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Originally published In Press as doi:10.1074/jbc.M105293200 on July 10, 2001

J. Biol. Chem., Vol. 276, Issue 36, 34105-34114, September 7, 2001
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ESDN, a Novel Neuropilin-like Membrane Protein Cloned from Vascular Cells with the Longest Secretory Signal Sequence among Eukaryotes, Is Up-regulated after Vascular Injury*

Kazuhiro KobukeDagger §, Yutaka Furukawa§, Manabu Sugai, Kenji TanigakiDagger , Naohiro Ohashi§, Akira Matsumori§, Shigetake Sasayama§, Tasuku HonjoDagger , and Kei Tashiro||

From the Dagger  Department of Medical Chemistry, the § Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan and the  Center for Molecular Biology and Genetics, Kyoto University Graduate School of Medicine, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan

Received for publication, June 8, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A novel cDNA has been isolated from primary culture of human coronary arterial cells by a signal sequence trap method, and designated ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). ESDN is a type-I transmembrane protein with the longest cleavable secretory signal sequence among eukaryotes. ESDN contains a CUB domain and a coagulation factor V/VIII homology domain, which reminds us of the structure of neuropilins. ESDN also harbors an LCCL domain, which is shared by Limulus factor C and Coch. Mouse and rat counterparts were also identified revealing >84% amino acid identity with human ESDN. The human ESDN gene was mapped between D3S1552 and D3S1271. Northern blot analysis showed that ESDN mRNA was expressed in various tissues; particularly highly expressed in cultured vascular smooth muscle cells. The ESDN expression was up-regulated in platelet-derived growth factor-BB-stimulated vascular smooth muscle cells in vitro and neointima of the balloon-injured carotid artery in vivo. Overexpression of ESDN in 293T cells suppressed their bromodeoxyuridine uptake. In addition, ESDN protein was strongly expressed in nerve bundles in rodents. Thus, ESDN is considered to play a role in regulation of vascular cell growth and may have a wide variety of functions in other tissues including the nervous system, like neuropilins.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The vascular system is distributed throughout a body of multicellular organisms like vertebrates and is essential for their life. Its disturbance is the bases of many human diseases including ischemic heart disease, cerebrovascular disease, and many renal disorders. Hypertension, hyperlipidemia, and diabetes mellitus all impair vascular integrity through the acceleration of atherosclerosis. Even tumor growth and metastasis require the development of new blood vessel growth (1). In the field of cardiovascular medicine, much therapeutic advance was made through the introduction of percutaneous transluminal coronary angioplasty for coronary heart disease which is one of the leading causes of human morbidity and mortality. Successful interventions, however, are followed by 20 to 50% of restenosis within 6 months (2-4), which demands considerable burden to patients and expense to the society due to the necessity of repeated diagnostic tests and therapeutic interventions.

Despite significance in human disorders, the basic study of the vascular system lagged behind those of the immune and the nervous system, whose initial major works were achieved by cell biological or anatomical approach. The introduction of molecular biological methods have changed such situations, and much progress has been made through the study of extracellular signal transducing molecules. Using the gene targeting strategy, the ligand and its receptor systems of VEGF1-VEGFR (5-8) and angiopoietin-Tie (9, 10) were shown to be essential for vasculogenesis and angiogenesis, respectively. Recently, several molecules originally studied in the nervous system have been shown to be involved in the vascular system: Ephrin-Eph are the until now the only surface markers discriminating between arteries and veins (11); and neuropilin-1 is found to be a functional co-receptor for VEGFR2 in endothelium (12) as well as for plexin in axon guidance repulsive activity (13).

Growth factors (e.g. PDGF (14, 15), basic fibroblast growth factor (16), and transforming growth factor-beta ) and inflammatory cytokines (e.g. monocyte chemoattractant protein-1 (17), interferon-gamma , and interleukin-1beta (18)), which had been extensively studied in other fields like cancer biology or immunology, have been shown to play important roles in restenosis or atherosclerosis as well. Many of them are aggravating factors, while some of them (transforming growth factor-beta (19), interferon-gamma (20, 21), bone morphogenetic protein-2 (22, 23)) have opposing reports as an aggravating as well as a protecting factor. The regenerating endothelium is considered to act protective in these pathological processes (24, 25), and several candidates of intervening molecules have been proposed which include nitric oxide (26), prostacyclin (27), and heparin/heparan sulfates (28, 29). However, little is known about the existence of other protective extracellular signal transducing proteins.

We presumed the existence of much more extracellular signal transducing molecules which are involved in the homeostasis and pathophysiology of the vascular system. Therefore, we tried to clone such molecules by the signal sequence trap method (30). Using this cDNA screening method, many molecules on the secretory pathway with a wide variety of functions have been efficiently isolated, for example, SDF-1 (31), ESOP-1/MD2 (32), DANCE (33), FKBP23 (34), calumenin (35), syncytin (36), and so on. We conducted a modified version of signal sequence trap screening (37) to vascular cells, and succeeded in cloning several novel transmembrane or secretory proteins. One of these, ESDN, is a novel type-I transmembrane protein, has a characteristic domain structure reminding us of neuropilins, and will be described here.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Cell Culture-- Human angiotensin II (AT-II), human recombinant PDGF-BB, and fetal calf serum (FCS) were purchased from Sigma, Life Technologies, and JRH Biosciences, respectively. Genomic DNAs of cow, rabbit, and Drosophila were purchased from CLONTECH, and that of yeast from Promega. The other genomic DNAs were isolated as previously described (38). Primary culture of normal human coronary artery endothelial cells or smooth muscle cells were purchased from Clonetics (San Diego, CA), cultured in EGM-2-MV or SmGM-2 (BioWhittaker) according to the manufacturer's instructions, and used between passages 5 and 8. To co-culture them, an equal number of endothelial and smooth muscle cells were mixed and maintained in EGM-2-MV for 2 days before RNA extraction. For smooth muscle cell stimulation experiments, they were serum-starved in DMEM supplemented with 2 mM L-glutamine for 48 h before the medium was exchanged to DMEM + 2 mM L-glutamine with or without PDGF-BB, AT-II, or FCS of indicated concentrations. Mouse cell lines, A9(Neo3) and A9(Neo12), were purchased from JCRB Cell Bank and cultured in DMEM with 10% FCS. HEK293T cells were cultured in DMEM + 10% FCS. Rat aortic vascular smooth muscle cells were prepared by the explant method (39) and used between passages 5 and 8.

Total RNA Extraction, Poly(A)+ RNA Purification, and the Preparation of RT-PCR Templates-- Total RNA was extracted with TRIzol or TRIzol LS (Life Technologies, Inc.). Oligotex(dT)30 Super (Roche Molecular Biochemicals) was used for the purification of poly(A)+ RNA. The first strand cDNA for the ordinary or quantitative RT-PCR was synthesized from total RNA using the SuperScript Preamplification System for First Strand cDNA Synthesis Kit (Life Technologies, Inc.).

Signal Sequence Trap Screening and cDNA Cloning of Human, Mouse, and Rat ESDN-- Poly(A)+ RNAs from human coronary artery endothelial cells, smooth muscle cells, and their co-cultured cells were used for the construction of the cDNA libraries, and the yeast signal sequence trap screening was carried out as described previously (40). 5'- and 3'-rapid amplification of cDNA ends methods were performed using the Marathon cDNA Amplification Kit (CLONTECH). Coding sequences except the 5' most region of mouse and rat were obtained with RT-PCR using two kinds of primer pairs based on human sequence data: 5'-CTGCTCCAACTCCTCCTCCTTC-3' and 5'-CTGCTTCATTCCTTTCCACCAACCTG-3'; 5'-TGTGCTGGTCATGGTCCTCACTACTCTC-3' and 5'-TGTGCTTTAAAACGATGCTTTG-3'. The N-terminal region of cDNA (nucleotide 453 to 800 of GenBankTM accession number AF387548) labeled with [alpha -32P]dCTP was used for subsequent mouse genomic library screening with Lambda FIX II library (Stratagene). A sense primer of 5'-CTGGCCGCTCATTGGTCTCAGC-3' located on the obtained genomic sequence was paired with an antisense primer of 5'-GGATGTAAGGGTTCCACTCTCAGG-3' situated in the downstream exon to acquire the first methionine-containing coding region of mouse ESDN. The above sense primer was replaced with another one, 5'-GCACTATGCGGGCGGATTGC-3', for cloning the first methionine-containing region of human and rat ESDNs.

Computer Analysis and Data Base Search-- Handling of all the nucleotide and amino acid sequence data and the construction of hydrophobicity profiles were performed with GenetyxMac Version 9.0 (Software Development Co., Ltd.). Sequence alignment was executed with MacVector (Oxford Molecular Group). BLAST and FASTA searches were performed at www.ncbi.nlm.nih.gov/blast/blast.cgi and www.fasta.genome.ad.jp/ideas/fasta/fasta_nr-aa.html, respectively. Motif search was done at www.motif.genome.ad.jp/. Prediction of signal peptides and transmembrane regions were based on the methods previously reported (41, 42).

Anti-ESDN Antibody Production and Western Blot Analysis-- Rabbit anti-CUB and anti-FV/VIII polyclonal antibodies were raised against keyhole limpet homocyanin-conjugated polypeptides of GERIRIKFGDFDIEDSD and QDKIFQGNKDYHKDVRNN, respectively, and affinity purified against each polypeptide by Sawady Technology. Western analysis was carried out following the ECL (Amersham Pharmacia Biotech) or Renaissance (PerkinElmer Life Sciences) Western blot protocols. The other primary antibodies used are anti-FLAG M2 monoclonal antibody (Sigma) and His-probe H-15 polyclonal antibody (Santa Cruz).

Overexpression of ESDN cDNA and Immunofluorescent Cell Staining-- Human ESDN constructs of hESDN-FL, -Delta EC, or -Delta Cy were cloned into pEF6V5-His (Invitrogen) with swapping the original V5 epitope to the FLAG tag. Mouse ESDN constructs of mESDN-Ex(Edns) or -Ex(CD5) were cloned into pCAGGS (43). An amino acid sequence of human CD5 is MPMGSLQPLATLYLLGMLVASVLA. These expression vectors were transfected into 293T and COS7 cells with CellPhect (Amersham Pharmacia Biotech) and LipofectAMINE (Life Technologies, Inc.), respectively, following their protocols. Transfected COS7 cells were stained essentially as described (44). Used secondary antibodies were Texas Red-conjugated anti-mouse IgG (Vector Laboratories) and fluorescein isothiocyanate-conjugated anti-rabbit IgG (Jackson Laboratories).

