BCL-2 Interacting Cell Death Suppressor Research Articles

Skeletal Muscle-Specific Bis Depletion Leads to Muscle Dysfunction and Early Death Accompanied by Impairment in Protein Quality Control.

Bcl-2-interacting cell death suppressor (BIS), also called BAG3, plays a role in physiological functions such as anti-apoptosis, cell proliferation, autophagy, and senescence. Whole-body Bis-knockout (KO) mice exhibit early lethality accompanied by abnormalities in cardiac and skeletal muscles, suggesting the critical role of BIS in these muscles. In this study, we generated skeletal muscle-specific Bis-knockout (Bis-SMKO) mice for the first time. Bis-SMKO mice exhibit growth retardation, kyphosis, a lack of peripheral fat, and respiratory failure, ultimately leading to early death. Regenerating fibers and increased intensity in cleaved PARP1 immunostaining were observed in the diaphragm of Bis-SMKO mice, indicating considerable muscle degeneration. Through electron microscopy analysis, we observed myofibrillar disruption, degenerated mitochondria, and autophagic vacuoles in the Bis-SMKO diaphragm. Specifically, autophagy was impaired, and heat shock proteins (HSPs), such as HSPB5 and HSP70, and z-disk proteins, including filamin C and desmin, accumulated in Bis-SMKO skeletal muscles. We also found metabolic impairments, including decreased ATP levels and lactate dehydrogenase (LDH) and creatine kinase (CK) activities in the diaphragm of Bis-SMKO mice. Our findings highlight that BIS is critical for protein homeostasis and energy metabolism in skeletal muscles, suggesting that Bis-SMKO mice could be used as a therapeutic strategy for myopathies and to elucidate the molecular function of BIS in skeletal muscle physiology.

Hepatocyte-specific deletion of Bis causes senescence in the liver without deteriorating hepatic function

Bcl-2-interacting cell death suppressor (BIS), also called as BAG3, regulates numerous physiological processes, such as apoptosis, protein quality control, and senescence. Whole-body Bis-knockout (KO) mice exhibit early lethality following cardiac and skeletal muscle dysfunction. The first attempt to generate organ-specific knockout mice resulted in constitutive or inducible heart-specific Bis-knockout mice, which exhibited cardiac dilation and underwent premature death. Here, we generated hepatocyte-specific Bis-knockout (Bis-HKO) mice and found no abnormalities in metabolic function and survival. However, depletion of HSPB8 and accumulation of p62 indicated impaired autophagy in Bis-HKO livers. Interestingly, the number of peroxisomes wrapped by phagophore membranes increased as evidenced by transmission electron microscopy analysis, indicating defects in the progression of pexophagy. In addition, increased dihydroethidine intensities and histone H3 K9me3-positive nuclei indicated increased oxidative stress and senescence induction in Bis-HKO livers. Mechanistically, p27 was upregulated in Bis-HKO livers. In SNU368 hepatocellular carcinoma cells, BIS depletion led to p27 upregulation, and increase in histone H3 K9me3 levels and senescence-associated β-galactosidase staining; therefore, reproducing the in vivo senescence phenotype. Despite the observation of no metabolic abnormalities, BIS depletion led to defective autophagy, increased oxidative stress, and senescence in Bis-HKO livers. Collectively, our results suggest a role for BIS in maintaining liver regeneration potential under pathological conditions.

An Adult Mouse Model of Dilated Cardiomyopathy Caused by Inducible Cardiac-Specific Bis Deletion.

BCL-2 interacting cell death suppressor (BIS) is a multifunctional protein that has been implicated in cancer and myopathy. Various mutations of the BIS gene have been identified as causative of cardiac dysfunction in some dilated cardiomyopathy (DCM) patients. This was recently verified in cardiac-specific knock-out (KO) mice. In this study, we developed tamoxifen-inducible cardiomyocyte-specific BIS-KO (Bis-iCKO) mice to assess the role of BIS in the adult heart using the Cre-loxP strategy. The disruption of the Bis gene led to impaired ventricular function and subsequent heart failure due to DCM, characterized by reduced left ventricular contractility and dilatation that were observed using serial echocardiography and histology. The development of DCM was confirmed by alterations in Z-disk integrity and increased expression of several mRNAs associated with heart failure and remodeling. Furthermore, aggregation of desmin was correlated with loss of small heat shock protein in the Bis-iCKO mice, indicating that BIS plays an essential role in the quality control of cardiac proteins, as has been suggested in constitutive cardiac-specific KO mice. Our cardiac-specific BIS-KO mice may be a useful model for developing therapeutic interventions for DCM, especially late-onset DCM, based on the distinct phenotypes and rapid progressions.

