Ionizing radiation increases adhesiveness of human aortic endothelial cells via a chemokine-dependent mechanism

Exposure to radiation from a variety of sources is associated with increased risk of heart disease and stroke. Since radiation also induces inflammation, a possible mechanism is a change in the adhesiveness of vascular endothelial cells, triggering pro-atherogenic accumulation of leukocytes. To investigate this mechanism at the cellular level, the effect of X rays on adhesiveness of cultured human aortic endothelial cells (HAECs) was determined. HAECs were grown as monolayers and exposed to 0 to 30 Gy X rays, followed by measurement of adhesiveness under physiological shear stress using a flow chamber adhesion assay. Twenty-four hours after irradiation, HAEC adhesiveness was increased, with a peak effect at 15 Gy. Radiation had no significant effect on surface expression of the endothelial adhesion molecules ICAM-1 and VCAM-1. Antibody blockade of the leukocyte integrin receptors for ICAM-1 and VCAM-1, however, abolished the radiation-induced adhesiveness. Since these leukocyte integrins can be activated by chemokines presented on the endothelial cell surface, the effect of pertussis toxin (PTX), an inhibitor of chemokine-mediated integrin activation, was tested. PTX specifically inhibited radiation-induced adhesiveness, with no significant effect on nonirradiated cells. Therefore, radiation induces increased adhesiveness of aortic endothelial cells through chemokine-dependent signaling from endothelial cells to leukocytes, even in the absence of increased expression of the adhesion molecules involved.     

N-glycosylation controls the function of junctional adhesion molecule-A

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells. JAM-A serves many roles and contributes to barrier function and cell migration and motility, and it also acts as a ligand for the leukocyte receptor LFA-1. JAM-A is reported to contain N-glycans, but the extent of this modification and its contribution to the protein’s functions are unknown. We show that human JAM-A contains a single N-glycan at N185 and that this residue is conserved across multiple mammalian species. A glycomutant lacking all N-glycans, N185Q, is able to reach the cell surface but exhibits decreased protein half-life compared with the wild- type protein. N-glycosylation of JAM-A is required for the protein’s ability to reinforce barrier function and contributes to Rap1 activity. We further show that glycosylation of N185 is required for JAM-A–mediated reduction of cell migration. Finally, we show that N-glycosylation of JAM-A regulates leukocyte adhesion and LFA-1 binding. These findings identify N-glycosylation as critical for JAM-A’s many functions.     

Effect of shear stress on migration and integrin expression in macaque trophoblast cells

During fetal development, trophoblast cells enter endometrial capillaries, migrate within the uterine vasculature, and eventually reside within spiral arteries of the uterus. This invasive activity is accompanied by upregulation of trophoblast beta1 integrin expression. Fluid mechanical shear stress regulates migration and expression of adhesion molecules in vascular endothelial cells, but nothing is known about the effects of shear stress on trophoblast cells. We tested the hypothesis that shear stress regulates the motility and beta1 integrin expression of trophoblast cells. Early gestation macaque trophoblast cells were cultured in 1 x 1-mm square cross-section capillary tubes within which the flow field was determined using three-dimensional computational fluid dynamic simulations. Trophoblast cells in the capillary tubes were exposed to a steady shear stress of 7.5, 15, or 30 dyn/cm2 for up to 24 h. In the absence of flow, trophoblast cells were highly dynamic with constant nondirectional positional shifts but with no net cell migration. Exposure of the cells to shear stress within 24-72 h of cell plating significantly increased the level of this activity and led to net cell migration in the direction of flow. Shear stress also increased the expression and altered the topography of beta1 integrin. These results suggest that shear stress regulates trophoblast motility and beta1 integrin expression in vitro.     

