Differential role of integrin beta 4 in HLA class-I induced endothelial cell activation

Abstract

Aim The development of anti-donor HLA class I (HLA-I) antibodies following solid organ transplant is associated with acute and chronic rejection and graft failure. Following ligation with antibody, HLA class I molecules form a molecular partnership with integrin β 4 (ITGB4) to transduce activation and proliferative signals in endothelial cells (EC) contributing to rejection. We hypothesize that determination of the domains on the integrin β 4 molecule that interact with HLA-I will provide critical information for the development of novel therapeutics for the treatment of chronic rejection. Methods To identify the ITGB4 intracellular domain(s) that associates with HLA-I, we used the ITGB4 full length (Wild Type) construct or deletion mutants that lack the Calx- β , fibronectin type III repeat FNIII 1-4 or connecting segment (CS) domain . These constructs were sub-cloned into an adenovirus-based vector and adenovirus encoding WT or mutants were generated. Endogenous ITGB4 expression was suppressed using siRNA transfection. Primary human aortic endothelial cells (HAEC) lacking endogenous ITGB4 were infected with adenovirus and then EC activation was determined by analyzing protein phosphorylation, stress fiber formation, cell migration and proliferation. Results Our results demonstrate that HLA-I binds to ITGB4 via the CS domain. The CS domain is required for the phosphorylation of Src, Paxillin, Akt, ERK1/2 and S6RP. The FNIII 3&4 domains are required for phosphorylating Paxillin, Akt, ERK1/2 and S6RP, but not Src. HLA-I phosphorylates ITGB4 at Tyr1494 and Tyr1526 in a Src dependent manner. Furthermore, deletion of CS, FNIII3&4, Calx- β inhibited HLA-I induced EC stress fiber formation, migration and proliferation. Moreover, HLA-I induces association between ITGB4 and the adaptor protein Shc which depends on CS and FNIII3&4 domains. Suppression of endogenous Shc protein by siRNA inhibited HLA-I induced EC migration, proliferation and phosphorylation of Akt, ERK1/2 and S6RP, but not Src. Conclusion Understanding the molecular mechanisms underlying HLA antibody-induced EC proliferation and migration should enable the development of novel therapeutic strategies for the treatment of antibody-mediated chronic transplant rejection.