N-acetylglucosaminyltransferase V negatively regulates integrin α5β1-mediated monocyte adhesion and transmigration through vascular endothelium

Changes in the expression of glycosyltransferases that branch N-linked glycans 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) and its product beta(1,6)-linked GlcNAc in monocytes were observed during inflammation. The effects of GnT-V and aberrant N-linked beta(1,6) branching on monocyte adhesion through vascular endothelium and transmigration were investigated. During IFN-gamma-induced inflammation, adhesion and transendothelial migration of THP-1 monocytes were enhanced, and the levels of GnT-V and beta(1,6)-linked GlcNAc in THP-1 monocytes were significantly decreased. Expression of the GnT-V shRNA vector in THP-1 cells reversed the abnormal IFN-gamma-induced characteristics, indicating direct involvement of N-glycosylation in these biological effects. The enhanced adhesion and transendothelial migration were significantly inhibited by functional blockade with antibodies against integrin alpha 5 or beta 1 in IFN-gamma-induced and GnT-V knockdown THP-1 cells, demonstrating the involvement of integrin alpha 5 beta 1 in the monocyte-endothelium interaction. However, IFN-gamma treatment and GnT-V knockdown in THP-1 cells lowered expression of N-linked beta(1,6) branching on integrin alpha 5 and beta 1, without affecting the total protein expression of the subunits. Decreased GnT-V expression caused marked enchancement of integrin-induced phosphorylation of focal adhesion kinase (FAK). The augmented FAK-mediated ERK phosphorylation and activation were observed in IFN-gamma-induced THP-1 cells. Furthermore, ERK inhibitor pre-treatment nearly abrogated the highly elevated IFN-gamma-induced monocyte adhesion and transmigration, concomitant with reversal of the decrease in GnT-V and beta(1,6) branching. Our results demonstrate for the first time that decreased GnT-V activity due to inflammatory cytokine induction in human monocytes resulted in enchancement of integrin alpha 5 beta 1-dependent monocyte-vascular endothelium adhesion and transmigration. Consequently, the activation of integrin caused elevation of FAK phosphorylation. These effects promoted FAK-mediated downstream signaling, including the ERK pathway, and indicate that GnT-V may be a potential therapeutic target for vascular inflammatory conditions.