April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Roles of Lumikine on the Healing of Corneal Epithelium Debridement in Vivo
Author Affiliations & Notes
  • Winston W Y Kao
    Ophthalmology, University of Cincinnati, Cincinnati, OH
  • Jianhua Zhang
    Ophthalmology, University of Cincinnati, Cincinnati, OH
  • Vivien Jane Coulson-Thomas
    Ophthalmology, University of Cincinnati, Cincinnati, OH
  • Mindy Kay Call
    Ophthalmology, University of Cincinnati, Cincinnati, OH
  • Yong Yuan
    Ophthalmology, University of Cincinnati, Cincinnati, OH
  • Chia-Yang Liu
    Ophthalmology, University of Cincinnati, Cincinnati, OH
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 4609. doi:
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      Winston W Y Kao, Jianhua Zhang, Vivien Jane Coulson-Thomas, Mindy Kay Call, Yong Yuan, Chia-Yang Liu; Roles of Lumikine on the Healing of Corneal Epithelium Debridement in Vivo. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4609.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Lumican (Lum) has multi matricellular functions. It is a constituent of the extracellular matrix and serves as a matrikine. We have recently shown that Lum binds ALK5 (type 1 receptor of TGFβ) via its C-terminal domain to promote epithelium wound healing. In this study, we attempt to elucidate the mechanism by which a Lumikine promotes epithelium wound healing.

Methods: C57BL/6J, Lum-/-, Alk5CEΔ/CEΔ and Tgfbr2CEΔ/CEΔ (CEΔ, corneal epithelium-specific deletion) mice were subjected to epithelium debridement. The injured corneas were treated and allowed to heal for 2 or 4 h by administering eye drops containing synthetic C-terminal peptides derived from Lum, e.g., LumC13, LumC13C-A (substitution of C with A), LumC33ΔC20, EGF and a neutralizing anti-TGFβ antibody or various kinase inhibitors for 2 h. The epithelium migration was determined by whole mount scanning of excised corneas stained with phalloidin and DAPI. In vitro, expression of EGFR ligands were determined by qRT-PCR of scratched HTCE cells (human telomerase immortalized corneal epithelial cells). shRNAi transduced HTCE cells were also used to determine the role of epiregulin (EREG) in the Lum/ALK5 signaling.

Results: Administration of LumC13 and LumC13C-A peptides promoted epithelium migration, but not LumC33ΔC20.The promoted epithelium migration was abrogated by the administration of the neutralizing anti-TGFβ antibody in Lum-/- and ablation of Alk5 and Tgfbr2. Interestingly, the effect of LumC13C-A was abrogated by administration of kinase inhibitors of ALK5 (SB431542, 10 µM); Src inhibitor (SrcI-1, 2 µM), EGFR inhibitor (AG1478, 10 nM); pERK1/2 inhibitor (PD98059, 5 µM); PI3K inhibitor (Wortmannin, 1 µM), while EGF effects were only abolished by AG1478, Wortmannin and partially by PD98059, but not by SB431542 and SrcI-1. The effects of LumC50 on wound healing of HTCE cells was abolished by transduction of EREG-shRNAi.

Conclusions: The polymerization of ALK5/TGFBR2 is required for the binding of Lum to ALK5 at the early phase of wound healing. Interaction of Lum/ ALK5 may lead to an alternative signaling pathway mediated via the Src-pERK1/2 axis for accelerated wound healing characterized by an increase in cell mobility and lifting of cell cycle suppression during the early phase of wound healing. The up-regulation of EREG may then serve as a feed-forward mechanism for sustained ERK1/2 activation.

Keywords: 765 wound healing • 519 extracellular matrix • 714 signal transduction  
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