Negative regulation of exogeneous polyl-4-hydroxylase domain proteins on hypoxia-inducible factor pathway in human RPE cells

Background

Anti-VEGF drugs are generally applied in the treatment of ocular neovascular diseases.However, the therapy effect is unsatisfactory in some patients.Studing the effect of hypoxia-inducible factor-1 (HIF-1), a upstream regulatory gene of VEGF, and its limiting enzyme prolyl-4-hydroxylase domain proteins (PHDs) is of important clinical significance.

Objective

This study was to investigate the negtive regulation of exogeneous PHDs on HIF-1 pathway in human RPE cells.

Methods

pFLAG-PHD₁, pFLAG-PHD₂ and pFLAG-PHD₃ plasmids were constructed by extracting RNA from Hela cell line and coloning PHD₁, PHD₂ and PHD₃ using reverse transcription PCR with restriction enzyme.The plasmids were identified by gene sequencing.ARPE-19 cells were cultured at 21% O₂ (normoxia group), 1% O₂ (hypoxia group), or in hypoxia-mimicking agents (CoCl₂, anoxia group), respectively, and then were transfected with plasmids encoding FLAG-tagged PHD₁, PHD₂, PHD₃ and pFLAG-CMV2 transfected cells served as blank control.The expressional intensities of PHD₁, PHD₂ and PHD₃ in the cells were detected and compared among different groups by using Western blot assay.The transcriptional activity of HIF-1 in the cells was evaluated with dual luciferase reporter assay.

Results

Western blot assay showed that PHD₁, PHD₂ and PHD₃ all were expressed in ARPE-19 cells in the normoxia group, hypoxia group and anoxia group.The expression was strong in PHD₂ protein and was weak in PHD₃protein, a statistically significant difference was found between PHD₂ protein expression and PHD₁ or PHD₃expressions (all at P<0.05). Endogenous HIF-1 activity was elevated in pFLAG-CMX transfected cells in the hypoxia group and anoxia group than that in the normoxia group.Compared with pFLAG-CMX transfected cells, no obvious change was seen in the endogenous HIF-1 activity in the normoxia group, however, HIF-1 activity was declined in the hypoxia group and anoxia group after pFLAG-PHD₁, pFLAG-PHD₂ or pFLAG-PHD₃ transfection.Under the same oxygen environment, HIF-1 activity was lower in the pFLAG-PHD₂ transfected cells than that in the pFLAG-PHD₁ or pFLAG-PHD₃ transfected cells (both at P<0.05).

Conclusions

PHDs play a negative regulation to HIF-1 activating pathway in human RPE cells, especially in hypoxia and anoxia cells.Among PHDs proteins, PHD₂ presents the strongest inhibition on HIF-1 activating pathway.

Cell hypoxia; Vascular endothelial growth factors; Gene expression regulation; Hypoxia-inducible factor-1; Procollagen-proline dioxygenase/metabolism; Humans; Transcription factors; Epithelial cells, retinal