DANCE, a Novel Secreted RGD Protein Expressed in Developing, Atherosclerotic, and Balloon-injured Arteries

Abstract

We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries andneural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1–5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis byin situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling. We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries andneural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1–5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis byin situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling. Vascular development in the growing embryo requires the controlled proliferation of smooth muscle cells, endothelial cells, and fibroblasts and their continuous remodeling to form larger vessels. In most adult tissues, these vascular component cells are normally quiescent. However, vascular remodeling also plays an important role in many cardiovascular disorders (1Gibbons G.H. Dzau V.J. N. Engl. J. Med. 1994; 330: 1431-1438Crossref PubMed Scopus (1351) Google Scholar). For example, in atherosclerosis and in restenosis after balloon angioplasty, vascular smooth muscle cells migrate and proliferate in the intima, resulting in the narrowing of the vascular lumen (2Ross R. Nature. 1993; 362: 801-809Crossref PubMed Scopus (9989) Google Scholar). In these conditions, cells are likely to reactivate fetal programs to enter the cell cycle. Indeed, the proliferating smooth muscle cells in the intima have changed from a contractile phenotype that can respond to vasoconstriction or vasodilation signals to a synthetic phenotype that can respond to growth stimulation. The synthetic phenotype may be regarded as an embryonic phenotype, as indicated by the switch in expression to an embryonic isoform of myosin heavy chain (3Kuro-o M. Nagai R. Nakahara K. Katoh H. Tsai R.C. Tsuchimochi H. Yazaki Y. Ohkubo A. Takaku F. J. Biol. Chem. 1991; 266: 3768-3773Abstract Full Text PDF PubMed Google Scholar). Extracellular matrix proteins are intimately involved in vascular remodeling, by affecting growth, migration, differentiation, and survival of vascular cell types (4Ruoslahti E. Engvall E. J. Clin. Invest. 1997; 99: 1149-1152Crossref PubMed Scopus (74) Google Scholar). Integrins constitute a large family of cell surface receptors for extracellular matrix proteins that mediate not only cell adhesion by cell-matrix and cell-cell interaction but also multiple outside-in signals that lead to activation of downstream pathways such as tyrosine kinases and phosphatidylinositol 3-kinase (5Shattil S.J. Ginsberg M.H. J. Clin. Invest. 1997; 100: S91-S95Crossref PubMed Google Scholar). The most common integrin recognition sites of ligands contain a consensus Arg-Gly-Asp (RGD) motif. This RGD motif is recognized by many integrins (α5β1, αIIbβ3, and all αvβ integrins), and synthetic peptides containing this RGD sequence are known to antagonize these integrins and inhibit angiogenesis or thrombosis (6Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (9014) Google Scholar). Another important motif frequently seen in secreted proteins, including diverse vascular extracellular matrix proteins, is an epidermal growth factor (EGF) 1The abbreviations used are: EGF, epidermal growth factor; cbEGF, calcium-binding EGF-like; RACE, rapid amplification of cDNA ends; BSA, bovine serum albumin; HUVEC, human umbilical vein endothelial cell; dpc, day(s) postcoitum -like motif (7Doolittle R.F. Feng D.F. Johnson M.S. Nature. 1984; 307: 558-560Crossref PubMed Scopus (126) Google Scholar). A subset of the EGF-like domains contain a distinctive amino acid sequence motif that is associated with calcium binding (8Selander-Sunnerhagen M. Ullner M. Persson E. Teleman O. Stenflo J. Drakenberg T. J. Biol. Chem. 1992; 267: 19642-19649Abstract Full Text PDF PubMed Google Scholar). This calcium-binding EGF-like (cbEGF) motif is found in several extracellular matrix proteins (fibrillin-1 and -2, fibulin-1 and -2, and nidogen) that are distributed in vessel walls, as well as in regulators of blood coagulation (factors IX and X, proteins C and S, and thrombomodulin), low density lipoprotein receptor, transforming growth