title: Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells.
abstract: OBJECTIVE
  The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells.
  
  
  METHODS
  HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression.
  
  
  RESULTS
  SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed.
  
  
  CONCLUSIONS
  Recently, we reported that nuclear β-catenin, but not HIF-2α, regulates the expression of fibronectin and α-SMA in atmospheric oxygen. In marked contrast, data from the hypoxic condition clearly establish that nuclear β-catenin plays little apparent role in the expression of EMT marker proteins. Instead, the loss of HIF-1α (but not HIF-2α) decreases the expression of the EMT marker proteins without sacrificing the levels of the prosurvival protein VEGF. These findings support the development of a potentially relevant therapeutic strategy to undermine the progression of normal cells to the mesenchymal phenotype in the naturally hypoxic lens without subverting cell viability.
authors.affiliation: Department of Cell Biology and Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX.
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Cammarata
authors.affiliation: Department of Cell Biology and Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX.
authors.forename: Sudha
authors.initials: S
authors.lastname: Neelam
authors.affiliation: Department of Cell Biology and Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX.
authors.forename: Morgan M
authors.initials: MM
authors.lastname: Brooks
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D051792:Basic Helix-Loop-Helix Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015687:Cell Hypoxia; D002461:Cell Line, Transformed; D002470:Cell Survival; D004220:Disulfides / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects* / Q000235:genetics; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008301:Maleimides / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology*; D014176:Protein Biosynthesis; D021381:Protein Transport; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D051176:beta Catenin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D000199:Actins; D051792:Basic Helix-Loop-Helix Proteins; D004220:Disulfides; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D006575:Heterocyclic Compounds, 3-Ring; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007211:Indoles; D008301:Maleimides; D010100:Oxygen; D013449:Sulfonamides; D042461:Vascular Endothelial Growth Factor A; D051176:beta Catenin; D000099162:Endothelial PAS Domain-Containing Protein 1; C541116:ACTA2 protein, human; C497442:HIF1A protein, human; C545729:KC7F2 compound; C417521:SB 216763; C467484:VEGFA protein, human; C000605506:GSK3B protein, human