IEX-1-induced cell death requires BIM and is modulated by MCL-1

MCL-1 (myeloid cell leukemia-1) is a distinguished and pivotal member of the pro-survival BCL-2 family of proteins, and we isolated IEX-1 (immediate early response gene X-1) as a MCL-1-interacting protein using the yeast two-hybrid system and confirmed their endogenous association in human cells. The underlying mechanisms by which IEX-1 affects cell survival and death are largely unknown. Ectopic expression of IEX-1-induced caspase-dependent apoptosis in 293T cells, and the response was significantly modulated by changes in the MCL-1 expression level in cells. Forced expression of IEX-1 was unable to induce cell death or to perturb mitochondrial membrane potential in BIM-depleted cells. Additionally, knockouts of NOXA or PUMA did not affect the activities of IEX-1, indicating that the pro-death action of IEX-1 specifically requires BIM. Our findings provide insight into a new regulatory circuit that controls cell death and survival by the coordinated action of MCL-1, IEX-1, and BIM.     

IEX-1相互作用蛋白的筛选与鉴定

骨肉瘤即原发于骨的恶性肿瘤,易发生早期肺转移且预后差,恶性程度高.本研究小组前期研究发现,IEX-1在骨肉瘤中具有重要作用.为了阐明其作用机制,本研究运用酵母双杂交技术筛选其相互作用蛋白,共鉴定出12个IEX-1相互作用蛋白,包括生物氧化相关酶类、分子伴侣、信号转导相关蛋白等.并首次证实clusterin(CLU,又名载脂蛋白J)与IEX-1存在相互作用,两者在细胞中具有很好的共定位.采用RNAi干扰减低骨肉瘤细胞中内源性CLU表达水平,显著抑制了细胞增殖与细胞侵袭能力.为阐明IEX-1在骨肉瘤发生发展中的作用机制提供了重要的线索,为骨肉瘤的早期诊治及预后监测提供了新的靶点.     

IEX-1 suppresses apoptotic damage in human intestinal epithelial Caco-2 cells induced by co-culturing with macrophage-like THP-1 cells

We have reported previously that apoptosis of intestinal epithelial Caco-2 cells is induced by co-culturing with human macrophage-like THP-1 cells, mainly via the action of TNFα (tumour necrosis factor α) secreted from THP-1 cells [Satsu, Ishimoto, Nakano, Mochizuki, Iwanaga and Shimizu (2006) Exp. Cell Res. 312, 3909-3919]. Our recent DNA microarray analysis of co-cultured Caco-2 cells showed that IEX-1 (immediate early-response gene X-1) is the most significantly increased gene during co-culture [Ishimoto, Nakai, Satsu, Totsuka and Shimizu (2010) Biosci. Biotechnol. Biochem. 74, 437-439]. Hence, we investigated the role of IEX-1 in the co-culture-induced damage of Caco-2 cells. We showed that IEX-1 expression induced in Caco-2 cells was suppressed by anti-TNFα antibody treatment. Experiments using IEX-1-overexpressing and -knockdown Caco-2 cells suggested that IEX-1 was involved in the suppression of Caco-2 cell damage. Increases in caspase 3 activity and TNFR1 (TNF receptor 1) mRNA expression were shown in IEX-1-knockdown Caco-2 cells, suggesting that IEX-1 plays a role in the suppression of apoptosis and protects cells by controlling sensitivity to TNFα under both normal and inflammatory conditions.     

Immediate early gene IEX-1 induces astrocytic differentiation of U87-MG human glioma cells

The immediate early response gene IEX-1 is involved in the regulation of apoptosis and cell growth. In order to increase the apoptotic sensitivity to chemotherapeutic drugs and gamma-ray, we attempted to establish U87-MG human glioma cell line expressing IEX-1. Unexpectedly, however, transfection of IEX-1 into U87-MG glioma cells resulted in morphological changes to astrocytic phenotype and increase in glial differentiation marker proteins, S-100 and glial fibrillary acidic protein (GFAP). Glial cell differentiation was used to examine in rat C6 glioma cell line, since this cell line express astrocytic phenotypes by increase in intracellular cAMP concentration. Stimulation of human U87-MG glioma cells by membrane-permeable dibutyryl cAMP (dbcAMP) not only elicited their morphological changes but also induced expression of IEX-1 as well as S-100 and GFAP. H89, an inhibitor of protein kinase A (PKA), blocked dbcAMP-induced morphological changes of U87-MG cells and expression of IEX-1. In contrast, morphological changes and expression of S-100 and GFAP induced by IEX-1 were not affected by H89. Morphological changes induced by dbcAMP were totally abolished by functional disruption of IEX-1 expression by anti-sense RNA. These results indicate that IEX-1 plays an important role in astrocytic differentiation of human glioma cells and that IEX-1 functions at downstream of PKA.     

