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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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Autophagy was induced in SH-SY5Y neuroblastoma cells by serum deprivation.A: Representative confocal images (5 µM scale bar) of GFP-LC3 in SH-SY5Y cells transfected with GFP-LC3 plasmid. Twenty-four hours after transfection, cells were treated with serum-free medium in the presence or absence of 100 nM Baf A1 for the indicated times. B: Quantification of autophagy in serum-starved SH-SY5Y cells transfected with GFP-LC3. Data represent mean ± SD for combined data from three independent experiments. C: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium in the presence or absence of 100 nM Baf A1. E: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium. Each time course has a control. D and F: Quantitative analysis of optical densities of LC3-II/LC3-I with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). G and H: Western blot analysis (G) and quantitative analysis (H) of Beclin1 expression in SH-SY5Y cells subjected to serum-free medium. Bars represent Mean ± SD (n = 5). I and J: Western blot analysis (I) and quantitative analysis (J) of p62 expression in SH-SY5Y cells subjected to serum-free medium with and without NH4Cl. Bars represent Mean ± SD (n = 3). K and L: Western blot analysis (K) and quantitative analysis (L) of cathepsin D expression in SH-SY5Y cells subjected to starvation. Bars represent Mean ± SD (n = 5). The optical densities of each protein band were quantified with Sigma Scan Pro 5 and normalized to the loading control. Statistical analysis was carried out with ANOVA followed by Dunnett t-test. (B and D) *p<0.05 and **p<0.01 vs control group. (I) #p<0.05 represent starvation vs starvation+NH4Cl.
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pone-0063232-g001: Autophagy was induced in SH-SY5Y neuroblastoma cells by serum deprivation.A: Representative confocal images (5 µM scale bar) of GFP-LC3 in SH-SY5Y cells transfected with GFP-LC3 plasmid. Twenty-four hours after transfection, cells were treated with serum-free medium in the presence or absence of 100 nM Baf A1 for the indicated times. B: Quantification of autophagy in serum-starved SH-SY5Y cells transfected with GFP-LC3. Data represent mean ± SD for combined data from three independent experiments. C: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium in the presence or absence of 100 nM Baf A1. E: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium. Each time course has a control. D and F: Quantitative analysis of optical densities of LC3-II/LC3-I with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). G and H: Western blot analysis (G) and quantitative analysis (H) of Beclin1 expression in SH-SY5Y cells subjected to serum-free medium. Bars represent Mean ± SD (n = 5). I and J: Western blot analysis (I) and quantitative analysis (J) of p62 expression in SH-SY5Y cells subjected to serum-free medium with and without NH4Cl. Bars represent Mean ± SD (n = 3). K and L: Western blot analysis (K) and quantitative analysis (L) of cathepsin D expression in SH-SY5Y cells subjected to starvation. Bars represent Mean ± SD (n = 5). The optical densities of each protein band were quantified with Sigma Scan Pro 5 and normalized to the loading control. Statistical analysis was carried out with ANOVA followed by Dunnett t-test. (B and D) *p<0.05 and **p<0.01 vs control group. (I) #p<0.05 represent starvation vs starvation+NH4Cl.