factor-β-binding protein, Notch, and its ligands Delta and Serrate, which are known to be involved in cell differentiation. Tandemly repeated cbEGF domains in the presence of calcium ion form structurally stable helices that allow protein-protein interaction by these domains (9Rao Z. Handford P. Mayhew M. Knott V. Brownlee G.G. Stuart D. Cell. 1995; 82: 131-141Abstract Full Text PDF PubMed Scopus (312) Google Scholar). To isolate novel secreted molecules involved in the control of cardiovascular development and disease, we have employed the signal sequence trap method (10Tashiro K. Tada H. Heilker R. Shirozu M. Nakano T. Honjo T. Science. 1993; 261: 600-603Crossref PubMed Scopus (638) Google Scholar, 11Jacobs K.A. Collins-Racie L.A. Colbert M. Duckett M. Golden-Fleet M. Kelleher K. Kriz R. LaVallie E.R. Merberg D. Spaulding V. Stover J. Williamson M.J. McCoy J.M. Gene (Amst.). 1997; 198: 289-296Crossref PubMed Scopus (0) Google Scholar), which is a newly developed cloning technique to isolate cDNAs of proteins containing signal sequences, including secreted, membrane, and endoplasmic reticulum proteins. We screened a cDNA library from mouse embryonic heart and isolated several new molecules. In this report, we describe the cloning and characterization of a novel secreted protein with an RGD motif and cbEGF domains. This molecule, named DANCE (developmentalarteries and neural crestEGF-like) protein after its embryonic distribution, is expressed mainly in developmental arteries and is re-expressed in atherosclerotic lesions and in balloon-injured arteries. DANCE promotes cell attachment via interaction of integrins and the RGD motif of the protein. DANCE is thus a new ligand of integrins that may play an important role in vascular remodeling. Poly(A) RNA from 9.5- and 18-dpc mouse embryonic heart was extracted with TRIzol reagent (Life Technologies, Inc.) and Oligotex-dT30 Super (Roche Molecular Biochemicals). The construction of the cDNA library and screening by yeast signal sequence trap was carried out as described previously (11Jacobs K.A. Collins-Racie L.A. Colbert M. Duckett M. Golden-Fleet M. Kelleher K. Kriz R. LaVallie E.R. Merberg D. Spaulding V. Stover J. Williamson M.J. McCoy J.M. Gene (Amst.). 1997; 198: 289-296Crossref PubMed Scopus (0) Google Scholar). Full-length cDNAs were cloned by screening a mouse 13.5-dpc embryonic heart cDNA library (Stratagene). Two positive clones were sequenced. 5′-Rapid amplification of cDNA ends (RACE) was performed using the Marathon cDNA amplification kit (CLONTECH) according to the manufacturer's protocol, and six clones were sequenced. The rat DANCE homologue clone was obtained by screening a rat heart cDNA library (Stratagene) and by 5′-RACE. The human DANCE clone was obtained from IMAGE (GenBankTM accession no. H17726). Each clone was sequenced and analyzed with the computer analysis program GeneWorks (IntelliGenetics, Inc.). A homology search was performed with BLAST and FASTA against public sequence data bases, and a motif search was performed on line at Prosite. Multiple sequence alignment was carried out using an on-line program. 2This program is available on the World Wide Web. For Northern blot analysis, a human multiple tissue Northern blot (CLONTECH) was hybridized to [32P]dCTP-labeled full-length human DANCE cDNA (2.5 kilobase pairs) using QuickHyb (Stratagene). For Northern analysis of rat carotid arteries, RNA was extracted with TRIzol (Life Technologies, Inc.), and [32P]dCTP-labeled full-length rat DANCE cDNA (2.5 kilobase pairs) was used as a probe. All blots were washed at 0.1× SSC, 65 °C, and exposed to autoradiography film overnight. Full-length human DANCE cDNA was used as a probe. The probe was labeled by standard nick translation using biotin-16-dUTP (Roche Molecular Biochemicals) and was purified using Sephadex G-50 spin columns. Hybridization and detection by fluorescein isothiocyanate was carried out as described previously (12Taniwaki M. Matsuda F. Jauch A. Nishida K. Takashima T. Tagawa S. Sugiyama H. Misawa S. Abe T. Kashima K. Blood. 