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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Pathology, Centre Medical
Universitaire, Geneva, Switzerland, and the Basel Institute for
Immunology, Switzerland.
Endothelial cells are linked to each other through intercellular
junctional complexes that regulate the barrier and fence function of
the vascular wall. The nature of these intercellular contacts varies
with the need for permeability: For example, in brain the impervious
blood-brain barrier is maintained by "tight" contacts between
endothelial cells. By contrast, in high endothelial venules (HEVs),
where lymphocytes continuously exit the bloodstream, the contacts are
generally leaky. The precise molecular components that define the type
of junction remain to be characterized. An immunoglobulin superfamily
molecule named JAM-2, specifically expressed in lymphatic endothelial
cells and HEVs, was recently identified. JAM-3 was cloned and
characterized in the current study, and JAM-1, -2, and -3 were shown to
form a novel protein family belonging to the larger cortical
thymocyte Xenopus (CTX) molecular family.
Using antibodies specific for each of the 3 family members,
their specific participation in different types of cell-cell contact in
vivo and their specific and differential localization in lateral
contacts or tight junctions were demonstrated. Furthermore, it was
shown that JAM-1 and JAM-2 differentially regulate paracellular
permeability, suggesting that the presence of JAM-1, -2, or -3 in
vascular junctions may play a role in regulating vascular function in vivo.
(Blood. 2001;98:3699-3707) The immune surveillance of the body is carried out
by lymphocytes constantly circulating from the blood to lymphoid or
peripheral organs and back to the blood. Most lymphocytes extravasate
through specialized vascular endothelium created by tissue
microenvironment or inflammation. This suggests that a regional
specialization of endothelial cells may control lymphocyte traffic.
Postcapillary high endothelial venules (HEVs) are specialized sites
along vessels where the migration of lymphocytes to lymphoid organs
occurs.1,2 Migration occurs in a multistep process
involving rolling of lymphocytes along the vessel wall, adhesion, and
transmigration.3 The first and the second steps are well
described and involve selectins, mucins, immunoglobulin (Ig)
superfamily molecules, and integrins.4,5 The last
transmigration step is less well understood and may occur through a
transcellular pathway or by the paracellular route.6
Evidence to support the latter model comes from numerous studies
suggesting that the transmigration of leukocytes involves the
disruption of interendothelial junctions in a specific and localized
manner.7-9 Nevertheless, the molecular events underlying the transmigration process are still controversial because other studies have demonstrated that transendothelial migration may occur
without a widespread disruption of tight or adherens
junctions.10,11 In this context, the molecules
participating specifically in intercellular junctional complexes of
endothelial cells may play a central role in regulating leukocyte
transmigration and vascular functions.12,13 Although all
endothelial cells are involved in exchanges of material from blood to
tissue, there is a great degree of tissue-specific specialization of
vascular junctions. This heterogeneity was described more than 20 years
ago when the first electron microscopy studies pointed out structural
differences in junctional complexes in different endothelial
cells.14,15 Recently, the molecular characterization of
proteins participating in intercellular junctional complexes has
increased our understanding of vascular junction heterogeneity. The interendothelial adhesive structures include tight,
adherens and gap junctions in which surface proteins such as occludin, claudins, cadherins, or connexins are specifically
incorporated.16-19 Interestingly, some members of these
protein families We recently cloned and characterized a molecule called JAM-2,
homologous to JAM and expressed by HEVs and lymphatic sinuses in murine
mesenteric lymph nodes.26 JAM-2 is a transmembrane type 1 protein with 2 immunoglobulin domains that share its overall protein
structure with JAM. The latter was shown to be present in tight
junctions of epithelial and vascular endothelial
cells.27,28 It has been found to interact with the
junctional proteins ZO-1 and AF-6 through its cytoplasmic PDZ-binding
domain.29,30 Furthermore, it was demonstrated that JAM
plays a central role in the regulation of paracellular permeability and
leukocyte transmigration across the endothelial
barrier.28,31 More recently, Palmeri et al32 characterized a novel human molecule, VE-JAM, which is structurally related to JAM and is expressed by vessels and HEVs. Independently, other authors have shown that VE-JAM supports leukocyte adhesion and
interacts with a 43-kd molecule expressed by human
leukocytes.33 For the sake of clarity, we will hereafter
refer to JAM as JAM-1 and VE-JAM as JAM-3.
