Opposite effects of tumor necrosis factor and soluble fibronectin on junctional adhesion molecule-A in endothelial cells
Ofelia M. Martinez-Estrada,1
Luca Manzi,1
Paolo Tonetti,1
Elisabetta Dejana,1,2,3 and
Gianfranco Bazzoni1
1Department of Immunology and Cell Biology, Istituto di Ricerche Farmacologiche Mario Negri, 2Department of Vascular Biology, FIRC Institute of Molecular Oncology, and 3Department of Biomolecular and Biotechnological Sciences, School of Sciences, University of Milan, Milan, Italy
Submitted 30 July 2004
; accepted in final form 9 February 2005
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ABSTRACT
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Junctional adhesion molecule-A (JAM-A) regulates key inflammatory responses, such as edema formation and leukocyte transmigration. Although it has been reported that the inflammatory cytokine tumor necrosis factor (TNF) causes the disassembly of JAM-A from the intercellular junctions, the mechanism has not been elucidated fully. Here, we report that TNF enhances the solubility of JAM-A in Triton X-100 and increases the amount of Triton-soluble JAM-A dimers at the cell surface but does not change the total levels of cellular JAM-A. Thus we hypothesized that TNF causes the redistribution of JAM-A from the junctions to the cell surface and that junction disassembly is sufficient to account for JAM-A redistribution. Intriguingly, however, even after complete disassembly of the junctions (with EDTA and trypsin), higher levels of JAM-A are detectable at the cell surface (by FACS analysis) in cells that had been previously incubated in the presence of TNF than in its absence. Thus we propose that TNF causes not only the disassembly of JAM-A from the junctions and its subsequent redistribution to the cell surface but also its dispersal in such a way that JAM-A becomes more easily accessible to the antibodies used for FACS analysis. Finally, we evaluated whether soluble fibronectin might attenuate the effects of TNF on JAM-A, as some inflammatory conditions are associated with the depletion of plasma fibronectin. We found that fibronectin reduces the effect of TNF on the disassembly of JAM-A, but not on its dispersal, thus further stressing that disassembly and dispersal can be functionally dissociated.
inflammation; junctions; permeability
JUNCTIONAL ADHESION MOLECULE-A (JAM-A) is an immunoglobulin-like glycoprotein (3) that was identified initially as a component of the tight junctions in endothelial and epithelial cells (26). JAM-A comprises an extracellular domain, a transmembrane segment, and a cytoplasmic tail. The extracellular domain forms parallel dimers (21) and binds several ligands, such as JAM-A itself (5, 24), the leukocyte integrin
L
2 (29), and the reovirus protein
-1 (2).
By means of its junctional localization and adhesive function, JAM-A contributes to restricting paracellular permeability (23, 24), which in turn regulates the extravasation of plasma proteins and circulating leukocytes during inflammation (13, 26). In addition, inflammatory stimuli regulate the localization of JAM-A at the intercellular junctions. Specifically, when given to human umbilical vein endothelial cells in combination with interferon-
, tumor necrosis factor (TNF) decreases the junctional localization of JAM-A (30, 38). However, despite the potential importance in inflammation, both the mechanism of action and the regulation of the TNF effect on JAM-A have not been elucidated fully.
As for the mechanism of action, it is known that the actin cytoskeleton is one of the numerous targets of TNF (40). In addition, TNF causes the disassembly of zonula occludens-1 (ZO-1) from the endothelial junctions (7). As ZO-1 associates with both JAM-A (6, 14) and actin (16, 19), ZO-1 may link JAM-A with cortical actin. Thus we hypothesized that TNF might induce the disassembly of JAM-A from the endothelial junctions by modulating the linkages of JAM-A with the actin cytoskeleton. In support of this view, we found that TNF enhances the solubility of JAM-A in the nonionic detergent Triton X-100. In addition, we found that, besides causing the redistribution of JAM-A from the junctions to the cell surface, TNF also causes its dispersal on the surface.
