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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
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 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.
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 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 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 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).
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.
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).
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).
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,
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.
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.
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,
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt10 HUVEC and
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.
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-
-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.
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
View larger version (53K):
[in a new window]
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.
View larger version (72K):
[in a new window]
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.
View larger version (91K):
[in a new window]
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.
View larger version (92K):
[in a new window]
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.
View larger version (83K):
[in a new window]
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.
-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
-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
-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.
View larger version (90K):
[in a new window]
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 -catenin or ZO-1. Anti-FLAG
monoclonal antibodies were visualized with Cy3 (red) and
antibodies to
-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
-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.
View larger version (88K):
[in a new window]
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
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).
![]() |
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.
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Edelman, G. M. (1986) Annu. Rev. Cell Biol. 2, 81-116[CrossRef] |
2. | Gumbiner, B. M. (1996) Cell 84, 345-357[Medline] [Order article via Infotrieve] |
3. | Hynes, R. O. (1996) Dev. Biol. 180, 402-412[CrossRef][Medline] [Order article via Infotrieve] |
4. | Williams, A. F., and Barclay, A. N. (1988) Annu. Rev. Immunol. 6, 381-405[CrossRef][Medline] [Order article via Infotrieve] |
5. | Huber, O., Bierkamp, C., and Kemler, R. (1996) Curr. Opin. Cell Biol. 8, 685-691[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Tedder, T. F.,
Steeber, D. A.,
Chen, A.,
and Engel, P.
(1995)
FASEB J.
9,
866-873 |
7. | Stromblad, S., and Cheresh, D. A. (1996) Chem. Biol. (Lond.) 3, 881-885[Medline] [Order article via Infotrieve] |
8. |
Bischoff, J.
(1997)
J. Clin. Invest.
99,
373-376 |
9. |
Martin-Padura, I.,
Lostaglio, S.,
Schneemann, M.,
Williams, L.,
Romano, M.,
Fruscella, P.,
Panzeri, C.,
Stoppacciaro, A.,
Ruco, L.,
Villa, A.,
Simmons, D.,
and Dejana, E.
(1998)
J. Cell Biol.
142,
117-127 |
10. | Folkman, J., and D'Amore, P. A. (1996) Cell 87, 1153-1155[Medline] [Order article via Infotrieve] |
11. | Risau, W. (1997) Nature 386, 671-674[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Drake, C. J.,
Cheresh, D. A.,
and Little, C. D.
(1995)
J. Cell Sci.
108,
2655-2661 |
13. |
Telo, P.,
Breviario, F.,
Huber, P.,
Panzeri, C.,
and Dejana, E.
(1998)
J. Biol. Chem.
273,
17565-17572 |
14. | Carmeliet, P., Lampugnani, M. G., Moons, L., Breviario, F., Compernolle, V., Bono, F., Balconi, G., Spagnuolo, R., Oostuyse, B., Dewerchin, M., Zanetti, A., Angellilo, A., Mattot, V., Nuyens, D., Lutgens, E., Clotman, F., de Ruiter, M. C., Gittenberger-de Groot, A., Poelmann, R., Lupu, F., Herbert, J. M., Collen, D., and Dejana, E. (1999) Cell 98, 147-157[Medline] [Order article via Infotrieve] |
15. | Robert, J., Brown, D. M., Pasquier, L. D., and Cohen, N. (1997) Exp. Cell Res. 235, 227-237[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Katevuo, K.,
Imhof, B. A.,
Boyd, R.,
Chidgey, A.,
Bean, A.,
Dunon, D.,
Gobel, T. W.,
and Vainio, O.
(1999)
J. Immunol.
162,
5685-5694 |
17. |
Huang, Y.,
Jellies, J.,
Johansen, K. M.,
and Johansen, J.
(1997)
J. Cell Biol.
138,
143-157 |
18. | Newman, P. J. (1997) J. Clin. Invest. 100 Suppl. 11, S25-29[Medline] [Order article via Infotrieve] |
19. | DeLisser, H. M., Christofidou-Solomidou, M., Strieter, R. M., Burdick, M. D., Robinson, C. S., Wexler, R. S., Kerr, J. S., Garlanda, C., Merwin, J. R., Madri, J. A., and Albelda, S. M. (1997) Am. J. Pathol. 151, 671-677[Abstract] |
20. |
Hidai, C.,
Zupancic, T.,
Penta, K.,
Mikhail, A.,
Kawana, M.,
Quertermous, E. E.,
Aoka, Y.,
Fukagawa, M.,
Matsui, Y.,
Platika, D.,
Auerbach, R.,
Hogan, B. L. M.,
Snodgrass, R.,
and Quertermous, T.
(1998)
Genes Dev.
12,
21-33 |
21. |
Hirata, K.,
Dichek, H. L.,
Cioffi, J. A.,
Choi, S. Y.,
Leeper, N. J.,
Quintana, L.,
Kronmal, G. S.,
Cooper, A. D.,
and Quertermous, T.
