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 TGF beta 2 (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 alpha B-crystallin plays an important role in TGF beta 2-mediated EMT of LECs. Here, we investigated the signaling mechanisms by which ECM-AGEs enhance TGF beta 2-mediated EMT in LECs. We found that in LECs cultured on AGE-modified basement protein extract (AGE-BME), TGF beta 2 treatment up-regulated the mesenchymal markers alpha-SMA (alpha-smooth muscle actin) and alpha B-crystallin and down-regulated the epithelial marker E-cadherin more than LECs cultured on unmodified BME and treated with TGF beta 2. 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 TGF beta 2-mediated EMT. This EMT response was strongly suppressed by inhibition of AKT and p38 MAPK phosphorylation. The AKT inhibitor LY294002 also suppressed TGF beta 2-induced up-regulation of nuclear Snail and reduced phosphorylation of GSK3 beta. Inhibition of Snail expression suppressed TGF beta 2-mediated alpha-SMA expression. alpha B-Crystallin was up-regulated in an AKT-dependent manner during AGE-BME/TGF beta 2-mediated EMT in LECs. The absence of alpha B-crystallin in LECs suppressed TGF beta 2-induced GSK3 beta phosphorylation, resulting in lower Snail levels. Taken together, these results show that ECM-AGEs enhance the TGF beta 2-mediated EMT response through activation of the AKT/Snail pathway, in which alpha B-crystallin plays an important role as a linker between the TGF beta 2 and AGE-mediated signaling pathways.