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M. L. Funderburgh, M. M. Mann, J. L. Funderburgh; TCF Activation Correlates With Downregulation of Keratan Sulfate Biosynthesis. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6205.
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© ARVO (1962-2015); The Authors (2016-present)
Keratan sulfate (KS) is uniquely abundant in the corneal stroma and is essential for corneal transparency. Biosynthesis of KS is a function of stromal keratocytes and is markedly downregulated during wound healing and when the cells are cultured in vitro. The expression of glycosyl- and sulfotransferase enzymes required for KS biosynthesis is rapidly regulated by exposure of keratocytes to cytokines or serum, but the signaling mechanism involved in this downregulation is not understood. In the current study we sought to determine if Wnt signaling is involved KS regulation.
Primary bovine keratocytes were treated in serum free media with cytokines and inhibitors regulating Wnt signaling pathways. Wnt signaling was examined by TCF-driven luciferase expression and cyclin D1 activation. KS biosynthesis was monitored by KS immunoblotting and by qPCR for mRNA levels of KS biosynthetic enzymes.
Synthetic Wnt agonist FH535 downregulated KS biosynthesis and mRNA for KS biosynthetic enzymes in a dose-dependent manner. Inhibitors which block degradation of beta catenin, BIO and M132, upregulated cyclin D1 indicating activation of TCF signaling. These inhibitors also produced rapid downregulation of glycosyl transferase B3GnT7, and the sulfotransferases CHST1 and CHST6, enzymes thought to be involved in KS synthesis.
Downregulation of KS and of the mRNA for KS biosynthetic enzymes correlates with activation of signaling via the canonical Wnt signaling pathway involving activation of the TCF transcription factor by nuclear translocation of beta catenin. These results lend support to the hypothesis that the Wnt signaling pathway is involved in control of KS biosynthesis.
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