September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
TGFβ-induced signaling leading to lens epithelial to mesenchymal transition (EMT): Implications for cataract induction
Author Affiliations & Notes
  • Magdalena Wojciechowski
    University of Sydney, Bosch Institute, Sydney, New South Wales, Australia
    Save Sight Institute, Sydney, New South Wales, Australia
  • Shannon Joel Das
    University of Sydney, Bosch Institute, Sydney, New South Wales, Australia
    Save Sight Institute, Sydney, New South Wales, Australia
  • Frank J Lovicu
    University of Sydney, Bosch Institute, Sydney, New South Wales, Australia
    Save Sight Institute, Sydney, New South Wales, Australia
  • Footnotes
    Commercial Relationships   Magdalena Wojciechowski, None; Shannon Das, None; Frank Lovicu, None
  • Footnotes
    Support  NHMRC
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Magdalena Wojciechowski, Shannon Joel Das, Frank J Lovicu; TGFβ-induced signaling leading to lens epithelial to mesenchymal transition (EMT): Implications for cataract induction. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose : Transforming Growth Factor β (TGFβ) is a potent inducer of lens epithelial to mesenchymal transition (EMT). The resultant mesenchymal cells resemble those found in plaques of human forms of subcapsular cataract and secondary cataract, such as posterior capsular opacification. Smad signaling has long been implicated as the sole driving force of TGFβ-mediated activity. This study examines the role of Smad-independent TGFβ signaling pathways, namely the ERK1/2 MAPK pathway, during TGFβ-induced EMT in the lens.

Methods : Rat lens epithelial explants were treated with up to 1ng/ml of TGFβ2 for up to 5 days, with or without 50µM UO126, a selective pharmacological inhibitor of ERK1/2-signaling or 5µM of Cis3, a selective inhibitor of Smad3 signaling. The TGFβ-induced EMT was evaluated through changes in cell morphology and the progression of TGFβ-induced cell loss using phase contrast microscopy. Immunolabeling of phosphorylated Smad2 and ERK1/2 in response to TGFβ, with or without UO126 or Cis3, was assessed, as were changes in α-smooth muscle actin (α-SMA) accumulation and E-Cadherin localization.

Results : ERK1/2 signaling is activated as early as 20 minutes in response to TGFβ, and this is effectively blocked by UO126. Blocking the phosphorylation of ERK1/2 prior to or at different stages of TGFβ-induced EMT demonstrated changes to Smad-signaling, α-SMA-labeled stress fiber accumulation, E-Cadherin localization, cell shape and numbers of cells lost. Overall, the earlier inhibition of ERK1/2 (prior to the addition of TGFβ, or 1 day after TGFβ addition) was more effective at suppressing features of EMT, than later inhibition (application of UO126, 2 days after TGFβ addition). Blocking the activation of Smad-signaling did not impact on TGFβ-induced ERK1/2 signalling; however blocking ERK1/2 did appear to effect Smad-signaling.

Conclusions : Our data implicates ERK1/2 signaling in both the initiation and early progression of TGFβ-induced EMT in rat lens epithelial cells, possibly through its crosstalk with Smad-signaling. The tight regulation of intracellular signaling pathways such as ERK1/2 and Smad2/3 are required for the maintenance of lens epithelial cell integrity and hence tissue transparency. A comprehensive understanding of the molecular mechanisms that drive the induction and progression of EMT in the lens may provide the basis for potential therapeutics for human cataract.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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