SRSF3 Is a Critical Requirement for Inclusion of Exon 3 of BIS Pre-mRNA.

BCL-2 interacting cell death suppressor (BIS), also known as BAG3, is a multifunctional protein. Aberrant expression and mutation of BIS have been implicated in cancers and myopathy. However, there have only been a few studies on the splicing of BIS pre-mRNA. In the present study, through RT-PCR and sequencing in various cell lines and mouse tissues, we identified for the first time the presence of BIS mRNA isomers in which exon 3 or exons 2–3 are skipped. We also demonstrated that the depletion of SRSF3 promoted the skipping of exon 3 of BIS pre-mRNA in endogenous BIS and the GFP-BIS minigene. SRSF3 specifically interacts with the putative binding sites in exon 3, in which deletion promoted the skipping of exon 3 in the GFP-BIS minigene, which was comparable to the effect of SRSF knockdown. Even though acceleration of exon 3 skipping was not observed in response to various stimuli, SRSF3 depletion, accompanied by the production of a truncated BIS protein, inhibited the nuclear translocation of HSF1, which was restored by the wild-type BIS, not by exon 3-depleted BIS. Therefore, our results suggested that the maintenance of SRSF3 levels and subsequent preservation of the intact BIS protein is an important factor in modulating HSF1 localization upon cellular stress.

Downregulation of BIS sensitizes A549 cells for digoxin-mediated inhibition of invasion and migration by the STAT3-dependent pathway

Digoxin, a compound of the cardiac glycoside family, was originally prescribed for heart failure but has recently been rediscovered for its potent antitumor activity. However, it has a narrow therapeutic margin due to its cardiotoxicity, limiting its safe use as an antitumor agent in clinical practice. To widen its therapeutic margin, we investigated whether the antitumor effect of digoxin is potentiated by the depletion of BCL-2-interacting cell death suppressor (BIS) in A549 lung cancer cells. BIS is a multifunctional protein that is frequently overexpressed in most human cancers including lung cancer. Our results demonstrated that the inhibitory potential of digoxin on the migratory behavior of A549 cells is significantly enhanced by BIS depletion as assessed by transwell assay and collagen-incorporated 3D spheroid culture. Western blotting revealed that combination treatment significantly reduces p-STAT3 expression. In addition, a STAT3 inhibitor substantially suppressed the aggressive phenotypes of A549 cells. Thus, our results suggest that loss of STAT3 activity is a possible molecular mechanism for the synergistic effect of digoxin and BIS depletion. Our findings suggest the sensitizing role of BIS silencing to reduce the dose of digoxin for treatment of lung cancer with a high metastatic potential.

Implication of BIS in the Migration and Invasion of A549 Non-small Cell Lung Cancer Cells.

High expression of the Bcl-2-interacting cell death suppressor (BIS), an anti-apoptotic protein, in various human cancers is linked to a poor outcome. The purpose of this study was to clarify whether BIS is associated with the migration and invasive characteristics of A549 cells. BIS-knockout (KO) cells were prepared by the CRISPR/Cas9 method. The aggressive behaviors of A549 cells were analyzed by wound healing and a transwell invasion assay as well as 3D spheroid culture. BIS depletion resulted in significant inhibition of the migration and invasive potential of A549 cells which was accompanied by an increased ratio of E-cadherin/N-cadherin and a decrease in the mRNA levels of Zeb1, Snail, Slug and MMP-2. NF-ĸB activity was suppressed in BIS-KO A549 cells due to the decrease in p65 protein levels, but not in mRNA levels. BIS regulates cell invasion and the induction of the epithelial-mesenchymal transition (EMT) phenotype in A549 cells probably via the NF-ĸB signaling pathway.

Implication of BIS in the Migration and Invasion of A549 Non-small Cell Lung Cancer Cells.

High expression of the Bcl-2-interacting cell death suppressor (BIS), an anti-apoptotic protein, in various human cancers is linked to a poor outcome. The purpose of this study was to clarify whether BIS is associated with the migration and invasive characteristics of A549 cells. BIS-knockout (KO) cells were prepared by the CRISPR/Cas9 method. The aggressive behaviors of A549 cells were analyzed by wound healing and a transwell invasion assay as well as 3D spheroid culture. BIS depletion resulted in significant inhibition of the migration and invasive potential of A549 cells which was accompanied by an increased ratio of E-cadherin/N-cadherin and a decrease in the mRNA levels of Zeb1, Snail, Slug and MMP-2. NF-κB activity was suppressed in BIS-KO A549 cells due to the decrease in p65 protein levels, but not in mRNA levels. BIS regulates cell invasion and the induction of the epithelial-mesenchymal transition (EMT) phenotype in A549 cells probably via the NF-κB signaling pathway.