Immediate early gene X1 (IEX-1) is organized in subnuclear structures and partially co-localizes with promyelocytic leukemia protein in HeLa cells

Immediate early gene X1 (IEX-1) represents a stress response gene involved in growth control and modulation of apoptosis. Here, we report a detailed analysis of IEX-1 with respect to its intracellular localization. By means of confocal laser scanning microscopy, a green fluorescent protein-IEX-1 fusion protein transfected into HeLa cells, as well as endogenous IEX-1, could be detected in distinct subnuclear structures. This particular subnuclear localization of IEX-1 was not observed with a green fluorescent protein-IEX-1 fusion protein lacking a putative nuclear localization sequence, along with a decreased effect on apoptosis. Double immunofluorescence staining revealed a partial co-localization of endogenous promyelocytic leukemia protein (PML) and IEX-1 in these subnuclear structures. Nuclear localization of IEX-1 is also enhanced upon treatment of cells with leptomycin B, an inhibitor of the nuclear exporter CRM1. These observations indicate that IEX-1 is specifically shuttled to and from the nucleus. Overexpression experiments using PML isoforms III and IV revealed distinct intranuclear interaction of IEX-1 and PML. Coprecipitation experiments showed physical interaction between IEX-1 and PML. The close structural relation of IEX-1-containing nuclear subdomains and PML nuclear bodies suggests a function of IEX-1 related to the multiple functions of these unique subnuclear regions, particularly during stress response and growth control.     

Distinct domains for anti- and pro-apoptotic activities of IEX-1

IEX-1 (immediate early response gene X-1) is a stress-inducible gene. Its overexpression can suppress or enhance apoptosis dependent on the nature of stress, yet the polypeptide does not possess any of the functional domains that are homologous to those present in well characterized effectors or inhibitors of apoptosis. This study using sequence-targeting mutagenesis reveals a transmembrane-like integrated region of the protein to be critical for both pro-apoptotic and anti-apoptotic functions. Substitution of the key hydrophobic residues with hydrophilic ones within this region impairs the capacity IEX-1 to positively and negatively regulate apoptosis. Mutations at N-linked glycosylation and phosphorylation sites or truncation of the C terminus of IEX-1 also abrogated its potential to promote cell survival. However, distinguished from the transmembrane-like domain, these mutants preserved pro-apoptotic activity of IEX-1 fully. On the contrary, mutation of nuclear localization sequence, despite its importance in apoptosis, did not impede IEX-1-mediated cell survival. Strikingly, all the mutants that lose their anti-apoptotic ability are unable to prevent acute increases in production of intracellular reactive oxygen species (ROS) at the initial onset of apoptosis, whereas those mutants that can sustain anti-death function also control acute ROS production as sufficiently as wild-type IEX-1. These findings suggest a critical role of IEX-1 in regulation of intracellular ROS homeostasis, providing new insight into the mechanism underlying IEX-1-mediated cell survival.     

APRIL, a new member of the tumor necrosis factor family, modulates death ligand-induced apoptosis

APRIL (a proliferation-inducing ligand) is a newly identified member of the tumor necrosis factor (TNF) family. Tumor growth-promoting as well as apoptosis-inducing effects of APRIL have been described. Here, we report that five of 12 human malignant glioma cell lines express APRIL. APRIL gene transfer experiments revealed that malignant glioma cells are refractory to growth-promoting activity of APRIL in vitro and in vivo. Interestingly, ectopic expression of APRIL confers minor protection from apoptotic cell death induced by the death ligands, CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand (Apo2L). This antiapoptotic activity is specific for death ligand/receptor-mediated apoptosis since APRIL does not protect glioma cells from the cytotoxicity of the drugs, teniposide, vincristine, lomustine or cisplatin. Ectopic expression of APRIL is associated with the upregulation of X-linked inhibitor of apoptosis protein (XIAP), providing a possible explanation for the antiapoptotic activity observed here. In contrast, APRIL does not regulate the expression levels of the antiapoptotic proteins FLICE-inhibitory protein (FLIP), Bcl-2 or Bcl-X(L). These findings suggest that APRIL is involved in the regulation of death ligand-induced apoptotic signaling in malignant glioma cells.     

Wogonin enhances antitumor activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo through ROS-mediated downregulation of cFLIPL and IAP proteins

