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First published online 17 January 2006
doi: 10.1242/jcs.02771
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Research Article |
vß3 specific
1 Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA
2 Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA
3 Delaware Biotechnology Institute, University of Delaware, Newark, DE 19716, USA
* Author for correspondence (e-mail: unaik{at}udel.edu)
Accepted 31 October 2005
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vß3 and not to integrin vß5. JAM-A-induced migration was inhibited by anti-JAM-A antibody. Additionally, overexpression of a JAM-A cytoplasmic domain deletion mutant failed to induce HUVEC migration. Addition of phosphoinositide 3-kinase and protein kinase C inhibitors blocked JAM-A-induced migration, suggesting that these kinases act downstream of JAM-A. Immunoprecipitation analysis showed that JAM-A interacts with integrin vß3, and this association was increased by engagement of the ligand-binding site of the integrin by Arg-Gly-Asp-Ser (RGDS) peptide. Furthermore, activation of both focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) on vitronectin was enhanced by JAM-A overexpression but not by its cytoplasmic domain deletion mutant. Taken together, these results suggest that signaling through JAM-A is necessary for vß3-dependent HUVEC migration and implicate JAM-A in the regulation of vascular function.
Key words: Junctional adhesion molecule, Endothelial cell migration, F11R, Integrin vß3, Vitronectin, MAPK
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and ß subunits that, together, have a specific affinity to different ECM proteins (Buck and Horwitz, 1987
; Hynes, 1992; Sonnenberg, 1993; Tozer et al., 1996). Regulation of these affinities through inside-out signaling events are key to the regulation of cellular dynamics, in that rapid adhesion and de-adhesion events promote a swift cellular motility (Dedhar, 1999). These signaling events, as well as those involved in depolymerization and repolymerization of the actin cytoskeleton, regulation of cell directionality, and transmission and reception of chemotactic or haptotactic cues, work together to regulate the motile dynamic (Abedi and Zachary, 1995; Christopher and Guan, 2000).
Among the members of the integrin family most widely studied in the migration of endothelial cells is integrin vß3, which is a receptor for vitronectin (Byzova et al., 2000). It has been reported that, in response to the inflammatory cytokine tumor necrosis factor- (TNF-), endothelial cells increase the activation and ligation of vß3, while decreasing the activation and ligation of 5ß1, to facilitate migration leading to vascular wound healing (Gao et al., 2002). Additionally, the angiopoietin family member ANGPTL3 also induces vß3-dependent endothelial cell migration by binding vß3, leading to the activation of mitogen-activated protein kinase (MAPK) and focal adhesion kinase (FAK), which are required for migration (Camenisch et al., 2002). It is known that activity of extracellular signal-related kinase-1 (ERK-1), a member of the MAPK family, is required for vß3-dependent migration of endothelial cells during angiogenesis (Klemke et al., 1997). More recently, it has been reported that ERK-1 associates with vß3 prior to focal complex formation, initiating cell spreading on vitronectin (Roberts et al., 2003).
In addition to integrins, cell adhesion and motility on substratum has also been attributed to signaling through transmembrane and membrane-associated proteins. Cross-talk between integrins and transmembrane proteins has long been implicated in the regulation of these processes (Barazi et al., 2002; Chellaiah and Hruska, 2003; Silvestri et al., 2002; Voura et al., 2001). Junctional adhesion molecule-A [JAM-A, previously referred as JAM-1 (Muller, 2003)], a recently identified member of the immunoglobulin superfamily (IgSF), has been reported to be a ligand of integrin Lß2, the leukocyte-function-associated antigen, and is involved in transendothelial migration of leukocytes by regulating the integrity and permeability of cell junctions (Ostermann et al., 2002). We have recently shown that JAM-A is important in the regulation of basic fibroblast growth factor (bFGF)-induced angiogenesis (Naik et al., 2003a). Inhibition of JAM-A signaling blocks bFGF-induced endothelial cell proliferation, tube formation and in vivo angiogenesis. It has been well established that JAM-A is specifically localized at the tight junctions of epithelial and endothelial cells (Liu et al., 2000; Martin-Padura et al., 1998). Thus, it was thought that, as a key tight junction component, overexpression of JAM-A would inhibit endothelial cell migration by enhancing cell-cell contacts, especially because JAM-A is known to participate in homotypic interaction (Bazzoni et al., 2000a; Kostrewa et al., 2001; Naik et al., 2001). Consistent with this notion, it has recently been shown that loss of JAM-A enhanced the random motility of mouse endothelial cells (Bazzoni et al., 2005). Surprisingly, our data presented here show that overexpression of JAM-A induces migration of human umbilical vein endothelial cells (HUVECs) on vitronectin, specifically through integrin vß3. Moreover, we find that JAM-A directly interacts with vß3, and regulates MAPK activation. Taken together, these data demonstrate that signaling through JAM-A and vß3 are required for HUVEC migration, and perhaps highlight a novel mechanism by which signaling through IgSF members and integrins influences endothelial cell physiology and vascular function.
