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Subtoxic levels of hydrogen peroxide induce brain-derived neurotrophic factor expression to protect PC12 cells.

Ogura Y, Sato K, Kawashima K, Kobayashi N, Imura S, Fujino K, Kawaguchi H, Nedachi T - BMC Res Notes (2014)

Bottom Line: We found, intriguingly, that gene expression of brain-derived neurotrophic factor (BDNF), but not PGRN was significantly induced by H2O2.This BDNF signaling appeared to contribute to PC12 cell protection, since PC12 cell death was significantly attenuated by BDNF treatment.Based on our results, we conclude that the induction of BDNF by subtoxic levels of H2O2 and its signaling may have roles in PC12 cell protection.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Oura-gun, Gunma 374-0193, Japan. nedachi@toyo.jp.

ABSTRACT

Background: Oxidative stress is one of the mechanisms underlying pathogenesis in neurodegenerative diseases such as Alzheimer's disease. Generally, oxidative stress represents cell toxicity; however, we recently found that oxidative stress promotes the expression of growth factor progranulin (PGRN) in HT22 murine hippocampus cells, thereby protecting the HT22 cells. In this study, we attempted to clarify whether a similar system exists in the other neuronal cell model, rat pheochromocytoma (PC12) cells.

Results: After confirming that high concentrations of hydrogen peroxide (H2O2; 100-250 μM) initiate PC12 cell death, we analyzed growth factor expressional changes after H2O2 treatment. We found, intriguingly, that gene expression of brain-derived neurotrophic factor (BDNF), but not PGRN was significantly induced by H2O2. Although little expression of the high affinity BDNF receptor tropomyosin-related kinase TrkB was observed in PC12 cells, expression of low affinity neurotrophin receptor, p75NTR, was clearly observed. This BDNF signaling appeared to contribute to PC12 cell protection, since PC12 cell death was significantly attenuated by BDNF treatment.

Conclusions: Based on our results, we conclude that the induction of BDNF by subtoxic levels of H2O2 and its signaling may have roles in PC12 cell protection.

No MeSH data available.


Related in: MedlinePlus

BDNF signaling in PC12 cells. (A) Total RNA was extracted from differentiated PC12 cells. The gene expression of each neurotrophin receptors was evaluated by RT-PCR analysis. (B) Differentiated PC12 cells were treated with the indicated amounts of BDNF for 30 min. Total and phosphorylated proteins were evaluated by western blotting analysis. Three independent experiments were performed and representative data are shown. (C, D, E) Densitometric analysis of (B). Data shown represent mean ± SEM, tested using one-way ANOVA with Tukey’s post-test (*p <0.05, **p <0.01, n = 3).
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Fig3: BDNF signaling in PC12 cells. (A) Total RNA was extracted from differentiated PC12 cells. The gene expression of each neurotrophin receptors was evaluated by RT-PCR analysis. (B) Differentiated PC12 cells were treated with the indicated amounts of BDNF for 30 min. Total and phosphorylated proteins were evaluated by western blotting analysis. Three independent experiments were performed and representative data are shown. (C, D, E) Densitometric analysis of (B). Data shown represent mean ± SEM, tested using one-way ANOVA with Tukey’s post-test (*p <0.05, **p <0.01, n = 3).

Mentions: Based on our hypothesis that BDNF induced by H2O2 may function in an autocrine/paracrine manner, we explored how PC12 cells responded against BDNF. Before evaluating the effects of BDNF on cellular functions, we analyzed whether BDNF receptor was expressed in PC12 cells, since it has been reported that high affinity BDNF receptor, TrkB, is not expressed in PC12 cells[15]. As shown in Figure 3A, we also confirmed that detectable levels of TrkB was not observed. On the contrary, TrkA, TrkC, and low affinity neurotrophin receptor p75NTR were expressed in PC12 cells (Figure 3A). BDNF-p75NTR signaling has been well studied and is especially characterized by prominent activation of NFκB signaling[16]. To test if BDNF treatment affects Trk signaling, we also analyzed Erk1/2 and Akt phosphorylation that are activated by the neurotrophin-Trk dependent signaling pathway. The amount of IκB, which inhibits NFκB nuclear translocation, was not changed by BDNF treatment (Figure 3B). The amounts and phosphorylation of NFκB were also not affected by BDNF (data not shown). In terms of Trk-dependent signaling, changes in Erk1/2 and GSK3β phosphorylation were not observed, but Akt phosphorylation was significantly decreased by BDNF treatment (Figure 3B–E). It was reported that pro-NGF induces expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative regulator for PI3K signaling, and thereby abolishes Akt activation in brain neurons[17]. However, our present results revealed that BDNF has an ability to dephosphorylate Akt acutely, within 30 min. Overall, our present data suggest that BDNF inactivated Akt perhaps via p75NTR, although little TrkB was expressed in PC12 cells.Figure 3


Subtoxic levels of hydrogen peroxide induce brain-derived neurotrophic factor expression to protect PC12 cells.