Northern and Southern Blot Analyses-- Human ESDN (nucleotide 193-585 of GenBankTM accession number AF387547), rat ESDN (332-830 of GenBankTM AF387549) and rat GAPDH (459-1001 of GenBankTM M17701) cDNAs were labeled with [alpha -32P]dCTP and used as a probe for Northern and Southern analyses. Southern zooblot was washed with 1 × SSC at 37 °C. The other blots were all washed with 0.2 × SSC at 65 °C.

A Rat Carotid Artery Balloon Injury Model and Immunohistochemical Staining-- Fourteen- to fifteen-week-old male Sprague-Dawley rats were anesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (50 mg/kg), and the endothelium of the left common carotid artery was denuded with a 2F Fogarty embolectomy catheter (Baxter Healthcare), as previously described (17). The carotid arteries were harvested at 0, 5, or 14 days after balloon injury and used for quantitative RT-PCR analysis or immunohistochemistry. The latter procedure was performed as previously described (45) with the following modifications. Cold 4% paraformaldehyde was used for perfusion-fixation; and as primary antibodies, rabbit anti-peptide polyclonal antibodies at a concentration of 5-10 µg/ml or the same concentration of normal rabbit IgG (DAKO) for negative controls were used.

Quantitative RT-PCR analysis-- The mRNAs for human or rat ESDN and GAPDH were measured by real-time quantitative RT-PCR using PE Applied Biosystems Prism Model 7700 Sequence Detection System. The nucleotide sequences of the dye-conjugated probe, forward and reverse primers are as follows: ESDN probe, 5'-(6-FAM)-CCTGAGAGTGGAACCCTTACATCCATAAAC-(TAMRA)-3'; ESDN forward, 5'-CCCAGCAAGGTGATGGATG-3'; ESDN reverse, 5'-CAAGAATCAGAATCTTCAATGTCAAAG-3'. These were based on the human sequence, but were confirmed to be applicable in quantitative measurements of rodent transcripts as well. TaqMan GAPDH Control Reagents and TaqMan Rodent GAPDH Control Reagents (PE Biosystems) were used for measurements of human and rat GAPDH, respectively. Standard curves were prepared from known amount of plasmids with ESDN or GAPDH amplicon subcloned into pBlueScript SK(-) (Stratagene), and the ESDN mRNA level normalized to that of GAPDH was used for further analyses.

BrdUrd Uptake Assay in 293T Cells-- 293T cells were transfected by CellPhect (Amersham Pharmacia Biotech) as the manufacturer's instructions. After 12 h of incubation with transfection solution, the medium was replaced with fresh DMEM + 10% FCS and incubated for 2 h, trypsinized, and replated to 96-well plates in duplicate. For one plate, a 2-h BrdUrd pulse was applied after 24 h of culture and incorporated BrdUrd was measured by Cell Proliferation ELISA, BrdUrd (colorimetric) (Roche Molecular Diagnostics) following the manufacturer's instructions. The other plate was used to estimate the cell number with tetrazolium/formazan assay using Premix WST-1 assay kit (TaKaRa Shuzo Co., Shiga, Japan) 2 h after replating.

Statistical Analysis-- Data are presented as mean ± S.E. and were analyzed by a paired t test with its p value corrected according to the Bonferroni's method. Normalization of mean1 ± S.E.1 (n = N) to mean2 ± S.E.2 (n = N) was performed as follows: normalized mean = mean1/mean2, normalized S.E. = (mean1/mean2) × [{(S.E.1/mean1)2 + (S.E.2/mean2)2}/N](1/2). In this case, the number of degrees of freedom ranges from N-1 to 2x(N-1) depending on the variances (this calculation can be performed with the Smith-Satterthwaite procedure), but we always assumed it to be N-1, which is the most conservative estimate for yielding statistical significance in subsequent analyses.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Signal Sequence Trap Screening from Primary Culture of Human Coronary Arterial Cells and Cloning of the Full-length Human, Mouse, and Rat ESDN cDNAs-- The three types of cDNA libraries constructed from primary culture of human coronary artery endothelial cells, smooth muscle cells, and their 2-day co-cultures yielded 3.2 × 106 Escherichia coli transformants, which were screened by the yeast signal sequence trap method described previously (40). Five thousand positive clones were obtained and all the base sequences were determined with redundant clones being removed by dot-blot hybridization. More than 90 different genes were identified; >10 of them were novel genes. One such clone derived from the co-culture library contained a CUB domain, which is found in an increasing number of secretory or transmembrane proteins with various functions (46), and was selected for a further study.

As the clones obtained by the signal sequence trap screening held only part of 5'-untranslated region and portion of the coding region, 5'- and 3'-rapid amplification of cDNA ends were performed to isolate the full-length cDNA. We also isolated mouse and rat counterparts by RT-PCR based on human sequence data. Alignment of human, mouse, and rat amino acid sequences deduced from their nucleotide sequences showed more than 84% amino acid identity between human and rodents, but revealed that the first methionine of human gene obtained by the signal sequence trap method was not conserved in rodents. Extensive 5'-rapid amplification of cDNA ends could not reach the definitive first methionine in any species. High GC content in the 5' region of ESDN (data not shown) could cause a high-order structure of mRNA hindering the performance of a reverse transcription in addition to the difficulty in PCR reaction. Therefore, we conducted a genomic library screening, which is dependent on neither process. Screening of a 1.2 × 106 mouse phage genomic library yielded two positive clones, and the determinated base sequence of the neighborhood of probe-hybridizing region revealed a candidate for the first methionine with an upstream stop codon. A sense primer was designed 5' side of this stop codon and paired with an antisense primer in the downstream exon to yield genuine RT-PCR product without an intron. Using human or rat RT-PCR templates, another primer pair covering mouse first methionine could also yield counterparts of these species. Alignment of three products showed conservation of the location of the first methionine which agreed very well with the Kozak consensus site (data not shown) (47). The overall primary structure was also well conserved (Fig. 1A), with an amino acid identity between human and rodents and between mouse and rat is 84-85 and 92%, respectively.


