Upregulation of connexin43 expression in corneal fibroblasts by corneal epithelial cells.
OBJECTIVE
The authors have previously shown that the expression of tight-junction proteins in corneal epithelial cells is regulated by corneal fibroblasts in a coculture system. They have now examined the effect of corneal epithelial cells on the expression of the gap-junction protein connexin43 in corneal fibroblasts.


METHODS
Human corneal fibroblasts and simian virus 40-transformed human corneal epithelial (HCE) cells were cultured on opposite sides of a collagen vitrigel membrane. Expression of junctional proteins in corneal fibroblasts was examined by reverse transcription-polymerase chain reaction, immunoblot, and immunofluorescence analyses. The effect of a small interfering RNA specific for insulin-like growth factor-1 (IGF-1) mRNA on connexin43 expression was determined by transfection.


RESULTS
The amounts of connexin43 mRNA and protein in corneal fibroblasts were increased by the presence of HCE cells. HCE cells had no effect on the expression of the tight-junction proteins ZO-1, occludin, and claudin in corneal fibroblasts. The effect of HCE cells on connexin43 expression was mimicked by exposure of corneal fibroblasts to IGF-1. Furthermore, depletion of IGF-1 in HCE cells by RNA interference largely abolished the effect of these cells on connexin43 expression in corneal fibroblasts. Finally, the upregulation of connexin43 expression in corneal fibroblasts by HCE cells was blocked by inhibitors of signaling by the mitogen-activated protein kinases ERK (PD98059) and p38 MAPK (SB203580).


CONCLUSIONS
The presence of corneal epithelial cells upregulated the expression of connexin43 in corneal fibroblasts, suggesting that corneal epithelial cells are important for the maintenance of gap junction-mediated communication among corneal fibroblasts.
Ko
Ji-Ae
JA
Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube City, Yamaguchi, Japan. jiae0831@yamaguchi-u.ac.jp
Yanai
Ryoji
R
Morishige
Naoyuki
N
Takezawa
Toshiaki
T
Nishida
Teruo
T
D000293:Adolescent; D000328:Adult; D000368:Aged; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D018920:Coculture Techniques; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism*; D003315:Cornea / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000502:physiology*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D007334:Insulin-Like Growth Factor I / Q000235:genetics; D008297:Male; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014019:Tissue Donors; D014162:Transfection; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology
D018031:Connexin 43; D004791:Enzyme Inhibitors; C500216:GJA1 protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D007334:Insulin-Like Growth Factor I; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases