May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
TGFß-1-Dependent Upregulation of Keratoepithelin Is Mediated via Smad
Author Affiliations & Notes
  • H. L. Berscheit
    Ophthalmology, University of Minnesota, Minneapolis, Minnesota
  • C. Yuan
    Ophthalmology, University of Minnesota, Minneapolis, Minnesota
  • A. J. W. Huang
    Ophthalmology, University of Minnesota, Minneapolis, Minnesota
  • Footnotes
    Commercial Relationships H.L. Berscheit, None; C. Yuan, None; A.J.W. Huang, None.
  • Footnotes
    Support Research to Prevent Blindness; Minnesota Medical Foundation
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 5860. doi:
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      H. L. Berscheit, C. Yuan, A. J. W. Huang; TGFß-1-Dependent Upregulation of Keratoepithelin Is Mediated via Smad. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5860.

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

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Purpose:: Keratoepithelin (KE or TGFß-inducible protein) has been implicated in the pathogenesis of various stromal corneal dystrophies and other systemic disorders. This study aims to investigate the molecular mechanism of the TGFß-1-mediated activation of KE gene.

Methods:: To study the expression and activation pathway of KE, A549 cells were treated with chemical inhibitors specific for mitogen-activated protein kinases (MAPK) and Smad proteins. Over-expression of Smad2, Smad3, and Smad4 cDNAs along with a dominant-negative mutant of Smad 2/3 in A549 cells were also performed to determine the roles of the Smad pathway in KE expression after TGFß-1 treatment. DNA pull-down experiments were used to confirm the interaction of Smad with KE gene promoter in vitro.Luciferase reporter assays were used to investigate Smad3 modulation of the KE gene promoter activity mediated by TGFß-1.

Results:: With chemical inhibitors, we found that Smad pathway, but not MAPK, was primarily affected in TGFß-1-mediated KE gene activation. Over-expression of Smad3 or a dominant negative mutant of Smad significantly affected the expression of KE in transfected cells by TGFß-1. Increased KE expression was noted in Smad3 transfected cells by Western blots (both media and cell lysates) and Northern blots. DNA-transcriptional factor pull-down demonstrated the direct interaction of Smad3 with the KE gene promoter. Furthermore, activation of KE by Smad3 showed a dose-dependent manner and was further confirmed by the promoter-luciferase assays. Together, these results suggest the essential role of Smad3 in the TGFß-1-mediated KE gene activation.

Conclusions:: Our results demonstrate that the Smad pathway, especially Smad3 is crucial for the TGFß-1-dependent activation of the human KE gene. Further investigations on the Smad activation will facilitate our understanding of KE and its pathogenic mechanism in related diseases.

Keywords: cornea: epithelium • degenerations/dystrophies • cornea: basic science 

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