Inhibition of hypoxia inducible factor-1? downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells.

ABSTRACT: The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, ?-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells.HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3? (GSK-3?) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of ?-catenin, as well as the total lysate content of fibronectin and ?-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1? (HIF-1?) translation inhibitor and an HIF-2? translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear ?-catenin and its downstream effect on EMT marker proteins and VEGF expression.SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3? activity prompting a significant increase in the translocation of cytoplasmic ?-catenin to the nucleus. The enhancement of nuclear ?-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and ?-SMA. In conjunction with SB216763, coadministration of an HIF-1? translation inhibitor, but not an HIF-2? translation inhibitor, markedly suppressed the expression of fibronectin and ?-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear ?-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed.Recently, we reported that nuclear ?-catenin, but not HIF-2?, regulates the expression of fibronectin and ?-SMA in atmospheric oxygen. In marked contrast, data from the hypoxic condition clearly establish that nuclear ?-catenin plays little apparent role in the expression of EMT marker proteins. Instead, the loss of HIF-1? (but not HIF-2?) decreases the expression of the EMT marker proteins without sacrificing the levels of the prosurvival protein VEGF. These findings support the development of a potentially relevant therapeutic strategy to undermine the progression of normal cells to the mesenchymal phenotype in the naturally hypoxic lens without subverting cell viability.

SUBMITTER: Cammarata PR 

PROVIDER: S-EPMC4556159 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature