Sprouty and Spred temporally regulate ERK1/2-signaling to suppress TGFß-induced lens EMT.

ABSTRACT

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 TGFß-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 TGFß2. Immunofluorescent labeling of explants for the epithelial membrane marker ß-catenin, and the mesenchymal marker alpha-smooth muscle actin (α-sma), were used to characterize the progression of EMT. Western blotting was used to quantify levels of α-sma and ERK1/2-signaling. Overexpression of Spry or Spred in LECs was sufficient to suppress EMT in response to TGFß, including a block to cell elongation, ß-catenin delocalization and α-sma accumulation. Spry and Spred were also shown to significantly block ERK1/2 phosphorylation for up to 18 h of TGFß 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 TGFß receptor, but may selectively target later ERK1/2-signaling driven by downstream RTK-mediated signaling. Taken together, our data establish Spry and Spred antagonists as potent negative regulators of TGFß-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 TGFß-induced EMT will be essential to facilitate the development of novel therapeutics for different pathologies driven by EMT, including fibrotic forms of cataract.