Abstract
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-δ, PKC-θ, and PKC-ϵ. 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-δ and PKC-θ but not PKC-ϵ. 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.
Footnotes
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This work was supported by Grants-in-Aid from National Project “knowledge Cluster Initiative” (2nd stage, “Sapporo Biocluster Bio-s”), the Ministry of Education, Culture, Sports Science, and Technology, and the Ministry of Health, Labor and Welfare of Japan, the Akiyama Foundation, and Japan Science and Technology Agency.
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ABBREVIATIONS: ZO, zona occludens; PKC, protein kinase C; DAG, diacylglycerol; nPKC, novel PKC; TPA, 12-O-tetradecanoylophorbol-13-acetate; MARCKS, myristoylated alanine-rich protein kinase C substrate; MAPK, mitogen-activated protein kinase; GF109203X, 3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride; PD98059, 2′-amino-3′-methoxyflavone; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PI3K, phosphatidylinositol 3-kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; EGFR, epidermal growth factor receptor; PD153035, 4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline; PBS, phosphate-buffered saline; siRNA, small interfering RNA; RT-PCR, reverse transcription-polymerase chain reaction; TER, transepithelial electrical resistance; FITC, fluorescein isothiocyanate.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received November 21, 2007.
- Accepted May 2, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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