The contribution of JAM-1 to epithelial tight junctions and its
expression by endothelial cells have been described,31,34 but little is known about the participation of JAM-2 and JAM-3 in
specialized interendothelial contacts. Therefore, the relative participation of JAM-1, JAM-2, and JAM-3 in different cellular junctions in vivo is central for understanding the specific function of
each molecule. In the current study, cloning of murine JAM-3 allowed us
to study and compare the tissue distribution of JAM-1, JAM-2, and
JAM-3. We define the JAM family as a novel subset of immunoglobulin
molecules that belong to the larger cortical thymocyte Xenopus (CTX) family.35,36 Furthermore, using
cells transfected with JAM-1, -2, or -3, we defined the respective
properties of each family member such as cell-cell contact
localization, participation in tight junctions, or receptor-ligand
interactions. These results suggest that JAM family members share a
similar structure and a common feature of homotypic interaction.
Nevertheless, the demonstration of differential function of JAM-1 and
JAM-2 in the control of paracellular permeability in vitro and the
important differences in tissue distribution or subcellular
localization observed in vivo suggest that they play specific roles in
the control of vascular functions in vivo.
Cell lines and antibodies
Sequence analysis
Reverse transcription-polymerase chain reaction on cell lines Specific PCR products were obtained using the following primer pairs: 5'-gtaactgtaatgggcaccgag-3' and 5'-cacagcatccatgtgtgcagcctc-3' for JAM-1, 5'-tcgacatggcgctgagc-3' and 5'-cagtgttgccgtcttgcctacag-3' for JAM-2, 5'-cctggactatcataaggcaaatgg-3' and 5'-catctttaaaccagatgtactccgga-3' for JAM-3. The PCR products were 704 base pairs (bp), 460 bp, and 457 bp long for JAM-1, JAM-2, and JAM-3, respectively. Control hypoxanthine phosphoribosyl transferase (HPRT) amplification was performed using 5'-gttggatacaggccagactttgttg-3' and 5'-gagggtaggctggcctataggct-3', a primer pair that resulted in a 350-bp-long PCR product.Cloning and expression of chimeric proteins Cloning of JAM-1 and JAM-2 in frame with enhanced green fluorescent protein (EGFP) was previously described.26 Briefly, it consisted of, respectively, the 3'HpaI or 3'ScaI of JAM-1 or JAM-2 sequences to fuse the coding sequence with EGFP in pcDNA3.43 Because no equivalent restriction sites were available in the sequence of JAM-3, we used a PCR approach to obtain the JAM-3-EGFP chimeric molecule using Pfu DNA polymerase and the following primers: 5'-tcagctaggcagccagct-3' as forward primer and 5'-cgaccggtgtgactttagatgcaggactgcc-3' as reverse primer. The reverse primer was modified by the AgeI site to clone the PCR product in frame with EGFP using the blunt EcoRI site on the 5' extremity and the AgeI site on the 3' extremity.To insert a FLAG-tag at the N-terminus of murine JAM-3, a 2-step PCR-based strategy on the cloned full-length cDNA was used. The first step consisted of amplification and cloning of the sequence encoding the leader peptide with 5'-tcagctaggcagccagct-3' and 5'-tcagaccggtcttatcgtcatcgtctttataatccccatttgccttatgatagtccagg-3' as forward and reverse primer, respectively. The reverse primer encoded the FLAG-tag sequence in frame with the sequence of JAM-3 leader peptide and was modified to insert an AgeI site. Therefore, a second PCR product was obtained by amplification with 5'-tcagaccggtttttctgcatcaaaagaccaccgt-3' and Sp6, and it encoded murine JAM-3 starting from the leader peptide. This PCR product was digested by AgeI and XhoI, and it was subcloned in the vector encoding the leader peptide fused to the FLAG-tag sequence. The integrity of the chimeric JAM-3 sequences was checked by sequencing the cloned product on both strands before transfection in MDCK cells using Fugene 6 (Roche Diagnostics, Rotkreuz, Switzerland). After 2 weeks of selection in medium containing 1 mg/mL G418, expressing cells were selected by fluorescence-activated cell sorter (Facstar; Becton Dickinson, Mountain View, CA) using EGFP fluorescence or immunostaining with appropriate antibody. Similar results were obtained with bulk sorted cells or clones. Northern blot analysis Total mRNA from cells or murine tissues was extracted using Trizol (Life Technologies AG, Basel, Switzerland) according to the manufacturer's instructions. Poly-A mRNA was extracted from 250 µg total RNA with the Oligotex mRNA Purification Kit (Qiagen, Zurich, Switzerland). Embryonic poly-A Northern blot was purchased from Clontech (PH Stehelin and Cie AG, Basel, Switzerland). Riboprobes were prepared from pcDNA3 vector (Invitrogen, Leek, Netherlands) and comprised the sequences encoding for the immunoglobulin domains of JAM-1, JAM-2, or JAM-3. Hybridization was performed at 62°C in buffer containing 50% formamide. Blots were washed twice (0.5 × standard sodium citrate [SSC], 0.1% sodium dodecyl sulfate [SDS], 67°C) and were autoradiographed on Kodak X-Omat at 80°C.
