Matrix-bound AGEs enhance TGFβ2-mediated mesenchymal transition of lens epithelial cells via the noncanonical pathway: implications for secondary cataract formation.

Advanced glycation end products (AGEs) are post-translational modifications formed from the reaction of reactive carbonyl compounds with amino groups in proteins. Our laboratory has previously shown that AGEs in extracellular matrix (ECM) proteins promote TGFβ2 (transforming growth factor-beta 2)-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs), which could play a role in fibrosis associated with posterior capsule opacification. We have also shown that αB-crystallin plays an important role in TGFβ2-mediated EMT of LECs. Here, we investigated the signaling mechanisms by which ECM-AGEs enhance TGFβ2-mediated EMT in LECs. We found that in LECs cultured on AGE-modified basement protein extract (AGE-BME), TGFβ2 treatment up-regulated the mesenchymal markers α-SMA (α-smooth muscle actin) and αB-crystallin and down-regulated the epithelial marker E-cadherin more than LECs cultured on unmodified BME and treated with TGFβ2. Using a Multiplex Assay, we found that AGE-BME significantly up-regulated the noncanonical pathway by promoting phosphorylation of ERK (extracellular signal-regulated kinases), AKT, and p38 MAPK (mitogen-activated protein kinases) during TGFβ2-mediated EMT. This EMT response was strongly suppressed by inhibition of AKT and p38 MAPK phosphorylation. The AKT inhibitor LY294002 also suppressed TGFβ2-induced up-regulation of nuclear Snail and reduced phosphorylation of GSK3β. Inhibition of Snail expression suppressed TGFβ2-mediated α-SMA expression. αB-Crystallin was up-regulated in an AKT-dependent manner during AGE-BME/TGFβ2-mediated EMT in LECs. The absence of αB-crystallin in LECs suppressed TGFβ2-induced GSK3β phosphorylation, resulting in lower Snail levels. Taken together, these results show that ECM-AGEs enhance the TGFβ2-mediated EMT response through activation of the AKT/Snail pathway, in which αB-crystallin plays an important role as a linker between the TGFβ2 and AGE-mediated signaling pathways.