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Kulandaiappan Varadaraj, Murali Varadaraj, George J. Baldo, Anil G. Menon, Sindhu S. Kumari; Spatial Expression, Protein Trafficking and Post-Translational Modification of AQP5 in Mammalian Cornea and Lens. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5342.
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© ARVO (1962-2015); The Authors (2016-present)
The present investigation studies the spatial expression, protein trafficking and post-translational modifications of AQP5 in the transparent tissues of eye, namely cornea and lens.
Spatial expression and protein trafficking were studied by immunostaining and western blotting analyses in the wild type (WT), and AQP5 knockout (AQP5-/-) mice. Western blotting was also performed to test for expression of AQP5 in rabbit. Protein trafficking and post-translational modifications were investigated using immunostaining and immunoblot analyses. Protein Kinase A (PKA) - induced phosphorylation effect on mouse AQP5 expression and trafficking was investigated in vitro using stably expressing MDCK cells and ex vivo by culturing mouse corneal cells.
Immunostaining of WT mouse cornea and lens sections showed expression of AQP5 in epithelial cells, and keratocytes of cornea, as well as in epithelial and fiber cells of lens. Immunoblotting of proteins from cornea, isolated lens epithelial cells, cortical fiber cells and nuclear fiber cells of both rabbit and mouse all showed the expression of AQP5. As expected, both immunostaining and immunoblotting of AQP5-/- mice cornea and lens did not show anti-AQP5 antibody binding. Both rabbit and mouse corneas and lenses expressed non-phosphorylated and phosphorylated forms of AQP5 protein. Expression of non-phosphorylated form of AQP5 was about ten-fold higher than the phosphorylated form. In vitro studies using mouse AQP5 expressed in MDCK cells also showed both non-phosphorylated and phosphorylated protein bands. These results suggest AQP5 may play a significant role in maintaining the transparency and homeostasis of the avascular cornea and lens. PKA agonist cAMP (100 µM), reduced AQP5 plasma membrane localization and promoted AQP5 internalization; in contrast, PKA antagonist H-89 (20 µM) retained AQP5 protein in the plasma membranes of the MDCK cells (transfected to express AQP5), corneal and lens epithelial cells. When the cells were pretreated with H-89 before exposure to cAMP there was not much internalization of AQP5 protein compared to the PKA activation, suggesting H-89 blocks the action of cAMP.
To our knowledge, this is the first report on the spatial expression of AQP5 protein in the corneal keratocytes, and lens epithelial and fiber cells. This study documents the role of PKA in the localization of AQP5 in the plasma membrane. Our data suggest AQP5 may be regulated by the PKA signaling pathway in the cornea to maintain water homeostasis to prevent dry eye syndrome, and in the lens for microcirculation to supply nutrients and to remove metabolic by-products to keep the lens transparent.
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