Ogura Y, Sato K, Kawashima K, Kobayashi N, Imura S, Fujino K, Kawaguchi H, Nedachi T - BMC Res Notes (2014)

BDNF signaling in PC12 cells. (A) Total RNA was extracted from differentiated PC12 cells. The gene expression of each neurotrophin receptors was evaluated by RT-PCR analysis. (B) Differentiated PC12 cells were treated with the indicated amounts of BDNF for 30 min. Total and phosphorylated proteins were evaluated by western blotting analysis. Three independent experiments were performed and representative data are shown. (C, D, E) Densitometric analysis of (B). Data shown represent mean ± SEM, tested using one-way ANOVA with Tukey’s post-test (*p <0.05, **p <0.01, n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4256810&req=5

Fig3: BDNF signaling in PC12 cells. (A) Total RNA was extracted from differentiated PC12 cells. The gene expression of each neurotrophin receptors was evaluated by RT-PCR analysis. (B) Differentiated PC12 cells were treated with the indicated amounts of BDNF for 30 min. Total and phosphorylated proteins were evaluated by western blotting analysis. Three independent experiments were performed and representative data are shown. (C, D, E) Densitometric analysis of (B). Data shown represent mean ± SEM, tested using one-way ANOVA with Tukey’s post-test (*p <0.05, **p <0.01, n = 3).
Mentions: Based on our hypothesis that BDNF induced by H2O2 may function in an autocrine/paracrine manner, we explored how PC12 cells responded against BDNF. Before evaluating the effects of BDNF on cellular functions, we analyzed whether BDNF receptor was expressed in PC12 cells, since it has been reported that high affinity BDNF receptor, TrkB, is not expressed in PC12 cells[15]. As shown in Figure 3A, we also confirmed that detectable levels of TrkB was not observed. On the contrary, TrkA, TrkC, and low affinity neurotrophin receptor p75NTR were expressed in PC12 cells (Figure 3A). BDNF-p75NTR signaling has been well studied and is especially characterized by prominent activation of NFκB signaling[16]. To test if BDNF treatment affects Trk signaling, we also analyzed Erk1/2 and Akt phosphorylation that are activated by the neurotrophin-Trk dependent signaling pathway. The amount of IκB, which inhibits NFκB nuclear translocation, was not changed by BDNF treatment (Figure 3B). The amounts and phosphorylation of NFκB were also not affected by BDNF (data not shown). In terms of Trk-dependent signaling, changes in Erk1/2 and GSK3β phosphorylation were not observed, but Akt phosphorylation was significantly decreased by BDNF treatment (Figure 3B–E). It was reported that pro-NGF induces expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative regulator for PI3K signaling, and thereby abolishes Akt activation in brain neurons[17]. However, our present results revealed that BDNF has an ability to dephosphorylate Akt acutely, within 30 min. Overall, our present data suggest that BDNF inactivated Akt perhaps via p75NTR, although little TrkB was expressed in PC12 cells.Figure 3

Bottom Line: We found, intriguingly, that gene expression of brain-derived neurotrophic factor (BDNF), but not PGRN was significantly induced by H2O2.This BDNF signaling appeared to contribute to PC12 cell protection, since PC12 cell death was significantly attenuated by BDNF treatment.Based on our results, we conclude that the induction of BDNF by subtoxic levels of H2O2 and its signaling may have roles in PC12 cell protection.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Life Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Oura-gun, Gunma 374-0193, Japan. nedachi@toyo.jp.

ABSTRACT

Background: Oxidative stress is one of the mechanisms underlying pathogenesis in neurodegenerative diseases such as Alzheimer's disease. Generally, oxidative stress represents cell toxicity; however, we recently found that oxidative stress promotes the expression of growth factor progranulin (PGRN) in HT22 murine hippocampus cells, thereby protecting the HT22 cells. In this study, we attempted to clarify whether a similar system exists in the other neuronal cell model, rat pheochromocytoma (PC12) cells.

Results: After confirming that high concentrations of hydrogen peroxide (H2O2; 100-250 μM) initiate PC12 cell death, we analyzed growth factor expressional changes after H2O2 treatment. We found, intriguingly, that gene expression of brain-derived neurotrophic factor (BDNF), but not PGRN was significantly induced by H2O2. Although little expression of the high affinity BDNF receptor tropomyosin-related kinase TrkB was observed in PC12 cells, expression of low affinity neurotrophin receptor, p75NTR, was clearly observed. This BDNF signaling appeared to contribute to PC12 cell protection, since PC12 cell death was significantly attenuated by BDNF treatment.

Conclusions: Based on our results, we conclude that the induction of BDNF by subtoxic levels of H2O2 and its signaling may have roles in PC12 cell protection.

No MeSH data available.


Related in: MedlinePlus