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  		Fig. 1.   Primary structure of ESDN with the longest secretory signal sequence. A, alignment of deduced amino acid sequences of human, mouse, and rat ESDNs. Identical and homologous amino acids are indicated by dark and light shading, respectively. Closed triangles mark potential N-linked glycosylation sites common to three species, and open triangles are those found only in human. Bi-directional arrows show the peptide region against which rabbit polyclonal antibodies were raised. Limits of the domains are also indicated. B, the domain structure of ESDN, neuropilins, and Coch. Signal sequences at the N terminus (S.S.), CUB domains (CUB), LCCL modules (LCCL), transmembrane regions (TM), coagulation factor V/VIII-homology domains (FV/VIII), a MAM domain (MAM), von Willebrand factor type A domains (vWFA), and cytoplasmic domains (Cytoplasmic) are indicated. C, alignment of LCCL modules from human, mouse, or rat ESDN, Limulus factor C, two LCCL domains of predicted rat Lgl-1, and human, mouse, or chicken Coch. Dark and light shading means identical and homologous amino acids, respectively. Four conserved cysteines are marked by asterisks (*). Number symbols (#) indicate the four positions on human Coch where mutations were found in DFNA9.

A Novel Clone, Named ESDN, Resembles Neuropilins in Its Domain Structure and Harbors a Recently Reported LCCL Domain-- Hydrophobicity profiles (Fig. 2A) of a novel clone showed an atypically long secretory signal sequence (a further analysis will be described below) and one transmembrane region, suggesting that it is a type-I transmembrane protein. A motif search revealed that the novel clone has two domains, a CUB domain and a coagulation factor V/factor VIII homology (FV/VIII) domain. This domain structure reminds us of neuropilins, which is a type-I transmembrane protein composed of two CUB domains, two FV/VIII domains, and one MAM domain in its extracellular portion (Fig. 1B). Based on the source and this structural similarity, we named this novel clone ESDN (endothelial and smooth muscle cell-derived neuropilin-like molecule). A sequence homology search of the other area revealed that the region between the CUB and FV/VIII domains has significant homology with Coch (48) and Limulus factor C (49). This region has been recently recognized and named as an LCCL module (50). The LCCL domain was not pointed out in the original paper reporting Lgl-1 (51), but Trexler et al. (50) demonstrated that a single base insertion revealing that Lgl-1 contains two tandem LCCL domains. Alignment of these proteins showed the conservation of four cysteines in this domain (Fig. 1C).


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  		Fig. 2.   Recombinant expression of ESDN, functional analysis of its signal sequence, and characterization of anti-peptide antibodies. A, hydrophobicity profiles of human and mouse ESDN calculated by the algorithm of Kyte and Doolittle (91). B, a schematic view of the expression vectors. Human ESDN of full-length (hESDN-FL), extracellular (hESDN-Delta EC), or cytoplasmic (hESDN-Delta Cy) constructs were all tagged with C-terminal FLAG and 6xHis. For construction of secretory type, the extracellular portion of mouse ESDN with C-terminal 6xHis tag was prepared with its signal sequence intact (mESDN-Ex(Edns)) or swapped by that of human CD5 (mESDN-Ex(CD5)). C, Western blot analysis of the recombinant full-length ESDN. 293T cells were transiently transfected with the human full-length construct. The cell lysates were applied to SDS-polyacrylamide gel electrophoresis analysis. Western blot detection was performed by anti-FLAG (left), anti-CUB (middle), or anti-FV/VIII (right) antibodies. D, surface expression of ESDN. COS7 cells were transiently transfected with the human full-length ESDN construct. The protein was detected at the cell surface both with an anti-FLAG (middle) and with anti-peptide (left) antibodies, indicating co-localization on the cell surface as observed in merged images (right) under confocal laser microscopy. As an anti-peptide antibody, anti-CUB, and anti-FV/VIII antibodies were used in upper and lower panels, respectively. E, the longest secretory signal sequence of ESDN is cleavable at the predicted site. 293T cells were transiently transfected with the constructs made up of extracellular portion of ESDN and C-terminal 6xHis, harboring endogenous or CD5-derived signal sequence. Culture media were harvested 2 days later and immunoblotted with the anti-6xHis antibody. The product of exactly the same size confirms the appropriateness of the predicted location of the signal sequence cleavage.

Expression of Recombinant ESDN Protein, Characterization of Anti-peptide Antibodies, and Confirmation of the Signal Sequence-- 293T cells were transiently transfected with an expression vector containing the full-length human ESDN cDNA with a C-terminal FLAG tag (and a 6xHis tag) driven by the human elongation factor-1alpha promoter (Fig. 2B). Cell lysate was analyzed by Western blot using anti-FLAG monoclonal antibody and two kinds of polyclonal antibodies raised against polypeptide within the CUB or the FV/VIII domain (bi-directional arrows in Fig. 1A). All three antibodies commonly recognized three sizes of protein bands, ~127, 106, and 93 kDa (Fig. 2C). These protein products were larger than the predicted molecular mass of 80 kDa, which was calculated from signal sequence-removed 709 amino acids plus 17 amino acids from the C-terminal FLAG and 6xHis tags. This size difference can be due to N-glycosylation, considering that 5-7 potential sites are there (Fig. 1A).