Putting the brakes on cancer cell migration

Junctional Adhesion Molecule A (JAM-A) is a member of the Ig superfamily of membrane proteins expressed in platelets, leukocytes, endothelial cells and epithelial cells. We have previously shown that in endothelial cells, JAM-A regulates basic fibroblast growth factor, (FGF-2)-induced angiogenesis via augmenting endothelial cell migration. Recently, we have revealed that in breast cancer cells, down-regulation of JAM-A enhances cancer cell migration and invasion. Further, ectopic expression of JAM-A in highly metastatic MDA-MB-231 cells attenuates cell migration, and down-regulation of JAM-A in low-metastatic T47D cells enhance migration. Interestingly, JAM-A expression is greatly diminished as breast cancer disease progresses. The molecular mechanism of this function of JAM-A is beyond its well-characterized barrier function at the tight junction. Our results point out that JAM-A differentially regulates migration of endothelial and cancer cells.     

Interplay between shear stress and adhesion on neutrophil locomotion

Leukocyte locomotion over the lumen of inflamed endothelial cells is a critical step, following firm adhesion, in the inflammatory response. Once firmly adherent, the cell will spread and will either undergo diapedesis through individual vascular endothelial cells or will migrate to tight junctions before extravasating to the site of injury or infection. Little is known about the mechanisms of neutrophil spreading or locomotion, or how motility is affected by the physical environment. We performed a systematic study to investigate the effect of the type of adhesive ligand and shear stress on neutrophil motility by employing a parallel-plate flow chamber with reconstituted protein surfaces of E-selectin, E-selectin/PECAM-1, and E-selectin/ICAM-1. We find that the level and type of adhesive ligand and the shear rate are intertwined in affecting several metrics of migration, such as the migration velocity, random motility, index of migration, and the percentage of cells moving in the direction of flow. On surfaces with high levels of PECAM-1, there is a near doubling in random motility at a shear rate of 180 s(-1) compared to the motility in the absence of flow. On surfaces with ICAM-1, neutrophil random motility exhibits a weaker response to shear rate, decreasing slightly when shear rate is increased from static conditions to 180 s(-1), and is only slightly higher at 1000 s(-1) than in the absence of flow. The random motility increases with increasing surface concentrations of E-selectin and PECAM-1 under static and flow conditions. Our findings illustrate that the endothelium may regulate neutrophil migration in postcapillary venules through the presentation of various adhesion ligands at sites of inflammation.     

Junctional Adhesion Molecule A Interacts with Afadin and PDZ-GEF2 to Activate Rap1A, Regulate β1 Integrin Levels, and Enhance Cell Migration

Junctional adhesion molecule-A (JAM-A) is a transmembrane tight junction protein that has been shown to regulate barrier function and cell migration through incompletely understood mechanisms. We have previously demonstrated that JAM-A regulates cell migration by dimerization of the membrane-distal immunoglobulin-like loop and a C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding motif. Disruption of dimerization resulted in decreased epithelial cell migration secondary to diminished levels of β1 integrin and active Rap1. Here, we report that JAM-A is physically and functionally associated with the PDZ domain-containing molecules Afadin and PDZ-guanine nucleotide exchange factor (GEF) 2, but not zonula occludens (ZO)-1, in epithelial cells, and these interactions mediate outside-in signaling events. Both Afadin and PDZ-GEF2 colocalized and coimmunoprecipitated with JAM-A. Furthermore, association of PDZ-GEF2 with Afadin was dependent on the expression of JAM-A. Loss of JAM-A, Afadin, or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreased cellular levels of activated Rap1, β1 integrin protein, and epithelial cell migration. The functional effects observed were secondary to decreased levels of Rap1A because knockdown of Rap1A, but not Rap1B, resulted in decreased β1 integrin levels and reduced cell migration. These findings suggest that JAM-A dimerization facilitates formation of a complex with Afadin and PDZ-GEF2 that activates Rap1A, which regulates β1 integrin levels and cell migration.     

