May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Epigenetic Silencing of Maspin Expression in Differentiated Human Corneal Stromal Cells
Author Affiliations & Notes
  • S. S. Twining
    Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin
  • M. A. Horswill
    Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin
  • M. Narayan
    Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin
  • D. J. Warejcka
    Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2742. doi:
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      S. S. Twining, M. A. Horswill, M. Narayan, D. J. Warejcka; Epigenetic Silencing of Maspin Expression in Differentiated Human Corneal Stromal Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2742.

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

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Abstract

Purpose:: DNA methylation is one of several processes that control gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis. Maspin, a 42 kDa non classical serpin (serine protease inhibitor) is expressed in many epithelial derived cells including corneal epithelial cells. In addition, corneal stromal keratocytes express maspin; however, maspin is down regulated upon conversion to fibroblasts and myofibroblasts. Maspin regulates motility and adhesion of corneal fibroblasts and inhibits angiogenesis during wound healing. To better understand maspin’s role in corneal wound healing and the mechanism of downregulation of maspin synthesis, the methylation state of the maspin promoter was assessed among various differentiated human corneal stroma cells.

Methods:: To study promoter methylation, human donor corneal stroma cells were cultured and treated using FBS, FGF, and TGF- to induce fibroblast, and myofibroblast phenotypes similar to those found in a wounded cornea. Following cell differentiation, cells were treated with the DNA demethylating agent, 5’-Aza-dc, and the histone deacetylase (HDAC) inhibitor, TSA. After treatment, cells were harvested and assayed for maspin mRNA, protein expression and DNA methylation using sodium bisulfite sequencing.

Results:: Freshly harvested corneal stromal cells expressed maspin but after cell differentiation, maspin mRNA and protein was undetected. Sodium bisulfite sequencing revealed that the maspin promoter was abundantly methylated in differentiated corneal stroma cells compared to freshly isolated or 5’-Aza-dc/TSA treated cells. These 5’-Aza-dc and TSA.treated fibroblasts and myofibroblasts re-expressed both maspin mRNA and protein.

Conclusions:: Our results suggest the loss of maspin expression is due to promoter methylation in differentiated corneal stromal cells. The ability of demethylating and HDAC inhibitors to induce re-expression of maspin further demonstrates that methylation of the maspin promoter is important in maspin expression in corneal stromal cells.

Keywords: cornea: basic science • cornea: stroma and keratocytes • gene/expression 
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