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The pro-survival role of autophagy depends on Bcl-2 under nutrition stress conditions.

Xu HD, Wu D, Gu JH, Ge JB, Wu JC, Han R, Liang ZQ, Qin ZH - PLoS ONE (2013)

Bottom Line: In contrast, overexpression of Bcl-2 inhibited autophagy and blocked cell death in response to serum deprivation.These data suggest that Bcl-2 plays an essential role in limiting autophagy activation and preventing initiation of programmed cell death.Thus Bcl-2 may be an important mechanism for balancing beneficial and detrimental impacts of autophagy on cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science, Suzhou, China.

ABSTRACT
Autophagy can be induced under nutrition stress conditions. Bcl-2 is a pro-survival protein which inhibits apoptosis and autophagy. However, the role of Bcl-2 in autophagy regulation and cell survival under nutrition deprivation has not been fully understood. This study sought to investigate if Bcl-2 upregulation is essential in limiting autophagic activity and prevent cell death under nutrition deprivation conditions. Autophagic activity was monitored by the changes in GFP-LC3 localization and protein levels of Beclin1, LC3-II, cathepsin D and p62 in neuroblastoma SH-SY5Y cells underwent serum deprivation. Manipulation of Bcl-2 function was achieved with siRNAs and small molecular inhibitors. The cell viability and apoptosis were assessed with MTT assay and Annexin V/PI staining. The results showed that serum starvation increased protein levels of LC3-II and Beclin1 but decreased autophagy substrate p62. Autophagy activation induced by serum deprivation and rapamycin was accompanied by an upregulation of Bcl-2 protein levels. When Bcl-2 was knocked down with siRNA or inhibited with HA 14-1 or ABT-737, serum starvation induced profound cell death and enhanced autophagic flux under nutrition deprivation conditions, while knockdown of autophagic gene Beclin1 or autophagy inhibitors (bafilomycin A1 and E64D), rescued cell death. In contrast, overexpression of Bcl-2 inhibited autophagy and blocked cell death in response to serum deprivation. These data suggest that Bcl-2 plays an essential role in limiting autophagy activation and preventing initiation of programmed cell death. Thus Bcl-2 may be an important mechanism for balancing beneficial and detrimental impacts of autophagy on cell survival.

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Bcl-2 was up-regulated in SH-SY5Y cells during serum starvation.A: Western blot analysis of Bcl-2 and p-Bcl-2 expression in SH-SY5Y cells subjected to starvation. B and C: Quantitative analysis of optical densities of the Bcl-2 and p-Bcl-2 protein bands with Sigma Scan Pro 5 and normalized to the loading control. Bars represent Mean ± SD (B, n = 7; C, n = 3). D: Representative confocal images (5 µm scale bar) of GFP-LC3 assay in SH-SY5Y cells transfected with GFP-LC3 plasmid. Twenty four hours after transfection, the cells were treated with rapamycin (50 nM) for 12 hrs in the present or absence of 100 nM Baf A1. E: Quantification of autophagy in rapamycin-treated SH-SY5Y cells transfected with GFP-LC3. Data represent mean ± SD from three independent experiments. F: Western blot analysis of Beclin1, Cathepsin D, Bcl-2, Bcl-xl and LC3 in SH-SY5Y cells treated with Rapamycin. G and H: Rapamycin induced upregulation of Beclin1 and Bcl-2 in SH-SY5Y cells. Quantitative analysis of optical densities of the Beclin1 and Bcl-2 protein bands with Sigma Scan Pro 5 and normalized to the loading control. Bars represent Mean ± SD (G, n = 3; H, n = 4). Statistical analysis was carried out with ANOVA followed by Dunnett t-test. *p<0.05 vs control group; **p<0.01 vs control group; ##p<0.001 vs control group.
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pone-0063232-g002: Bcl-2 was up-regulated in SH-SY5Y cells during serum starvation.A: Western blot analysis of Bcl-2 and p-Bcl-2 expression in SH-SY5Y cells subjected to starvation. B and C: Quantitative analysis of optical densities of the Bcl-2 and p-Bcl-2 protein bands with Sigma Scan Pro 5 and normalized to the loading control. Bars represent Mean ± SD (B, n = 7; C, n = 3). D: Representative confocal images (5 µm scale bar) of GFP-LC3 assay in SH-SY5Y cells transfected with GFP-LC3 plasmid. Twenty four hours after transfection, the cells were treated with rapamycin (50 nM) for 12 hrs in the present or absence of 100 nM Baf A1. E: Quantification of autophagy in rapamycin-treated SH-SY5Y cells transfected with GFP-LC3. Data represent mean ± SD from three independent experiments. F: Western blot analysis of Beclin1, Cathepsin D, Bcl-2, Bcl-xl and LC3 in SH-SY5Y cells treated with Rapamycin. G and H: Rapamycin induced upregulation of Beclin1 and Bcl-2 in SH-SY5Y cells. Quantitative analysis of optical densities of the Beclin1 and Bcl-2 protein bands with Sigma Scan Pro 5 and normalized to the loading control. Bars represent Mean ± SD (G, n = 3; H, n = 4). Statistical analysis was carried out with ANOVA followed by Dunnett t-test. *p<0.05 vs control group; **p<0.01 vs control group; ##p<0.001 vs control group.

