IP3R2 levels dictate the apoptotic sensitivity of diffuse large B-cell lymphoma cells to an IP3R-derived peptide targeting the BH4 domain of Bcl-2

Disrupting inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP₃R-derived peptide (TAT-IDP^S)) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca²⁺ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP₃R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDP^S in a more heterogeneous Bcl-2-dependent cancer model using a set of ‘primed to death'' diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDP^S with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDP^S to promote IP₃R-mediated Ca²⁺ release. Although total IP₃R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP₃R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDP^S-induced Ca²⁺ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP₃R2-protein level, but not with IP₃R1-, IP₃R3- or total IP₃R-expression levels. Inhibiting or knocking down IP₃R2 activity in SU-DHL-4-reduced TAT-IDP^S-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP₃R2 and to promote IP₃-induced pro-apoptotic Ca²⁺ signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP₃R hyperactivity. In particular, cancer cells expressing high levels of IP₃R2 are addicted to IP₃R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca²⁺ signaling and cell death.     

Targeting Bcl-2-IP3 receptor interaction to treat cancer: A novel approach inspired by nearly a century treating cancer with adrenal corticosteroid hormones

Bcl-2 inhibits cell death by at least two different mechanisms. On the one hand, its BH3 domain binds to pro-apoptotic proteins such as Bim and prevents apoptosis induction. On the other hand, the BH4 domain of Bcl-2 binds to the inositol 1,4,5-trisphosphate receptor (IP₃R), preventing Ca²⁺ signals that mediate cell death. In normal T-cells, Bcl-2 levels increase during the immune response, protecting against cell death, and then decline as apoptosis ensues and the immune response dissipates. But in many cancers Bcl-2 is aberrantly expressed and exploited to prevent cell death by inhibiting IP₃R-mediated Ca²⁺ elevation. This review summarizes what is known about the mechanism of Bcl-2's control over IP₃R-mediated Ca²⁺ release and cell death induction. Early insights into the role of Ca²⁺ elevation in corticosteroid-mediated cell death serves as a model for how targeting IP₃R-mediated Ca²⁺ elevation can be a highly effective therapeutic approach for different types of cancer. Moreover, the successful development of ABT-199 (Venetoclax), a small molecule targeting the BH3 domain of Bcl-2 but without effects on Ca²⁺, serves as proof of principle that targeting Bcl-2 can be an effective therapeutic approach. BIRD-2, a synthetic peptide that inhibits Bcl-2-IP₃R interaction, induces cell death induction in ABT-199 (Venetoclax)-resistant cancer models, attesting to the value of developing therapeutic agents that selectively target Bcl-2-IP₃R interaction, inducing Ca²⁺-mediated cell death.     

The BH4 Domain of Anti-apoptotic Bcl-XL,but Not That of the Related Bcl-2, Limits the Voltage-dependent Anion Channel 1 (VDAC1)-mediated Transfer of Pro-apoptotic Ca2+ Signals to Mitochondria

Excessive Ca²⁺ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting Ca²⁺-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP₃Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca²⁺-dependent apoptosis by binding and inhibiting IP₃Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP₃Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca²⁺ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1^−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca²⁺ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca²⁺ release in the cytosol or to prevent apoptosis, as done instead by an IP₃R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca²⁺ uptake into the mitochondria.     

A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation

Sustained elevation of intracellular calcium by Ca²⁺ release–activated Ca²⁺ channels is required for lymphocyte activation. Sustained Ca²⁺ entry requires endoplasmic reticulum (ER) Ca²⁺ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP₃R)/Ca²⁺ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca²⁺, and the mechanism that enables the prolonged activation of IP₃R1 required for lymphocyte activation is unclear. We show that IP₃R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP₃R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP₃ and renders the channel less sensitive to Ca²⁺-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca²⁺ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca²⁺ levels during lymphocyte activation.     

The BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as an evolutionary conserved inhibitor of IP3 receptor channels

Ca²⁺ signalling plays an important role in various physiological processes in vertebrates. In mammals, the highly conserved anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein is an important modulator of the inositol 1,4,5-trisphosphate receptor (IP₃R), i.e. the main intracellular Ca²⁺ - release channel located at the endoplasmic reticulum (ER). The Bcl-2 Homology (BH) 4 domain of Bcl-2 (BH4-Bcl-2) is a critical determinant for inhibiting IP₃Rs, by directly targeting a region in the modulatory domain of the receptor (domain 3). In this paper, we aimed to track the evolutionary history of IP₃R regulation by the BH4 domain of Bcl-2 orthologues from different classes of vertebrates, including Osteichthyes, Amphibia, Reptilia, Aves and Mammalia. The high degree of conservation of the BH4 sequences correlated with the ability of all tested peptides to bind to the domain 3 of mouse IP₃R1 in GST-pull downs and their overall ability to inhibit IP₃-induced Ca²⁺ release (IICR) in permeabilized cells. Nevertheless, the BH4 domains differed in their potency to suppress IICR. The peptide derived from X. laevis was the least potent inhibitor. We identified a critical residue in BH4-Bcl-2 from H. sapiens, Thr7, which is replaced by Gly7 in X. laevis. Compared to the wild type X. laevis BH4-Bcl-2, a “humanized” version of the peptide (BH4-Bcl-2 Gly7Thr), displayed increased IP₃R-inhibitory properties. Despite the differences in the inhibitory efficiency, our data indicate that the BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as a binding partner and inhibitor of IP₃R channels.     

Tracing the evolutionary history of Ca2+-signaling modulation by human Bcl-2: Insights from the Capsaspora owczarzaki IP3 receptor ortholog

Recently, a functional IP₃R ortholog (CO.IP₃R-A) capable of IP₃-induced Ca²⁺ release has been discovered in Capsaspora owczarzaki, a close unicellular relative to Metazoa. In contrast to mammalian IP₃Rs, CO.IP₃R-A is not modulated by Ca²⁺, ATP or PKA. Protein-sequence analysis revealed that CO.IP₃R-A contained a putative binding site for anti-apoptotic Bcl-2, although Bcl-2 was not detected in Capsaspora owczarzaki and only appeared in Metazoa. Here, we examined whether human Bcl-2 could form a complex with CO.IP₃R-A channels and modulate their Ca²⁺-flux properties using ectopic expression approaches in a HEK293 cell model in which all three IP₃R isoforms were knocked out. We demonstrate that human Bcl-2 via its BH4 domain could functionally interact with CO.IP₃R-A, thereby suppressing Ca²⁺ flux through CO.IP₃R-A channels. The BH4 domain of Bcl-2 was sufficient for interaction with CO.IP₃R-A channels. Moreover, mutating the Lys17 of Bcl-2's BH4 domain, the residue critical for Bcl-2-dependent modulation of mammalian IP₃Rs, abrogated Bcl-2's ability to bind and inhibit CO.IP₃R-A channels. Hence, this raises the possibility that a unicellular ancestor of animals already had an IP₃R that harbored a Bcl-2-binding site. Bcl-2 proteins may have evolved as controllers of IP₃R function by exploiting this pre-existing site, thereby counteracting Ca²⁺-dependent apoptosis.     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

Stimulation of Inositol 1,4,5-Trisphosphate (IP3) Receptor Subtypes by Adenophostin A and Its Analogues

Inositol 1,4,5-trisphosphate receptors (IP₃R) are intracellular Ca²⁺ channels. Most animal cells express mixtures of the three IP₃R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP₃R and it shares with IP₃ the essential features of all IP₃R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP₃. The two essential phosphate groups contribute to closure of the clam-like IP₃-binding core (IBC), and thereby IP₃R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP₃R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP₃R. We measured Ca²⁺ release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP₃R and stably expressing single subtypes of mammalian IP₃R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP₃R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP₃R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP₃. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP₃R subtypes. They demonstrate that differences in the Ca²⁺ signals evoked by AdA analogues are unlikely to be due to selective regulation of IP₃R subtypes.     

