Limits...
Differential dependence on Beclin 1 for the regulation of pro-survival autophagy by Bcl-2 and Bcl-xL in HCT116 colorectal cancer cells.

Priault M, Hue E, Marhuenda F, Pilet P, Oliver L, Vallette FM - PLoS ONE (2010)

Bottom Line: Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway.This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival.Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome.

View Article: PubMed Central - PubMed

Affiliation: CNRS IBGC UMR 5095, Bordeaux, France. muriel.priault@ibgc.cnrs.fr

ABSTRACT
Autophagy is described to be involved in homeostasis, development and disease, both as a survival and a death process. Its involvement in cell death proceeds from interrelationships with the apoptotic pathway. We focused on survival autophagy and investigated its interplays with the apoptotic machinery. We found that while Mcl-1 remained ineffective, Bcl-2 and Bcl-xL were required for starved cells to display a fully functional autophagic pathway as shown by proteolysis activity and detection of autophagic vesicles. Such pro-autophagic functions of Bcl-2 and Bcl-xL were independent of Bax. However they appeared to operate through non redundant mechanisms as Bcl-xL wielded a tighter control than Bcl-2 over the regulation of autophagy: unlike Bcl-2, Bcl-xL and Atg7 manipulation yielded identical phenotypes suggesting they could be components of the same signalling pathway; Bcl-xL subcellular localisation was modified upon starvation, and importantly Bcl-xL acted independently of Beclin 1. Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway. This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival. Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome.

Show MeSH

Related in: MedlinePlus

HCT116 and HCT116-BaxKO cells display comparable autophagic capacities.(A) HCT116 and HCT116-BaxKO cells were incubated with L-[14C]valine, and chased for 9 hours in complete medium (white bars), HBSS (black bars) or HBSS supplemented with either 0,1 µM Baf A1 (dark gray bars), or 10 mM 3-MA (light gray bars), or in complete medium +20 µM etoposide (hatched bars). Results report the stimulation of proteolysis relative to the respective basal levels measured under non-starving conditions (white bars). The values are the means of at least 3 independent experiments ± s.d. (B) Western blot and quantification of the conversion of LC3-I into phosphatidylethanolamine-conjugated LC3-II. Cells were grown in complete medium or starved for 9 h in the presence of E-64d (20 µg/ml) and leupeptine (20 µg/ml) to prevent the degradation of intra-autophagosomal LC3-II. 150 µg whole cell lysate were loaded on 15% SDS-PAGE. *Student test p<0,02.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2807451&req=5

pone-0008755-g002: HCT116 and HCT116-BaxKO cells display comparable autophagic capacities.(A) HCT116 and HCT116-BaxKO cells were incubated with L-[14C]valine, and chased for 9 hours in complete medium (white bars), HBSS (black bars) or HBSS supplemented with either 0,1 µM Baf A1 (dark gray bars), or 10 mM 3-MA (light gray bars), or in complete medium +20 µM etoposide (hatched bars). Results report the stimulation of proteolysis relative to the respective basal levels measured under non-starving conditions (white bars). The values are the means of at least 3 independent experiments ± s.d. (B) Western blot and quantification of the conversion of LC3-I into phosphatidylethanolamine-conjugated LC3-II. Cells were grown in complete medium or starved for 9 h in the presence of E-64d (20 µg/ml) and leupeptine (20 µg/ml) to prevent the degradation of intra-autophagosomal LC3-II. 150 µg whole cell lysate were loaded on 15% SDS-PAGE. *Student test p<0,02.

Mentions: The previous experiment suggested that when cells can execute both apoptosis and autophagy, apoptotic phenotypes prevail over autophagic features. We were therefore concerned by the possibility that the autophagic response could indeed be impaired by the execution of apoptosis. To address this question, we have compared the autophagic capacity of HCT116 and HCT116-BaxKO cells under conditions where macroautophagy is the predominant form of protein degradation, namely after 6–9 hours of starvation [34]. Fig. 2A shows the degradation of L-[14C]valine-labeled long-lived proteins. The basal proteolysis measured under control conditions was comparable in both cell lines and respectively set as the internal reference (white bars). Starvation similarly resulted in a two-fold increased proteolysis in both cell lines (black bars). This proteolysis was (i) essentially lysosomal since Bafilomycin A1 (Baf A1, an inhibitor of lysosomal ATPase proton pump) completely prevented its augmentation (dark gray bars), and (ii) associated with autophagy since its stimulation was significantly reversed by 3-MA (light gray bars). As a control, an apoptotic induction by etoposide over the same period of time did not stimulate any proteolysis (hatched bars). We also assayed the conversion of cytosolic Atg8/LC3-I into the autophagosome-bound phosphatidylethanolamine conjugate LC3-II, which is one of the hallmarks of autophagy. Western blots (Fig. 2B) confirmed that HCT116 and HCT116-BaxKO both produced comparable amounts of LC3-II upon starvation. The diffuse cytosolic of punctate localisation of mCherry-LC3 was also monitored in mouse embryonic fibroblasts (MEFs) as an alternative cell line, and showed that starvation triggered the same autophagosomal relocalisation of LC3 in wild-type MEFs and Bax knocked-out MEFs (Fig. S1). Taken together, our experiments show that apoptosis execution does not impair the autophagic response, and we conclude that the autophagic capacity is independent of Bax.