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; Bazzoni et al., 2000b; Kostrewa et al., 2001; Naik et al., 2001), but its physiological role in these cells beyond that of a tight junction protein has yet to be established. We sought to study the functional relevance of JAM-A in endothelial cells and thus examined the effect of its overexpression in HUVECs. To differentiate between endogenous and exogenous JAM-A, we took advantage of the HA-tag that is engineered at the N-terminus of the JAM-A construct. We have previously shown that this construct expresses JAM-A on the cell surface and that tagging with HA does not interfere with the tight junctional localization of JAM-A (Naik et al., 2001). However, the overexpressed JAM-A was uniformly distributed all over the cell membrane, including cell-cell junctions, as opposed to the restricted localization of endogenous JAM-A only to the cell-cell junctions. Western blot analysis of HUVECs overexpressing JAM-A showed a substantial increase in the level of JAM-A expression in these cells compared with mock-transfected cells (Fig. 1A). Densitometric analysis indicated that we were able to overexpress about three times that of endogenous levels of JAM-A (Fig. 1B). Morphological analysis indicated that JAM-A overexpression induced long cytoplasmic extensions similar to those observed when HUVECs were stimulated with bFGF (data not shown) (Naik et al., 2003a). We next reasoned that, similar to bFGF activation, JAM-A overexpression might also induce endothelial cell migration. To explore this possibility, we performed a qualitative wound-induced migration assay on vitronectin under serum-free conditions (Fig. 1C). Within 24 hours, JAM-A-transfected cells migrated to fill about three-quarters of the wounded area as compared with mock-transfected cells, which showed very little migration. Because the assay was performed in the absence of growth factors or serum, it is apparent that the filling was caused by cell migration and not simply as a result of proliferation of cells at the wound edge. These data preliminarily suggested that overexpression of JAM-A might induce cell migration.
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JAM-A-induced migration is specific to vitronectin
To analyze the above observation more thoroughly, we next asked whether this migration was ECM specific using a more quantitative haptotactic transwell motility assay. We found that JAM-A induced increased migration on vitronectin as expected (Fig. 2A). In fact, quantitation of these data revealed that, as compared with mock-transfected cell migration, a two- to fivefold increase in migration of JAM-A-overexpressing cells occurred on vitronectin (Fig. 2B,C). In examining migration on other ECM proteins, we found less than a twofold increase in JAM-A-induced migration occurred on gelatin, and no significant increase in migration occurred on collagen (Fig. 2B,C). Although we observed maximum migration on fibronectin, no significant difference was observed between the two populations (Fig. 2B,C). These data thus conclude that JAM-A-induced cell migration is vitronectin specific.
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JAM-A-induced migration on vitronectin is integrin vß3 specific
Two integrin receptors for vitronectin are expressed on endothelial cells: integrin vß3 and integrin vß5. To determine which of these is involved in JAM-A-induced motility, we analyzed HUVEC migration in the presence of specific function-blocking antibodies of these integrins. Both endogenous and JAM-A-induced cell motility on vitronectin were dose-dependently and significantly inhibited by anti-vß3 antibody (Fig. 4A, *P<0.05). As expected, anti-vß3 had no effect on HUVEC migration on fibronectin (data not shown). By contrast, when anti-vß5 antibody was used, it only modestly inhibited endogenous migration, but did not affect JAM-A-enhanced migration at any antibody concentration tested (Fig. 4B). In addition, XT199, a specific small molecule antagonist of integrin vß3 (Mousa, 1999), dose-dependently and completely inhibited JAM-A-induced motility on vitronectin (Fig. 4C) but had no effect on JAM-A-induced migration on fibronectin even when used at 100 µM concentration (Fig. 4D). These results indicate that JAM-A-induced cell motility on vitronectin is integrin vß3 specific.
