From the Department of Pathology, Centre Medical Universitaire, 1 rue Michel-Servet, CH-1211 Geneva, Switzerland
Received for publication, June 22, 2000, and in revised form, September 28, 2000
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ABSTRACT |
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Cell-cell contacts are essential for
morphogenesis and tissue function and play a vital role in mediating
endothelial cohesion within the vascular system during vessel growth
and organization. We identified a novel junctional adhesion molecule,
named JAM-2, by a selective RNA display method, which allowed
identification of transcripts encoding immunoglobulin superfamily
molecules regulated during coculture of endothelial cells with tumor
cells. The JAM-2 transcript is highly expressed during embryogenesis
and is detected in lymph node and Peyer's patches RNA of adult mice.
Accordingly, antibodies specific for JAM-2 stain high endothelial
venules and lymphatic vessels in lymphoid organs, and vascular
structures in the kidney. Using real time video microscopy, we show
that JAM-2 is localized within minutes to the newly formed cell-cell contact. The role of the protein in the sealing of cell-cell contact is
further suggested by the reduced paracellular permeability of cell
monolayer transfected with JAM-2 cDNA, and by the localization of
JAM-2 to tight junctional complexes of polarized cells. Taken together,
our results suggest that JAM-2 is a novel vascular molecule, which
participates in interendothelial junctional complexes.
Adhesion molecules play essential roles in the overall tissue
organization and the proper physiological function of organs by
establishing and organizing cell-cell contacts (1). The function of
endothelial cells lining the vessel walls relies in part on their
ability to express different adhesion molecules in a coordinated and
regulated way. It has been demonstrated that endothelial cells regulate
vascular permeability and leukocyte emigration from the blood into the
surrounding tissues. Leukocyte emigration occurs in a multistep
adhesion process involving different classes of adhesion molecules,
namely selectins, integrins, and cell adhesion molecules of the
immunoglobulin superfamily (Ig Sf)1 (2-4). The members of
the Ig Sf possess structural domains
similar to the variable (V type) or
constant (C type) immunoglobulin domains found in T or B cell receptor
(5, 6). JAM (hereafter referred to as JAM-1), a recently described
immunoglobulin superfamily molecule with two VH domains,
was shown to regulate monocyte transmigration across endothelial cell
layers (7, 8). The protein specifically localizes to tight junctions of
epithelial and endothelial cells, indicating that molecules
participating in intercellular junctions may regulate vascular
functions (9, 10). To date, the cell surface molecules known to
participate specifically in tight junctions include JAM-1, occludin,
and the claudins (8, 11, 12). JAM-1 and occludin are expressed by both
endothelial and epithelial cells, while some members of the claudin
family display tissue-specific distribution (7, 13, 14). Interestingly,
claudin-5 was found in the tight junctions of endothelial cells (15),
whereas claudin-11 was present in tight junctions of myelin sheaths in the brain and sertoli cells in the testis (16), reflecting some heterogeneity in the molecular composition of tight junctions found in
different cellular system (17). This suggests that the function of
tight junctions and their regulation may involve specific expression of
junctional proteins or distinct activation events, which regulate the
stability of tight junctions (18).
Hence, we started to search for novel surface molecules of the Ig Sf,
which may be more specific for vascular endothelium than JAM-1. We took
advantage of the fact that blood vessels in tumors are often leaky,
indicating that the barrier function of angiogenic endothelial cells
may be lost, due to regulation of tight junctional molecules in
endothelial cells growing in the presence of tumor cells (19, 20). Our
experimental approach consisted in the coculture of endothelial cell
lines with melanoma cells to identify regulated transcripts by
differential screening (21). For this purpose, we developed a method
based on the principle of RNA display that allowed the preferential
identification of transcripts encoding molecules of the Ig Sf (22, 23).
This method led to the identification of JAM-2, which encoded for a transmembrane protein specifically incorporated in tight junctions, with a structural organization into V-C2 domains. The
protein is not found on epithelial cells and is expressed by
endothelial and lymphatic cells in vivo. Our results suggest
that JAM-2, together with JAM-1, may constitute the prototypes of a
novel junctional adhesion molecule family.
Cell Lines--
The murine thymic (tEnd.1) and embryonic
(eEnd.2) endothelioma cell lines (24) were provided by Dr. W. Risau and
Dr. B. Engelhardt (Max Planck Institute, Bad Nauheim, Germany). The
murine SV40 transformed lymph node endothelial cell line TME was
provided by Dr. A. Hamann (25). The murine squamous cell carcinoma KLN 205, CHO cells, MDCK cells, and myeloma cell line Sp2/0 were obtained from the American Type Tissue Culture Collection (ATCC). All cells, except CHO, were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Paisley, Scotland), supplemented with 10% fetal
calf serum (PAA Laboratories, Linz, Austria), 2 mM
glutamine, 100 units/ml penicillin, and 100 units/ml streptomycin (all
from Life Technologies, Inc.). CHO cells were grown in nutrient mixture F-12 (Ham's) medium supplemented as above. Adherent cells were detached by washing with PBS and 0.15 mM EDTA, followed by
a 5-min incubation in trypsin/EDTA at 37 °C.
Display, Cloning, and Sequence Analysis--
For co-culture
experiments, 5 × 105 tEnd.1 cells were grown together
with 2.5 × 104 B16 F10 melanoma cells for 64 h
in 10-cm tissue culture dishes. As control, 5 × 105
tEnd.1 and 2.5 × 105 B16 F10 cells were grown
separately under the same conditions, resulting in confluent monolayers
after 64 h. Total RNA was directly extracted in Petri dishes with
Trizol reagent following the manufacturer's instructions (Life
Technologies, Inc.). The cDNA was prepared from 5 µg of total
RNA, employing oligo(dT) (16-mer) primer and Superscript reverse
transcriptase (Life Technologies, Inc.). The quality and the quantity
of cDNA were checked by running 27 cycles of PCR on 1 µl of
cDNA diluted 1:5, using primers specific for the housekeeping
hypoxanthine phosphoribosyltransferase cDNA. We then
performed the differential PCR with the following degenerated primers:
5'-TAYAGNTGYNNNGCYTCYAA-3', 5'-TAYCRGTGYNNNGCYTCYAA-3', and
5'-TAYTAYTGYNNNGCYTCYAA-3' encoding for the most frequent amino acid
sequences encountered in C2 domains: YRCXAS,
YQCXAS, and YYCXAS. The PCR conditions were as
follows: 2 µl of diluted cDNA, 2.5 µl of 10× Goldstar PCR
buffer, 2 µl of MgCl2, 2 µl of degenerated primers (0.3 mM), 0.5 µl of dNTP (0.1 mM), 0.1 µl of
[
The QD10 PCR product was identical to three murine ESTs encoding for a
new putative Ig superfamily molecule (accession no. AA726206, AA052463,
and AA175925). All ESTs were incomplete, and the remaining 5'-coding
sequence was obtained by rapid amplification of cDNA ends using
three primers designed on the basis of the EST sequences (5'-rapid
amplification of cDNA ends PCR system, Life Technologies, Inc.).