Combination of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) with other agents is a promising strategy to overcome TRAIL resistance in malignant cells. Wogonin, a flavonoid originated from Scutellaria baicalensis Georgi, has been shown to enhance TRAIL-induced apoptosis in malignant cells in in vitro studies. However, whether wogonin enhances TRAIL’s antitumor activity in vivo has never been studied. In this study, the effect of combination of TRAIL and wogonin was tested in a non-small-cell lung cancer xenografted tumor model in nude mice. Consistent with the in vitro study showing that wogonin sensitized A549 cells to TRAIL-induced apoptosis, wogonin greatly enhanced TRAIL-induced suppression of tumor growth, accompanied with increased apoptosis in tumor tissues as determined by TUNEL assay. The expression levels of antiapoptotic proteins including long form of cellular FLICE-like inhibitory protein (cFLIP_L), X-linked inhibitor of apoptosis protein (XIAP), and cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) were markedly reduced in both cultured cells and xenografted tumor tissues after co-treatment with wogonin and TRAIL. The down-regulation of these antiapoptotic proteins was likely mediated by proteasomal degradation that involved intracellular reactive oxygen species (ROS), because wogonin robustly induced ROS accumulation and ROS scavengers butylated hydroxyanisole (BHA) and N-acetyl-l-cysteine (NAC) and the proteasome inhibitor MG132 restored the expression of these antiapoptotic proteins in cells co-treated with wogonin and TRAIL. These results show for the first time that wogonin enhances TRAIL’s antitumor activity in vivo, suggesting this strategy has an application potential for clinical anticancer therapy.     

Roscovitine sensitizes breast cancer cells to TRAIL-induced apoptosis through a pleiotropic mechanism

The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO2L) is a member of the TNF gene superfamily that induces apoptosis upon engagement of cognate death receptors. While TRAIL is relatively non-toxic to normal cells, it selectively induces apoptosis in many transformed cells. Nevertheless, breast tumor cells are particularly resistant to the effects of TRAIL. Here we report that, in combination with the cyclin-dependent kinase inhibitor roscovitine, exposure to TRAIL induced marked apoptosis in the majority of TRAIL-resistant breast cancer cell lines examined. Roscovitine facilitated TRAIL death-inducing signaling complex formation and the activation of caspase-8. The cFLIP(L) and cFLIP(S) FLICE-inhibitory proteins were significantly down-regulated following exposure to roscovitine and, indeed, the knockdown of cFLIP isoforms by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. In addition, we demonstrate that roscovitine strongly suppressed Mcl-1 expression and up-regulated E2F1 protein levels in breast tumor cells. Significantly, the silencing of Mcl-1 by siRNA sensitized breast tumor cells to TRAIL-induced apoptosis. Furthermore, the knockdown of E2F1 protein by siRNA reduced the sensitizing effect of roscovitine in TRAIL-induced apoptosis. In summary, our results reveal a pleitropic mechanism for the pro-apoptotic influence of roscovitine, highlighting its potential as an antitumor agent in breast cancer in combination with TRAIL.     

A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferase by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.     

IEX-1 deficiency protects against colonic cancer

The immediate early response gene X-1 (IEX-1) is involved in regulation of various cellular processes including proliferation, apoptosis in part by controlling homeostasis of reactive oxygen species (ROS) at mitochondria. The present study shows reduced inflammatory responses and colorectal cancer in IEX-1 knockout (KO) mice treated with azoxymethane/dextran sulfate sodium (DSS). However, DSS induced worse colitis in RAG(-/-)IEX-1(-/-) double KO mice than in RAG and IEX-1 single KO mice, underscoring an importance of T cells in IEX-1 deficiency-induced protection against colon inflammation. Lack of IEX-1 promoted the differentiation of interleukin (IL)-17-producing T cells, concomitant with upregulation of Gαi2 expression, a gene that is well-documented for its role in the control of inflammation in the colon. In accordance with this, T-helper 17 (T(H)17) cell differentiation was compromised in the absence of Gαi2, and deletion of Gαi2 in T cells alone aggravated colon inflammation and colorectal cancer development after azoxymethane/DSS treatment. Null mutation of IEX-1 also enhanced both proliferation and apoptosis of intestinal epithelial cells (IEC) after injury. A potential impact of this altered IEC turnover on colon inflammation and cancer development is discussed. These observations provide a linkage of IEX-1 and Gαi2 expression in the regulation of T(H)17 cell differentiation and suggest a previously unappreciated role for IEX-1 in the control of colon epithelial homeostasis.     

Three adenovirus E3 proteins cooperate to evade apoptosis by tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2

Adenovirus encodes multiple gene products that regulate proapoptotic cellular responses to viral infection mediated by both the innate and adaptive immune systems. The E3-10.4K and 14.5K gene products are known to modulate the death receptor Fas. In this study, we demonstrate that an additional viral E3 protein, 6.7K, functions in the specific modulation of the two death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 6.7K protein is expressed on the cell surface and forms a complex with the 10.4K and 14.5K proteins, and this complex is sufficient to induce down-modulation of TRAIL receptor-1 and -2 from the cell surface and reverse the sensitivity of infected cells to TRAIL-mediated apoptosis. Down-modulation of TRAIL-R2 by the E3 complex is dependent on the cytoplasmic tail of the receptor, but the death domain alone is not sufficient. These results identify a mechanism for viral modulation of TRAIL receptor-mediated apoptosis and suggest the E3 protein complex has evolved to regulate the signaling of selected cytokine receptors.