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JAM-A interacts with integrin vß3
JAM-A has previously been shown to interact with integrin Lß2 (Ostermann et al., 2002). Because JAM-A-induced migration on vitronectin is vß3 specific, we reasoned that integrin vß3 might also associate with JAM-A. To investigate whether JAM-A interacts with integrin vß3, coimmunoprecipitation experiments were performed. We found that JAM-A indeed coimmunoprecipitated with vß3, indicating a physical interaction (Fig. 5A). Furthermore, when cells were incubated with RGDS peptide, thus engaging the ligand-binding site of vß3, the amount of JAM-A bound to the integrin was increased (Fig. 5A). These data indicate that, when its ligand-binding site is engaged, vß3 associates with JAM-A.
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vß3 with JAM-A. We theorized that, if RGD-bound vß3 promotes association with JAM-A, then antagonist-bound vß3 should accumulate at the cell-cell junction of live cells, where JAM-A predominantly resides. To investigate this, we treated live HUVECs with a FITC-labeled RGD-based antagonist of vß3, XT199, and monitored FITC-XT199 accumulation in real-time by confocal microscopy (Fig. 5B). We observed that FITC-labeled XT199 specifically accumulated in a time-dependent manner at the cell-cell junctions, reaching maximum within 20 minutes of incubation (Fig. 5Bi-iv). Molar excess of RGDS peptide ablated FITC-XT199 accumulation due to competitive inhibition, indicating that it is integrin specific (Fig. 5Bv). To investigate whether the accumulated XT199 is in fact a result of the accumulation of integrin vß3, we pre-treated the live HUVECs with RGDS peptide for 20 minutes to allow its accumulation at the junctions. These cells were then fixed and stained with anti-vß3 antibody. We found that integrin vß3 was concentrated at cell-cell junctions (Fig. 5Bvi), confirming that the above-observed XT199 accumulation was indeed vß3 specific. These data suggest that engagement of the vß3 ligand-binding site by RGD-based ligand-mimetic XT199 causes vß3 to associate with JAM-A at the cell-cell junctions.
Signaling through JAM-A, phosphoinositide 3-kinase and protein kinase C is required for JAM-A-induced migration
JAM-A is a cell adhesion molecule primarily localized at the tight junction. Its role in cell signaling remains unexplored. It is known that the cytoplasmic domain of JAM-A contains several phosphorylation sites as well as a motif that binds the PDZ (for `postsynaptic density-95, discs large, ZO-1') domain (Bazzoni et al., 2000b; Naik et al., 1995; Naik et al., 2001; Ozaki et al., 2000). However, the functional significance of these sites is not known. To investigate whether the cytoplasmic domain of JAM-A plays any role in JAM-A-induced cell migration, we generated a cytoplasmic domain deletion mutant (
-257). Overexpression of this mutant in HUVECs to similar levels as that of wild-type JAM-A, but several-fold higher than the endogenous JAM-A, was confirmed by western blot analysis (Fig. 6A). As shown in Fig. 6A, the deletion of the cytoplasmic domain decreased the size of JAM-A. We reasoned that, if signaling through the cytoplasmic domain is required, then this mutant would fail to induce HUVEC migration. As expected, overexpression of the -257 mutant failed to induce HUVEC motility, indicating that signaling through JAM-A is necessary for this event (Fig. 6B). These results establish a functional significance to transmembrane signaling through JAM-A during HUVEC migration on vitronectin. In order to obtain insight into the mechanism and significance of the association of JAM-A with integrin vß3, we overexpressed -257 in HUVECs and asked whether the mutant would interact with integrin vß3. We were unable to coimmunoprecipitate JAM-A -257 with vß3 (Fig. 6C). These results suggest that the cytoplasmic domain of JAM-A might be important for its association with vß3 and thus intracellular signaling. In addition, pre-treatment of cells with anti-JAM-A function-blocking antibody dose-dependently and significantly inhibited HUVEC motility on vitronectin caused by JAM-A overexpression (Fig. 6D, *P<0.05). However, addition of this antibody had no effect on HUVEC migration on fibronectin (data not shown). These data indicate that the engagement of the extracellular domain of JAM-A is also necessary for this event.
