The Study Of Regulation Of High Glucose On Osteopontin Expression In Macrophages

Hyperglycemia is an important role to promote the development of vascular complications in diabetic patients,and vascular complications are responsible for most of the excess mutilation and mortality observed in diabetic patients.Osteopontin(OPN)is an important factor involved in atherogenesis of vascular complications,and enhanced expression of OPN was observed in plasma and atherosclerotic plaques from diabetes.Although hyperglycemia upregulates OPN levels in vascular smooth muscle cell and endothelial cell,the regulation of OPN expression is not clear.Fox O1 is a member of the FoxO subfamily in the forkhead box(FOX)transcription factor family,which is closely association with insulin resistance and diabetes.In this study,we examined the mRNA and protein expression levels of OPN,as well as the phosphorylated levels of FoxO1,in mouse macrophage-like cell line RAW 264.7 cells after cultured in high-glucose medium or overexpression of FoxO1.Then we detected the regulation of OPN expression by knockdown of FoxO1 using 3 pairs of shRNA.We cloned mouse OPN promoter and constructed luciferase reporter vector to analyze the activity change of OPN promoter after cultured in high-glucose medium or downexpression of Fox O1.The binding of FoxO1 to the OPN promoter was detected by EMSA and ChIP.Our results showed that high glucose induced up-rugulated of OPN expression and inhibited phosphorylation of FoxO1 in RAW 264.7.Overexpression of wild type or the constitutively active mutant Fox O1-ADA promoted the OPN expression levels,while the dominant-negative mutant FoxO1-(35)256 decreased the expression of OPN.Furthermore,knockdown of Fox O1 mediated by shRNA down-regulated the expression of OPN.To futher elucidate the mechanism,we cloned the mouse OPN promoter,and found that the promoter region between-1918 and-713 showed high activity,which contains 10 FBE binding sites,while the region between-713 and +79 without FBE binding site showed very low activity.High glucose promoted the activity of OPN promoter region between-1918 and-713,while knockdown of Fox O1 inhibited the acitivity.The binding of Fox O1 to the OPN promoter was confirmed by EMSA and Ch IP.Our results provide a new clue for the pathogenesis of diabetic vascular complication and a risk evaluation index.