G-protein-coupled Receptor Kinase-interacting Proteins Inhibit Apoptosis by Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Signal Regulation

The inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) is an intracellular IP₃-gated calcium (Ca²⁺) release channel and plays important roles in regulation of numerous Ca²⁺-dependent cellular responses. Many intracellular modulators and IP₃R-binding proteins regulate the IP₃R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP₃R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP₃R. The interaction was favored by the cytosolic Ca²⁺ concentration and it functionally inhibited IP₃R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP₃R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP₃R-mediated Ca²⁺ signal through a direct interaction with IP₃R in a cytosolic Ca²⁺-dependent manner.The inositol 1,4,5-trisphosphate (IP₃)³ receptor (IP₃R) consisting of three subtypes, IP₃R1, IP₃R2, and IP₃R3, is a tetrameric intracellular IP₃-gated calcium (Ca²⁺) release channel localized at the endoplasmic reticulum (ER) with its NH₂ terminus and COOH-terminal tail (CTT) exposed to the cytoplasm (1, 2; see Fig. 1A). IP₃Rs are composed of five functional domains. The long NH₂-terminal cytoplasmic region contains three domains, a coupling/suppressor domain, an IP₃-binding core domain, and an internal coupling domain. The COOH-terminal region has a six-membrane spanning channel domain and a short cytoplasmic CTT “gatekeeper domain” that is critical for IP₃R channel opening (2, 3). Ca²⁺ release activity of the IP₃R channel is regulated by many intracellular modulators (ATP, calmodulin, and Ca²⁺), protein kinases, and IP₃R-binding proteins (2, 4), and the tight regulation of IP₃R channel activity by these factors generates various spatial and temporal intracellular Ca²⁺ patterns such as Ca²⁺ spikes and Ca²⁺ oscillations, leading to numerous cellular responses (1, 2, 5, 6).Open in a separate windowFIGURE 1.GIT1 and GIT2 bind to all three subtypes of IP₃R. A, schematic of ER residential IP₃R. The CTT of IP₃R1 is used as bait in a yeast two-hybrid screen. B, schematic representation of GIT1, GIT2, and two GIT1 fragments identified from the yeast two-hybrid screen. Functional domains are indicated. ARF-GAP, ARF-specific GTPase-activating protein domain; ANK-REP, ankyrin repeats; CC, coiled-coil domains; SHD, the Spa2-homology domain; EF, EF-hand; IQ, IQ-like motifs; aa, amino acid. C, GIT1 binds to IP₃R1 in vitro. GST and GST-IP₃R1/CTT were incubated with mouse brain lysate for a pull-down assay. The input and pulled-down samples were probed with α-GIT1. D and E, GIT1 binds to IP₃R1 in vivo. Mouse brain lysates were processed to control IgG and α-IP₃R1 (D) or α-GIT1 (E) for IP. The input and IP samples were probed with α-GIT1 and α-IP₃R1. F and G, both GIT1 and GIT2 bind to all three IP₃R subtypes. HeLa cells coexpressing GFP-fused IP₃R1, IP₃R2, or IP₃R3 and mRFP-fused GIT1 (F) or GIT2 (G) were processed for IP using α-RFP. The input and IP samples were blotted with α-GFP (top) and α-RFP (bottom).One of the physiological roles of IP₃R-mediated Ca²⁺ signaling is a pro-apoptotic regulator during apoptosis. Ca²⁺ released from ER can stimulate several key enzymes activated during apoptosis such as endonucleases (7) and calpain (8). In addition, the close proximity of ER to mitochondria may facilitate the mitochondrial overload of Ca²⁺ released from the IP₃Rs with certain apoptotic stimuli, triggering the opening of the mitochondrial permeability transition pore and the release of apoptotic signaling molecules, such as cytochrome c and apoptosis-inducing factor, which leads to the activation of caspases (5, 6). Moreover, several key components of apoptotic cascades, such as cytochrome c (9) and anti-apoptosis proteins Bcl-2 (10, 11) and Bcl-X_L (12), have been reported to interact with the internal coupling domain and/or the CTT of IP₃R and enhance the Ca²⁺-release activity of IP₃Rs during apoptosis. In this study, we identified the ubiquitously expressed G-protein-coupled receptor kinase-interacting proteins (GIT) (13), GIT1 and GIT2, as novel IP₃R-binding proteins that bind to the CTT of IP₃R and inhibit apoptosis by regulation of IP₃R-mediated Ca²⁺ signal.     

Stroma Cell-Derived Factor-1α Signaling Enhances Calcium Transients and Beating Frequency in Rat Neonatal Cardiomyocytes

Stroma cell-derived factor-1α (SDF-1α) is a cardioprotective chemokine, acting through its G-protein coupled receptor CXCR4. In experimental acute myocardial infarction, administration of SDF-1α induces an early improvement of systolic function which is difficult to explain solely by an anti-apoptotic and angiogenic effect. We wondered whether SDF-1α signaling might have direct effects on calcium transients and beating frequency.Primary rat neonatal cardiomyocytes were culture-expanded and characterized by immunofluorescence staining. Calcium sparks were studied by fluorescence microscopy after calcium loading with the Fluo-4 acetoxymethyl ester sensor. The cardiomyocyte enriched cellular suspension expressed troponin I and CXCR4 but was vimentin negative. Addition of SDF-1α in the medium increased cytoplasmic calcium release. The calcium response was completely abolished by using a neutralizing anti-CXCR4 antibody and partially suppressed and delayed by preincubation with an inositol triphosphate receptor (IP₃R) blocker, but not with a ryanodine receptor (RyR) antagonist. Calcium fluxes induced by caffeine, a RyR agonist, were decreased by an IP₃R blocker. Treatment with forskolin or SDF-1α increased cardiomyocyte beating frequency and their effects were additive. In vivo, treatment with SDF-1α increased left ventricular dP/dtmax.These results suggest that in rat neonatal cardiomyocytes, the SDF-1α/CXCR4 signaling increases calcium transients in an IP₃-gated fashion leading to a positive chronotropic and inotropic effect.     

Calcium Release at Fertilization in Starfish Eggs Is Mediated by Phospholipase Cγ

Although inositol trisphosphate (IP₃) functions in releasing Ca²⁺ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP₃. To distinguish between a role for PLCγ, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCβ, which is activated by a G protein, we injected starfish eggs with a PLCγ SH2 domain fusion protein that inhibits activation of PLCγ. In these eggs, Ca²⁺ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCγSH2 protein is a specific inhibitor of PLCγ in the egg, since it did not inhibit PLCβ activation of Ca²⁺ release initiated by the serotonin 2c receptor, or activation of Ca²⁺ release by IP₃ injection. Furthermore, injection of a PLCγ SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca²⁺ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cγ by an SH2 domain-mediated process stimulates the production of IP₃ that causes intracellular Ca²⁺ release.     

Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

A uniform extracellular stimulus triggers cell-specific patterns of Ca²⁺ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP₃) concentration changes using a fluorescent IP₃ sensor in single HeLa cells showing different patterns of histamine-induced Ca²⁺ oscillations in terms of the time constant of Ca²⁺ spike amplitude decay and the Ca²⁺ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca²⁺ signals and we found that there were cell-specific IP₃ dynamics depending on the patterns of Ca²⁺ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP₃ increases in HeLa cells. Modulation of IP₃ dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca²⁺ oscillations, such as the time constant of the temporal changes in the Ca²⁺ spike amplitude and the Ca²⁺ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP₃ production, rather than IP₃-induced Ca²⁺ release, can cause cell-to-cell variability in the patterns of Ca²⁺ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca²⁺ signals evoked by G protein-coupled receptor stimulation.     

