Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults

Wnt proteins are involved in tissue development and their signaling pathways play an important role during embryogenesis. Wnt signaling can promote cell survival, which is beneficial for neurons, but could also lead to tumor development in different tissues. The present study investigated the effects of a Wnt protein on the susceptibility of a neural tumor cell line (PC12 cells) to the cytotoxic compounds ferrous sulfate (10 mM), staurosporine (100 and 500 nM), 3-nitropropionic acid (5 mM), and amyloid β-peptide (Aβ25-35; 50 µM). Cells (1 x 10(6) cells/mL) were treated with the Wnt-3a recombinant peptide (200 ng/mL) for 24 h before exposure to toxic insults. The Wnt-3a protein partially protected PC12 cells, with a 6-15% increase in cell viability in the presence of toxic agents, similar to the effect measured using the MTT and lactate dehydrogenase cell viability assays. The Wnt-3a protein increased protein expression of β-catenin by 52% compared to control. These findings suggest that Wnt signaling can protect neural cells against apoptosis induced by toxic agents, which are relevant to the pathogenesis of Alzheimer’s and Huntington’s diseases.

Wnt-3a; PC12 cells; Beta-catenin; NF-κB

Authorship

E.M. Kawamoto

Universidade de São Paulo, Instituto de Ciências Biomédicas , Departamento de Farmacologia, São Paulo, São Paulo, BrazilUniversidade de São PauloBrazilSão Paulo, São Paulo, BrazilUniversidade de São Paulo, Instituto de Ciências Biomédicas , Departamento de Farmacologia, São Paulo, São Paulo, Brazil

National Institute on Aging Intramural Research Program, Laboratory of Neurosciences , Baltimore, MD, USANational Institute on Aging Intramural Research ProgramUSABaltimore, MD, USANational Institute on Aging Intramural Research Program, Laboratory of Neurosciences , Baltimore, MD, USA

M. Gleichmann

L.M. Yshii

L. de Sá Lima

M.P. Mattson

C. Scavone

SCIMAGO INSTITUTIONS RANKINGS

Figures

Figures (8)

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Figure 1.
mRNA expression of Frizzled (Fz) receptors (1-10) in PC12 cells by regular RT-PCR. Gel electrophoresis of the PCR product was performed using a 2% ethidium bromide-containing agarose gel and the resulting bands were visualized under UV light. lane 1: Fz receptor 1; lane 2: Fz receptor 2; lane 3: Fz receptor 3; lane 4: Fz receptor 4; lane 5: Fz receptor 5; lane 6: Fz receptor 6; lane 7: Fz receptor 7; lane 8: Fz receptor 8; lane 9: Fz receptor 9; lane 10: Fz receptor 10.

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Figure 2.
Effects of Wnt-3a (100 ng/mL) on β-catenin expression by immunoblot assay. A, A representative immunoblot shows increased β-catenin expression in PC12 cells incubated with Wnt-3a recombinant peptide. β-actin was used as internal control. B, The respective densitometric analyses (arbitrary units, AU) of the β-catenin/β-actin ratio bands presented in Panel A is shown. Data are reported as means ± SEM for 5 independent experiments. *P < 0.02 compared to control (unpaired Student t-test).

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Figure 3.
Wnt-3a peptide reduced the loss of cell viability induced by staurosporine. A,B, PC12 cells were incubated with 100 nM (A) and 500 nM (B) staurosporine 24 h after the addition of mouse recombinant Wnt-3a peptide (100 ng/mL). C,D, PC12 cells were incubated with 500 nM staurosporine 24 h after the addition of three different concentrations of mouse recombinant Wnt-3a peptide (50, 100, and 200 ng/mL). Twenty-four hours later, cell viability was measured by MTT and lactate dehydrogenase (LDH). Data are reported as means ± SEM for 6 independent experiments. *P <0.001 vs control (vehicle: 0.08% DMSO); **P < 0.05 vs staurosporine; ⁺P < 0.05 vs staurosporine + 50 ng/mL Wnt or staurosporine + 100 ng/mL WNT (one-way ANOVA followed by the Newman-Keuls test).

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Figure 4.
Wnt-3a peptide reduced the loss of cell viability induced by 10 mM FeSO₄ (Fe). A,B, PC12 cells were incubated with 10 mM FeSO₄ 24 h after the addition of mouse recombinant Wnt-3a peptide (100 ng/mL). Twenty-four hours later, cell viability was measured by A, MTT reduction (570 nm) and B, by lactate dehydrogenase (LDH) release (490 nm). Data are reported as means ± SEM for 6 independent experiments. *P < 0.001 vs control (vehicle: water); **P < 0.05 vs Fe (one-way ANOVA followed by the Newman-Keuls test).

