Ishii Yumiko, Saeki Kazuko, Liu Min, Sasaki Fumiyuki, Koga Tomoaki, Kitajima Keiko, Meno Chikara, Okuno Toshiaki, Yokomizo Takehiko
*Department of Medical Biochemistry, Research Institute for Diseases of the Chest, and Department of Developmental Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan; and Department of Endocrinology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
*Department of Medical Biochemistry, Research Institute for Diseases of the Chest, and Department of Developmental Biology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan; and Department of Endocrinology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
FASEB J. 2016 Feb;30(2):933-47. doi: 10.1096/fj.15-279653. Epub 2015 Nov 2.
GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPCRs and epithelial barrier function remains unknown. Leukotriene B4 (LTB4) receptor type 2 (BLT2), mainly expressed in epithelial cells, is a GPCR for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. In our study, BLT2 localized at the lateral membrane in BLT2-overexpressing Madin-Darby canine kidney (MDCK) II cells and in the small intestine of BLT2-transgenic mice. BLT2-deficient mice exhibited higher transepidermal water loss and were more sensitive to epicutaneous sensitization. MDCK-BLT2 cells recovered transepithelial electrical resistance (TER) after a calcium switch faster than did MDCK-Mock cells, and 12-HHT stimulation accelerated TER recovery only in MDCK-BLT2 cells. Quantitative PCR and immunoblot analyses revealed that the 12-HHT/BLT2 axis up-regulated claudin-4 (CLDN4) expression in MDCK-BLT2 cells and human primary keratinocytes, and CLDN4 knockdown abolished 12-HHT-dependent TER recovery. Acceleration of TER recovery and induction of CLDN4 expression by 12-HHT stimulation were abolished by inhibition of Gαi protein or p38 MAPK. These results show that 12-HHT/BLT2 enhances epithelial barrier function by increasing CLDN4 expression via the Gαi protein-p38 MAPK pathway.
G蛋白偶联受体（GPCRs）参与众多生理功能，是重要的药物靶点。尽管上皮屏障对于抵御病原体入侵很重要，但GPCRs与上皮屏障功能之间的相关性仍不清楚。白三烯B4（LTB4）受体2型（BLT2）主要在上皮细胞中表达，是12（S）-羟基十七碳-5Z,8E,10E-三烯酸（12-HHT）和LTB4的GPCR。在我们的研究中，BLT2定位于过表达BLT2的麦氏犬肾（MDCK）II细胞的侧膜以及BLT2转基因小鼠的小肠中。BLT2基因缺陷型小鼠表现出更高的经表皮水分流失，并且对表皮致敏更敏感。MDCK-BLT2细胞在钙转换后恢复跨上皮电阻（TER）的速度比MDCK-Mock细胞快，并且12-HHT刺激仅在MDCK-BLT2细胞中加速了TER的恢复。定量PCR和免疫印迹分析表明，12-HHT/BLT2轴上调了MDCK-BLT2细胞和人原代角质形成细胞中紧密连接蛋白4（CLDN4）的表达，并且CLDN4基因敲低消除了12-HHT依赖性的TER恢复。抑制Gαi蛋白或p38丝裂原活化蛋白激酶（MAPK）可消除12-HHT刺激对TER恢复的加速作用以及CLDN4表达的诱导。这些结果表明，12-HHT/BLT2通过Gαi蛋白-p38 MAPK途径增加CLDN4表达来增强上皮屏障功能。