1994; 83: 2962-2969Crossref PubMed Google Scholar). Chromosomes were identified using counterstaining with 4′,6′-diaminido-2-phenolindole dihydrochloride. An EcoRI fragment including the full coding sequence of rat DANCE cDNA was cloned into a pCXN expression vector (gift from Dr. M. Kinoshita at Kyoto University), where expression is driven by the chick β-actin promoter and cytomegalovirus enhancer. Transfections of COS7 and 293T cells were performed using Lipofectamine Plus (Life Technologies, Inc.), according to the manufacturer's protocol. 16 h after transfection, the medium was changed to 8 ml of serum-free Dulbecco's modified Eagle's medium per 10-cm plate. 24 h later, the medium was harvested, and cells from 10-cm plates were lysed in 8 ml of radioimmune precipitation assay buffer (1× phosphate-buffered saline, 1% Nonidet P-40, 0.1% SDS). 25 μl of medium and cell lysate were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting. Rabbit anti-mouse/rat DANCE polyclonal antibodies were raised against KLH-conjugated polypeptide CMTRPIKGPRDIQLDLEMITVN, which corresponds to amino acids 406–426 of mouse and rat DANCE protein by TANA laboratories, LC. Western blotting was carried out following the ECL Western blot (Amersham Pharmacia Biotech) protocol. For in situ hybridization of mouse tissues, a XbaI–PstI fragment (616 base pairs) was subcloned into pBlueScript in both directions. The sense and antisense RNA probe was transcribed in vitro. 7-μm paraffin sections from either mouse tissues or embryos were rehydrated, treated with 10 mg/ml proteinase K for 7.5 min, and hybridized with 10,000 cpm of 35S-labeled riboprobe/ml in hybridization solution containing 50% formamide, 30 mm NaCl, 20 mm EDTA, 10 mm NaH2PO4, 10% dextran sulfate, 1× Denhardt's solution, and 10 mmdithiothreitol. Hybridization was carried out for 14 h at 60 °C. Samples were subsequently washed at 60 °C in 5× SSC, 10 mm dithiothreitol, 2× SSC, 50% formamide, 10 mm dithiothreitol; digested with RNase A (10 mg/ml) for 30 min at 37 °C; and dehydrated. Slides were dipped in Ilford-K5 photographic emulsion (Polysciences, Warrington, PA), exposed for 3 weeks, and developed in Kodak D19 solution. Counterstaining was performed in 0.02% toluidin blue/hematoxylin. In situ hybridization of rat balloon-injured aortae was carried out on en face preparations of vessel segments as described (13Lindner V. Reidy M.A. Circ. Res. 1993; 73: 589-595Crossref PubMed Scopus (172) Google Scholar), using [35S]UTP-labeled sense and antisense rat DANCE riboprobes. In this model, endothelial regeneration and smooth muscle cell proliferation were studied in the thoracic aorta of which the endothelium had been removed with a 2F Fogarty balloon catheter. In this vessel, endothelial regrowth occurs from intercostal arteries, and rapidly proliferating smooth muscle cells migrate onto the denuded surface from the underlying tunica media starting at approximately 6–8 days after denudation. Once smooth muscle cells have migrated into the intima, form an intimal the of several the cells on the this the of smooth muscle cells at after cells are and at late proliferation segments of arteries were identified by of ml of solution in 10 min to All were with For en face vessel segments were and the tissue was out on the were as described (13Lindner V. Reidy M.A. Circ. Res. 1993; 73: 589-595Crossref PubMed Scopus (172) Google Scholar). DANCE cDNA the were subcloned to to a and to 293T protein was purified from the serum-free medium of stable using according to protocol, and into solution containing 10 mm was by of SDS-polyacrylamide gel and Western was by Plus reagent using as a assay plates were with of either DANCE protein or in solution for h at °C. The plates were for h with a solution of phosphate-buffered containing 10 mg/ml umbilical vein endothelial cells from were by and in adhesion buffer solution containing 10 mm mm and 1% were to well in the presence or of and control from Technologies, Inc.) at of were with cells for 30 min in were at 37 °C for min, and were washed several μl of medium was to and cell was with the reagent We screened yeast from mouse 18-dpc embryonic heart and yeast from mouse embryonic heart, and positive clones were obtained by the yeast signal sequence trap as described previously T. K. M. Nakano T. S. Honjo T. 1995; PubMed Scopus Google Scholar). All