The full-length coding sequence for JAM-2 was assembled in the pGemt
vector (Promega Corp.) from the cloned 5'-rapid amplification of
cDNA ends PCR product and the EST (accession no. AA726206) using
the internal HpaI restriction site. The cDNA encoding
JAM-1 was kindly provided by Dr. P. Naquet (CIML, Marseille-Luminy,
France). The nucleic acid sequences were determined using the Thermo
Sequence fluorescent labeled primer cycle sequencing kit (Amersham
Pharmacia Biotech) and the LI-COR DNA analysis system (MWG-Biotech).
Further sequence analyses were performed via the applications available
on the ExPASy Molecular Biology Server (Blast, Prosite, Swiss-Prot, and
signal peptide prediction).
Northern Blot--
Total mRNA from cells or murine tissues
was extracted using Trizol (Life Technologies AG, Basel, Switzerland)
according to manufacturer's instructions. Poly(A) mRNA was
extracted from 250 µg of total RNA with the Oligotex mRNA
purification kit (Qiagen, Zurich, Switzerland). Embryonic poly(A)
Northern blot was purchased from CLONTECH (P. H
Stehelin and Cie AG, Basel, Switzerland). The riboprobes were prepared
from pcDNA3 vector (Invitrogen, Leek, Netherlands) and comprised
the sequences encoding for the immunoglobulin domains of JAM-1 and
JAM-2, or the full-length coding sequence for Construction of Expression Vectors--
The sequence encoding
EGFP was subcloned from pEGFP-1 vector (CLONTECH,
P. H. Stehelin and Cie AG) into pcDNA3 using
HindIII and NotI sites, therefore named
pcDNA3/EGFP. The 3' restriction sites, HpaI and
ScaI, found in the sequence encoding, respectively, the
cytoplasmic domain of JAM-2 and JAM-1, were used to fuse the two
sequences at the N terminus of the EGFP in pcDNA3 vector
(Invitrogen). The inserts encoding JAM-2 or JAM-1 were excised from
pGemt or pRc/CMV using SacII/HpaI or
HindIII/ScaI digestions, respectively. The coding
sequences were then cloned in pcDNA3/EGFP vector digested with
AgeI, blunted by fill-in, and further digested with
HindIII or SacII enzymes. This resulted in fusion
sites at amino acid positions DGV291 for JAM-2 and
QPS285 for JAM-1. The transfection of CHO cells was
performed as described previously (26). Stable transfectants used for
permeability assays were selected by growing transfected CHO cells for
2 weeks in medium containing 1 mg/ml G418. Resistant colonies were
isolated and checked for EGFP fluorescence intensity by flow cytometry (FACScalibur apparatus; Becton Dickinson, Mountain View, CA) and fluorescence localization by microscopy (Axiovert; Zeiss, Oberkochen, Germany). Time-lapse video microscopy was performed using an Axiovert fluorescence microscope and Openlab software for image acquisition. To
produce soluble molecules, the sequence encoding extracellular domains
of JAM-1 or JAM-2 were amplified by PCR from plasmids containing the
full-length coding sequences using, respectively, T7 and
5'-gctctagacacagcatccatgtgtgcagcctc-3' for JAM-1, or T7 and
5'-gctctagaatagacttccatgtcctgcc-3' for JAM-2. The reverse primers were
modified by XbaI site to clone the PCR products in frame with Flag Tag
sequence in pcDNA3 vector, as described previously (27). The
absence of PCR-induced errors was confirmed by sequencing the
constructs on both strands. 293T cells were then transfected using
calcium phosphate precipitation method, and supernatants were collected
every 2 days over a period of 10 days. Isolation of the recombinant
soluble molecules (namely sJAM-1 or sJAM-2) was achieved on a M2 column
(Sigma-Aldrich Co., FlukaChemie AG, Buchs, Switzerland), and purity was
checked by SDS-PAGE and Coomassie Blue staining (data not shown).
Reagents, Enzyme-linked Immunosorbent Assay, and
Immunofluorescence Analysis--
The following monoclonal antibodies
were used: anti-PECAM (GC51, rat IgG2a; EA-3, rat
IgG1), anti-JAM-1 (H2O2.106.7.4, rat IgG1; (7,
28), anti-occludin (Zymed Laboratories Inc., Gebr Mächler AG, Basel, Switzerland), anti-ZO-1 (R40/76; Ref. 29), and
anti-E-cadherin (Arc-1; Ref. 30). The panel of CRAM antibodies against
murine JAM-2 was generated in the laboratory using standard techniques,
and recombinant soluble molecule as immunogen in rats (31). Briefly,
100 µg of purified recombinant soluble JAM-2 molecule (sJAM-2) mixed
with Titermax adjuvant (Sigma) was used to immunize male Fischer rats
intraperiteonally. Two days after a final intravenous injection of 50 µg of sJAM-2, splenocytes were fused to Sp2/0 cells, and hybridoma
were selected in HAT-containing medium. Resistant clones were screened
by ELISA for the production of mAbs recognizing specifically sJAM-2.
