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Ram H Nagaraj, Mi-Hyun Nam; AGEs promote TGFβ2-mediated EMT response through the non-canonical pathway. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5634. doi: https://doi.org/.
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Basement proteins are long-lived and accumulate Advanced Glycation End products (AGEs) during aging. We have previously shown that AGEs in extracellular matrix (ECM) proteins promote TGFβ2 mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs). Such EMT could play a role in fibrosis associated with posterior capsule opacification (PCO). We investigated the signaling mechanisms by which ECM-AGEs enhance TGFβ2-mediated EMT in LECs.
Microplate wells were coated with a basement protein extract (BME) and glycated with a mixture of glucose, ascorbate and methylglyoxal. Human FHL124 cells and mouse lens epithelial cells were seeded on AGE-modified BME (AGE-BME) or unmodified BME, serum-starved overnight and stimulated with 10 ng/ml of TGFβ2. The TGFβ signaling pathway was assessed using intracellular bead-based multiplex assay kits (except for p38MAPK). To investigate the role of Snail in EMT, FHL124 cells were transfected with non-silencing or Snail-specific siRNA before treatment with TGFβ2. Western blotting and qPCR methods measured specific protein and mRNA levels.
In LECs cultured on AGE-BME, TGFβ2 treatment upregulated the mesenchymal markers α-SMA and αB-crystallin, and downregulated the epithelial marker E-cadherin more than LECs cultured on BME. In a Multiplex Assay we found that AGE-BME significantly upregulated the non-canonical pathway by promoting phosphorylation of ERK, AKT, and p38 MAPK 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 upregulation of nuclear Snail and reduced phosphorylation of GSK3β. Inhibition of Snail expression suppressed TGFβ2-mediated α-SMA expression. αB-Crystallin was upregulated 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.
The 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.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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