Endophilin A2-mediated alleviation of endoplasmic reticulum stress-induced cardiac injury involves the suppression of ERO1α/IP3R signaling pathway

Cardiac injury upon myocardial infarction (MI) is the leading cause of heart failure. The present study aims to investigate the role of EndoA2 in ischemia-induced cardiomyocyte apoptosis and cardiac injury. In vivo, we established an MI mouse model by ligating the left anterior descending (LAD) coronary artery, and intramyocardial injection of adenoviral EndoA2 (Ad-EndoA2) was used to overexpress EndoA2. In vitro, we used the siRNA and Ad-EndoA2 transfection strategies. Here, we reported that EndoA2 expression was remarkably elevated in the infarct border zone of MI mouse hearts and neonatal rat cardiomyocytes (NRCMs) stimulated with oxygen and glucose deprivation (OGD) which mimicked ischemia. We showed that intramyocardial injection of Ad-EndoA2 attenuated cardiomyocyte apoptosis and reduced endoplasmic reticulum (ER) stress in response to MI injury. Using siRNA for knockdown and Ad-EndoA2 for overexpression, we validated that knockdown of EndoA2 in NRCMs exacerbated OGD-induced NRCM apoptosis, whereas overexpression of EndoA2 attenuates OGD-induced cardiomyocyte apoptosis. Mechanistically, knockdown of EndoA2 activated ER stress response, which increases ER oxidoreductase 1α (ERO1α) and inositol 1, 4, 5-trisphosphate receptor (IP₃R) activity, thus led to increased intracellular Ca²⁺ accumulation, followed by elevated calcineurin activity and nuclear factor of activated T-cells (NFAT) dephosphorylation. Pretreatment with the IP₃R inhibitor 2-Aminoethoxydiphenylborate (2-APB) attenuated intracellular Ca²⁺ accumulation, and pretreatment with the Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) or the calcineurin inhibitor Cyclosporin A (CsA) inhibited EndoA2-knockdown-induced NRCM apoptosis. Overexpression of EndoA2 led to the opposite effects by suppressing ER-stress-mediated ERO1α/IP₃R signaling pathway. This study demonstrated that EndoA2 protected cardiac function in response to MI via attenuating ER-stress-mediated ERO1α/IP₃R signaling pathway. Targeting EndoA2 is a potential therapeutic strategy for the prevention of postinfarction-induced cardiac injury and heart failure.     

JNK1/2 regulates ER–mitochondrial Ca2+ cross-talk during IL-1β–mediated cell death in RINm5F and human primary β-cells

Elevated interleukin-1β (IL-1β) induces apoptosis in pancreatic β-cells through endoplasmic reticulum (ER) stress induction and subsequent c-jun-N-terminal kinase 1/2 (JNK1/2) activation. In earlier work we showed that JNK1/2 activation is initiated before ER stress and apoptotic induction in response to IL-1β. However, the detailed regulatory mechanisms are not completely understood. Because the ER is the organelle responsible for Ca²⁺ handling and storage, here we examine the effects of IL-1β on cellular Ca²⁺ movement and mitochondrial dysfunction and evaluate the role of JNK1/2. Our results show that in RINm5F cells and human primary β-cells, IL-1β alters mitochondrial membrane potential, mitochondrial permeability transition pore opening, ATP content, and reactive oxygen species production and these alterations are preceded by ER Ca²⁺ release via IP₃R channels and mitochondrial Ca²⁺ uptake. All these events are prevented by JNK1/2 small interfering RNA (siRNA), indicating the mediating role of JNK1/2 in IL-1β–induced cellular alteration. This is accompanied by IL-1β–induced apoptosis, which is prevented by JNK1/2 siRNA and the IP₃R inhibitor xestospongin C. This suggests a regulatory role of JNK1/2 in modulating the ER-mitochondrial-Ca²⁺ axis by IL-1β in apoptotic cell death.     