Effect of BIS depletion on HSF1-dependent transcriptional activation in A549 non-small cell lung cancer cells.

The expression of BCL-2 interacting cell death suppressor (BIS), an anti-stress or anti-apoptotic protein, has been shown to be regulated at the transcriptional level by heat shock factor 1 (HSF1) upon various stresses. Recently, HSF1 was also shown to bind to BIS, but the significance of these protein-protein interactions on HSF1 activity has not been fully defined. In the present study, we observed that complete depletion of BIS using a CRISPR/Cas9 system in A549 non-small cell lung cancer did not affect the induction of heat shock protein (HSP) 70 and HSP27 mRNAs under various stress conditions such as heat shock, proteotoxic stress, and oxidative stress. The lack of a functional association of BIS with HSF1 activity was also demonstrated by transient downregulation of BIS by siRNA in A549 and U87 glioblastoma cells. Endogenous BIS mRNA levels were significantly suppressed in BIS knockout (KO) A549 cells compared to BIS wild type (WT) A549 cells at the constitutive and inducible levels. The promoter activities of BIS and HSP70 as well as the degradation rate of BIS mRNA were not influenced by depletion of BIS. In addition, the expression levels of the mutant BIS construct, in which 14 bp were deleted as in BIS-KO A549 cells, were not different from those of the WT BIS construct, indicating that mRNA stability was not the mechanism for autoregulation of BIS. Our results suggested that BIS was not required for HSF1 activity, but was required for its own expression, which involved an HSF1-independent pathway.

KRIBB11 accelerates Mcl-1 degradation through an HSF1-independent, Mule-dependent pathway in A549 non-small cell lung cancer cells

The Bcl-2 family protein, Mcl-1 is known to have anti-apoptotic functions, and depletion of Mcl-1 by cellular stresses favors the apoptotic process. Moreover, Mcl-1 levels are frequently increased in various cancer cells, including non-small cell lung cancer (NSCLC), and is implicated in resistance to conventional chemotherapy and in cancer metastasis. In this study, we demonstrated that KRIBB11 accelerates the proteasomal degradation of Mcl-1 in the NSCLC cell line, A549. While KRIBB11 is an inhibitor of HSF1, we found that KRIBB11 induced Mcl-1 degradation in an HSF1-independent manner. Furthermore, this process was triggered via increase ubiquitination by the E3 ligase, Mule, rather than via de-ubiquitination by USP9X. Additionally, we found that Mcl-1 levels were only transiently reduced by KRIBB11: Mcl-1 levels were gradually restored as KRIBB11 activity diminished. However, we found that this effect was blocked in BIS (Bcl-2 interacting cell death suppressor, also called BAG3)-depleted cells, and that BIS prevents Mcl-1 from undergoing HSP70-driven proteasomal degradation, through an interaction with HSP70. Taken together, our results suggest that targeting Mcl-1 with KRIBB11 treatment, while simultaneously downregulating BIS, could be a therapeutic strategy in NSCLC.

BIS overexpression does not affect the sensitivity of HEK 293T cells against apoptosis

BCL-2 interacting cell death suppressor (BIS), also known as BAG3, has been known to function as an anti-stress or anti-apoptotic protein. Due to high levels of BIS expression in most of cancer cell lines, strategies to suppress BIS and any subsequent increased sensitivity to apoptosis are usually employed in conjunction with the pro-survival activity of BIS. To overcome the limitations in verifying a substantial level of expression for BIS, here we established BIS-overexpressing 293T cells following retroviral transfection and subsequent selection under puromycin. In a western assay, BIS expression levels were increased about 2.2-fold in selected cells (BIS-293T) compared with that in control cells (CON-293T). There was no difference in either cell growth rate or in cell cycle progression between the two groups. In addition, the susceptibilities of BIS-293T cells were not noticeably different from those of CON-293T cells following treatment with several apoptotic stimuli: cisplatin, VP-16, hydrogen peroxide, KRIBB11 (HSF1 inhibitor), AT-101 (pan-Bcl-2 inhibitor) or staurosporine (PKC inhibitor). Both cells revealed similar expression profiles of Bcl-2 family proteins, cleaved PARP and lipidation of LC3 upon AT-101 treatment, which indicate that BIS overexpression affect neither apoptosis nor autophagy induction upon AT-101 treatment in 293T cells. We further showed that inhibition of autophagy by chemical inhibitor attenuates AT-101-induced apoptosis, which is inconsistent with previous reports of the cytoprotective effect of autophagy that is induced by AT-101 in several types of cancer cells. Taken together, our results suggest that the cellular phenotypes determined by BIS and AT-101 treatment might be different between cancer cells and non- cancer cells and defining the molecular basis for such differences will require further study.

