Introduction Acute lung injury (ALI) syndromes are highly morbid consequences of systemic inflammatory conditions such as sepsis. Inflammation-induced disruption during ALI of the endothelial cell (EC) barrier that lines the pulmonary vasculature results in leakage of fluid, protein, and cells into the airspaces of the lung, resulting in respiratory failure. We have previously described (Dudek et al. J Biol Chem 2004;279:24692-700) a critical role for the actin-binding protein cortactin in mediating EC cytoskeletal rearrangements that regulate in vitro barrier function.
Methods/Results We now report immunofluorescence and coimmunoprecipitation data demonstrating increased association of cortactin with the junctional proteins β-catenin, focal adhesion kinase, and vinculin during the recovery phase following thrombin-induced permeability. Thus, cortactin interacts at critical cell-cell and cell-matrix junctional sites as the EC monolayer recovers after inflammation-induced disruption. As a translational approach building upon these in vitro observations, single-nucleotide polymorphism (SNP) discovery of the cortactin gene was performed using direct fluorescence-based resequencing. Cortactin SNP discovery in 36 patients, subdivided into those with sepsis-induced ALI, sepsis alone and healthy controls, identified 26 SNPs within the human cortactin gene, including a single novel coding variant. This SNP at amino acid position 484 results in a serine to asparagine change (Ser484Asn), which was enriched in patients with ALI and sepsis compared with controls. Since this S484N site is in close proximity to a critical p60src-targeted tyrosine residue (Y486), a major regulatory site of cortactin function, we generated wild-type and S484N mutant constructs for overexpression in human pulmonary ECs. These studies revealed greatly increased phosphorylation of cortactin at Y486 in the S484N mutant under baseline and EC barrier-altering conditions. In addition, overexpression of S484N in ECs inhibited the potent barrier-promoting effects of sphingosine 1-phosphate as well as delayed barrier recovery after the barrier disruption by thrombin.
Summary Cortactin plays a critical role in mediating cytoskeletal and junctional protein rearrangements that regulate EC barrier function. Given that phosphorylation of Y486 modulates cortactin function, the S484N cortactin SNP may increase ALI susceptibility via this mechanism.
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