Synergy between Src Family Kinases and TNF-alpha signaling in the regulation of transendothelial migration

Project: Research


  • Alejandro Pablo Adam (PI)


The first step in localized leukocyte infiltration at the site of injury is achieved by cytokine-induced local increases in cell adhesion receptors such as E-selectin and ICAM-1. However, the mechanisms that govern TEM after initial attachment are less well understood. My long term goal is to identify the signals required by the EC to allow the passage of cells across the endothelial barrier. In this proposal, I will specifically investigate the role of signaling pathways that utilize both ICAM and integrins in mediating TEM. Proinflammatory cytokines such as TNF-a induce ICAM-1 expression without generating large changes in EC permeability in vivo. Leukocyte binding signals to promote loosening of endothelial cell-cell junctions and to allow TEM through activation of Src and subsequent VE-cad phosphorylation and adherens junctions (AJ) disassembly. However, in our recent publication we demonstrated that SFK activation and AJ phosphorylation is not sufficient to induce AJ disassembly and alter barrier function. Preliminary data show that SFK activation induces tyr phosphorylation of b3 integrin and other focal adhesion (FA) components. Exposure to a low dose of TNF-a does not change TEER by itself but allows SFKs to alter the EC barrier through a p38-dependent pathway, together with a drastic actin rearrangement that was dependent on b3 integrin clustering. The inability of Src or low dose TNF-a to decrease EC barrier function individually but to drastically alter EC barrier when signaling together led me to hypothesize a model in which the EC requires two concurrent signaling events to allow TEM. First, TNF-a primes the EC by the activation of p38 pathway leading to an increase in thin actin stress fibers. The second signal is provided by ICAM-1-induced, SFK-dependent phosphorylation of b3 integrin and integrin inside-out activation. I hypothesize that this may also reduce integrin binding to tetraspanins CD9, 81 and 151. Together, TNF-a and SFKs lead to a cortical integrin loss, FA aggregation and actin bundling required for the contractile forces involved in EC deformation during leukocyte diapedesis.I propose to (1) determine if ICAM-1 ligation promotes loss of TEER and TEM independently of cytokine-mediated priming, (2) test the hypothesis that tetraspanins mediate the crosstalk between SFKs, TNF-a, ICAM-1, b3 integrin and FAs and (3) identify the role of b3 integrin and FA aggregation in in vivo TNF-a-mediated vascular permeability and TEM.
Award amount$308,000.00
Award date07/01/2013
Program typeScientist Development Grant
Award ID13SDG17100110
Effective start/end date07/01/201306/30/2017