For this purpose, Maxisorb Immunoplates (Nunc) were coated overnight at
4 °C with M2 antibody diluted at 2 µg/ml in 150 mM
Nacl, 50 mM borate buffer, pH = 9. Wells were washed,
blocked for 1 h with serum-containing medium, and incubated for
1 h with supernatants of transfected 293T cells. After three
washes with PBS plus 0.2% bovine serum albumin, hybridoma supernatants
were added to the wells and incubated for 1 h at 4 °C. After
washing, bound antibodies were detected using mouse anti-rat peroxidase
(Jackson Immunoresearch, Milan AG, La Roche, Switzerland), and
ABTS (Sigma). Optical densities at 405 nm were read using a
kinetic microplate reader and SoftMAXPro software (Molecular Devices
Corp). Positive clones were subcloned twice, rescreened, and tested by
cytofluorimetry on CHO cells stably transfected with plasmids encoding
EGFP alone (mock), JAM-1-EGFP, or JAM-2-EGFP. All CRAM antibodies were
of IgG1 or IgG2a isotype except CRAM-25F24,
which is of the IgG2b subclass. Antibodies were purified on
protein G-Sepharose columns (Amersham Pharmacia Biotech) according to
the manufacturer instructions. CRAM-19H36 mAb was used for
immunoprecipitation and CRAM-18F26 for immunohistochemistry. Immunofluorescence analysis was performed using secondary reagents coupled to FITC or Texas Red (Jackson Immunoresearch) for
cytofluorimetry and immunohistochemistry, respectively. For
immunohistochemistry, samples were fixed 5 min with cooled ( Immunoprecipitations--
Immunoprecipitations were performed as
described previously (33) using 10 mM Tris-HCl buffer, pH
7.4, 150 mM NaCl, 0.5% Triton X-100, 0.5% Nonidet P-40,
protease inhibitor mixture (Roche Diagnostics Ltd, Rotkreuz,
Switzerland) for lysis. After immunoprecipitation, SDS-PAGE, and
transfer to nitrocellulose membrane, the biotinylated proteins were
revealed using streptavidin coupled to peroxidase (Jackson
Immunoresearch) and ECL (Amersham Pharmacia Biotech). Deglycosylations
were performed after four washes of the beads in lysis buffer.
Immunoprecipitates were incubated with 10 µl of PBS, 0.5% SDS and
boiled for 3 min to ensure denaturation of the proteins before
deglycosylation. Thereafter, 90 µl of PBS, 10 mM EDTA,
0.5% Triton X-100, protease inhibitors (Complete, Roche), and 10 units
of N-glycosidase F (Roche) were added and incubated
overnight at 37 °C. Reaction was stopped by adding loading buffer,
samples were submitted to SDS-PAGE, blotted, and visualized with
streptavidin peroxidase.
Permeability Assays--
Permeability was measured using
Transwell chambers (6.5-mm diameter; PC filters, 0.4-µm pore size,
Costar Corp). In brief, 1 × 104 transfected or
nontransfected CHO cells were cultured to confluence on filters
previously coated for 30 min with 0.2% gelatin. After 5 days, the
medium was changed for prewarmed nutrient/F-12 medium without
fetal calf serum (500 µl in the lower chamber and 200 µl in the
upper chamber). FITC-dextran (Mr 38,900, Sigma)
was added in the upper chamber at 1 mg/ml final concentration. After 1 h, chambers were removed and fluorescence was read directly in
the lower chamber using Cytofluor II. The mean fluorescence intensity
of five independent chambers was calculated and compared using Statview
software and t test unpaired comparisons. To normalize experiments, the value of mean fluorescence intensity obtained with
wild type CHO cells was taken as 100%.
Identification of JAM-2 by Selective RNA Display--
During the
coculture of endothelial cells with tumor cells, endothelial properties
are modified by transcriptional regulations of adhesion molecules (21).
Such a model is therefore ideal to search for novel regulated adhesion
molecules by differential screening method. Due to the lack of
specificity of RNA display, we modified the technique to selectively
identify transcripts encoding adhesion molecules of the Ig Sf. For this
purpose, we used degenerated primers targeting the sequences encoding
molecules with C2 domains. This was achieved by the
alignment of C2 domains of several Ig Sf adhesion molecules
(data not shown) and the identification of a linear amino acid
consensus, surrounding the cysteine residue participating to the
C2 domain structure:
Y-(RQYS)-C-X-A-S-N-X2-G. Therefore,
we used the reverse translation of the most frequent consensus
sequences (YRCXAS, YQCXAS, YYCXAS) to
design the degenerated primers used for differential display. The level
of degeneracy, between 2048 and 4096 different forms, was sufficient to
obtain discrete PCR products without the use of a second reverse
primer. The display of one representative radioactive reverse
transcription-PCR with three different degenerated primers is shown on
Fig. 1A. The comparison of the
radioactive PCR products obtained from cDNA prepared from the
endothelial cell line alone (lane 1), from the mix of endothelial and tumor cell lines (lane 2),
or from the tumor cell line alone (lane 3),
allowed the selection of four differentially expressed transcripts.
After cloning and sequencing the four PCR products, it appeared that
RU9 and QU11 encoded respectively for PAI-1 and RalGDS-like factors.
The sequences of the two remaining products, QU14 and QD10, were
unknown, but were good candidates as coding for partial sequences of
novel Ig Sf transcripts since they presented a partial open reading
frame containing the canonical sequence of C2 domain,
YYCXAS. Furthermore, the QD10 sequence showed the
C2 domain canonical residues Asn and Gly at positions +4
and +7 relative to the targeted cysteine.
Sequence comparison of the QD10 PCR product with sequence data bases
revealed several identical sequences in the murine EST data base.
Unfortunately, none of these comprised the initiating methionine, and
so the complete coding 5' region was obtained by using a rapid
amplification of cDNA ends approach. As shown in Fig.
1B, the cDNA sequence contained an open reading frame encoding a protein of 310 amino acids of a predicted molecular mass of
34.8 kDa for the core protein. The initiating methionine was followed
by a signal peptide (underlined) that may be cleaved between
Ala31 and Val32 (34). The protein structure was
typical of a type I protein comprising one membrane-distal
VH and one proximal C2 immunoglobulin domain.
