A BAX/BAK and cyclophilin D-independent intrinsic apoptosis pathway.

Most intrinsic death signals converge into the activation of pro-apoptotic BCL-2 family members BAX and BAK at the mitochondria, resulting in the release of cytochrome c and apoptosome activation. Chronic endoplasmic reticulum (ER) stress leads to apoptosis through the upregulation of a subset of pro-apoptotic BH3-only proteins, activating BAX and BAK at the mitochondria. Here we provide evidence indicating that the full resistance of BAX and BAK double deficient (DKO) cells to ER stress is reverted by stimulation in combination with mild serum withdrawal. Cell death under these conditions was characterized by the appearance of classical apoptosis markers, caspase-9 activation, release of cytochrome c, and was inhibited by knocking down caspase-9, but insensitive to BCL-X(L) overexpression. Similarly, the resistance of BIM and PUMA double deficient cells to ER stress was reverted by mild serum withdrawal. Surprisingly, BAX/BAK-independent cell death did not require Cyclophilin D (CypD) expression, an important regulator of the mitochondrial permeability transition pore. Our results suggest the existence of an alternative intrinsic apoptosis pathway emerging from a cross talk between the ER and the mitochondria.

A pharmacologic inhibitor of the protease Taspase1 effectively inhibits breast and brain tumor growth.

The threonine endopeptidase Taspase1 has a critical role in cancer cell proliferation and apoptosis. In this study, we developed and evaluated small molecule inhibitors of Taspase1 as a new candidate class of therapeutic modalities. Genetic deletion of Taspase1 in the mouse produced no overt deficiencies, suggesting the possibility of a wide therapeutic index for use of Taspase1 inhibitors in cancers. We defined the peptidyl motifs recognized by Taspase1 and conducted a cell-based dual-fluorescent proteolytic screen of the National Cancer Institute diversity library to identify Taspase1 inhibitors (TASPIN). On the basis of secondary and tertiary screens the 4-[(4-arsonophenyl)methyl]phenyl] arsonic acid NSC48300 was determined to be the most specific active compound. Structure-activity relationship studies indicated a crucial role for the arsenic acid moiety in mediating Taspase1 inhibition. Additional fluorescence resonance energy transfer-based kinetic analysis characterized NSC48300 as a reversible, noncompetitive inhibitor of Taspase1 (K(i) = 4.22 µmol/L). In the MMTV-neu mouse model of breast cancer and the U251 xenograft model of brain cancer, NSC48300 produced effective tumor growth inhibition. Our results offer an initial preclinical proof-of-concept to develop TASPINs for cancer therapy.

p53 controls radiation-induced gastrointestinal syndrome in mice independent of apoptosis.

Acute exposure to ionizing radiation can cause lethal damage to the gastrointestinal (GI) tract, a condition called the GI syndrome. Whether the target cells affected by radiation to cause the GI syndrome are derived from the epithelium or endothelium and whether the target cells die by apoptosis or other mechanisms are controversial issues. Studying mouse models, we found that selective deletion of the proapoptotic genes Bak1 and Bax from the GI epithelium or from endothelial cells did not protect mice from developing the GI syndrome after sub-total-body gamma irradiation. In contrast, selective deletion of p53 from the GI epithelium, but not from endothelial cells, sensitized irradiated mice to the GI syndrome. Transgenic mice overexpressing p53 in all tissues were protected from the GI syndrome after irradiation. These results suggest that the GI syndrome is caused by the death of GI epithelial cells and that these epithelial cells die by a mechanism that is regulated by p53 but independent of apoptosis.

The VDAC2-BAK rheostat controls thymocyte survival.

The proapoptotic proteins BAX and BAK constitute the mitochondrial apoptotic gateway that executes cellular demise after integrating death signals. The lethal BAK is kept in check by voltage-dependent anion channel 2 (VDAC2), a mammalian-restricted VDAC isoform. Here, we provide evidence showing a critical role for the VADC2-BAK complex in determining thymocyte survival in vivo. Genetic depletion of Vdac2 in the thymus resulted in excessive cell death and hypersensitivity to diverse death stimuli including engagement of the T cell receptor. These phenotypes were completely rescued by the concurrent deletion of Bak but not that of Bax. Thus, the VDAC2-BAK axis provides a mechanism that governs the homeostasis of thymocytes. Our study reveals a sophisticated built-in rheostat that likely fine-tunes immune competence to balance autoimmunity and immunodeficiency.

