title: Epigenetic modulation of the muscarinic type 3 receptor in salivary epithelial cells.
abstract: Muscarinic receptors, particularly the type 3 subtype (M3R), have an important role in exocrine secretion. M3R normally function in HSG cells originated from human submandibular gland ducts, but not in A253 and SGT cells, derived from human submandibular carcinoma and salivary gland adenocarcinoma. However, the underlying mechanism of this suppression has remained elusive. In this study, we examined whether M3R function is suppressed by epigenetic modulation of the receptor. To this end, we investigated the mRNA transcript and protein levels of the M3R using reverse transcriptase-PCR, western blot, and confocal microscopy analyses. Global DNA methylation assays, methylation-specific PCR, and bisulfite sequencing were also performed to understand the epigenetic status of the M3R CpG island. We found that A253 cells expressed all subtypes of muscarinic receptors, except M3R, on the mRNA level. However, treatment of cells with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA-demethylating agent, increased the expression levels of both M3R mRNA transcript and protein in proportion to the incubation period. 5-Aza-CdR completely restored the carbachol-induced calcium response, which was not observed in untreated A253 cells. In untreated A253 cells, all CG pairs from the 1st to 14th were methylated and 5-Aza-CdR treatment demethylated one of the methylated CG pairs. We also examined the methylation pattern of M3R CpG island in human cancer tissue. Interestingly, the result was very similar to those of A253 cells. All CG pairs in M3R CpG island were also methylated. Another salivary gland tumor cell line, SGT, also showed the similar methylation pattern, heavy methylation in M3R CpG island. It is concluded that CpG island in M3R is hypermethylated in cancer cell lines and human cancer. Our results further suggest that 5-Aza-CdR could potentially be used to restore the function of M3R, suppressed in some cancer cell types.
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Yong-Hwan
authors.initials: YH
authors.lastname: Shin
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Meihong
authors.initials: M
authors.lastname: Jin
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Sung-Min
authors.initials: SM
authors.lastname: Hwang
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Seul-Ki
authors.initials: SK
authors.lastname: Choi
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Eun
authors.initials: E
authors.lastname: Namkoong
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Minkyoung
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Moon-Yong
authors.initials: MY
authors.lastname: Park
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Se-Young
authors.initials: SY
authors.lastname: Choi
authors.affiliation: Oral and Maxillofacial Surgery, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Jong-Ho
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul, Korea.
authors.forename: Kyungpyo
authors.initials: K
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012469:Salivary Glands / Q000166:cytology* / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D043587:Receptor, Muscarinic M3; D000077209:Decitabine; D001374:Azacitidine