title: Upregulation of ZO-1 in cultured human corneal epithelial cells by a peptide (PHSRN) corresponding to the second cell-binding site of fibronectin.
abstract: 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.
authors.affiliation: Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan. yanai@yamaguchi-u.ac.jp
authors.forename: Ryoji
authors.initials: R
authors.lastname: Yanai
authors.forename: Ji-Ae
authors.initials: JA
authors.lastname: Ko
authors.forename: Norimasa
authors.initials: N
authors.lastname: Nomi
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Morishige
authors.forename: Tai-ichiro
authors.initials: T
authors.lastname: Chikama
authors.forename: Atsushi
authors.initials: A
authors.lastname: Hattori
authors.forename: Kentaro
authors.initials: K
authors.lastname: Hozumi
authors.forename: Motoyoshi
authors.initials: M
authors.lastname: Nomizu
authors.forename: Teruo
authors.initials: T
authors.lastname: Nishida
mesh_terms: 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
chemical_list: 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