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It is well known that signaling molecules such as phosphoinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC) play an important role in cell migration. It is therefore possible that JAM-A-induced cell migration also requires signaling through these molecules. In order to explore this possibility, we used pharmacological inhibitors of PI 3-kinase and PKC. Using mock- and JAM-A-transfected HUVECs, a migration assay was performed on vitronectin in the presence or absence of wortmannin, PI 3-kinase and bisindolymaleimide 1 (Bis), a PKC inhibitor, with DMSO used as a vehicle (Fig. 6E). We found DMSO had no effect on JAM-A-induced cell migration, but was inhibited by wortmannin and Bis, thus suggesting that JAM-A-induced cell migration requires signaling through PI 3-kinase and PKC. Together, these results establish a functional link between signaling through JAM-A, PI 3-kinase and PKC during HUVEC migration on vitronectin through vß3.
JAM-A regulates vß3-dependent MAPK activation
It is well documented that activation of vß3 leads to MAPK activation and endothelial cell migration (Camenisch et al., 2002; Degryse et al., 2001; Sajid et al., 2002). Because JAM-A overexpression leads to HUVEC migration, we thereby reasoned that JAM-A might also be involved in MAPK activation. We therefore investigated the effect of JAM-A overexpression on vitronectin-induced MAPK activation, and found that ERK1/2 activity (P-ERK1/2) was substantially enhanced in JAM-A-overexpressing cells adhered to vitronectin as compared with mock-transfected cells (Fig. 7A). This JAM-A-enhanced ERK1/2 phosphorylation was sustained for up to 60 minutes of adhesion (Fig. 7A). Densitometric analysis of normalized data from three separate experiments indicated that, at every time point tested, JAM-A expression enhanced ERK activation (Fig. 7B). Taken together, the data presented here suggest that signaling through JAM-A results in MAPK activation leading to vß3-dependent endothelial cell migration on vitronectin.
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Because -257 failed to induce HUVEC migration, we asked whether this mutant also fails to activate MAPK. We found that wild-type JAM-A was able to stimulate ERK1/2 activation as expected, but that -257 failed to induce their activation (Fig. 7C). Densitometric quantitation of MAPK activation further suggested that expression of -257 reduces the endogenous activation of MAPK (Fig. 7D). These results indicate that signaling through JAM-A appears to be important for the process of HUVEC migration.
FAK is known to be an important intracellular signaling molecule that regulates cell adhesion and migration. It is known that phosphorylation of Tyr397 of FAK results in its activation. We therefore reasoned that if overexpression of JAM-A, but not its mutant, results in cell migration then it should parallel FAK activation. We found that significant activation of FAK occurred in mock-transfected cells within 10 minutes of attachment to vitronectin (Fig. 8A,B). JAM-A-overexpressing cells showed a robust increase in FAK phosphorylation over the mock-transfected cells. By contrast, -257-overexpressing cells failed to show this increase of FAK phosphorylation over mock-transfected cells (Fig. 8A,B). These results suggest that JAM-A, a cell adhesion molecule that normally resides at the tight junction, is capable of inducing intracellular signaling that regulates cell migration.
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vß3, but is independent of vß5, as indicated by the use of function-blocking antibodies. Immunoprecipitation experiments indicate that JAM-A associates with integrin vß3, and this association is enhanced upon engagement of the ligand-binding site. In addition, our studies indicate that overexpression of JAM-A enhances vitronectin-dependent MAPK activation. These results thus support and provide a mechanistic insight to our previous finding that JAM-A is a key regulator of bFGF-induced angiogenesis (Naik et al., 2003a).
