Abstract
Purpose: :
Lumican, a member of Small Leucine-rich Proteoglycan family, plays an important role in corneal wound healing. We examined the signaling pathway by which lumican promotes wound healing via TGFβ type I receptor (TBR1/ALK5) signal and identified the lumican derived peptides that are effective in promoting wound healing using human telomerase immortalized corneal epithelial (HTCE) cells.
Methods: :
Recombinant glutathione S-transferase-lumican fusion protein (GST-Lum) and GST-Lum C terminal peptide (GST-LumC) were isolated from E. coli transformed with pGEX-2T-Lum plasmids, and purified. Additionally several lumican peptides (13 amino acids long) were synthesized. GST-column pull down assay was performed to confirm the binding of lumican to TBR1. Immunohistochemisty and western blot analysis were performed to detect the signaling pathway by which lumican promoted wound healing. We evaluated the effects of TBR1 chemical inhibitor and ShRNA on the promoted wound healing of HTCE by Lumican and its derivative C-terminal peptides.
Results: :
GST pull down assay showed that Flag-TBR1 was co-purified with GST-Lum but not with GST. In the scratch wound model, phosphorylation of ERK1/2 was observed in 30 min of adding GST-Lum but those of p38MAPK and Smad2 were not detected. Blockage of TBR1 by TBRR1 inhibitor and ShRNA abolished the enhanced wound healing by GST-Lum. GST-LumC, Lumican peptides and GST-Lum promoted wound healing with a biphasic healing kinetics, slow phase (Lum; Δ5.8±1.2%X100/h, LumC; Δ6.15±0.8%X100/h), and fast phase (Lum, Δ14.2±1.9%X100/h; LumC, Δ14.2±1.5%X100/h). In contrast, basic medium (Δ3.7±0.5%X100/h) or GST treated (Δ3.8±0.4%X100/h) cells showed a monophasic healing rate. Ki67 immunostaining showed there was an increased cell proliferation at wound edge of lumican treated cells in 2 h, whereas no proliferative cell was detected in control. The observations suggest that lumican and its derivative peptides may promote cell locomotion and cell proliferation as well.
Conclusions: :
Our data showed that lumican exerted its effect on cell proliferation and healing of scratched wound of HTCE cells via its binding to TBR1/ALK5. Lumican peptides have the biological function in wound healing. This suggests the great possibility of lumican peptide for clinical application in the future.
Keywords: cornea: epithelium • wound healing • growth factors/growth factor receptors