Occludin is required for TRPV1-modulated paracellular permeability in the submandibular gland.
Occludin plays an important role in maintaining tight junction barrier function in many types of epithelia. We previously reported that activation of transient receptor potential vanilloid subtype 1 (TRPV1) in rabbit submandibular gland promoted salivary secretion, partly by an increase in paracellular permeability. We have now explored the role of occludin in TRPV1-modulated paracellular permeability in a rat submandibular gland cell line SMG-C6. Both TRPV1 and occludin were expressed in SMG-C6 cells, and capsaicin induced redistribution of occludin, but not claudin-3, claudin-4 or E-cadherin, from the cell membrane into the cytoplasm. Capsaicin also decreased transepithelial electrical resistance (TER) and increased the Trypan Blue and FITC-dextran flux. Capsazepine (CPZ), a TRPV1 antagonist, inhibited the capsaicin-induced occludin redistribution and TER decrease. Moreover, occludin knockdown by shRNA suppressed, whereas occludin re-expression restored, the TER response to capsaicin. Mechanistically, TRPV1 activation increased ERK1/2 and MLC2 phosphorylation. PD98059, an ERK1/2 kinase inhibitor, abolished the capsaicin-induced MLC2 phosphorylation, whereas ML-7, an MLC2 kinase inhibitor, did not affect ERK1/2 phosphorylation, suggesting that ERK1/2 is the upstream signaling molecule of MLC2. Capsaicin also induced F-actin reorganization, which was abolished by CPZ, PD98059 and ML-7, indicating that TRPV1 activation altered F-actin organization in an ERK1/2- and MLC2-dependent manner. Furthermore, either PD98059 or ML-7 could abolish the capsaicin-induced TER response and occludin redistribution, whereas knockdown of ERK1/2 further confirmed that the TRPV1-modulated paracellular permeability was ERK1/2 dependent. Taken together, these results identified a crucial role of occludin in submandibular epithelial cells, and more importantly, demonstrated that occludin was required to mediate TRPV1-modulated paracellular permeability.
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, China.
Xin
X
Cong
Yan
Y
Zhang
Ning-Yan
NY
Yang
Jing
J
Li
Chong
C
Ding
Qian-Wen
QW
Ding
Yun-Chao
YC
Su
Mei
M
Mei
Xiao-Hong
XH
Guo
Li-Ling
LL
Wu
Guang-Yan
GY
Yu
D000818:Animals; D015153:Blotting, Western; D002211:Capsaicin / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D005419:Flavonoids / Q000494:pharmacology; D047468:Immunoprecipitation; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D062793:Occludin / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013363:Submandibular Gland / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism
D005419:Flavonoids; D062793:Occludin; D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; C071423:capsazepine; D002211:Capsaicin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one