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Yujin Zhang, Lung-Kun Yeh, Winston Kao, Chia-Yang Liu, Edith J. Crawley Vision research Center, Department of Ophthalmology, University of Cininn School of Medicineati; Wnt/b-catenin canonical signaling in corneal keratocytes regulates epithelium stratification through repressing Bmp4/Mapk pathway. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4555.
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Mesenchyme-epithelial interaction plays pivotal roles on corneal morphogenesis during development. Our previous data showed that loss of β-catenin (Ctnnb1) in corneal stromal keratocytes (Ctnnb1cskΔ/cskΔ) triggers precocious epithelium stratification in mice. In this study, attempts were made to explore the mechanisms controlling this aberrant developmental event in the absence of Ctnnb1.
To ablate Lrp5 and Lrp6 gene in keratocytes, KR/TC/Lrp5f/f/Lrp6f/f tetratransgenic mice were fed Dox-chow from embryonic 12.5 (E12.5) to postnatal day 0 (P0) or from P0 to P10. Embryos and neonates were collected and subjected to histology and immunohistochemistry examination. Primary corneal stroma fibroblasts from the Ctnnb1f/f mice were cultured and infected by Ad-cre or Ad-EGFP virus. Expression profiles of cytokines after virus infection were determined by cDNA micro-array, RT-PCR and western blotting. MAPK signaling was examined in HTCE treated with BMP4 protein. Specimens from WT mice treated with BMP4 protein from P0 to P10 by subcutaneous injection were subjected to immunohistochemistry examination.
Lrp5cskΔ/cskΔ/Lrp6cskΔ/cskΔ double knock-out mice manifested precocious corneal epithelium stratification similar to the phenotype observed in Ctnnb1cskΔ/cskΔ mice, while single Lrp5cskΔ/cskΔ or Lrp6cskΔ/cskΔ mice were normal. Interestingly, BMP4-treated neonates displayed phenotype resembling Lrp5cskΔ/cskΔ/Lrp6cskΔ/cskΔ and Ctnnb1cskΔ/cskΔ mice. Moreover, SEM showed the collagen fibrils and stromal cells are better organized in neonates of aforementioned mutant and BMP4-treated mice. Cytokine cDNA array screening showed that Bmp4 was up-regulated in cultured corneal fibroblasts from Ctnnb1cskΔ/cskΔ mice. Furthermore, MAPK pathway was activated via ERK in HTCE cell by BMP4 treatment.
The precocious corneal epithelium stratification observed in Ctnnb1cskΔ/cskΔ and Lrp5cskΔ/cskΔ/Lrp6cskΔ/cskΔ mutants may result from the up-regulated expression of BMP4 in developing corneal stroma, which enhanced MAPK signaling in corneal epithelial, leading to precocious epithelium stratification. Our data supports the notion that cross-talk between Wnt/β-catenin/Bmp4 axis (in stroma) and MAPK signaling (in epithelium) play critical roles in corneal morphogenesis during development.
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