Effect of dexamethasone and aquaporin-1 antisense oligonucleotides on the aquaporin-1 expression in cultured human trabecular meshwork cells

Abstract

The changes in the expression of aquaporin-1 (AQP1) mRNA and protein in cultured human trabecular meshwork (HTM) cells treated with dexamethasone and transfected with antisense oligonucleotides (AS-ODN) were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cellsin vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group (0 μg/mL). In the 4 μg/mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.

Keywords

trabecular meshwork cells / aquaporin-1 / dexamethasone / antisense oligonucleotides

References

[1] VerkmanA S. Role of aquaporin water channels in eye function. Exp Eye Res, 2003, 76: 137-143

[2] MulderJ, ChakrayartyS, HaddadM N, et al.. Glucocorticoids increase osmotic water permeability (Pf) of neonatal rabbit renal brush border membrane vesicles. Am J Physiol Regul Integr Comp Physiol, 2005, 288: 1417-1421

[3] StoenoiuM S, NiJ, VerkaerenC, et al.. Corticosteroids induce expression of aquaporin-1 and increase transcellular water transport in rat peritoneum. J Am Soc Nephrol, 2003, 14: 555-565

[4] FukushimaM, KitaharaT, FuseY, et al.. Changes in aquaporin expression in the inner ear of the rat after i. p. injection of steroids. Acta Otolaryngol Suppl, 2004, 553: 13-18

[5] HanZ, PatilR V. Protein kinase A-dependent phosphorylation of aquaporin-1. Biochem Biophys Res Commun, 2000, 273: 328-332

[6] SakuradaT, KuboshimaS, OgimotoG, et al.. Aquaporin-1 is recruited to the plasma membrane by hyperosmotic stimuli via a protein kinase A-dependent pathway in rat peritoneal mesothelial cells. Adv Perit Dial, 2004, 20: 37-42

[7] ZhangD, VetrivelL, VerkmanA S. Aquaporin deletion in mice reduces intraocular pressure and aqueous fluid production. J Gen Physiol, 2002, 119: 561-569

[8] SunX C, AllenK T, XieQ, et al.. Effect of AQP1 expression level on Co(2) permeability in bovine corneal endothelium. Invest Ophthalmol Vis Sci, 2001, 42: 417-423