Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

Toxicol Appl Pharmacol. 2012 Oct 15;264(2):246-54. doi: 10.1016/j.taap.2012.08.005. Epub 2012 Aug 15.

Abstract

Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H(2)O(2) across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H(2)O(2) release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p<0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H(2)O(2) release, assessed by Amplex Red, was reduced by about 45% (p<0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+120%, p<0.05) and loss of mitochondrial membrane potential (-80%, p<0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H(2)O(2) release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Aquaporins / genetics*
  • Aquaporins / physiology*
  • Blotting, Western
  • Cell Survival / genetics*
  • Cell Survival / physiology*
  • Cyclosporine / pharmacology
  • Hep G2 Cells
  • Humans
  • Hydrogen Peroxide / metabolism
  • L-Lactate Dehydrogenase / metabolism
  • Membrane Potential, Mitochondrial / genetics*
  • Microscopy, Fluorescence
  • Mitochondria, Liver / metabolism*
  • Oxidants / metabolism
  • Permeability
  • RNA, Small Interfering / biosynthesis
  • RNA, Small Interfering / genetics
  • Reactive Oxygen Species / metabolism*
  • Tetrazolium Salts
  • Thiazoles
  • Trypan Blue

Substances

  • Aquaporins
  • Oxidants
  • RNA, Small Interfering
  • Reactive Oxygen Species
  • Tetrazolium Salts
  • Thiazoles
  • aquaporin 8
  • Cyclosporine
  • Adenosine Triphosphate
  • Hydrogen Peroxide
  • L-Lactate Dehydrogenase
  • thiazolyl blue
  • Trypan Blue