GlcNAc inhibits MAPKs pathway in rat corpus cavernosum smooth muscle cells via O-glycosylation-dependent pathway

Abstract

Objective To investigate the effect of β-N-acetylglucosamine (GlcNAc) on the mitogen-activated protein kinases (MAPKs) pathway in rat penile cavernosum smooth muscle cells. Methods After penile tissue was collected from healthy adult rats, cavernous smooth muscle cells (CSMC) were isolated and cultured under high glucose medium. Immunofluorescence (IF) assay and immunocytochemical (ICC) assay were used to identify CSMC. Then the cells were treated with GlcNAc for 12 h, the cell lysate was collected at 0, 10 min, and 1, 2, 4, 8 and 12 h. Western blotting was employed to detect the total glycosylation level of protein and phosphorylation of MAPKs pathway-associated proteins after the cells were treated with O-N-acetylglucosamine transferase (OGT) inhibitor, Alloxan, O-GlcNAcase (OGA) inhibitor, Thiamet G, and GlcNAc. Results The rat CMSC were successfully isolated. Compared with the control cells, GlcNAc treatment significantly increased the O-glycosylation level of CSMC (P 0.05), which was the same effect of Thiamet G. In addition, GlcNAc and Thiamet G synergistically inhibited the phosphorylation levels of ERK1/2, JNK1/2/3 and p38 (P < 0.05). Conclusion GlcNAc inhibits MAPKs pathway in CSMC via O-glycosylation-dependent pathway.

Keywords

• cavernous smooth muscle cells
• β-n-acetylglucosamine
• o-glycosylation
• mitogen-activated protein kinases