Modulation of apoptotic pathways in intestinal mucosa during hibernation

Mammalian hibernation is associated with several events that can affect programmed cell death (apoptosis) in nonhibernators, including marked changes in blood flow, extended fasting, and oxidative stress. However, the effect of hibernation on apoptosis is poorly understood. Here, we investigated apoptosis and expression of proteins involved in apoptotic pathways in intestinal mucosa of summer and hibernating ground squirrels. We used terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) to identify possible apoptotic enterocytes in small intestine of summer squirrels and hibernating squirrels throughout the winter. Nuclear TUNEL staining increased as hibernation progressed, but the staining pattern was diffuse and not accompanied by chromatin condensation or apoptotic bodies. Electrophoresis of mucosal DNA revealed no ladders typical of apoptosis. Nuclear levels of proapoptotic p53 protein were fourfold less in hibernators compared with summer squirrels. A 12-fold increase in anti-apoptotic Bcl-x(L) compared with a 2-fold increase in proapoptotic Bax suggested a balance in favor of antiapoptotic signaling in hibernators. There was no change in Bcl-2 protein expression but phospho-Bcl-2 increased in mucosa of hibernators. Hibernation had minimal effects on expression of active caspase-8 or -9, whereas caspase-3-specific activity was lower in hibernators during an interbout arousal compared with summer squirrels. Expression of the prosurvival protein Akt increased 20-fold during hibernation, but phospho-Akt was not altered. These data provide evidence for enhanced expression of antiapoptotic proteins during hibernation that may promote enterocyte survival in a pro-oxidative, proapoptotic environment.     

Transforming growth factor-beta 1 induces apoptosis independently of p53 and selectively reduces expression of Bcl-2 in multipotent hematopoietic cells

Transforming growth factor-beta1 (TGF-beta1) can inhibit cell proliferation or induce apoptosis in multipotent hematopoietic cells. To study the mechanisms of TGF-beta1 action on primitive hematopoietic cells, we used the interleukin-3 (IL-3)-dependent, multipotent FDCP-Mix cell line. TGF-beta1-mediated growth inhibition was observed in high concentrations of IL-3, while at lower IL-3 concentrations TGF-beta1 induced apoptosis. The proapoptotic effects of TGF-beta1 occur via a p53-independent pathway, since p53(null) FDCP-Mix demonstrated the same responses to TGF-beta1. IL-3 has been suggested to enhance survival via an increase in (antiapoptotic) Bcl-x(L) expression. In FDCP-Mix cells, neither IL-3 nor TGF-beta1 induced any change in Bcl-x(L) protein levels or the proapoptotic proteins Bad or Bax. However, TGF-beta1 had a major effect on Bcl-2 levels, reducing them in the presence of high and low concentrations of IL-3. Overexpression of Bcl-2 in FDCP-Mix cells rescued them from TGF-beta1-induced apoptosis but was incapable of inhibiting TGF-beta1-mediated growth arrest. We conclude that TGF-beta1-induced cell death is independent of p53 and inhibited by Bcl-2, with no effect on Bcl-x(L). The significance of these results for stem cell survival in bone marrow are discussed.     

Single-point mutations of a lysine residue change function of Bax and Bcl-xL expressed in Bax- and Bak-less mouse embryonic fibroblasts: novel insights into the molecular mechanisms of Bax-induced apoptosis

Members of the Bcl-2 family play key roles as proapoptotic (e.g., Bax) and antiapoptotic (e.g., Bcl-x(L)) regulators of programmed cell death. We previously identified the mitochondrial potassium channel Kv1.3 as a novel target of Bax. Incubating Kv1.3-positive isolated mitochondria with Bax triggered apoptotic events, whereas Kv1.3-deficient mitochondria were resistant to this stimulus. Mutation of Bax at lysine 128 (BaxK128E) abrogated its effects on Kv1.3 and the induction of apoptotic changes in mitochondria. These data indicate a toxin-like action of Bax on Kv1.3 to trigger at least some of the mitochondrial changes typical for apoptosis. To gain insight into the mechanism of Bax-Kv1.3 interaction, we mutated Glu158 of Bcl-x(L) (corresponding to K128 in Bax) to lysine. This substitution turned Bcl-x(L) proapoptotic. Transfection of double knockout (Bax(-/-)/Bak(-/-)) mouse embryonic fibroblasts (DKO MEFs) with either wild-type Bax, BaxK128E, or Bcl-x(L)E158K showed that apoptosis induced by various stimuli was defective in DKO MEFs and BaxK128E-transfected cells, but was recovered upon transfection with Bcl-xLE158K or wild-type Bax. Both wild-type Bax and BaxK128E can form similar ion-conducting pores upon incorporation into planar lipid bilayers. Our results point to a physiologically relevant interaction of Bax with Kv1.3 and further indicate a crucial role of a distinct lysine in determining the proapoptotic character of Bcl2-family proteins.     

