Cloning of an Immunoglobulin Family Adhesion Molecule Selectively Expressed by Endothelial Cells*

Ken-ichi HirataDagger§, Tatsuro IshidaDagger, Kalyani Penta, Mehrdad Rezaee, Eugene Yang, Jay Wohlgemuth, and Thomas Quertermous

From the Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, California, 94305 and § The First Department of Internal Medicine, Kobe University School of Medicine, Kobe 650-0017, Japan

Received for publication, January 23, 2001, and in revised form, February 8, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To gain fundamental information regarding the molecular basis of endothelial cell adhesive interactions during vascular formation, we have cloned and characterized a unique cell adhesion molecule. This molecule, named endothelial cell-selective adhesion molecule (ESAM), is a new member of the immunoglobulin superfamily. The conceptual protein encoded by cDNA clones consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Northern blot analysis showed ESAM to be selectively expressed in cultured human and murine vascular endothelial cells and revealed high level expression in lung and heart and low level expression in kidney and skin. In situ hybridization analysis indicated that ESAM is primarily expressed in the developing vasculature of the embryo in an endothelial cell-restricted pattern. Epitope-tagged ESAM was shown to co-localize with cadherins and catenins in cell-cell junctions. In aggregation assays employing ESAM-expressing Chinese hamster ovary cells, this novel molecule was shown to mediate cell-cell adhesion through homophilic interactions. The endothelial cell-selective expression of this immunoglobulin-like adhesion molecule coupled with its in vitro functional profile strongly suggests a role in cell-cell interactions that is critical for vascular development or function.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adhesion to neighboring cells and extracellular matrix is crucial for cells to initiate and maintain contacts that are essential for specialized tissue structure and function (1, 2). Recognition and adhesion are mediated by cell surface molecules that bind to proteins expressed on the surface of neighboring cells or deposited in the extracellular matrix. In addition to the mechanical adhesive properties of such receptors, there is accumulating evidence that these molecules can also activate cytoplasmic signaling cascades, thus supporting autocrine and juxtacrine activation pathways. Adhesion molecules have been classified into groups on the basis of their primary structure, and these families include integrin receptors (3), immunoglobulin superfamily receptors (4), cadherins (5), and selectins (6). Members of these groups of adhesion receptors play pivotal roles in morphogenesis and organogenesis (1).

The process of blood vessel formation requires cell-cell adhesion and communication, and members of each adhesion receptor family have been implicated in this complex process (7-9). During embryogenesis, blood vessels form by vasculogenesis and angiogenesis, whereas physiological and pathophysiological blood vessel formation in the adult occurs strictly by angiogenesis (10, 11). Vasculogenesis is a process during which migratory endothelial progenitor cells, angioblasts, differentiate in situ and form connections with existing endothelial cells to contribute to primary capillary plexus formation. In contrast, angiogenesis depends on budding and sprouting from preexisting vessels. Angiogenic endothelial cells degrade the extracellular matrix, migrate into the perivascular space, proliferate, and align themselves to coordinate their shape to provide a patent vascular channel. Integrin receptors have been shown to support angiogenesis and vasculogenesis in the embryo and to be essential for cytokine and tumor-induced angiogenesis in the adult (7, 12). Specifically, the alpha vbeta 3 receptor supports endothelial cell division in response to growth factor stimulation and enhances tissue degradation through its interactions with matrix metalloproteinase 2 (7). The cadherin family of adhesion molecules is represented in endothelial cells by expression of the cell-specific vascular endothelial (VE)1-cadherin. VE-cadherin is a component of the adherens junctions between endothelial cells and mediates homophilic interactions of this cell type (13). Selective deletion of VE-cadherin through gene targeting reveals an early embryonic role for this molecule in endothelial cell differentiation as well as the organization of endothelial cells into vessels and the general process of angiogenesis (14). Cell-associated and soluble forms of the selectin family of adhesion molecules have been linked to angiogenesis (8).

The rapidly growing family of immunoglobulin-like adhesion molecules appears to mediate diverse biological processes, including vascular growth and function. The receptors are transmembrane glycoproteins that contain extracellular immunoglobulin-like domains that mediate homophilic or heterophilic cell-cell adhesion. A number of these adhesion molecules have been cloned, and some show cell- or tissue-restricted expression patterns. Through their regulation of T cell function, immune cell maturation, and neuronal growth and targeting, the fundamental importance of this family of receptors has been established (15-17). Extensive studies of one endothelial cell-restricted family member, PECAM/CD31, has suggested a role in blood vessel development as well as other fundamental endothelial cell functions (18, 19).

