title: Lenticular cytoprotection, part 2: link between glycogen synthase kinase-3β, epithelial to mesenchymal transition, and mitochondrial depolarization.
abstract: OBJECTIVE
  The inhibition of GSK-3β blocks mitochondrial membrane permeability transition (mMPT) for HLE-B3 cells in atmospheric oxygen. GSK-3β, as part of a multifactorial complex, also regulates nuclear levels of β-catenin, a known coordinator of cell survival and adhesion. The purpose of these studies was to demonstrate a novel, but likely disadvantageous, link between β-catenin's influence on the expression of the pro-survival protein, vascular endothelial growth factor (VEGF), resulting in enhanced lens epithelial cell mitochondrial protection against depolarization and nuclear β-catenin as an inducer of epithelial to mesenchymal transition (EMT).
  
  
  METHODS
  Virally transformed human lens epithelial cells (HLE-B3) were treated with SB216763, a specific inhibitor of GSK-3β catalytic activity and XAV939, a specific β-catenin inhibitor that bars the translocation of β-catenin from cytoplasm to the nucleus. Western blot analysis was employed to detect the levels of cytoplasmic and nuclear β-catenin and phospho-β-catenin, pBcl-2 and the EMT proteins, α-smooth muscle actin (α-SMA), and fibronectin. ELISA was used to measure the levels of VEGF in cell culture supernatants. JC-1 analysis was performed to analyze the influence of either SB216763 or XAV939 on mitochondrial depolarization.
  
  
  RESULTS
  Cultured lens epithelial cells maintained in hypoxia (1% oxygen) and subsequently reintroduced into atmospheric oxygen and treated with the GSK-3β inhibitor SB216763 illustrated a marked inhibition of phosphorylation of glycogen synthase (downstream substrate of GSK-3β) and significant increase in nuclear translocation of β-catenin. The augmented nuclear β-catenin levels positively correlated with increased expression of α-SMA and fibronectin, both marker proteins indicative of EMT. The enhanced nuclear β-catenin activity also elicited increased VEGF and pBcl-2 expression, resulting in increased resistance to mitochondrial depolarization. Treatment of the cells with the β-catenin inhibitor XAV939 resulted in decreased expression of nuclear β-catenin, VEGF levels, pBcl-2, and EMT proteins, as well as increased mitochondrial depolarization.
  
  
  CONCLUSIONS
  The data support a model whereby the onset of epithelial to mesenchymal transition may circuitously benefit from the enhanced synthesis of VEGF by setting up a potentially harmful situation whereby the resulting mesenchymal cell population may be more resistant to mitochondrial depolarization than the lens epithelial cell population from which it originated. These findings support the potential therapeutic relevance of developing strategies to undermine the progression of normal cells to mesenchymal transition 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: 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
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
mesh_terms: D000818:Animals; D002417:Cattle; D015687:Cell Hypoxia; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D008301:Maleimides / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008954:Models, Biological; D021381:Protein Transport / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
chemical_list: C495270:CTNNB1 protein, human; D004791:Enzyme Inhibitors; D006575:Heterocyclic Compounds, 3-Ring; D007211:Indoles; D008301:Maleimides; D019253:Proto-Oncogene Proteins c-bcl-2; C417521:SB 216763; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C544261:XAV939; D051176:beta Catenin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3