Targeting γ-Herpesvirus 68 Bcl-2-mediated Down-regulation of Autophagy

γ-herpesviruses (γHVs) are common human pathogens that encode homologs of the anti-apoptotic cellular Bcl-2 proteins, which are critical to viral reactivation and oncogenic transformation. The murine γHV68 provides a tractable in vivo model for understanding general features of these important human pathogens. Bcl-X_L, a cellular Bcl-2 homolog, and the murine γHV68 Bcl-2 homolog, M11, both bind to a BH3 domain within the key autophagy effector Beclin 1 with comparable affinities, resulting in the down-regulation of Beclin 1-mediated autophagy. Despite this similarity, differences in residues lining the binding site of M11 and Bcl-X_L dictate varying affinities for the different BH3 domain-containing proteins. Here we delineate Beclin 1 differential specificity determinants for binding to M11 or Bcl-X_L by quantifying autophagy levels in cells expressing different Beclin 1 mutants and either M11 or Bcl-X_L, and we show that a G120E/D121A Beclin 1 mutant selectively prevents down-regulation of Beclin 1-mediated autophagy by Bcl-X_L, but not by M11. We use isothermal titration calorimetry to identify a Beclin 1 BH3 domain-derived peptide that selectively binds to M11, but not to Bcl-X_L. The x-ray crystal structure of this peptide bound to M11 reveals the mechanism by which the M11 BH3 domain-binding groove accommodates this M11-specific peptide. This information was used to develop a cell-permeable peptide inhibitor that selectively inhibits M11-mediated, but not Bcl-X_L-mediated, down-regulation of autophagy.     

Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons

Progesterone (P4) is a steroid hormone that plays multiple roles in the central nervous system (CNS) including promoting neuroprotection. However, the precise mechanisms involved in its neuroprotective effects are still unknown. Given that the regulation of the intracellular calcium (Ca²⁺) concentration is critical for cell survival, we determined if inositol 1, 4, 5-trisphosphate receptors (IP₃Rs) are relevant targets of P4. Using primary hippocampal neurons, we tested the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca²⁺ signaling in neurons and characterized the subcellular distribution and phosphorylation of potential signaling intermediates involved in P4s actions. Our results reveal that P4 treatment altered the intensity and distribution of IP3R immunoreactivity and induced the nuclear translocation of phosphorylated Akt. Further, P4 potentiated IP₃R-mediated intracellular Ca²⁺ responses. These results suggest a potential involvement of P4 in particular and of steroid hormone signaling pathways in general in the control of intracellular Ca²⁺ signaling and its related functions.     

A non-canonical role for pyruvate kinase M2 as a functional modulator of Ca2+ signalling through IP3 receptors

Pyruvate kinase isoform M2 (PKM2) is a rate-limiting glycolytic enzyme that is widely expressed in embryonic tissues. The expression of PKM2 declines in some tissues following embryogenesis, while other pyruvate kinase isozymes are upregulated. However, PKM2 is highly expressed in cancer cells and is believed to play a role in supporting anabolic processes during tumour formation. In this study, PKM2 was identified as an inositol 1,4,5-trisphosphate receptor (IP₃R)-interacting protein by mass spectrometry. The PKM2:IP₃R interaction was further characterized by pull-down and co-immunoprecipitation assays, which showed that PKM2 interacted with all three IP₃R isoforms. Moreover, fluorescence microscopy indicated that both IP₃R and PKM2 localized at the endoplasmic reticulum. PKM2 binds to IP₃R at a highly conserved 21-amino acid site (corresponding to amino acids 2078–2098 in mouse type 1 IP₃R isoform). Synthetic peptides (denoted ‘TAT-D5SD’ and ‘D5SD’), based on the amino acid sequence at this site, disrupted the PKM2:IP₃R interaction and potentiated IP₃R-mediated Ca²⁺ release both in intact cells (TAT-D5SD peptide) and in a unidirectional ⁴⁵Ca²⁺ flux assay on permeabilized cells (D5SD peptide). The TAT-D5SD peptide did not affect the enzymatic activity of PKM2. Reducing PKM2 protein expression using siRNA increased IP₃R-mediated Ca²⁺ signalling in intact cells without altering the ER Ca²⁺ content. These data identify PKM2 as an IP₃R-interacting protein that inhibits intracellular Ca²⁺ signalling. The elevated expression of PKM2 in cancer cells is therefore not solely connected to its canonical role in glycolytic metabolism, rather PKM2 also has a novel non-canonical role in regulating intracellular signalling.     

