Fig. 5.

Down-regulation of HIF-2α contributes to IH-induced oxidative stress as a result of decreased SOD2 promoter trans-activation and enzyme activity. (A) Over-expression of HIF-2α prevents IH-evoked oxidative stress and HIF-2α down-regulation in PC12 cells. TBARS levels were monitored as an index of oxidative stress in cells transiently transfected with an empty vector (pCDNA3.1; control) or pHIF-2α plasmid, and then exposed to normoxia (N) or to IH₆₀. (B) Transcriptionally active HIF-2α prevents IH-induced inhibition of SOD2 promoter activity. SOD2 promoter activity was determined in PC12 cells transiently transfected with the reporter plasmid pGL3-SOD-Luciferase and co-transfected with pIRES-WT HIF2 (transcriptionally active) and pIRES-IA HIF2 (transcriptionally inactive) expression plasmids. Cells were exposed to normoxia (N) or IH₆₀ and SOD2 promoter activity was determined by luciferase activity in cell lysates. (C and D) Treatment of cells with the calpain inhibitor ALLM (10 μM) prevents the IH₆₀-evoked increase in TBARS (index of oxidative stress) as well as the decrease in SOD2 enzyme activity. Control cells were exposed to normoxia (N). Data presented are mean ± SEM. from 3 to 5 experiments. *P < 0.05; n.s., not significant, P > 0.05.