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Inhibition Of Glycogen Synthase Kinase (gsk-3) Affects Markers Of Oxidative Stress And Attenuates Apoptosis In Human Lens Epithelial Cells

Paper i proceeding
Författare Jan-Olof Karlsson
Anne Petersen
Madeleine Zetterberg
Johan Sjöstrand
Publicerad i Invest. Ophthalmol. Vis. Sci.
Volym 45
Nummer/häfte 5
Sidor 3508-
Publiceringsår 2004
Publicerad vid Institutionen för klinisk neurovetenskap, Sektionen för oftalmologi
Institutionen för anatomi och cellbiologi
Sidor 3508-
Språk en
Länkar abstracts.iovs.org/cgi/content/abst...
Ämneskategorier Cell- och molekylärbiologi

Sammanfattning

Purpose: GSK-3, an evolutionary conserved S/T kinase which regulates cell fate determination in diverse organisms have been implicated in the formation of amyloid b-peptides and the phosphorylation of tau and catenin. 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: Confluent human lens epithelial cells (HLEC) 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, 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 with Li or Kenpaullone for 24h. No change was observed for peroxide production. The basal (low) level of caspase-3 activity was decreased by 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. Conclusions:Inhibition of GSK-3 may protect against oxidative damage and attenuate apoptosis in HLEC. No changes of the other major proteolytic systems in the cell were detected. These data may be important for the interpretation of Wnt signaling and cell growth in HLEC as well as for the formation of amyloid in the lens.

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