Insulin growth factor promotes human corneal fibroblast network formation in vitro.
OBJECTIVE
Corneal fibroblast cell (CFC) reticulation is involved in the structural development of corneal stroma and in wound healing. In an earlier paper, it was reported that the expression of VEGFR-1 by CFCs is related to their reticulogenic properties in vitro and decreases with the age of the donors. The present study was focused on the nonreticulogenic corneal fibroblast population and explored whether these cells can be induced to form cell networks in vitro.


METHODS
The network formation was analyzed using an array of signaling pathway inhibitors: wortmannin for PI3 kinase, U0126 for MEK-1/2 kinase, Rottlerin for PKC, farnesyl transferase inhibitor (FTI-277) for Ras, and picropodophyllin (PPP) for IGFR-1. Among the several growth factors studied, IGF seemed to be crucial to cell network formation. The presence of IGF signaling was demonstrated using gene array analysis and was confirmed by RT-PCR and immunocytochemistry and by cell network formation on reduced synthetic basement membrane arrays. The pleiotropic effect of IGF-1 on the cells was analyzed by protein cytokine array.


RESULTS
The genesis of reticulation was found to occur via MEK-1/2 and IGFR pathways, since inhibitors of these signaling pathways reduced or prevented cell network formation. The addition of exogenous IGF-1 generated a cell network structure in corneal fibroblasts obtained from healthy donors, indicating the involvement of IGF-1.


CONCLUSIONS
IGF signaling and the MEK-1/2 pathway are involved in the cell network formation of corneal fibroblast cells from aged donors.
Berthaut
Alexandre
A
INSERM (Institut National de la Santé et de la Recherche Médicale) UMRS (Unité Mixte de Recherche Scientifique) 872, Faculté de Médecine Paris VI, Paris, France.
Mirshahi
Pezhman
P
Benabbou
Nadia
N
Ducros
Elodie
E
Agra
Aureliou
A
Therwath
Amu
A
Legeais
Jean Marc
JM
Mirshahi
Massoud
M
D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D060527:Corneal Keratocytes / Q000166:cytology* / Q000378:metabolism; D003319:Corneal Stroma / Q000166:cytology* / Q000378:metabolism; D016207:Cytokines / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D048369:MAP Kinase Kinase 1 / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014945:Wound Healing / Q000502:physiology
D016207:Cytokines; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D007334:Insulin-Like Growth Factor I; C482128:MAP2K2 protein, human; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human