Recent studies have highlighted a new paradigm whereby the maintenance of normal tissue homeostasis and the prevention of chronic inflammation are dependent on the active process(es) of inflammation resolution. Pro-resolution is mediated by endogenous regulatory intracellular signaling networks that, to date, are poorly defined and the subject of extensive investigation. Since endothelial cells (EC) are the primary targets of cellular and humoral alloimmune responses, we hypothesized that regulatory signaling in this cell type is critical to attenuate the effects of the immune response on allograft injury. In these studies, we found that DEPTOR, a novel endogenous regulator of mTOR, is expressed in EC from different vascular beds. Using a siRNA knockdown approach and phosphokinase protein arrays, we found that DEPTOR inhibits the mTORC1/pS6K1, ERK1/2 and STAT1 signaling pathways in EC. In addition, using pharmacological inhibitors of mTOR (Rapamycin, Torin1) and ERK1/2 (U0126) in DEPTOR siRNA-transfected EC, we found that it regulates mTORC1/pS6K1 and ERK1/2 activity through independent mechanisms. Moreover, we found that DEPTOR inhibits STAT1 signaling in part via crosstalk among intermediaries within both the ERK1/2 and the mTOR pathways. DEPTOR knockdown in EC resulted in marked activation responses as assessed by mRNA gene arrays and qPCR analyses; it notably increased (up to ∼1000 fold) the expression of the T cell chemoattractant chemokines CXCL9, CXCL10, CXCL11, CX3CL1, CCL5 and CCL20, as well as the adhesion molecules VCAM-1 and ICAM-1 (P<0.05). In addition, DEPTOR knockdown in EC resulted in increased adhesion of PBMC (n=5, P<0.005) and CD3+ T cells (n=5, P<0.005) vs. control siRNA-transfected EC, and led to increased migratory and angiogenic responses in vitro in wound-healing (n=4, P<0.01) and spheroid sprouting (n=3, P<0.01) assays. Collectively, these findings identify DEPTOR as a first-in-kind upstream inhibitor of EC activation responses that elicits anti-inflammation and pro-resolution. Our data highlight a new paradigm for anti-rejection therapeutics, and suggest that agents that stabilize DEPTOR expression will have implications for the prevention of chronic allograft rejection.

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