368 IGF via la voie de signalisation ERK induit la formation de réseau in vitro par les fibroblastes cornéens

Introduction The cornea is kept transparent due to a mechanism which implicates corneal fibroblasts loosely arrayed between collagen lamellae. This mechanism involved in the formation of this network is not clear. Our study was performed to provide an explanation concerning this phenomenon into the stroma. In this aim, the role of IGF in the capacity of human fibroblasts corneal to form a network was investigated in vitro . Material and Methods Corneal fibroblasts, obtained from cornea gifts, were then immortalized by the SV40 virus. The presence of IGFI, IGFII and their receptors on immortalized cells was evaluated by a genomic profile of total RNA using microarray analysis with DNA of 1300 genes, by RT-PCR and by immunocytochemistry. Additionally, IGF-1 effect (100ng/mL) on corneal fibroblast network formation was analyzed on semi solid matrix of Matrigel™ and growth factor reduced Matrigel™. Cellular signalisation involved in this process was analysed by testing the network formation on Matrigel™ in presence of several signaling inhibitors: U0126 (inhibitor of MEK1/2, tranilast® and wortmannin (PI3 kinase C inhibitors), rottlerin (an inhibitor of PKCδ) and FTI (Ras cell inhibitor). Results Microarray shows a significant homology between primary cultured human corneal fibroblasts and those immortalized by the SV40 virus. It was evidenced that: 1) IGF receptors (IGFRI, RII) and IGFR binding proteins (IGFRBP3, 4, 9, 10) are expressed on corneal fibroblasts. IGF receptors on corneal fibroblasts were also detected by RT-PCR and by immunocytochemical analysis using specific antibodies. 2) IGF added to corneal fibroblasts induces the formation of a network on growth factor reduced-Matrigel™. This finding suggests that IGFRI is involved in the formation of corneal network. 3) The ERK signal pathway signalisation is implicated in the network formation on Matrigel™ of IGF- activated corneal fibroblasts, by U 0126 inhibits the network formation. Discussion Our resultats show an IGFR1 implication into the network formation. Conclusion IGF via IGFR-1 regulates the formation of “ in vitro ” corneal fibroblast network by ERK signalization suggesting crucial role of IGF in corneal wound healing.