Immunostaining For immunohistochemistry with anti-JAM-1 (H202-106) or double staining for JAM-1 and JAM-2, samples were fixed for 5 minutes with cooled methanol (20°C), dried, and rehydrated in phosphate-buffered saline (PBS), 0.2% gelatin, and 0.05% Tween 20 (PGT). For single staining with polyclonal antibody against JAM-3 or anti-JAM-2 (XVIIIF26), acetone fixation for 5 minutes at 20°C was used. Stainings were visualized using secondary reagent coupled to Texas Red
or to horseradish peroxidase (Jackson Immunoresearch Laboratories, West
Grove, PA). Peroxidase activity was revealed using AEC as substrate,
and sections were counterstained for 1 minute with Hemalum before they
were mounted in Aquatex (Merck, Germany). For double staining,
anti-JAM-2 was revealed using antirat Texas Red, followed by staining
with biotinylated anti-JAM-1 in the presence of normal rat serum and
streptavidin-fluorescein isothiocyanate (FITC). For triple staining,
sections were fixed with 70% methanol-30% acetone at 20°C,
dried, rehydrated, and incubated overnight with a mix of
anti-JAM-1-Alexa584 (H202-106), anti-JAM-2-Alexa488 (CRAM-18F26), and rabbit polyclonal antibody against JAM-3 in PGT. After washing, JAM-3 staining was visualized with secondary reagent against rabbit coupled to DAMCA incubated for 1 hour in PGT in the presence of 0.2%
normal rat serum. Pictures were acquired using an Axiocam or Axiovert
fluorescence microscope (Zeiss, Oberkochen, Germany). For
immunocytochemistry, 11 days before the experiment, cells were plated
at 1 × 104 cells/cm2 on coated coverslips
(1% fetal calf serum in PBS), and the medium was changed every day
starting on day 4. Cells were fixed for 5 minutes with cooled methanol
and were washed 3 times in PBS and 0.2% bovine serum albumin before
incubation for 1 hour at room temperature with anti-JAM-1, JAM-2, or
antiFLAG antibody (M2; Sigma). After 3 washes, primary staining was
revealed using appropriate secondary reagent coupled to FITC (Jackson
Immunoresearch Laboratories). After 3 washes, rabbit anti-ZO-1 (Zymed)
was incubated for 1 hour at room temperature in the presence of 0.2%
normal rat or mouse serum. After washing, ZO-1 staining was visualized using a probe antirabbit coupled to Texas Red (Jackson Immunoresearch Laboratories) incubated in the presence of 0.2% normal rat or mouse
and 0.2% goat serum. Under these conditions, no signal in the red
channel was observed when the anti-ZO-1 reagent was omitted, and the
absence of leakage from red in green channel was checked by single
staining for ZO-1. Pictures were acquired using LSM510 confocal microscope and LSM510 software (Zeiss).
