dami and HeLa cells were hybridized with the riboprobe at 45 C for 20 h and digested with 100-fold diluted RNase solution at 37 C for 30 min. The protected products were analyzed by 8 M urea-7% PAGE with ³²P-labeled and HaeIII-digested X174 RFI DNA to determine the sizes of the products.

View larger version (16K): [in this window] [in a new window]   Figure 7. RNA analysis by Northern blotting hybridization and transient expression analysis of the hNPR-C promoter-luciferase reporter chimeric gene. A, Poly(A)+ RNA was purified from HeLa cells. Five micrograms of poly(A)+ RNA were subjected to Northern blot analysis. Hybridization was carried out with the hNPR-C cDNA fragment as a probe, as described in Materials and Methods. B and C, Various lengths of the hNPR-C 5'-flanking sequences were inserted upstream of the firefly luciferase gene, and the nucleotide boundaries were confirmed by nucleotide sequencing. The chimeric constructs shown to the left were introduced into HeLa cells. Normalized luciferase activity, relative to the activities of phNPCRluc1 (B) and phNPCRluc3 (C), as 100% is shown to the right. These results presented here are the average from the experiments using plasmid DNAs prepared at least three times independently.

Transient cell transfection
Transient cell transfection was performed according to the calcium phosphate/DNA precipitate method. HeLa cells were seeded in 12-well plates at a density producing 10–20% confluence on the next day. After 18–24 h, 3 µg of each test plasmid and 0.2 µg of the control plasmid pRL-CMV carrying the Renilla luciferase gene downstream of the CMV promoter were added to the medium, incubated for an additional 16 h, then washed three times with PBS, and cultured again in the fresh medium. After 24 h, the cells were washed twice with PBS, collected in a microcentrifuge, and disrupted by a freeze-throw cycle in 300 µl cell lysis solution (Promega). The supernatant obtained by centrifugation for 5 min was pooled to measure firefly and Renilla luciferase activities. Luciferase activity was measured by TD20e (Turner, Sunnyvale, CA) with 15 µl cell extract using a dual luciferase reporter assay system (Promega).

Preparation of nuclear miniextracts
Cultured cells were suspended in 400 µl buffer A [10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonylfluoride]. The homogenates were chilled on ice for 15 min, and then 25 µl 10% Nonidet P-40 were added. After vigorous vortex for 10 sec, the nuclear fraction was precipitated by centrifugation at 15,000 x g for 5 min and suspended in 100 µl buffer B [20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM phenylmethylsulfonylfluoride]. The mixture was left on ice for 15 min with frequent agitation. Nuclear extracts were prepared by centrifugation at 15,000 x g for 5 min and stored at -80 C. The protein concentration was determined by the DC protein assay reagent (Bio-Rad, Richmond, CA).

DNA mobility shift assay
DNA-protein binding reactions were performed for 30 min at 30 C in a mixture (20 µl) containing 20 mM HEPES (pH 7.9), 0.3 mM EDTA, 0.2 mM EGTA, 80 mM NaCl, 1 mM DTT, 2 µg poly(deoxyinosine-deoxycytidine) (dI-dC), 0.1–0.4 ng ³²P-labeled oligonucleotide probe, and nuclear miniextracts (8 µg protein). Where indicated, the reaction was performed in the presence of unlabeled oligonucleotide competitors. Sequences of oligonucleotide within the P1 sequence were as follows: fragment A, 5'-AGACGAACAGGTTTACACCCGGTG-3'; fragment B, 5'-AGACGAACAGGTTTACACCCGGTGAACTTTTT-3'; and fragment C, 5'-AGGTTTACACCCGGTGAACTTTTT-3'. Sequences of oligonucleotide corresponding to P2, P3, and E2F binding sequences were as follows: P2, 5'-ACTAGTGACATTGCAGAGAAGGACGCTTCCTC-3'; P3, 5'-CTCTCTATCTTTTGGCGCATTAGTGAAGG-3'; and E2F-binding element, 5'-TCTTTTGGCGCATT-3'. The DNA-protein complexes were electrophoresed on 4% PAGE containing 6.7 mM Tris-HCl (pH 7.5), 3.3 mM sodium acetate, 2.5% glycerol, and 0.1 mM EDTA. Electrophoresis was carried out for 2 h in a cold room. After electrophoresis, the gel was dried and exported to a x-ray film.