Mentions: Autophagy can be induced by starvation. To confirm if the serum-free medium induced autophagy, the human SH-SY5Y neuroblastoma cells were treated with serum-free medium for different time courses and the changes in the levels of autophagy regulatory proteins were determined. Serum starvation caused overall reduction in cell size but no robust cell death as observed with inverted microscopy (data not shown). The microtubule-associated protein 1 light chain 3 (LC3) was initially identified as a protein co-purified with microtubule-associated protein A1 and B1 from rat brain [26]. LC3 is a homologue of Apg8p essential for autophagy in yeast [27]. The LC3-II is localized in autophagosomes to help elongation of autophagosomal membranes [28]. Thus, LC3-II has been defined as a marker of autophagosomes in mammalian cells. To monitor the formation of autophagosomes, the SH-SY5Y cells were transfected with GFP-LC3 for 24 hours, the cells were then treated with serum-free medium for various length of time as indicated. As shown in figure 1, serum deprivation induced the redistribution of GFP-LC3 from a diffuse pattern to punctate structures (Fig. 1, A and B). To detect autophagic flux, bafilomycin A1 (Baf A1, a potent and specific inhibitor of vacuolar H+ ATPase [29]), was used to inhibit the acidification of the lysosome and autophagosome-lysosome. Baf A1 significantly enhanced GFP-LC3 patches under serum deprivation conditions (Fig. 1A). Western blot analysis of LC3 also observed that the upregulation of LC3-II induced by serum deprivation dramatically accumulated in the presence of Baf A1 (Fig. 1, C and D). These results indicate that autophagy is stimulated by serum starvation. Autophagy is sensitive to and can be influenced by many other factors. In order to exclude the possibility that the changes in LC3-II levels were caused simply by medium change, we harvested samples with a control at each time point. The data showed that there was a decrease in LC3-I and an increase in LC3-II at each time point examined, as compared with their respective controls (Fig. 1, E and F), indicating that autophagy was indeed induced. After serum deprivation, Beclin1 was also up-regulated (Fig. 1, G and H). Beclin1, the mammalian orthologue of Atg6, promotes autophagy in human breast carcinoma MCF-7 cells [30], by localizing at the trans-Golgi network and participates in the initial steps of vesicle nucleation through forming a complex with the class III phosphatidylinositol 3-kinase (PI3K) [31]. p62/sequestosome 1 (SQSTM1) is a protein involved in the formation of autophagosomes which is constitutively degraded by the autophagic pathway through specific binding to LC3 [32], [33]. Serum starvation also resulted in a transient down-regulation of p62 at 6 hours, followed by a quick recovery at other time points (Fig. 1I). To further assess autophagic flux, we treated the cells with NH4CL, an inhibitor of lysosomal acidification that can inhibit autophagic degradation of autophagic substrates. Addition of NH4CL recovered the levels of p62 at 6 hours, indicating p62 was indeed degraded by the autophagy process (Fig. 1, I and J). Autophagy is a constitutive process involving activation of lysosomal enzymes and subsequent degradation of substrates [34]. Therefore, we measured levels of cathepsin D, an aspartic protease localized inside the lysosomes. Cathepsin D was up-regulated upon serum starvation (Fig. 1, K and L). Overall, these data demonstrated that serum starvation enhanced autophagy flux, led to increase in autophagosome formation, activation of lysosomes and degradation of autophagic substrates.


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)

Autophagy was induced in SH-SY5Y neuroblastoma cells by serum deprivation.A: Representative confocal images (5 µM scale bar) of GFP-LC3 in SH-SY5Y cells transfected with GFP-LC3 plasmid. Twenty-four hours after transfection, cells were treated with serum-free medium in the presence or absence of 100 nM Baf A1 for the indicated times. B: Quantification of autophagy in serum-starved SH-SY5Y cells transfected with GFP-LC3. Data represent mean ± SD for combined data from three independent experiments. C: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium in the presence or absence of 100 nM Baf A1. E: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium. Each time course has a control. D and F: Quantitative analysis of optical densities of LC3-II/LC3-I with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). G and H: Western blot analysis (G) and quantitative analysis (H) of Beclin1 expression in SH-SY5Y cells subjected to serum-free medium. Bars represent Mean ± SD (n = 5). I and J: Western blot analysis (I) and quantitative analysis (J) of p62 expression in SH-SY5Y cells subjected to serum-free medium with and without NH4Cl. Bars represent Mean ± SD (n = 3). K and L: Western blot analysis (K) and quantitative analysis (L) of cathepsin D expression in SH-SY5Y cells subjected to starvation. Bars represent Mean ± SD (n = 5). The optical densities of each protein band were quantified with Sigma Scan Pro 5 and normalized to the loading control. Statistical analysis was carried out with ANOVA followed by Dunnett t-test. (B and D) *p<0.05 and **p<0.01 vs control group. (I) #p<0.05 represent starvation vs starvation+NH4Cl.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643928&req=5