Permeability assays Permeability was measured using Transwell chambers (6.5-mm diameter polycarbonate filters, 0.4-µm pore size [Costar] or 10-mm diameter polycarbonate filters, 3-µm pore size [Nunc]). Briefly, 5 × 104 transfected or nontransfected MDCK cells were cultured to confluence on filters for 4 days. Medium was changed for prewarmed nutrient-F-12 medium (Life Technologies) without fetal calf serum (500 µL in the lower chamber and 350 µL containing 1 mg/mL FITC-dextran, Mr 42 000; Sigma-Aldrich Fluka Chemie AG, Buchs, Switzerland) in the upper chamber. After 4 hours, the chambers were removed and fluorescence was read directly in the lower chamber using Cytofluor II. Mean fluorescence intensity of 4 control wells (nontransfected MDCK cells) was calculated and referred to as 100% permeability. Values obtained with different transfected cells were then expressed as a percentage of control.
JAM-1, JAM-2, and JAM-3 form a subset of the immunoglobulin family of CTX We recently identified JAM-2, a novel immunoglobulin Sf molecule expressed by HEVs and lymphatic endothelial cells in mice.26 By sequence comparison, we identified 2 related sequences JAM-1 and a mouse EST (accession number, AA445150), encoding
an incomplete protein sequence identified earlier as a member of the
CTX gene family.36 The missing 5' region was
obtained with the rapid amplification of cDNA ends to give the
full-length coding sequence that we called JAM-3. JAM-3 was
subsequently identified as the murine equivalent of human VE-JAM,
recently identified by Palmeri et al.32 The 3 JAM proteins
were closely related: amino acid identities ranged from 31% for JAM-2
and JAM-1, up to 35% for JAM-2 and JAM-3, and the homologies were 51%
and 54%, respectively (Figure 1). The 3 proteins were type 1 proteins with 2 extracellular immunoglobulin Sf
domains, a transmembrane, and a short cytoplasmic segment. It is
noteworthy that the cytoplasmic membrane proximal sequence
(A-Y/Q-S/R-R/K-GYF) and the C-terminal sequence (T/K-S/K-SF-L/V/I-V/I) were almost identical for the 3 proteins. In JAM-2 and JAM-3, the
extracellular domain of the protein distal to the membrane was of V
type, and the proximal domain was of C2 type. Based on our
analysis, we propose a similar structure for JAM-1. Nevertheless, this
is still controversial because JAM-1 was originally described as a
molecule comprising 2 V domains and was more recently proposed to
present 2 C2 domains.27,44 This latter
analysis was solely based on the number of amino acids between the
cysteine residues contributing to the structure of the immunoglobulin
domains. However, this criterion is invalid because the spacing between
cysteine residues can vary in both V and C2
domains.45,46 In addition, JAM-2 and JAM-3but not
JAM-1contain an extra pair of cysteines in the A and G strands of
their C2 domains, at the same position described in all CTX
family members. This has been described as one of the characteristic
features of C2 domains from CTX family members47; therefore, we postulate that the JAM family may
be part of the larger CTX molecular family. Until now, this family of
proteins comprised the following members: CTX (Xenopus);
ChT1 (Gallus); CTH; A33; CAR, Coxsackie virus receptor; and CTHx (all in mammals). As illustrated by the subdivisions of the phylogenetic tree of the V+C domains (Figure 1B), JAM members form a subgroup within
the CTX family. These subdivisions are valid for mouse and human
molecules. The cytoplasmic tails of the CTX family members are
heterogeneous. Some members have ITIM motifs (CTX, ChT1, CAR), others
are not, and the length of the cytoplasmic tail varies. However, in the
case of JAM-1, JAM-2, and JAM-3, this part of the molecule is
relatively conserved in length and composition, and it contributes to
the feature of the JAM subset. This suggests that members of the JAM
subset may perform similar functions inside the cells once their
external ligands are engaged. Other features of this subfamily seem to
be conserved during evolution; 3 molecules of zebra fish and
Xenopus had been identified in the database, with the C
strand (P/S-R-V/I/L-EWK-F/K) of their V domain remarkably similar to
that of JAM-1, JAM-2, and JAM-3.47
Relative tissue distribution of JAM family members To further understand the relationship between JAM-1, JAM-2, and JAM-3, we explored the relative expression of the transcripts by Northern blot analysis. As shown in Figure 2A, JAM-3 gives 2 hybridization signals at 1.8 kb and 4 kb, with mRNA obtained from different murine tissues (Figure 2Ai), whereas JAM-1 and JAM-2 give single signals at 2 kb (Figure 2Aii-iii). When the distribution of the 3 family members is compared, JAM-1 is abundantly expressed in liver, whereas JAM-2 and -3 are weakly expressed. In contrast, JAM-2 and JAM-3 are highly expressed in lymph nodes, which express only limited amounts of JAM-1 (Figure 2A, Table 1). Notably, differences in transcriptional expression of the 3 molecules were also observed during embryogenesis (Table 1). However, these relative differences must be carefully interpreted because the results were obtained with mRNA from whole embryos in which the ratio between developing organs and the entire embryo varied with age. Similarly, differences between the relative expression of JAM members in adult tissues might result from cell type-specific expression of the individual JAMs.