Dual role of proapoptotic BAD in insulin secretion and beta cell survival.

The proapoptotic BCL-2 family member BAD resides in a glucokinase-containing complex that regulates glucose-driven mitochondrial respiration. Here, we present genetic evidence of a physiologic role for BAD in glucose-stimulated insulin secretion by beta cells. This novel function of BAD is specifically dependent upon the phosphorylation of its BH3 sequence, previously defined as an essential death domain. We highlight the pharmacologic relevance of phosphorylated BAD BH3 by using cell-permeable, hydrocarbon-stapled BAD BH3 helices that target glucokinase, restore glucose-driven mitochondrial respiration and correct the insulin secretory response in Bad-deficient islets. Our studies uncover an alternative target and function for the BAD BH3 domain and emphasize the therapeutic potential of phosphorylated BAD BH3 mimetics in selectively restoring beta cell function. Furthermore, we show that BAD regulates the physiologic adaptation of beta cell mass during high-fat feeding. Our findings provide genetic proof of the bifunctional activities of BAD in both beta cell survival and insulin secretion.

Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival.

We investigated the mechanism by which B lymphocyte stimulator (BLyS)/BAFF, a tumor necrosis factor superfamily ligand, promotes B-cell survival and resistance to atrophy. BLyS stimulation activates 2 independent signaling pathways, Akt/mTOR and Pim 2, associated with cell growth and survival. BLyS blocks the cell volume loss (atrophy) that freshly isolated B cells normally undergo when maintained in vitro while concurrently increasing glycolytic activity and overall metabolism. This atrophy resistance requires Akt/mTOR. We used a genetic approach to resolve the contributions of Akt/mTOR and Pim kinase pathways to BLyS-mediated survival. Pim 2-deficient B cells are readily protected from death by BLyS stimulation, but this protection is completely abrogated by treatment with the mTOR inhibitor rapamycin. Furthermore, rapamycin treatment in vivo significantly reduces both follicular and marginal zone B cells in Pim-deficient but not healthy hosts. BLyS-dependent survival requires the antiapoptotic protein Mcl-1. Mcl-1 protein levels rise and fall in response to BLyS addition and withdrawal, respectively, and conditional deletion of the Mcl-1 gene renders B cells refractory to BLyS-mediated protection. Because BlyS is required for the normal homeostasis of all B cells, these data suggest a therapeutic strategy simultaneously inhibiting mTOR and Pim 2 could target pathogenic B cells.

A spatially and temporally restricted mouse model of soft tissue sarcoma.

Soft tissue sarcomas are mesenchymal tumors that are fatal in approximately one-third of patients. To explore mechanisms of sarcoma pathogenesis, we have generated a mouse model of soft tissue sarcoma. Intramuscular delivery of an adenovirus expressing Cre recombinase in mice with conditional mutations in Kras and Trp53 was sufficient to initiate high-grade sarcomas with myofibroblastic differentiation. Like human sarcomas, these tumors show a predilection for lung rather than lymph node metastasis. Using this model, we showed that a prototype handheld imaging device can identify residual tumor during intraoperative molecular imaging. Deletion of the Ink4a-Arf locus (Cdkn2a), but not Bak1 and Bax, could substitute for mutation of Trp53 in this model. Deletion of Bak1 and Bax, however, was able to substitute for mutation of Trp53 in the development of sinonasal adenocarcinoma. Therefore, the intrinsic pathway of apoptosis seems sufficient to mediate p53 tumor suppression in an epithelial cancer, but not in this model of soft tissue sarcoma.

Proapoptotic histone H1.2 induces CASP-3 and -7 activation by forming a protein complex with CYT c, APAF-1 and CASP-9.

