Intracellular Signaling Pathways Involved in ex vivo Expansion of Human Limbal Epithelial Cells on Intact Amniotic Membrane

Abstract

Purpose:: Previous studies have demonstrated that matrix metalloproteinase-9 (MMP-9) participated, at least in part, in the outgrowth of expanded limbal epithelial cells on intact amniotic membrane (AM). We further try to investigate the intracellular signaling pathways involved in the limbal epithelial proliferation on intact AM as well as the relationships of these pathways to MMP-9 expression.

Methods:: Human corneoscleral buttons obtained after conventional penetrating keratoplasty surgery were cut into 1.5 x 2 x 3 mm³ pieces, cultured and expanded on intact AM with basement membrane side-up for 3 weeks. To elucidate the distinct signaling pathways necessary for limbal outgrowth in this model, various concentrations of pharmacological inhibitors and neutralizing antibodies were added at day 14. The extent of each outgrowth was monitored with a phase contrast microscope and the area at day 21 was calculated. MMP-9 expression was studied by gelatin zymography and reverse transcription-polymerase chain reaction (RT-PCR).

Results:: Limbal epithelial outgrowths were significantly suppressed by 1µM AG1478 (EGFR inhibitor), 30µM Genistein or 10µM PP1 (tyrosine kinase inhibitors), 8µg/ml ß4-integrin and focal adhesion kinase (FAK) antibodies, 10µM Ro318220 and 1µM GÖ6976 (PKC inhibitors), 30µM LY294002 or Wortmannin (PI3-K inhibitors), 10µM Apigenin (Ras/MAPK inhibitor), 10µM U0126 (MEK1/2 inhibitor) and 10µM SP600125 (c-Jun N-terminal kinase (JNK) inhibitor) as compared to the control groups, but not inhibited by 30µM SB202190 (p38 MAPK inhibitor). However, gelatin zymography demonstrated that the enzymatic activity of MMP-9 was attenuated by the aforementioned inhibitors except for ß4-integrin antibody, FAK antibody, U0126 and SB202190, the results of which were confirmed by RT-PCR.

Conclusions:: We established the significant roles of integrin- and EGF-related signals in this culture system. However, these data also suggested a MMP-9-independent signaling pathway regulating the proliferation of limbal epithelial cells on intact AM.

Keywords: cornea: epithelium • proliferation • signal transduction