Abstract
Purpose :
p38 is a downstream MAPK important for non-canonical transforming growth factor β- (TGF-β) signaling associated with epithelial-mesenchymal transition (EMT). While past studies have implicated p38 in lens EMT associated with fibrotic cataract, the inconsistency in the effects of inhibiting p38, along with the lack of selectivity of the p38 inhibitors employed, requires further investigation. Here we use inhibitors that are reportedly more selective for p38α, the main p38 isoform involved in fibrosis, to elucidate the functional role of p38 in TGFβ-induced lens EMT leading to fibrotic cataract.
Methods :
Postnatal 21-day-old rat lens epithelial explants were treated for up to 5 days with TGF-β2 (200 pg/ml) to induce EMT, with or without selective p38α inhibitors, VX-745 (2µM) and Skepinone-L (1µM). Changes in different EMT markers, as well as p38- and Smad2/3-signaling were assessed using immunofluorescence and Western blotting. TGF-β-overexpressing transgenic mice were used to validate some of our in vitro findings.
Results :
Activation (phosphorylation) of p38 by TGF-β in lens epithelia presented as a biphasic response, initiated as early as 2 hours and peaking at 6 hours, before dropping and increasing again by 18 hours. This was consistent with elevated levels of phospho-p38 in fibrotic plaques of lenses of transgenic mice. Pre-treatment with VX-745 or Skepinone-L was able to block the morphological changes and apoptosis associated with TGF-β-induced lens EMT in vitro. Consistent with this, the EMT marker tropomyosin (Tpm1.6/1.7 and Tpm2.1) was blocked; however, the hallmark EMT marker, α-SMA, was not. The nuclear translocation of Smad2/3 induced by TGF-β was found not to be dependent on p38 activity when lens epithelial cells were pre-treated with VX-745 ahead of TGF-β.
Conclusions :
Our findings validate a role for p38 in regulation of cell morphology leading to myofibroblasts associated with TGF-β-induced lens EMT, as well as apoptosis. We can uncouple this lens EMT process, given that α-SMA accumulation was independent of p38 activity, and more likely directly dependent on Smad2/3-signaling. These findings support a key role for p38 in lens EMT, and may lead to the development of potential therapeutics for preventing fibrotic cataract.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.