Deoxyribonuclease I (DNase I) footprinting assay
To obtain a DNA fragment (from +10 to +80) used in the DNase I footprinting assay, pUC/hNPR-C11 was digested with EcoT14I and end labeled with [-³²P]ATP and T4 polynucleotide kinase. The resulting fragment was digested with SpeI and separated on PAGE. Forty micrograms of nuclear extracts from HeLa cells and 20 µg nuclear extracts from human coronary artery SMCs were preincubated in a 20-µl reaction mixture containing 25 mM HEPES (pH 7.6), 40 mM KCl, 3 mM MgCl₂, 0.1 mM EDTA, 10% glycerol, 1 mM DTT, and 1 µg poly(dI-dC). After 15 min on ice, 30,000 cpm of the endo-labeled fragment were added, and incubation was continued for 90 min at 4 C. Two microliters of DNase I (Takara), freshly diluted to a final concentration of 0.2 µg/µl in 25 mM CaCl₂, were added, and the digestion was allowed to proceed for 5 min at 4 C. The reaction was stopped by the addition of 4 µl 125 mM Tris-HCl (pH 8.0), 125 mM EDTA, and 3% SDS. Forty micrograms of proteinase K and 5 µg transfer RNA (Sigma) were added, and the reaction mixtures were incubated for 30 min at 65 C. The DNAs were extracted once with 100 µl phenol-chloroform, precipitated with 2.0 vol ethanol, resuspended in 98% formamide dye, and electrophoresed on 8 M urea-8% PAGE.

	Results

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NPR-C expression in the rat balloon-injured artery
In response to vessel wall injury, vascular SMCs undergo a phenotypic change from a differentiated, contractile state to a dedifferentiated, proliferative state. Balloon injury of the rat aorta is a well characterized model for studying this change in phenotype in vivo. The specificity of the antibody prepared in this study was tested by Western blot analysis using a rat lung membrane fraction. Under reducing conditions, the solubilized NPR-C protein was detected as a single band of approximately 63 kDa (data not shown). Immunohistological analysis demonstrated that NPR-C was strongly expressed in the SMCs in arteriosclerotic neointimas in the rat balloon-injured artery 11 and 21 days after surgery (Fig. 1). The SMCs in the intimal area 11 days after surgery were also revealed to be proliferating cell nuclear antigen positive (data not shown), strongly indicating that NPR-C is a useful marker molecule representing the phenotype of proliferative SMCs.

View larger version (87K): [in this window] [in a new window]   Figure 1. The expression of NPR-C at the histological level. Aortic balloon injury was performed on anesthetized rats as previously reported (31). Animals were killed 11 days (A and B) or 21 days (C and D) after surgery. Immunostaining was performed with a primary rabbit anti-NPR-C antibody (diluted 1:1000) and the avidin-biotin-peroxidase complex kit as described in Materials and Methods. B and D, Control staining with preabsorbed antibody and hematoxylin. I and M indicate intima and media, respectively. Original magnification, x40.

View larger version (29K): [in this window] [in a new window]   Figure 2. Southern blot analysis of the human genomic DNA. A, Restriction map of the human genomic DNA containing the hNPR-C 5'-flanking region. Arrows represent a DNA fragment used as a probe for Southern blot analysis. B, Human genomic DNA (10 µg) was double digested with XhoI, HindIII, XhoI/HindIII, KpnI/HindIII, and KpnI/XhoI. Hybridization was carried out using a 32P-labeled fragment of the 5'-flanking region in the NPR-C gene as a probe, as described in Materials and Methods.

View larger version (83K): [in this window] [in a new window]   Figure 3. Nucleotide sequence and putative regulatory elements of the 5'-flanking region of the hNPR-C gene. The nucleotide sequence from -1352 to +300 is given. Potential consensus sequences for DNA-binding proteins are underlined, with possible functions noted below.

View larger version (16K): [in this window] [in a new window]   Figure 4. RNA analysis by Northern blotting hybridization and ANP binding assay. A, Poly(A)+ RNA was purified from Dami cells. Five micrograms of poly(A)+ RNA were subjected to Northern blot analysis. Hybridization was carried out with the hNPR-C cDNA fragment as a probe, as described in Materials and Methods. B, Dami cells were incubated with 1 nM [125I]hANP in the absence or presence of unlabeled human ANP or C-ANF-(4–23), as described in Materials and Methods.