Myosin heavy chain is stabilized by BCL-2 interacting cell death suppressor (BIS) in skeletal muscle

BCL-2 interacting cell death suppressor (BIS), which is ubiquitously expressed, has important roles in various cellular processes, such as apoptosis, the cellular stress response, migration and invasion and protein quality control. In particular, BIS is highly expressed in skeletal and cardiac muscles, and BIS gene mutations result in human myopathy. In this study, we show that mRNA and protein levels of BIS were markedly increased during skeletal myogenesis in C2C12 cells and mouse satellite cells. BIS knockdown did not prevent the early stage of skeletal myogenesis, but did induce muscle atrophy and a decrease in the diameter of myotubes. BIS knockdown significantly suppressed the expression level of myosin heavy chain (MyHC) without changing the expression levels of myogenic marker proteins, such as Mgn, Cav-3 and MG53. In addition, BIS endogenously interacted with MyHC, and BIS knockdown induced MyHC ubiquitination and degradation. From these data, we conclude that molecular association of MyHC and BIS is necessary for MyHC stabilization in skeletal muscle.

Depletion of BIS sensitizes A549 cells to treatment with cisplatin

Bcl-2 interacting cell death suppressor (BIS), an anti-stress and ant-apoptotic protein, has been reported to be expressed at high levels in various cancers. In a previous study, we reported on a high level of expression of BIS in non small cell lung cancer tissues. To explore the significance this finding in lung cancer, in this study, we investigated the effect of BIS depletion on the survival of A549 cells upon treatment with anti-tumor agents. BIS knock out A549 cells, prepared by the CRISPR/Cas9 system, revealed a substantial decrease in survival to cisplatin treatment. Western blotting and quantitative real time PCR assays indicated that, among the anti-apoptotic Bcl-2 family proteins, the expression of Mcl-1 was decreased by BIS depletion at the protein level not at the mRNA level. Since BIS expression has been shown to be regulated by HSF1, we subsequently illustrated the sensitization effect of KRIBB11, a HSF1 inhibitor, on cisplatin-induced toxicity in A549 cells, accompanied by a decrease in both BIS and Mcl-1 expression. Our results suggest that BIS-mediated Mcl-1 stabilization represents a potential therapeutic target for cancer therapy.

BIS targeting induces cellular senescence through the regulation of 14-3-3 zeta/STAT3/SKP2/p27 in glioblastoma cells.

Cellular senescence is an important mechanism for preventing tumor progression. The elevated expression of Bcl-2-interacting cell death suppressor (BIS), an anti-apoptotic and anti-stress protein, often correlates with poor prognosis in several cancers including glioblastoma; however, the role of BIS in the regulation of senescence has not been well defined. Here, we describe for the first time that the depletion of BIS induces G1 arrest and cellular senescence through the accumulation of p27 that is independent of p53, p21 or p16. The increase in p27 expression in BIS-depleted cells was attributable to an impairment of the ubiquitin-mediated degradation of p27, which was caused by a decrease in S-phase kinase-associated protein 2 (SKP2) at the transcriptional level. As an underlying molecular mechanism, we demonstrate that the loss of activity of signal transducer and activator of transcription 3 (STAT3) was specifically linked to the suppression of SKP2 expression. Despite a reduction in phospho-STAT3 levels, total STAT3 levels were unexpectedly increased by BIS depletion, specifically in the insoluble fraction. Our results show that 14-3-3ζ expression is decreased by BIS knockdown and that 14-3-3ζ depletion per se significantly induced senescence phenotypes. In addition, the ectopic expression of 14-3-3ζ blocked senescence caused by BIS depletion, which was paralleled with a decrease in insoluble STAT3 in A172 glioblastoma cells. These findings indicate that the impairment of the protein quality control conferred by BIS and/or 14-3-3ζ is critical for BIS depletion-induced senescence. Moreover, BIS knockdown also induced senescence along with an accumulation of total STAT3 and p27 in several different cell types as well as embryonic fibroblasts derived from Bis-knock out mice with/without variations in 14-3-3ζ levels. Therefore, our findings suggest that a downregulation of BIS expression could serve as a potential strategy for restricting tumor progression via an induction of senescence through the regulation of STAT3/SKP2/p27 pathway.

