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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 7, 2002 as doi:10.1096/fj.01-0969fje. |
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Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
2Correspondence: Department of Pathology, Vanderbilt University School of Medicine, U4202 Medical Center North, 1161 21st Ave. South, Nashville, TN 37232, USA. E-mail: richard.hoover{at}mcmail.vanderbilt.edu
SPECIFIC AIMS
Adhesion molecule clustering on the cellular surface is an important step in mediating firm adhesion by regulating the formation of a signaling complex on the cytoplasmic side of the plasma membrane at sites of adhesion. We sought to identify molecular signaling pathways downstream of the adhesion molecules on endothelial cells, E-selectin, and intercellular adhesion molecule-1 (ICAM-1), which are necessary for leukocyte adhesion to the blood vessel wall at sites of inflammation.
PRINCIPAL FINDINGS
1. Cortactin is tyrosine phosphorylated after adhesion molecule engagement in endothelial cells
Tyrosine phosphorylation is known to play a role in the formation of focal adhesion complexes after adhesion molecule clustering. To identify proteins that become tyrosine phosphorylated after adhesion molecule engagement, paraformaldehyde-fixed THP-1 monocytic cells were allowed to adhere to tumor necrosis factor (TNF) -treated human umbilical vein endothelial cells (HUVECs) and total cell lysates were analyzed by Western blotting for phosphotyrosine. An 85 kDa protein was phosphorylated after THP-1 cell adhesion. This phosphoprotein originated from the HUVECs, because the THP-1 cells were fixed and incapable of internal phosphorylation events. This 85 kDa band was also detected on phosphotyrosine blots of endothelial cell lysates after antibody-mediated cross-linking of E-selectin or ICAM-1, indicating that this protein becomes phosphorylated as a result of adhesion molecule clustering on endothelial cells after leukocyte adhesion. Hypothesizing that this protein was cortactin, a cytoskeleton binding protein and a prominent substrate of the nonreceptor tyrosine kinase src, we immunoprecipitated cortactin from endothelial cell lysates after adhesion molecule clustering. Cortactin was phosphorylated in the cells that were subjected to E-selectin or ICAM-1 cross-linking, demonstrating that cortactin is a downstream target of a tyrosine kinase signaling pathway initiated by adhesion molecule engagement on the endothelial cells.
2. Phosphorylated cortactin is associated with E-selectin and ICAM-1 after adhesion of antibody-coated beads
Since tyrosine-phosphorylated proteins are known to be involved in the formation of focal adhesion complexes on the cytoplasmic side of the plasma membrane at sites of adhesion, we hypothesized that phosphorylated cortactin could play a role in focal adhesion formation in endothelial cells at sites of leukocyte adhesion to E-selectin and ICAM-1. To test the association of cortactin with clustered adhesion molecules, paramagnetic beads were coated with antibodies against E-selectin or ICAM-1 and allowed to adhere to TNF-treated endothelial cells. The beads were isolated by a magnetic side-pull system to separate the beads from insoluble cellular debris, and the bound proteins were analyzed by Western blotting. Probing with a phosphotyrosine antibody revealed several phosphorylated proteins ranging between 60 and 230 kDa that were associated with the anti-E-selectin and anti-ICAM-1 beads (Fig. 1
A). The most prominent phosphoprotein had an apparent molecular mass of 85 kDa. Stripping and reprobing the blot with an anti-cortactin antibody identified this protein as cortactin (Fig. 1B
), indicating that phosphorylated cortactin is associated with E-selectin and ICAM-1 when these adhesion molecules are engaged. Since cortactin binds to src and is a src substrate, we also probed the blot with an anti-src antibody, revealing the presence of src on the anti-E-selectin and anti-ICAM-1 beads (Fig. 1C
). These results suggest that a complex including src and cortactin forms at sites of adhesion molecule clustering after leukocyte adhesion.
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3. Src activity is required for cortactin phosphorylation after adhesion molecule engagement
Since cortactin and src are both localized to sites of adhesion molecule clustering, we next sought to determine whether src activity was necessary for cortactin phosphorylation and its association with E-selectin and ICAM-1. HUVECs were treated with 10 µM PP2, a specific inhibitor of src family kinases, before E-selectin or ICAM-1 cross-linking. PP2 completely inhibited cortactin phosphorylation in these cells. To determine the effect of this src inhibition on cortactin association with adhesion molecules, we repeated the magnetic bead assay described above in the presence of PP2. HUVECs were treated with 10 µM PP2 before the addition of the beads, and the proteins bound to the beads were analyzed by Western blotting for phosphotyrosine and cortactin. Although PP2 again completely inhibited cortactin phosphorylation, it did not inhibit the association of cortactin with the anti-E-selectin and anti-ICAM-1 beads. These results suggest that cortactin is phosphorylated by a src family kinase after adhesion molecule engagement; however, this phosphorylation event is not necessary for the association of cortactin with adhesion molecules.
4. The clustering of E-selectin and ICAM-1 around adhered THP-1 cells is dependent on src activity
Because phosphorylated cortactin is associated with clustered adhesion molecules, we postulated that cortactin phosphorylation by src is involved in leukocyte adhesion to endothelial cells. To test the effect of src inhibition on leukocyte/endothelial interactions, we performed adhesion assays using paraformaldehyde-fixed THP-1 cells. The THP-1 cells were allowed to adhere to TNF-treated HUVEC monolayers for 1 h in the absence or presence of 10 µM PP2. Since fixed THP-1 cells were used, the effects of PP2 were limited to the endothelial cells. PP2 inhibited THP-1 adhesion by 
50%, suggesting that src activity in endothelial cells is necessary for efficient adhesion between leukocytes and endothelial cells.