View larger version (45K): [in this window] [in a new window]   Figure 5. Determination of the transcription start point of the hNPR-C gene. Primer extension reactions (A and B) were performed with 10 µg poly(A)+ RNA isolated from Dami cells using 20-mer synthetic oligonucleotides, primers A and primer B, respectively, as described in Materials and Methods. The four left lanes, CTAG, are nucleotide sequencing using hNPR-C cDNA as a template. The right lane shows the extended products, and the most likely end point of the extension reaction is indicated by an arrow on the right and by stars on the sense strand shown on theleft. C, RNase protection analysis was performed with 5 µg poly(A)+ RNA as described in Materials and Methods. The riboprobe (523 bases) used in this experiment is shown in the left lane. The protected products and most likely the signals of the reaction are indicated by arrows on the right, and the sizes of the protected products are 250 (a) and 200 (b) bases. D, Positions of extended and protected products on the 5'-flanking region of the hNPR-C gene are shown.

View larger version (42K): [in this window] [in a new window]   Figure 6. Comparison of the nucleotide sequences around the transcription start site between the human and mouse NPR-C genes. The nucleotide sequences of human (h) and mouse (m) NPR-C genes are aligned for the best match. Identical nucleotides are indicated by dots, and the conserved AP-2-like elements and TATA box are underlined. Transcription start points are indicated by arrows. The three cis-acting DNA sequences (P1, P2, and P3) are underlined with bold lines.

View larger version (43K): [in this window] [in a new window]   Figure 8. DNA mobility shift assay and competition analysis of the P1 sequence in the hNPR-C gene. The 32P-labeled oligonucleotides corresponding to three sequences (fragments A, B, and C) within the P1 sequence, located at -60/-33, were incubated with 8 µg nuclear extracts from HeLa cells for 30 min at 30 C. The specificity of the shift was determined by shift competition with 500-fold higher concentrations of the unlabeled oligonucleotides, respectively. The most specific signal of the reaction is indicated by the arrow on the left. The DNA-protein complexes were analyzed by electrophoresis on 4% polyacrylamide gels as described in Materials and Methods.

View larger version (27K): [in this window] [in a new window]   Figure 9. DNA mobility shift assay and competition analysis of the P2 and P3 sequences in the hNPR-C gene. A 32P-labeled oligonucleotide corresponding to the P2 sequence, located at +14/+40 (A), or the P3 sequence, located at +41/+66 (B), was incubated with 8 µg nuclear extracts from HeLa cells for 30 min at 30 C. The specificity of the shift was determined by shift competition with 500-fold higher concentrations of the unlabeled oligonucleotides of the P2 sequence (A) and with 500-fold higher concentrations of the unlabeled oligonucleotides of the P3 sequence or the E2F binding sequence (B), respectively. The most specific signals of the reaction are indicated by the arrows on the left. The DNA-protein complexes were analyzed by electrophoresis on 4% polyacrylamide gels as described in Materials and Methods.

View larger version (46K): [in this window] [in a new window]   Figure 10. DNase I footprinting analysis of the hNPR-C gene. DNase I footprinting analysis was performed using a DNA fragment labeled on the 3'-end from +10 to +80 of the hNPR-C gene. The labeled DNA fragment was incubated with nuclear extracts as described in Materials and Methods. Lane 1, No nuclear extract; lanes 2 and 3, 40 µg nuclear extracts from HeLa cells; lane 4, 20 µg nuclear extracts from cultured human coronary artery SMCs. The nuclear extracts used in lanes 2 and 3 were independently prepared from HeLa cells. The numbers beside them refer to the nucleotide positions relative to the transcription start site. The protected DNAs were analyzed by electrophoresis on an 8-M urea-8% PAGE as described in Materials and Methods.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

On the other hand, TATA box-like elements, TATAT sequences (at -90, -80, -78, -76, -74, and -68), and TATTA sequence (at -66) were identified. We previously demonstrated similar tandem repeated TATA-box-like elements, five TATAT sequences, and TATTA sequence in the 5'-flanking region of the mouse NPR-C gene, suggesting a promoter-like sequence. Recent studies have shown that a poly(deoxyadenosine-deoxythymidine)-rich sequence was involved in expression of the various genes (40, 41). The AT-rich sequence in the 5'-flanking region of the Na⁺/H⁺ exchanger (NHE1) gene could enhance the promoter activity (40). Primer extension and RNase protection analyses in this study have shown that the transcription start points were located in the corresponding region; the AT-rich region is expected to have another promoter activity. However, lack of the region (from -273 to -34) containing these TATA box-like sequences and an additional consensus TATA box, identified at -107, did not affect the basal transcriptional activity of the hNPR-C gene in HeLa cells, indicating that the DNA sequences do not play a significant role in the promoter activity of the hNPR-C gene or that this transcriptional activity requires another cis-acting DNA sequence, located further upstream or downstream in the NPR-C gene locus.