To test this, we analyzed the expression of the 3 molecules by reverse transcription-PCR in murine endothelial and epithelial cell lines. Transcripts of JAM-1, JAM-2, and JAM-3 are found in most endothelial lines (Figure 2B). Interestingly, JAM-3 is absent from the te-V+ cell line, which represents a variant of t-end cells with phenotypic and morphologic properties of angiogenic endothelial cells (Aurrand-Lions et al, manuscript in preparation). The transcript encoding JAM-2 is detected in all tested endothelial cells but is absent from the epithelial carcinoma cell line KLN 205, which expresses reasonable levels of JAM-1 and JAM-3 transcripts. This indicates that JAM-1 and JAM-3 expression are not exclusive to vascular endothelial cells. Specificity of anti-JAM-1, -2, and -3 antibodies To further address the question of the relative tissue distribution of the 3 JAM family members at the protein level, antibodies against each molecule were used. As shown in Figure 3A, MDCK cells transfected with plasmids encoding murine JAM-1, JAM-2, or N-terminal FLAG-tagged JAM-3 (FgJAM-3) were specifically stained by antibodies to JAM-1 (H202-106), JAM-2 (XVIIIF26), JAM-3 (polyclonal antibody), or FLAG-tag (M2) (plain profiles), respectively. In addition, results showed that none of these reagents cross-reacted on another JAM family member.
A further specificity control was obtained with the KLN 205 squamous carcinoma cell line, which expressed high levels of JAM-1 and JAM-3 transcript and no detectable JAM-2 mRNA. Immunocytochemistry was performed on this cell line using monoclonal antibodies against JAM-1 and JAM-2 or affinity-purified polyclonal antibody against JAM-3.26,32,41 As expected, monoclonal antibody against JAM-1 stains the intercellular border with a sharp line resembling the pattern of tight junctions (Figure 3B, i). Anti-JAM-3 also stains cell borders but the pattern is hazy, indicating that this molecule is distributed over the entire lateral surface (Figure 3B, iii). In agreement with the absence of JAM-2 transcript in KLN 205, we did not detect a signal with anti-JAM-2 monoclonal antibody (Figure 3B, ii). Endothelial expression of JAM family members in vivo To further analyze the heterogeneity of JAM expression on endothelial cells in vivo, we performed immunohistochemical analysis of JAM-1, -2, and -3 expression on sections of brain or kidney. These organs are known to contain highly specialized vascular structures, such as glomerular endothelial cells in kidney or tightly sealed vascular beds of the blood barrier in the brain. Analysis of brain sections shows a restricted expression of JAM-1 and JAM-3 to vessels, whereas JAM-2 is not detected (Figure 4, left panel). In the kidney JAM-1, -2, and -3 are expressed in glomeruli (gl), whereas surrounding epithelial cells of tubules (tu) are solely stained for JAM-1 (Figure 4, right panel). This is in agreement with the previously reported expression of JAM-1 in tight junctions of epithelial cells.34,49
However, because the transcripts encoding JAM-1, -2, and -3 were
also detected in RNA preparations of lymph nodes, we were interested in
defining the cellular localization of the molecules in these organs.
Peroxidase staining was performed on sections of murine mesenteric
lymph nodes with antibodies directed against JAM-1, JAM-2, JAM-3,
PECAM, and MAdCAM to identify the cellular compartment
expressing the different family members. As expected, all these
molecules are expressed in mesenteric lymph nodes with differences in
their patterns of expression (Figure 5A).
PECAM is used as a positive control to visualize the entire endothelial compartment, comprising the lymphatic sinuses, and MAdCAM is used to
distinguish specifically the HEVs (arrowheads). When the staining obtained for JAM-1, -2, or -3 is compared to these 2 markers, it
appears that all 3 molecules are expressed relatively weakly by HEVs
(Figure 5A, arrowheads) and that JAM-1 and JAM-2 are expressed on
lymphatic sinuses where JAM-3 is not detected. To confirm the coexpression of JAM-1, JAM-2, and JAM-3 by some endothelial cells of
mesenteric lymph nodes, we performed triple immunofluorescence staining. Some vascular structures, presumably HEVs, expressed all 3 JAM family members (Figure 5B), whereas other endothelial structures
such as lymphatic sinuses were solely stained with anti-JAM-1 and
anti-JAM-2. The micrograph presented in Figure 5C shows a reticular
network of lymphatic medullary sinus surrounding a blood vessel
(arrowhead), diffusely stained for JAM-1, whereas JAM-2 is distributed
in sharp lines at cell-cell borders. This characteristic staining
pattern for the 2 molecules was also observed on lymphatic vessels
located in the cortical area of Peyer patches (not shown).
Altogether, these results show that JAM-1 expression on brain endothelial cells and most epithelial cells is in agreement with its putative function in tight junctions. In contrast, JAM-2 is not detected on brain vasculature and is expressed by lymphatic sinuses known to be highly permeable. This suggests that JAM-1 and JAM-2 may play opposite roles in interendothelial junctional sealing. JAMs differentially regulate paracellular permeability To directly test the hypothesis of opposite roles of JAM-1 and JAM-2 in interendothelial junctions and vascular functions, we performed paracellular permeability assays on MDCK cells stably transfected with JAM-1 or JAM-2. As shown in Figure 6, the stable expression of JAM-1 did not affect the paracellular permeability assessed by FITC dextran (42 kd) diffusion across monolayer. Interestingly, the expression of JAM-2 protein on the surfaces of MDCK cells, with an expression level comparable to that of JAM-1, resulted in a 5-fold increase in FITC-dextran flux. These results indicate that JAM-1 and JAM-2, though highly related, may perform distinct functions in the regulation of intercellular sealing and vascular function.
Localization of JAM family members to cell-cell contacts Because the barrier function and the control of permeability are known to depend on adherens and tight junctions, we addressed the issue of specific junctional and subcellular localization of JAM-1, JAM-2, and JAM-3. For this purpose, we used MDCK II cells that show the classical arrangement of apical tight and lateral adherens junctions. MDCK cells, transfected with full-length JAM-1, JAM-2, or FgJAM-3, were doubly stained with antibodies against each JAM in green and an antibody to ZO-1 in red to visualize the tight junctional regions (Figure 7A). Differences in the subcellular localization of the 3 family members were immediately apparent. JAM-1 colocalizes with ZO-1 at the most apical part of cell-cell contact, but it is also present in more basal locations (Figure 7A, z-axis). In contrast, JAM-2 is solely enriched in apical ZO-1-positive regions and is almost absent from more basal cell-cell contacts as depicted in yellow on the z-axis reconstitution. JAM-3 appears more diffuse and is not specifically enriched in ZO-1-positive tight junctional regions. The same results were obtained when experiments were performed on cells transfected with JAM molecules fused to EGFP on their carboxy terminal side (Figure 7B). Interestingly, this indicates that the C-terminal cytoplasmic PDZ binding motif is not required for the differential targeting of the 3 JAMs to different cell-cell contact regions of polarized cells because this motif was removed in the chimeric constructs. This prompted us to test the type of extracellular protein interactions required to target JAM family members to intercellular contacts. For this purpose, we mixed nontransfected MDCK cells with cells transfected with JAM-1-EGFP, JAM-2-EGFP, or JAM-3-EGFP chimeras. As illustrated in Figure 8, all JAM members are enriched at cell-cell borders between transfected cells but are absent from contacts between transfected and nontransfected cells (depicted by the black line on phase-contrast images). Although we cannot exclude that other ligands for JAM family members exist, these results indicate that each JAM member interacts in a homophilic way.
