Abstract
Anoctamin1 (ANO1), a calcium activated chloride channel, is known to play a critical role in salivary secretion. In the salivary gland, ANO1 is expressed exclusively in the acinar cells, with no expression in the ductal cells. However, the mechanisms that determine this distinctive cell type-dependent expression pattern of ANO1 remain unknown. In this study, we discovered that the cell-dependent expression of ANO1 during salivary gland organogenesis is regulated by DNA methylation of ANO1 CpG islands. ANO1 CpG islands in e12 embryonic submandibular glands (eSMG) are highly methylated, but those in e14 eSMG or adult SMG are significantly unmethylated. The differential expression pattern of ANO1 in duct and acini is defined at e14. Artificial demethylation by treatment with the demethylating agent 5-aza-2’-deoxycytidine (5-Aza-CdR), induced the expression of ANO1 in both the ductal cell line Human Submandibular Gland (HSG) and in the duct cells of adult mouse SMG. During the trans-differentiation in Matrigel of duct-origin HSG cells into acinar-like phenotype, significant demethylation of ANO1 CpG islands is observed. This may be due to the reduced expression of DNA methyltransferase (DNMT) 3a and 3b. These results suggest that the differential expression of ANO1 in salivary glands during organogenesis and differentiation is mainly regulated by epigenetic demethylation of the ANO1 gene.
Highlights
ANO1 is a Ca2+ activated Cl− channel (CaCC), which is widely expressed in tissues such as sensory neurons, smooth muscle cells, and salivary glands
ANO1 is mainly expressed in the apical membrane of the secretory acinar cells, but not in the epithelial cells that line the ducts of the gland [2]
A previous study reported that ANO1 expression is regulated by epigenetic mechanisms involving DNA methylation of the ANO1 gene promoter region in head and neck squamous cell carcinomas, but similar studies have not been conducted on normal salivary gland tissue [3]
Summary
ANO1 is a Ca2+ activated Cl− channel (CaCC), which is widely expressed in tissues such as sensory neurons, smooth muscle cells, and salivary glands. A previous study reported that ANO1 expression is regulated by epigenetic mechanisms involving DNA methylation of the ANO1 gene promoter region in head and neck squamous cell carcinomas, but similar studies have not been conducted on normal salivary gland tissue [3]. The epigenetic regulation of ANO1 in salivary gland duct and acinar cells is examined under more physiologic conditions by using Matrigel-induced trans-differentiation of the Human Salivary Gland (HSG) cell line as model of development. The HSG cell line is derived from intercalated ducts of irradiated human submandibular glands, and is known to trans-differentiate into acinar cells when cultured on basement membrane extracts such as Matrigel or laminin-1 [18,19,20] By using this and other experimental models, we demonstrate that the differential expression of ANO1 in acini and duct is regulated by epigenetic mechanisms
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