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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 GSK-3β and GS phosphorylation in HLE-B3 cells in the presence or absence of SB216763. Total cell lysates were collected from >85% confluent HLE-B3 cell cultures that were incubated for 90 min in serum-free minimal essential media (MEM) containing either 12 µM SB216763 or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h. At the end of the incubation period, the hypoxic medium was removed, and fresh, oxygenated serum-free MEM with SB216763 or dimethyl sulfoxide (DMSO) was added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after continuous normoxic exposure (approximately 21% oxygen), hypoxic exposure (approximately 1% oxygen), or after reintroduction of atmospheric oxygen (approximately 21%) for 1, 2, or 3 h. Total cell lysates were analyzed by immunoblots using 25 µg of protein per lane. Antiactin was used to normalize the bands to ensure equivalent lane loading. Note: These data were taken from a prior publication [9] but are typical of SB216763 treatment. Note to reader: Look at the hypoxic exposure lanes to verify that glycogen synthase, a substrate of glycogen synthase kinase-3β (GSK-3β) treated with SB216763 fails to be phosphorylated. This is indicative of inactivation of GSK-3β catalytic activity that also prevents phosphorylation of β-catenin.
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f1: Western blot analysis of GSK-3β and GS phosphorylation in HLE-B3 cells in the presence or absence of SB216763. Total cell lysates were collected from >85% confluent HLE-B3 cell cultures that were incubated for 90 min in serum-free minimal essential media (MEM) containing either 12 µM SB216763 or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h. At the end of the incubation period, the hypoxic medium was removed, and fresh, oxygenated serum-free MEM with SB216763 or dimethyl sulfoxide (DMSO) was added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after continuous normoxic exposure (approximately 21% oxygen), hypoxic exposure (approximately 1% oxygen), or after reintroduction of atmospheric oxygen (approximately 21%) for 1, 2, or 3 h. Total cell lysates were analyzed by immunoblots using 25 µg of protein per lane. Antiactin was used to normalize the bands to ensure equivalent lane loading. Note: These data were taken from a prior publication [9] but are typical of SB216763 treatment. Note to reader: Look at the hypoxic exposure lanes to verify that glycogen synthase, a substrate of glycogen synthase kinase-3β (GSK-3β) treated with SB216763 fails to be phosphorylated. This is indicative of inactivation of GSK-3β catalytic activity that also prevents phosphorylation of β-catenin.

Mentions: Our experimental approach involved the intentional bypass of the physiologic influence of activated TGF-β/Wnt by oxygen exposure (as might occur during cataract surgery) by going directly to inhibition of GSK-3β catalytic activity with pharmaceutical intervention. The direct inhibition of GSK-3β catalytic activity prevents phosphorylation of the downstream substrates, β-catenin and HIF-1α. Non-phosphorylated GSK-3β is the active form of the enzyme. The active form of the enzyme phosphorylates its downstream substrate, glycogen synthase (GS). Phosphorylation of GS is a useful indicator of the inactivation of GSK-3β activity. Treatment of HLE-B3 cells with SB216763 resulted in inhibition of phosphorylation of GS compared to the untreated controls (Figure 1). There was no significant change in the levels of GSK-3β and phosphoglycogen synthase kinase-3β (pGSK-3β) between the control and the SB216763-treated cells (Figure 1). Many incorrectly use the autophosphorylation of GSK-3β as an indicator of GSK-3β activity. Note that the autophosphorylation of GSK-3β is unaffected by the treatment with SB216763, whereas inhibition of the catalytic site prevented downstream phosphorylation of glycogen synthase. The data presented are reproduced from a previous publication [9]. Cultures of HLE-B3 cells were grown on 100 mm dishes until >85% confluence. Cells were treated with 12 µM SB216763 or mock-treated with DMSO (control). After 90 min in ambient oxygen, the cells were placed under hypoxic conditions (about 1% O2) for 3 h and then switched back to atmospheric oxygen (about 21% O2) for 3 h. Samples were collected from cells consistently maintained in atmospheric oxygen (control) immediately after hypoxic exposure and 1, 2, and 3 h of reexposure to atmospheric oxygen. The reader should note the effective blockage of phosphoglycogen synthase (pGS) with the SB216763 treatment irrespective of whether the cells were maintained in atmospheric oxygen (control) or taken through 3 h of hypoxic exposure or reintroduced to atmospheric oxygen subsequent to hypoxic exposure.


