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Ryoji Yanai, Ji-Ae Ko, Norimasa Nomi, Naoyuki Morishige, Tai-ichiro Chikama, Atsushi Hattori, Kentaro Hozumi, Motoyoshi Nomizu, Teruo Nishida; Upregulation of ZO-1 in Cultured Human Corneal Epithelial Cells by a Peptide (PHSRN) Corresponding to the Second Cell-Binding Site of Fibronectin. Invest. Ophthalmol. Vis. Sci. 2009;50(6):2757-2764. doi: 10.1167/iovs.08-2341.
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purpose. To investigate the effect of a peptide (PHSRN) corresponding to the second cell-binding site of fibronectin on the expression of ZO-1 in cultured human corneal epithelial (HCE) cells.
methods. The effects of the PHSRN peptide on the expression of ZO-1, -2, and -3; claudin; and occludin were determined by reverse transcription-polymerase chain reaction (RT-PCR), immunoblot, and immunofluorescence analyses. Phosphorylation of mitogen-activated protein kinases (MAPKs) and the transcription factor c-Jun was assessed with a multiplex analysis system and immunoblot analysis. The barrier function of cultured HCE cells was evaluated by measurement of transepithelial electrical resistance.
results. RT-PCR and immunoblot analyses revealed the PHSRN peptide increased the amounts of ZO-1 mRNA and protein in HCE cells in a concentration- and time-dependent manner. The PHSRN peptide had no effect on the expression of ZO-2, ZO-3, claudin, or occludin. Immunofluorescence microscopy showed that the PHSRN peptide did not affect the localization of ZO-1 at the interfaces of neighboring cells. The PHSRN peptide induced the phosphorylation of the MAPKs ERK, p38, and JNK as well as that of c-Jun. The upregulation of ZO-1 expression by the PHSRN peptide was blocked by inhibitors of signaling by ERK (PD098059), p38 (SB203580), or JNK (JNK inhibitor II). The PHSRN peptide had no effect on the transepithelial electrical resistance of cultured HCE cells.
conclusions. The PHSRN peptide upregulated the expression of ZO-1 through activation of MAPK signaling pathways in HCE cells. This effect of the PHSRN sequence of fibronectin may contribute to the formation of tight junctions and play an important role in the differentiation of corneal epithelial cells.
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