May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Effects of Sp1 Overexpression on Cultured Human Corneal Fibroblasts
Author Affiliations & Notes
  • B. Yue
    Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL
  • X. Shen
    Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL
  • J. Sugar
    Ophthalmology and Visual Sciences, University of Illinois Chicago, Chicago, IL
  • Footnotes
    Commercial Relationships  B. Yue, None; X. Shen, None; J. Sugar, None.
  • Footnotes
    Support  NIH grants EY03890, EY05628, and EY01792; RPB Senior Scientific Investigator Award
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 4960. doi:
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      B. Yue, X. Shen, J. Sugar; Effects of Sp1 Overexpression on Cultured Human Corneal Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4960.

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Abstract

Abstract: : Purpose: To assess the effects of Sp1 overexpression on human corneal fibroblasts in culture. Sp1, a transcription factor, has been shown to be upregulated in keratoconus, a cornea–thinning disease. Evidence has also been presented that in keratoconus corneas, levels of degradative enzymes such as cathepsin B are increased and those of proteinase inhibitors such as α1–proteinase inhibitor (α1–PI) are decreased. Methods: Sp1 construct was transfected into corneal fibroblasts using Fugene 6 reagent. Stably transfected cells were selected by G418 treatment. Overexpression of Sp1 was verified. Protein levels of cathepsin B and and α1–PI inhibitor were evaluated by Western blotting and the activity of cathepsin B was measured by enzymatic assays. Gelatinases were examined by zymography. Cell proliferation and apoptosis in Sp1 transfectants were investigated by proliferation and apoptosis assay kits and by immunostaining. Results: The Sp1 transfected cells contained a greater amount of cathepsin B than did mock transfected controls. The activity of cathepsin B was also increased, while the gelatinase level was unaffected. By contrast, the protein level of α1–PI was lowered. Cell proliferation in corneal fibroblasts was found inhibited by Sp1 transfection. Moreover, the Sp1 transfectants displayed a heightened apoptotic activity compared to mock controls. Conclusions: Sp1 overexpression results in an elevated level of cathepsin B but a decreased α1–PI, mimicking alterations found in keratoconus corneas. The Sp1–mediated events may thus be factors contributing directly to the disease development. Overexpression of Sp1 in addition induces apoptosis, which has also been proposed as a possible pathogenic mechanism for keratoconus.

Keywords: cornea: basic science • transcription factors • cell death/apoptosis 
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