Mapping the dynamics of shear stress-induced structural changes in endothelial cells

Hemodynamic shear stress regulates endothelial cell biochemical processes that govern cytoskeletal contractility, focal adhesion dynamics, and extracellular matrix (ECM) assembly. Since shear stress causes rapid strain focusing at discrete locations in the cytoskeleton, we hypothesized that shear stress coordinately alters structural dynamics in the cytoskeleton, focal adhesion sites, and ECM on a time scale of minutes. Using multiwavelength four-dimensional fluorescence microscopy, we measured the displacement of rhodamine-fibronectin and green fluorescent protein-labeled actin, vimentin, paxillin, and/or vinculin in aortic endothelial cells before and after onset of steady unidirectional shear stress. In the cytoskeleton, the onset of shear stress increased actin polymerization into lamellipodia, altered the angle of lateral displacement of actin stress fibers and vimentin filaments, and decreased centripetal remodeling of actin stress fibers in subconfluent and confluent cell layers. Shear stress induced the formation of new focal complexes and reduced the centripetal remodeling of focal adhesions in regions of new actin polymerization. The structural dynamics of focal adhesions and the fibronectin matrix varied with cell density. In subconfluent cell layers, shear stress onset decreased the displacement of focal adhesions and fibronectin fibrils. In confluent monolayers, the direction of fibronectin and focal adhesion displacement shifted significantly toward the downstream direction within 1 min after onset of shear stress. These spatially coordinated rapid changes in the structural dynamics of cytoskeleton, focal adhesions, and ECM are consistent with focusing of mechanical stress and/or strain near major sites of shear stress-mediated mechanotransduction.     

Abrogation of junctional adhesion molecule-A expression induces cell apoptosis and reduces breast cancer progression

Intercellular junctions promote homotypic cell to cell adhesion and transfer intracellular signals which control cell growth and apoptosis. Junctional adhesion molecule-A (JAM-A) is a transmembrane immunoglobulin located at tight junctions of normal epithelial cells of mammary ducts and glands. In the present paper we show that JAM-A acts as a survival factor for mammary carcinoma cells. JAM-A null mice expressing Polyoma Middle T under MMTV promoter develop significantly smaller mammary tumors than JAM-A positive mice. Angiogenesis and inflammatory or immune infiltrate were not statistically modified in absence of JAM-A but tumor cell apoptosis was significantly increased. Tumor cells isolated from JAM-A null mice or 4T1 cells incubated with JAM-A blocking antibodies showed reduced growth and increased apoptosis which paralleled altered junctional architecture and adhesive function. In a breast cancer clinical data set, tissue microarray data show that JAM-A expression correlates with poor prognosis. Gene expression analysis of mouse tumor samples showed a correlation between genes enriched in human G3 tumors and genes over expressed in JAM-A +/+ mammary tumors. Conversely, genes enriched in G1 human tumors correlate with genes overexpressed in JAM-A-/- tumors. We conclude that down regulation of JAM-A reduces tumor aggressive behavior by increasing cell susceptibility to apoptosis. JAM-A may be considered a negative prognostic factor and a potential therapeutic target.     

Induction of JAM-A during differentiation of human THP-1 dendritic cells

Junctional adhesion molecule (JAM)-A is not only localized at tight junctions of endothelial and epithelial cells but is also expressed on circulating leukocytes and dendritic cells (DCs). In the present study, to investigate the regulation of JAM-A in DCs, mature DCs were differentiated from the human monocytic cell THP-1 by treatment with IL-4, GM-CSF, TNF-α, and ionomycin, and some cells were pretreated with the PPAR-γ agonists. In the THP-1 monocytes, mRNAs of tight junction molecules, occludin, tricellulin, JAM-A, ZO-1, ZO-2 and claudin-4, -7, -8, and -9 were detected by RT-PCR. In mature DCs that had elongated dendrites, mRNA and protein of JAM-A were significantly increased compared to the monocytes. PPAR-γ agonists prevented the elongation of dentrites but not upregulation of JAM-A in mature DCs. These findings indicated that the induction of JAM-A occurred during differentiation of human THP-1 DCs and was independent of PPAR-γ and the p38 MAPK pathway.     

Deletion of JAM-A causes morphological defects in the corneal epithelium

Junctional adhesion molecule-A (JAM-A, JAM-1, F11R) is an Ig domain containing transmembrane protein that has been proposed to function in diverse processes including platelet activation and adhesion, leukocyte transmigration, angiogenesis, epithelial cell shape and endothelial cell migration although its function in vivo is less well established. In the mouse eye, JAM-A protein expression is first detected at 12.5 dpc in the blood vessels of the tunica vasculosa, while it is first detected in both the corneal epithelium and lens between 13.5 and 14.5 dpc. In the corneal epithelium, JAM-A levels remain appreciable throughout life, while JAM-A immunostaining becomes stronger in the lens as the animals age. Both the cornea and lens of mice lacking an intact JAM-A gene are transparent until at least a year of age, although the cells of the JAM-A null corneal epithelium are irregularly shaped. In wild-type mice, JAM-A protein is found at the leading edge of repairing corneal epithelial wounds, however, corneal epithelial wound repair was qualitatively normal in JAM-A null animals. In summary, JAM-A is expressed in the corneal epithelium where it appears to regulate cell shape.     

A comparative molecular force spectroscopy study of homophilic JAM-A interactions and JAM-A interactions with reovirus attachment protein sigma1

JAM-A belongs to a family of immunoglobulin-like proteins called junctional adhesion molecules (JAMs) that localize at epithelial and endothelial intercellular tight junctions. JAM-A is also expressed on dendritic cells, neutrophils, and platelets. Homophilic JAM-A interactions play an important role in regulating paracellular permeability and leukocyte transmigration across epithelial monolayers and endothelial cell junctions, respectively. In addition, JAM-A is a receptor for the reovirus attachment protein, sigma1. In this study, we used single molecular force spectroscopy to compare the kinetics of JAM-A interactions with itself and sigma1. A chimeric murine JAM-A/Fc fusion protein and the purified sigma1 head domain were used to probe murine L929 cells, which express JAM-A and are susceptible to reovirus infection. The bond half-life (t(1/2)) of homophilic JAM-A interactions was found to be shorter (k(off)(o) = 0.688 +/- 0.349 s(-1)) than that of sigma1/JAM-A interactions (k(off)(o) = 0.067 +/- 0.041 s(-1)). These results are in accordance with the physiological functions of JAM-A and sigma1. A short bond lifetime imparts a highly dynamic nature to homophilic JAM-A interactions for regulating tight junction permeability while stable interactions between sigma1 and JAM-A likely anchor the virus to the cell surface and facilitate viral entry.     

Tetraspanin CD9 links junctional adhesion molecule-A to αvβ3 integrin to mediate basic fibroblast growth factor–specific angiogenic signaling

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin family with diverse functions in epithelial cells, including cell migration, cell contact maturation, and tight junction formation. In endothelial cells, JAM-A has been implicated in basic fibroblast growth factor (bFGF)-regulated angiogenesis through incompletely understood mechanisms. In this paper, we identify tetraspanin CD9 as novel binding partner for JAM-A in endothelial cells. CD9 acts as scaffold and assembles a ternary JAM-A-CD9-αvβ3 integrin complex from which JAM-A is released upon bFGF stimulation. CD9 interacts predominantly with monomeric JAM-A, which suggests that bFGF induces signaling by triggering JAM-A dimerization. Among the two vitronectin receptors, αvβ3 and αvβ5 integrin, which have been shown to cooperate during angiogenic signaling with bFGF and vascular endothelial growth factor (VEGF), respectively, CD9 links JAM-A specifically to αvβ3 integrin. In line with this, knockdown of CD9 blocks bFGF- but not VEGF-induced ERK1/2 activation. JAM-A or CD9 knockdown impairs endothelial cell migration and tube formation. Our findings indicate that CD9 incorporates monomeric JAM-A into a complex with αvβ3 integrin, which responds to bFGF stimulation by JAM-A release to regulate mitogen-activated protein kinase (MAPK) activation, endothelial cell migration, and angiogenesis. The data also provide new mechanistic insights into the cooperativity between bFGF and αvβ3 integrin during angiogenic signaling.     

Opposite effects of tumor necrosis factor and soluble fibronectin on junctional adhesion molecule-A in endothelial cells

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.     

Activation of GPR4 by acidosis increases endothelial cell adhesion through the cAMP/Epac pathway

Endothelium-leukocyte interaction is critical for inflammatory responses. Whereas the tissue microenvironments are often acidic at inflammatory sites, the mechanisms by which cells respond to acidosis are not well understood. Using molecular, cellular and biochemical approaches, we demonstrate that activation of GPR4, a proton-sensing G protein-coupled receptor, by isocapnic acidosis increases the adhesiveness of human umbilical vein endothelial cells (HUVECs) that express GPR4 endogenously. Acidosis in combination with GPR4 overexpression further augments HUVEC adhesion with U937 monocytes. In contrast, overexpression of a G protein signaling-defective DRY motif mutant (R115A) of GPR4 does not elicit any increase of HUVEC adhesion, indicating the requirement of G protein signaling. Downregulation of GPR4 expression by RNA interference reduces the acidosis-induced HUVEC adhesion. To delineate downstream pathways, we show that inhibition of adenylate cyclase by inhibitors, 2',5'-dideoxyadenosine (DDA) or SQ 22536, attenuates acidosis/GPR4-induced HUVEC adhesion. Consistently, treatment with a cAMP analog or a G(i) signaling inhibitor increases HUVEC adhesiveness, suggesting a role of the G(s)/cAMP signaling in this process. We further show that the cAMP downstream effector Epac is important for acidosis/GPR4-induced cell adhesion. Moreover, activation of GPR4 by acidosis increases the expression of vascular adhesion molecules E-selectin, VCAM-1 and ICAM-1, which are functionally involved in acidosis/GPR4-mediated HUVEC adhesion. Similarly, hypercapnic acidosis can also activate GPR4 to stimulate HUVEC adhesion molecule expression and adhesiveness. These results suggest that acidosis/GPR4 signaling regulates endothelial cell adhesion mainly through the G(s)/cAMP/Epac pathway and may play a role in the inflammatory response of vascular endothelial cells.     

Coronin 1C negatively regulates cell-matrix adhesion and motility of intestinal epithelial cells

Coronins, WD-repeat actin-binding proteins, are known to regulate cell motility by coordinating actin filament turnover in lamellipodia of migrating cell. Here we report a novel mechanism of Coronin 1C-mediated cell motility that involves regulation of cell-matrix adhesion. RNAi silencing of Coronin 1C in intestinal epithelial cells enhanced cell migration and modulated lamellipodia dynamics by increasing the persistence of lamellipodial protrusion. Coronin 1C-depleted cells showed increased cell-matrix adhesions and enhanced cell spreading compared to control cells, while over-expression of Coronin 1C antagonized cell adhesion and spreading. Enhanced cell-matrix adhesion of coronin-deficient cells correlated with hyperphosphorylation of focal adhesion kinase (FAK) and paxillin, and an increase in number of focal adhesions and their redistribution at the cell periphery. siRNA depletion of FAK in coronin-deficient cells rescued the effects of Coronin 1C depletion on motility, cell-matrix adhesion, and spreading. Thus, our findings provide the first evidence that Coronin 1C negatively regulates epithelial cell migration via FAK-mediated inhibition of cell-matrix adhesion.     

