title: Claudin-4 is required for modulation of paracellular permeability by muscarinic acetylcholine receptor in epithelial cells.
abstract: The epithelial cholinergic system plays an important role in water, ion and solute transport. Previous studies have shown that activation of muscarinic acetylcholine receptors (mAChRs) regulates paracellular transport of epithelial cells; however, the underlying mechanism is still largely unknown. Here, we found that mAChR activation by carbachol and cevimeline reduced the transepithelial electrical resistance (TER) and increased the permeability of paracellular tracers in rat salivary epithelial SMG-C6 cells. Carbachol induced downregulation and redistribution of claudin-4, but not occludin or ZO-1 (also known as TJP1). Small hairpin RNA (shRNA)-mediated claudin-4 knockdown suppressed, whereas claudin-4 overexpression retained, the TER response to carbachol. Mechanistically, the mAChR-modulated claudin-4 properties and paracellular permeability were triggered by claudin-4 phosphorylation through ERK1/2 (also known as MAPK3 and MAPK1, respectively). Mutagenesis assay demonstrated that S195, but not S199, S203 or S207, of claudin-4, was the target for carbachol. Subsequently, the phosphorylated claudin-4 interacted with β-arrestin2 and triggered claudin-4 internalization through the clathrin-dependent pathway. The internalized claudin-4 was further degraded by ubiquitylation. Taken together, these findings suggested that claudin-4 is required for mAChR-modulated paracellular permeability of epithelial cells through an ERK1/2, β-arrestin2, clathrin and ubiquitin-dependent signaling pathway.
authors.affiliation: Center for Salivary Gland Diseases of Peking University School and Hospital of Stomatology, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, China Neuroscience Research Institute and Department of Neurobiology, The Key Laboratory for Neuroscience of the Ministry of Education and Health, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Cong
authors.affiliation: Center for Salivary Gland Diseases of Peking University School and Hospital of Stomatology, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing 100081, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology, Beijing 100081, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Mei
authors.affiliation: Central Laboratory, Peking University School and Hospital of Stomatology, Beijing 100081, China.
authors.forename: Chong
authors.initials: C
authors.lastname: Ding
authors.affiliation: Center for Salivary Gland Diseases of Peking University School and Hospital of Stomatology, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, China.
authors.forename: Ruo-Lan
authors.initials: RL
authors.lastname: Xiang
authors.affiliation: Department of Oral Medicine and Traditional Chinese Medicine, Peking University School and Hospital of Stomatology, Beijing 100081, China.
authors.forename: Li-Wei
authors.initials: LW
authors.lastname: Zhang
authors.affiliation: Neuroscience Research Institute and Department of Neurobiology, The Key Laboratory for Neuroscience of the Ministry of Education and Health, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Center for Salivary Gland Diseases of Peking University School and Hospital of Stomatology, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing Key Laboratory of Cardiovascular Receptors Research, Beijing 100191, China pathophy@bjmu.edu.cn gyyu@263.net.
authors.forename: Li-Ling
authors.initials: LL
authors.lastname: Wu
authors.affiliation: Neuroscience Research Institute and Department of Neurobiology, The Key Laboratory for Neuroscience of the Ministry of Education and Health, Peking University Health Science Center, Beijing 100191, China pathophy@bjmu.edu.cn gyyu@263.net.
authors.forename: Guang-Yan
authors.initials: GY
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured; D062506:Claudin-4 / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D017097:Electric Impedance; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010539:Permeability; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012469:Salivary Glands / Q000166:cytology / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D019108:Tight Junctions; D062826:Zonula Occludens-1 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D062506:Claudin-4; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011976:Receptors, Muscarinic; D062826:Zonula Occludens-1 Protein; D000071557:beta-Arrestins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: Claudin-4; Muscarinic acetylcholine receptor; Paracellular permeability; Tight junction