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The Src-cortactin pathway is required for clustering of E-selectin and ICAM-1 in endothelial cells
Robert W Tilghman1, Richard L Hoover
1Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Insights
Src and cortactin mediate leukocyte-endothelial cell interactions during inflammation. They regulate adhesion molecule clustering on endothelial cells, impacting leukocyte recruitment to inflamed tissues.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Endothelial cells express adhesion molecules like E-selectin and ICAM-1 at inflammation sites.
- These molecules are crucial for recruiting leukocytes from blood to tissues.
- Signaling pathways in endothelial cells after adhesion molecule engagement are not well understood.
Purpose of the Study:
- To investigate signaling pathways in endothelial cells upon adhesion molecule engagement.
- To identify proteins involved in leukocyte-endothelial cell interactions.
- To elucidate the role of src family kinases and cortactin in these interactions.
Main Methods:
- Immunoprecipitation to identify phosphorylated proteins.
- Western blotting to detect tyrosine phosphorylation.
- Inhibition studies using src family kinase inhibitor PP2.
- Microscopy to observe adhesion molecule clustering and cell attachment.
Main Results:
- An 85-kDa protein, identified as cortactin, becomes tyrosine phosphorylated in endothelial cells after leukocyte adhesion or E-selectin/ICAM-1 clustering.
- Src kinase and tyrosine-phosphorylated cortactin associate with E-selectin and ICAM-1.
- Src inhibition by PP2 blocks cortactin phosphorylation and adhesion molecule clustering.
- PP2 treatment reduces THP-1 cell adhesion to endothelial cells, correlating with inhibited clustering.
Conclusions:
- Src and cortactin are key mediators of leukocyte-endothelial cell interactions at inflammatory sites.
- Src signaling regulates cortactin phosphorylation and adhesion molecule clustering on endothelial cells.
- Targeting src and cortactin pathways may offer therapeutic strategies for inflammatory diseases.
Abstract:
Adhesion molecules such as E-selectin and intercellular adhesion molecule-1 (ICAM-1) expressed on endothelial cells (ECs) at sites of inflammation play an important role in the recruitment of leukocytes from the bloodstream into extravascular tissue. However, little is known about the signaling pathways that are initiated in ECs following adhesion molecule engagement. Here, we report that an 85-kDa protein becomes tyrosine phosphorylated in human ECs following leukocyte adhesion or upon antibody-induced clustering of E-selectin or ICAM-1. Through immunoprecipitation experiments, this protein was identified as cortactin, a cytoskeleton-binding molecule and prominent src substrate involved in cell adhesion. Following adhesion molecule clustering, cortactin phosphorylation was inhibited by the src family kinase inhibitor PP2. Both src and tyrosine-phosphorylated cortactin were found to be associated with E-selectin and ICAM-1 following adhesion of antibody-coated beads to ECs. PP2 did not inhibit the association of cortactin with E-selectin and ICAM-1; however, PP2 inhibited adhesion between paraformaldehyde-fixed THP-1 cells and ECs. This decrease in adhesion correlated with inhibition of adhesion molecule clustering on PP2-treated ECs at sites of THP-1 attachment. These findings implicate src and cortactin as mediators of leukocyte/EC interactions at sites of inflammation by regulating adhesion molecule clustering on ECs.