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Figure 5.
Effect of two concentrations of Wnt-3a peptide against 3-nitropropionic acid (3-NP) toxicity. PC12 cells were incubated with Wnt-3a (100 or 200 ng/mL) for 24 h followed by incubation with 5 mM 3-NP for 24 h. Twenty-four hours later, cell viability was measured by A, MTT reduction (570 nm) and by B, lactate dehydrogenase (LDH) release (490 nm). Data are reported as means ± SEM for 6 independent experiments. *P < 0.001 vs control (vehicle: 0.08% ethanol), **P < 0.05 vs 3-NP (one-way ANOVA followed by the Newman-Keuls test).

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Figure 6.
Effect of recombinant Wnt-3a protein on loss of cell viability induced by Aβ_25-35. PC12 cells were incubated with Wnt-3a (100 ng/mL) for 24 h followed by 50 µM Aβ_25-35for 24 h. Twenty-four hours later, cell viability and protein expression were evaluated by A, MTT reduction (570 nm) and by B, lactate dehydrogenase (LDH) release (490 nm) assays. Data are reported as means ± SEM for 6 independent experiments. *P < 0.01 vs control (vehicle: PBS); **P < 0.05 vs Aβ (one-way ANOVA followed by the Newman-Keuls test).

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Figure 7.
Effect of Wnt-3a on NF-κB suppression induced by staurosporine and FeSO₄ on PC12 cells. Nuclear proteins (2.5 µg) were extracted from PC12 cells pre-incubated (24 h) with mouse recombinant peptide Wnt-3a (100 ng/mL) followed by 24-h incubation with A, 500 nM staurosporine or B, 10 mM FeSO₄. Densitometric analyses (arbitrary units, AU) of the NF-κB band are presented in Panels A and B. Data are reported as means ± SEM for 3 independent experiments *P < 0.001 vs control; **P < 0.05 vs staurosporine and FeSO₄ (one-way ANOVA followed by the Newman-Keuls test). C, Competition studies were performed using nuclear extracts (2.5 µg) from PC12 cells pre-incubated (24 h) with mouse recombinant protein Wnt-3a (100 ng/mL) followed by 24-h incubation with saline in the absence and in the presence of unlabeled specific oligonucleotide (2.5- and 10-fold molar excess), as indicated. D, Supershift assays were performed with nuclear proteins (2.5 µg) extracted from PC12 cells pre-incubated (24 h) with mouse recombinant peptide Wnt-3a (100 ng/mL) followed by 24-h incubation with FeSO₄ (10 mM) in the absence or presence of antibodies against subunits p50 (1:10 dilution), p65 (1:10 dilution), as indicated. Antibodies were added 20 min prior to the addition of the radiolabeled NF-κB consensus oligonucleotide. The position of the specific NF-κB/DNA binding complex p50/p65 (band) is indicated. The localization of the probe is also indicated. Results are representative of 3 independent experiments.

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Figure 8.
Effect of recombinant Wnt-3a protein on β-catenin protein suppression induced by staurosporine and FeSO₄. PC12 cells were pre-incubated with Wnt-3a recombinant protein (100 ng/mL) 24 h before treatment with 500 nM staurosporine and 10 mM FeSO₄. Twenty-four hours later, cytosolic proteins (10 µg) were isolated and β-catenin levels were determined by immunoblot. The respective densitometric analyses (arbitrary units, AU) of the β-catenin/β-actin ratio bands are presented in Panels A and B. Data are reported as means ± SEM for 3 independent experiments (one-way ANOVA followed by the Newman-Keuls test). *P < 0.05 vs control; ⁺P < 0.01 vs staurosporine + Wnt-3a; ^#P < 0.05 vs control and FeSO₄. β-actin was used as internal control.

Figure 1. mRNA expression of Frizzled (Fz) receptors (1-10) in PC12 cells by regular RT-PCR. Gel electrophoresis of the PCR product was performed using a 2% ethidium bromide-containing agarose gel and the resulting bands were visualized under UV light. lane 1: Fz receptor 1; lane 2: Fz receptor 2; lane 3: Fz receptor 3; lane 4: Fz receptor 4; lane 5: Fz receptor 5; lane 6: Fz receptor 6; lane 7: Fz receptor 7; lane 8: Fz receptor 8; lane 9: Fz receptor 9; lane 10: Fz receptor 10.

Figure 2. Effects of Wnt-3a (100 ng/mL) on β-catenin expression by immunoblot assay. A, A representative immunoblot shows increased β-catenin expression in PC12 cells incubated with Wnt-3a recombinant peptide. β-actin was used as internal control. B, The respective densitometric analyses (arbitrary units, AU) of the β-catenin/β-actin ratio bands presented in Panel A is shown. Data are reported as means ± SEM for 5 independent experiments. *P < 0.02 compared to control (unpaired Student t-test).

