LPA protects intestinal epithelial cells from apoptosis by inhibiting the mitochondrial pathway

We previously showed (Gastroenterology 123: 206-216, 2002) that lysophosphatidic acid (LPA) protects and rescues rat intestinal epithelial cells (IEC-6) from apoptosis. Here, we provide evidence for the LPA-elicited inhibition of the mitochondrial apoptotic pathway leading to attenuation of caspase-3 activation. Pretreatment of IEC-6 cells with LPA inhibited campothecin-induced caspase-9 and caspase-3 activation and DNA fragmentation. A caspase-9 inhibitor peptide mimicked the LPA-elicited antiapoptotic activity. LPA elicited ERK1/ERK2 and PKB/Akt phosphorylation. The LPA-elicited antiapoptotic activity and inhibition of caspase-9 activity were abrogated by pertussis toxin, PD 98059, wortmannin, and LY 294002. LPA reduced cytochrome c release from mitochondria and prevented activation of caspase-9. LPA prevented translocation of Bax from cytosol to mitochondria and increased the expression of the antiapoptotic Bcl-2 mRNA and protein. LPA had no effect on Bcl-xl, Bad, and Bak mRNA or protein expression. These data indicate that LPA protects IEC-6 cells from camptothecin-induced apoptosis through G(i)-coupled inhibition of caspase-3 activation mediated by the attenuation of caspase-9 activation due to diminished cytochrome c release, involving upregulation of Bcl-2 protein expression and prevention of Bax translocation.     

Antiapoptotic property of human alpha-synuclein in neuronal cell lines is associated with the inhibition of caspase-3 but not caspase-9 activity

Abnormalities of alpha-synuclein (alpha-Syn) are mechanistically linked to Parkinson's disease (PD) and other alpha-synucleinopathies. To gain additional insights into the relationships between alpha-Syn expression and cell death, we examined the effects of expressing human alpha-Syn (Hualpha-Syn) variants on the cellular vulnerability to apoptotic stimuli. We show that the expression of wild-type (WT) and A30P mutant, but not A53T mutant, Hualpha-Syn leads to the protection of neuronal cell lines from apoptosis but not necrosis. Significantly, Hualpha-Syn did not protect non-neuronal cell lines from apoptosis. We also show that A53T mutant is a loss of function in regards to the antiapoptotic property since the expression of WT Hualpha-Syn with an excess of A53T mutant Hualpha-Syn leads to protection of the cells from apoptosis. The antiapoptotic property is specific to human alpha-Syn as neither beta-Syn nor mouse alpha-Syn protected cells from apoptosis, and the carboxy-terminal 20 amino acids are required for the antiapoptotic property. Analyses of capase-3 and caspase-9 activation reveal that the antiapoptotic property of Hualpha-Syn in neuronal cell lines is associated with the attenuation of caspase-3 activity without affecting the caspase-9 activity or the levels of cleaved, active caspase-3. We conclude that Hualpha-Syn modulates the activity of cleaved caspase-3 product in neuronal cell lines.     

Rosmarinic acid and siRNA combined therapy represses Hsp27 (HSPB1) expression and induces apoptosis in human glioma cells

High expression of Hsp27 in glioma cells has been closely associated with tumor cell proliferation and apoptosis inhibition. The aim of the present study was to asses the effects of rosmarinic acid (RA) on Hsp27 expression and apoptosis in non-transfected and transfected human U-87 MG cells. The effect of rosmarinic acid was compared to quercetin, which is known to be a good Hsp27 inhibitor. In order to block the expression of Hsp27 gene (HSPB1), transfection with specific siRNAs was performed. Western blotting technique was used to assess the Hsp27 expression, and caspase-3 colorimetric activity assay was performed to determine apoptosis induction. According to the results, it was found that RA and quercetin effectively silenced Hsp27 and both agents induced apoptosis by activating the caspase-3 pathway. Eighty and 215 μM RA decreased the level of Hsp27 by 28.8 and 46.7% and induced apoptosis by 30 and 54%, respectively. For the first time, we reported that rosmarinic acid has the ability to trigger caspase-3 induced apoptosis in human glioma cells. As a result of siRNA transfection, the Hsp27 gene was silenced by ~?50% but did not cause a statistically significant change in caspase-3 activation. It was also observed that apoptosis was induced at a higher level as a result of Hsp27 siRNA and subsequent quercetin or RA treatment. siRNA transfection and 215 μM RA treatment suppressed Hsp27 expression level by 90.5% and increased caspase-3 activity by 58%. Herein, we demonstrated that RA administered with siRNA seems to be a potent combination for glioblastoma therapy.     

