Enhanced EGF receptor-signaling potentiates TGFβ-induced lens epithelial-mesenchymal transition

Abstract

The ocular lens is exposed to numerous growth factors that influence its behavior in diverse ways. While many of these, such as FGF and EGF promote normal cell behavior, TGFβ is unique in that it can also induce lens cell pathology, namely, the epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) leading to fibrotic cataract formation. The present study explores how EGF impacts on TGFβ-induced EMT in the lens. LECs in explants prepared from 21-day-old Wistar rats were treated with either 200 pg/ml TGFβ2, 5 ng/ml EGF, or a combination of these, with or without a 2-h pre-treatment of an EGFR inhibitor (PD153035), MEK inhibitor (U0126) or Smad3 inhibitor (SIS3). Co-treatment of LECs with TGFβ2 and EGF, compared with TGFβ2 alone, resulted in a more pronounced elongation and transdifferentiation of the LECs into myofibroblastic cells, with higher protein levels of mesenchymal cell markers (α-SMA and tropomyosin). Combining EGF with a less potent lower dose of TGFβ2 (50 pg/ml) induced LECs to undergo EMT equivalent to treatment with a higher dose of TGFβ2 (200 pg/ml) within 5 days of culture. EGF alone, nor the lower dose of TGFβ2, were able to induce EMT in LECs within 5 days. Co-treatment of LECs with EGF and TGFβ2 induced a temporal shift in the phosphorylation levels of Smad2/3, ERK1/2 and EGFR and changed the expression patterns of downstream EMT target genes, compared to treatment of LECs with either growth factor alone. Inhibition of EGFR-signaling with PD153035 blocked the EMT response induced by co-treatment with EGF and TGFβ2. Taken together, our data demonstrate that EGF can potentiate TGFβ2 activity to enhance EMT in LECs, further highlighting the importance of EGFR-signaling in cataract formation. By directly blocking EGFR signaling, the activity of both EGF and TGFβ2 can be simultaneously reduced, thereby serving as a potential target for cataract prevention.

Keywords: Cataract; ERK1/2; Epidermal growth factor (EGF); Epidermal growth factor receptor (EGFR); Epithelial-mesenchymal transition (EMT); Fibrosis; Lens; Myofibroblast; Posterior capsular opacification; Smad; Transforming growth factor-beta (TGFβ).