Cytochrome c (CYT c) is a protein that employs the caspase recruitment domain (CARD)-containing proteins APAF-1 and CASP-9 to activate effectors CASP-3 and -7. By using affinity labeling techniques and mass spectrometry analysis, we show that histone H1.2 is a regulator of caspases upon UV irradiation. We demonstrated that histone H1.2 forms a protein complex with APAF-1, CASP-9 and CYT c upon UV irradiation. In cell-free systems, we show that histone H1.2 triggers activation of CASP-3 and -7 via APAF-1 and CASP-9. We therefore conclude that upon DNA damage histone H1.2 acts as a positive regulator of apoptosome formation.

A stapled BID BH3 helix directly binds and activates BAX.

BAX is a multidomain proapoptotic BCL-2 family protein that resides in the cytosol until activated by an incompletely understood trigger mechanism, which facilitates BAX translocation to mitochondria and downstream death events. Whether BAX is activated by direct contact with select BH3-only members of the BCL-2 family is highly debated. Here we detect and quantify a direct binding interaction between BAX and a hydrocarbon-stapled BID BH3 domain, which triggers the functional activation of BAX at nanomolar doses in vitro. Chemical reinforcement of BID BH3 alpha helicity was required to reveal the direct BID BH3-BAX association. We confirm the specificity of this BH3 interaction by characterizing a stapled BAD BH3 peptide that interacts with antiapoptotic BCL-X(L) but does not bind or activate BAX. We further demonstrate that membrane targeting of stapled BID BH3 optimizes its ability to activate BAX, supporting a model in which BID directly engages BAX to trigger mitochondrial apoptosis.

Proteolysis of MLL family proteins is essential for taspase1-orchestrated cell cycle progression.

Taspase1 was identified as the threonine endopeptidase that cleaves mixed-lineage leukemia (MLL) for proper Hox gene expression in vitro. To investigate its functions in vivo, we generated Taspase1(-/-) mice. Taspase1 deficiency results in noncleavage (nc) of MLL and MLL2 and homeotic transformations. Remarkably, our in vivo studies uncover an unexpected role of Taspase1 in the cell cycle. Taspase1(-/-) animals are smaller in size. Taspase1(-/-) mouse embryonic fibroblasts (MEFs) exhibit impaired proliferation, and acute deletion of Taspase1 leads to a marked reduction of thymocytes. Taspase1 deficiency incurs down-regulation of Cyclin Es, As, and Bs and up-regulation of p16(Ink4a) . We show that MLL and MLL2 directly target E2Fs for Cyclin expression. The uncleaved precursor MLL displays a reduced histone H3 methyl transferase activity in vitro. Accordingly, chromatin immunoprecipitation assays demonstrate a markedly decreased histone H3 K4 trimethylation at Cyclin E1 and E2 genes in Taspase1(-/-) cells. Furthermore, MLL(nc/nc;2nc/nc) MEFs are also impaired in proliferation. Our data are consistent with a model in which precursor MLLs, activated by Taspase1, target to Cyclins through E2Fs to methylate histone H3 at K4, leading to activation. Lastly, Taspase1(-/-) cells are resistant to oncogenic transformation, and Taspase1 is overexpressed in many cancer cell lines. Thus, Taspase1 may serve as a target for cancer therapeutics.

A membrane-targeted BID BCL-2 homology 3 peptide is sufficient for high potency activation of BAX in vitro.

The multidomain pro-apoptotic proteins BAX and BAK constitute an essential gateway to mitochondrial dysfunction and programmed cell death. Among the "BCL-2 homology (BH) 3-only" members of pro-apoptotic proteins, truncated BID (tBID) has been implicated in direct BAX activation, although an explicit molecular mechanism remains elusive. We find that BID BH3 peptide alone at submicromolar concentrations cannot activate BAX or complement BID BH3 mutant-tBID in mitochondrial and liposomal release assays. Because tBID contains structurally defined membrane association domains, we investigated whether membrane targeting of BID BH3 peptide would be sufficient to restore its pro-apoptotic activity. We developed a Ni(2+)-nitrilotriacetic acid liposomal assay system that efficiently conjugates histidine-tagged peptides to a simulated outer mitochondrial membrane surface. Strikingly, nanomolar concentrations of a synthetic BID BH3 peptide that is chemically tethered to the liposomal membrane activated BAX almost as efficiently as tBID itself. These results highlight the importance of membrane targeting of the BID BH3 domain in tBID-mediated BAX activation and support a model in which tBID engages BAX to trigger its pro-apoptotic activity.

