Up-regulated Aquaporin-5 in human salivary gland cells treated with 5-aza-2`-deoxycytidine

Objective: Sjögrens syndrome (SjS) is characterized by dry eyes and dry mouth. Defective secretion in SjS is believed to be caused by impaired trafficking of aquaporin-5 (AQP-5), a water channel protein, in acinar cells in the presence of anti-muscarinic type-3-receptor (M3R) autoantibodies. Human salivary gland cell line (HSG) utilized for SjS research, however, exhibits minimal endogenous Aqp-5 expression potentially due to hypermethylation of CpG islands within the promoter and 5` regions of the gene. The aim of our study was to investigate if HSG ductal cell line could acquire increased Aqp-5 gene expression in response to 5-aza-2`-deoxycytidine (5-aza-CdR) treatment, a DNA demethylating agent.

Methods: HSG cells were grown in 6-well plates with each well containing 1x106 cells in the presence of 2µM concentration of 5-aza-CdR. Total cellular RNA was isolated at 0, 48, 72, 96, and 120 hours after treatment. The expression of Aqp-5 was analyzed by RT-PCR and Western Blot Analysis.

Results: Densitometer analysis revealed a fifteen-fold (p < 0.01) increase in Aqp-5 in the treated HSG cells compared to that of the negative control or non-treated cells after 48 hour incubation. There was a gradual increase over time even after 72 hours with a two-fold increase from the previous. A similar trend was observed in the western blot analysis of AQP-5 with about two-fold increase after 72 hour treatment compared to that of non-treated. However, densitometer analysis on M3R gene expression revealed a similar expression level between the non-treated and treated cells.

Conclusions: DNA demethylating agent 5-aza-CdR up-regulated Aqp-5 expression at both DNA and protein level, which will allow further research on Aqp-5 in ductal HSG cells. However, the effect on AQP5 was not the same for M3R potentially due to a difference in methylation in the promoter region of M3R (supported by NIH/NIDCR grants U24DE016509 and R21DE016705).