To search for new molecular pathways of vascular development, we have characterized a new member of the immunoglobulin superfamily of adhesion receptors. The protein encoded by this gene, named endothelial cell-selective adhesion molecule (ESAM), is a putative transmembrane type I glycoprotein bearing an immunoglobulin V-type and C2-type domain. Northern blot and in situ hybridization analyses revealed that expression of ESAM mRNA was restricted to vascular endothelial cells in development and adulthood. In vitro functional studies have suggested that it mediates homophilic cell-cell interactions that are likely important for endothelial cell development and angiogenesis.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Human umbilical vein endothelial cells (HUVEC), human coronary artery endothelial cells, human coronary artery smooth muscle cells, human aortic smooth muscle cells, and normal human epidermal keratinocytes were obtained from Clonetics, Inc. (San Diego, CA). Namalwa (human B-cell malignancy), Molt4 (human T-cell malignancy), JEG-3 (human choriocarcinoma cells), HeLa (human epithelial cell tumor), 143B (human osteosarcoma cell line), A549 (human lung epithelial cell line), MEL (mouse erythroleukemia cell line), NIH3T3 (mouse embryonic fibroblast cell line), C2C12 (mouse myoblast cell line), RAW 264.7 (mouse monocyte macrophage cell line), HepG2 (human hepatoma cell line), MEG01 (human megakaryocytic leukemia cell line), MH-S (mouse alveolar macrophage cell line), WEHI-78/24 (mouse macrophage/monocyte cell line), TCMK (mouse kidney epithelial cell line), SV40 MES13 (mouse mesangial cell line), Neuro-2a (mouse neuroblastoma cell line), 4MBr-5 (monkey lung epithelial cell line), aortic smooth muscle cells, and CHO cells were obtained from American Type Culture Collection (Manassas, VA). These cells were cultured under recommended conditions. Py-4-1 cells (mouse endothelial cell line) and endothelioma cells were cultured as described previously (20). Rat cardiac myocytes (Cardm) and non-myocytes (Cardnm) were a gift from Dr. Seigo Izumo (Harvard Medical School). Madin-Darby canine kidney (MDCK) clone II cells were provided by Dr. W. James Nelson (Stanford University).

Cloning by Suppression Subtraction Hybridization-- Suppression subtraction hybridization, a polymerase chain reaction-based cDNA subtraction method (CLONTECH, Palo Alto, CA), was employed to identify genes preferentially expressed in tube-forming endothelial cells as previously described (21). Matrigel, a basement membrane extract (Becton Dickinson, Bedford, MA), was applied at 480 µl/35-mm tissue culture dish. HUVEC at 3.9 × 104 cells/cm2 were plated on the Matrigel and incubated at 37 °C for 3 h. After incubation, the HUVEC elongated and migrated to form a network-like structure (22). The media was removed, and the adherent layer of cells on the Matrigel was rinsed three times with cold phosphate-buffered saline (PBS). MatriSperse solution (Becton Dickinson) was added at 2 ml/35-mm dish, and the cell/gel layer was scraped into 50-ml conical tubes. An additional 2 ml of MatriSperse solution was added to the dishes and then transferred to the tubes. The Matrigel was dissolved at 4 °C for 1 h to release the HUVEC. The cells were washed with PBS three times and used for isolation of mRNA with a MicroFast Track kit (Invitrogen, San Diego, CA). Initially, cDNA was synthesized from the RNA of two cell lines. Tester DNA was derived from 2 µg of tube-forming HUVEC poly(A)+ RNA, and driver DNA was derived from 2 µg of poly(A)+ RNA from growth-arrested cobblestone HUVEC. Subtraction hybridization was performed according to established methodology (21). The products of secondary polymerase chain reactions were inserted into the pT7-Blue T vector (Novagen, Madison, WI) and sequenced by the dideoxy method.

cDNA clones representing the entire open reading frame of the human and mouse genes were isolated from lambda gt10 HUVEC and lambda gt11 embryonic day-11 mouse libraries employing standard methodology. Positive clones were sequenced automatically (model 373; Applied Biosystems, Inc., Foster City, CA) and manually by dideoxy sequencing. The resulting nucleotide and conceptual protein sequences were aligned to data bases of existing sequences through the BLAST network server and through a local Genetics Computer Group (GCG, Madison, WI) package.

Northern Blot Analysis-- Total RNA was isolated from murine tissues and cultured cells by the acid guanidinium thiocyanate-phenol chloroform extraction method. For Northern blot analysis, 20 µg of total RNA was size-fractionated on 1.3% agarose gels containing 2.2 M formaldehyde and transferred to nylon membranes. Membranes were then hybridized with a 908-base pair EcoRI-KpnI human cDNA fragment or a 498-base pair EcoRI-PstI mouse cDNA fragment radiolabeled with [32P]dCTP by random priming. Blots were hybridized at 42 °C for 16-24 h in the presence of 48% formamide and 10% dextran sulfate. After hybridization, the membranes were washed at high stringency conditions, 65 °C in the presence of 0.2× SSC buffer (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate) and 0.5% SDS. Visualization was achieved by exposure to Kodak Biomax MS film (Eastman Kodak Co.).

In Situ Hybridization-- Whole-mount in situ hybridization of mouse embryos with cRNA digoxigenin-labeled probes was performed as described with 9.5-day embryos treated with proteinase K for 15 min (23). In situ hybridization of sections was performed with paraformaldehyde-fixed, paraffin-embedded mouse embryos according to the established methodology. A 514-base pair SacI mouse ESAM cDNA fragment was cloned into pBluescript KS(+) and used to generate sense and antisense 35S-labeled probes by in vitro RNA transcription. Hybridization, washing, and probe detection were performed as described (23). After hybridization and washes, slides were dipped into Kodak NTB2 emulsion, exposed at 4 °C for 1-2 weeks, and after standard development, stained with nuclear fast red.

