11 The role of N-glycosylation of the NOTCH1 receptor in jagged1-stimulated myogenic differentiation in vitro

Resident stem cell fate decisions within the vasculature are crucial to the pathogenesis of vascular diseases, including, arteriosclerosis, atherosclerosis and in-stent restenosis after angioplasty. The Notch signalling pathway regulates stem cell fate and is highly regulated by a number of mechanisms including glycosylation, a post-translational modification. Our main objective was to define a putative role for N-glycosylation of Notch1 receptor in controlling resident vascular stem cell fate in vitro . Utilising ligand-induced Notch signalling assay with Jagged1, qRT-PCR, immunocytochemistry, ectopic expression of Notch1 receptor, siRNA knockdown, pharmacological inhibition and enzyme linked lectin assay (ELLA), alterations in N-glycan decoration of the Notch1 receptor were assessed before evaluation of their effects on Notch signalling and Notch ligand promotion of myogenic differentiation. N-glycosylation of the Notch1 receptor was assessed using a combination of the HPLC and ELLA assays and confirmed the presence of N-glycans on the receptor, an effect that was abrogated following inhibition of glycosyltransferase activity with tunicamycin and lunatic fringe (Lfng) knockdown. Jagged1- induced Notch activation increased Notch target gene expression and promoted myogenic differentiation of bone-marrow derived mesenchymal stem cells and resident vascular stem cells. Selective knockdown of the Notch1 receptor in stem cells resulted in a significant decrease in Jagged1 stimulated Hey1 expression, a Notch1 target gene, concomitant with a reduction in myogenic differentiation due to decreased smooth muscle differentiation marker expression (CNN1 and MYH11 mRNA and protein levels). Inhibition of N-glycosylation with tunicamycin lead to a down regulation of smooth muscle differentiation markers, CNN1 and MYH11 independent of a reduction in Notch target gene expression. Lfng knockdown lead to a similar significant reduction in Jagged1 induced myogenic differentiation (reduced CNN1 expression). Collectively, these results suggest that N-glycosylation of Notch1 receptor is involved in Notch signalling leading to altered resident vascular stem cell fate.