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Xuan Li, Martha Funderburgh, Mary Mann, Yiqin Du, James Funderburgh; Genes Controlling Keratan Sulfate Biosynthesis During Differentiation Of Corneal Stromal Stem Cells To Keratocytes. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3424.
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Keratan sulfate (KS) is an abundant stromal glycosaminoglycan essential for corneal transparency. KS synthesis is lost during wound healing and in stromal scars, however this process is not well understood. Enzymes involved in corneal KS elongation and sulfation have not all been identified. Corneal stromal stem cells do not express KS but as they differentiate to keratocytes KS synthesis is upregulated. These cells provide a novel opportunity to identify enzymes involved in regulation of corneal KS biosynthesis. In this study, we sought to determine the enzymes used by stromal stem cells in the up-regulation of corneal KS synthesis during differentiation to keratocytes.
Primary stromal cells were isolated from bovine corneas and human corneal stromal stem cells were isolated from donor corneas. Differentiation to keratocytes was induced in serum-free medium containing ascorbate. Expression of mRNA for specific glycosyltransferase (GTase) and sulfotransferase (STase) genes was examined by RT-PCR. Participation of specific genes in KS biosynthesis was determined by mRNA knockdown with siRNA. Gene expression of GTase and STase was characterized using quantitative real time PCR (qPCR) and western blotting. KS expression was detected using immunoblotting.
Expression of mRNA for 15 GTase and STase genes was detected in uncultured corneal stromal cells. Knockdown of mRNA for two STase genes (CHST6 and CHST1) and one GTase gene (B3GnT7) strongly inhibited corneal KS biosynthesis. In stromal stem cells, mRNA expression of both CHST6 and B3GnT7 was markedly increased in conjunction with induction of KS synthesis. Immunoblotting showed expression of both CHST6 and B3GnT7 proteins as the cells began synthesis of KS.
Multiple enzymes may participate in KS biosynthesis but two of these (CHST6 and B3GNT7) appear primarily to control expression of KS by corneal stromal cells. This observation is important for understanding stromal wound healing and has implications for bioengineering of corneal stroma.
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