May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Regulation of Maspin Expression by DNA and Histone Methylation During Corneal Wound Healing
Author Affiliations & Notes
  • M. Narayan
    Medical College of Wisconsin, Milwaukee, Wisconsin
    Biochemistry,
  • M. A. Horswill
    Medical College of Wisconsin, Milwaukee, Wisconsin
    Biochemistry,
  • D. J. Warejcka
    Medical College of Wisconsin, Milwaukee, Wisconsin
    Biochemistry,
  • S. S. Twining
    Medical College of Wisconsin, Milwaukee, Wisconsin
    Biochemistry and Ophthalmology,
  • Footnotes
    Commercial Relationships  M. Narayan, None; M.A. Horswill, None; D.J. Warejcka, None; S.S. Twining, None.
  • Footnotes
    Support  NIH Grant EY014168
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3371. doi:https://doi.org/
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      M. Narayan, M. A. Horswill, D. J. Warejcka, S. S. Twining; Regulation of Maspin Expression by DNA and Histone Methylation During Corneal Wound Healing. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3371. doi: https://doi.org/.

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

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Abstract

Purpose: : Epigenetic mechanisms such as methylation of DNA and histones regulate gene expression. Maspin, a 42-kDa non classical serine protease inhibitor that controls cell adhesion and migration, is expressed by corneal epithelial cells and stromal keratocytes. Maspin expression is downregulated by keratocytes upon differentiation into fibroblasts during corneal wound healing. The purpose of this study was to determine whether DNA methylation and histone H3 dimethylation are involved in regulation of maspin expression in human corneal stromal phenotypes.

Methods: : Human donor corneal stromal cell cultures were established in serum-free medium or in the presence of FBS and passed into serum-free medium. Medium containing FBS or FGF-2 was used to induce the fibroblast phenotype or TGF-β1 for the myofibroblast phenotype. RNA and protein were extracted for semi-quantitative or quantitative RT-PCR and western analysis. Differentiated cells or fibroblasts cultured in FBS to passage 4 (P4) were treated with the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin promoter in the P4 fibroblasts was determined using a ChIP assay.

Results: : Freshly harvested stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter and the first intron in freshly isolated keratocytes were hypomethylated while keratocytes cultured in serum-free medium or FBS containing medium, and the P1 cells cultured in serum free defined medium, FGF-2 or TGF-β1 were hypermethylated; 81-85% of the CpG methylation sites were modified. Down regulation of maspin synthesis was also associated with histone H3 dimethylation at Lysine 9. Maspin mRNA and protein were reexpressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3.

Conclusions: : Maspin promoter hypermethylation and histone dimethylation occur with down regulation of maspin synthesis in corneal stromal cells. This suggests regulation of genes upon conversion of keratocytes to wound healing phenotypes can involve DNA and histone methylation.

Keywords: cornea: stroma and keratocytes • gene/expression • differentiation 
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