JAM-A is endogenously expressed on epithelial and endothelial cells, and predominantly localizes to the tight junctions of these cells. The extracellular domain of JAM-A is known to be involved in homotypic interactions at these junctions (Bazzoni et al., 2000a; Bazzoni et al., 2000b; Kostrewa et al., 2001; Naik et al., 2001) and, through its PDZ-domain-binding motif, the JAM-A cytoplasmic domain is involved in the recruitment of, and association with, PDZ-domain-containing proteins such as ZO-1, AF-6 and PAR3 (Bazzoni et al., 2000b; Ebnet et al., 2000; Itoh et al., 2001). On the basis of this knowledge, the role of JAM-A in these cells was believed to be confined to the formation of tight junctions and the regulation of their integrity. Along these lines, it was therefore presumed that overexpression of JAM-A would enhance tight junction formation. Surprisingly, we found that, when overexpressed, JAM-A localization is no longer limited to tight junctions, and is instead uniformly distributed along the cell membrane. When plated on vitronectin, these cells exhibit long cytoplasmic extensions, which are reminiscent of filopodia of migratory cells. In studying the ability of these cells to migrate on vitronectin, our data indicated that this overexpression in fact promotes HUVEC migration, an observation that was counterintuitive to our original hypothesis that overexpression would enhance cell-cell contacts and thus inhibit migration. Though surprising, these data are consistent with the finding that activation of endothelial cells with inflammatory cytokines such as TNF- and interferon-
(IFN-) redistributes JAM-A from the tight junctions to areas all along the cell membrane (Ostermann et al., 2002; Ozaki et al., 1999; Shaw et al., 2001). In addition, it has also been shown in a variety of angiogenic models that TNF- induces angiogenesis (Koolwijk et al., 1996; Niedbala and Stein, 1991), a process that requires cell migration. It is therefore possible that JAM-A involvement in cell migration is physiological such that, in quiescent endothelial cells, JAM-A is sequestered at the cell-cell junction. When released and redistributed along the cell membrane, either upon cytokine activation or upon overexpression (as we find), JAM-A is then free to participate in the adhesion and de-adhesion events required for cell migration. It is important to mention that overexpression of JAM-A as seen in Fig. 3, or as previously reported following knockdown of endogenous JAM-A (Naik et al., 2003b), both slightly reduce cell adhesion to vitronectin, suggesting that surface expression of JAM-A regulates the adhesive function of these cells. In regard to this, it has recently been shown that knockdown of JAM-A results in reduced activity of the small GTPase Rap1 and thus affects epithelial cell morphology and ß1 integrin expression (Mandell et al., 2005). By contrast, endothelial cells from a JAM-A-null mouse show enhanced spontaneous and random motility through activation of glycogen synthase kinase-3ß (Bazzoni et al., 2005). The later experiments were performed using fibronectin as a substrate, which is consistent with our finding that overexpression of JAM-A has no effect on HUVEC motility on fibronectin.
We have previously reported that the cytoplasmic domain of JAM-A contains several tyrosine and serine residues that are capable of being phosphorylated (Naik et al., 1995; Naik et al., 2001). It has been shown that JAM-A is phosphoryated by PKC on Ser284 upon activation of platelets by agonists (Naik et al., 1995; Ozaki et al., 2000). This is further supported by our mutational data in which the cytoplasmic domain deletion mutant of JAM-A failed to initiate either morphological changes or migration on vitronectin. These findings thus suggest that the JAM-A cytoplasmic domain is involved in the signaling cascade required for these events. Recently, JAM-A has been shown to bind integrin Lß2 in a heterotypic manner, and play a role in leukocyte transmigration (Ostermann et al., 2002). Our results show that JAM-A interacts with integrin vß3 and induces vß3-dependent HUVEC migration on vitronectin. Integrin vß3 has been previously shown to interact with several transmembrane and membrane-associated proteins, and these interactions are important for its function (Barazi et al., 2002; Chellaiah and Hruska, 2003; Hapke et