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

Cammarata PR, Neelam S, Brooks MM - Mol. Vis. (2015)

Bottom Line: XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression.Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed.Instead, the loss of HIF-1α (but not HIF-2α) decreases the expression of the EMT marker proteins without sacrificing the levels of the prosurvival protein VEGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX.

ABSTRACT

Purpose: 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, α-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.

Methods: HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an 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.

Results: SB216763-treated HLE-B3 cells caused 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 looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed.

Conclusions: 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 from the hypoxic condition clearly establish that nuclear β-catenin plays little apparent role in the expression of EMT marker proteins. Instead, the loss of HIF-1α (but not HIF-2α) 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.

No MeSH data available.


Related in: MedlinePlus

Western blot analysis of fibronectin and α-SMA in HLE-B3 cells treated with XAV939. HLE-B3 cells were cultured in 100 mm2 culture dishes and incubated with 1 µm XAV939 for 3 h in hypoxia. At the end of 3 h the total cell lysate was collected and analyzed with western blot. The experiment was repeated three times with independent cell populations and was quantified using ImageJ analysis. The asterisk (*) signifies there was a small but significant increase in fibronectin and alpha smooth muscle actin (α-SMA) in the XAV939-treated cells compared with the controls (p<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4556159&req=5

f6: Western blot analysis of fibronectin and α-SMA in HLE-B3 cells treated with XAV939. HLE-B3 cells were cultured in 100 mm2 culture dishes and incubated with 1 µm XAV939 for 3 h in hypoxia. At the end of 3 h the total cell lysate was collected and analyzed with western blot. The experiment was repeated three times with independent cell populations and was quantified using ImageJ analysis. The asterisk (*) signifies there was a small but significant increase in fibronectin and alpha smooth muscle actin (α-SMA) in the XAV939-treated cells compared with the controls (p<0.05).

Mentions: To further confirm whether there is an association between nuclear β-catenin and mesenchymal marker protein expression with hypoxic lens epithelial cells, HLE-B3 cells were cultured in 100 mm2 culture dishes and incubated with 1 µm XAV939 for 3 h in hypoxia. The total cell lysates were collected and used to detect the fibronectin and α-SMA levels with western blot analysis. The decrease in nuclear β-catenin with the XAV939 treatment (Figure 5) correlated with a small but statistically significant increase in the expression of the EMT proteins, fibronectin and α-SMA, compared to the control cells (Figure 6). The experiment was repeated three times, each time with independent cell populations of HLE-B3 cells.


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

Cammarata PR, Neelam S, Brooks MM - Mol. Vis. (2015)

Western blot analysis of fibronectin and α-SMA in HLE-B3 cells treated with XAV939. HLE-B3 cells were cultured in 100 mm2 culture dishes and incubated with 1 µm XAV939 for 3 h in hypoxia. At the end of 3 h the total cell lysate was collected and analyzed with western blot. The experiment was repeated three times with independent cell populations and was quantified using ImageJ analysis. The asterisk (*) signifies there was a small but significant increase in fibronectin and alpha smooth muscle actin (α-SMA) in the XAV939-treated cells compared with the controls (p<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556159&req=5

f6: Western blot analysis of fibronectin and α-SMA in HLE-B3 cells treated with XAV939. HLE-B3 cells were cultured in 100 mm2 culture dishes and incubated with 1 µm XAV939 for 3 h in hypoxia. At the end of 3 h the total cell lysate was collected and analyzed with western blot. The experiment was repeated three times with independent cell populations and was quantified using ImageJ analysis. The asterisk (*) signifies there was a small but significant increase in fibronectin and alpha smooth muscle actin (α-SMA) in the XAV939-treated cells compared with the controls (p<0.05).
Mentions: To further confirm whether there is an association between nuclear β-catenin and mesenchymal marker protein expression with hypoxic lens epithelial cells, HLE-B3 cells were cultured in 100 mm2 culture dishes and incubated with 1 µm XAV939 for 3 h in hypoxia. The total cell lysates were collected and used to detect the fibronectin and α-SMA levels with western blot analysis. The decrease in nuclear β-catenin with the XAV939 treatment (Figure 5) correlated with a small but statistically significant increase in the expression of the EMT proteins, fibronectin and α-SMA, compared to the control cells (Figure 6). The experiment was repeated three times, each time with independent cell populations of HLE-B3 cells.

Bottom Line: XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression.Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed.Instead, the loss of HIF-1α (but not HIF-2α) decreases the expression of the EMT marker proteins without sacrificing the levels of the prosurvival protein VEGF.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX.

ABSTRACT

Purpose: 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, α-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.

Methods: HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an 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.

Results: SB216763-treated HLE-B3 cells caused 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 looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed.

Conclusions: 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 from the hypoxic condition clearly establish that nuclear β-catenin plays little apparent role in the expression of EMT marker proteins. Instead, the loss of HIF-1α (but not HIF-2α) 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.

No MeSH data available.


Related in: MedlinePlus