Immunofluorescence Microscopy-- For antibody detection, ESAM was tagged with the FLAG epitope at the C terminus. To generate a FLAG-tagged mouse ESAM, polymerase chain reaction was performed with primers 5'- TATGAATTCATGATTCTTCAGGCTGGAACCCCCGAGACC-3' and 5'-CTCTCTAGACTACTTGTCATCGTCGTCCTTGTAGTCCACCAAGAGACCCAGCCTGACT-3'. The EcoRI/XbaI-digested polymerase chain reaction products were ligated into pcDNA3 and verified by nucleotide sequence. MDCK cells were used for transfection. ESAM-pcDNA3 was introduced into cells, and transfectants were selected in the presence of 300 µg/ml G418. Stable transfectants plated on polycarbonate filters (Costar, Cambridge, MA) were extracted with 1% Triton X-100/PBS as described previously (24). The filters were then fixed with methanol at -20 °C for 10 min and blocked with 1% bovine serum albumin, 0.5% Triton X-100/PBS for 30 min. Anti-FLAG M2 antibody (Sigma), anti-E-cadherin antibody, anti-beta -catenin (both Santa Cruz Biotechnology, Santa Cruz, CA), and/or anti-ZO-1 antibody (Zymed Laboratories Inc. Laboratories, South San Francisco, CA) were used as primary antibodies and incubated for 1 h at room temperature. After washing three times with PBS, cells were incubated with secondary antibody for 1 h at room temperature. Cy3-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch Laboratory, West Grove, PA) and/or fluorescein isothiocyanate-conjugated anti-rabbit or anti-goat IgG (Santa Cruz) were used as secondary antibodies. Cells were washed three times and then mounted with FluoroGuard Antifade Reagent (Bio-Rad). The samples were viewed with a Molecular Dynamics laser confocal-imaging system. For confocal images, x-z views were obtained by collecting sections over a line at each z position in 0.2-µm steps.

Cell Aggregation Assay-- The coding region of mouse ESAM (EcoRI-XbaI fragment) was subcloned into pcDNA3 vector, and this expression construct or the empty vector was transfected into CHO cells with TransIT-LT1 (PanVera Co, Madison, WI). The cells were selected in the presence of 1000 µg/ml G418. Expression levels of murine ESAM were determined by Northern blot analysis. Three high expressing and two low expressing clones were expanded for study. Negative control clones were randomly selected from the transfection with the empty pcDNA3 vector. Transfected CHO cells were detached by incubation with PBS with 1 mM EDTA at 37 °C for 10 min. After washing with Ca2+-free Hanks' balanced salt solution (HBSS) twice, cells were resuspended in Ca2+ free HBSS, 2% fetal calf serum (predialyzed against Ca2+-free HBSS) to a final concentration of 2 × 106 cells/ml by 3 passages through an 18-gauge syringe. Single-cell suspensions were incubated in 1.5-ml siliconized polypropylene tubes (Fisher) with gentle rotation on the platform rotator for 30-90 min at 37 °C. Cells were finally placed on glass slides and observed under the microscope. All clones were evaluated and found to provide consistent results within each group in at least three separate experiments. For the statistics shown in Fig. 7H, single high, low, and nonexpressing clones were employed in aggregation studies that were repeated in five independent experiments. In similar experiments, cells were labeled with different fluorescent lipophilic dyes (Sigma) before aggregation assays. pcDNA3-ESAM-transfected CHO cells were labeled with PKH2 (green fluorochrome), and vector-transfected CHO cells were labeled with PKH26 (red fluorochrome). These labeled cells were then subsequently mixed for analyzing cell aggregation. The aggregation index was calculated as D = (N0 - Nt)/ N0, where N0 is the initial number of particles corresponding to the total number of cells, and Nt is the number of remaining particles at the incubation time point t (25).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning and Sequence Analysis-- We performed subtractive suppression hybridization to isolate genes preferentially expressed in HUVEC undergoing tube formation on Matrigel compared with growth-arrested HUVEC. Sequence analysis of these clones identified a number of previously uncharacterized genes. Data base searches with one of these sequences and corresponding human expressed sequence tags (GenBankTM/ EBI accession numbers R79923, T98690, AI624748) suggested that the encoded protein had homology to cell adhesion molecules. One EST clone (GenBankTM/EBI accession number R79923) was used as a probe to screen HUVEC and murine 11-day embryonic cDNA libraries. Nucleotide sequence analysis of clones thus obtained allowed characterization of the entire open reading frame of the human and murine ESAM proteins. Although the sequence was not found in the public data bases, it was subsequently apparent that this gene had previously been cloned and partially sequenced (26). Because there was no further information about this molecule, the following studies were pursued.

The conceptual human ESAM protein was found to contain 390 amino acids, and the murine protein was found to contain 394 amino acids (Fig. 1A). The primary ESAM sequence was noted to be highly conserved between mouse and human. Analysis of the predicted amino acid sequence indicated that ESAM encodes a type I transmembrane glycoprotein (Fig. 1, A and B) with a presumptive cleavable 29-amino acid signal peptide and a putative 25-amino acid transmembrane region (human, residues 247-271; mouse, residues 250-274). The mature human and murine ESAM proteins thus consist of 361 and 365 amino acids, respectively. Four potential glycosylation sites and a putative serine phosphorylation site were observed (Fig. 1A). Four of the glycosylation sites were predicted by the presence of the universal acceptor sequence Asn-X-(Thr/Ser). The complete ESAM nucleotide sequence did not match any data base sequences other than ESTs (GenBankTM/EBI).