al., 2001; Silvestri et al., 2002; Voura et al., 2001). We find that the interaction of JAM-A with integrin vß3 seems to be enhanced upon engagement of its ligand-binding site, as shown by the increased association seen during immunoprecipitation in the presence of RGDS peptide. RGDS peptides bind to the ligand-binding pocket (Hantgan et al., 1999; Xiong et al., 2002), and may mimic ligand binding, thus inducing the same conformational changes and signaling events as would the ligand. However, it is also possible that a monovalent ligand such as RGDS will not be able to induce the same integrin avidity that would occur upon binding of a multivalent ligand such as vitronectin. Increased integrin avidity has been shown to be key to propagation of signaling events leading to cell adhesion and migration (Stewart and Hogg, 1996; van Kooyk and Figdor, 2000) and, hence, binding of RGDS might in fact inhibit such increased avidity and the subsequent signaling events occurring as a result. In this regard, we can interpret our results as follows: if RGDS mimics ligand binding and induces changes in integrin conformation, enhancement of the vß3–JAM-A interaction by RGDS suggests that interaction with JAM-A might enhance vß3 outside-in signaling. However, if RGDS binding inhibits the integrin avidity required for these events, association of JAM-A and vß3 might be inhibitory to subsequent signaling events. By these means, a second possibility might be that uncomplexed JAM-A and vß3 is more favorable for the enhanced signaling that leads to cell migration. Although our data favors the second possibility, further experimentation as to whether this complex is inhibitory or advantageous to propagation of these signals is ongoing.
Integrin vß3 has also been implicated in tumor-induced angiogenesis (Brooks et al., 1994; Friedlander et al., 1995). In fact, the integrin family members most widely studied in the angiogenic process are the vitronectin receptors vß3 and vß5 (Brooks et al., 1994; Drake et al., 1995; Eliceiri and Cheresh, 2000; Friedlander et al., 1995). It is known that these two integrins regulate two distinct pathways of angiogenesis (Friedlander et al., 1995). Angiogenesis initiated by vascular endothelial growth factor (VEGF), transforming growth factor- (TGF-) or phorbol ester is dependent upon integrin vß5 (Friedlander et al., 1995), whereas bFGF and TNF- signal through vß3 (Brooks et al., 1994; Friedlander et al., 1995). In addition, expression of vß3 is upregulated on endothelial cells involved in new blood vessel formation, and is required for sustained MAPK activity leading to neovascularization induced by bFGF and TNF- (Eliceiri et al., 1998). We have recently shown that JAM-A is an important regulator of bFGF-induced angiogenesis (Naik et al., 2003a). In light of our findings, it is also possible that JAM-A will play an important role in bFGF- or TNF--induced activation of vß3. Just as in bFGF-induced signaling (Carron et al., 1998; Pintucci et al., 2002), our results indicate that JAM-A-induced HUVEC migration is vß3 specific but not vß5 specific (Naik et al., 2003a). In addition, activation of endothelial cells with TNF- or bFGF redistributes JAM-A along the cell surface (Naik et al., 2003a; Ozaki et al., 1999), just as we find after JAM-A overexpression in these cells. In this manner, JAM-A might in fact regulate bFGF- or TNF--induced activation of vß3, leading to endothelial cell migration and eventually angiogenesis.
Upon engagement of the integrin vß3 ligand-binding site by vitronectin, a signaling cascade that leads to MAPK activation ensues, which is necessary for cell migration (Eliceiri et al., 1998; Tanaka et al., 1999). Our data suggest that overexpression of JAM-A enhances sustained MAPK activation on vitronectin. It is therefore possible that interaction of JAM-A with vß3 regulates signaling events that lead to MAPK activation. Along the same lines as migration, bFGF-induced MAPK activation also signals through vß3 (Eliceiri et al., 1998; Tanaka et al., 1999), and this MAPK activation is JAM-A dependent (Naik et al., 2003a). Thus, JAM-A and vß3 might be intermediate in the bFGF-induced pathway leading to MAPK activation. This is further supported by our finding that FAK, PI 3-kinase and PKC are intermediates in the signaling pathway induced by JAM-A that leads to cell migration.
