Silencing Mannosylphospho Dolichol Synthase with shRNA Impacts Differentiation of Capillary Endothelial Cells

Mannosylphospho dolichol synthase (DPMS) is down stream of N‐acetylglucosaminyl 1‐phosphate transferase (GPT) in the dolichol pathway of asparagine‐linked (N‐linked) protein glycosylation. When capillary endothelial cells are treated with a GPT inhibitor tunicamycin, angiogenesis is inhibited quantitatively reducing the breast tumor growth in nude mice by inducing unfolded protein response ( upr )‐mediated apoptosis. There was also a quantitative loss of DPMS catalytic activity. We, therefore, hypothesized that there is a cross‐talk between the GPT and DPMS, and DPMS independently plays a significant role in angiogenesis. To evaluate, we have isolated a stable capillary endothelial cell line in which DPMS has been knocked down after transfecting the cells with DPMS shRNA plasmid in pSilencer 4.1‐CMV neo vector. The control used a scrambled shRNA sequence. DPMS expression in each cell clone was monitored by measuring catalytic activity, qRT‐PCR for DPMS gene expression, western blotting and immunofluorescence microscopy for DPMS protein expression. They all have down regulated in the knockdown clone. Western blotting with Concanavalin A also indicated the presence of reduced level of N‐linked glycoproteins with terminally linked mannose. Interestingly, when the cellular morphology was followed as a function of time, the DPMS knockdown clone exhibited lumen‐like structure formation more frequently than the control. Clonogenic assay indicated 0% survival of DPMS knockdown cells after 48 hours of tunicamycin (1 μg/mL) treatment. We, therefore, conclude that DPMS silencing makes cells less angiogenic, more susceptible to the anti‐angiogenic glycotherapy tunicamycin and importantly impacts the differentiation characteristics of the cell. Support or Funding Information ).Supported in part by grants from NIH/NIMHD 2G12MD007583 (KB) and NSF‐EPSCoR RII Track 1 Grant # EPS‐1002410 (DKB)