Synergistic killing of human small cell lung cancer cells by the Bcl-2-inositol 1,4,5-trisphosphate receptor disruptor BIRD-2 and the BH3-mimetic ABT-263

Small cell lung cancer (SCLC) has an annual mortality approaching that of breast and prostate cancer. Although sensitive to initial chemotherapy, SCLC rapidly develops resistance, leading to less effective second-line therapies. SCLC cells often overexpress Bcl-2, which protects cells from apoptosis both by sequestering pro-apoptotic family members and by modulating inositol 1,4,5-trisphosphate receptor (IP₃R)-mediated calcium signaling. BH3-mimetic agents such as ABT-263 disrupt the former activity but have limited activity in SCLC patients. Here we report for the first time that Bcl-2-IP₃ receptor disruptor-2 (BIRD-2), a decoy peptide that binds to the BH4 domain of Bcl-2 and prevents Bcl-2 interaction with IP₃Rs, induces cell death in a wide range of SCLC lines, including ABT-263-resistant lines. BIRD-2-induced death of SCLC cells appears to be a form of caspase-independent apoptosis mediated by calpain activation. By targeting different regions of the Bcl-2 protein and different mechanisms of action, BIRD-2 and ABT-263 induce cell death synergistically. Based on these findings, we propose that targeting the Bcl-2–IP₃R interaction be pursued as a novel therapeutic strategy for SCLC, either by developing BIRD-2 itself as a therapeutic agent or by developing small-molecule inhibitors that mimic BIRD-2.Lung cancer accounts for 12% of all new cancers worldwide and is a leading cause of cancer-related mortality in the United States.^{1, 2, 3} Although small cell lung cancer (SCLC) comprises only 15% of lung cancer cases,^{2, 3} it has an annual mortality rate approaching that of breast and prostate cancer.⁴ Compared with the more common non-small cell lung cancer (NSCLC), SCLC is more aggressive and associated with rapid development of metastasis.² Moreover, although SCLC is more responsive to chemotherapy and radiation therapy initially, it typically relapses quickly with treatment-resistant disease.² In contrast to dramatic advances in chemotherapy and personalized medicine in other malignancies, the life expectancy of SCLC patients has remained <2 years for decades and is <1 year for patients with extensive disease.^{5, 6} The lethality of SCLC is attributed in part to the development of resistance to standard combination chemotherapies, underscoring the need to develop novel therapeutic approaches based on understanding the molecular and cellular biology of SCLC.^{5, 6}Evasion from apoptosis is a major hallmark of cancer and a prominent factor underlying drug resistance in SCLC.³ Multiple mechanisms contribute to apoptosis resistance in SCLC, including elevated expression of the antiapoptotic Bcl-2 protein³ (Supplementary Figure S1). Tsujimoto and colleagues discovered elevated levels of Bcl-2 mRNA and protein in SCLC cells not long after their identification of Bcl-2 as the protein product of the bcl-2 gene in follicular lymphoma.^{7, 8} Subsequently, immunohistochemistry of 164 primary SCLC samples revealed 76% were positive for Bcl-2, a finding substantiated by microarray detection of increased BCL-2 mRNA levels in 84% of SCLC samples^{9, 10} and by genomic sequencing of circulating SCLC tumor cells.¹¹ Moreover, proteomic profiling documented that Bcl-2 is more highly expressed in SCLC than in NSCLC, reflecting the vastly different biology of these lung cancer subtypes.¹²The major known function of Bcl-2 is to bind and sequester BH3-only proteins such as Bim, preventing these proteins from inducing apoptosis.^{13, 14, 15} Therefore, a major investment has been made in targeting this interaction for cancer treatment. The interaction takes place in a hydrophobic groove on Bcl-2 and the therapeutic strategy for targeting this interaction has been to develop small molecules, BH3-mimetic agents, which bind in the hydrophobic groove and induce apoptosis by displacing the BH3-only proteins. This approach has been reviewed in detail.