In summary, results from this study provide evidence for the involvement of JAM-A in endothelial cell migration through integrin vß3. These data implicate JAM-A, a tight junction protein, in transmembrane signaling, as well as in the regulation of vascular function, thereby adding novel functions to the JAM-A repertoire. Ongoing efforts aimed at knockout of JAM-A are expected to cast light on the molecular relationship between JAM-A and vß3 so that their roles in endothelial cell function and angiogenesis can be more clearly defined.
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). HUVECs were transfected using SuperFect reagent (Qiagen). Transfectants were selected in 500 µg/ml G418-containing growth medium, and owing to a limited passage number, pools of stably transfected cells between passages 3 and 7 were used instead of a clonal population. Several clones were selected, isolated and further maintained in 300 µg/ml G418-containing growth medium. CHO cells stably expressing HA-tagged human JAM-A were described previously (Naik et al., 2001). The level of protein expression was determined by western blotting as described (Naik et al., 2001). Densitometric analysis of band intensity was quantified using a BioRad Gel Doc 2000.
Antibodies and ECM reagents
Monoclonal antibody (mAb) J3F.1 (anti-JAM-A antibody) was a gift from Charles Parkos (Emory University, Atlanta, GA). Additional anti-JAM-A antibodies were purchased from BD Bioscience. LM609 (anti-vß3 antibody), P1F6 (anti-vß5 antibody) and anti-ß3 antibody were purchased from Chemicon. Phosphospecific FAK (p397) antibody was purchased from BioSource. Isotype-matched control antibody HB67 (cIgG) was obtained from the American Type Culture Company. XT199, a ligand-mimetic antagonist of integrin vß3 were a generous gift from Shaker Mousa (Bishop et al., 2001). Wortmannin, a PI 3-kinase inhibitor, and bisindolymaleimide 1 (Bis), a PKC inhibitor, were purchased from Calbiochem. Rabbit polyclonal antibodies against ERK1/2 and phospho-ERK1/2 were purchased from New England Biolabs. Rat-tail collagen type I and gelatin were purchased from Sigma. Human vitronectin and fibronectin were obtained from Collaborative Biomedical Research. Polyclonal antibody against FAK was obtained from Santa Cruz Biotechnology.
Immunofluorescence
Immunofluorescence studies were performed as previously described (Naik et al., 2001). Briefly, live adherent HUVECs were allowed to spread on 8-chambered glass coverslides (Nalge Nunc International) either uncoated or coated overnight with indicated ECM. Cells were incubated for various time intervals with 100 nM FITC-labeled XT199 compound alone, or in combination with 1 mM Arg-Gly-Asp-Ser (RGDS), and then observed live using confocal microscopy. In another set of experiments, live cells were pre-treated for 40 minutes with 100 µM RGDS at 37°C, and then fixed with freshly prepared 4% paraformaldehyde in phosphate buffer saline (PBS). Following fixation, cells were permeabilized with 0.2% Triton X-100 for 5 minutes, then rinsed and blocked with 3% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Cells were incubated with indicated primary antibody overnight at 4°C, and then followed with appropriate fluorescently labeled secondary antibody. Slowfade (Molecular Probes) was added to prevent quenching. In order to highlight cell-cell junctions, horizontal optical sections were taken through the middle of the cells using a Zeiss LSM510 laser-scanning microscope.
Cell migration assays
A wound-induced migration assay (scratch assay) was performed as described previously (Angers-Loustau et al., 1999). Briefly, mock- and JAM-A-transfected HUVECs were plated to form a monolayer. Using a sterile yellow tip, a scratch was made in triplicate. Detached cells were removed, and the scratches were monitored for 24 hours under serum-free conditions. Photographs were taken at the time of the wound (0 hours) and after 24 hours. In addition, a more quantitative haptotactic migration assay was performed essentially as described previously (Keely et al., 1995). Briefly, transwell inserts (8 µm pore size) in triplicates were coated on the underside with gelatin (1%), collagen I (30 µg/ml), vitronectin (10 µg/ml) or fibronectin (10 µg/ml) as indicated. In certain experiments, cells in suspension were pre-treated with specific antibodies or inhibitors as indicated for 15 minutes before migration. Cells were allowed to migrate across inserts for 5 hours at 37°C. Unmigrated cells from the upper chamber were removed, and migrated cells in the lower side of the membrane were fixed, stained and dried. The average number of migrated cells in ten randomly chosen fields of view per insert was taken to quantify the extent of migration. In addition, each set of experiments was performed in triplicate. Statistical analysis of the data was performed using the paired Student's t-test.