(1999)
J. Biol. Chem.
274,
14170-14175 |
22. | Kubota, Y., Kleinman, H. K., Martin, G. R., and Lawley, T. J. (1988) J. Cell Biol. 107, 1589-1598[Abstract] |
23. | Hogan, B. L. M., Beddington, R., Constantini, F., and Lacy, E. (1994) Manipulating the Mouse Embryo , pp. 344-367, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
24. | Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., Rodriguez-Bowlan, E., Scheller, R. H., and Nelson, W. J. (1998) Cell 93, 731-740[Medline] [Order article via Infotrieve] |
25. | Shimoyama, Y., Nagafuchi, A., Fujita, S., Gotoh, M., Takeichi, M., Tsukita, S., and Hirohashi, S. (1992) Cancer Res. 52, 5770-5774[Abstract] |
26. | Chretien, I., Marcuz, A., Courtet, M., Katevuo, K., Vainio, O., Heath, J. K., White, S. J., and Du Pasquier, L. (1998) Eur. J. Immunol. 28, 4094-4104[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Heath, J. K.,
White, S. J.,
Johnstone, C. N.,
Catimel, B.,
Simpson, R. J.,
Moritz, R. L.,
Tu, G. F.,
Ji, H.,
Whitehead, R. H.,
Groenen, L. C.,
Scott, A. M.,
Ritter, G.,
Cohen, L.,
Welt, S.,
Old, L. J.,
Nice, E. C.,
and Burgess, A. W.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
469-474 |
28. | Chretien, I., Robert, J., Marcuz, A., Garcia-Sanz, J. A., Courtet, M., and Du Pasquier, L. (1996) Eur. J. Immunol. 26, 780-791[Medline] [Order article via Infotrieve] |
29. |
Palmieri, D.,
van Zante, A.,
Huang, C.-C.,
Hemmerich, S.,
and Rosen, S. D.
(2000)
J. Biol. Chem.
275,
19139-19145 |
30. |
Bergelson, J. M.,
Cunningham, J. A.,
Droguett, G.,
Kurt-Jones, E. A.,
Krithivas, A.,
Hong, J. S.,
Horwitz, M. S.,
Crowell, R. L.,
and Finberg, R. W.
(1997)
Science
275,
1320-1323 |
31. |
Newman, P. J.
(1999)
J. Clin. Invest.
103,
5-9 |
32. |
Baldwin, H. S.,
Shen, H. M.,
Yan, H. C.,
DeLisser, H. M.,
Chung, A.,
Mickanin, C.,
Trask, T.,
Kirschbaum, N. E.,
Newman, P. J.,
Albelda, S. M.,
and Buck, C. A.
(1994)
Development
120,
2539-2553 |
33. | Millauer, B., Wizigmann-Voos, S., Schnurch, H., Martinez, R., Moller, N. P., Risau, W., and Ullrich, A. (1993) Cell 72, 835-846[Medline] [Order article via Infotrieve] |
34. | Pimenta, A. F., Zhukareva, V., Barbe, M. F., Reinoso, B. S., Grimley, C., Henzel, W., Fischer, I., and Levitt, P. (1995) Neuron 15, 287-297[Medline] [Order article via Infotrieve] |
35. | Bevilacqua, M. P. (1993) Annu. Rev. Immunol. 11, 767-804[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Duncan, G. S.,
Andrew, D. P.,
Takimoto, H.,
Kaufman, S. A.,
Yoshida, H.,
Spellberg, J.,.,
Luis de la Pompa, J.,
Elia, A.,
Wakeham, A.,
Karan-Tamir, B.,
Muller, W. A.,
Senaldi, G.,
Zukowski, M. M.,
and Mak, T. W.
(1999)
J. Immunol.
162,
3022-3030 |
37. |
Gale, N. W.,
and Yancopoulos, G. D.
(1999)
Genes Dev.
13,
1055-1066 |
38. |
Zhou, Y.,
Fisher, S. J.,
Janatpour, M.,
Genbacev, O.,
Dejana, E.,
Wheelock, M.,
and Damsky, C. H.
(1997)
J. Clin. Invest.
99,
2139-2151 |
39. | Connolly, A. J., Ishihara, H., Kahn, M. L., Farese, R. V., Jr., and Coughlin, S. R. (1996) Nature 381, 516-519[CrossRef][Medline] [Order article via Infotrieve] |
40. | Carmeliet, P., Mackman, N., Moons, L., Luther, T., Gressens, P., Van Vlaenderen, I., Demunck, H., Kasper, M., Breier, G., Evrard, P., Muller, M., Risau, W., Edgington, T., and Collen, D. (1996) Nature 383, 73-75[CrossRef][Medline] [Order article via Infotrieve] |
41. | Bosenberg, M. W., and Massague, J. (1993) Curr. Opin. Cell Biol. 5, 832-838[Medline] [Order article via Infotrieve] |
42. | Pawson, T. (1995) Nature 373, 573-580[CrossRef][Medline] [Order article via Infotrieve] |
43. | Dustin, M. L., Olszowy, M. W., Holdorf, A. D., Li, J., Bromley, S., Desai, N., Widder, P., Rosenberger, F., van der Merwe, P. A., Allen, P. M., and Shaw, A. S. (1998) Cell 94, 667-677[Medline] [Order article via Infotrieve] |
44. | von Heijne, G. (1987) Sequence Analysis in Molecular Biology: Treasure Trove or Trivial Pursuit , p. 112, Academic Press, Inc., San Diego, CA |
45. | Vaughn, D. E., and Bjorkman, P. J. (1996) Neuron 16, 261-273[Medline] [Order article via Infotrieve] |