N-acetylglucosaminytransferase Ⅴ Negatively Regulates Integrin α5β1-mediated Monocyte Adhesion And Transmigration Trough Vascular Endothelium

Changes in expression of glycosyltransferases that branch N-linkedglycans are associated with many physiological and pathological events,such as cell adhesion, migration, proliferation and tumor cell malignancy.Here, the altered levels of N-acetylglucosaminyltransferase V (GnT-V) andits product β(1,6)-linked GlcNAc in monocytes were observed duringinflammation. The effects of GnT-V and aberrant N-linked β(1,6)branching on monocyte adhesion through vascular endothelium andtransmigration were investigated. During IFN-γ-induced inflammation,adhesion and transendothelial migration of THP-1monocytes wereenhanced, and the levels of GnT-V and β(1,6)-linked GlcNAc in THP-1monocyte cells were significantly decreased. Expression of the GnT-VshRNA vector in THP-1cells reversed the abnormal IFN-γ-inducedcharacteristics, indicating direct involvement of N-glycosylation in these biological effects. The enhanced adhesion and transendothelial migrationwere significantly inhibited by functional blockade with antibodies againstintegrin α5or β1in IFN-γ-induced and GnT-V knockdown THP-1cells,demonstrating the involvement of integrin α5β1in themonocyte-endothelium interaction. However, IFN-γ treatment and GnT-Vknockdown in THP-1cells lowered expression of N-linked β(1,6) branchingon integrin α5and β1, without affecting the total protein expression ofthose subunits. Decreased GnT-V expression caused marked enchancementof integrin-induced phosphorylation of focal adhesion kinase (FAK). Theaugmented FAK-mediated ERK phosphorylation and activation wereobserved in IFN-γ-induced THP-1cells. Furthermore, ERK inhibitorpre-treatment nearly abrogated the highly elevated IFN-γ-induced monocyteadhesion and transmigration, concomitant with reversal of the decrease inGnT-V and β(1,6) branching. Our results demonstrate for the first time thatdecreased GnT-V activity due to inflammatory cytokine induction in humanmonocytes resulted in enchancement of integrin α5β1-dependentmonocyte-vascular endothelium adhesion and transmigration. Consequently,the activation of integrin caused elevation of FAK phosphorylation. Theseeffects promoted FAK-mediated downstream signaling, including the ERKpathway, and suggest GnT-V as a potential therapeutic target for vascularinflammatory conditions.