positive clones were and clones were were or to in or other of the novel an EGF-like the clones obtained by the screening only cDNAs for were isolated from a 13.5-dpc embryonic heart cDNA cDNAs containing the were obtained by 5′-RACE. The sequence a acid protein The translation was at of the presence of an signal and the with a consensus sequence M. Res. PubMed Scopus Google Scholar). The amino acid sequence an which is to be the signal sequence and is to be after at but other that can as domains and not have a endoplasmic reticulum signal and that is a secreted protein. was named as DANCE (developmentalarteries and neural crestEGF-like) to its expression of the six clones from revealed that only one a and coding sequence a clone containing the of this sequence was found to downstream of not sequence and amino acid sequence of mouse are on the sequence of the and that of the form of the signal sequence using a program on data J. Biol. PubMed Scopus Google is shown by An RGD motif found in the cbEGF Two sites are indicated alignment of six cbEGF domains of mouse DANCE with that of mouse Notch, protein and fibulin-1 amino acid including and for calcium binding are alignment of mouse, rat, and human DANCE amino acid For rat and human only amino acid from of the mouse clone are and amino acid are indicated by and S1–5 signal are indicated and cbEGF domains are shown The RGD which is only found in is also acid of the cbEGF and domains human clones of the family are shown alignment of the domains of mouse DANCE and mouse and amino acid that are to DANCE amino acid The DANCE a protein with six EGF-like domains is at the and the other are distributed at the of the domains contain a consensus sequence associated with calcium and are and an Mayhew M. M. Brownlee G.G. Nature. 1991; PubMed Scopus Google Scholar). This subset of the with a calcium-binding been as cbEGF and is well a of proteins Tandemly repeated cbEGF domains form a to the of the proteins. only important as cbEGF domains are also known to mediate protein-protein P. S. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). The amino acid sequence of the DANCE also contains sites and an RGD motif in the cbEGF which is known to be a common ligand sequence motif of integrin ligands E. Cell. Full Text PDF PubMed Scopus Google Scholar). The rat homologue of DANCE was obtained by screening a rat heart cDNA library and by 5′-RACE. sequence of a human DANCE homologue was found in data this human clone out to contain the coding of mouse, rat, and human DANCE amino acid that DANCE is well acid mouse and rat and mouse and human is and The RGD motif and sites are A sequence homology search against the protein and data base revealed that proteins in are to DANCE S1–5 was cloned from fibroblasts of a with and was to be in and to S. Cell. Biol. 1995; PubMed Scopus Google Scholar). is a rat homologue of S1–5 and amino acid with and are found in the data and amino acid All of these have six cbEGF domains, one at the and repeated in the of these molecules. proteins have homology both in the cbEGF DANCE human DANCE S1–5 and DANCE human DANCE these proteins are to comprise a new EGF-like protein which we to the and family. the only DANCE an RGD motif in the cbEGF domains of DANCE and other family have a homology with of and which are extracellular matrix proteins with cbEGF H. K. J. Biol. PubMed Scopus Google Scholar, T. R. R. J. Biol. 1993; PubMed Scopus Google Scholar, H. M. S. Biol. 1997; PubMed Scopus Google but larger family The of is to be involved in binding to S. H. R. J. Biol. 1997; PubMed Scopus Google Scholar), but for of and are not The multiple of DANCE protein that may to multiple receptors or integrin and extracellular or integrin and other cell surface COS7 cells and 293T cells were with a full-length rat DANCE cDNA in an expression lysate and media of cells were analyzed by Western blot using a polyclonal raised against polypeptide of rat DANCE DANCE protein was in both cell lysate and that DANCE is a secreted protein. The of the expressed DANCE protein is approximately or of DANCE was with gel electrophoresis and Western blot not Northern blot analysis using RNA from human tissues and a full-length