Prosurvival Bcl-2 family members affect autophagy only indirectly,by inhibiting Bax and Bak

Antiapoptotic B-cell lymphoma 2 (Bcl-2) family members such as Bcl-2, myeloid cell leukemia 1 (Mcl-1), and B-cell lymphoma-X large (Bcl-xL) are proposed to inhibit autophagy by directly binding to the BH3 domain of Beclin 1/Atg6. However, these Bcl-2 family proteins also block the proapoptotic activity of Bcl-2–associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), and many inducers of autophagy also cause cell death. Therefore, when the mitochondrial-mediated apoptosis pathway is functional, interpretation of such experiments is complicated. To directly test the impact of the endogenous antiapoptotic Bcl-2 family members on autophagy in the absence of apoptosis, we inhibited their activity in cells lacking the essential cell death mediators Bax and Bak. We also used inducible lentiviral vectors to overexpress Bcl-2, Bcl-xL, or Mcl-1 in cells and subjected them to treatments that promote autophagy. In the absence of Bax and Bak, Bcl-2, Bcl-xL, and Mcl-1 had no detectable effect on autophagy or cell death in myeloid or fibroblast cell lines. On the other hand, when Bax and Bak were present, inhibiting the prosurvival Bcl-2 family members stimulated autophagy, but this correlated with increased cell death. In addition, inhibition of autophagy induced by amino acid starvation, etoposide, or interleukin-3 withdrawal did not affect cell death in the absence of Bax and Bak. These results demonstrate that the antiapoptotic Bcl-2 family members do not directly inhibit components of the autophagic pathway but instead affect autophagy indirectly, owing to their inhibition of Bax and Bak.Autophagy is a process in which cellular material is degraded so that homeostasis can be maintained when nutrients are scarce. During macroautophagy (henceforth referred to as autophagy), cytoplasm is enveloped by the formation of the autophagosome, which when fused to the lysosome forms the autophagolysosome. This organelle degrades the enclosed cellular material and returns “building blocks” such as amino acids back to the cytoplasm. Autophagy was initially studied in yeast and subsequently in mammalian cells, where it has been proposed to be not only a mechanism to promote cell survival in conditions of starvation but also a mechanism by which cells can commit suicide (1).Much of our understanding of the molecular mechanisms of autophagy has come from studying the highly conserved Atg (autophagy-related) proteins. As autophagy progresses, microtubule-associated protein 1 light chain 3 beta (LC3B)-I (Atg8 in yeast) in the cytoplasm is conjugated with phosphatidylethanolamine to form LC3B-II, which becomes associated with the autophagosomal membrane and is involved in its elongation. An increase in LC3B-II (and concomitant decrease in LC3B-I) is commonly used as a marker of autophagy. Because LC3B is also one of the only autophagy-associated proteins that remain attached to the autophagosome throughout the entire process, it is commonly used to visualize autophagosomes and autophagolysosomes.In yeast, Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a core component of the class III phosphatidylinositol 3-kinase (Vsp34) complex required for nucleation and assembly of the autophagosomal membrane. In a similar way, the mammalian Atg6 homolog, Beclin 1, is important for the formation of a complex with the mammalian PI3K Vps34 and nucleation of the autophagosome membrane. Beclin 1 was identified in yeast two-hybrid experiments using the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) as bait (2). It has been proposed that when nutrients are abundant, Bcl-2 and the related proteins Bcl-xL and myeloid cell leukemia 1 (Mcl-1) bind to Beclin 1 via Beclin 1’s BH3 domain and thereby inhibit induction of autophagy (Fig. 1A) (3–5). According to this model, when nutrients are scarce, Bcl-2 is phosphorylated by JNK1, which prevents its binding to Beclin 1 and allows it to initiate formation of autophagosomes (6).Open in a separate windowFig. 1.Inhibiting the prosurvival Bcl-2 family members does not promote nonapoptotic cell death or LC3B lipidation in the absence of Bax and Bak. (A) A simplified model illustrating the proposed role of the Bcl-2 family members. (B) MEFs were cotreated with 34 μM etoposide (VP-16) and indicated concentrations of ABT-737 for 96 h. Viability relative to cells not treated with ABT-737 was measured by the absence of PI uptake. The mean ± SD of two independent cell lines are shown relative (n = 4). (C) ABT-737 only promotes LC3B lipidation when Bax or Bak are present. Western blot of MEFs after a 4-h treatment with 1 μM ABT-737 or HBSS. (D) Induction of Bim_s does not alter LC3B levels. Bax^−/−Bak^−/− MEFs were treated with 1 μg/mL dox for 48 h or were cultured in HBSS for 4 h.In apoptosis, the roles of Bcl-2, Bcl-2–like protein W (Bcl-w), Mcl-1, and B-cell lymphoma-X large (Bcl-xL) are well established. They inhibit apoptosis in two ways: first by directly binding the proapoptotic effector proteins Bcl-2–associated X (Bax) and Bcl-2 homologous antagonist/killer (Bak), and second by binding to BH3-only proteins such as Bim, thereby preventing them from activating Bax and Bak (7, 8). The prosurvival Bcl-2 family members bind to Bim, Bak, and Bax and the BH3 mimetic compound ABT-737 via the BH3 binding groove, the same region as the proposed binding site for Beclin 1, and they bind competitively (Fig. 1A). In contrast, the roles for prosurvival Bcl-2 family members in the regulation of autophagy have been less well characterized, not least because their inhibition or knockdown can also trigger Bax/Bak-dependent apoptosis. Furthermore, many of the pivotal studies on the role of Bcl-2 in autophagy were performed using overexpression of one or both binding partners, putting into question the physiological relevance of the interactions. Because of these caveats, we decided to further investigate whether the prosurvival Bcl-2 family members can inhibit autophagy in cells unable to undergo mitochondrial-mediated apoptosis owing to deletion of genes for the essential apoptosis effector proteins Bax and Bak.To definitively determine whether the prosurvival Bcl-2 family members can regulate autophagy, we investigated whether inhibiting endogenous Bcl-2, Bcl-xL, and Mcl-1 could block autophagy in cells lacking Bax and Bak. We found that in both fibroblast and interleukin-3 (IL-3) dependent myeloid cell lines, treatment with the BH3 mimetic ABT-737, or altering the levels of Bcl-2, Bcl-xL, or Mcl-1, had no discernable effects on autophagy. Indeed, the prosurvival Bcl-2 proteins only affected LC3B lipidation when Bax and Bak were present and cells were undergoing apoptosis. These results do not support the model that direct interactions between Beclin 1 and the antiapoptotic Bcl-2 family members inhibit autophagy. Instead, they suggest that previously reported cellular effects are likely to be a consequence of inducing or modifying apoptotic events triggered by Bax or Bak (4, 9–11).     

