[A study on the regulatory mechanisms of mitogen-activated protein kinase phosphatase-1 in hypoxia inducible factor-1 trans-activation].

TLDR: In SGC7901 cells, the function of MKP-1 is involved in regulation of HIF-1 trans-activation via inactivation of the ERK pathway, and the VEGF protein concentration in the culture fluid was examined.

Abstract: OBJECTIVE To investigate the regulatory mechanisms of mitogen-activated protein kinase phosphatase-1 (MKP-1) in hypoxia inducible factor (HIF)-1 trans-activation. METHODS (1) Gastric cancer cells of the line SGC7901 were cultured, then continued to be cultured in hypoxic environment, and was lysed. The supernatant was collected. Western blotting was used to detect the content of total extracellular signal-regulated kinase (ERK) and phosphorylated ERK. (2) Another SGC7901 cells were cultured with PD98059, inhibitor of ERK passway, or SB203580, inhibitor of p38 passway, in the same manner as above-mentioned. Dual luciferase reporter (DLR) was used to detect the luciferase activity so as to measure the HIF-1 trans-activation. (3) siRNA vector U6M2 plasmid against MKP-1 mRNA was constructed. In another experiment SGC7901 cells were cultured and U6M2 and blank vector U6 were transfected into the cells respectively. 24 hours later, the cells were cultured in hypoxic environment with added PD98059 of different concentrations for 12 hours. Dual luciferase reporter (DLR) was used to detect the luciferase activity HIF-1 trans-activation. (4) Another SGC7901 cells were co-transfected with U6M2, pGL-3SV40HRE vector containing promoter SV40, and pRL-TK (internal control vector). Then PD98059 was added, the cells were lysed, and the activity of fluorescein was tested. (5) SGC7901 cells were cultured, transfected with UdM2 or U6 respectively, and 24 hours later cultured under hypoxia with PD98059 of different concentrations for 12 hours. ELISA was used to examine the VEGF protein concentration in the culture fluid. RESULTS (1) The content of phosphorylated ERK in the SGC7901 cells increased along with the time of hypoxia, peaked at the 12th hour, and then decreased. However, there was no difference in total ERK expression. (2) After 12 hours of hypoxia, different concentrations of PD98059 inhibited the luciferase activity, however, SB203580 of different concentrations had no effect. (3) 24 hours after transfection, the expression of phosphorylated form of ERK in the SGC7901cells transfected with siRNA plasmid against MKP-1 mRNA was higher compared with that in cells transfected with blank vectors after 12 hour of exposure to hypoxia. (4) PD98059 inhibited the luciferase activity either in U6 cells or in U6M2 cells. Notably, when the PD98059 concentration was above 50 micromol/L, there was no difference in HIF-1 activity between the U6 and U6M2 cells. (5) PD98059 of different concentrations all inhibited the VEGF expression either in U6 cells or in U6M2 cells, and when the concentration of PD98059 was over 50 micromol/L there was no difference in VEGF expression level between the U6 cells and the U6M2 cells. CONCLUSION In SGC7901 cells, the function of MKP-1 is involved in regulation of HIF-1 trans-activation via inactivation of the ERK pathway.