Inhibition of Glycogen Synthase Kinase (GSK-3) Protects Against Oxidative Stress and Attenuates Apoptosis in Human Lens Epithelial Cells and the Mouse Lens in Organ Culure

• Purpose: GSK-3 may regulate Wnt signaling, gene expression, the cell cycle, cell differentiation and apoptosis. Inhibition of GSK-3 can be obtained via the structurally unrelated substances lithum or Kenpaullone. The lens and the lens epithelial cells are excellent models to study the role of this enzyme. Methods: Primary cultures of human lens epithelial cells (HLEC) or the mouse lens in organ culture were exposed to the GSK-3 inhibitors lithium (2 mM) or Kenpaullone (2 {micro}M) for times upp to 24h.The cells were, before or after treatment, placed in medium containing fluorogenic indicators of oxidative damage. DCFH-DA was used to assay peroxides, mitochondrial function was evaluated with Rhodamine 123 and JC-1, monochlorobimane was used to assay intracellular glutathione (GSH) levels, Proteolytic activities were assayed, on line, with cell-permeable fluorogenic substrates.Proteasome and calpain activities were assayed with LLVY-AMC, Cathepsin B with RR-AMC or FR-AMC. Metalloproteases were assayed with AAF-AMC. Caspase-3, 8 and 9 were assayed in cell extracts with DEVD-, IETD- or LEHD-AMC, respectively. Results: The mitochondrial membrane potential and the level of GSH increased by 10% after treatment of HLEC with Li or Kenpaullone for 24h. No change was observed for peroxide production. The basal (low) level of caspase-3 activity was decreased by at least 20%. No significant effects were found concerning caspase-8 or 9 activities. No effect was observed on the activity of calpain, the proteasome, metalloproteases and cathepsin D/E activity.The whole mouse lens in organ culture showed essentially the same elevated mitochondrial potential. The GSH increase was even more evident in the whole lens preparation. Conclusions: Inhibition of GSK-3 may protect against oxidative stress (and cataract) via prevention of MPT induction and attenuate apoptosis in HLEC.

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MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Cell- och molekylärbiologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Cell and Molecular Biology (hsv//eng)

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