Clustering of adhesion molecules involves the formation of an adhesion complex containing tyrosine-phosphorylated proteins on the cytoplasmic side of the plasma membrane. Therefore, we hypothesized that cortactin phosphorylation by src may play a role in the clustering of E-selectin and ICAM-1 at sites of leukocyte adhesion. To test this, we allowed paraformaldehyde-fixed THP-1 cells to adhere to TNF-activated HUVECs for 1 h in the absence or presence of 10 µM PP2. The endothelial monolayers were fixed and stained for E-selectin (Fig. 2
A, B) or ICAM-1 (Fig. 2C, D
) using fluorescently labeled antibodies. Both E-selectin and ICAM-1 formed clusters around the adhered THP-1 cells, as evidenced by a bright ring of fluorescence surrounding the attached THP-1 cell (Fig. 2A, C
, arrows). However, this clustering was inhibited by PP2 (Fig. 2B, D
), confirming that src activity is necessary for the aggregation of adhesion molecules on the surface of endothelial cells. This also suggests that the inhibition of THP-1 cell adhesion by PP2 is a result of impaired clustering of E-selectin and ICAM-1.
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CONCLUSIONS AND SIGNIFICANCE
Inflammation plays an important role in many human diseases. Chronic leukocyte recruitment from the bloodstream to the extravascular tissue leads to disease states such as atherosclerosis, arthritis, and multiple sclerosis. Endothelial cell adhesion molecules that bind to leukocytes in the bloodstream are necessary for leukocyte adhesion to the vessel wall. There are two major types of endothelial adhesion molecules: the selectins (E-selectin, P-selectin), which mediate low-affinity interactions between endothelial cells and leukocytes, and the Ig-like adhesion molecules (ICAM-1, VCAM-1), which mediate firm adhesion of the leukocyte to the endothelium. After adhesion, the leukocyte migrates across the endothelium and enters the extravascular tissue. This entire process of leukocyte adhesion and transmigration requires intimate interactions between the leukocytes and endothelial cells mediated by the endothelial adhesion molecules and their respective ligands on leukocytes. Whereas other adhesion molecules (i.e., integrins) are known to be important mediators of signal transduction in cells, little is known about the events that occur in endothelial cells after leukocyte adhesion to endothelial cell adhesion molecules. The goal of the studies described here was to identify signaling events initiated by endothelial adhesion molecules after leukocyte adhesion. The results indicate that a multimolecular complex forms at sites of adhesion molecule clustering, resulting in the activation of a tyrosine phosphorylation-dependent signaling pathway that is critical not only for this clustering event to occur but also for efficient leukocyte adhesion to endothelial cells.
Focal adhesion complexes have long been known to be important mediators of adhesion and signal transduction in adherent cells. Much work has been accomplished dealing with the formation of adhesion complexes at sites of integrin clustering on cells attached to extracellular matrix proteins. These complexes are believed to be important not only for adhesion, but also for cellular migration by providing foci for contraction of the actin cytoskeleton. In this model, focal complexes are the footholds for adhesion to the substratum as the cell ‘pulls’ itself over them to propel itself forward. This model can also be extended to include endothelial/leukocyte interactions during an inflammatory response. Leukocyte-specific adhesion molecules, such as E-selectin and ICAM-1, are expressed on endothelial cells at sites of inflammation. Like integrins adhering to the extracellular matrix, these adhesion molecules become clustered after adhesion to leukocytes; however, little is known about the regulation or consequences of this clustering. If endothelial adhesion molecules behave similarly to integrins, then focal adhesion complexes would form at the plasma membrane as the adhesion molecules cluster. These complexes would then serve as foci for contraction as the endothelial cells ‘pull’ apart to allow transmigration of the leukocyte through the vessel wall. Thus, adhesion molecules would provide endothelial cells with a signal to contract only at sites of leukocyte adhesion, preventing the formation of any undue ‘holes’ in the endothelial layer and thereby maintaining an otherwise intact barrier along the vessel wall. This model underscores the importance of focal adhesion complex formation in endothelial cells at sites of leukocyte adhesion.
Cortactin has long been overlooked as an important mediator of cellular adhesion. Only recently has it been identified as a stimulator of actin filament elongation at the cell periphery, an event that is crucial for the linkage of adhesion molecules to the cytoskeleton. The presence of cortactin at sites of leukocyte adhesion suggests that it is important in linking E-selectin and ICAM-1 to cortical actin filaments. Cortactin phosphorylation by the membrane-bound tyrosine kinase src occurs after adhesion molecule clustering, but its phosphorylation is not necessary for its association with the clusters. This suggests that cortactin phosphorylation is a result of, rather than a requirement for, adhesion molecule clustering. The tyrosine phosphorylation of proteins in focal adhesion complexes is believed to be important for providing binding sites for SH2 domain-containing proteins, thereby performing a scaffolding role in multimolecular complex formation. Cortactin phosphorylation could therefore be necessary for the recruitment of other proteins to sites of leukocyte adhesion. In fact, previous work has shown that the SH2 domain of src binds to cortactin. This interaction would stabilize src in its active state and link cortactin to the plasma membrane at the site of leukocyte adhesion. The recruitment of src and other SH2 domain-containing proteins would lead to a large-scale clustering of adhesion molecules after the initial multimerization of E-selectin and ICAM-1 induced by leukocyte adhesion (Fig. 3
).
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Our results identify an endothelial-dependent signaling pathway that is induced by leukocyte adhesion and is necessary for adhesion molecule clustering on endothelial cells. Future studies are necessary to determine the role of this pathway in leukocyte recruitment in vivo, especially where this aspect of inflammation is deregulated, leading to disease.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0969fje; to cite this article, use FASEB J. (June 7, 2002) 10.1096/fj.01-0969fje 
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