Down-regulation of AQP5 is not correlated with significant changes in promoter methylation
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During mammary gland differentiation it has been observed that the membrane water channel aquaporin 5 (AQP5) is down-regulated. This down-regulation can also be achieved in an in vitro system with the lactogenic hormone mix: dexamethasone (Dex), insulin and prolactin, which induces cell differentiation. Separate evidence has shown that AQP5 can be regulated by promoter methylation. We wanted to test whether AQP5 expression is regulated by DNA methylation in mammary gland differentiation.
Mouse mammary gland epithelial cells (EpH4) were treated with Dex in the presence of solubilised basement membrane matrix Matrigel, providing a non-dividing cell system. Changes in AQP5 expression levels were measured at the protein and mRNA level using Western blots and quantitative PCR, respectively. In addition, we also assessed changes in DNA methylation using three different methods: bacterial bisulfite sequencing, bead emulsion amplification and restriction fragment length polymorphism analysis allowing the investigation of DNA methylation at the level of single cells, at the level of single sites in up to hundreds of thousands of cells and at the level of cell pools. We observed a 5 fold decrease of the AQP5 mRNA already after 2 hours of Dex addition. No further decrease was observed with longer treatment periods. At the protein level, we also measured a decrease but with a delay of several hours compared to mRNA expression. The observed decrease in expression levels of AQP5 was not coupled to changes in AQP5 promoter methylation. Interestingly, CpG sites at the AQP5 promoter were highly methylated before Dex treatment in spite of the high AQP5 expression levels.
We could show that in non-dividing cells, down regulation of AQP5 mRNA and protein as a result of Dex treatment is independent of changes in DNA methylation. It is possible that the role of promoter methylation is different in dividing cells than in non-dividing cells, at least in AQP5 expression.