| Summary: | 博士 === 高雄醫學大學 === 醫學研究所博士班 === 95 === Hyperglycemia is a major cause of diabetic vascular disease. High glucose is associated with reduce [3H] thymidine incorporation into DNA and cell proliferation in human umbilical vein endothelial cell (HUVECs). Although high glucose may inhibit cell growth and several serine-threonine kinase, the exact mechanism by which it promotes cell cycle arrest is still unclear. In this study, we demonstrated that high glucose at a range of concentrations (18.5-38.5 mM) dose and time-dependently inhibited [3H] thymidine incorporation and decreased cell number in culture HUVECs. Flow cytometry analyses demonstrated that hyperglycemia arrested the cell at the G0/G1 phase of the cell cycle. High glucose also induced collagen and TGF-?? mRNA. N-acetylcysteine (an antioxidant, NAC) attenuated high glucose-inhibited cell proliferation and high glucose-induced collagen. Both mRNA and protein of the CDK inhibitor p15 INK4b and p15.5INK4b in HUVECs were time-dependently induced by high glucose (28.5 mM). In addition, pretreatment of HUVECs with 0.5 ?嵱 p15INK4b antisense oligonucleotide reversed the hyperglycemia-induced inhibition of [3H] thymidine incorporation and up-regulation of p15 INK4b and p15.5 INK4b protein into HUVECs for 24 h. The phosphorylation of ERK1/2 was increased by high glucose exposure 12 h and gradually reduced after long exposure in HUVECs. Pretreatment of 15 ?嵱 PD98095, an inhibitor of MEK which is the upstream kinase of ERK1/2, reverse high glucose-induced G0/G1 phase of cell cycle arrest in HUVECs. PD98095 also blocked induction of p15INK4b and p15.5INK4b by high glucose for 24 h in HUVECs. These results indicate that high glucose-induced HUVECs growth inhibition are associated with p15INK4b and p15.5INK4b and stimulation of ERK pathway can result in an induction of CDK inhibitor p15INK4b and p15.5INK4b and cell cycle arrest. High glucose increased transforming growth factor-?? (TGF-??) gene transcriptional activity and mRNA expression. However, neither SB431542 (type I TGF-?? receptor blocker) nor TGF-??1 antibody affected high glucose-induced p15INK4b and p15.5INK4b protein expression. NAC and calphostin C could decrease high glucose-induced ERK1/2 activity. Thus, high glucose induced p15INK4b and p15.5INK4b via PKC/ROS/ERK1/2 pathway. Similary, high glucose induced p21 WAF1, p53 and p53(ser15) and inhibited cyclinB1 via PKC/ROS/ERK1/2 pathway. High glucoe induced p15INK4b and p15.5INK4b via p53 pathway. Taken together, these result suggest that high glucose-induced growth arrest is dependent on p15 INK4b、 p15.5 INK4b、 p21 WAF1/CIP1、 p53 and cyclin B1 via PKC/ROS/ERK1/2 pathway.
|
|---|