Because the predicted signal sequence of ESDN is very long and atypical in its hydrophobicity profile (Fig. 2A), we analyzed the suitability of this point by two ways. First, COS7 cells were transiently transfected with human full-length ESDN cDNA, double-stained with anti-FLAG and anti-ESDN antibodies, and observed under a confocal laser microscope. Fig. 2D confirmed the cell-surface expression of this protein. Then, we tried to confirm the location of a signal sequence cleavage site. The extracellular portion of mouse ESDN was recloned into an expression vector under the CAG promoter with the C-terminal 6xHis tag. We prepared another construct whose signal sequence was swapped with that of human CD5 (Fig. 2B). 293T cells were transiently transfected with these constructs, and culture media were analyzed by Western blot using the anti-6xHis antibody. Both constructs yielded the protein product of exactly the same size (Fig. 2E). As the size difference of the signal sequences between human CD5 and mouse ESDN is 39 amino acid residues, corresponding approximately to a difference of 4 kDa, this result supports the same location of signal sequence cleavage sites.

Conservation and the Chromosomal Localization of the Human ESDN Gene-- To characterize the evolutionary conservation of ESDN, a Southern zooblot analysis was performed with human ESDN probe (Fig. 3A). In addition to strong bands observed in all mammals (mouse, rat, rabbit, cow, and human), weak bands were detected in Xenopus. No bands were observed in fly and yeast. BLAST search also revealed that there exist several ESTs from zebrafish (data not shown). Thus the conservation of ESDN is high in mammals, with extension to vertebrates.


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  		Fig. 3.   Evolutionary conservation of ESDN and its chromosomal localization in human. A, Southern zooblot analysis of ESDN was performed with 32P-labeled human ESDN cDNA as a probe. The filter was loaded with 200 ng (Saccharomyces cerevisiae), 1 µg (D. melanogaster), or 10 µg (the other species) of EcoRI-digested genomic DNA as normalized to genome size. B, human ESDN gene is on chromosome 3. Genomic DNAs from mouse cell lines, A9(Neo3) and A9(Neo12), were loaded and hybridized with the 32P-labeled human ESDN cDNA probe.

Search for STS in human ESDN sequence revealed that it contains two independent STS clones, stSG29921 and sts-D29024, which are mapped to D3S1603-D3S1271 and D3S1552-D3S1603, respectively (52). To confirm this data base result experimentally, mouse cell lines A9(Neo3) and A9(Neo12), which contain human chromosome 3 and 12, respectively, were used for genomic Southern hybridization. Human ESDN probe identified a cross-hybridized mouse band in both lanes, but two additional human bands were present in the lane of A9(Neo3) but not in the negative control lane of A9(Neo12) (Fig. 3B). Thus the human ESDN gene is located around D3S1603 within the range between D3S1552 and D3S1271, which corresponds to the cytogenetic region of chromosome 3p12-q11 (53).

Expression Profile of ESDN in Human and Rat Cells or Tissues-- Northern blot analysis using total RNA from cultured human coronary arterial cells revealed that the major and minor transcripts of 6.4 and 3 kilobases, respectively, were highly expressed in smooth muscle cells, and significantly, but to a lesser degree, in endothelial cells (Fig. 4A). Co-culture of the cells neither up-regulated nor down-regulated its expression. Northern blot using poly(A)+ RNA from various rat cells or tissues revealed that ESDN was expressed in many organs, with the highest expression in cultured aortic smooth muscle cells, undetectable in whole blood cells, and very faint in liver (Fig. 4B).


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  		Fig. 4.   Northern blot analysis of ESDN. 15 µg of total RNA from human coronary arterial cells (panel A) and 3 µg of poly(A)+ RNA from rat organs or cultured aortic smooth muscle cells (panel B) were subjected to Northern blot analysis with human and rat cDNA, respectively, as a probe. Equivalent loading of RNA was confirmed by ethidium bromide staining of 18 S and 28 S rRNA (A) or rehybridization of the same filter with rat GAPDH probe (B). hCAEC, human coronary artery endothelial cells; hCASMC, human coronary artery smooth muscle cells; HeLa, HeLa cells; RASMC, rat aortic smooth muscle cells.

Augmented Expression of ESDN in PDGF-BB- but Not AT-II-stimulated Human Coronary Artery Smooth Muscle Cells-- The result of Northern blot analysis directed our attention to the regulation of ESDN expression in vascular smooth muscle cells. First as an in vitro study, we examined the effect of the stimulation with PDGF-BB, AT-II, or FCS on primary culture of human coronary artery smooth muscle cells. The stimulation was continued for 48 h because AT-II, which is only a weak mitogen for vascular smooth muscle cells in a short-term stimulation (<24 h), is reported to up-regulate their DNA synthesis by 48 h stimulation (54). The results of quantitative RT-PCR normalized to GAPDH (Fig. 5A) showed that ESDN was up-regulated with PDGF-BB dose-dependently, but not with AT-II stimulation. FCS also up-regulated ESDN expression dose-dependently, but much less than PDGF-BB. Thus, ESDN mRNA expression is positively and specifically controlled by PDGF-BB stimulation.