Cognitive Impairment and Psychiatric Morbidity in HIV+ Zambians with New-Onset Seizure

A prospective cohort study of new-onset seizure in people with human immunodeficiency virus (HIV) in Zambia is ongoing to determine the incidence of subsequent epilepsy and risk factors for epileptogenesis in this population. At enrollment, we evaluated this cohort for cognitive impairment and psychiatric morbidity. Over 50% of participants had cognitive impairment and significant psychiatric morbidity. Most participants had advanced HIV disease based on CD4+ T-cell count and World Health Organization stage, but we found no association between cognitive impairment or psychiatric morbidity and HIV disease staging.     

Seroprevalence of Seven Zoonotic Pathogens in Pregnant Women from the Caribbean

Studies examining the prevalence of zoonotic agents in the Caribbean are very limited. The objective of this study was to examine the seroprevalence of seven zoonotic agents among individuals residing on 10 English-speaking Caribbean countries. Sera from healthy, pregnant women were collected from Antigua-Barbuda, Belize, Bermuda, Dominica, Grenada, Jamaica, Montserrat, St. Kitts-Nevis, St. Lucia, and St. Vincent-Grenadines and tested for the presence of IgG antibodies to dengue virus, hepatitis E virus, hantaviruses, leptospiral agents, spotted fever group rickettsiae (SFGR), typhus group rickettsiae (TGR), and Coxiella burnetii (Q fever). The highest seroprevalence values were observed for dengue virus, SFGR, and leptospirosis, although the lowest seroprevalence values were observed for hepatitis E virus, C. burnetii, and TGR. Antibodies to hantaviruses were not detected in any individuals.     

Fracture risk in diabetic elderly men: the MrOS study

Aims/hypothesis

Diabetes mellitus is associated with increased fracture risk in women but few studies are available in men. To evaluate the relationship between diabetes and prospective non-vertebral fractures in elderly men, we used data from the Osteoporotic Fractures in Men (MrOS) study.

Methods

The MrOS enrolled 5,994 men (aged ≥65 years). Diabetes (ascertained by self-report, the use of medication for diabetes or an elevated fasting glucose level) was reported in 881 individuals, 80 of whom were using insulin. Hip and spine bone mineral density (BMD) was measured using dual x-ray absorptiometry (DXA). After recruitment, the men were followed for incident non-vertebral fractures using a triannual (3 yearly) questionnaire for an average of 9.1 (SD 2.7) years. The Cox proportional hazards model was used to assess the incident risk of fractures.

Results

In models adjusted for age, race, clinic site and total hip BMD, the risk of non-vertebral fracture was higher in men with diabetes compared with normoglycaemic men (HR 1.30, 95% CI 1.09, 1.54) and was elevated in men using insulin (HR 2.46, 95% CI 1.69, 3.59). Men with impaired fasting glucose did not have a higher risk of fracture compared with normoglycaemic men (HR 1.04, 95% CI 0.89, 1.21). After multivariable adjustment, the risk of non-vertebral fracture remained higher only among men with diabetes who were using insulin (HR 1.74, 95% CI 1.13, 2.69).

Conclusions/interpretation

Men with diabetes who are using insulin have an increased risk of non-vertebral fracture for a given age and BMD.