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Fig. 1.   Primary sequence of murine ESAM. The deduced amino acid sequence of murine ESAM (mESAM) and murine VE-JAM and the murine coxsackievirus adenovirus receptor (mCAR) proteins are compared. Identical amino acids are boxed, and conserved amino acid substitutions are shaded. A vertical arrow indicates the putative signal sequence cleavage site of ESAM, as predicted by the rule of von Heijne (44). Conserved cysteines are indicated by asterisks. Potential N-linked glycosylation sites are indicated by dots. A putative transmembrane domain is indicated by dashed underlines, and a core SH3 binding motif is indicated by ellipses. These sequence data are available under GenBankTM/EBI/DDBJ accession numbers human ESAM, AF361746 and mouse ESAM, AF361882. B, predicted secondary structure of ESAM, as predicted by homology to other immunoglobulin protein family members (45). C, dendogram indicating the relationship between ESAM and other immunoglobulin-like receptors with greatest amino acid sequence similarity.

Data base search results and amino acid sequence alignments revealed putative functional motifs in this molecule. Extensive computerized and manual alignment of ESAM conceptual protein sequence to that of other immunoglobulin-like molecules suggests that this receptor contains one V-type and one C2-type immunoglobulin domain (Fig. 1B) (4). This analysis is based on the presence of highly conserved residues in each of these domains. Residues such as glycines (human amino acids 45 and 122; mouse amino acids 47 and 125), tryptophan (human 120, mouse 123), and tyrosine (human 124, mouse 127) are typical features of immunoglobulin V regions. Residues such as glycine (human 162 and 167, mouse 165 and 170), alanine (human 226, mouse 229) and asparagine (human 228, mouse 231) correspond to conserved amino acids in C2 domains. The V region has sequence similarity primarily with variable regions of rearranging genes such as T-cell receptor genes. The C2 domain of ESAM has sequence similarity with C2 domains of a number of putative adhesion molecules, including the recently characterized intestinal A33 antigen and CTX, a marker for cortical thymocytes of Xenopus (Fig. 1C) (27, 28). By comparison to other immunoglobulin superfamily members, four cysteine residues identified in the C2 domain are likely to contribute to intrachain disulfide bonds (4). The predicted transmembrane domain (human amino acids 248-271, murine amino acids 251-274) was identified by hydrophobicity analysis according to Kyte and Doolittle. The cytoplasmic domain contains proline-rich sequences, including a core motif (PXXP) that is known to interact with SH3 domains of signaling molecules. Interestingly, the cytoplasmic domain of ESAM is longer than the corresponding domain of other recently characterized endothelial cell receptors of this family, JAM and VE-JAM (Fig. 1A) (9, 29). In addition, the C-terminal 20-amino acid region is highly conserved between human and mouse and homologous to the coxsackievirus and adenovirus receptor (CAR) (30) (Fig. 1, A and C). These findings suggest that the cytoplasmic domain might interact with intracellular proteins to initiate signaling pathways that regulate endothelial function. Interestingly, ESAM does not have cytoplasmic tyrosine residues, eliminating the potential for use of signaling pathways employed by other immunoglobulin family members such as PECAM/CD31 (31).

Northern Blot Analysis of ESAM Expression-- Northern blot analysis was performed with a variety of mouse tissues to examine the pattern of ESAM expression. Analysis of adult mouse tissues revealed a band representing a 2.1-kilobase mRNA in two tissues containing a high component of endothelial cells, the heart and lung (Fig. 2). Weaker signals were observed with RNA samples from the kidney and skin, and very weak signals were found with RNA from the brain and gut.


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Fig. 2.   Northern blot analysis of ESAM expression in murine tissues and cultured cells. Murine and human cDNA fragments were employed as probes for hybridization to blots containing total RNA isolated from murine adult tissues and murine and human cultured cells. A dominant band corresponding to a primary ESAM transcript was observed at 2.1 kilobases in both murine and human tissues, with size determined by comparison to the mobility of 28 S and 18 S ribosomal RNA. A second larger band was observed in cultured cells that expressed the 2.1-kilobase ESAM transcript and may represent unspliced RNA species, alternatively spliced mRNAs from the ESAM locus, or transcripts from different genes with significant nucleotide similarity. Blots were made with total RNAs (20 µg) isolated from murine tissue and from HUVEC, human coronary artery endothelial cells (HCAEC), Namalwa (human B-cell malignancy), Molt4 (human T-cell malignancy), JEG-3 (human choriocarcinoma cells), HeLa (human epithelial cell tumor), 143B (human osteosarcoma cells), A549 (human lung epithelial cell), HepG2 (human hepatocyte) cells, MEG01 (human megakaryocytic leukemia cell line), human aortic smooth muscle cells (HAoSMC), human coronary artery smooth muscle cells (HCASMC), human epidermal keratinocyte (NHEK), 4MBr-5 (lung epithelial cell line), hemangioendothelioma (EOMA), Py-4-1 (mouse endothelial cell line), MEL (mouse erythroleukemia cell line), RAW (mouse monocyte/macrophage cell line), NIH3T3 (mouse embryonic fibroblast cell line), C2C12 (mouse myoblast cell line), MH-S (mouse alveolar macrophage), WEHI (mouse monocyte/macrophage cell line), MES13 (mouse mesangial cell line), TCMK (mouse kidney epithelial cell line), Neuro2a (mouse neuroblastoma cells), mouse aortic smooth muscle cells (AoSMC). A cyclophilin (Cyph) probe was used as a control for assessing RNA loading.