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 GSK-3β and GS phosphorylation in HLE-B3 cells in the presence or absence of SB216763. Total cell lysates were collected from >85% confluent HLE-B3 cell cultures that were incubated for 90 min in serum-free minimal essential media (MEM) containing either 12 µM SB216763 or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h. At the end of the incubation period, the hypoxic medium was removed, and fresh, oxygenated serum-free MEM with SB216763 or dimethyl sulfoxide (DMSO) was added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after continuous normoxic exposure (approximately 21% oxygen), hypoxic exposure (approximately 1% oxygen), or after reintroduction of atmospheric oxygen (approximately 21%) for 1, 2, or 3 h. Total cell lysates were analyzed by immunoblots using 25 µg of protein per lane. Antiactin was used to normalize the bands to ensure equivalent lane loading. Note: These data were taken from a prior publication [9] but are typical of SB216763 treatment. Note to reader: Look at the hypoxic exposure lanes to verify that glycogen synthase, a substrate of glycogen synthase kinase-3β (GSK-3β) treated with SB216763 fails to be phosphorylated. This is indicative of inactivation of GSK-3β catalytic activity that also prevents phosphorylation of β-catenin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f1: Western blot analysis of GSK-3β and GS phosphorylation in HLE-B3 cells in the presence or absence of SB216763. Total cell lysates were collected from >85% confluent HLE-B3 cell cultures that were incubated for 90 min in serum-free minimal essential media (MEM) containing either 12 µM SB216763 or 0.05% DMSO vehicle. Cells were then exposed to hypoxia for 3 h. At the end of the incubation period, the hypoxic medium was removed, and fresh, oxygenated serum-free MEM with SB216763 or dimethyl sulfoxide (DMSO) was added to the cultures. Cells were then placed in atmospheric oxygen for up to 3 h. Cultures were collected after continuous normoxic exposure (approximately 21% oxygen), hypoxic exposure (approximately 1% oxygen), or after reintroduction of atmospheric oxygen (approximately 21%) for 1, 2, or 3 h. Total cell lysates were analyzed by immunoblots using 25 µg of protein per lane. Antiactin was used to normalize the bands to ensure equivalent lane loading. Note: These data were taken from a prior publication [9] but are typical of SB216763 treatment. Note to reader: Look at the hypoxic exposure lanes to verify that glycogen synthase, a substrate of glycogen synthase kinase-3β (GSK-3β) treated with SB216763 fails to be phosphorylated. This is indicative of inactivation of GSK-3β catalytic activity that also prevents phosphorylation of β-catenin.
Mentions: Our experimental approach involved the intentional bypass of the physiologic influence of activated TGF-β/Wnt by oxygen exposure (as might occur during cataract surgery) by going directly to inhibition of GSK-3β catalytic activity with pharmaceutical intervention. The direct inhibition of GSK-3β catalytic activity prevents phosphorylation of the downstream substrates, β-catenin and HIF-1α. Non-phosphorylated GSK-3β is the active form of the enzyme. The active form of the enzyme phosphorylates its downstream substrate, glycogen synthase (GS). Phosphorylation of GS is a useful indicator of the inactivation of GSK-3β activity. Treatment of HLE-B3 cells with SB216763 resulted in inhibition of phosphorylation of GS compared to the untreated controls (Figure 1). There was no significant change in the levels of GSK-3β and phosphoglycogen synthase kinase-3β (pGSK-3β) between the control and the SB216763-treated cells (Figure 1). Many incorrectly use the autophosphorylation of GSK-3β as an indicator of GSK-3β activity. Note that the autophosphorylation of GSK-3β is unaffected by the treatment with SB216763, whereas inhibition of the catalytic site prevented downstream phosphorylation of glycogen synthase. The data presented are reproduced from a previous publication [9]. Cultures of HLE-B3 cells were grown on 100 mm dishes until >85% confluence. Cells were treated with 12 µM SB216763 or mock-treated with DMSO (control). After 90 min in ambient oxygen, the cells were placed under hypoxic conditions (about 1% O2) for 3 h and then switched back to atmospheric oxygen (about 21% O2) for 3 h. Samples were collected from cells consistently maintained in atmospheric oxygen (control) immediately after hypoxic exposure and 1, 2, and 3 h of reexposure to atmospheric oxygen. The reader should note the effective blockage of phosphoglycogen synthase (pGS) with the SB216763 treatment irrespective of whether the cells were maintained in atmospheric oxygen (control) or taken through 3 h of hypoxic exposure or reintroduced to atmospheric oxygen subsequent to hypoxic exposure.

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