Epigenetic regulation of CFTR in salivary gland.
Cystic fibrosis transmembrane conductance regulator (CFTR) plays a key role in exocrine secretion, including salivary glands. However, its functional expression in salivary glands has not been rigorously studied. In this study, we investigated the expression pattern and regulatory mechanism of CFTR in salivary glands using immunohistochemistry, western blot analysis, Ussing chamber study, methylation-specific PCR, and bisulfite sequencing. Using an organ culture technique, we found that CFTR expression was first detected on the 15th day at the embryonic stage (E15) and was observed in ducts but not in acini. CFTR expression was confirmed in HSG and SIMS cell lines, which both originated from ducts, but not in the SMG C-6 cell line, which originated from acinar cells. Treatment of SMG C-6 cells with 5-aza-2'-deoxycytidine (5-Aza-CdR) restored the expression level of CFTR mRNA in a time-dependent manner. Restoration of CFTR was further confirmed by a functional study. In the Ussing chamber study, 10 μM Cact-A1, a CFTR activator, did not evoke any currents in SMG C-6 cells. In contrast, in SMG C-6 cells pretreated with 5-Aza-CdR, Cact-A1 evoked a robust increase of currents, which were inhibited by the CFTR inhibitor CFTRinh-172. Furthermore, forskolin mimicked the currents activated by Cact-A1. In our epigenetic study, SMG C-6 cells showed highly methylated CG pairs in the CFTR CpG island and most of the methylated CG pairs were demethylated by 5-Aza-CdR. Our results suggest that epigenetic regulation is involved in the development of salivary glands by silencing the CFTR gene in a tissue-specific manner.
Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Republic of Korea.
Yong-Hwan
YH
Shin
Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Republic of Korea.
Sang-Woo
SW
Lee
Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Republic of Korea.
Minkyoung
M
Kim
Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Republic of Korea.
Se-Young
SY
Choi
Center for Salivary Gland Diseases, Peking University School of Stomatology, Beijing 100081, China.
Xin
X
Cong
Center for Salivary Gland Diseases, Peking University School of Stomatology, Beijing 100081, China.
Guang-Yan
GY
Yu
Department of Physiology, School of Dentistry, Seoul National University and Dental Research Institute, Seoul 110-749, Republic of Korea. Electronic address: kppark@snu.ac.kr.
Kyungpyo
K
Park
D061354:Acinar Cells / Q000502:physiology*; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D012469:Salivary Glands / Q000196:embryology* / Q000502:physiology*
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
5-Aza-2′-deoxycytidine; CFTR; DNA methylation; Epigenetic regulation; Organ culture; Salivary glands