Northern blot experiments with a diverse group of murine and human cells suggested an endothelial restricted pattern of expression of this gene. ESAM was expressed at high levels in all four endothelial cell types investigated (HUVEC, human coronary artery endothelial cells (HCAEC), hemangioendothelioma, and Py-4-1) (Fig. 2). Although ESAM mRNA was identified as abundant in adult lung tissue, ESAM was not found be expressed by lung parenchymal cells such as alveolar macrophages (MH-S), lung epithelial cells (A549 and 4MBr-5), fibroblasts (NIH3T3) or any type of hematopoietic cells (Namalwa B cell line, Molt4 T cell line, MEL mouse erythroleukemia line). Similarly, although ESAM was expressed in adult murine heart, there was no expression by cultured cardiac myocytes (data not shown). In the kidney, ESAM was not expressed in parenchymal cells including kidney epithelial cells (TCMK), mesangial cells (SV40 MES13), fibroblasts (NIH3T3 and CHO), or macrophages (WEHI-78/24 and RAW 264.7). Notably, this molecule was not expressed by vascular smooth muscle cells (human coronary artery smooth muscle cells, human aortic smooth muscle cells, and mouse aortic smooth muscle cells), suggesting that vascular expression would be restricted to the endothelium. Additional cell lines derived from skeletal muscle (C2C12), liver tumor (HepG2), osteosarcoma (143B), and choriocarcinoma (JEG-3) were negative for ESAM expression, further suggesting a highly restricted pattern of cellular expression.

In Situ Hybridization Analysis of ESAM Expression-- To determine the developmental-specific pattern of ESAM expression, we performed in situ hybridization with murine embryos with both whole mount and tissue section methodology. First we analyzed expression of ESAM in E8.5-E9.5 embryos by whole-mount in situ hybridization. Expression was observed in embryonic blood vessels including the dorsal aorta, intersomitic arteries, and the forming vascular plexus in the head as well as the cardiac outflow tract at this early stage of development (Fig. 3, A-C). In situ hybridization on sections of much later stage embryos, at E13.5, revealed staining of a subset of cells in virtually all organs. This pattern was most consistent with that seen for genes considered to be endothelial cell-specific, including flk-1 and CD31 (Fig. 3, D and E), and suggested that by this time of development all endothelial cells were expressing ESAM (32, 33). Further in situ hybridization studies of staged mouse embryos defined the developmental pattern of expression of this gene. Early in development, ESAM expression was limited to large vessels and the outflow tract. By E10.5-11.5, ESAM expression was clearly found in association with the all recognizable blood vessels and in association with cells lining the heart chambers (Fig. 4, A-F). Although it is difficult to prove the vascular cells expressing ESAM were solely endothelial, expression was documented in even the smallest vascular structures identified, which would be unlikely to have smooth muscle or other adventitial components at this stage of development. Also, hybridization to heart tissues was clearly restricted to the endocardial layer and not associated with the developing myocardial tissue (Fig. 4E). By mid-gestation, expression was identified in all organs developing a microcirculation, including the nervous system, the lung, liver, and heart (Fig. 3, D and E, and Fig. 4, G and H). Although we have not attempted to identify these ESAM-expressing cells by hybridization to other probes, the patterns observed with ESAM labeling are identical to those achieved with well characterized endothelial cell markers (32, 33). Interestingly, expression appeared to decline after mid-gestation, as evaluated in sections from 15.5-day embryos (data not shown).


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Fig. 3.   Whole mount and section in situ hybridization analysis of ESAM expression. Digoxigenin-labeled cRNA sense and antisense probes were hybridized to E9.5-day embryos, with only the sense probe showing a signal above background. A, whole mount in situ hybridization of murine embryo at 9.5 days of development shows an intense signal over the vascular endothelial cells including dorsal aorta, branchial arches, and intersomitic vessels. B, high power view of the head, consistent with expression by large vessels and forming a capillary plexus around the developing brain (arrows). C, high power view of dorsal aorta (arrows) and intersomitic arteries (arrowheads). D and E, 35S-labeled cRNA sense and antisense probes were hybridized to sections of 13.5-day murine embryos, with only the antisense probes showing a signal above background. Sections were photographed with brightfield (D) and darkfield (E) illumination. Specific hybridization was observed in association with large vascular structures, endocardium and outflow tract, and tissues with high density of vasculature.


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Fig. 4.   In situ hybridization analysis of ESAM expression in the embryo. Detailed analysis of mid-gestation embryos revealed ESAM expression restricted to the endothelial cell lineage. Labeled cRNA sense and antisense probes were hybridized to sections of staged murine embryos. Sections were photographed with brightfield (A, C, E, and G) and darkfield (B, D, F, and H) illumination. A and B, aorta at E10.5, with labeling specifically noted over the vessel wall, most likely confined to the endothelial cell layer. Arrows point to the endothelial component of the vessel wall. C and D, labeling over a vessel cut in the cross-section at E10.5 showing labeling over the sole component of the vessel wall, the endothelium. E and F, the atrium of the heart at E11.5; labeling is confined to the single endocardial (end) layer of lining cells. G and H, at E13.5, intense continuous labeling is observed over the endocardium of the atrium (at) and ventricle (v) as well as speckled staining over the trabeculae of the heart, the developing lung (lu), nervous system (ns), and liver (li). This speckled pattern most likely marks the forming microcirculation in these organs.

ESAM was also expressed in extraembryonic tissues. At 10.5 days of mouse development, labeling was associated with syncytiotrophoblasts and cytotrophoblasts as well as endothelial cells associated with blood vessels in the decidua (Fig. 5, A and B). By mid-gestation, expression was associated with vascular channels, and a speckled pattern was observed over the placental tissues (Fig. 5, C and D).