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  		Fig. 5.   Up-regulation of ESDN mRNA in the setting of vascular stimuli. A, an induction of ESDN mRNA in human coronary artery smooth muscle cells in response to PDGF-BB or FCS stimulation, but not to AT-II. Human coronary artery smooth muscle cells were serum depleted for 2 days, and the medium was exchanged to basal medium of DMEM supplemented with 2 mM L-glutamine and either of PDGF-BB, AT-II, FCS, or the mixture of PDGF-BB and AT-II at the indicated concentrations. After 2 days stimulation, total RNA was extracted and quantitative RT-PCR was performed. ESDN and GAPDH were measured in triplicate and the ESDN means and S.E. normalized to those of GAPDH were shown. Similar response was observed in another experiment. A, p < .01 versus basal medium. B, p < .01 versus PDGF 2 ng/ml. C, p < .01 versus AT-II 1 µM. B, up-regulation of ESDN mRNA in balloon-injured rat carotid arteries. Quantitative RT-PCR was performed to calculate ESDN mRNA normalized to GAPDH mRNA. Value of each time point represents the mean ± S.E. (n = 5 at day 0 or 5 and n = 4 at day 14). At day 0, the results obtained from the samples of normal control rats were shown. *, p < .03 versus day 0.

Augmented Expression of ESDN in Balloon-injured Rat Vessels-- To explore the significance of such in vitro expression regulation in the in vivo system, an ESDN expression profile in balloon-injured rat carotid arteries was studied. Quantitative RT-PCR analysis revealed that rat ESDN mRNA expression normalized to GAPDH mRNA showed a tendency of up-regulation at day 5, and an significant increase at day 14 by 30% (Fig. 5B). To elucidate the expression and localization of ESDN at the protein level, an immunohistochemical study was performed using anti-peptide antibodies characterized in Fig. 2C. Two antibodies raised against different portions of ESDN were used in parallel to rule out the nonspecific staining. The anti-CUB and anti-FV/VIII antibodies yielded the comparable staining results, and only the data obtained with the former was shown in Fig. 6. Nerve bundles surrounding a carotid artery (a vagus nerve and ansae cervicales) were strongly stained with these two antibodies (Fig. 6, A and B). At day 0, tunica media, which is mainly composed of smooth muscle cells, was positively but weakly stained (Fig. 6E). At day 14, stronger staining is observed throughout the neointima as well as in tunica media (Fig. 6, B and F). Thus ESDN expression is up-regulated in rat-injured arteries both at mRNA and at protein levels.


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  		Fig. 6.   Up-regulation of ESDN protein in the rat carotid artery following balloon injury. Immunohistochemical staining of rat carotid arteries with anti-CUB antibody (A, B, E, and F). Negative control results were shown just below of each sample (C, D, G, and H) obtained by using an equal concentration of normal rabbit IgG as a primary antibody. E through H are the higher magnification (×400) images of A through D (×100), respectively. The tunica media (M) and neointima (N) are shown in E-F. Arrows and arrowheads indicate the surrounding vagus nerve and ansae cervicales, respectively.

ESDN Down-regulates BrdUrd Uptake in 293T Cells-- Up-regulation of ESDN in the neointima, where vascular smooth muscle cells transform from resting to proliferating phenotype, then returning finally to resting, suggests the possibility that ESDN has some function related to cell growth regulation. We analyzed, through BrdUrd uptake experiments, the effect on 293 T-cell growth of the overexpressed full-length or deletion mutants of ESDN driven by human elongation factor-1alpha promoter. 293T cells were chosen because of the high transfection efficiency (more than 50%). Preliminary experiments showed that ESDN had a small growth-suppressive effect which was easily masked by rapidly growing non-transfected cells and smaller than the error of cell-counting manually by a Burker-Turk hemocytometer. Thus we measured the BrdUrd uptake 1.5 days after transfection and normalized cell number at replating to 96-well plates by measurement of an aqueous soluble tetrazolium/formazan assay. The cell number can be more accurately estimated in a variety of cell lines by this assay (55) than by counting manually with the Burker-Turk hemocytometer in our experience. Full-length ESDN down-regulated BrdUrd uptake significantly. Deletion of an extracellular portion alleviated this effect, and deletion of an intracellular portion completely and significantly eliminated the suppressive effect (Fig. 7). The same results were reproducibly obtained in three independent experiments. Thus ESDN down-regulates the BrdUrd uptake in 293T cells in this experimental protocol.


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  		Fig. 7.   Suppression of BrdUrd uptake in 293T cells overexpressed with full-length ESDN. 293T cells were transfected with the upper three constructs shown in Fig. 2B, and the BrdUrd uptake was measured 1 day later. Values were expressed as mean ± S.E. of octaplicate results in one representative experiment. The same results were obtained in another two independent experiments. *, p < .01 versus mock; #, p < .025 versus hESDN-FL.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have cloned a novel cDNA from human coronary arterial cells, termed ESDN, with its mouse and rat counterparts. Data base search revealed that there exist several matching sequences. GenBankTM accession number D29810 partially covers the human ESDN coding region with some errors (discussed later), and GenBankTM AK001362 and AF104313 (56) both correspond to the 3'-untranslated region of human ESDN. The 3' region of GenBankTM AK006805 matches the transmembrane and cytoplasmic domains of mouse ESDN. This paper, however, is the first report about identifying the full-length ESDN-coding region and its characterization.