As expected, the sequence of the initial QD10 PCR product encoded for
the end of the C2 domain (italic nucleic acid
sequence). Two putative N-glycosylation (Asn104
and Asn192) and four putative cytoplasmic phosphorylation
sites (Ser274, Ser281, Tyr293, and
Ser297) were identified. Comparison of the protein sequence
with nonredundant sequence data bases identified the tight junctional
protein, JAM-1 (8), as the closest related sequence with 31% identity
at the protein level (Fig. 1C). The transcript corresponding
to the QD10 PCR product was therefore named JAM-2. The similarities in
the extracellular part of the molecules were largely confined to the residues participating in the structure of the Ig domains (5), and to sequences surrounding the cysteines, the tryptophan residues of
c strands, or the N-glycosylation site within the
C2 domain (NSSF195). Interestingly, two
additional cysteines are found within the C2 domain of
JAM-2, while they are absent from the JAM-1 sequence. In the
cytoplasmic domain, three stretches of homology are identified: the
AYRRGYF motif, the SPGK sequence, and the C-terminal parts of the proteins.
JAM-2 Is Highly Expressed during Embryogenesis, and Restricted to
Endothelial Cell Subpopulations in Adult Tissues--
The tissue
distribution of JAM-2 transcript was explored by Northern blotting and
compared with JAM-1 (Fig. 2). The JAM-2 transcript was 2 kilobases long, highly expressed in embryonic tissue
and in Peyer's patches, lymph nodes, kidney, and testis of adult
animals. A putative splice variant of 1.8 kilobases was detected in
testis. The expression of the JAM-2 transcript was low in lung, liver,
spleen, and thymus. The relative abundance of JAM-1 and JAM-2 were
compared during embryogenesis; the mRNA encoding JAM-2 was
detectable as early as day 7.5 post coitum, whereas JAM-1 mRNA was
not detected during embryogenesis. This suggested that the JAM-2
transcript might be abundantly expressed in tissues undergoing
remodeling with restricted expression in specialized compartments in
adult tissues.
To analyze the expression of JAM-2 in more details, we produced a panel
of monoclonal antibodies (CRAM) against a soluble form of JAM-2
(sJAM-2). The specificity of CRAM mAbs was addressed by performing
ELISA on soluble forms of JAM-1 and JAM-2. All CRAM mAbs recognized
specifically sJAM-2 (Fig. 3A).
To further confirm the specificity of the selected mAbs,
cytofluorimetric analysis was performed on CHO cells stably transfected
with EGFP or chimeric EGFP fusion proteins of JAM-1 or JAM-2 (Fig.
3B). These results confirmed the specificity of the mAbs for
JAM-2 (CRAM panel), and the absence of
cross-reactivity on JAM-1 transfected cells, which were specifically
recognized by the already described H2O2-106 mAb (7). Therefore, we
performed immunohistological analysis of JAM-2 expression on kidney and
mesenteric lymph node sections (Fig. 4),
using the CRAM-18F26 mAb. In the cortical region of the kidney, a weak
staining of intertubular structures was observed with CRAM-18F26 in
areas brightly stained with anti-PECAM (GC51), whereas anti-ZO-1 or
anti-JAM-1 stained predominantly the tubular epithelial cells (data not
shown). We therefore focused our attention on the medulla of the kidney
and found a staining of sparse linear structures with CRAM-18F26 mAb
against JAM-2. To ask whether this staining concerned vascular
structures, we performed serial sections and identified the vessels by
their PECAM expression (Fig. 4d). On the identical region of
serial sections, linear interendothelial staining was detected using
antibodies to JAM-2, JAM-1, or ZO-1 (Fig. 4, a,
b, and c, respectively). On mesenteric lymph node sections, the CRAM-18F26 mAb stained high endothelial venules (HEV)
(Fig. 4e). The HEVs also expressed JAM-1, ZO-1, and PECAM (Fig. 4, f, g, and h), although with
differences in the subcellular localization of the stainings (Fig. 4,
e-h, insets). In the cortical area of the
mesenteric lymph nodes, all antibodies labeled cells in subcapsular
sinuses (Fig. 4, i-l), which corresponded to cells of
afferent lymphatic vessels. Thus, the staining with anti-JAM-2 mAb
seemed to be restricted to lymphatic vessels and a subset of
endothelial cells.
Although the staining with anti-JAM-2 antibody appeared to be
endothelial, we wished to confirm this endothelial specific expression
of JAM-2. For this purpose, we performed cytofluorimetric analysis of
JAM-2 expression on various cell lines or on freshly isolated
endothelial cells from dissociated tissues using CRAM-4H31 mAb.
Identical results were obtained with the different anti-JAM-2 mAbs of
the CRAM panel described in Fig. 3. Endothelial cell lines (tEnd.1,
eEnd.2, and TME) expressed low levels of JAM-2 on the cell surface and
variable levels of JAM-1 (Fig.
5A). We were unable to detect
JAM-2 on any murine epithelial cell lines (data not shown), whereas the
majority of these expressed JAM-1 with similar levels to the carcinoma
cell line, KLN 205. This is consistent with the expression of JAM-1 by
various epithelial cells such as enterocytes, (8), and the restricted
expression of JAM-2 to HEVs and lymphendothelium in lymphoid organs or
vascular structures of the kidney. Therefore, flow cytometric analysis
of freshly isolated endothelial cells was performed following
collagenase/dispase organ dissociation of Peyer's patches, lymph node,
and kidneys. Endothelial cells were identified by their staining with
both PECAM/CD31 and acetylated LDL (36). The double positive cell population was analyzed for JAM-1 or JAM-2 expression, and the results
are shown in Fig. 4B. In kidney and Peyer's patches, all cells that were positive for CD31 and acetylated LDL also expressed JAM-2 or JAM-1, demonstrating that, at least in these organs, endothelial cells express JAM-2 and JAM-1 in vivo. When the
staining was performed on cells obtained from lymph node, JAM-2 and
JAM-1 expression were weaker and only found on a subpopulation of
PECAM/LDL double-positive cells, reflecting a possible heterogeneity of endothelial cell phenotypes within this tissue. Taken together, the
results of cytometric and immunohistochemical analysis show that JAM-2
expression is restricted to subpopulations of endothelial cells found
in kidney, Peyer's patches, and lymph nodes.
