Koizumi Jun-ichi, Kojima Takashi, Ogasawara Noriko, Kamekura Ryuta, Kurose Makoto, Go Mitsuru, Harimaya Atsushi, Murata Masaki, Osanai Makoto, Chiba Hideki, Himi Tetsuo, Sawada Norimasa
Department of Otolaryngology, Sapporo Medical University School of Medicine, S1. W17. Sapporo 060-8556, Japan.
Mol Pharmacol. 2008 Aug;74(2):432-42. doi: 10.1124/mol.107.043711. Epub 2008 May 13.
The epithelium of upper respiratory tissues such as human nasal mucosa forms a continuous barrier via tight junctions, which is thought to be regulated in part through a protein kinase C (PKC) signaling pathway. To investigate the mechanisms of the regulation of PKC-mediated tight junction barrier function of human nasal epithelium in detail, primary human nasal epithelial cells were treated with the PKC activator 12-O-tetradecanoylophorbol-13-acetate (TPA). In primary human nasal epithelial cells, treatment with TPA led not only to activation of phosphorylation of PKC, myristoylated alanine-rich C kinase substrate, and mitogen-activated protein kinase but also expression of novel PKC-delta, PKC-theta, and PKC-epsilon. Treatment with TPA increased transepithelial electrical resistance, with tight junction barrier function more than 4-fold that of the control, together with up-regulation of tight junction proteins, occludin, zona occludens (ZO)-1, ZO-2 and claudin-1 at the transcriptional level. Furthermore, it affected the subcellular localization of the tight junction proteins and the numbers of tight junction strands. The up-regulation of barrier function and tight junction proteins was prevented by a pan-PKC inhibitor, and the inhibitors of PKC-delta and PKC-theta but not PKC-epsilon. In primary human nasal epithelial cells, transcriptional factors GATA-3 and -6 were detected by reverse transcription-polymerase chain reaction. The knockdown of GATA-3 using RNA interference resulted in inhibition of up-regulation of ZO-1 and ZO-2 by treatment with TPA. These results suggest that TPA-induced PKC signaling enhances the barrier function of human nasal epithelial cells via transcriptional up-regulation of tight junction proteins, and the mechanisms may contribute to a drug delivery system.
人类鼻粘膜等上呼吸道组织的上皮细胞通过紧密连接形成连续屏障，据认为部分是通过蛋白激酶C（PKC）信号通路进行调节的。为了详细研究PKC介导的人类鼻上皮紧密连接屏障功能的调节机制，将原代人鼻上皮细胞用PKC激活剂12 - O - 十四酰佛波醇 - 13 - 乙酸酯（TPA）处理。在原代人鼻上皮细胞中，用TPA处理不仅导致PKC、富含肉豆蔻酰化丙氨酸的C激酶底物和丝裂原活化蛋白激酶的磷酸化激活，还导致新型PKC - δ、PKC - θ和PKC - ε的表达。用TPA处理可增加跨上皮电阻，紧密连接屏障功能是对照的4倍多，同时紧密连接蛋白occludin、闭合蛋白（ZO）-1、ZO - 2和claudin - 1在转录水平上调。此外，它影响紧密连接蛋白的亚细胞定位和紧密连接链的数量。泛PKC抑制剂以及PKC - δ和PKC - θ的抑制剂可阻止屏障功能和紧密连接蛋白的上调，但PKC - ε的抑制剂则不能。在原代人鼻上皮细胞中，通过逆转录 - 聚合酶链反应检测到转录因子GATA - 3和 - 6。使用RNA干扰敲低GATA - 3导致TPA处理对ZO - 1和ZO - 2上调的抑制。这些结果表明，TPA诱导的PKC信号通过紧密连接蛋白的转录上调增强了人类鼻上皮细胞的屏障功能，其机制可能有助于药物递送系统。