The DNA damage response pathway regulates the alternative splicing of the apoptotic mediator Bcl-x

Alternative splicing often produces effectors with opposite functions in apoptosis. Splicing decisions must therefore be tightly connected to stresses, stimuli, and pathways that control cell survival and cell growth. We have shown previously that PKC signaling prevents the production of proapoptotic Bcl-x(S) to favor the accumulation of the larger antiapoptotic Bcl-x(L) splice variant in 293 cells. Here we show that the genotoxic stress induced by oxaliplatin elicits an ATM-, CHK2-, and p53-dependent splicing switch that favors the production of the proapoptotic Bcl-x(S) variant. This DNA damage-induced splicing shift requires the activity of protein-tyrosine phosphatases. Interestingly, the ATM/CHK2/p53/tyrosine phosphatases pathway activated by oxaliplatin regulates Bcl-x splicing through the same regulatory sequence element (SB1) that receives signals from the PKC pathway. Convergence of the PKC and DNA damage signaling routes may control the abundance of a key splicing repressor because SB1-mediated repression is lost when protein synthesis is impaired but is rescued by blocking proteasome-mediated protein degradation. The SB1 splicing regulatory module therefore receives antagonistic signals from the PKC and the p53-dependent DNA damage response pathways to control the balance of pro- and antiapoptotic Bcl-x splice variants.     

Interaction between the antiapoptotic protein Nr-13 and cytochrome c. Antagonistic effect of the BH3 domain of Bax

Mitochondria act as a focal point for upstream apoptosis signals by releasing cytochrome c into the cytosol, leading to the activation of caspases and subsequent cell death. Members of the Bcl-2 protein family regulate this phenomenon by heterodimerization via the BH3 domain of proapoptotic members opposing their pro- and antiapoptotic functions. The mechanism of cytochrome c release from mitochondria and of its regulation remains controversial. In vitro binding studies of purified and biologically active proteins should help in understanding the molecular mechanism of interactions and protein functions. In this work, the Bcl-2-related antiapoptotic chicken protein Nr-13 was overexpressed as a highly soluble recombinant protein which showed correct folding as judged by circular dichroism and fluorescence spectroscopy. Purified Nr-13 inhibits caspase-3 activation in a Xenopus egg-derived cell-free system, and neutralizes the proapoptotic activity of a synthetic peptide containing the BH3 domain of Bax. The latter effect correlates with the high-affinity binding of the BH3 peptide to Nr-13 as monitored by the intrinsic tryptophan fluorescence. On the basis of the structural similarity with Bcl-x(L), putative residues involved in this interaction were identified. Nr-13 exhibits a high-affinity interaction with cytochrome c which is prevented by preincubation with the BH3-Bax peptide. These findings are discussed with respect to a model for the regulation of apoptosis in which a direct interaction between the antiapoptotic protein and cytochrome c may prevent the apoptosis.     

Bak but not Bax is essential for Bcl-xS-induced apoptosis

Bcl-x(S), a proapoptotic member of the Bcl-2 protein family, is localized in the mitochondria and induces apoptosis in a caspase- and BH3-dependent manner by a mechanism involving cytochrome c release. The way in which Bcl-x(S) induces caspase activation and cytochrome c release, as well as the relationship between Bcl-x(S) and other proapoptotic members of the Bcl-2 family, is not known. Here we used embryonic fibroblasts derived from mice deficient in the multidomain proapoptotic members of the Bcl-2 family (Bax and Bak) and the apoptotic components of the apoptosome (Apaf-1 and caspase-9) to unravel the cascade of events by which Bcl-x(S) promotes apoptosis. Our results show that Bak but not Bax is essential for Bcl-x(S)-induced apoptosis. Bcl-x(S) induced activation of Bak, which in turn promoted apoptosis by apoptosome-dependent and -independent pathways. These findings provide the first evidence that a proapoptotic Bcl-2 family protein induces apoptosis exclusively via Bak.     