JAM-2, a 45-kDa Protein, Interacting Homophilically--
Since the
TME cell line derived from high endothelial venules expressed the
highest level of JAM-2, we used this murine cell line to further study
the subcellular localization of JAM-2, and compare it with that of
JAM-1. The localization of the JAM-2 protein on the surface of the
endothelial cells was restricted to cell-cell contacts (Fig.
6A, a). The
staining for JAM-2 was weaker than that observed for JAM-1 and less
prominent in the membrane extensions between cells. Next, we wished to
investigate whether JAM-2 present at cell-cell contacts interacted
homophilically with JAM-2 or whether it interacted heterophilically
with another molecule on the neighboring cell. For this purpose we
fused the JAM-2 protein to green fluorescent protein (JAM-2-EGFP), and
transfected the construct into CHO cells. When CHO cells transfected
with JAM-2-EGFP cDNA reached confluence, JAM-2 was observed in
cell-cell contacts between two cells, exclusively when both cells
expressed the protein (Fig. 6B). The contacts between
expressing and nonexpressing cells were devoid of JAM-2 (Fig.
6B, a, indicated by arrowhead). The same result was obtained when cells were transfected with the chimeric
molecule JAM-1-EGFP (Fig. 6B, b). This indicated
that either JAM-2 or JAM-1 needed homophilic interactions to be
localized at cell-cell contacts. Furthermore, experiments with mixed
cells transfected with both constructs indicated that JAM-1 was unable to interact with JAM-2 (data not shown).
For a biochemical characterization of JAM-2, we performed
immunoprecipitation with CRAM-19H36 mAb. First, we wanted to confirm the reactivity of CRAM-19H36 mAb and the predicted molecular weight of
JAM-2 using lysates of MDCK cells transfected with plasmid encoding the
full-length sequence of JAM-2. As shown in Fig. 6C, the
CRAM-19H36 mAb immunoprecipitated a single band of Dynamic Localization of JAM-2 to Cell-Cell Contacts--
To
understand the mechanism by which JAM-2 was specifically localized to
cell-cell contacts, time-lapse video microscopy was performed. CHO
cells, stably transfected with the fluorescent chimeric molecule, were
trypsinized and plated into chamber slides for imaging. After cell
spreading, surface expression of JAM-2-EGFP was not uniform, but was
rather clusterized at existing cell-cell contacts (Fig.
7A, cells depicted by
asterisks). During the formation of new cell-cell contacts,
localization of JAM-2-EGFP to cell junctions was observed and an
intense fluorescence signal was detected at the novel contact point
between the cells forming the new cell-cell contact
(arrows). The chimeric protein was enriched in the membrane
protrusions between contacting cells, leading to the "zipper like"
pictures seen after 12 or 18 min. Interestingly, the localization of
JAM-2 at the primary cell-cell contacts was not lost during the
formation of the new membrane contact (see upper
left corner cell contacts). This finding
indicated that, once JAM-2-EGFP was specifically localized to the new
cell contact, its localization was stable. To further address the
requirements for JAM-2 localization, time-lapse video microscopy was
performed after wounding the cell monolayer (Fig. 7B). Cells
at the wounded edge maintained JAM-2 at their intact contact sites
(arrowhead), but lost JAM-2 localization at the wounded side
(arrows), indicating that JAM-2 engagement was necessary to
maintain its membrane localization. Over a period of 90 min following
wounding, cells bordering the wound began to migrate into the wounded
area. Interestingly, these cells maintained contacts with neighboring
cells via membrane protrusions that were brightly fluorescent,
i.e. JAM-2-positive (arrowhead). These results
support the hypothesis that JAM-2 homophilic interactions may play a
role in the establishment or maintenance of cell-cell contacts.
JAM-2 Increases Monolayer Tightness and Participates in Tight
Junctional Complexes--
Since a number of molecules participating in
cell-cell contacts have been shown to regulate the paracellular
permeability of cell monolayers, we tested whether JAM-2 might also
affect this function. Transfection of JAM-2-EGFP reduced the
paracellular permeability to FITC-dextran and improved sealing of CHO
cell monolayers by 42.5%, whereas transfection of the unrelated
molecule Tac (interleukin-2 receptor We herein describe the cloning and characterization of JAM-2, a
novel junctional adhesion molecule of endothelial cells. In adult
murine tissues, JAM-2 expression is restricted to lymphendothelial cells, endothelial cells in the kidney, and HEVs of lymphoid organs.
The identification of JAM-2 is achieved by a selective method of RNA
display, which uses degenerated primers to selectively amplify
transcripts of interest. The sequence of the degenerated primers used
in this technique is based on the amino acid sequence participating to
the structure of C2 immunoglobulin domains. Therefore, JAM-2 is a type I protein with two Ig domains: one V domain and one
C2 domain. The molecule is homologous to the recently
described JAM-1 (7, 8). In its membrane-proximal domains, JAM-2
contains two cysteine residues more than JAM-1, which leads us to
conclude that it is organized as a VH/C2
molecule unlike JAM-1, which was classified as a
VH/VH molecule (8, 37). Several stretches of
sequence conservation between JAM-1 and JAM-2 are also identified within their cytoplasmic domains. Since the C terminus PDZ binding domain of JAM-1 was recently shown to bind and recruit PDZ-containing proteins, it will be interesting to address whether these shared sequences may control the specific localization of both molecules to
the tight junctions of polarized cells (38, 39). The specific localization of the EGFP chimeric proteins, in which the conserved C-terminal sequence SSFVI was removed, excluded a role for this conserved sequence in the targeting of the proteins (38). Indeed, the
protein sequence motifs identified as putative phosphorylation sites
for PKCs (Ser274, Ser281) may be sufficient to
guide JAM-2 to cell-cell contacts, since a role for PKCs in the
establishment of cell-cell contacts was recently suggested (40, 41).
However, we cannot exclude a role of the extracellular domains in the
specific localization of the proteins since JAM-2 and JAM-1 were solely
enriched in the membrane contacts between transfected cells.