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Fig. 5.   In situ hybridization analysis of ESAM expression in extraembryonic tissues. The embryo and surrounding supporting structures were sectioned and hybridized to labeled sense and antisense ESAM cRNA probes. A and B, specific hybridization was observed with the antisense probe to section regions corresponding to large trophoblastic cells at E10.5 (arrows). C and D, at mid-gestation, a prominent starry pattern of labeling was observed over the placenta, with less prominent labeling also associated with cells lining vascular channels and vessels.

In situ hybridization experiments were conducted with adult tissues found to express ESAM by Northern blot experiments. A diffuse hybridization signal was observed over the lung parenchyma that could not be localized to individual cells but was most consistent with vascular endothelial cell labeling (data not shown). Other adult tissues, including heart and kidney, did not show specific labeling.

Subcellular Localization of ESAM-- Experiments were conducted with cultured epithelial (MDCK) cells transfected with an expression construct encoding FLAG-tagged murine ESAM. Monolayers of cells were evaluated by immunofluorescence for subcellular localization of the ESAM protein, and a comparison was made to the localization of ZO-1, beta -catenin, and E-cadherin proteins. The anti-FLAG M2 antibody revealed accumulation of ESAM protein in cell-cell junctions in a distribution that was similar to that observed with the beta -catenin (Fig. 6, A-C), E-cadherin (data not shown), and ZO-1 (Fig. 6, G-I) antibodies. Since ZO-1 is known to be localized in tight junctions and cadherin/catenin proteins are known to be localized in adherens junctions, we performed confocal microscopy to determine the intracellular localization of ESAM in the x-z views. ESAM was broadly distributed along the length of the lateral membrane in a distribution similar to that of the adherens junction-associated proteins beta -catenin (Fig. 6, D-F) and E-cadherin (data not shown). These data suggest that ESAM co-localizes with these proteins in adherens junctions as well as their extrajunctional distribution in the lateral membrane. In contrast, ZO-1 was restricted to the apex of the lateral membrane (Fig. 6K), and ESAM distribution in these cells did not overlap with ZO-1 (Fig. 6, J-L). Thus, ESAM did not appear to be present in tight junctions in these cells.


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Fig. 6.   ESAM is found in cell-cell junctions where it colocalizes with catenin proteins. MDCK cells expressing FLAG-tagged ESAM were fixed/permeabilized in methanol. ESAM expression was compared with that of either beta -catenin or ZO-1. Anti-FLAG monoclonal antibodies were visualized with Cy3 (red) and antibodies to beta -catenin and ZO-1 were visualized with fluorescein isothiocyanate (green). Confocal images in the upper panels were acquired along the x-y axis (enface view) of the cell monolayer (A, B and G, H). The x-z views in the lower panels (D, E, and J, K), were constructed by collecting sections over a line at each z position in 0.2-µm steps. Merging of images allowed the visualization of colocalization by the yellow color (C, F, I, and L). ESAM was broadly distributed along the length of the lateral membrane in a distribution similar to that of the adherens junction-associated protein beta -catenin (D-F). In contrast, ZO-1 was restricted to the apex of the lateral membrane (K), and ESAM distribution in these cells did not overlap with this component of the tight junction (J-L). The scale bar indicates 20 µm.

Homophilic Interaction of ESAM Receptors-- To gain insight into the functional role of ESAM in vascular development, experiments were conducted to evaluate the ability of this molecule to mediate cell adhesion. We performed an aggregation assay with CHO cells transfected with an ESAM expression construct or an empty pcDNA3 vector. A number of clones expressing different levels of ESAM mRNA were chosen for aggregation assays. Three clones were found to express relatively high levels of ESAM message, and two clones expressed relatively low levels (data not shown). These clones were used for the aggregation assay. Single-cell suspensions were allowed to aggregate, and the presence of cell clusters was monitored by microscopic examination. Nonrecombinant pcDNA3-transfected CHO cells were scattered as a single-cell suspension, whereas ESAM-expressing CHO cells formed large cell clusters after 60 min of incubation (Fig. 7, A-C). Large aggregates (>10 cells) were observed only in the case of ESAM-transfected cells, and the aggregation index appeared to be 10-fold higher with cells expressing ESAM versus those transfected with the empty pcDNA3 vector (Fig. 7, B, C, and H). Although low expressing clones formed some cell aggregates, the aggregation index was lower than for those expressing high levels of ESAM mRNA.


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Fig. 7.   ESAM-mediated cell aggregation. CHO cells transfected with an ESAM expression construct or a negative control pcDNA3 plasmid were employed in aggregation assays. After dissociation, the ESAM-expressing and control cell lines were allowed to aggregate in suspension culture and then analyzed for 60 min after incubation in low calcium buffer. Vector-transfected cells remained scattered as a single cell suspension (A), whereas large cell clusters were formed by ESAM-expressing cells (B and C). In similar experiments, ESAM- and control-transfected cells were labeled separately with fluorescent lipophilic dyes and then mixed for aggregation assays. ESAM-transfected CHO cells were labeled with PKH2 (green fluorochrome), and vector-transfected CHO cells were labeled with PKH26 (red fluorochrome). When mixed, ESAM-expressing cells aggregated, with only occasional incorporation of non-ESAM-expressing CHO cells into the cellular aggregate (D, E, F, and G). H, extent of aggregation as expressed as an aggregation index, calculated as D = (N0 - Nt)/N0. Three high expression clones and two low expression clones were used in these experiments. The aggregation index in the high and low expression clones appeared to be 10- and 3-fold higher, respectively, than those observed with pcDNA3-transfected cells. The index in high expressing clones was 67.8 ± 6.4% (**, p < 0.001) and the index in low expressing clones was 18.9 ± 3.4% (*, p < 0.005) versus vector transfectants, 6.6 ± 1.4%. Values are mean ± S.E. of eight independent determinations. Statistical analysis was performed using one-way analysis of variance with Bonferroni correction.

