BI-1 enhances Fas-induced cell death through a Na+/H+-associated mechanism

The role of Bax inhibitor-1 (BI-1) in the protective mechanism against apoptotic stimuli has been studied; however, as little is known about its role in death receptor-mediated cell death, this study was designed to investigate the effect of BI-1 on Fas-induced cell death, and the underlying mechanisms. HT1080 adenocarcinoma cells were cultured in high concentration of glucose media and transfected with vector alone (Neo cells) or BI-1-vector (BI-1 cells), and treated with Fas. In cell viability, apoptosis, and caspase-3 analyses, the BI-1 cells showed enhanced sensitivity to Fas. Fas significantly decreased cytosolic pH in BI-1 cells, compared with Neo cells, and this decrease correlated with BI-1 oligomerization, mitochondrial Ca²⁺ accumulation, and significant inhibition of sodium-hydrogen exchanger (NHE) activity. Compared with Neo cells, a single treatment of BI-1 cells with the NHE inhibitor EIPA or siRNA against NHE significantly increased cell death, which suggests that the viability of BI-1 cells is affected by the maintenance of intracellular pH homeostasis through NHE. [BMB Reports 2014; 47(7): 393-398]     

Enhanced lysosomal activity is involved in Bax inhibitor-1-induced regulation of the endoplasmic reticulum (ER) stress response and cell death against ER stress: involvement of vacuolar H+-ATPase (V-ATPase)

Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H(+)-ATPase(V-ATPase), a proton pump, was activated, suggesting high H(+) uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.     

Bax Inhibitor-1 Is a pH-dependent regulator of Ca2+ channel activity in the endoplasmic reticulum

In this study, Bax inhibitor-1 (BI-1) overexpression reduces the ER pool of Ca(2+) released by thapsigargin. Cells overexpressing BI-1 also showed lower intracellular Ca(2+) release induced by the Ca(2+) ionophore ionomycin as well as agonists of ryanodine receptors and inositol trisphosphate receptors. In contrast, cells expressing carboxyl-terminal deleted BI-1 (CDelta-BI-1 cells) displayed normal intracellular Ca(2+) mobilization. Basal Ca(2+) release rates from the ER were higher in BI-1-overexpressing cells than in control or CDelta-BI-1 cells. We determined that the carboxyl-terminal cytosolic region of BI-1 contains a lysine-rich motif (EKDKKKEKK) resembling the pH-sensing domains of ion channels. Acidic conditions triggered more extensive Ca(2+) release from ER microsomes from BI-1-overexpressing cells and BI-1-reconstituted liposomes. Acidic conditions also induced BI-1 protein oligomerization. Interestingly subjecting BI-1-overexpressing cells to acidic conditions induced more Bax recruitment to mitochondria, more cytochrome c release from mitochondria, and more cell death. These findings suggest that BI-1 increases Ca(2+) leak rates from the ER through a mechanism that is dependent on pH and on the carboxyl-terminal cytosolic region of the BI-1 protein. The findings also reveal a cell death-promoting phenotype for BI-1 that is manifested under low pH conditions.     

An acidic pH environment increases cell death and pro-inflammatory cytokine release in osteoblasts: the involvement of BAX inhibitor-1

BAX Inhibitor-1 (BI-1), a transmembrane protein on the endoplasmic reticulum, has been studied previously in various physio/pathological conditions, but not in bone cells. In this study, using the MG63 osteoblast cell line and osteoblasts differentiated from stem cells, the role of BI-1 was studied. First, expression of BI-1 was confirmed in osteoblasts, as well as osteoclasts, in mouse tibiae bone immunohistochemistry. For evaluation of a recently published property of BI-1, an acidic pH-dependent Ca2? channel-like effect in osteoblasts, acidic pH-associated cell death, and pro-inflammatory cytokine release were examined. In MG63 osteoblasts, acidic pH induced a pH-dependent increase in cell death and ER stress, as determined by elevated expression of GRP78, CHOP, phospho-eIF2α, IRE-1α, spliced XBP-1, and phospho-JNK. In osteoblasts, mitochondrial Ca2? also showed a strong pH-dependent increase. BI-1 knock-down using siRNA protected cells against acidic pH, regulating mitochondrial Ca2? accumulation, possibly via the acidic pH-dependent Ca2? channel-like effect of BI-1. BI-1 knock-down also resulted in inhibition of acidic pH-induced release of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. In addition, bone marrow stem cells were differentiated into human osteoblasts, which showed increased expression of BI-1 mRNA and protein. In differentiated primary human osteoblasts, acidic pH-associated cell death, mitochondrial Ca2? accumulation, and pro-inflammatory cytokine release were more significant than in non-differentiated stem cells. In summary, endogenous expression of BI-1 is associated with acidic pH-induced Ca2? release, cell death, and pro-inflammatory cytokine release in human osteoblasts.     

Bax inhibitor-1 enhances survival and neuronal differentiation of embryonic stem cells via differential regulation of mitogen-activated protein kinases activities

Bax inhibitor-1 (BI-1), a member of the BI-1 family of integral membrane proteins, was originally identified as an inhibitor of stress-induced cell death in mammalian cells. Previous studies have shown that the withdrawal of leukemia inhibitory factor (LIF) results in differentiation of the majority of mouse embryonic stem (mES) cells into various cell lineages, while some ES cells die within 3days. Thus, to investigate the function of BI-1 in ES cell survival and neuronal differentiation, we generated mES cell lines that overexpress BI-1 or a carboxy-terminal BI-1ΔC mutant. Overexpression of BI-1 in mES cells significantly increased cell viability and resistance to apoptosis induced by LIF withdrawal, while the control vector or BI-1ΔC-overexpressing mES cells had no effect. Moreover, overexpression of BI-1 produced significant inhibition of the p38 mitogen-activated protein kinases (MAPK) pathway in response to LIF withdrawal, while activity of the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) MAPK pathway was increased. Interestingly, we found that BI-1-overexpressing cells showed higher expression levels of neuroectodermal markers (Otx1, Lmx1b, En1, Pax2, Wnt1, Sox1, and Nestin) and greater neuronal differentiation efficiency than control or BI-1ΔC-overexpressing mES cells did. Considering these findings, our results indicated that BI-1-modulated MAPK activity plays a key role in protecting mES cells from LIF-withdrawal-induced apoptosis and in promoting their differentiation toward neuronal lineages.     

The barley apoptosis suppressor homologue BAX inhibitor-1 compromises nonhost penetration resistance of barley to the inappropriate pathogen Blumeria graminis f. sp. tritici

BAX inhibitor-1 (BI-1) proteins have been characterized as suppressors of programmed cell death in mammals and plants. The barley BI-1 is a suppressor of nonspecific background resistance and mlo-mediated penetration resistance to the biotrophic fungal pathogen Blumeria graminis f. sp. hordei when overexpressed in epidermal cells of barley. We report here that BI-1 expression is also slightly up-regulated during interaction with the inappropriate wheat pathogen Blumeria graminis f. sp. tritici. Significantly, overexpression of BI-1 in single epidermal cells of barley by microprojectile-mediated transformation rendered cells susceptible to penetration by inappropriate B. graminis f. sp. tritici. The degree of transgene-induced accessibility to B. graminis f. sp. tritici was thereby similar to the effect achieved by overexpression of the defense suppressor gene Mlo and could not be further enhanced by double expression of both BI-1 and Mlo. Confocal laser scanning microscopy was used to locate a functional green fluorescing GFP:BI-1 fusion protein in endomembranes and the nuclear envelope of barley epidermal cells. Together, enhanced expression of barley BI-1 suppresses penetration resistance to B. graminis f. sp. tritici, linking barley nonhost resistance with cell death regulation.     

Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell death suppressor

In spite of fundamental differences between plant and animal cells, it is remarkable that some cell death regulators that were identified to control cell death in metazoans can also function in plants. The fact that most of these proteins do not have structural homologs in plant genomes suggests that they may be targeting a highly conserved 'core' mechanism with conserved functions that is present in all eukaryotes. The ubiquitous Bax inhibitor-1 (BI-1) is a common cell death suppressor in eukaryotes that has provided a potential portal to this cell death core. In this review, we will update the current status of our understanding on the function and activities of this intriguing protein. Genetic, molecular and biochemical studies have so far suggested a consistent view that BI-1 is an endoplasmic reticulum (ER)-resident transmembrane protein that can interact with multiple partners to alter intracellular Ca(2+) flux control and lipid dynamics. Functionally, the level of BI-1 protein has been hypothesized to have the role of a rheostat to regulate the threshold of ER-stress inducible cell death. Further, delineation of the cell death suppression mechanism by BI-1 should shed light on an ancient cell death core-control pathway in eukaryotes, as well as novel ways to improve stress tolerance.     