Finally, while searching for mechanisms of JAM-A regulation that might be relevant to inflammation, we focused on soluble fibronectin. Severe inflammatory conditions (e.g., burns, trauma, and bacteremia) are associated with initial decrease of fibronectin levels in plasma, which can be followed (e.g., in severe sepsis) by persistent depletion (1). In addition, soluble fibronectin attenuates TNF-induced increase of vascular permeability both in vivo (11) and in vitro (42). Thus we hypothesized that soluble fibronectin, besides affecting permeability, might also affect the subcellular distribution of endothelial JAM-A. Actually, we found that soluble fibronectin reduces the TNF-dependent disassembly of JAM-A from the intercellular junctions.
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MATERIALS AND METHODS
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Cell lines, antibodies, and reagents.
The murine endothelial cell line H5V, derived from the heart microcirculation (17), was kindly provided by Dr. A. Vecchi (Mario Negri Institute, Milan, Italy) and was cultured in DMEM (GIBCO-BRL) supplemented with 10% fetal calf serum (HyClone Laboratories). Rat anti-JAM-A MAbs BV12, BV11, and BV19, anti-platelet/endothelial cell adhesion molecule (PECAM)-1 MEC 13.3, and anti-vascular endothelial (VE)-cadherin MAb BV13 have been described (5, 12, 26, 41). Rabbit ZO-1 and
-catenin antisera were from Zymed and Sigma, respectively. Rat Texas red isothiocyanate-labeled anti-IgG antibodies were from Jackson ImmunoResearch Laboratories. Recombinant human TNF (6.6 x 106 U/mg) was from Genzyme. Human fibronectin and FITC-phalloidin were from Sigma.
Immunofluorescence.
H5V cells were seeded onto glass coverslips (13-mm-diameter) that had been coated with human plasma fibronectin (7 µg/ml). Cells were grown in DMEM plus 10% serum. When cells reached confluence, the medium was replaced with DMEM plus 1% serum in either the presence or absence of 400 U/ml of TNF. Cells were then incubated for 24 h at 37°C before fixation. To test the effect of soluble fibronectin, after 18 h of incubation with TNF, human plasma fibronectin was added (at a final concentration of 600 µg/ml), and cells were incubated for an additional 6 h at 37°C. Finally, cells were fixed with either 4% formaldehyde (for 15 min at room temperature) or methanol (for 3 min at 20°C), stained with antibodies for indirect immunofluorescence microscopy, mounted in Mowiol 4-88 (Hoechst), and analyzed as described (27).
Fluorescence flow cytometric analysis.
Fluorescence flow cytometric analysis was performed with a FACStar Plus apparatus (Becton Dickinson, Mountain View, CA). Cells were detached by incubation with a solution containing 0.5 mM EDTA and 0.05% (wt:vol) trypsin (GIBCO). Then, aliquots (100 µl) of cell suspensions (106 cells/ml) were incubated with 2% (wt:vol) BSA in PBS (for 60 min on ice), washed with PBS plus 0.2% BSA, and incubated with primary antibodies (for 45 min on ice) at a concentration of 20 µg/ml. Cells were then washed three times, incubated (for 45 min on ice) with secondary FITC-conjugated goat F(ab)2 anti-rat IgG (Caltag Laboratories, Burlingame, CA), and washed three times before analysis.
Immunoprecipitation.
Confluent monolayers of H5V cells were washed twice with PBS and then lysed for 30 min with lysis buffer containing 0.5% Triton X-100, 150 mM NaCl, 50 mM Tris·HCl, and protease inhibitors. In some experiments, before cell lysis, cells were incubated for 2 h at room temperature in PBS with 5 mM bis(sulfosuccinimidyl)suberate (BS3; Pierce). The lysate was then centrifuged (14,000 g for 10 min) to collect the supernatant ("soluble" fraction). The pellet was incubated with lysis buffer supplemented with 0.02% SDS and centrifuged. The resulting supernatant represents the "insoluble" fraction. Immunoprecipitation was performed as described (5).
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RESULTS
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TNF reduces the junctional localization of JAM-A.