A notable feature of this gene is its domain structure resembling that of neuropilins. Neuropilin-1 was originally identified as an antigen expressed in the developing optic neurons in Xenopus embryo (57). The cloning of this gene revealed a unique domain structure made up of two CUB domains, two FV/VIII domains, one MAM domain, and a transmembrane segment attached to a short cytoplasmic tail (58). Another family member, neuropilin-2 has the same domain structure (59). The unique feature of neuropilins is the promiscuity for ligands and co-receptors. They function as a receptor for class 3 semaphorins (60, 61) in a complex with a co-receptor partner of plexins (13), and mediate chemorepulsive (59, 62) as well as chemoattractive (63) signals to axons and the latter effect to dendrites (64). Neuropilin-1 is also a co-receptor of VEGFR2 for VEGF165 and enhances its biological function in vascular endothelial cells (12). The mapping study of the ligand-binding region has advanced in the study of neuropilins revealing that the CUB domain is the primary binding site and specificity determining region for semaphorins (65), whereas the FV/VIII domain is responsible for binding with VEGF165 (66). The interaction with plexin A1 is mediated through the extracellular portion of neuropilin-1 (13). The extracellular portion of neuropilin-1 does not directly bind solely to VEGFR2, but it shows a strong and reversible interaction with VEGFR-1, depriving VEGFR-1 of its affinity for VEGF (67). Although the exact partner has not been identified, the existence of another heterophilic cell adhesion system has also been suggested for neuropilin-1 (68). The CUB domain is shared by a number of extracellular proteins, most of which are involved in the developmental processes (46), and some of which are reported to be involved in ligand binding (69, 70). The FV/VIII domain is also known as a discoidin-like domain. Discoidin was originally identified in the slime mold Dictyostelium discoideum and is thought to facilitate cellular aggregation and migration by functioning as a lectin (71, 72). It is also found in over 20 kinds of eukaryotic proteins (73). Although it is presently unknown whether any of the mammalian proteins with the discoidin-like domain could act as lectins as well, many of them appear to be implicated in cell surface-mediated regulatory events, as exemplified in blood coagulation factors (74), discoidin domain receptor (75), or neurexin IV (76). As a molecule containing both CUB and FV/VIII domains and because of its structural similarity with neuropilins, ESDN can be considered to have a great potential for binding with more than a kind of ligand or co-receptor.

Another structural feature of ESDN is its long cytoplasmic tail, whereas neuropilins contain a short cytoplasmic domain of about 40 amino acids long with no known catalytic or binding motif. Indeed, it was shown that neuropilin-1 does not require its transmembrane and cytoplasmic domains to mediate the biological activity of semaphorin 3A, implying that another transmembrane protein(s) transduces the signals across the cell membrane (65). Such co-receptors for VEGF165 and semaphorin 3A are VEGFR-2 and plexin A1, respectively. VEGFR-2 harbors a kinase domain, whereas the long cytoplasmic domain of plexin A1 is devoid of an apparent catalytic domain or any known motifs. However, plexin A1 was shown to harbor a phosphorylation site(s) and associating kinase activity (77). The cytoplasmic domains of human, mouse, and rat ESDN conserve 15 serines, 15 threonines, and 11 tyrosines, and 9, 5, and 5, respectively, are candidate sites of phosphorylation (data not shown) according to the predicting tool (78). Therefore, ESDN potentially makes complexes with other transmembrane or soluble kinase receptors and offers phosphoprotein as a docking site for adapter protein(s) to induce an intracellular signal transduction.

An LCCL domain is a recently recognized motif which is shared by and whose name is derived from Limulus factor C, Coch, and Lgl-1 (50). Factor C is an initiator in Limulus amoebocytes of the coagulation cascade against lipopolysaccharide, and its LCCL domain is located in the H chain which is responsible for lipopolysaccharide binding (49, 79). Lgl-1 was cloned as a glucocorticoid-inducible gene that surges in the late gestation lung and is highly expressed in its mesenchyme (51). Coch is considered to be a secretory protein and a causal gene for one human hereditary deafness disorder, DFNA9 (80). The pathogenesis of this disease has not completely been elucidated, but the deposition of acidophilic mucopolysaccharide-containing substance is considered to cause strangulation of vestibular and cochlear neurons (80). All four kinds of missense mutations identified to date in DFNA9 patients converge in this domain (Fig. 1C) (80, 81), implicating the importance of this domain in Coch. Another LCCL domain containing protein, cub1 predicted in (GenBankTM accession number D29810)2 is identical to human ESDN with some sequence mistakes, which spoil the alignment of LCCL domain (50). With correct sequence, the alignment as shown in Fig. 1C revealed perfect conservation of four cysteines. Although we cannot reach the exact role of the LCCL domain in ESDN at present, its possession by ESDN should add scientific interest to the functional analysis of the LCCL domain.

ESDN has a very long N-terminal secretory signal sequence of 67 (human) or 64 (rodents) amino acid residues. While there are reports on viral (82) or bacterial (83) proteins bearing a longer signal peptide for secretory pathway with a long n-region (84), the signal peptide of ESDN is to our knowledge the longest among eukaryotic proteins reported to date (85). Therefore, we functionally assayed the activity of this long signal peptide to guide mature protein to cell surface in Fig. 2D as well as the cleavage of this peptide in Fig. 2E.