The dynamics of JAM-2 localization to cell-cell contacts was explored
using JAM-2 protein fused to green fluorescent protein and time-lapse
video microscopy. Time-lapse imaging of JAM-2-EGFP during the formation
of cell-cell contacts showed that the protein was located within
minutes to the contacts between cells. Once partial cell-cell contacts
were formed, the JAM-2 enriched membranes closed in a "zipper-like"
fashion, indicating that JAM-2 homophilic interaction is an early event
in the establishment of cell connections and may play a role in
cell-cell contact organization. The latter hypothesis gains further
support from paracellular permeability experiments in CHO cells
transfected with JAM-1-EGFP or JAM-2-EGFP. A similar reduced
paracellular permeability was described following transfection of
VE-cadherin and JAM-1, two molecules participating in adherens and
tight junctions, respectively (8, 42). Accordingly, when expressed in
MDCK cells, JAM-2 was colocalized with ZO-1 and occludin, two
components of tight junctions (11, 29), indicating that JAM-2 is
specifically associated with subcellular junctional complexes in
epithelial cells. Together, these results show that JAM-1 and JAM-2 not
only have sequence similarities, but also have similar properties in
terms of subcellular localization, permeability regulation, and
homophilic interactions.
The major difference between JAM-2 and JAM-1 appears to be in their
tissue distribution. Although the morphogenesis of tight junctions
during vasculogenesis or angiogenesis is not known, the absence of the
JAM-1 transcript and the prominence of the JAM-2 transcript during
embryogenesis may reflect a sequentially ordered role of the proteins
in the biogenesis of tight junctions. More interestingly, the
expression of JAM-2 in adult murine tissue is restricted to endothelial
cell subpopulations such as HEVs or lymphatic cells, whereas JAM-1 is
expressed by platelets, antigen-presenting cells, endothelial cells,
and epithelial cells (7, 8). However, due to the lack of tight
junctions in cultured endothelial cells (43), the participation of
JAM-2 to tight junctional complexes was examined in epithelial cells
upon transfection. Therefore, its participation to epithelial tight
junctions may indicate its participation to similar compartments in
endothelial cells expressing it. Nevertheless, this raises the question
of the presence of endothelial tight junctional complexes in HEVs or
lymphatic vessels, two endothelial compartments specialized in
leukocyte recirculation. According to the specific function of HEVs in
the recirculation of leukocytes, many studies demonstrated the absence
of tight junctions in HEVs, whereas others suggested that partial or
normal tight junctions may be present (44-46). In the present study we demonstrated that HEVs express ZO-1, JAM-1, and JAM-2, three molecules that incorporate into tight junctions. Since the presence of such structures in HEVs is still a matter of debate, it remains to be seen
whether ZO-1, JAM-1, and JAM-2 participate in "tight
junctional-like" domains in HEVs. More interestingly, JAM-2 was not
expressed by adult brain vasculature (data not shown), which is known
to express ZO-1, occludin, and JAM-1, and which forms one of the
tightest blood tissue barrier of the body (43, 47, 48). It is therefore tempting to speculate that JAM-2 may be present in tight
junctional-like structures, which undergo continuous remodeling to
allow the passage of transmigrating leukocytes. An alternative
hypothesis is that JAM-2 expression is induced by microenvironmental
factors, resulting in loosening of the existing endothelial tight
junctions. The negative regulation of endothelial tight junctions may
result from the competition of JAM-2 and JAM-1 for intracellular
proteins involved in the stabilization of tight junctional complexes.
Interestingly, the JAM-1 staining on HEVs, where JAM-2 was highly
expressed, seemed to be diffuse on the cell surface (Fig. 4,
insets). We therefore believe that the relative
participation of JAM molecules to subcellular compartment such as tight
junctions may regulate the properties of the vascular bed (49-51). A
future challenge will be to identify the molecular architecture of
junctional complexes in situations such as chronic inflammation or
angiogenesis known to affect the function of the vascular barrier
(49-51).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-33P]dATP (10 mCi/ml; Amersham Pharmacia Biotech,
Dübendorf, Switzerland), 15.65 µl of H2O, 0.25 µl
of Goldstar Taq polymerase (Eurogentech, Seraing, Belgium).
The parameters for the PCR were as follows: 45 s at 94 °C,
90 s at 50 °C, and 45 s at 72 °C for 40 cycles. Formamide/EDTA loading buffer was added, and samples were denatured for
2 min at 94 °C. The PCR products were then separated on a 6%
polyacrylamide gel and autoradiographed using Kodak OM-Mat. Band
intensity was compared, differentially expressed bands were cut from
the dried polyacrylamide gel, and fragments were retrieved by boiling
and ethanol precipitation as described previously (22). The PCR
products were then reamplified using increased concentrations of dNTPs
(0.2 mM instead of 2 µM) without
[33P]ATP. The products of re-amplification were cloned
into pGem-T Easy Vector (Promega Corp., Wallisellen, Switzerland).
Nucleic acid sequences of two independent clones were determined using the Thermo Sequence fluorescence-labeled primer cycle sequencing kit
(Amersham Pharmacia Biotech) and the LI-COR DNA analysis system (MWG-Biotech GmbH, Ebersberg, Germany).
-actin. Hybridization
was performed at 62 °C in buffer containing 50% formamide. The
blots were washed twice (0.5× SSC, 0.1% SDS, 67 °C) and
autoradiographed on Kodak X-Omat at
80 °C.
20 °C)
methanol. Samples were rehydrated in PBS, 0.2% gelatin, 0.05% Tween
20, incubated overnight with the primary antibodies before washing, and
revealed with the appropriate secondary reagent coupled to Texas Red.
For the analysis of fresh endothelial cells, dissociation of freshly dissected murine tissues was performed using collagenase/dispase digestion, according to established procedures (32). The dissociated cells were stained for 2 h at 37 °C with diI-acetylated LDL
(Molecular Probe Europe BV, Leiden, Netherlands) before staining with
mAbs to JAM-1 or JAM-2, and goat anti-rat FITC probe. After this step, normal rat serum diluted 1/50 was added for washes and incubations. After wash, cells were stained with biotinylated anti-CD31 (PharMingen) and streptavidin red 670 (Life Technologies AG). JAM-1 or JAM-2 expression was analyzed on cells positive for the two endothelial cell
markers: acetylated LDL (FL-2) and CD31 (FL-3). Negative controls were
obtained by omitting primary antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (93K):
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Fig. 1.
Display, nucleotide sequence of murine JAM-2,
and alignment with JAM-1. A, one representative display
experiment with the three indicated degenerated primers
(YRCxAS, YYCxAS, YQCxAS) is shown.