Down-modulation of Bis reduces the invasive ability of glioma cells induced by TPA, through NF-κB mediated activation of MMP-9

Bcl-2 interacting cell death suppressor (Bis) has been shown to have anti-apoptotic and anti-stress functions. Recently, increased Bis expression was reported to correlate with glioma aggressiveness. Here, we investigated the effect of Bis knockdown on the acquisition of the invasive phenotype of A172 glioma cells, induced by 12-O-Tetradecanoylphorbol- 3-acetate (TPA), using a Transwell assay. Bis knockdown resulted in a significant decrease in the migration and invasion of A172 cells. Furthermore, Bis knockdown notably decreased TPAinduced matrix metalloproteinase-9 (MMP-9) activity and mRNA expression, as measured by zymography and quantitative real time PCR, respectively. A luciferase reporter assay indicated that Bis suppression significantly down-regulated NF-κB-driven transcription. Finally, we demonstrated that the rapid phosphorylation and subsequent degradation of IκB-α induced by TPA was remarkably delayed by Bis knockdown. These results suggest that Bis regulates the invasive ability of glioma cells elicited by TPA, by modulating NF-κB activation, and subsequent induction of MMP-9 mRNA. [BMB Reports 2014; 47(5): 262-267]

Down-modulation of Bis sensitizes cell death in C6 glioma cells induced by oxygen–glucose deprivation

Bcl-2 interacting cell death suppressor (Bis), also known as Bag3, has been implicated in anti-stress and anti-apoptotic pathways. In a previous study, we observed a significant induction of Bis in reactive astrocytes of the rat hippocampus after transient forebrain ischemia. To investigate the significance of this induction in ischemic injury, the expression of Bis was reduced with siRNA in C6 glioma cells and exposed to oxygen–glucose deprivation (OGD) conditions. Bis knock-down resulted in an increase in the cell death rate of the C6 cells after OGD, accompanied by accumulation of reactive oxygen species. Among the cellular antioxidants, the induction of superoxide dismutase (SOD) activity was significantly interfered within the cells treated with bis siRNA treated cells ( bis-kd C6). A Western blot assay revealed that SOD1 expression gradually increased in control cells, which was not observed in bis-kd cells upon OGD treatment. A quantitative analysis of Sod1 and Sod2 transcripts indicated that the induction of Sod1 was more evidently suppressed by the reduction of Bis. As a transcription factor candidate for the Sod1 gene, the activity of NF-κB was determined the nuclear translocation of p65, showing that the activation of NF-κB was attenuated in bis-kd C6. Supporting this, an overexpression of Bis augments the activation of NF-κB and Sod1 mRNA with an increased cell survival under OGD conditions. These results suggest that one of physiological significances of Bis induction in reactive astrocytes after ischemia in vivo is to protect glial cells from oxidative stress, probably via the induction of SOD1, which is related to the activation of NF-κB.

Bis deficiency results in early lethality with metabolic deterioration and involution of spleen and thymus

Bcl-2 interacting cell death suppressor (Bis), also known as Bag3 or CAIR-1, is involved in antistress and antiapoptotic pathways. In addition to Bcl-2, Bis binds to several proteins, suggesting it has diverse functions in normal and pathological conditions. To better define the physiological function of Bis in vivo, we developed bis-deficient mice with a cre-loxP system. Targeted disruption of exon 4 of the bis gene was demonstrated by Southern blotting and PCR, and Western blotting showed that no intact or truncated Bis protein was synthesized in bis(-/-) mice. While heterozygotes were fertile and appeared normal, Bis-deficient mice showed growth retardation and died by 3 wk after birth. The relative weight of the thymus and spleen was reduced and the total numbers of white blood cells, splenocytes, and thymocytes were significantly reduced compared with wild-type littermates. Serum profiles indicated significant hypoglycemia as well as decrease in triglyceride and cholesterol levels. Expression profiles of metabolic genes indicated that gluconeogenesis and beta-oxidation are activated in the liver of bis(-/-) mice. This activation, as well as a decrease in peripheral fat and an induction of fatty liver, appears to be an adaptive response to hypoglycemia. Our study reveals that the absence of Bis has considerable influences on postnatal growth and survival, possibly due to a nutritional impairment.