#47-OR
ANTIBODY MEDIATED CLASS I SIGNAL TRANSDUCTION IN HUMAN ENDOTHELIAL CELLS IS REGULATED BY THE DEGREE OF MOLECULAR AGGREGATION.
Yi-Ping Jin, M.D., Michael S. Bennett, Dennis J. Montoya, Kelsey N. Retting and Elaine F. Reed, Ph.D.. Los Angeles CA, USA, David Geffen School of Medicine at UCLA, 90095, Pathology
Laboratory Medicine.
Anti-HLA antibodies (Ab) may contribute to chronic rejection by binding to endothelial cells (EC) and smooth muscle cells and stimulating cell proliferation. Characterization of the intracellular signaling pathway shows that binding of Ab to class I molecules induces phosphorylation of Src kinase and the focal adhesion protein FAK. We investigated the impact of anti-HLA Ab concentration on the regulation of FAK activation by Src. EC were treated with the anti-HLA class I mAb W6/32 at different concentrations and cell lysates were immunoprecipitated with Src or FAK mAb and immunoblotted with anti-phospho Ab. Treatment of EC with anti-class I Ab induced a dose dependent phosphorylation of Src at Tyr-416 in the activation loop of the kinase domain with the lowest dose inducing the highest level of phosphorylation. In contrast, the highest level of dephosphorylation of Tyr-416 was achieved using the highest dose of Ab. Similarly, low titers of Ab stimulated the highest level of FAK phosphorylation at residues Tyr-577, Tyr-576 and Tyr-925. Treatment with PP2, a pharmacological inhibitor of Src, blocked class I mediated FAK phosphorylation indicating a critical role for Src kinase in this process. Ligation of class I molecules also triggered a dose dependent assembly of molecular complexs of Shc/Grb2 and Grb2/Sos providing a link between class I signaling and the MAP kinase pathway. In conclusion, the intracellular events initiated by Ab ligation of class I molecules are regulated by the concentration of Ab and may have a detrimental effect on graft survival by stimulating cell proliferation and increasing risk for transplant arteriosclerosis.