Lanes 1, 2, and 3 correspond, respectively, to PCR products obtained from cDNA of
t-end cell line (lane 1), coculture of t-end with
B16 (lane 2), and B16 (lane
3). Cloned and sequenced differential PCR products are
indicated by asterisks. B, nucleotidic and
deduced amino acid sequence of JAM-2. The original QD10 PCR product
sequence is shown in italic characters in the nucleic acid sequence. On
amino acid sequence, the putative hydrophobic signal peptide and the
transmembrane region are underlined. The putative
N-glycosylation and phosphorylation sites are marked,
respectively, by bold and italic characters. The
cysteine residues participating in the Ig domain structure are
circled. These sequence data are available from
GenBank/EBI/DDBJ under accession number AJ300304. C,
alignment of the closest murine related sequences, moJAM-2 and
moJAM-1.
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Fig. 2.
Northern blot analysis of JAM-2
(a), JAM-1 (b), or
-actin (c) transcripts in mouse
tissues. Results on embryonic post coitum (pc) and
adult mRNA preparations are shown. The sizes of the hybridization
signals are indicated on the right.
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Fig. 3.
Characterization of mAbs against murine
JAM-2: the CRAM panel. A, soluble recombinant JAM-1
(sJAM-1, white bars) or JAM-2 (sJAM-2) were
detected by enzyme linked immunosorbent assay using the indicated
monoclonal antibody of the CRAM panel against JAM-2. Negative control
was obtained using GC51 mAb directed against PECAM. B,
cytofluorimetric analysis of CHO cells transfected with EGFP fusion
proteins. Histograms show the staining profiles obtained with the
indicated mAb on cells transfected with EGFP alone (Mock,
dashed line), JAM-1-EGFP (JAM-1,
filled profiles), or JAM-2-EGFP
(JAM-2, plain line). Instrument
compensations were set to avoid EGFP fluorescence signal in FL-2 chanel
used to detect mAb stainings.
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Fig. 4.
Immunohistological analysis of JAM-2, JAM-1,
ZO-1 and PECAM expression. Serial sections of kidney
(a-d) or sections from mesenteric lymph node
(e-l) were stained with the following antibodies:
CRAM-18F26, H2O2-106, R40/76, or GC51 recognizing, respectively, JAM-2
(a, e, and i), JAM-1 (b,
f, and j), ZO-1 (c, g, and
k), or PECAM (d, h, and l).
Each series of pictures (a-d, e-h, and
i-l) were acquired with identical settings for the CCD.
Using the same settings, no staining was detectable if primary mAb was
omitted.
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Fig. 5.
JAM-2 expression on endothelial cells.
A, cytofluorimetric analysis of JAM-2, JAM-1, and PECAM
expression on endothelial cell lines (tEnd.1,
eEnd.2, and TME) or squamous carcinoma cell line
(KLN 205). mAbs used for staining are indicated
on the right. Dashed profiles represent the
negative controls obtained with an antibody directed against CD4.
B, cytofluorimetric analysis of JAM-2 on freshly isolated
endothelial cells. Indicated organs were dissociated by
collagenase/dispase digestion, stained with diI-acetylated LDL, CD31,
and anti-JAM-2 or anti-JAM-1 as indicated. Histogram profiles were
obtained by gating endothelial cell population positive for
diI-acetylated LDL (FL-2) and CD31 (FL-3).
Negative controls were obtained by omitting the primary mAbs against
JAM-1 or JAM-2.
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Fig. 6.
Localization of the JAM-2 protein to
cell-cell contacts. A, immunocytochemistry was
performed on TME cells with CRAM-18F26 anti-JAM-2 (a) or
H2O2-106 anti-JAM-1 (b) antibodies. Arrows
indicate the specific localization of the proteins to cell-cell
contacts. Bar, 10 µm. B, JAM-2-EGFP
(a) and JAM-1-EGFP (b) chimeric molecules were
specifically localized to cell contacts between transfected cells. The
enrichment in EGFP recombinant proteins was not observed between
transfected and nontransfected cells (arrowhead).
Bar, 20 µm. C, biochemical characterization of
JAM-2 protein using CRAM-19H36 mAb and biotinylated lysates of
JAM-2-transfected (lanes 1 and 3) or
nontransfected MDCK cells (lane 2). In
lane 3, immunoprecipitated material was submitted
to N-glycosidase F treatment. D,
immunoprecipitation of JAM-2 after surface biotinylation of TME
endothelial cells. GC51 anti-PECAM (lane 1) and
H2O2-106 anti-JAM-1 (lane 2) antibodies were
used respectively as negative and positive controls for the
immunoprecipitation with CRAM-19H36 anti-JAM-2 antibody
(lane 3). E, immunoprecipitation of
EGFP recombinant proteins from CHO-transfected cells. CRAM-19H36
(lanes 1 and 4) or H2O2-106
(lanes 2 and 3) were used to
immunoprecipitate the biotinylated lysates from CHO cells transfected
with JAM-1-EGFP (lanes 1 and 2) or
JAM-2-EGFP (lanes 3 and 4). Molecular
sizes are indicated on the right.
45 kDa from
lysate of transfected cells (lane 1), and no
signal was detectable when lysate of nontransfected cells were used
(lane 2). After treatment of the
immunoprecipitated material with N-glycosidase F, we
observed a reduction of the apparent molecular mass from 45 to 34-36
kDa (lane 3), the faint upper band resulting
probably from a partial digestion with the enzyme. This was in
agreement with the predicted molecular weight of the core JAM-2
protein. When immunoprecipitations were performed on TME cell lysate,
the molecular mass of 45 kDa for JAM-2 protein was confirmed (Fig. 6D, lane 3), and JAM-1 protein was
resolved as a single band of lower molecular mass (Fig. 6D,
lane 2). This difference in size between JAM-1
and JAM-2 was further confirmed by immunoprecipitation of the EGFP
recombinant fusion proteins after surface biotinylation of transfected
cells. As shown in Fig. 6E, single broad bands of 70 and 73 kDa were, respectively, obtained for JAM-1-EGFP (lane 2) and JAM-2-EGFP (lane 4). This was
expected since EGFP has a molecular mass of 28 kDa.
