Abstract
Purpose :
Purpose: Transforming growth factor (TGF-β2) mediated pathways are integral to the epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) during secondary cataract formation. Previous studies have suggested that hypoxia inducible factor-1α (HIF-1α) could play a role in EMT. In this study we investigated the role of HIF-1α in TGF-β2-induced EMT of human LECs.
Methods :
Methods: Human lens epithelial cells (FHL124) were cultured at 21% or 1% oxygen and treated with TGF-β2 (10 ng/ml) for 48 h to induce the EMT response. The HIF-1α and VEGF-A levels were measured by western blotting and ELISA, respectively. To determine the direct effect of HIF-1α on the EMT of LECs, FHL124 cells were treated with either a prolyl hydroxylase inhibitor, dimethyloxaloylglycine (DMOG, 0 to 10 μM or 500 μM) or it was overexpressed by transfecting with a HIF-1α plasmid. To further study the role of HIF-1α in TGF-β2 induced EMT, we used 200 nM of KC7F2, a HIF-1α translation inhibitor. To check the direct effect of VEGF-A on EMT, cells were cultured with 0 to 100 ng/ml of recombinant human VEGF-A. Expression of various EMT markers was analyzed by western blotting.
Results :
Results: TGF-β2 treatment resulted in the upregulation of EMT markers (αSMA, αV and β1 -Integrin), which was largely independent of the oxygen levels. TGF-β2 treatment increased the HIF-1α levels in cells, along with the VEGF-A levels in the conditioned media. HIF-1α upregulation either by DMOG treatment or forced overexpression (without TGF-β2 treatment) failed to induce the EMT response. Direct VEGF-A treatment also showed no EMT response. However, the TGF-β2 mediated EMT response was significantly reduced in cells treated with KC7F2, and also in cells treated with high levels of DMOG (500 μM).
Conclusions :
Conclusion: Our results suggest that the mild upregulation of HIF-1α might be necessary for the TGF-β2-mediated EMT response in lens epithelial cells, but that is independent of VEGF-A.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.