ARF tumor suppressor induces mitochondria-dependent apoptosis by modulation of mitochondrial Bcl-2 family proteins

A tumor suppressor gene product, ARF, sensitizes cells to apoptosis in the presence of appropriate collateral signals. In this study, we analyzed the mechanism of ARF-dependent apoptosis and demonstrated that ARF induces mitochondria-dependent apoptosis in p53 wild-type, ARF/p16-null cells. We also found that ARF evokes cytochrome c release from mitochondria, decreases mitochondrial membrane potential, and activates pro-caspase-9 to induce apoptosis. Our findings suggest that this apoptotic cellular modulation is brought about by up-regulation of the proapoptotic Bcl-2 family proteins Bax and Bim and down-regulation of antiapoptotic Bcl-2 in mitochondrial fractions. Additionally, ARF seems to down-regulate Bcl-2 in a p53-dependent manner while up-regulating Bax/Bim via a p53-independent pathway.     

Mycobacterium tuberculosis promotes apoptosis in human neutrophils by activating caspase-3 and altering expression of Bax/Bcl-xL via an oxygen-dependent pathway

In addition to direct bactericidal activities, such as phagocytosis and generation of reactive oxygen species (ROS), neutrophils can regulate the inflammatory response by undergoing apoptosis. We found that infection of human neutrophils with Mycobacterium tuberculosis (Mtb) induced rapid cell death displaying the characteristic features of apoptosis such as morphologic changes, phosphatidylserine exposure, and DNA fragmentation. Both a virulent (H37Rv) and an attenuated (H37Ra) strain of Mtb were equally effective in inducing apoptosis. Pretreatment of neutrophils with antioxidants or an inhibitor of NADPH oxidase markedly blocked Mtb-induced apoptosis but did not affect spontaneous apoptosis. Activation of caspase-3 was evident in neutrophils undergoing spontaneous apoptosis, but it was markedly augmented and accelerated during Mtb-induced apoptosis. The Mtb-induced apoptosis was associated with a speedy and transient increase in expression of Bax protein, a proapoptotic member of the Bcl-2 family, and a more prominent reduction in expression of the antiapoptotic protein Bcl-x(L). Pretreatment with an inhibitor of NADPH oxidase distinctly suppressed the Mtb-stimulated activation of caspase-3 and alteration of Bax/Bcl-x(L) expression in neutrophils. These results indicate that infection with Mtb causes ROS-dependent alteration of Bax/Bcl-x(L) expression and activation of caspase-3, and thereby induces apoptosis in human neutrophils. Moreover, we found that phagocytosis of Mtb-induced apoptotic neutrophils markedly increased the production of proinflammatory cytokine TNF-alpha by human macrophages. Therefore, the ROS-dependent apoptosis in Mtb-stimulated neutrophils may represent an important host defense mechanism aimed at selective removal of infected cells at the inflamed site, which in turn aids the functional activities of local macrophages.     

Expression of Bcl-2 family proteins in thyrocytes from young patients with immune and nonimmune thyroid diseases

