title: Lenticular mitoprotection. Part B: GSK-3β and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.
abstract: OBJECTIVE
  Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mitochondrial electrochemical gradient that leads to mitochondrial membrane permeability transition (mMPT). This study emphasizes the role of glycogen synthase kinase 3beta (GSK-3β) in maintaining mitochondrial membrane potential, thus preventing mitochondrial depolarization (hereafter termed mitoprotection). Using 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), an inhibitor of GSK-3β, and drawing a distinction between it and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), an inhibitor of extracellular-signal-regulated kinase (ERK) phosphorylation, the means by which GSK-3β influences mitoprotection in cultured human lens epithelial (HLE-B3) cells and normal, secondary cultures of bovine lens epithelial cells, maintained in atmospheric oxygen, was investigated.
  
  
  METHODS
  Virally transfected human lens epithelial cells (HLE-B3) and normal cultures of bovine lens epithelial cells were exposed to acute hypoxic conditions (about 1% O2) followed by exposure to atmospheric oxygen (about 21% O2). Specific antisera and western blot analysis was used to examine the state of phosphorylation of ERK and GSK-3β, as well as the phosphorylation of a downstream substrate of GSK-3β, glycogen synthase (GS, useful in monitoring GSK-3β activity). The potentiometric dye, 1H-benzimidazolium-5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1,3-diethyl-iodide (JC-1), was used to monitor mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Caspase-3 activation was scrutinized to determine whether mitochondrial depolarization inevitably leads to apoptosis.
  
  
  RESULTS
  Treatment of HLE-B3 cells with SB216763 (12 µM) inactivated GSK-3β activity as verified by the enzyme's inability to phosphorylate its substrate, GS. SB216763-treated cells were not depolarized relative to the control cells as demonstrated with JC-1 fluorescent dye analysis. The HLE-B3 cells treated with UO126, which similarly blocked phosphorylation of GS, were nevertheless prone to mMPT relative to the control cells. Western blot analysis determined that Bcl-2-associated X (BAX) levels were unchanged for SB216763-treated or UO126-treated HLE-B3 cells when compared to their respective control cells. However, unlike the SB216763-treated cells, the UO126-treated cells showed a marked absence of Bcl-2, as well as phosphorylated Bcl-2 relative to the controls. UO126 treatment of bovine lens epithelial cells showed similar results with pBcl-2 levels, while the Bcl-2 content appeared unchanged relative to the control cells. HLE-B3 and normal bovine lens cell cultures showed susceptibility to mMPT associated with the loss of pBcl-2 by UO126 treatment.
  
  
  CONCLUSIONS
  MITOCHONDRIAL DEPOLARIZATION MAY OCCUR BY ONE OF TWO KEY OCCURRENCES: interruption of the electrochemical gradient across the inner mitochondrial membrane resulting in mMPT or by disruption of the integrity of the inner or outer mitochondrial membrane. The latter scenario is generally tightly regulated by members of the Bcl-2 family of proteins. Inhibition of GSK-3β activity by SB216763 blocks mMPT by preventing the opening of the mitochondrial permeability transition pore. UO126, likewise, inhibits GSK-3β activity, but unlike SB216763, inhibition of ERK phosphorylation induces the loss of intracellular pBcl-2 levels under conditions where intracellular BAX levels remain constant. These results suggest that the lenticular mitoprotection normally afforded by the inactivation of GSK-3β activity may, however, be bypassed by a loss of pBcl-2, an anti-apoptotic member of the Bcl-2 family. Bcl-2 prevents the translocation of BAX to the mitochondrial outer membrane inhibiting depolarization by disrupting the normal electrochemical gradient leading to mMPT.
authors: Department of Cell Biology and Anatomy, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX.
authors: Morgan M
authors: MM
authors: Brooks
authors: Sudha
authors: S
authors: Neelam
authors: Patrick R
authors: PR
authors: Cammarata
mesh_terms: D000818:Animals; D001562:Benzimidazoles; D002070:Butadienes / Q000494:pharmacology; D002232:Carbocyanines; D002417:Cattle; D015687:Cell Hypoxia / Q000235:genetics; D002460:Cell Line; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D005786:Gene Expression Regulation; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008301:Maleimides / Q000494:pharmacology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000235:genetics; D008928:Mitochondria / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051025:bcl-Associated Death Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C494819:BAD protein, human; C494826:BAX protein, human; D001562:Benzimidazoles; D002070:Butadienes; D002232:Carbocyanines; D005456:Fluorescent Dyes; D007211:Indoles; D008301:Maleimides; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D009570:Nitriles; D047428:Protein Kinase Inhibitors; C417521:SB 216763; C113580:U 0126; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D020928:Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3; D010100:Oxygen