Post-translational modifications are decisive in providing functional maturation and diversity of many proteins. Leukocyte-specific protein 1 (LSP1) plays an important role in the regulation of endothelial permeability, leukocyte chemotaxis and transendothelial migration during inflammation. Unlike leukocyte-expressed LSP1 which is predominantly a cytosolic protein, endothelial cell-expressed LSP1 is mainly localized in the nucleus. It’s translocation to extranuclear compartments is promoted by tumor necrosis factor-α (TNF-α). There is a discrepancy between the predicted molecular weight of LSP1 (37 kDa) and its actual appearance in SDS-PAGE (˃ 50 kDa). All of these discrepancies indicate possible post-translational modifications. In spite of its important role in acute inflammation and these apparent indications, very little is known about post-translational modifications of endothelial cell-expressed LSP1. To address this, we investigated whether endothelial cell-expressed LSP1 activation is linked to its phosphorylation by p38 mitogen activated protein kinase (p38 MAPK) and protein kinase C (PKC) upon chemokine treatment of endothelial cells and whether it requires additional signaling such as leukocyte-endothelial cell interaction. The present study also addresses how post-translational modifications help maintain the functions of endothelial cell-expressed LSP1.
Using in vitro adhesion assay and immunoblotting, we showed that endothelial cell-expressed LSP1 was phosphorylated only in the presence of adherent neutrophils or engagement of endothelial intercellular cell adhesion moelecule-1 (ICAM-1), but not cytokine or chemokine alone. Pharmacological inhibition of p38 MAPK by SB 203580, a selective MAPK inhibitor, significantly blunted the phosphorylation of endothelial LSP1. In endothelial cells, ICAM-1 engagement, in vitro, also caused a novel interaction between LSP1 and moesin which was mediated by p38 MAPK and Rho kinase. Absence of either LSP1 or moesin blunted the ICAM-1 engagement-induced endothelial permeability changes.
Using scratched wound healing and in vitro transmigration assay, we found that silencing of LSP1 reduced migratory behavior of endothelial cells. Selective deficiency of endothelial cell-expressed LSP1 also resulted in a marked reduction of neutrophil transendothelial migration and a subtle reduction in endothelial cell proliferation and neutrophil adhesion. The study demonstrates that LSP1-deficiency resulted in GATA-2-mediated selective reduction of endothelial cell-expressed platelet endothelial cell adhesion molecule-1 (PECAM-1) that altered the endothelial cell functions.
Using murine primary endothelial cells and recombinant LSP1 expressed in mouse endothelial cell line (SVEC4-10EE2) cells and human embryonic kidney (HEK293T) cells, we demonstrate that LSP1 is post-translationally modified by small ubiquitin-like modifier 1 (SUMO1). Single lysine-to-alanine mutation at K270 and K318 of murine LSP1 prevented the SUMOylation of recombinant LSP1 in HEK293T cells. DeSUMOylation resulted in marked reduction in steady-state LSP1 levels. It is shown that reduced steady-state level of SUMOylation-deficient LSP1 was due to enhanced ubiquitination and subsequent rapid proteasomal degradation and that deSUMOylation of LSP1 impaired its translocation from nucleus to extranuclear compartments in endothelial cells in response to TNF-α stimulation.
This study confirms that adhesion-mediated phosphorylation is required for endothelial cell-expressed LSP1 functions whereas SUMOylation helps to maintain its functions by protecting LSP1 from proteasomal degradation and facilitating its translocation from nucleus to cytoskeleton in endothelial cells