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Fig. 7.
A, JAM-2-EGFP localization during
cell-cell contact formation. Single fluorescence pictures were
collected every 3 min for 1 h during the monolayer formation of
CHO cells transfected with JAM-2-EGFP. Pictures obtained during the
first 18 min are shown. At time 0, asterisks identify the
three cells present on the field. At 6, 12, and 18 min,
arrows highlight the relocalization of JAM-2-EGFP to the
newly formed cell-cell contact. B, JAM-2-EGFP localization
after wounding. Arrows indicate the wounded side, and
arrowheads highlight the membrane processes rich in
JAM-2-EGFP. Elapsed time is indicated on the pictures. Bar,
10 µm.
) did not significantly reduce
the paracellular permeability (Fig. 8).
The transfection of JAM-1-EGFP also reduced the paracellular
permeability of the cells monolayer as described previously for wild
type JAM-1 (8). Since CHO cells do not present tight junctions, and
because paracellular permeability has been shown to be regulated by
molecules participating in tight and adherens junctions, we wished to
explore the participation of JAM-2 to such subcellular junctional
complexes. To address this issue, we transfected the JAM-2-EGFP
chimeric protein in MDCK cells, known to possess well defined tight and
adherens junctions. As shown in Fig.
9A, when serial pictures taken
at 0.9-µm intervals were analyzed for EGFP fluorescence and compared
with occludin staining, JAM-2-EGFP was specifically enriched in
cell-cell contacts at the level of the tight junction (Fig.
9A, fourth and fifth pictures to the right). At the basal level, we
observed intracellular dots of EGFP fluorescence, probably due to the
overexpression of the protein. The restricted localization of
JAM-2-EGFP to tight junctions was more striking when 40 pictures taken
at 0.3-µm intervals were stacked, and rotated along the
x-y axis (Fig. 9B). Colocalization with ZO-1 was
observed (Fig. 9B, a), whereas JAM-2-EGFP was not found in the adherens junctions stained with Arc-1 anti-E-cadherin antibody (Fig. 9B, b). Similar results were
obtained when nonmodified JAM-2 was transfected (data not shown),
indicating that EGFP does not affect JAM-2 localization to tight
junctions. These results demonstrate that JAM-2 presents the properties
of a novel junctional adhesion molecule, targeted to tight
junctions.
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Fig. 8.
JAM-2 expression decreases paracellular
permeability. A, paracellular permeability was
evaluated by FITC-dextran diffusion across nontransfected CHO cell
monolayers, CHO cells transfected with Tac (huIL2R ), or
with the indicated EGFP fusion protein (JAM-1 or
JAM-2). Transfection of JAM-2-EGFP or JAM-1-EGFP in CHO
cells led to a significant decrease in paracellular permeability
(57.8 ± 4.9% and 70.8 ± 3.6%, respectively;
p < 0.0001), whereas transfection of Tac did not
significantly affect the paracellular permeability (100.4 ± 4.4%, p = 0.9872). Results were normalized to
nontransfected CHO cells.
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Fig. 9.
Localization of JAM-2-EGFP to tight
junctions. A, confluent MDCK cells, stably
transfected with JAM-2-EGFP, were stained with anti-occludin and
anti-rabbit-Texas Red. Series of pictures every 0.9 µm from basal to
apical levels are shown for EGFP fluorescence (a) or
occludin staining (b). The basal level on the
left was arbitrarily defined such as the serial pictures
comprise the tight junctional level on focus at +3.6 and +4.5 µm
(fourth and fifth pictures to the
right). B, composition obtained from overlay of
40 pictures every 0.3 µm (upper panels) and
rotation along x-y axis are shown (lower
panels). The regions used for z axis projection
are depicted by white squares on
pictures a and b. Staining obtained
with R40/76 mAb against ZO-1 (a) or Arc-1 mAb against
E-cadherin (b) are depicted in red, whereas EGFP
fluorescence of JAM-2-EGFP is shown in green. The
subcellular localizations of tight and adherens junctions are indicated
by arrowheads and brackets, respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are grateful to D. Gay-Ducrest and C. Magnin for their technical expertise, advice, and enthusiastic support. We thank Dr. P. Naquet for providing us the JAM-1 cDNA, Prof. P. Meda for providing anti-ZO-1 mAb, and Drs. C. Johnson-Leger and P. Cosson for helpful discussions and for critical reading of the manuscript.
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Addendum |
---|
In conclusion, the characterization of JAM-2, its structural relation to JAM-1, and the overlapping but distinct tissue distribution of both molecules suggest the existence of a novel junctional adhesion molecule family. This is further supported by the fact that, while the present report was under review, a related molecule named VE-JAM was characterized by Palmeri and colleagues (35). This molecule is different from the presently described JAM-2 molecule, and is not recognized by our CRAM panel of anti-JAM-2 mAbs (data not shown). Therefore, it is tempting to speculate that more members of the junctional adhesion molecule family have to be expected, and that the specific expression of each member is of central importance for studying the interendothelial junctions in respect to permeability control and leukocyte trafficking.
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FOOTNOTES |
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* This work was supported by Swiss National Science Foundation Grant 31-49241.96), Foundation Gabriella Giorgi-Cavaglieri, and Grant LT 0218/1998-M from the Human Frontier Science Program Organization (to M. A. L.).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 EBI/DDBJ Data Bank with acession number(s) AJ300304.
To whom correspondence should be addressed. Tel.: 41-22-702-57-56;
Fax: 41-22-702-57-46; E-mail:
michel.aurrand-lions@medecine.unige.ch.
§ Present address: Addenbrookes Hospital, Cambridge CB2 2XY, United Kingdom.
Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M005458200
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ABBREVIATIONS |
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The abbreviations used are: Sf, superfamily; JAM, junctional adhesion molecule; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; EGFP, enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; LDL, low density lipoprotein; HEV, high endothelial venule; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; EST, expressed sequence tag; CRAM, Confluency Regulated Adhesion Molecules; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PECAM, Platelet Endothelial Cell Adhesion Molecule; ABTS, 2,2'-Azino-Bis (3-ethybenz Thiazoline 6-Sulfonic acid) diammonium.
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