Upregulation of ZO-1 in cultured human corneal epithelial cells by a peptide (PHSRN) corresponding to the second cell-binding site of fibronectin.
OBJECTIVE
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.
Yanai
Ryoji
R
Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan. yanai@yamaguchi-u.ac.jp
Ko
Ji-Ae
JA
Nomi
Norimasa
N
Morishige
Naoyuki
N
Chikama
Tai-ichiro
T
Hattori
Atsushi
A
Hozumi
Kentaro
K
Nomizu
Motoyoshi
M
Nishida
Teruo
T
D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry* / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D062826:Zonula Occludens-1 Protein; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D005353:Fibronectins; D008565:Membrane Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; C495978:prolyl-histidyl-seryl-arginyl-asparagine; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4