pone-0063232-g001: Autophagy was induced in SH-SY5Y neuroblastoma cells by serum deprivation.A: Representative confocal images (5 µM scale bar) of GFP-LC3 in SH-SY5Y cells transfected with GFP-LC3 plasmid. Twenty-four hours after transfection, cells were treated with serum-free medium in the presence or absence of 100 nM Baf A1 for the indicated times. B: Quantification of autophagy in serum-starved SH-SY5Y cells transfected with GFP-LC3. Data represent mean ± SD for combined data from three independent experiments. C: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium in the presence or absence of 100 nM Baf A1. E: Western blot analysis of LC3 expression in SH-SY5Y cells subjected to serum-free medium. Each time course has a control. D and F: Quantitative analysis of optical densities of LC3-II/LC3-I with Sigma Scan Pro 5. Bars represent Mean ± SD (n = 3). G and H: Western blot analysis (G) and quantitative analysis (H) of Beclin1 expression in SH-SY5Y cells subjected to serum-free medium. Bars represent Mean ± SD (n = 5). I and J: Western blot analysis (I) and quantitative analysis (J) of p62 expression in SH-SY5Y cells subjected to serum-free medium with and without NH4Cl. Bars represent Mean ± SD (n = 3). K and L: Western blot analysis (K) and quantitative analysis (L) of cathepsin D expression in SH-SY5Y cells subjected to starvation. Bars represent Mean ± SD (n = 5). The optical densities of each protein band were quantified with Sigma Scan Pro 5 and normalized to the loading control. Statistical analysis was carried out with ANOVA followed by Dunnett t-test. (B and D) *p<0.05 and **p<0.01 vs control group. (I) #p<0.05 represent starvation vs starvation+NH4Cl.
Mentions: Autophagy can be induced by starvation. To confirm if the serum-free medium induced autophagy, the human SH-SY5Y neuroblastoma cells were treated with serum-free medium for different time courses and the changes in the levels of autophagy regulatory proteins were determined. Serum starvation caused overall reduction in cell size but no robust cell death as observed with inverted microscopy (data not shown). The microtubule-associated protein 1 light chain 3 (LC3) was initially identified as a protein co-purified with microtubule-associated protein A1 and B1 from rat brain [26]. LC3 is a homologue of Apg8p essential for autophagy in yeast [27]. The LC3-II is localized in autophagosomes to help elongation of autophagosomal membranes [28]. Thus, LC3-II has been defined as a marker of autophagosomes in mammalian cells. To monitor the formation of autophagosomes, the SH-SY5Y cells were transfected with GFP-LC3 for 24 hours, the cells were then treated with serum-free medium for various length of time as indicated. As shown in figure 1, serum deprivation induced the redistribution of GFP-LC3 from a diffuse pattern to punctate structures (Fig. 1, A and B). To detect autophagic flux, bafilomycin A1 (Baf A1, a potent and specific inhibitor of vacuolar H+ ATPase [29]), was used to inhibit the acidification of the lysosome and autophagosome-lysosome. Baf A1 significantly enhanced GFP-LC3 patches under serum deprivation conditions (Fig. 1A). Western blot analysis of LC3 also observed that the upregulation of LC3-II induced by serum deprivation dramatically accumulated in the presence of Baf A1 (Fig. 1, C and D). These results indicate that autophagy is stimulated by serum starvation. Autophagy is sensitive to and can be influenced by many other factors. In order to exclude the possibility that the changes in LC3-II levels were caused simply by medium change, we harvested samples with a control at each time point. The data showed that there was a decrease in LC3-I and an increase in LC3-II at each time point examined, as compared with their respective controls (Fig. 1, E and F), indicating that autophagy was indeed induced. After serum deprivation, Beclin1 was also up-regulated (Fig. 1, G and H). Beclin1, the mammalian orthologue of Atg6, promotes autophagy in human breast carcinoma MCF-7 cells [30], by localizing at the trans-Golgi network and participates in the initial steps of vesicle nucleation through forming a complex with the class III phosphatidylinositol 3-kinase (PI3K) [31]. p62/sequestosome 1 (SQSTM1) is a protein involved in the formation of autophagosomes which is constitutively degraded by the autophagic pathway through specific binding to LC3 [32], [33]. Serum starvation also resulted in a transient down-regulation of p62 at 6 hours, followed by a quick recovery at other time points (Fig. 1I). To further assess autophagic flux, we treated the cells with NH4CL, an inhibitor of lysosomal acidification that can inhibit autophagic degradation of autophagic substrates. Addition of NH4CL recovered the levels of p62 at 6 hours, indicating p62 was indeed degraded by the autophagy process (Fig. 1, I and J). Autophagy is a constitutive process involving activation of lysosomal enzymes and subsequent degradation of substrates [34]. Therefore, we measured levels of cathepsin D, an aspartic protease localized inside the lysosomes. Cathepsin D was up-regulated upon serum starvation (Fig. 1, K and L). Overall, these data demonstrated that serum starvation enhanced autophagy flux, led to increase in autophagosome formation, activation of lysosomes and degradation of autophagic substrates.

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