The Bcl-2 family proteins that control homeostasis of cells play an important role in apoptosis. This group consists of antiapoptotic (Bcl-2, Bcl-XL) and proapoptotic (Bcl-2 associated protein X, Bax; B-cell homologous antagonist/killer, Bak) molecules. In the thyroid, abnormal apoptotic activity may be involved in a variety of diseases such as autoimmune thyroid diseases. The aim of the current study was to estimate the expression of pro- and antiapoptotic proteins in thyroid tissues from young patients with Graves' disease (GD), nontoxic nodular goiter and toxic nodular goiter using Western Blot and immunohistochemistry. Identification of the antiapoptotic Bcl-2 and Bcl-XL molecules in the thyrocytes revealed higher expression of both proteins in patients with GD (assessed as +++/++ and ++/+, respectively). In adolescents with toxic and nontoxic nodular goiter, this expression was lower (Bcl-2 ++/+ , ++/+; Bcl-XL +, +). The tissue material was additionally subjected to Western Blot analysis, which in GD patients showed the presence of Bcl-2 and Bcl-XL in one band p26 kDa. In patients with toxic and nontoxic nodular goiter, the intensity of expression for these two antiapoptotic proteins was lower (referred to band 26 kDa for Bcl-2 and Bcl-XL). Identification of the proapoptotic proteins Bax and Bak revealed their predominance in thyrocytes of GD patients (+, ++/+, respectively) as compared to patients with toxic and nontoxic nodular goiter (0/+, 0/+ for Bax and 0/+, 0/+ for Bak). In GD patients, Western Blot analysis showed Bax expression in one band 21 kDa and Bak in two bands p50, p24 kDa. In patients with nodular goiter, the degree of expression of both proapoptotic proteins was lower and referred to band 21 kDa for Bax (toxic and nontoxic goiter) and 24 kDa for Bak (toxic goiter only). Patients with GD showed a statistically significant correlation between Bcl-2 expression and antibodies against receptor for thyroid stimulating hormone (R = 0.47, p < 0.03); however, such a correlation was not observed in patients with nodular goiter. In conclusion, our findings suggest that the changes in the expression of regulatory proteins of the Bcl-2 family in the thyroid follicular cells indicate the involvement of apoptosis in the pathogenesis of GD.     

Mutual regulation of Bcl-2 proteins independent of the BH3 domain as shown by the BH3-lacking protein Bcl-x(AK)

The BH3 domain of Bcl-2 proteins was regarded as indispensable for apoptosis induction and for mutual regulation of family members. We recently described Bcl-x(AK), a proapoptotic splice product of the bcl-x gene, which lacks BH3 but encloses BH2, BH4 and a transmembrane domain. It remained however unclear, how Bcl-x(AK) may trigger apoptosis.For efficient overexpression, Bcl-x(AK) was subcloned in an adenoviral vector under Tet-OFF control. The construct resulted in significant apoptosis induction in melanoma and nonmelanoma cell lines with up to 50% apoptotic cells as well as decreased cell proliferation and survival. Disruption of mitochondrial membrane potential, and cytochrome c release clearly indicated activation of the mitochondrial apoptosis pathways. Both Bax and Bak were activated as shown by clustering and conformation analysis. Mitochondrial translocation of Bcl-x(AK) appeared as an essential and initial step. Bcl-x(AK) was critically dependent on either Bax or Bak, and apoptosis was abrogated in Bax/Bak double knockout conditions as well by overexpression of Bcl-2 or Bcl-x(L). A direct interaction with Bcl-2, Bax, Bad, Noxa or Puma was however not seen by immunoprecipitation. Thus besides BH3-mediated interactions, there exists an additional way for mutual regulation of Bcl-2 proteins, which is independent of the BH3. This pathway appears to play a supplementary role also for other proapoptotic family members, and its unraveling may help to overcome therapy resistance in cancer.     

Structure of M11L: A myxoma virus structural homolog of the apoptosis inhibitor, Bcl-2

Apoptosis of virally infected cells is an innate host mechanism used to prevent viral spread. However, viruses have evolved a number of proteins that function to modulate the apoptotic cascades and thereby favor productive viral replication. One such antiapoptotic protein, myxoma virus M11L, has been shown to inhibit mitochondrial-dependent apoptosis by binding to and blocking the two executioner proteins Bak and Bax. Since M11L has no obvious sequence homology with Bcl-2 or Bcl-x(L), the normal cellular inhibitors for Bak and Bax, and the structure of M11L has not been solved, the mode of binding to Bak and Bax is not known. In order to understand how M11L functions, the crystal structure of M11L was solved to 2.91 A. Despite the lack of sequence similarity, M11L is a structural homolog of Bcl-2. Studies using a peptide derived from Bak indicate that M11L binds to Bak with a similar affinity (4.9 +/- 0.3 microM) to the published binding affinities of Bcl-2 and Bcl-x(L) to the same peptide (12.7 microM and 0.5 microM, respectively), indicating that M11L inhibits apoptosis by mimicking and competing with host proteins for the binding of Bak and Bax. The structure provides important insight into how myxoma virus and other poxviruses facilitate viral dissemination by inhibiting mitochondrial dependent apoptosis.     

