OR23: THE INTEGRIN β4 CONNECTING SEGMENT DOMAIN IS REQUIRED FOR HLA CLASS I-MEDIATED ENDOTHELIAL CELL ACTIVATION

Abstract

Endothelial cell (EC) activation is a major consequence of HLA class I (HLA-I) antibody-mediated rejection. Previous studies have shown that the molecular association between HLA-I molecule and integrin β 4 (ITGB4) is required for EC migration and proliferation by regulating ERK1/2 signaling pathway. However, the proximal signaling pathway that regulates HLA-I and ITGB4 association remains unclear. We determined the domains of the ITGB4 cytoplasmic tail required for HLA-I mediated phosphorylation pathways and correlated these findings with functional studies. Primary human aortic endothelial cells (HAEC) were used for this study. To identify the ITGB4 intracellular domain that associates with HLA-I, we generated constructs encoding ITGB4 full length (WT) or deletion mutants that lack the Calx- β , fibronectin type III repeat (FNIII 1–4) or connecting segment (CS). These constructs were sub-cloned into adenovirus-based vector and adenovirus encoding WT or mutants were generated. Endogenous ITGB4 expression was suppressed using siRNA transfection. HAEC lacking endogenous ITGB4 were infected with adenovirus and protein phosphorylation and cell migration were further analyzed. Flow cytometry and Western blot analysis showed that both the WT and deletion mutant-infected HAEC had similar levels of cell surface or total expression of ITGB4. Coimmunoprecipitation studies showed that stimulation with the F(ab′)2 fragment of HLA-I antibody increased HLA-I association with ITGB4 in a time dependent manner in WT adenovirus infected HAEC. In contrast, HAEC infected with the CS deletion mutant significantly inhibited HLA-I association with ITGB4 compared to WT, Calx- β or FNIII mutant infected cells. In addition, the CS deletion mutant decreased HLA-I induced ERK1/2 phosphorylation as measured by Western blot. Using in vitro wound healing assay, we found that over expression of ITGB4 increased EC migration compared to LacZ infected cells, whereas CS deletion decreased HLA-I induced EC migration. Furthermore, HLA-I antibody increased the association of ITGB4 with the adaptor protein Shc. This association was decreased by CS deletion. Our results demonstrate that HLA-I binds to ITGB4 via the connecting segment domain that is required for ERK1/2 signaling and EC activation.