Sprouty and Spred Temporally Regulate ERK1/2-signaling to Suppress TGFβ-induced Lens EMT.

Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) principally contributes to the pathogenesis of fibrotic cataract. Sprouty (Spry) and Spred proteins are receptor tyrosine kinase (RTK) antagonists that can regulate RTK-mediated signaling pathways, such as the MAPK/ERK1/2-signaling pathway. The present study examines the ability of Spry and Spred to inhibit TGF beta-induced EMT in LECs. LECs explanted from postnatal-day 21 Wistar rats were transduced with adenoviral vectors coding for Spry1, Spry2 or Spred2, and subsequently treated with or without TGF beta 2. Immunofluorescent labeling of explants for the epithelial membrane marker beta-catenin, and the mesenchymal marker alpha-smooth muscle actin (alpha-sma), were used to characterize the progression of EMT. Western blotting was used to quantify levels of alpha-sma and ERK1/2-signaling. Overexpression of Spry or Spred in LECs was sufficient to suppress EMT in response to TGF beta, including a block to cell elongation, beta-catenin delocalization and alpha-sma accumulation. Spry and Spred were also shown to significantly block ERK1/2 phosphorylation for up to 18 h of TGF beta treatment but did not impair the earlier activation of ERK1/2 at 20 min. These findings suggest that Spry and Spred may not directly impact ERK1/2-signaling activated by the serine/ threonine kinase TGF beta receptor, but may selectively target later ERK1/2-signaling driven by downstream RTKmediated signaling. Taken together, our data establish Spry and Spred antagonists as potent negative regulators of TGF beta-induced EMT that can regulate ERK1/2-signaling in a temporal manner. A greater understanding of how Spry and Spred regulate the complex signaling interactions that underlie TGF beta-induced EMT will be essential to facilitate the development of novel therapeutics for different pathologies driven by EMT, including fibrotic forms of cataract.