Mentions: Previous studies have shown that Bcl-2 can bind essential autophagic protein Beclin1 and inhibit autophagy activation under acute nutrient deprivation conditions [20]. The present study found that serum starvation induced the up-regulation of total and phosphorylated Bcl-2 (Fig. 2, A and B). However, quantitative analysis of the ratio between phosphorylated Bcl-2 and total Blc-2 had no significant change (Fig. 2C). Previous studies have shown that phosphorylated Bcl-2 localizes predominantly to the ER [35], and that starvation can induce phosphorylation of the ER-localized pool of Bcl-2 [22]. The present study is consistent with the previous observation that Bcl-2 was phosphorylated under acute starvation conditions [22]. This study found that total Bcl-2 protein levels were up-regulated, thus, the increase of phosphorylated Bcl-2 could be due to the increased pool of total Bcl-2. To further test if an increase in autophagy activity through inhibition of mammalian target of rapamycin (now called the mechanistic target of rapamycin within higher eukaryotes) would cause an up-regulation of Bcl-2, the present study also used rapamycin to induce autophagy in SH-SY5Y cells. Rapamycin is a putative autophagy activator which inhibits mTOR and mimics nutrient deprivation condition in yeast, Drosophila, and mammalian cells [36], [37]. The present results showed that rapamycin treatment induced an increase in punctate structures in SH-SY5Y cells transfected with GFP-LC3 plasmid (Fig. 2, D and E). Moreover, more number of GFP-LC3 patches accumulated in the presence of Baf A1 (Fig. 2D). Other autophagy regulatory proteins were also detected after rapamycin treatment. LC3 was slightly increased at 12 hours, following a decrease at 24 and 48 hours. This may be due to the continuous activation and degradation of autophagosomes, which depleted the LC3 protein pool in the cytosol. Cathepsin D was persistently increased (Fig. 2F). Rapamycin also induced a significant upregulation of Beclin1 (Fig 2, F and G) and Bcl-2 (Fig. 2, F and H), suggesting autophagy activation is accompanied by a Bcl-2 up-regulation in these conditions.


The pro-survival role of autophagy depends on Bcl-2 under nutrition stress conditions.

Xu HD, Wu D, Gu JH, Ge JB, Wu JC, Han R, Liang ZQ, Qin ZH - PLoS ONE (2013)

Bcl-2 was up-regulated in SH-SY5Y cells during serum starvation.A: Western blot analysis of Bcl-2 and p-Bcl-2 expression in SH-SY5Y cells subjected to starvation. B and C: Quantitative analysis of optical densities of the Bcl-2 and p-Bcl-2 protein bands with Sigma Scan Pro 5 and normalized to the loading control. Bars represent Mean ± SD (B, n = 7; C, n = 3). D: Representative confocal images (5 µm scale bar) of GFP-LC3 assay in SH-SY5Y cells transfected with GFP-LC3 plasmid. Twenty four hours after transfection, the cells were treated with rapamycin (50 nM) for 12 hrs in the present or absence of 100 nM Baf A1. E: Quantification of autophagy in rapamycin-treated SH-SY5Y cells transfected with GFP-LC3. Data represent mean ± SD from three independent experiments. F: Western blot analysis of Beclin1, Cathepsin D, Bcl-2, Bcl-xl and LC3 in SH-SY5Y cells treated with Rapamycin. G and H: Rapamycin induced upregulation of Beclin1 and Bcl-2 in SH-SY5Y cells. Quantitative analysis of optical densities of the Beclin1 and Bcl-2 protein bands with Sigma Scan Pro 5 and normalized to the loading control. Bars represent Mean ± SD (G, n = 3; H, n = 4). Statistical analysis was carried out with ANOVA followed by Dunnett t-test. *p<0.05 vs control group; **p<0.01 vs control group; ##p<0.001 vs control group.
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Related In: Results  -  Collection

