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UBC Theses and Dissertations

The role of caveolin-1 phosphorylation in AQP4 membrane expression in a model of oxidative stress in primary astrocyte cultures Bi, Chongshan


Astrocytes play an important role in a wide variety of physiological processes and in disease states such as ischemia. Ischemic damage in the brain involves apoptotic cell death of neurons as well as astrocytes and it has been suggested that reactive oxygen species (ROS) generated as a consequence of ischemia are a major factor in triggering cell death. The waterpermeable channel, aquaporin 4 (AQP4), which is expressed at high concentrations in astrocytes, is an important determinant of mortality and morbidity in mice subjected to ischemia, however the effects of ROS on AQP4 expression and the underlying mechanisms are still obscure. In the present study, we used primary astrocyte cultures to examine the expression of AQP4 under oxidative stress using hydrogen peroxide. First, we showed that H₂O₂ induces a significant increase of both AQP4 mRNA and protein levels and that this effect is inhibited by the antioxidant N-acetylcysteine. Second, we demonstrated using cell surface biotinylation that H₂O₂ increases AQP4 plasma membrane expression independently of the newly synthesized pool of AQP4. In parallel, we found that caveolin-1 undergoes a dose- and time-dependent phosphorylation in astrocytes treated with H₂O₂ and that this effect is inhibited by the src kinase inhibitor PP2. More importantly, PP2 inhibits the H₂O₂-induced increase in AQP4 cell surface expression, suggesting that the phosphorylation of caveolin-1 and possibly other proteins may play a role in this process. To investigate this further, we used MDA-435 cells expressing Y14F and Y14D caveolin-1 mutants transfected with AQP4 and found that these cells exhibit a decrease and an increase in AQP4 membrane expression, respectively. Furthermore, caveolin-1 knock down in astrocytes inhibits H₂O₂-induced increase in AQP4 cell surface expression. Together these findings show that the phosphorylation of caveolin-1 Y14 is a key regulator of AQP4 cell surface expression in oxidative stress possibly by altering AQP4 internalization and trafficking resulting in its redistribution within different compartments of the cell.

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