PP2A regulates BCL-2 phosphorylation and proteasome-mediated degradation at the endoplasmic reticulum.

Anti-apoptotic activity of BCL-2 is mediated by phosphorylation at the endoplasmic reticulum (ER), but how this phosphorylation is regulated and the mechanism(s) by which it regulates apoptosis are unknown. We purified macromolecular complexes containing BCL-2 from ER membranes and found that BCL-2 co-purified with the main two subunits of the serine/threonine phosphatase, PP2A. The association of endogenous PP2A and BCL-2 at the ER was verified by co-immunoprecipitation and microcystin affinity purification. Knock down or pharmacological inhibition of PP2A caused degradation of phosphorylated BCL-2 and led to an overall reduction in BCL-2 levels. We found that this degradation was due to the action of the proteasome acting selectively at the ER. Conversely, overexpression of PP2A caused elevation in endogenous BCL-2. Most importantly, we found that PP2A knock down sensitized cells to several classes of death stimuli (including ER stress), but this effect was abolished in a genetic background featuring knock in of a non-phosphorylatable BCL-2 allele. These studies support the hypothesis that PP2A-mediated dephosphorylation of BCL-2 is required to protect BCL-2 from proteasome-dependent degradation, affecting resistance to ER stress.

Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1alpha.

Accumulation of misfolded protein in the endoplasmic reticulum (ER) triggers an adaptive stress response-termed the unfolded protein response (UPR)-mediated by the ER transmembrane protein kinase and endoribonuclease inositol-requiring enzyme-1alpha (IRE1alpha). We investigated UPR signaling events in mice in the absence of the proapoptotic BCL-2 family members BAX and BAK [double knockout (DKO)]. DKO mice responded abnormally to tunicamycin-induced ER stress in the liver, with extensive tissue damage and decreased expression of the IRE1 substrate X-box-binding protein 1 and its target genes. ER-stressed DKO cells showed deficient IRE1alpha signaling. BAX and BAK formed a protein complex with the cytosolic domain of IRE1alpha that was essential for IRE1alpha activation. Thus, BAX and BAK function at the ER membrane to activate IRE1alpha signaling and to provide a physical link between members of the core apoptotic pathway and the UPR.

Uncleaved TFIIA is a substrate for taspase 1 and active in transcription.

In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single TFIIAalphabeta gene and posttranslationally cleaved into alpha and beta subunits. The molecular mechanisms and biological significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate of taspase 1 as reported for the trithorax group mixed-lineage leukemia protein. We demonstrate that recombinant taspase 1 cleaves TFIIA in vitro. Transfected taspase 1 enhances cleavage of TFIIA, and RNA interference knockdown of endogenous taspase 1 diminishes cleavage of TFIIA in vivo. In taspase 1-/- MEF cells, only uncleaved TFIIA is detected. In Xenopus laevis embryos, knockdown of TFIIA results in phenotype and expression defects. Both defects can be rescued by expression of an uncleavable TFIIA mutant. Our study shows that uncleaved TFIIA is transcriptionally active and that cleavage of TFIIA does not serve to render TFIIA competent for transcription. We propose that cleavage fine tunes the transcription regulation of a subset of genes during differentiation and development.

Traumatic brain injury in mice deficient in Bid: effects on histopathology and functional outcome.

