Upregulation of tight-junctional proteins in corneal epithelial cells by corneal fibroblasts in collagen vitrigel cultures.
OBJECTIVE
To investigate the effects of corneal fibroblasts on the differentiation of corneal epithelial cells in a coculture system based on a collagen vitrigel membrane.


METHODS
Simian virus 40-transformed human corneal epithelial (HCE) cells and human corneal fibroblasts were cultured on opposite sides of a collagen vitrigel membrane. The distribution of HCE cells and corneal fibroblasts on the collagen membrane was determined by immunofluorescence staining and immunoblot analysis of marker proteins. Expression of the tight-junctional proteins ZO-1, occludin, and claudin and of the adherens-junctional proteins E- and N-cadherin in HCE cells was determined at the mRNA and protein levels by reverse transcription-polymerase chain reaction analysis and immunoblot analysis, respectively.


RESULTS
The abundance of ZO-1, occludin, and claudin mRNA and proteins in HCE cells was markedly increased by coculture with corneal fibroblasts. The expression of E- or N-cadherin did not differ between HCE cells cultured with corneal fibroblasts and those cultured without them. PD98059, a specific inhibitor of signaling by extracellular signal regulated kinase (ERK), prevented the upregulation of tight-junctional proteins in HCE cells by corneal fibroblasts.


CONCLUSIONS
Human corneal fibroblasts regulated the expression of tight-junctional proteins in HCE cells, suggesting that corneal fibroblasts may play an important role in the differentiation of corneal epithelial cells.
Ko
Ji-Ae
JA
Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, 111 Minami-Kogushi, Ube City, Yamaguchi, Japan. jiae0831@yamaguchi-u.ac.jp
Liu
Yang
Y
Yanai
Ryoji
R
Chikama
Tai-ichiro
T
Takezawa
Toshiaki
T
Nishida
Teruo
T
D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000494:pharmacology; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D062445:Claudin-1; D018920:Coculture Techniques; D003094:Collagen; D003315:Cornea / Q000166:cytology; D019573:Epithelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D005347:Fibroblasts / Q000502:physiology*; D005419:Flavonoids / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D062793:Occludin; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013539:Simian virus 40; D015854:Up-Regulation / Q000502:physiology*; D062826:Zonula Occludens-1 Protein
D015703:Antigens, CD; C050361:CDH2 protein, human; C568108:CLDN1 protein, human; D015820:Cadherins; D062445:Claudin-1; D005419:Flavonoids; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D012333:RNA, Messenger; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D003094:Collagen; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one