Overexpression of Bax inhibitor suppresses the fungal elicitor-induced cell death in rice (Oryza sativa L) cells

Treatment of suspension-cultured cells of rice (Oryza sativa L.) with cell wall extract of rice blast fungus (Magnaporthe grisea) elicits a rapid generation of H2O2, alkalinization of culture medium, and eventual cell death. To elucidate genes involved in these processes, we exploited SAGE (Serial Analysis of Gene Expression) technique for the molecular analysis of cell death in suspension-cultured cells treated with the elicitor. Among the downregulated genes in the elicitor-treated cells, a BI-1 gene coding for Bax inhibitor was identified. Transgenic rice cells overexpressing Arabidopsis BI-1 gene showed sustainable cell survival when challenged with M. grisea elicitor. Thus, the plant Bax inhibitor plays a functional role in regulating cell death in the rice cell culture system.     

BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress

Bax inhibitor-1 (BI-1) is an evolutionarily conserved endoplasmic reticulum (ER) protein that suppresses cell death in both animal and plant cells. We characterized mice in which the bi-1 gene was ablated. Cells from BI-1-deficient mice, including fibroblasts, hepatocytes, and neurons, display selective hypersensitivity to apoptosis induced by ER stress agents (thapsigargin, tunicamycin, brefeldin A), but not to stimulators of mitochondrial or TNF/Fas-death receptor apoptosis pathways. Conversely, BI-1 overexpression protects against apoptosis induced by ER stress. BI-1-mediated protection from apoptosis induced by ER stress correlated with inhibition of Bax activation and translocation to mitochondria, preservation of mitochondrial membrane potential, and suppression of caspase activation. BI-1 overexpression also reduces releasable Ca(2+) from the ER. In vivo, bi-1(-/-) mice exhibit increased sensitivity to tissue damage induced by stimuli that trigger ER stress, including stroke and tunicamycin injection. Thus, BI-1 regulates a cell death pathway important for cytopreservation during ER stress.     

Melatonin restricts Pb-induced PCD by enhancing BI-1 expression in tobacco suspension cells

Melatonin is a conserved substance, which was discovered in the evolutionary distant organisms like bacteria, plants, invertebrates and vertebrates. Recent studies have shown that melatonin despite its possible role in photoperiod processes, has been found to be a direct free radical scavenger and an indirect antioxidant. In this report the impact of exogenous melatonin on the Bax inhibitor-1 (BI-1) expression level in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells exposed to lead was examined. BI-1 is a well-conserved protein in plants and animals that serves as the inhibitor of mammalian proapoptotic proteins as well as plant ROS-induced cell death. Our results showed that pretreatment with 200 nm melatonin, expressing BI-1 and fortified tobacco suspension cells against damages induced by lead. The obtained results revealed, that melatonin significantly increases BY-2 cells proliferation and protects BY-2 cells against death. Moreover, the conducted analyses showed for the first time that the protective effect of melatonin may be connected not only with its antioxidant properties but also with its direct impact on elevating BI-1 expression and lead-induced programmed cell death (PCD) restriction.     

Effects of phenylalanine on the survival and neurite outgrowth of rat cortical neurons in primary cultures: possible involvement of brain-derived neurotrophic factor

Bax inhibitor-1 (BI-1), a newly identified apoptosis inhibitor, has recently been found to be overexpressed in several human carcinomas and its specific down-regulation by RNA interference (RNAi) could lead to cell death. The purpose of this study is to investigate the role of BI-1 in apoptosis-resistance and the underlying mechanisms in human nasopharyngeal carcinoma (NPC) cells. Our results showed that BI-1 was expressed in two different human NPC cell lines, CNE-2Z and CNE-1, and specific inhibition of BI-1 expression by siRNA caused a significant increase in spontaneous apoptosis in both cell lines. Mechanistically, we demonstrated that down-regulation of BI-1 protein expression decreased the ratio of Bcl-X_L/Bcl-2 with Bax protein as determined by Western blot and increased the activity of caspase-3 by colorimetric analysis, thus leading to the activation of the associated cell death pathways. Taken together, these results have provided evidence that BI-1 could serve as an important molecular target gene for the development of new therapeutic strategy against human NPCs.     