Two independent STSs matched human ESDN sequence, and combined with genomic Southern blot analysis of mouse cell lines containing a human chromosome, human ESDN is mapped between D3S1552 and D3S1271 (chromosome 3p12-q11) (52, 53). One genetic disorder linked to this region is familial nonspecific dementia, mapped between D3S1284 and D3S1603 (chromosome 3p11.1-q11.2) (86). Considering the high expression of ESDN in the nervous system (discussed later), further investigation is needed to determine whether ESDN is involved in this disease.

Northern blot analysis revealed relatively ubiquitous expression of ESDN, but the highest expression was observed in cultured vascular smooth muscle cells in both human and rat, which directed our attention to scrutinize the expression profile of ESDN in vascular smooth muscle cells. Because cultured vascular smooth muscle cells are considered to be in the synthetic phenotype in contrast to the contractile phenotype observed in the physiologic adult vessel wall, we examined the effect of growth factors on the expression regulation of ESDN in cultured vascular smooth muscle cells. We tested PDGF-BB, AT-II, and FCS for this purpose. PDGF-BB and, to a lesser extent, FCS up-regulated DNA synthesis, whereas AT-II did not. PDGF-BB and AT-II partly share many downstream signaling pathways (87, 88), but there is also some difference, for example, a delayed mitogenic effect of the latter (54). The gene expression of ESDN is controlled specifically under the PDGF-BB signaling pathway. Next we investigated the ESDN expression in a balloon-injured rat carotid artery as an in vivo model where the growth of vascular smooth muscle cells is involved in the pathogenesis of the expansion of neointima (89). At the mRNA level, ESDN was up-regulated by about 30% at day 14, but the up-regulation was more strikingly shown by the immunohistochemical study. ESDN protein was more highly expressed in neointima than in tunica media at this later stage of vascular injury (Fig. 6F).

For further characterization of its function, we tried to examine the effect of overexpression of ESDN in 293T cells, which endogenously express a limited amount of ESDN at the mRNA level (data not shown). As shown in Fig. 7, the full-length construct significantly down-regulated the BrdUrd uptake, while constructs deleted with the extracellular or intracellular portion were abrogated with this suppressive effect. Because the principal difficulty in the detection of the growth-suppressive effect by transient transfection assays, ignorance of its ligand(s) and the results of small effects, we must refine the system like the adoption of inducible-expression system or check in other cells like vascular smooth muscle cells before concluding its function. However, we could present a model that ESDN is up-regulated in neointimal smooth muscle cells after vascular injury to counteract the growth-promoting signal as a "brake." This is reminiscent of our previous report about DANCE, which was cloned from embryonic mouse heart as a novel secretory molecule by the yeast signal sequence trap screening (33). Detailed expression analysis in the balloon-injured rat carotid artery showed a similar expression profile, which led to the speculation that DANCE may have the growth-suppressive effect. The study of expression profile of ESDN in human atherosclerotic lesion is needed to further clarify the significance of this gene in vascular pathology.

Another important feature of ESDN is its expression in the nervous system. It is highly expressed in a vagus nerve or ansae cervicales in adult rats (Fig. 6, A and B) and in brain or vagus nerves of mouse embryos (data not shown). It is interesting that the structurally related neuropilins also have dual roles in the nervous system as well as the vascular system. Another example of dual activity is the Ephrin-Eph system. Eph is the largest family of receptor tyrosine kinase studied extensively in the nervous system (90), and this ligand-receptor system has been re-identified as the marker discriminating arteries and veins (11).

Considering the unique domain structure and rather ubiquitous expression in addition to vascular cells and nerve bundles, it is possible that ESDN has wide functions not confined in the setting of vascular injury. Identification of its ligands, and possibly its co-receptors, should add a new field in the extracellular signal transduction network involving vascular, neural, and other systems.

    ACKNOWLEDGEMENTS

We thank Dr. Reiko Shinkura for helpful advice in genomic DNA preparation and DNA sequencing, Dr. Nobuo Kanazawa for the manipulation of mouse cell lines A9(Neo3) and A9(Neo12), Dr. Tomoyuki Nakamura for modified pEF6V5-His plasmid, Prof. Hiroshi Nakata (Kyoto Sangyo University) for discussion about glycosylation, and Prof. Junichi Miyazaki (Osaka University) for pCAGGS. We also thank Naoko Tomikawa and Masami Tanaka for excellent technical assistance.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF387547 (human ESDN), AF387548 (mouse ESDN), and AF387549 (rat ESDN).

|| To whom correspondence should be addressed: Center for Molecular Biology and Genetics, Kyoto University Graduate School of Medicine, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507 Japan. Tel.: 81-75-751-4192; Fax: 81-75-751-4190; E-mail: ktashiro@mfour.med. kyoto-u.ac.jp.

Published, JBC Papers in Press, July 10, 2001, DOI 10.1074/jbc.M105293200

2 T. Shibata, unpublished data.

    ABBREVIATIONS

The abbreviations used are: VEGF, vascular endothelial growth factor; AT-II, angiotensin II; BrdUrd, 5-bromo-2'-deoxyuridine; CUB domain, domain found in complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1; cDNA, complementary DNA; DMEM, Dulbecco's modified Eagle's medium; EST, expressed sequence tag; FCS, fetal calf serum; FV/VIII domain, coagulation factor V/factor VIII-homology domain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LCCL domain, domain found in Limulus factor C, Coch and Lgl-1; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; rRNA, ribosomal RNA; RT-PCR, reverse transcriptase-polymerase chain reaction; STS, sequence-tagged site; VEGFR, vascular endothelial growth factor receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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