To investigate the effect of TNF on JAM-A, we incubated confluent monolayers of the endothelial cell line H5V in either the absence or presence of 400 U/ml of TNF (for 24 h at 37°C). TNF induced no major changes in cell morphology, as assessed by phase-contrast microscopy (data not shown). However, TNF affected the distribution of JAM-A and ZO-1, as evaluated by indirect immunofluorescence microscopy using anti-JAM-A MAb BV12 and a ZO-1 antiserum (Fig. 1). In the absence of TNF, JAM-A and ZO-1 localized to the intercellular junctions. At variance, in the presence of TNF, the junctional localization of JAM-A and ZO-1 was considerably reduced.

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Fig. 1. Tumor necrosis factor (TNF) reduces the junctional localization of junctional adhesion molecule-A (JAM-A) and zonula occludens-1 (ZO-1). Confluent H5V cells were incubated for 24 h in either the absence (control) or presence (TNF) of 400 U/ml of TNF. Samples were then fixed and stained with either anti-JAM-A MAb BV12 (A and B) or a ZO-1 antiserum (C and D), followed by Texas red isothiocyanate-conjugated secondary antibodies.
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TNF enhances the solubility of JAM-A in Triton X-100.
As TNF was reported to enhance the solubility of ZO-1 in nonionic detergents (7), we examined whether TNF might enhance the Triton solubility of JAM-A as well. For this purpose, H5V cells were incubated in either the absence or presence of 400 U/ml of TNF (for 24 h at 37°C) and then lysed with 0.5% (vol:vol) Triton X-100. The resulting supernatant was saved (Triton-soluble fraction), while the pellet was further solubilized with 0.02% (wt:vol) SDS (Triton-insoluble fraction). Finally, JAM-A was immunoprecipitated from both fractions with MAb BV12 (Fig. 2A).

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Fig. 2. TNF enhances the solubility of JAM-A in Triton X-100. A: confluent H5V cells were incubated for 24 h in either the absence () or presence (+) of 400 U/ml of TNF. Triton X-100-soluble and -insoluble JAM-A was analyzed by immunoprecipitation and Western blotting with anti-JAM-A MAbs BV12 and BV19, respectively. The 45-kDa molecular marker is indicated at right. As loading control, the amount of -tubulin in the preimmune lysate did not differ in either the absence or presence of TNF (not shown). In B, the values of optical density (in arbitrary units) are shown. Results are expressed as means ± SE and are derived from 3 experiments (*P < 0.05 and **P < 0.01, TNF+ vs. TNF; 2-sample t-test assuming unequal variances). Numbers in parentheses represent percent of JAM-A, with 100% corresponding to total (i.e., soluble + insoluble) JAM-A. Sol, soluble; Insol, insoluble.
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In H5V cells, JAM-A is immunoprecipitated as a major band with an apparent relative molecular mass of 35 kDa and additional minor bands of slightly higher mass, which reflect variable patterns of N-linked glycosylation, as assessed by PNGaseF treatment (not shown). In both the absence (Fig. 2A, lanes 1 and 3) and presence (lanes 2 and 4) of TNF, Triton-soluble JAM-A was more abundant than Triton-insoluble JAM-A. However, more Triton-soluble JAM-A was detectable in the presence than in the absence of TNF (lanes 2 and 1). Accordingly, less Triton-insoluble JAM-A was detectable in the presence than in the absence of TNF (lanes 4 and 3). Densitometric analysis showed that the ratio of soluble-to-insoluble JAM-A was 2.5-fold higher in the presence than in the absence of TNF (Fig. 2B). Thus TNF increases the solubility of JAM-A in Triton X-100.
TNF redistributes Triton X-100-soluble JAM-A dimers to the cell surface.
JAM-A forms dimers, which in turn mediate homophilic adhesion (5, 21, 23). Thus we surmised that the TNF-induced disassembly of JAM-A from the junctions might result in the redistribution of junctional JAM-A dimers to the cell surface. To specifically detect JAM-A dimers at the cell surface, we deployed the membrane-impermeable cross-linker BS3, which was used already to detect dimeric JAM-A (5) and PECAM-1 (46). Briefly, cell monolayers were incubated (for 24 h at 37°C) in either the absence or presence of 400 U/ml of TNF. Then, cells were incubated with 5 mM BS3 (for 2 h at room temperature) and lysed with 0.5% (vol:vol) Triton X-100, as described above. Finally, JAM-A was immunoprecipitated with MAb BV12 from the Triton-soluble and -insoluble fractions, to determine the amount of BS3 cross-linkable (i.e., dimeric and cell surface bound) JAM-A (Fig. 3A).

