Prolonged Hypoxia Differentially Regulates Hypoxia Inducible Factor (HIF)-1 And HIF-2 Expression In Lung Epithelial Cell Line SPCA-1

ObjectiveTo study whether hypoxia could induce the expression of HIF-2αin lung epithelial cells SPCA-1 compare the characteristics of induction of HIF-2αby hypoxia with HIF-1αand investigate the underlying mechanismsMethodsLung epithelial cells SPCA-1 cells were cultured in vitro under hypoxia or normoxia conditions, with different times, then we used RT-PCR and Western blot to determine the activation of HIF-2αand HIF-1α.An inhibitor of Mitochondrial reactive oxygen species,such as Rotenone, diphenyleneiodonium and myxothiazol were added to the medium of the cells to investigate the expression of HIF-2αand HIF-1αin the hypoxia, then the underlying mechanisms of induction by hypoxia was investigatedResult1 Under normoxia conditions, in the extract of SPCA-1 the whole cell and the nuclear, the protein levels of HIF-1αwere scarcely expressed HIF-2αwere low in the whole cell, but nuclear extracts had not significantly express, after SPCA-1 were cultured 4h of hypoxia (0.5% O₂), the protein levels of HIF-2αand HIF-1αhad obviously increase.2 Under normal oxygen conditions, the lower of the cell density, the less of HIF-2αand HIF-1αprotein were significantly produced. When the oxygen concentration dropped to below 3%, HIF-2αand HIF-1αprotein were significantly increased.3 After in hypoxia 4h, HIF-1αprotein were increased, then dramatically decreased after 6h, at 12h decreased to the level of the control group. HIF-2αprotein in 4 ¹2h had no significant change. HIF-2αand HIF-1αmRNA at 4h hypoxia did not change significantly. But with the extention in hypoxia, hypoxia could significantly vary HIF-1αand HIF-2αmRNA expression, HIF-1αmRNA levels from 6h to 12h in hypoxia dramatically reduced, while the H1F-2αmRNA level was significantly rised.4 When the SPCA-1 cells were handled 4h with Mitochondrial reactive oxygen species inhibitor,such as Rotenone, diphenyleneiodonium and myxothiazol, HIF-2αand HIF-1αprotein significantly decreasedConclusion1 Hypoxia can induce the expression of HIF-1αand HIF-2αin lung epithelial cells;2 Post-translational modification plays all important roles in the induction of HIF-1αand HIF-2αin hypoxia;3 HIF-2αand HIF-1αmRNA and their protein levels were different at different times of the hypoxic adaptation;4 Hypoxia maybe play the roles of induction and regulation through the same signaling pathway on HIF-2αand HIF-1α.