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Patrick R Cammarata, Sudha Neelam, Morgan M Brooks; Inhibition of HIF-1α Downregulates EMT Early Marker Proteins Without Undermining Cell Survival in Hypoxic Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2650.
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© ARVO (1962-2015); The Authors (2016-present)
To identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins without sacrificing the pro-survival protein, VEGF, in cultured hypoxic lens epithelial cells.
HLE-B3 cells, maintained in continuous hypoxic environment (1% oxygen) were treated with SB216763, a specific inhibitor of GSK-3β catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin and phospho-β-catenin, as well as the total lysate content of fibronectin and α-smooth muscle actin (α-SMA). ELISA was used to measure the levels of VEGF in cell culture supernatants. A HIF-1α translation inhibitor and a HIF-2α 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 β-catenin and its downstream effect on EMT marker proteins and VEGF expression.
SB216763-treated HLE-B3 cells caused a marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin resulted in significant increase of basal expression of VEGF as well as increased expression of fibronectin and α-SMA. Administration of a HIF-1α translation inhibitor, but not a HIF-2α translation inhibitor, markedly suppressed expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin whereas neither the levels of the EMT marker proteins nor VEGF were changed.
Recently, we reported that nuclear β-catenin but not HIF-2α, regulates the expression of fibronectin and α-SMA in atmospheric oxygen. In marked contrast, data stemming from the hypoxic condition clearly establishes that nuclear β-catenin plays no apparent role in the expression of EMT marker proteins. Rather, it is the loss of HIF-1α (but not HIF-2α), that lowers the expression of the EMT marker proteins without sacrificing the levels of the pro-survival protein, VEGF. These findings support the development of a potentially relevant therapeutic strategy to undermine the progression of normal cells to mesenchymal phenotype in the naturally hypoxic lens without subverting cell viability.
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