The homology among JAM-1, JAM-2, and JAM-3 allowed their classification in a novel subfamily of adhesion molecules. The structural organization in the extracellular portions of JAM-2 and JAM-3 consists of one V-like and one C2 domain with 2 disulfide bridges. This structure has been shown to be specific for molecules of the CTX family, which present a typical exon-intron structure, an extracellular organization in V-C2 domains with J features, and an extra disulfide bridge within the C domain. Although JAM-1 was originally described as a protein with 2 V domains27 and later as a 2 C2-containing protein,44 our sequence alignments suggested better matching to a V-C2 structure with a single disulfide bridge. The structural data indicate that the 3 JAMs represent a subgroup of the CTX family. Furthermore, by database search, human JAM-1,48 JAM-2, and JAM-3 genomic sequences were localized, respectively, on chromosomes 1, 11, and 21 already outlined for their immunoglobulin Sf CTX subset contents.47 This may suggest that CTX and JAMs are present in gene clusters and that they originate from gene duplication. However, the high homology between the 3 JAMs argues for a recent functional adaptation and the existence of a unique, functionally specialized linkage group. This hypothesis is further supported by the fact that the homology between JAM-1, JAM-2, and JAM-3 concerns not only the overall protein structure but also the transmembrane and the cytoplasmic domains indicating functional adaptation. The novelty of the JAM family consists in the fact that each member of a unique protein family participates specifically in different junctional compartments. When expressed in polarized MDCK cells, JAM-1, -2, or 3 is incorporated in different types of junctions. JAM-2 colocalizes with ZO-1, whereas JAM-1 is only partially incorporated in tight junctions as demonstrated by partial colocalization with ZO-1 in MDCK cells. Although we cannot exclude that JAM-1 localization was caused by overexpression, a similar result of JAM-1 localization was reported by Liu et al34 in nontransfected T84 cells. More surprisingly, in transfected MDCK cells, JAM-3 appeared more diffuse and not particularly enriched at a specific subcellular compartment of intercellular junctions. This result argued for the participation of JAM-3 in cell-cell contact in structures distinct from tight junctions, which is in agreement with the diffuse lateral staining observed on the KLN205 cell line. The function of the 3 JAMs in the intercellular junctions was further addressed using permeability assays with MDCK cells transfected with the different JAM isoforms. When compared with control cells, JAM-1 stable expression did not change FITC dextran diffusion across the MDCK monolayer, whereas JAM-2 increased the paracellular permeability. This was in agreement with the expression of JAM-2 found in permeable endothelial cells such as lymphatic sinuses or HEVs. However, this result contradicted the presumed barrier function of tight junctions and the previously described effect of JAM-1 or JAM-2-EGFP on paracellular permeability of Chinese hamster ovary cells.26,27 Although we cannot exclude that the differential effects of JAM-1 and JAM-2 observed in MDCK cells are attributed to differences in junctional organization between CHO and MDCK intercellular contacts, studies comparing both models remain to be conducted. According to this, the overexpression of JAM-1 in Chinese hamster ovary cells was shown to be sufficient to recruit ZO-1 in cell-cell contacts,29 whereas we did not observe a relocalization of ZO-1 in MDCK cells expressing JAM-1 in basal cell-cell contacts. These results indicate that the specific function of JAM family members may depend on the cell type and the composition of the intercellular junctional complexes. Therefore, it is not surprising to find heterogeneous expression of JAM family members on endothelial cells linked to each other through adhesive complexes resembling those of epithelial cells but presenting distinct molecular composition and structure. This probably reflects the heterogeneity of interendothelial junctions. The highest vascular expression of JAM-1 was found in adult brain, which is known to develop a network of tight junctional strands unique to the vascular system.50,51 In contrast, endothelial cells of HEVs and lymphatic