The Study Of VEGF165 Up-regulates The Expression Of UPA Protein Through MAPK Signaling Pathways

Objective:In this study,we measured the phosphorylation of ERK, p38 and JNK and the expression of urokinase-type plasminogen activator(uPA)in human umbilical vein endothelial cells(HUVECs) cultured with vascular endothelial growth factor 165( VEGF165), in order to investigate the role of MAPK signal transdution pathways in the effect of VEGF165 on uPA protein expression in HUVECs.Methods:1. Prove the influence of VEGF165 to HUVECs’ viability : HUVECs were cultured in different densities of VEGF165(0 ng/mL, 5 ng/mL, 10 ng/mL, 30 ng/mL and 50 ng/mL), then observed the cell proliferation by CCK-8.2. Validate the effect exerted by VEGF165 on uPA protein expression: Divided HUVECs into two groups, one was incubated with 10 ng/ml VEGF165 and the other one with no VEGF165 as control. Then analysed the expression of uPA protein of each group at 24 h, 48 h, and 72 h by Western blot(WB).3. Demonstrate the role of VEGF165 on MAPK signal pathway: Divided HUVECs into two groups, one was cultured with VEGF165 and the other was for contrast with no VEGF165.Then analysed the phosphorylation of ERK, p38 and JNK in these two groups at 0 h, 6 h, 12 h and 24 h by Western blot.4. Confirm the related MAPK signaling pathways to the uPA protein expression:In order to identify whether the expression of uPA was ERK signaling pathway dependent, we used PD98059, a specific blocking agent of ERK signaling pathway. Groups were like below: control group, add VEGF165, PD98059 pretreated for 1h, PD98059 pretreated for 1h then add VEGF165. Analysed the expression of uPA protein and phosphorylation of ERK, p38 and JNK by WB. In order to identify whether the expression of uPA protein was p38 signaling pathway dependent, we used SB203580, a specific blocking agent of P38 signaling pathway.Groups and the analyses were like above.Also in order to identify whetherthe expression of uPA protein was JNK signaling pathway dependent, we used SP600125, a specific blocking agent of JNK signaling pathway. Groups and the analyses were as above.5. Verify the cross-talk among MAPK signaling pathways: HUVECs were disposed with 50 uM PD98059, 20 uM SB203580 and 10 uM SP600125, then analysed the phosphorylation of ERK, p38 and JNK by Western blot to identify the cross-talk of these three signaling molecules.Results:1. The OD values increased as the plus of VEGF165 concentrations. Compared with the control group, the differences were statistically significant(P < 0.05). The OD value was maximum with 10 ng/ml VEGF165, and no longer increased as the plus of VEGF165(P>0.05).2. The relative intensity of uPA divided by GAPDH at VEGF165 treated group increased gradually at 24 h, 48 h, 72 h with significantly differences(P < 0.05), while no difference among control groups(p>0.05). Comparision of VEGF165 group and control group, the uPA protein expression had no difference at 24 h, while differences were significant at 48 h and 72 h.3. Cultivated HUVECs with VEGF165, and detected at 0 h, 6 h, 12 h and 24 h.Statistical analysis suggested p-ERK level increased with differences(p < 0.05),except for 0 h with 6 h(p > 0.05). p-p38 and p-JNK level increased with statistical differences at each time point(p < 0.05), while total ERK, p38 and JNK protein had no difference(p > 0.05). In control groups p-ERK, p-p38 and p-JNK protein had no obvious changes at each time point(p > 0.05).4. Comparision of control group and VEGF165 cultivated HUVECs that pretreatd with PD98059、SB203580 and SP600125 separately for 1 h, the uPA protein expression was lower than the control group(p < 0.05) only by SB203580.5. ERK, p38 and JNK signaling pathways were separately cut off with PD98059、SB203580 and SP600125, and there were no statistical differences in the phosphorylation of other kinases(P>0.05).Conclusion:1. Exogenous VEGF165 could promote the viability of HUVECs and the uPAprotein expression.2. VEG165 stimulates MAPK signal transduction pathways. p38 MAPK signaling pathway was identified to play a very important role upon up-regulation of uPA protein expression after HUVECs cultured with VEGF165.