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Jianhua Zhang, Vivien Jane Coulson-Thomas, Yong Yuan, Yujin Zhang, Chia-Yang Liu, Winston W Y Kao, None; Lum binds Alk5 to promote healing of corneal epithelium debridement in vivo. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4686. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Lumican is essential for the healing of corneal epithelium debridement, little is known regarding how this is achieved. Using in silico and pull down assays, we previously showed that TGFβ type I receptor (TGFBR1/ALK5) is a novel Lum receptor and identified that the interaction occurs between the last C-terminal 50 amino acids of Lum (LumC50) and ALK5. In the present study, we examined the effects of Lum/Alk5 binding in the healing of epithelium debridement and further demonstrate the co-localization of Lum/Alk5 in epithelial cells at the wound edge in vivo.
Wild type C57BL/6J and Lum-/- mice were subjected to epithelium debridement using an Algerbrush® followed by treatment with eye drops containing recombinant protein GST-LumC50 and synthetic LumC-peptides. Injured corneas were allowed to heal for various periods of time 2, 4 or 6 h. The epithelium migration was determined by whole mount scanning of excised corneas stained with phalloidin and DAPI using a Zeiss ApoTome microscope (Observer Z1). To determine the co-localization of Lum and Alk5, excised corneas healed for 6 h were subjected to proximity ligation assay (PLA) using goat anti-LumC and rabbit anti-Alk5 antibodies by a ZEISS LSM710 confocal microscope.
A cut view of Z-stacked images of debrided corneas from wild type mice in which the epithelium migrated about 103.4±26.7 µm in 2 h. The epithelium migration was greatly retarded in injured corneas of Lum-/- mouse (55.22±6.44 µm), treatment of LumC13C-A improved the epithelium migration (157.68±22.14 µm), whereas administration of LumC33ΔC20 had little effect on wound healing (51.32±8.31 µm). Administration of LumC13C-A also promoted wound healing of epithelium debridement in wild type mice to the same extent as injured corneas of Lum-/- mice treated with LumC13C-A.The confocal images of PLA demonstrate that LumC50 is co-localized with Alk5 in the cytoplasm and nuclei of epithelial cells at the wound edge.
The C-terminal domain of Lum containing the last 13 amino acids (LumC13) and and its derivative (LumC13C-A, substitution of cysteine with alanine) is sufficient to promote healing of epithelium debridement in vivo. Lum binds Alk5 via it C-terminal domain and Lum/Alk5 complex may assume nuclear translocation, albeit the significance of such nuclear translocation is not known.
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