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Winston W Y Kao, Fei Dong, Jianhua Zhang, Yong Yuan, Yueh-Chiang HU; Role of TGF-β Receptor 1 (Tgfbr1/Alk5) on The Healing of Corneal Epithelium Debridement. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4329. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Lumican and its C-terminal peptides promote healing of corneal epithelium debridement via binding to Tgfbr1/Alk5. Present studies aim to determine Alk5 domains, e.g., GS (glycine serine), KD (kinase domain), iKD (intracellular kinase domain), which are essential for wound healing.
Transgenic mice carrying TRE-driven FLAG-tagged transgenes encoding full length Alk5 cDNA (Tet-O-Alk5WT), mutant -Alk5dGS and -Alk5dKD, were created by target insertion into H11 site in chromosome 11 of C57BL/6 mice by TARGATT technique, and intracellular kinase domain (-Alk5iKD) by transgenesis. Founder mice were identified by PCR genotyping with tail DNA and were crossed with Krt14-rtTa (K14R) to obtain bi-transgenic K14R/Alk5 mice. The expression of transgenes in bi-transgenic F1 mice via doxycycline chow induction was confirmed by immunofluorescence staining with anti-FLAG antibodies. Adult bi-transgenic mice (both sex, 10 weeks old) were induced by doxycycline for two weeks, then subjected to epithelium debridement. The injured corneas were treated with eye drops containing LumC13C-A (lumikine, OS) and LumC18ΔC5 peptides (OD) for 6 hours, the migration of healing epithelium was measured by whole mount scanning of 4% PFA fixed corneas stained with phalloidin and DAPI.
Two to three founder mice were obtained for each of Tet-O-Alk5WT, -Alk5dGS and -Alk5dKD transgene, PCR with primers flanking the TARGATT site and within transgenes confirmed only a single insertion of each of individual mouse lines. Only one tet-O-Alk5iKD founder was obtained by transgenesis, the chromosome location of the transgene is not known. The expression of Alk5 transgenes was confirmed by immunofluorescence staining in cryosections from tails of bi-transgenic neonates with anti-FLAG. Lumikine (LumC13C-A peptide) promotes epithelium sheet migration after debridement in wild type mice: Lumikine/control, 103±2.4/45.9±3.0 μm, P, 0.001, n=8. Expression of Alk5 transgenes abolishes the effects of Lumikine on promoting epithelium migration: Alk5WT, Lumikine/control, 51.5±3.9/36.1±3.3 μm (P, 0.03, n=3); Alk5dGS, 27.2±5.2/46.6±13.9 μm (P 0.04, n=5); Alk5iKD, 30.9±2.4/38.9±7.8 (P 0.13, n=5).
Excess wild type and mutant Alk5 proteins may perturb the formation of stoichiometric Tgfbr2/Tgfbr1(Alk5) polymers essential for lumican/lumikine promoted wound healing.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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