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pone-0063232-g002: Bcl-2 was up-regulated in SH-SY5Y cells during serum starvation.A: Western blot analysis of Bcl-2 and p-Bcl-2 expression in SH-SY5Y cells subjected to starvation. B and C: Quantitative analysis of optical densities of the Bcl-2 and p-Bcl-2 protein bands with Sigma Scan Pro 5 and normalized to the loading control. Bars represent Mean ± SD (B, n = 7; C, n = 3). D: Representative confocal images (5 µm scale bar) of GFP-LC3 assay in SH-SY5Y cells transfected with GFP-LC3 plasmid. Twenty four hours after transfection, the cells were treated with rapamycin (50 nM) for 12 hrs in the present or absence of 100 nM Baf A1. E: Quantification of autophagy in rapamycin-treated SH-SY5Y cells transfected with GFP-LC3. Data represent mean ± SD from three independent experiments. F: Western blot analysis of Beclin1, Cathepsin D, Bcl-2, Bcl-xl and LC3 in SH-SY5Y cells treated with Rapamycin. G and H: Rapamycin induced upregulation of Beclin1 and Bcl-2 in SH-SY5Y cells. Quantitative analysis of optical densities of the Beclin1 and Bcl-2 protein bands with Sigma Scan Pro 5 and normalized to the loading control. Bars represent Mean ± SD (G, n = 3; H, n = 4). Statistical analysis was carried out with ANOVA followed by Dunnett t-test. *p<0.05 vs control group; **p<0.01 vs control group; ##p<0.001 vs control group.
Mentions: Previous studies have shown that Bcl-2 can bind essential autophagic protein Beclin1 and inhibit autophagy activation under acute nutrient deprivation conditions [20]. The present study found that serum starvation induced the up-regulation of total and phosphorylated Bcl-2 (Fig. 2, A and B). However, quantitative analysis of the ratio between phosphorylated Bcl-2 and total Blc-2 had no significant change (Fig. 2C). Previous studies have shown that phosphorylated Bcl-2 localizes predominantly to the ER [35], and that starvation can induce phosphorylation of the ER-localized pool of Bcl-2 [22]. The present study is consistent with the previous observation that Bcl-2 was phosphorylated under acute starvation conditions [22]. This study found that total Bcl-2 protein levels were up-regulated, thus, the increase of phosphorylated Bcl-2 could be due to the increased pool of total Bcl-2. To further test if an increase in autophagy activity through inhibition of mammalian target of rapamycin (now called the mechanistic target of rapamycin within higher eukaryotes) would cause an up-regulation of Bcl-2, the present study also used rapamycin to induce autophagy in SH-SY5Y cells. Rapamycin is a putative autophagy activator which inhibits mTOR and mimics nutrient deprivation condition in yeast, Drosophila, and mammalian cells [36], [37]. The present results showed that rapamycin treatment induced an increase in punctate structures in SH-SY5Y cells transfected with GFP-LC3 plasmid (Fig. 2, D and E). Moreover, more number of GFP-LC3 patches accumulated in the presence of Baf A1 (Fig. 2D). Other autophagy regulatory proteins were also detected after rapamycin treatment. LC3 was slightly increased at 12 hours, following a decrease at 24 and 48 hours. This may be due to the continuous activation and degradation of autophagosomes, which depleted the LC3 protein pool in the cytosol. Cathepsin D was persistently increased (Fig. 2F). Rapamycin also induced a significant upregulation of Beclin1 (Fig 2, F and G) and Bcl-2 (Fig. 2, F and H), suggesting autophagy activation is accompanied by a Bcl-2 up-regulation in these conditions.

Bottom Line: In contrast, overexpression of Bcl-2 inhibited autophagy and blocked cell death in response to serum deprivation.These data suggest that Bcl-2 plays an essential role in limiting autophagy activation and preventing initiation of programmed cell death.Thus Bcl-2 may be an important mechanism for balancing beneficial and detrimental impacts of autophagy on cell survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science, Suzhou, China.

ABSTRACT
Autophagy can be induced under nutrition stress conditions. Bcl-2 is a pro-survival protein which inhibits apoptosis and autophagy. However, the role of Bcl-2 in autophagy regulation and cell survival under nutrition deprivation has not been fully understood. This study sought to investigate if Bcl-2 upregulation is essential in limiting autophagic activity and prevent cell death under nutrition deprivation conditions. Autophagic activity was monitored by the changes in GFP-LC3 localization and protein levels of Beclin1, LC3-II, cathepsin D and p62 in neuroblastoma SH-SY5Y cells underwent serum deprivation. Manipulation of Bcl-2 function was achieved with siRNAs and small molecular inhibitors. The cell viability and apoptosis were assessed with MTT assay and Annexin V/PI staining. The results showed that serum starvation increased protein levels of LC3-II and Beclin1 but decreased autophagy substrate p62. Autophagy activation induced by serum deprivation and rapamycin was accompanied by an upregulation of Bcl-2 protein levels. When Bcl-2 was knocked down with siRNA or inhibited with HA 14-1 or ABT-737, serum starvation induced profound cell death and enhanced autophagic flux under nutrition deprivation conditions, while knockdown of autophagic gene Beclin1 or autophagy inhibitors (bafilomycin A1 and E64D), rescued cell death. In contrast, overexpression of Bcl-2 inhibited autophagy and blocked cell death in response to serum deprivation. These data suggest that Bcl-2 plays an essential role in limiting autophagy activation and preventing initiation of programmed cell death. Thus Bcl-2 may be an important mechanism for balancing beneficial and detrimental impacts of autophagy on cell survival.

Show MeSH
Related in: MedlinePlus