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Fig. 3. TNF redistributes soluble JAM-A dimers to the cell surface. A: confluent H5V cells were incubated for 24 h in either the absence (; lanes 1 and 2) or presence (+; lanes 3 and 4) of 400 U/ml of TNF. Before lysis with Triton X-100, cells were incubated with bis(sulfosuccinimidyl)suberate (BS3). Then, lysates were divided into Triton-soluble (S) and -insoluble (I) fractions. Finally, JAM-A was analyzed by immunoprecipitation and Western blotting with anti-JAM-A MAbs BV12 and BV19, respectively. Molecular markers are indicated at right. In B, the values of optical density (in arbitrary units) are shown. Numbers in parentheses represent percent of JAM-A, with 100% corresponding to total (i.e., soluble + insoluble) JAM-A. Results are derived from 1 (of 2) experiment.
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In the absence of TNF, only low amounts of JAM-A dimers (either soluble or insoluble) could be cross-linked with BS3 (Fig. 3A, lanes 1 and 2), probably because dimers localized to sites of the cell surface (e.g., the junctions) that may not be easily accessible to BS3. However, in the presence of TNF, the amount of Triton-soluble and BS3 cross-linkable JAM-A was increased (lane 3) and correspondingly the amount of insoluble and BS3 cross-linkable JAM-A was decreased (lane 4). Interestingly, in the presence of TNF, the pool of soluble and non-cross-linkable (i.e., monomeric) JAM-A was also decreased (lane 3), likely because some monomers assembled together and formed soluble dimers. Densitometric quantification is shown in Fig. 3B. Thus TNF enhances the localization of soluble JAM-A dimers on the cell surface.
TNF induces the dispersal of JAM-A on the cell surface.
As TNF induced both the disassembly of JAM-A (from the junctions) and its redistribution (to the cell surface), we evaluated whether junction disassembly was sufficient to account for a complete redistribution of JAM-A to the surface. To this aim, monolayers of confluent cells were incubated for 24 h in either the absence or presence of 400 U/ml of TNF. Then, cells were treated with 0.5 mM EDTA and 0.05% (wt:vol) trypsin to obtain single cell suspensions. Finally, the levels of cell surface JAM-A were measured by fluorescence-activated cell sorting (FACS) analysis using MAb BV12. As the EDTA/trypsin treatment induces complete disassembly of the junctions (regardless of the previous incubation with or without TNF), we expected that the cell surface levels of JAM-A would not differ between cells incubated in the absence or presence of TNF, if the TNF-dependent redistribution of JAM-A is solely due to junction disassembly. Unexpectedly, however, we detected higher levels of cell surface JAM-A in cells that had been incubated in the presence of TNF than in its absence (Table 1). Also, TNF increased the levels of JAM-A dimers, which are specifically detected with anti-JAM-A MAb BV11 (5), but not the levels of PECAM-1. The increased surface levels of JAM-A were not due to increased total levels of cellular JAM-A, as the sum of soluble plus insoluble JAM-A (Fig. 2B) was not significantly different in the absence or presence of TNF.
Although these results indicate that junction disassembly is not sufficient to account for a complete redistribution of JAM-A to the cell surface, we examined whether junctions are nonetheless necessary to account for the increased levels of cell surface JAM-A that are detected by FACS analysis. For this purpose, we compared contacting cells (which form junctions) with noncontacting cells (which lack junctions). As above, cells were incubated for 24 h in either the absence or presence of 400 U/ml of TNF, and then cell surface JAM-A was determined by FACS analysis using MAb BV12. We found that TNF increased JAM-A levels in contacting cells (129 ± 3 and 172 ± 5) but not in noncontacting cells (126 ± 4 and 128 ± 2 arbitrary units of mean fluorescence intensity, in the absence and presence of TNF, respectively), thus indicating that, on TNF treatment, the increased levels of cell surface JAM-A derive from the junctions. Based on these results, we propose that TNF induces not only the disassembly of JAM-A from the junctions and its redistribution to the cell surface but also its dispersal in such a way that JAM-A becomes more easily accessible to the antibodies used for FACS analysis.