Further analysis of the promoter region provided some interesting information. Promoter analysis demonstrated that lack of the P1 region (from -60 to -29) significantly decreased transcriptional activity. Primer extension analysis showing no extended product in the corresponding region indicated that the P1 sequence functions in a cis-acting positive manner. DNA mobility shift studies using nuclear extracts from HeLa cells demonstrated the nuclear factor(s) specifically binding to the DNA element from -60 to -37 within the P1 sequence. The promoter regions of the human and mouse NPR-C genes that showed high similarity to the DNA sequences downstream of the transcription start points suggested the essential role in transcriptional control of the downstream sequences. As we expected, two distinct sequences, the P2 and P3 sequences, were demonstrated to modulate the minimal promoter activity and to play an important regulatory role in NPR-C gene expression. DNA mobility shift assay revealed that a protein(s) in nuclear extracts from human cells expressing NPR-C transcripts specifically binds to these regions, respectively, and that no shifted DNA was seen using those from human chondrosarcoma SW1353 expressing no or little NPR-C transcripts (data not shown). Additionally, promoter analysis indicated that the P2 and P3 sequences were not active in a synergistic manner for enhancement of NPR-C gene transcription. DNase I footprinting analysis of the region containing these sequences revealed that a DNA element from +48 to +60 within the P3 sequence was preferentially protected, suggesting that this element within the P3 sequence actively regulates NPR-C gene expression and that interaction of nuclear extracts with the P3 sequence inhibits the binding of nuclear factor(s) to the P2 sequence (or reduces their affinity). However, the mechanism underlying this interaction remains unclear. Furthermore, we have demonstrated that nuclear extracts from human cultured vascular SMCs expressing NPR-C transcripts also specifically bind to this element from +48 to +60. A transcription factor motif search has shown that the E2F consensus element (42) is located within the P3 sequence. The retinoblastoma protein (Rb), an important nuclear factor in cell cycle control, is shown to be hyperphosphorylated around the G1/S boundary, leading to release of the E2F transcription factor that activates the transcription of genes required for DNA synthesis in S phase (43). The roles of E2F and Rb in vascular SMCs are now discussed. Nuclear factor(s) from HeLa cells binding to the P3 sequence did not recognize or require the E2F element within the P3 sequence. Although we investigated whether the P3 sequence can activate a foreign promoter, transcriptional activity of the simian virus 40 promoter carrying the P3 sequence downstream was slightly increased compared with that inserting it into the NPR-C minimum promoter (data not shown), suggesting its cis-acting effect in a promoter-specific manner. Further detailed studies will be needed to elucidate the mechanisms involved in this promoter-specific activation.

NPR-C mRNA is known to be detectable, but at a low level, in a wide variety of human tissues; lung, placenta, kidney, and heart (6, 44). One finding in this study was that NPR-C mRNA is expressed abundantly in HeLa cells and the human megakaryocyte-like cell line (Dami cells). Previous studies (45, 46, 47) have shown that the NPR-C level in platelets was significantly down-regulated in diabetic nephropathy, heart failure, and obese patients, mediating the availability of NPs to their biological receptors with guanylyl cyclase. Dami cells were useful in further studies to gain a better understanding of the regulation of NPR-C expression in platelets and its pathophysiological roles. Additionally, modulated expression of the NPR-C gene in vivo has been described in various experimental models, as described above. The pathophysiological loss of NPR-C is suggested to reduce not only the elimination rate of NPs with a protective role in cardiovascular disorder, but also the unidentified biological action via NPR-C.

Our present study is a first report regarding the structural and functional characterization of the 5'-flanking region of the hNPR-C gene and an initial step toward understanding the molecular mechanisms governing the regulation of NPR-C gene expression. It provides a basis for further understanding the transcriptional regulation of the NPR-C gene during the development of various diseases and its tissue-specific expression. We are planning to investigate the relationship between the NPR-C gene expression during the phenotypic change in vascular SMCs and the function of these cis-acting positive elements.

	Acknowledgments

 
We thank H. Sakurai, N. Shigemori, and K. Fujishige for their technical support.

	Footnotes

 
¹ The nucleotide sequence reported in this paper has been submitted to the GenBank TM/EMBL DataBank under accession no. AB001286.

Received August 11, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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