Adhesion receptor-ligand interactions can influence vascularized organ allografts at multiple levels, including targeted trafficking of host leukocytes into the graft, costimulation of alloreactive T cells through leukocyte integrins, or directed killing of donor target cells by conjugate formation with cytotoxic, allospecific effector cells. We have previously demonstrated that LFA-1 (al B2) plays a critical role in lymphocyte-EC adhesion and lymphocyte migration, is dynamically regulated and, as a transmembrane signaling molecule, stabilizes Thi cytokine transcripts including IFN-g, TNF-a, and LL-2. This RNA stabilization is, in part, cytoskeleton dependent, is directed at modulation of transcript degradation through AU-rich target sequences, and requires the LFA-1 engagement-induced nuclear-to-cytoplasmic translocation of the critical RNA binding protein HuR. This has led to our overall hypothesis as follows: T cell co-activation, through LFA-1, drives actin cytoskeleton dependent modulation of HuR which, in turn, facilitates stabilization of labile T cell activation transcripts for multiple pro-inflammatory and pro-proliferative key effector molecules in acute and chronic vascular rejection. Specific proposals now include to (1) define proximal signaling components of LFA-1-mediated HuR translocation, by using actin cytoskeleton-disrupting agents and rho family GTPase mutants in T cell or LFA-1+ stable transfectant HuR translocation assays; (2) assess LFA-1-induced biochemical modifications on, and induced protein-protein interactions with, HuR in I cells, using phosphopeptide mapping and coimmunoprecipitation proteomics; (3) evaluate the effect of LFA-1 engagement on ARE-bearing transcripts encoding cell cycle regulatory molecules, including G1-, S- and G2-specific cyclins; and (4) test the role of HuR function in heterotopic and aortic allograft models in vivo with a T cell-specific, tetracycline regulable HuR knock out mouse. This work may provide insight into cellular and molecular triggers of allograft rejection, including those directed at the vasculature, and immune activation more broadly.
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