Finally, we tested the role of actin in JAM-A dispersal (i.e., the increased levels of JAM-A, as assessed by FACS analysis with either MAb BV12 or MAb BV11). For this purpose, at the end of the 24-h incubation period (in either the absence or presence of TNF), cell monolayers were treated with 2 µM cytochalasin D (30 min at 37°C) to depolymerize actin. In the absence of TNF, cytochalasin D had no effect on the dispersal. However, in the presence of TNF, cytochalasin D amplified the dispersal (Table 1). Thus weakened linkage with actin may contribute to the TNF-dependent dispersal of JAM-A.
Soluble fibronectin reduces the TNF-dependent disassembly of JAM-A from the junctions.
We then evaluated whether fibronectin might reduce the effects of TNF on JAM-A. For this purpose, confluent monolayers were incubated for 18 h in either the absence or presence of 400 U/ml of TNF. Then, soluble fibronectin (600 µg/ml) was added, and cells were incubated for additional 6 h before analysis by fluorescence microscopy. Under these conditions (Fig. 4), JAM-A was detectable at the junctions in both the absence (Fig. 4A) and presence (Fig. 4B) of TNF. At variance, ZO-1 was detectable at the junctions in the absence (Fig. 4C), but not in the presence (Fig. 4D), of TNF. Similarly to JAM-A, we found that the junctional localization of other junctional molecules, such as VE-cadherin and
-catenin (Fig. 5A, a and d), was lost in cells treated with TNF alone (Fig. 5A, b and e) but was detectable in cells treated with both TNF and fibronectin (Fig. 5A, c and f).

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Fig. 4. Soluble fibronectin reduces the effect of TNF on the junctional localization of JAM-A. Confluent H5V cells were incubated for 18 h in either the absence (control) or presence (TNF) of 400 U/ml of TNF. Then, soluble fibronectin (600 µg/ml) was added, and cells incubated for an additional 6 h. Finally, cells were fixed with formaldehyde and stained with either anti-JAM-A MAb BV12 (A and B) or a ZO-1 antiserum (C and D), followed by TRITC-conjugated secondary antibodies.
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Fig. 5. Vascular endothelial (VE)-cadherin, -catenin, actin filaments, and JAM-A solubility in cells treated with TNF and fibronectin. Confluent H5V cells were incubated for 24 h in either the absence (control; a, d, g, and j) or presence (TNF; b, e, h, and k) of 400 U/ml of TNF. After 18 h, some samples were incubated with 600 µg/ml of fibronectin for an additional 6 h (TNF + fibronectin; c, f, i, and l). Then, in A and B, cells were fixed with formaldehyde, whereas in C, cells were extracted with Triton X-100 before fixation. Finally, cells were stained with anti-VE-cadherin MAb BV13 (A; ac) and -catenin antiserum (A; df), FITC-phalloidin (B; gi), and anti-JAM-A MAb BV12 (C; jl). F-actin, filamentous actin.
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As negative control, we found that (unlike fibronectin) albumin (600 µg/ml) had no effect in all conditions tested (not shown). Also, FACS analysis of VCAM-1 expression ruled out that 600 µg/ml of fibronectin exerted a general inhibition of cell responsiveness to TNF. The levels of VCAM-1 were increased to a similar extent upon treatment with either 400 U/ml of TNF for 24 h (5.6 and 82.2% VCAM-1-positive cells, in the absence and presence of TNF, respectively) or with TNF for 18 h plus fibronectin for an additional 6 h (5.2 and 80.9% positive cells).