Loss of Calmodulin Binding to Bax Inhibitor-1 Affects Pseudomonas-mediated Hypersensitive Response-associated Cell Death in Arabidopsis thaliana

Bax inhibitor-1 (BI-1) is a cell death suppressor protein conserved across a variety of organisms. The Arabidopsis atbi1-1 plant is a mutant in which the C-terminal 6 amino acids of the expressed BI-1 protein have been replaced by T-DNA insertion. This mutant BI-1 protein (AtBI-CM) produced in Escherichia coli can no longer bind to calmodulin. A promoter-reporter assay demonstrated compartmentalized expression of BI-1 during hypersensitive response, introduced by the inoculation of Pseudomonas syringae possessing the avrRTP2 gene, Pst(avrRPT2). In addition, both BI-1 knockdown plants and atbi1-1 showed increased sensitivity to Pst(avrRPT2)-induced cell death. The results indicated that the loss of calmodulin binding reduces the cell death suppressor activity of BI-1 in planta.Bax inhibitor-1 (At5g47120, BI-1)² is a highly conserved cell death suppressor protein that resides in the endoplasmic reticulum (ER) membranes of a range of organisms. BI-1 is important in the response of organisms to abiotic and biotic stresses. Down-regulation of BI-1 in tobacco suspension cells (BY2) induced sensitivity against starvation (1), whereas overexpression in barley induced the breakdown of mlo-mediated penetration resistance to the fungal pathogen, powdery mildew (Blumeria graminis) (2). Cultured rice cells overexpressing Arabidopsis BI-1 (AtBI-1) showed increased resistance to Magnaporthe grisea-induced hypersensitive response (HR)-like cell death, potentially confirming the role of BI-1 in HR regulation (3). Recent studies on animal and plant BI-1 indicated a close relationship with ER stress response (4–6). BI-1-deficient mice are hypersensitive to apoptosis induced by ER stress agents such as thapsigargin, tunicamycin, and brefeldin A (4). Such events correlate with decreased calcium release from the ER, and our previous study demonstrated an association of BI-1 with calcium signaling in stress-treated plant cells (7). However, the molecular mechanism by which BI-1 suppresses cell death is still unclear.Recently, Watanabe et al. (5, 8) demonstrated that an Arabidopsis T-DNA-tagged mutant, atbi1-1, was more susceptible to fungal toxin-, heat-shock-, and tunicamycin-induced cell death. The atbi1-1 plant has T-DNA inserted into the AtBI-1 protein C-terminal region, which contains potential coiled-coil structures and is essential for inhibiting both Bax-induced lethality in yeast and oxidative stress-induced cell death in plant cells as we had demonstrated earlier (9). We also found that the C-terminal 14 amino acids of AtBI-1 were capable of binding to the calmodulin molecule, a mediator of calcium signaling (7). Here, the present study directly proved the functional interaction between the highly conserved calmodulin molecule and BI-1 using a genomic mutation of the AtBI-1 gene. Such a genomic mutant showed accelerated sensitivity against Pseudomonas-induced HR cell death. The results indicated that the C-terminal-less BI-1 protein, which lost the CaM binding, was associated with reduced cell death suppression activity in vivo.     

The Bax lnhibitor-1 needs a functional electron transport chain for cell death suppression

Bax inhibitor-1 (BI-1) is an evolutionarily conserved cell death suppresser in animals, yeast, and plants. In this study, yeast strains carrying single-gene deletions were screened for factors related to cell death suppression by Arabidopsis BI-1 (AtBI-1). Our screen identified mutants that failed to survive Bax-induced lethality even with AtBI-1 coexpression (Bax suppressor). The Deltacox16 strain was isolated as a BI-1-inactive mutant; it was disrupted in a component of the mitochondrial cytochrome c oxidase. Other mutants defective in mitochondrial electron transport showed a similar phenotype. ATP levels were markedly decreased in all these mutants, suggesting that BI-1 requires normal electron transport activity to suppress cell death in yeast.     

Plant Bax Inhibitor-1 interacts with ATG6 to regulate autophagy and programmed cell death

Autophagy is an evolutionarily conserved catabolic process and is involved in the regulation of programmed cell death during the plant immune response. However, mechanisms regulating autophagy and cell death are incompletely understood. Here, we demonstrate that plant Bax inhibitor-1 (BI-1), a highly conserved cell death regulator, interacts with ATG6, a core autophagy-related protein. Silencing of BI-1 reduced the autophagic activity induced by both N gene-mediated resistance to Tobacco mosaic virus (TMV) and methyl viologen (MV), and enhanced N gene-mediated cell death. In contrast, overexpression of plant BI-1 increased autophagic activity and surprisingly caused autophagy-dependent cell death. These results suggest that plant BI-1 has both prosurvival and prodeath effects in different physiological contexts and both depend on autophagic activity.     