Differential dependence on Beclin 1 for the regulation of pro-survival autophagy by Bcl-2 and Bcl-xL in HCT116 colorectal cancer cells.

Priault M, Hue E, Marhuenda F, Pilet P, Oliver L, Vallette FM - PLoS ONE (2010)

HCT116 and HCT116-BaxKO cells display comparable autophagic capacities.(A) HCT116 and HCT116-BaxKO cells were incubated with L-[14C]valine, and chased for 9 hours in complete medium (white bars), HBSS (black bars) or HBSS supplemented with either 0,1 µM Baf A1 (dark gray bars), or 10 mM 3-MA (light gray bars), or in complete medium +20 µM etoposide (hatched bars). Results report the stimulation of proteolysis relative to the respective basal levels measured under non-starving conditions (white bars). The values are the means of at least 3 independent experiments ± s.d. (B) Western blot and quantification of the conversion of LC3-I into phosphatidylethanolamine-conjugated LC3-II. Cells were grown in complete medium or starved for 9 h in the presence of E-64d (20 µg/ml) and leupeptine (20 µg/ml) to prevent the degradation of intra-autophagosomal LC3-II. 150 µg whole cell lysate were loaded on 15% SDS-PAGE. *Student test p<0,02.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2807451&req=5

pone-0008755-g002: HCT116 and HCT116-BaxKO cells display comparable autophagic capacities.(A) HCT116 and HCT116-BaxKO cells were incubated with L-[14C]valine, and chased for 9 hours in complete medium (white bars), HBSS (black bars) or HBSS supplemented with either 0,1 µM Baf A1 (dark gray bars), or 10 mM 3-MA (light gray bars), or in complete medium +20 µM etoposide (hatched bars). Results report the stimulation of proteolysis relative to the respective basal levels measured under non-starving conditions (white bars). The values are the means of at least 3 independent experiments ± s.d. (B) Western blot and quantification of the conversion of LC3-I into phosphatidylethanolamine-conjugated LC3-II. Cells were grown in complete medium or starved for 9 h in the presence of E-64d (20 µg/ml) and leupeptine (20 µg/ml) to prevent the degradation of intra-autophagosomal LC3-II. 150 µg whole cell lysate were loaded on 15% SDS-PAGE. *Student test p<0,02.
Mentions: The previous experiment suggested that when cells can execute both apoptosis and autophagy, apoptotic phenotypes prevail over autophagic features. We were therefore concerned by the possibility that the autophagic response could indeed be impaired by the execution of apoptosis. To address this question, we have compared the autophagic capacity of HCT116 and HCT116-BaxKO cells under conditions where macroautophagy is the predominant form of protein degradation, namely after 6–9 hours of starvation [34]. Fig. 2A shows the degradation of L-[14C]valine-labeled long-lived proteins. The basal proteolysis measured under control conditions was comparable in both cell lines and respectively set as the internal reference (white bars). Starvation similarly resulted in a two-fold increased proteolysis in both cell lines (black bars). This proteolysis was (i) essentially lysosomal since Bafilomycin A1 (Baf A1, an inhibitor of lysosomal ATPase proton pump) completely prevented its augmentation (dark gray bars), and (ii) associated with autophagy since its stimulation was significantly reversed by 3-MA (light gray bars). As a control, an apoptotic induction by etoposide over the same period of time did not stimulate any proteolysis (hatched bars). We also assayed the conversion of cytosolic Atg8/LC3-I into the autophagosome-bound phosphatidylethanolamine conjugate LC3-II, which is one of the hallmarks of autophagy. Western blots (Fig. 2B) confirmed that HCT116 and HCT116-BaxKO both produced comparable amounts of LC3-II upon starvation. The diffuse cytosolic of punctate localisation of mCherry-LC3 was also monitored in mouse embryonic fibroblasts (MEFs) as an alternative cell line, and showed that starvation triggered the same autophagosomal relocalisation of LC3 in wild-type MEFs and Bax knocked-out MEFs (Fig. S1). Taken together, our experiments show that apoptosis execution does not impair the autophagic response, and we conclude that the autophagic capacity is independent of Bax.

Bottom Line: Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway.This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival.Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome.

View Article: PubMed Central - PubMed

Affiliation: CNRS IBGC UMR 5095, Bordeaux, France. muriel.priault@ibgc.cnrs.fr

ABSTRACT
Autophagy is described to be involved in homeostasis, development and disease, both as a survival and a death process. Its involvement in cell death proceeds from interrelationships with the apoptotic pathway. We focused on survival autophagy and investigated its interplays with the apoptotic machinery. We found that while Mcl-1 remained ineffective, Bcl-2 and Bcl-xL were required for starved cells to display a fully functional autophagic pathway as shown by proteolysis activity and detection of autophagic vesicles. Such pro-autophagic functions of Bcl-2 and Bcl-xL were independent of Bax. However they appeared to operate through non redundant mechanisms as Bcl-xL wielded a tighter control than Bcl-2 over the regulation of autophagy: unlike Bcl-2, Bcl-xL and Atg7 manipulation yielded identical phenotypes suggesting they could be components of the same signalling pathway; Bcl-xL subcellular localisation was modified upon starvation, and importantly Bcl-xL acted independently of Beclin 1. Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway. This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival. Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome.

Show MeSH
Related in: MedlinePlus