To investigate whether cellular aggregation was mediated by homophilic or heterophilic ESAM interactions, monocellular suspensions of the two ESAM-expressing and control cells were labeled with different fluorescent dyes and subsequently mixed in a 1:1 ratio. Aggregates were found to contain only ESAM-expressing cells (green fluorescence) in a 60-min assay, which was conducted in the absence of Ca2+ (Fig. 7, D-G). Incorporation of control transfected cells (red fluorescence) into aggregates was rare, with the vast majority remaining as single cells or two-cell clusters. These results demonstrate that ESAM expression can mediate cell aggregation, most likely through a homophilic molecular interaction, and suggest that the adhesion activity of ESAM is Ca2+-independent.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have cloned and characterized a new member of the growing number of adhesion molecules in the immunoglobulin supergene family. We have assigned this new molecule to the immunoglobulin family because of its characteristic primary structure and predicted secondary and tertiary structure as well as the conservation of key residues in the immunoglobulin repeat domains. The ESAM gene encodes a type I transmembrane glycoprotein with a signal peptide, characteristic V-type and C2-type domains, and a cytoplasmic domain. Because of its distinct endothelial cell-selective pattern of expression and its ability to mediate cell-cell adhesion, we have named this protein endothelial cell-selective adhesion molecule (ESAM). Because of the early developmental expression of ESAM and its ability to mediate endothelial cell interaction, we hypothesize that it has a role in vascular morphogenesis.

Database searches and amino acid comparisons have revealed significant sequence similarity to diverse members of this family of receptors. The greatest sequence similarity was to an immunoglobulin-like molecule that serves as the cellular receptor for coxsackievirus and adenovirus (30). Other molecules with a high degree of similarity include the A33 antigen (27), CTX (28), and ChT1 (16). All three of these molecules share the V-C2 immunoglobulin domain organization with ESAM. The A33 antigen is a recently cloned intestine-specific member of this family, identified through its association with a high percentage of colon carcinomas (27). CTX is a marker of cortical thymocytes of Xenopus, and expression of this molecule is highly restricted to the thymus (28). CTX-cross-linking at the cell surface with an anti-CTX monoclonal antibody inhibited cell division and induced abnormal mitosis (15). Given the restricted pattern of expression and these functional data, CTX has been postulated to be involved in cortical microenvironment/thymocyte interactions and to mediate T-cell selection through its adhesion or signaling functions. ChT1 is a related molecule that is required for T-cell differentiation (16). Molecules with a lower level of similarity include leechCAM (17) and limbic system-associated membrane protein (LAMP) (34). LeechCAM is a leech homologue of NCAM, FasII, and ApCAM, and regulates neurite extension and fascicle formation of sensory neurons. Another related member of the immunoglobulin receptor family, limbic system-associated membrane protein (LAMP), serves as an important mediator of axon patterning in the developing limbic system in the vertebrate brain. Thus, the primary sequence of ESAM places it in this interesting group of adhesion molecules that appear to mediate important and diverse biological processes. Characterization of ESAM adds to the growing literature suggesting that these receptors are expressed in a tissue-restricted manner to provide lineage-specific activities that regulate fundamental processes for the assembly and function of specialized tissues.

A number of immunoglobulin-like cell adhesion molecules have been characterized in association with endothelial cell function and linked to fundamental processes such as angiogenesis, immune response, and vascular permeability. ESAM has amino acid and secondary structural similarity to JAM and VE-JAM molecules. JAM is expressed by endothelial and epithelial cells where it has been identified in tight junctions and shown to have a role in monocyte recruitment (9). VE-JAM is a related molecule whose expression is restricted to endothelial cells but with no clearly defined functional role in this cell type (29). Intercellular adhesion molecules 1, 2, and 3 and vascular endothelial cell adhesion molecule 1 employ immunoglobulin repeats to mediate leukocyte adhesion to the vascular wall (35). A well-characterized member of this family, PECAM/CD31, has homophilic and heterophilic activity and is expressed at endothelial cell-cell junctions. This adhesion molecule has been shown to mediate endothelial cell survival through integrin activation, help maintain vascular integrity through homophilic interactions, and to be involved in the transit of polymorphonuclear leukocytes through the vessel wall (18). PECAM/CD31 is one of the earliest endothelial cell markers and has been implicated in both vasculogenic and angiogenic vascular formation in the developing embryo (32). Recent gene-targeting experiments have indicated that mice functionally lacking PECAM/CD31 develop normally, suggesting functional redundancy for cellular adhesion processes that are important for vascular formation in the embryo (36).

The structure, expression pattern, and initial functional characterization of ESAM suggests that it may have one or more functional roles similar to those served by these other immunoglobulin receptors expressed on endothelial cells. One obvious possibility is that ESAM supports embryonic vascular development, providing an overlapping role to that of PECAM/CD31. ESAM has a similar embryonic endothelial cell-selective pattern of expression, and functional overlap would explain the lack of developmental phenotype in PECAM/CD31 gene-targeted mice. Another possibility is that ESAM provides a barrier function, helping to maintain a functional circulation in the embryo and perhaps later in the adult. Although we have shown that ESAM has homophilic binding activity, which might be important for endothelial cell alignment in vascular development and the maintenance of vascular integrity, other molecular interactions and functions are plausible. PECAM/CD31, for instance, has been shown to mediate heterophilic as well as homophilic interactions (18). ESAM could interact with other immunoglobulin family adhesion receptors expressed on endothelial cells, and the known molecules JAM and VE-JAM become candidates in this regard. ESAM might also bind an integrin countereceptor to support transit of a subgroup of leukocytes across the vascular wall or one of a variety of other cell-cell-mediated adhesion functions.

