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Daisy Shu, Brooke P Mao, Frank J Lovicu; Non-canonical TGFβ-signaling pathways in lens EMT: interplay between EGFR and p38 MAPK. Invest. Ophthalmol. Vis. Sci. 2019;60(9):6445.
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TGFβ induces lens epithelial cells (LECs) to undergo an epithelial-mesenchymal transition (EMT) resulting in fibrotic forms of cataract, such as anterior subcapsular cataract and posterior capsular opacification. EGFR-signaling is essential for this process. While the canonical TGFβ/Smad signaling pathway plays a key role in mediating lens EMT, MAPK-signaling, such as EGFR-induced ERK1/2 is also involved, but not required for all aspects of the EMT. As other MAPKs, such as the stress-activated p38 pathway is reported to regulate apoptosis and EMT, the present study aimed to explore its role in TGFβ-induced lens EMT, and its relation to EGFR-signaling.
Lens epithelial explants from 21-day-old Wistar rats were cultured for up to five days with 200 pg/ml TGFβ2 and/or 5 ng/ml EGF, with or without 100 nM PD153035 (EGFR-inhibitor) or 10μM SB203580 (p38-inhibitor). Cell morphology was examined using phase-contrast microscopy. Labeling of β-catenin, α-smooth muscle actin (α-SMA), phospho-p38 and p38 was assessed using immunofluorescence and/or western blotting.
TGFβ induced LECs to transdifferentiate into myofibroblastic cells by day 2, and subsequently undergo apoptosis by day 5 of culture. Treatment of LECs with a combination of TGFβ and EGF resulted in a more pronounced EMT response compared to TGFβ alone. TGFβ induced the phosphorylation of p38 in LECs, with higher levels of phospho-p38 observed in cells treated with TGFβ and EGF. EGF alone did not induce phospho-p38, nor did it induce EMT in our lens explants. Inhibition of EGFR-signaling using PD153035 suppressed TGFβ-induced activation of p38 signaling. Selective inhibition of p38 activity using SB203580 completely blocked the TGFβ-induced EMT response, with retention of the epithelial phenotype (membranous β-catenin labeling), absence of α-SMA-positive stress fibers and no evidence of cell loss due to apoptosis over the five-day culture period.
p38-signaling is essential for TGFβ-induced EMT of LECs and contributes to the more pronounced EMT response observed when co-treating cells with TGFβ and EGF. TGFβ-driven p38 activation appears to involve EGFR-signaling, highlighting the interplay between these non-canonical TGFβ pathways. Targeting p38-signaling may be an effective strategy for the pharmacological treatment of fibrotic cataract.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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