human DANCE cDNA probe revealed that the of kilobase is expressed mainly in heart, ovary, and colon expression is found in most of the tissues However, is in and blood This expression profile is largely from that of in which expression in heart is S. T. K. Y. PubMed Scopus Google Scholar). family is is likely that of the family may have a developmental or the homology the The of the human DANCE gene was by in situ hybridization using full-length human DANCE cDNA as a probe. All 24 cells a hybridization signal with at A and known thus been mapped to this The S1–5 is chromosome S. T. K. Y. PubMed Scopus Google Scholar), and fibulin-1 and have been mapped on and R. 1994; PubMed Scopus Google Scholar, R. 1994; PubMed Scopus Google family and family have DANCE expression in mouse development was studied by in situ dpc, expression was to endothelial cells of A and signal was in embryonic tissues not In neural crest cells and the DANCE and mesenchymal cells from neural crest cells to the DANCE expression is in the neural the and aorta but expression is in several other tissues A and In the heart, the DANCE is in endocardial cushion tissues, which from endocardial cells by an J. PubMed Scopus Google and with DANCE expression in neural crest cells, these that one developmental of DANCE may be to play a role in tissues, and several other mesenchymal tissues also DANCE In the DANCE expression is seen both in endothelial cells and in smooth muscle In neural tissues such as and to but neural crest tissues such as not not tissues in to developing also DANCE mRNA not The aorta the expression of DANCE at of DANCE mRNA in a embryo as shown by in situ hybridization with and neural In adult DANCE expression is largely However, expression is found at intercostal in the thoracic aorta This is in to of at been out to at these Full Text PDF PubMed Scopus Google Scholar, S. J. 1993; PubMed Scopus Google Scholar). DANCE expression was studied in atherosclerotic vessels using with a S. M.S. J. J. Clin. Invest. 1994; PubMed Scopus Google Scholar). cells the a in DANCE mRNA expression with of the vessel To DANCE is re-expressed in other of vascular remodeling, DANCE expression in balloon-injured rat carotid arteries and aortae was studied Reidy M.A. Invest. Google Scholar). Northern blot analysis revealed that DANCE mRNA expression was following balloon with the seen at 14 with smooth muscle cell in situ hybridization carried out with en of vessel expression of DANCE mRNA is in both endothelial cells and smooth muscle cells from aorta expression 8 days following endothelial expression is seen at the of the endothelium but expression is the where cells to smooth muscle cells that migrated from the underlying media onto the surface of the denuded aorta at 8 days after DANCE mRNA but of expression are seen at 14 days smooth muscle is This that DANCE may cell growth as a in or proliferation of DANCE mRNA in endothelial cells and smooth muscle cells of rat In situ hybridization was carried out on en of vessel segments as recently described (13Lindner V. Reidy M.A. Circ. Res. 1993; 73: 589-595Crossref PubMed Scopus (172) Google using DANCE sense and and antisense and endothelial and endothelial cells 8 days after denudation. The of endothelium is indicated by the line in smooth muscle cells that migrated to intima at 8 days after and 14 days after and DANCE an RGD which is known as an we studied a role in cell For this recombinant human DANCE protein was purified from the medium of 293T on DANCE plates cells on plates 10 This attachment to DANCE protein was 10 A synthetic and attachment of to the control 10 This RGD of the cell attachment of DANCE that the attachment is by binding of cell surface integrins to DANCE protein. the multiple and expression in embryonic arteries, atherosclerotic lesions, and balloon-injured arteries, an is that DANCE may to vascular remodeling via interaction with integrins and other extracellular molecules. We Dr. S. and Dr. for low density lipoprotein with a We Dr. T. Nakano of Molecular for University), Dr. H. Dr. S. and Dr. J. McCoy for in and screening of the signal sequence trap library and Dr. S. and Dr. J. of of at for many in the We also N. and S. for