Figure 3. Wnt-3a peptide reduced the loss of cell viability induced by staurosporine. A,B, PC12 cells were incubated with 100 nM (A) and 500 nM (B) staurosporine 24 h after the addition of mouse recombinant Wnt-3a peptide (100 ng/mL). C,D, PC12 cells were incubated with 500 nM staurosporine 24 h after the addition of three different concentrations of mouse recombinant Wnt-3a peptide (50, 100, and 200 ng/mL). Twenty-four hours later, cell viability was measured by MTT and lactate dehydrogenase (LDH). Data are reported as means ± SEM for 6 independent experiments. *P <0.001 vs control (vehicle: 0.08% DMSO); **P < 0.05 vs staurosporine; ⁺P < 0.05 vs staurosporine + 50 ng/mL Wnt or staurosporine + 100 ng/mL WNT (one-way ANOVA followed by the Newman-Keuls test).

Figure 4. Wnt-3a peptide reduced the loss of cell viability induced by 10 mM FeSO₄ (Fe). A,B, PC12 cells were incubated with 10 mM FeSO₄ 24 h after the addition of mouse recombinant Wnt-3a peptide (100 ng/mL). Twenty-four hours later, cell viability was measured by A, MTT reduction (570 nm) and B, by lactate dehydrogenase (LDH) release (490 nm). Data are reported as means ± SEM for 6 independent experiments. *P < 0.001 vs control (vehicle: water); **P < 0.05 vs Fe (one-way ANOVA followed by the Newman-Keuls test).

Figure 5. Effect of two concentrations of Wnt-3a peptide against 3-nitropropionic acid (3-NP) toxicity. PC12 cells were incubated with Wnt-3a (100 or 200 ng/mL) for 24 h followed by incubation with 5 mM 3-NP for 24 h. Twenty-four hours later, cell viability was measured by A, MTT reduction (570 nm) and by B, lactate dehydrogenase (LDH) release (490 nm). Data are reported as means ± SEM for 6 independent experiments. *P < 0.001 vs control (vehicle: 0.08% ethanol), **P < 0.05 vs 3-NP (one-way ANOVA followed by the Newman-Keuls test).

Figure 6. Effect of recombinant Wnt-3a protein on loss of cell viability induced by Aβ_25-35. PC12 cells were incubated with Wnt-3a (100 ng/mL) for 24 h followed by 50 µM Aβ_25-35for 24 h. Twenty-four hours later, cell viability and protein expression were evaluated by A, MTT reduction (570 nm) and by B, lactate dehydrogenase (LDH) release (490 nm) assays. Data are reported as means ± SEM for 6 independent experiments. *P < 0.01 vs control (vehicle: PBS); **P < 0.05 vs Aβ (one-way ANOVA followed by the Newman-Keuls test).

Figure 7. Effect of Wnt-3a on NF-κB suppression induced by staurosporine and FeSO₄ on PC12 cells. Nuclear proteins (2.5 µg) were extracted from PC12 cells pre-incubated (24 h) with mouse recombinant peptide Wnt-3a (100 ng/mL) followed by 24-h incubation with A, 500 nM staurosporine or B, 10 mM FeSO₄. Densitometric analyses (arbitrary units, AU) of the NF-κB band are presented in Panels A and B. Data are reported as means ± SEM for 3 independent experiments *P < 0.001 vs control; **P < 0.05 vs staurosporine and FeSO₄ (one-way ANOVA followed by the Newman-Keuls test). C, Competition studies were performed using nuclear extracts (2.5 µg) from PC12 cells pre-incubated (24 h) with mouse recombinant protein Wnt-3a (100 ng/mL) followed by 24-h incubation with saline in the absence and in the presence of unlabeled specific oligonucleotide (2.5- and 10-fold molar excess), as indicated. D, Supershift assays were performed with nuclear proteins (2.5 µg) extracted from PC12 cells pre-incubated (24 h) with mouse recombinant peptide Wnt-3a (100 ng/mL) followed by 24-h incubation with FeSO₄ (10 mM) in the absence or presence of antibodies against subunits p50 (1:10 dilution), p65 (1:10 dilution), as indicated. Antibodies were added 20 min prior to the addition of the radiolabeled NF-κB consensus oligonucleotide. The position of the specific NF-κB/DNA binding complex p50/p65 (band) is indicated. The localization of the probe is also indicated. Results are representative of 3 independent experiments.

Figure 8. Effect of recombinant Wnt-3a protein on β-catenin protein suppression induced by staurosporine and FeSO₄. PC12 cells were pre-incubated with Wnt-3a recombinant protein (100 ng/mL) 24 h before treatment with 500 nM staurosporine and 10 mM FeSO₄. Twenty-four hours later, cytosolic proteins (10 µg) were isolated and β-catenin levels were determined by immunoblot. The respective densitometric analyses (arbitrary units, AU) of the β-catenin/β-actin ratio bands are presented in Panels A and B. Data are reported as means ± SEM for 3 independent experiments (one-way ANOVA followed by the Newman-Keuls test). *P < 0.05 vs control; ⁺P < 0.01 vs staurosporine + Wnt-3a; ^#P < 0.05 vs control and FeSO₄. β-actin was used as internal control.

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Effect of activation of canonical Wnt signaling by the Wnt-3a protein on the susceptibility of PC12 cells to oxidative and apoptotic insults

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