Membrane-insertion fragments of Bcl-xL, Bax, and Bid

Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria and the endoplasmic reticulum, where they perform their function. The activity of these proteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm, probably through the formation of ion and/or protein transport channels. Most of these proteins contain a C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic alpha helices (alpha5-alpha6) similar to the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here, we report on the membrane-insertion properties of different segments from antiapoptotic Bcl-x(L) and proapoptotic Bax and Bid, that correspond to defined alpha helices in the structure of their soluble forms. According to prediction methods, there are only two putative TM fragments in Bcl-x(L) and Bax (the C-terminal alpha helix and alpha-helix 5) and one in activated tBid (alpha-helix 6). The rest of their sequence, including the second helix of the pore-forming domain, displays only weak hydrophobic peaks, which are below the prediction threshold. Subsequent analysis by glycosylation mapping of single alpha-helix segments in a model chimeric system confirms the above predictions and allows finding an extra TM fragment made of helix alpha1 of Bax. Surprisingly, the amphipathic helices alpha6 of Bcl-x(L) and Bax and alpha7 of Bid do insert in membranes only as part of the alpha5-alpha6 (Bcl-x(L) and Bax) or alpha6-alpha7 (Bid) hairpins but not when assayed individually. This behavior suggests a synergistic insertion and folding of the two helices of the hairpin that could be due to charge complementarity and additional stability provided by turn-inducing residues present at the interhelical region. Although these data come from chimeric systems, they show direct potentiality for acquiring a membrane inserted state. Thus, the above fragments should be considered for the definition of plausible models of the active, membrane-bound species of Bcl-2 proteins.     

DeubiKuitylation: A novel DUB enzymatic activity for the DNA repair protein,Ku70

The Ku70 protein was shown to be involved in multiple cellular pathways including DNA repair, telomere maintenance, V(D)J recombination and Bax mediated apoptosis. Yet, despite this wide spectrum of pathways, till recently the enzymatic activity of Ku70 was elusive. Recent findings demonstrate that Ku70 is associated with the proapoptotic protein Bax and possesses a deubiquitin enzyme (DUB) activity on it. These data suggest a dual role for Ku70 in apoptotic regulation; on one hand the association with Ku70 sequestered Bax away from the mitochondria and plays an antiapoptotic function. On the other hand, this association mediates and promotes Bax deubiquitination which will block its labeling for proteasomal degradation and in this manner Ku70 will have a proapoptotic role. The exciting finding of Ku70’s DUB activity opens numerous avenues for future research. Here we suggest candidate substrate proteins and indicate how the DUB activity of Ku70 on these, might affect the known Ku70’s related pathways.     

Depression of proteasome activities during the progression of cardiac dysfunction in pressure-overloaded heart of mice

The ubiquitin-proteasome system contributes to regulation of apoptosis degrading apoptosis-regulatory proteins. Marked accumulation of ubiquitinated proteins in cardiomyocytes of human failing hearts suggested impaired ubiquitin-proteasome system in heart failure. Since cardiomyocyte apoptosis contributes to the progression of cardiac dysfunction in pressure-overloaded hearts, we investigated the role of ubiquitin-proteasome system in such conditions. We found that proteasome activities already depressed before the onset of cardiac dysfunction in pressure-overloaded hearts of mice. Cardiomyocyte apoptosis was observed along with depression of proteasome activities and elevation of proapoptotic/antiapoptotic protein ratio in failing hearts. In cultured cardiomyocytes, pharmacological inhibition of proteasome accumulated proapoptotic proteins such as p53 and Bax. Gene silencing of these proapoptotic proteins by RNA interference prevented the accumulation of respective proteins and attenuated cardiomyocyte apoptosis induced by proteasome inhibition. We conclude that depression of proteasome activities contributes to cardiac dysfunction resulting from cardiomyocyte apoptosis through accumulation of proapoptotic proteins by impaired degradation.