Finally, we evaluated whether fibronectin had any effect on actin filaments, JAM-A solubility, and JAM-A dispersal. As above, cells were treated with 400 U/ml of TNF (for 18 h) and then incubated (for an additional 6 h) in either the absence or presence of 600 µg/ml of fibronectin. First, to examine actin filaments, we used FITC-phalloidin (Fig. 5B) and observed no major differences in either the absence (Fig. 5B, g) or presence (Fig. 5B, h) of TNF or in the presence of TNF plus fibronectin (Fig. 5B, i). Second, to examine the Triton solubility of JAM-A, cells were extracted with 0.2% Triton X-100 (for 2 min at room temperature) and then fixed with 4% formaldehyde (Fig. 5C). Under these conditions, a pool of Triton-resistant JAM-A was detectable at the intercellular junctions in the absence of TNF (Fig. 5C, j) and was almost completely lost in its presence (Fig. 5C, k). At variance with results reported in Fig. 4B (in which cells were only fixed but not extracted), the junctional staining of JAM-A was incomplete and discontinuous in the presence of TNF plus fibronectin. Third, to examine the dispersal of JAM-A on the cell surface, we prepared single cell suspensions and measured the levels of cell surface JAM-A by FACS analysis. As shown in Table 1, fibronectin had no effect on the levels of cell surface JAM-A in either the absence or presence of TNF. Thus when added to TNF-treated cells, soluble fibronectin enhanced JAM-A localization to the junctions (Fig. 4) without concurrently affecting actin filaments (Fig. 5B), restoring the Triton insolubility of JAM-A (Fig. 5C) and reducing its dispersal on the cell surface (Table 1).
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DISCUSSION
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The major findings of this manuscript are as follows. 1) TNF, which was reported to disassemble JAM-A from the junctions (30, 38), enhances the solubility of JAM-A in Triton X-100. In addition, 2) TNF induces not only the redistribution, but also the dispersal, of JAM-A on the cell surface. Finally, 3) in TNF-treated cells, soluble fibronectin reduces the disassembly of JAM-A from the junctions but not its dispersal on the cell surface.
Electron microscopy of thin (26) and freeze-fracture sections (20) indicates that JAM-A localizes in proximity of the tight junction strands. This location likely contributes to sealing the intercellular spaces and controlling paracellular permeability (4). For instance, anti-JAM-A antibodies prevent the calcium-driven resealing of epithelial junctions, thus causing persistently low levels of electrical resistance (24). Also, soluble mediators of inflammation target junctions and increase paracellular permeability. Among such agents, TNF (in combination with interferon-
) reduces the junctional localization of JAM-A in human umbilical vein endothelial cells (30, 38). In addition, TNF alone reduces the junctional localization of ZO-1 (7) as well as the transcription of occludin (37). Finally, TNF abolishes the localization of the junctional molecules VE-cadherin and PECAM-1 when TNF is used either alone (34, 44) or in combination with interferon-
(22, 33, 39). Our system, which deploys the heart microvascular H5V endothelial cells, differs from the one described by Ozaki et al. (30) and by Shaw et al. (38), as TNF alone was sufficient to induce the junctional disassembly of JAM-A. In this respect, H5V cells may allow a more specific analysis of the effect of TNF.
To date, however, the mechanism whereby TNF affects JAM-A distribution has not been elucidated fully. The cytoplasmic domain of JAM-A binds several proteins that contain PSD95/dlg/ZO-1 (PDZ) domains, such as ZO-1 (6, 14), ALL-1 fusion partner from chromosome 6 (AF-6) (14), calcium/calmodulin-dependent serine protein kinase (CASK)/lineage-defective (Lin)2 (27), partitioning-defective (PAR)3 (15, 20), and multi-PDZ protein (MUPP)1 (18). In turn, some of these molecules link (either directly or indirectly) JAM-A to cortical actin. For instance, ZO-1 (16) and AF-6/Afadin (8) bind directly filamentous actin and profilin, respectively. In addition, CASK/Lin2 interacts with protein 4.1, which nucleates actin and spectrin filaments (10). Results from our study indicate that TNF increases the solubility of endothelial JAM-A in Triton X-100, thus suggesting that TNF disassembles JAM-A from the junctions by weakening its association with cortical actin. Similar to our findings, it has been shown recently that interferon-
(either alone or in association with TNF) enhances the Triton solubility of JAM-A in epithelial cells (9). According to the model we propose (Fig. 6), TNF-induced disassembly of JAM-A from the junctions is followed by its redistribution to the cell surface. In this location, it is likely that the JAM-A molecules become sparse and scattered, as suggested by the increased availability for antibody binding in FACS analysis. We refer to the latter effect as dispersal, to distinguish it from the mere redistribution to the cell surface that follows the disassembly from the junctions. Similar to the disassembly, the TNF-induced dispersal is another likely consequence of reduced linkage with cortical actin, as the extent of the dispersal is augmented by cytochalasin D.