^{14, 15, 16}Among BH3-mimetic agents advancing through clinical trials for both hematological malignancies^{15, 17} and solid tumors¹⁸ are ABT-737 and its orally bioavailable derivative ABT-263 (Navitoclax). Reported studies of ABT-199, a selective inhibitor of Bcl-2, are at present limited to hematological malignancies.¹⁸ In screening a large number of cancer cell lines, the pioneering work of Oltersdorf et al.¹⁹ demonstrated potent single-agent activity of ABT-737 against cell lines representative of lymphoid malignancies and SCLC. Clinical trials of ABT-263, an orally bioavailable version of ABT-737, achieved overall response rates ranging from as high as 35% in relapsed/refractory chronic lymphocytic leukemia (CLL) and 22% in follicular lymphoma.¹⁷ Reported responses are generally less in solid tumors with the notable exception of SCLC.¹⁸ But even in SCLC, activity of ABT-263 is limited in comparison to hematological malignancies, with 1 of the 39 (3%) of patients achieving a partial response to ABT-263 and 9 of the 37 (23%) achieving stable disease in a phase I clinical trial.²⁰ This experience suggests a need to develop additional ways of targeting Bcl-2 for cancer treatment.A potential alternative therapeutic target for Bcl-2-positive malignancies involves interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP₃R), an IP₃-gated Ca²⁺ channel located on the endoplasmic reticulum (ER). Bcl-2 is located not only on the outer mitochondrial membrane but also on the ER, and at both of these locations, it functions as a potent inhibitor of apoptosis.^{21, 22, 23} ER-localized Bcl-2 interacts with IP₃Rs and inhibits apoptosis by preventing excessive IP₃R-mediated Ca²⁺ transfer from the ER lumen into the cytoplasm and nearby mitochondria.^{24, 25, 26} Notably, regions of Bcl-2 involved in binding BH3-only proteins and IP₃Rs are entirely different. Whereas BH3-only proteins and their BH3-mimetic counterparts bind in a hydrophobic groove composed of BH3 domains 1–3 of Bcl-2,^{13, 14} the BH4 domain of Bcl-2 is necessary for interaction with IP₃Rs.²⁷ To develop a peptide inhibitor of Bcl-2–IP₃R interaction, we identified the Bcl-2-binding region on the IP₃R and developed a small synthetic 20 amino-acid peptide corresponding to this region.²⁸ This peptide, when fused to the cell-penetrating peptide of HIV TAT, binds to the BH4 domain of Bcl-2 and functions as a decoy peptide, inhibiting Bcl-2–IP₃R interaction.^{29, 30} We currently refer to this peptide as BIRD-2 (Bcl-2-IP₃ Receptor Disruptor-2), having formerly named it TAT-IDP_DD/AA.³¹ By disrupting the Bcl-2–IP₃R interaction, BIRD-2 abrogates Bcl-2 control over IP₃R-mediated Ca²⁺ elevation and induces Ca²⁺-mediated apoptosis in primary human CLL cells²⁹ and diffuse large B-cell lymphoma cells.³² Notably, BIRD-2 does not kill normal cells, including human lymphocytes isolated from peripheral blood²⁹ and normal murine embryonic fibroblasts (F Zhong and C Distelhorst, unpublished data).The present investigation was undertaken to determine whether Bcl-2–IP₃R interaction is a potentially useful therapeutic target in SCLC. In support of this concept, we find the majority of SCLC lines tested are sensitive to BIRD-2-induced apoptosis and that BIRD-2 induces apoptosis in several ABT-263-resistant SCLC lines. BIRD-2, we find, lacks generalized cytotoxicity as it does not induce cell death in NSCLC lines or a normal lung epithelial line. On the other hand, we find that BIRD-2 and ABT-263 synergize in killing SCLC cells. These findings for the first time provide preclinical evidence of the potential value of targeting both antiapoptotic mechanisms of Bcl-2 for the treatment of SCLC.