endothelial cells, which have highly specialized cell-cell junctions,52-54 expressed high levels of JAM-2 and low amounts of JAM-1. Finally, JAM-3 was not expressed in lymphatic sinuses but was found in most endothelial contacts ranging from brain vasculature to HEVs. This argues for a role for JAM-3 in constitutive functions of vascular endothelial cells, consistent with its putative function as an adhesion molecule for circulating leukocytes.33 Taken together, our results suggest that the JAM family members may be involved in different interendothelial junctions reflecting the tissue specificity of vascular beds. By analogy with the function of JAM-1 as an organizer of occludin clustering in epithelial cells,34,49 one may speculate about a similar function in endothelial cells. Such a regulatory role for JAM-1 on endothelial tight junction is further supported by its expression in brain vasculature and its role in leukocyte recruitment in the cerebrospinal fluid of mice with experimental meningitis.28,31 We cannot exclude a similar regulatory function for JAM-2 and JAM-3, but their participation in endothelial junctional complexes devoid of occludin expression suggests their involvement in different mechanisms. Although the morphologic heterogeneity of endothelial junctions is well established, the specific properties of the different interendothelial junctional complexes are unknown. It has been suggested that interendothelial junctions should be considered as adherens junctions in which gap and tight junctions may be inserted.55 These "mixed junctional complexes" would be more easily regulated than morphologically defined junctions found in epithelial cells, and their molecular composition could regulate their "leakiness." Therefore, the specific route of leukocyte recirculation in noninflammatory conditions will depend on the establishment and stability of interendothelial junctions. The specific expression of JAM-1, -2, or -3 in different vascular compartments brings new insights in the molecular nature of such junctional complexes. Their structural and molecular characterization is probably the next step in our understanding of specific vascular functions that contribute to the maintenance of homeostasis. Sequences used in the current study are accessible under the following GenBank accession numbers: CD 2, M16445; PVR (poliovirus receptor), M24406; CD 22, X59350; MCAR (mouse Coxsackie virus receptor), U90715; A33, U79725; CTH, AF061022; CTX, (cortical thymocyte molecule Xenopus), U43330; CTHX (CTX human homologue), hs889n15; CHT1 (cortical thymocyte molecule chicken), Y14063; moJAM-1, U89915; huJAM-1, NM_016 946; moJAM-2, AJ300304; moJAM-3, AJ291757; amalgam Drosophila, M23561.
We thank Dr S. Rosen for kindly providing us with polyclonal antibody against JAM-3. We also thank D. Gay-Ducrest and C. Magnin for their technical expertise in molecular biology and immunohistochemistry.
Submitted February 26, 2001; accepted August 3, 2001.
Supported by Krebsforschung schweiz (KFS 981-02-2000), Foundation (31-49241.96), Foundation Gabriella Giorgi-Cavaglieri, and Human Frontier Science Program Organization (LT 0218/1998-M) (M.A.L.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michel Aurrand-Lions, Department of Pathology, Centre Medical Universitaire, 1 rue Michel-Servet, CH-1211, Geneva, Switzerland; e-mail: michel.aurrand-lions{at}medecine.unige.ch.
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S. Santoso, U. J.H. Sachs, H. Kroll, M. Linder, A. Ruf, K. T. Preissner, and T. Chavakis The Junctional Adhesion Molecule 3 (JAM-3) on Human Platelets is a Counterreceptor for the Leukocyte Integrin Mac-1 J. Exp. Med., September 2, 2002; 196(5): 679 - 691. [Abstract] [Full Text] [PDF]
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S. A. Cunningham, J. M. Rodriguez, M. P. Arrate, T. M. Tran, and T. A. Brock JAM2 Interacts with alpha 4beta 1. FACILITATION BY JAM3 J. Biol. Chem., July 26, 2002; 277(31): 27589 - 27592. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-strand-loop immunoglobulin
domain organization is indicated, and the letters refer to the strands
of the V and C2 domains. Murine sequences for JAM-1 and
JAM-2 are accessible from the database under the respective accession
numbers, 
1.5 and
4.4 kb, whereas JAM-1 (ii) and JAM-2 (iii) probes give a single
hybridization signal at 
