Placental growth factor promotes epithelial-mesenchymal transition-like changes in ARPE-19 cells under hypoxia

Mol Vis. 2018 Apr 26;24:340-352. eCollection 2018.

Abstract

Purpose: To investigate the role of placental growth factor (PGF) in the epithelial-mesenchymal transition (EMT) of ARPE-19 cells under hypoxia, and whether the NF-κB signaling pathway is involved in this process.

Methods: ARPE-19 cells were treated in five groups: a control group, hypoxia group, PGF group, hypoxia+PGF group, and NF-κB-blocked group. A chemical hypoxia model was established in the ARPE-19 cells by adding CoCl2 to the culture medium. The morphological changes after treatment were observed. The proliferation rates were measured with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The migration abilities were measured with scratch assay. The EMT biomarkers were measured with quantitative real-time PCR (qRT-PCR), western blotting, and immunofluorescence. The relative protein expression of components of the NF-κB signaling pathway was measured with western blotting and immunofluorescence.

Results: Cells treated with PGF under hypoxia exhibited morphological changes consistent with the transition from an epithelial to a mesenchymal phenotype. In the ARPE-19 cells, exogenous PGF under hypoxia increased the proliferation rate compared to the rate under hypoxia alone (p<0.05) and increased the migration rate (p<0.05). Treatment of hypoxia-exposed cells with PGF caused decreased expression of the epithelial biomarkers E-cadherin and ZO-1 (both p<0.05) and increased expression of the mesenchymal marker α-SMA (p<0.05) by enhancing the phosphorylation of NF-κB p65 of the total protein, promoting the translocation of p65 to the nucleus, and inducing the degradation of IκB-α (a negative regulator of the NF-κB pathway) in the ARPE-19 cells. Additionally, the effect of PGF-induced EMT in the ARPE-19 cells under hypoxia was counteracted with BAY 11-7082 (a selective NF-κB inhibitor).

Conclusions: Exogenous PGF promotes EMT-like changes in ARPE-19 cells under hypoxia by activating the NF-κB signaling pathway. The study results suggest that PGF may play a role in scar formation in neovascular age-related macular degeneration (AMD) and that the inhibition of PGF may be a promising target for the prevention and treatment of AMD.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / genetics
  • Actins / metabolism
  • Antigens, CD
  • Cadherins / genetics
  • Cadherins / metabolism
  • Cell Hypoxia
  • Cell Line
  • Cell Movement / drug effects
  • Cell Proliferation / drug effects
  • Cobalt / pharmacology*
  • Epithelial Cells / cytology
  • Epithelial Cells / drug effects*
  • Epithelial Cells / metabolism
  • Epithelial-Mesenchymal Transition / drug effects*
  • Gene Expression Regulation
  • Humans
  • NF-KappaB Inhibitor alpha / genetics
  • NF-KappaB Inhibitor alpha / metabolism
  • Nitriles / pharmacology
  • Placenta Growth Factor / pharmacology*
  • Retinal Pigment Epithelium / cytology
  • Retinal Pigment Epithelium / drug effects
  • Retinal Pigment Epithelium / metabolism
  • Signal Transduction / drug effects*
  • Sulfones / pharmacology
  • Transcription Factor RelA / agonists
  • Transcription Factor RelA / antagonists & inhibitors
  • Transcription Factor RelA / genetics*
  • Transcription Factor RelA / metabolism
  • Zonula Occludens-1 Protein / genetics
  • Zonula Occludens-1 Protein / metabolism

Substances

  • 3-(4-methylphenylsulfonyl)-2-propenenitrile
  • ACTA2 protein, human
  • Actins
  • Antigens, CD
  • CDH1 protein, human
  • Cadherins
  • Nitriles
  • PGF protein, human
  • Sulfones
  • TJP1 protein, human
  • Transcription Factor RelA
  • Zonula Occludens-1 Protein
  • NF-KappaB Inhibitor alpha
  • Placenta Growth Factor
  • Cobalt
  • cobaltous chloride