Bid is a proapoptotic member of the Bcl-2 family that mediates cell death by caspase-dependent and -independent pathways. We tested mice genetically deficient in Bid in a controlled cortical impact (CCI) model to examine the hypothesis that Bid contributes to cell death and functional outcome after traumatic brain injury. After CCI, truncated Bid (15 kDa) was robustly detected in cortical brain homogenates of wild-type mice. Bid-/- mice had decreased numbers of cortical cells with acute plasmalemma injury at 6 h (wild type (WT), 1721+/-124; Bid-/-, 1173+/-129 cells/ x 200 field; P<0.01), decreased numbers of cells expressing cleaved caspase-3 in the dentate gyrus at 48 h (WT, 113+/-15; Bid-/-, 65+/-9 cells/ x 200 field; P<0.05), and reduced lesion volume at 12 days (Bid-/-, 5.9+/-0.4 mm(3); WT, 8.4+/-0.4 mm(3); P<0.001), but did not differ from WT mice at later times after injury regarding lesion size (30 days) or brain tissue atrophy (40 days). Compared with naïve mice, injured mice in both groups performed significantly worse on motor and Morris water maze (MWM) tests; however, mice deficient in Bid did not differ from WT in postinjury motor and MWM performance. The data show that Bid deficiency decreases early posttraumatic brain cell death and tissue damage, but does not reduce functional outcome deficits after CCI in mice.

Bax channel inhibitors prevent mitochondrion-mediated apoptosis and protect neurons in a model of global brain ischemia.

Ischemic injuries are associated with several pathological conditions, including stroke and myocardial infarction. Several studies have indicated extensive apoptotic cell death in the infarcted area as well as in the penumbra region of the infarcted tissue. Studies with transgenic animals suggest that the mitochondrion-mediated apoptosis pathway is involved in ischemia-related cell death. This pathway is triggered by activation of pro-apoptotic Bcl-2 family members such as Bax. Here, we have identified and synthesized two low molecular weight compounds that block Bax channel activity. The Bax channel inhibitors prevented cytochrome c release from mitochondria, inhibited the decrease in the mitochondrial membrane potential, and protected cells against apoptosis. The Bax channel inhibitors did not affect the conformational activation of Bax or its translocation and insertion into the mitochondrial membrane in cells undergoing apoptosis. Furthermore, the compounds protected neurons in an animal model of global brain ischemia. The protective effect in the animal model correlated with decreased cytochrome c release in the infarcted area. This is the first demonstration that Bax channel activity is required in apoptosis.

p600, a unique protein required for membrane morphogenesis and cell survival.

In this article, we identify and characterize p600, a unique 600-kDa retinoblastoma protein- and calmodulin-binding protein. In the nucleus, p600 and retinoblastoma protein seem to act as a chromatin scaffold. In the cytoplasm, p600 and clathrin form a meshwork structure, which could contribute to cytoskeletal organization and membrane morphogenesis. Reduced expression of p600 with interference RNA abrogates integrin-mediated ruffled membrane formation and, furthermore, prevents activation of integrin-mediated survival pathways. Consequently, knockdown of p600 sensitizes cells to apoptosis induced by cell detachment. These findings provide mechanistic insight into the regulation of membrane-proximal events in tumorigenesis.

Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia.

Mitochondrial permeability transition (PT) is a phenomenon induced by high levels of matrix calcium and is characterized by the opening of the PT pore (PTP). Activation of the PTP results in loss of mitochondrial membrane potential, expansion of the matrix, and rupture of the mitochondrial outer membrane. Consequently, PT has been implicated in both apoptotic and necrotic cell death. Cyclophilin D (CypD) appears to be a critical component of the PTP. To investigate the role of CypD in cell death, we created a CypD-deficient mouse. In vitro, CypD-deficient mitochondria showed an increased capacity to retain calcium and were no longer susceptible to PT induced by the addition of calcium. CypD-deficient primary mouse embryonic fibroblasts (MEFs) were as susceptible to classical apoptotic stimuli as the WT, suggesting that CypD is not a central component of cell death in response to these specific death stimuli. However, CypD-deficient MEFs were significantly less susceptible than their WT counterparts to cell death induced by hydrogen peroxide, implicating CypD in oxidative stress-induced cell death. Importantly, CypD-deficient mice displayed a dramatic reduction in brain infarct size after acute middle cerebral artery occlusion and reperfusion, strongly supporting an essential role for CypD in an ischemic injury model in which calcium overload and oxidative stress have been implicated.