May 2004
Volume 45, Issue 13
ARVO Annual Meeting Abstract  |   May 2004
Zinc Affects Cell Proliferation and KSPG Expression of Corneal Keratocytes
Author Affiliations & Notes
  • E.S. Tasheva
    Division of Biology, Kansas State University, Manhattan, KS
  • K. An
    Division of Biology, Kansas State University, Manhattan, KS
  • G.W. Conrad
    Division of Biology, Kansas State University, Manhattan, KS
  • Footnotes
    Commercial Relationships  E.S. Tasheva, None; K. An, None; G.W. Conrad, None.
  • Footnotes
    Support  NIH Grant EY13395 to GWC and EST
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3836. doi:
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      E.S. Tasheva, K. An, G.W. Conrad; Zinc Affects Cell Proliferation and KSPG Expression of Corneal Keratocytes . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3836.

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

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Abstract: : Purpose:Zinc is the second most abundant essential element in the human body that influences cellular metabolism, blocks apoptosis in many systems, and plays roles in neuronal signaling. Both its deficiency and abnormal accumulation contribute to the pathogenesis of many ocular diseases. In this study we evaluate effects of zinc on cell growth and KSPG (keratan sulfate proteoglycan) expression of bovine corneal keratocytes. We also characterize metal responsive regulatory elements (MRE) present in promoters of KSPG genes. Methods: Primary cultures of bovine corneal keratocytes were treated with 25microM, 50microM and 100microM ZnSO4 for1 to 8 hours. mRNA expression was analyzed by reverse transcription– polymerase chain reaction (RT–PCR) and quantitative real–time PCR. Transcriptional activity of human mimecan and keratocan promoters was evaluated, before and after zinc treatment, using transient transfections of promoter/luciferase reporter constructs into corneal keratocytes. Site–directed mutagenesis and corresponding functional assays were used to determine the contribution of MRE to mimecan and keratocan transcription. The cDNA encoding bovine MRE–binding transcription factor, MTF–1, was cloned by RT–PCR, and a MTF–1 role demonstrated in co–transfection experiments. Results:Treatment of cultured corneal keratocytes with 25microM ZnSO4 had no effect on cellular growth, whereas 50microM and 100microM led to keratocyte apoptosis. Mimecan and keratocan exhibited approx. 2–fold increased expression 6hours after treatment with 50microM ZnSO4, whereas lumican expression was not significantly affected. 100microM ZnSO4 led to initial increase followed by decrease in mimecan and keratocan expression. Mimecan and keratocan promoter constructs activated following 50microM ZnSO4 treatment contained MRE–binding site. Deletion of this site considerably diminished the zinc response of the promoter. Roles of the MRE and MTF–1 in zinc responses of the human mimecan promoter were confirmed by co–transfection experiments using cloned bovine MTF–1 transcription factor. Conclusions: Zinc affects cellular growth and modulates transcription of mimecan and keratocan in bovine corneal keratocytes. MRE–binding sites in promoters of these genes and transcription factor MTF–1 are mediators of zinc effects on corneal KSPG transcription.

Keywords: cornea: stroma and keratocytes • gene/expression • cell death/apoptosis 

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