The embryonic expression pattern of ESAM is unique and provides clues regarding its potential role in vascular development. ESAM expression is first observed at ~9.5 days of mouse development. This is well after the onset of expression of endothelial cell factors such as flk-1 and VE-cadherin, which appear to be essential for differentiation, proliferation, or survival of the endothelial cell lineage. This timing of expression of ESAM is more similar to the tek/tie family of receptor tyrosine kinases which have been linked to angiogenic and remodeling processes in the embryo (37). ESAM expression coincides with the requisite functional activation of the embryonic circulation, suggesting that it may have a role in establishing or maintaining the integrity of the vasculature. Initiation of ESAM expression is ~1 day later than that observed for PECAM/CD31, and the coordinate embryonic endothelial cell-restricted pattern of expression suggests that these proteins have similar functional roles in vascular development. Unfortunately, the embryonic pattern of expression of JAM and VE-JAM has not been well studied, so it is not yet possible to compare the developmental patterns of expression of these related molecules.

Like a number of other endothelial cell adhesion molecules, ESAM is expressed at high levels by extraembryonic cytotrophoblast cells. During differentiation, cytotrophoblasts aggregate and invade both the uterine interstitium and vasculature and establish blood flow in the placenta. In this process, cytotrophoblasts lose epithelial adhesion receptors and express adhesion receptors characteristic of endothelial cells, such as PECAM/CD31, alpha vbeta 3 integrin, vascular endothelial cell adhesion molecule 1, and VE-cadherin (38). However, the lack of ESAM expression in the yolk sac is distinct from PECAM/CD31 and other embryonic endothelial cell-restricted markers. ESAM expression was not detected in endothelial cells associated with vessels or blood islands of the yolk sac. Clues from recent gene-targeting experiments suggest that formation of the yolk sac vasculature is unique, and this difference in ESAM expression between the embryonic and yolk sac endothelial cells supports this notion (39, 40).

There remains some uncertainty regarding the tissue and cell-specific expression of ESAM in the adult. Although RNA blot analysis suggested ESAM expression in adult lung and heart, there was no positive signal with in situ hybridization of these tissues. The lack of signal in the in situ studies is unlikely to be a technical problem as we employed positive controls for the slides and the probe. There are two possible explanations. One is that ESAM is expressed by endothelial cells in these adult tissues but at such a low level that it is not detected by in situ hybridization, with the Northern blot being a more sensitive indicator of this expression. Alternatively, ESAM is expressed by multiple cell types in these adult tissues, producing a diffuse background that is not identifiable as cell-specific expression. At this time it is impossible to distinguish between these two possibilities. A number of tissues such as the nervous system demonstrated ESAM expression in the embryo, with no apparent expression detected by either Northern blot or in situ studies in adult tissues. The most likely interpretation of these findings is that ESAM is developmentally regulated in endothelial cells in these tissues, suggesting a role in vascular development.

Adhesion molecules have the potential to mediate cell-cell signaling as well as adhesion through activation of intracellular molecules, a process that has been termed juxtacrine signaling (41). The cytoplasmic domain of ESAM has a proline-rich peptide sequence that contains the consensus PXXP motif. These amino acids are capable of forming a polyproline type II helix and mediating interactions with SH3 binding domains (42). Some adapter proteins containing SH3 domains may bind the cytoplasmic domain of ESAM and induce intracellular signaling. Recently, the CD2-interacting adapter protein CD2AP was cloned (43). CD2AP binds the proline-rich cytoplasmic sequence of CD2 and induces clustering of CD2 as well as cytoskeletal polarization. In addition, the C-terminal amino acid sequence of ESAM is highly conserved between mouse and man and shows high similarity to the corresponding region of the cytoplasmic domain of the coxsackievirus and adenovirus receptor (30). These observed sequences suggest that ESAM ligation may activate intracellular signaling pathways and thus modulate the endothelial cell phenotype and vascular formation through regulation of gene expression.

    ACKNOWLEDGEMENTS

We thank Dr. W. James Nelson and members of his laboratory for kind assistance with experiments reported in this manuscript. We gratefully acknowledge the assistance of Dr. Susan Palmieri and the Cell Sciences Imaging Facility at the Beckman Center, Stanford University.

    FOOTNOTES

* This work was supported by a grant from the American Heart Association.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) human ESAM, AF361746 and mouse ESAM, AF361882.

Dagger These authors contributed equally to this work and should be considered co-first authors.

To whom correspondence should be addressed: Division of Cardiovascular Medicine, Falk CVRC, Stanford University Medical School, 300 Pasteur Dr., Stanford, CA 94305. Tel.: 650-723-5013; Fax: 650-725-2178; E-mail: tomq1@leland.stanford.edu.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M100630200

    ABBREVIATIONS

The abbreviations used are: VE, vascular endothelial; JAM, junctional adhesion molecule; ESAM, endothelial cell-selective adhesion molecule; HUVEC, human umbilical vein endothelial cells; MDCK cells, Madin-Darby canine kidney cells; PBS, phosphate-buffered saline; CHO, Chinese hamster ovary.

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