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Fig. 6. Schematic model of the TNF effect on JAM-A. TNF loosens the linkages of JAM-A with actin filaments. In addition, TNF induces disassembly of JAM-A from the junctions as well as redistribution and dispersal on the cell surface. As a consequence, following complete disruption of the junctions with EDTA and trypsin, more cell surface JAM-A becomes available for antibody binding, as assessed by fluorescence-activated cell sorting analysis.
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While searching for mechanisms of JAM-A regulation that might be relevant to inflammation, we focused on fibronectin because of clinical evidence in humans as well as in vivo and in vitro studies. First, early acute decrease of plasma fibronectin occurs in patients undergoing burn, trauma, and major surgery (11), whereas sustained depletion occurs in severe sepsis (1) and adult respiratory distress syndrome (28). Second, infusion of gelatin in rats induces deficiency of fibronectin and worsens the systemic effects of endotoxin (45). Conversely, administration of fibronectin in sheep ameliorates the effects of postsurgical bacteremia (36), even though it should be stressed that the efficacy of fibronectin administration in humans has not been proven (25). Third, addition of soluble fibronectin to cultured endothelial cells attenuates TNF-induced increase in permeability (42). To date, the mechanism whereby soluble fibronectin attenuates the effect of TNF and endotoxin is not fully understood. After intravenous administration, soluble fibronectin is incorporated into the subendothelial matrix (31). In addition, fibronectin ability to counteract TNF-induced permeability requires binding of fibronectin to the subendothelial matrix (43). Hence, fibronectin might attenuate TNFs effect by facilitating adhesion of endothelial cells to the matrix (32).
Here, we propose that fibronectin may also favor endothelial integrity by enhancing the junctional localization of JAM-A and thus strengthening cell-cell cohesion. Interestingly, the fibronectin-driven localization of JAM-A is not accompanied by concomitant recruitment of ZO-1 and by fully restored resistance of JAM-A to Triton X-100. We and others (14, 27) reported that the junctional recruitment of JAM-A that follows addition of calcium in the "calcium switch" assay is an early event that precedes the recruitment of ZO-1. Similarly, our described recruitment of JAM-A that follows addition of soluble fibronectin does not require concomitant mobilization of ZO-1. In addition, in our system, fibronectin did not reduce the dispersal of JAM-A at the cell surface, thus indicating that JAM-A disassembly and dispersal can be functionally dissociated. As for the cause of such dissociation, we speculate that reduction of disassembly may just require adhesive interactions (e.g., between the extracellular domains of JAM-A on adjacent cells). At variance, reduction of dispersal may require additional interactions (e.g., between the cytoplasmic domains of JAM-A and actin-associated proteins).
In conclusion, these results reveal the existence of a functional interplay between JAM-A linkage to actin, disassembly from the junctions, and dispersal on the cell surface in response to TNF. In addition, our data highlight a fibronectin-based regulatory mechanism that may be relevant to some severe forms of inflammation that are associated with reduction and depletion of plasma fibronectin.
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GRANTS
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G. Bazzoni was supported by AICR (Association International for Cancer Research, UK; 04-095), AIRC (Italian Association for Cancer Research), and MIUR (Italian Ministry of University and Research; FIRB RBNE01T8C8004, FIRB RBAU01E5F5). E. Dejana was supported by AIRC and MIUR (RBNE01F8LT-007, RBNE01MAWA-009), Italian Ministry of Health (Ricerca Finalizzata 2002; Convenzione 192), Italian NRC (04.00149.ST 97), Fondazione Cariplo (2003.1697/10.6399), ISS (CS36), and European Community (LSHG-CT-2004-503254, 502935, 503573).
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FOOTNOTES
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Address for reprint requests and other correspondence: G. Bazzoni, Laboratory of Systems Biology, Istituto di Ricerche Farmacologiche Mario Negri, via Eritrea 62, I-20157 Milan, Italy (E-mail: bazzoni{at}marionegri.it)
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.
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