A novel Arabidopsis gene causes Bax-like lethality in Saccharomyces cerevisiae

Overexpression of the mammalian proapoptotic protein Bax induces cell death in plant and yeast cells. The Bax inihibitor-1 (BI-1) gene rescues yeast and plant from Bax-mediated lethality. Using the Arabidopsis BI-1 (AtBI-1) gene controlled by the GAL1 promoter as a cell death suppressor in yeast, Cdf1 (cell growth defect factor-1) was isolated from Arabidopsis cDNA library. Overexpression of Cdf1 caused cell death in yeast, whereas such an effect was suppressed by co-expression of AtBI-1. The Cdf1 protein fused with a green fluorescent protein was localized in the mitochondria and resulted in the loss of mitochondrial membrane potential in yeast. The Bax-resistant mutant BRM1 demonstrated tolerance against Cdf1-mediated lethality, whereas the Deltaatp4 strain was sensitive to Cdf1. Our results suggest that Cdf1 and Bax cause mitochondria-mediated yeast lethality through partially overlapped pathways.     

Role of BI-1 (TEGT)-mediated ERK1/2 activation in mitochondria-mediated apoptosis and splenomegaly in BI-1 transgenic mice

Bax Inhibitor-1 (BI-1) is an evolutionally conserved apoptotic suppressor and belongs to the BI-1 family of proteins, which contain BI-1-like transmembrane domains. As their cellular functions and regulatory mechanisms remain incompletely understood, we compared their anti-apoptotic properties. Forced expression of BI-1 resulted in the most effective suppression of stress-induced apoptosis, compared with other family members, together with significant extracellular signal-regulated kinase (ERK)1/2 activation. BI-1-mediated ERK1/2 activation led to the suppression of mitochondria-mediated reactive oxygen species (ROS) production. Involvement of the ERK signaling pathway in BI-1-induced anti-apoptotic effects was confirmed by knockdown studies with ERK- or BI-1-specific siRNA. Moreover, we produced transgenic (TG) mice overexpressing BI-1, and the relationship between ERK1/2 activation and the suppression of ROS production or apoptosis was confirmed in mouse embryonic fibroblast (MEF) cells derived from these mice. Interestingly, we found that BI-1 TG mice showed splenomegaly and abnormal megakaryopoiesis. Taken together, our results suggest that BI-1-induced ERK1/2 activation plays an important role in the modulation of intracellular ROS generation and apoptotic cell death and may also affect autoimmune response.     

The possible role of BAX and BI-1 genes in chilling-induced cell death in cucumber fruit

The aim of the present study was to investigate the possible role of BAX and BI-1 genes in chilling injury of cucumber fruit. BAX and BI-1 gene expressions were assayed under 2 ± 1 °C. Meanwhile, cell death, cellular integrity, specific chromatin fragmentation and nucleus morphology in cucumber (Cucumis sativus L. cv. Zhexiu-1) fruits were determined. Results indicated that BAX and BI-1 genes were activated by low temperature and the expression level of the BAX was much higher than BI-1. At the same time, electrolyte leakage and cell death were increased coupled with nuclear envelope disassembly and DNA fragmentation during the occurrence of chilling injury. In addition, characteristic features of programmed cell death were induced as well as the initiation of chilling injury. The interaction of BAX and BI-1 might predetermine the cell life or death in response to cold stimulus.     

Molecular characterization of two plant BI-1 homologues which suppress Bax-induced apoptosis in human 293 cells

To date, few homologues of animal programmed cell death (PCD) regulators have been identified in plants. Among these is the plant Bax Inhibitor-1 (BI-1) protein, which possesses, like its human counterpart, the ability to suppress Bax-induced lethality in yeast cells. As the role of BI-1 in the regulation of plant PCD remains to be elucidated, we cloned BnBI-1 and NtBI-1 from cDNA libraries of oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.). The analysis of the deduced amino acid sequences of BnBI-1 and NtBI-1 indicated that these proteins share a relatively high level of identity with other plant BI-1 proteins (73-95%) as well as with animal BI-1 proteins (26-42%). Comparative analysis with other available plant BI-1 proteins allowed the establishment of a structural model presenting seven transmembrane domains. Moreover, transient co-transfection of Bax with BnBI-1 or NtBI-1 in human embryonic kidney 293 cells revealed that both proteins can substantially inhibit apoptosis induced by Bax overexpression. Localization studies were also conducted using stable transformation of tobacco BY-2 cells and Saccharomyces cerevisiae, or transient expression in tobacco leaves, with the fusion protein BnBI-1GFP under control of the cauliflower mosaic virus 35S promoter. All transformants showed a fluorescence pattern of distribution typical of an endoplasmic reticulum (ER) protein. Results from differential permeabilization experiments in BY-2 cells expressing BnBI-1GFP also showed that the C-terminus is located on the cytosolic side of the ER. Taken altogether, our results suggest that BI-1 is evolutionarily conserved and could act as a key regulator of a death pathway common to plants and animals.