Trans-dimerization of JAM-A regulates Rap2 and is mediated by a domain that is distinct from the cis-dimerization interface

Junctional adhesion molecule-A (JAM-A) is a tight junction–associated signaling protein that regulates epithelial cell proliferation, migration, and barrier function. JAM-A dimerization on a common cell surface (in cis) has been shown to regulate cell migration, and evidence suggests that JAM-A may form homodimers between cells (in trans). Indeed, transfection experiments revealed accumulation of JAM-A at sites between transfected cells, which was lost in cells expressing cis- or predicted trans-dimerization null mutants. Of importance, microspheres coated with JAM-A containing alanine substitutions to residues 43NNP45 (NNP-JAM-A) within the predicted trans-dimerization site did not aggregate. In contrast, beads coated with cis-null JAM-A demonstrated enhanced clustering similar to that observed with wild-type (WT) JAM-A. In addition, atomic force microscopy revealed decreased association forces in NNP-JAM-A compared with WT and cis-null JAM-A. Assessment of effects of JAM-A dimerization on cell signaling revealed that expression of trans- but not cis-null JAM-A mutants decreased Rap2 activity. Furthermore, confluent cells, which enable trans-dimerization, had enhanced Rap2 activity. Taken together, these results suggest that trans-dimerization of JAM-A occurs at a unique site and with different affinity compared with dimerization in cis. Trans-dimerization of JAM-A may thus act as a barrier-inducing molecular switch that is activated when cells become confluent.     

Talin 1 and paxillin facilitate distinct steps in rapid VLA-4-mediated adhesion strengthening to vascular cell adhesion molecule 1

VLA-4 (alpha4beta1) is a key integrin in lymphocytes, interacting with endothelial vascular cell adhesion molecule 1 (VCAM-1) on blood vessels and stroma. To dissect the contribution of the two cytoskeletal VLA-4 adaptor partners paxillin and talin to VLA-4 adhesiveness, we transiently knocked them down in Jurkat T cells and primary resting human T cells by small interfering RNA silencing. Paxillin was required for VLA-4 adhesiveness to low density VCAM-1 under shear stress conditions and was found to control mechanical stability of bonds mediated by the alpha4 subunit but did not affect the integrin affinity or avidity to VCAM-1 in shear-free conditions. Talin 1 maintained VLA-4 in a high affinity conformation, thereby promoting rapid VLA-4 adhesion strengthening to VCAM-1 under both shear stress and shear-free conditions. Talin 1, but not paxillin, was required for VLA-4 to undergo optimal stimulation by the prototypic chemokine, CXCL12, under shear stress conditions. Interestingly, talin 1 and paxillin played the same distinct roles in VLA-4 adhesions of primary T lymphocytes, although VLA-4 affinity to VCAM-1 was at least 200-fold lower in these cells than in Jurkat cells. Collectively, our results suggest that whereas paxillin is a mechanical regulator of VLA-4 bonds generated in the absence of chemokine signals and low VCAM-1 occupancy, talin 1 is a versatile VLA-4 affinity regulator implicated in both spontaneous and chemokine-triggered rapid adhesions to VCAM-1.     

Junctional adhesion molecules are required for melanoma cell lines transendothelial migration in vitro

One of the main steps of metastasis is extravasation, a phenomenon well described in lymphocytes but remaining to be fully uncovered for melanoma. Junctional adhesion molecules (JAMs) control the transendothelial migration of leukocytes. To date, the role of the JAM proteins, notably JAM-A and JAM-C, has not been examined in melanoma. Here, we compared two melanoma tumor cell lines, A375 and SLM8 cells, the A375 cell line being four times more efficient than the SLM8 cells in the crossing of the endothelial monolayer. We show evidence of the differential expression of JAM-A and JAM-C in these cell lines with JAM-C mainly expressed in the A375 cell line, and JAM-A detected preferentially in the SLM8 cells. To further dissect the respective roles of these proteins, we used both siRNA and blocking antibodies to decrease JAM-A and JAM-C expression.