Cell adhesion assay
Cell adhesion assay was performed as described (Ponce et al., 2001). HUVECs stably transfected with JAM-A or empty vector were serum starved. 50,000 cells in 100 µl per well were added to a vitronectin pre-coated plate and allowed to adhere for 1 hour at 37°C. Unbound cells were washed, and the attached cells were fixed and stained with 0.2% Crystal Violet. After extensively washing with distilled water, bound dye was lysed with 2% sodium dodecyl sulfate (SDS) and quantified in an ELISA Plate reader at 600 nm.
In vitro binding assay
An in vitro binding assay was performed as described previously (Naik et al., 1997). Briefly, microtiter wells were coated with various concentrations (1-10 µg/ml) of vitronectin, recombinant JAM-A or BSA. After blocking with 1% BSA, JAM-A protein was added to wells pre-coated with BSA and/or vitronectin and incubated for 1 hour. Wells were washed four times with PBS. Wells containing recombinant JAM-A, vitronectin and BSA were incubated with anti-JAM-A antibody overnight at 4°C. The amount of JAM-A bound was determined using anti-mouse secondary antibody conjugated with alkaline phosphatase, and quantified in an ELISA Plate reader at 600 nm.
MAPK activation
MAPK activation was assessed essentially as described (Aplin et al., 1999). In brief, 35 mm petri dishes were coated with 10 µg/ml vitronectin in PBS overnight at 4°C. Mock-transfected cells and cells overexpressing either JAM-A or the JAM-A cytoplasmic domain deletion mutant -257 were serum starved overnight. Cells at a density of 3x105 cells/ml were kept in suspension on a rotator at 37°C for 45 minutes, and then plated on vitronectin-coated dishes, as indicated. Laemmli sample buffer was then added immediately, and proteins (50 µg) from total cell lysates were separated by 10% SDS-PAGE gel electrophoresis, then immunoblotted as described (Naik et al., 2001), and probed with anti-phospho-ERK1/2. Blots were then reprobed with anti-ERK1/2 antibodies to show total ERK1/2. Band intensity was quantified using BioRad Gel Doc 2000 software.
FAK activation
Lysates were prepared from serum-starved mock-transfected HUVECs and HUVECs overexpressing either JAM-A or the JAM-A cytoplasmic deletion mutant, as described previously (Naik and Naik, 2003). Briefly, cells (3x105) were plated on vitronectin (10 µg/ml) pre-coated dishes for various time points at 37°C. Lysates (50 µg) were then subjected to western blotting and the relative levels of FAK phosphorylated at Tyr397 were determined. The blots were then further reprobed for total FAK. Band intensity was quantified using BioRad Gel Doc 2000 software. Each set of experiments was performed more than three times. Statistical analysis of the data was performed using the paired Student's t-test.
Immunoprecipitation
Serum-starved JAM-A- and/or -257-overexpressing cells (3x106) were lysed with lysis buffer [1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl pH 7.5, 10 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM phenylmethanesulfonyl fluoride (PMSF) and 10 mM NaF] for 30 minutes on ice, and then centrifuged at 4°C. Where indicated, cells were pre-treated with 100 µg/ml RGDS peptide for 20 minutes at 37°C and then lysed as above. Lysates (500 µg/ml) were immunoprecipitated with anti-ß3 antibody or HB67 (cIgG) along with protein G-Sepharose beads (Amersham Biosciences). Immunocomplex-captured beads were washed with lysis buffer without inhibitors, and boiled in Laemmli sample buffer. The proteins were separated by 10% SDS-PAGE and western blotted using anti-JAM-A antibody and the corresponding blots were reprobed with anti-ß3 antibody as described previously (Naik et al., 2001).
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Acknowledgments |
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-257 construct; and F. Chowdhary and K. Maddox for their technical support. This work was supported by grants (to U.P.N.) from the American Heart Association Pennsylvania and Delaware Affiliate (9906203U), and the National Institutes of Health (HL63960). |
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