Dickkopf‐3 alters the morphological response to retinoic acid during neuronal differentiation of human embryonal carcinoma cells

Dickkopf‐3 (Dkk‐3) and Dkkl‐1 (Soggy) are secreted proteins of poorly understood function that are highly expressed in subsets of neurons in the brain. To explore their potential roles during neuronal development, we examined their expression in Ntera‐2 (NT2) human embryonal carcinoma cells, which differentiate into neurons upon treatment with retinoic acid (RA). RA treatment increased the mRNA and protein levels of Dkk‐3 but not of Dkkl‐1. Ectopic expression of both Dkk‐3 and Dkkl‐1 induced apoptosis in NT2 cells. Gene silencing of Dkk‐3 did not affect NT2 cell growth or differentiation but altered their response to RA in suspension cultures. RA treatment of NT2 cells cultured in suspension resulted in morphological changes that led to cell attachment and flattening out of cell aggregates. Although there were no significant differences in the expression levels of cell adhesion molecules in control and Dkk‐3‐silenced cells, this morphological response was not observed in Dkk‐3‐silenced cells. These findings suggest that Dkk‐3 plays a role in the regulation of cell interactions during RA‐induced neuronal differentiation. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1243–1254, 2014     

E1A oncogene enhancement of caspase-2-mediated mitochondrial injury sensitizes cells to macrophage nitric oxide-induced apoptosis

The adenovirus E1A oncogene induces innate immune rejection of tumors by sensitizing tumor cells to apoptosis in response to injuries, such as those inflicted by macrophage-produced TNF alpha and NO. E1A sensitizes cells to TNF by repressing its activation of NF-kappaB-dependent, antiapoptotic defenses. This suggested the hypothesis that E1A blockade of the NF-kappaB activation response might be the central mechanism of E1A induced cellular sensitivity to other proapoptotic injuries, such as macrophage-produced NO. However, creation of E1A-positive NIH-3T3 mouse cell variants with high-level, NF-kappaB-dependent resistance to TNF did not coselect for resistance to apoptosis induced by either macrophage-NO or chemical-NO, as the hypothesis would predict. E1A expression did block cellular recovery from NO-induced mitochondrial injury and converted the reversible, NO-induced cytostasis response of cells to an apoptotic response. This viral oncogene-induced phenotypic conversion of the cellular injury response of mouse and human cells was mediated by an E1A-related increase in NO-induced activation of caspase-2, an apical initiator of intrinsic apoptosis. Blocking caspase-2 activation or expression eliminated the NO-induced apoptotic response of E1A-positive cells. These results define an NF-kappaB-independent pathway through which the E1A gene of human adenovirus sensitizes mouse and human cells to apoptosis by enhancement of caspase-2-mediated mitochondrial injury.     

Divergent modulation of neuronal differentiation by caspase-2 and -9

Human Ntera2/cl.D1 (NT2) cells treated with retinoic acid (RA) differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2) or -9 (si-Casp9) was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM), microtubule associated protein-2 (MAP2) and tyrosine hydroxylase (TH) mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ～100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved) in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells.     

ANG II induces apoptosis of human vascular smooth muscle via extrinsic pathway involving inhibition of Akt phosphorylation and increased FasL expression

In addition to well-documented vascular growth-promoting effects, ANG II exerts proapoptotic effects that are poorly understood. IGF-1 is a potent survival factor for human vascular smooth muscle cells (hVSMC), and its antiapoptotic effects are mediated via the IGF-1 receptor (IGF-1R) through a signaling pathway involving phosphatidylinositol 3-kinase and Akt. We hypothesized that there would be cross talk between ANG II proapoptotic effects and IGF-1 survival effects in hVSMC. To investigate ANG II-induced apoptosis and the potential involvement of IGF-1, we exposed quiescent and nonquiescent hVSMC to ANG II. ANG II induced apoptosis only in nonquiescent cells but stimulated hypertrophy in quiescent cells. ANG II-induced apoptosis was characterized by marked inhibition of Akt phosphorylation and stimulation of membrane Fas ligand (FasL) expression, caspase-8 activation, and a reduction in soluble FasL expression. Adenovirally mediated overexpression of Akt rescued hVSMC from ANG II-induced apoptosis. IGF-1R activation increased Akt phosphorylation and soluble FasL expression, and these effects were completely blocked by coincubating hVSMC with ANG II. In conclusion, ANG II-induced apoptosis of hVSMC is characterized by marked inhibition of Akt phosphorylation and stimulation of an extrinsic cell death signaling pathway via upregulation of membrane FasL expression, caspase-8 activation, and a reduction in soluble FasL expression. Furthermore, ANG II antagonizes the antiapoptotic effect of IGF-1 by blocking its ability to increase Akt phosphorylation and soluble FasL. These findings provide novel insights into ANG II-induced apoptotic signaling and have significant implication for understanding ANG II-induced remodeling in hypertension and atherosclerosis.     

Cloning of a novel human caspase-9 splice variant containing only the CARD domain

Caspase-9 plays a key role in the intrinsic apoptotic pathway and currently two splice variants (caspase-9-alpha and -beta) have been identified. The present study cloned and characterized a novel caspase-9 splice variant, hereby designated Casp9-gamma. Casp9-gamma is generated from an additional alternative 3' splice site in the fourth exon of caspase-9, resulting in a 58-nucleotide fragment insertion compared with the full-length caspase-9-alpha. The fragment introduces an in-frame stop codon, and the resulting open reading frame (ORF) is preterminated. The Casp9-gamma comprises the deduced 154 amino acid residues containing only the caspase recruitment domain (CARD) and does not contain the large and small subunits. The Casp9-gamma does not promote apoptosis when overexpressed in mammalian cells. Moreover, it inhibits the cleavage of procaspase-3 mediated by proapoptotic member Bax or apoptosis inductor staurosporine. Therefore, Casp9-gamma may function as an endogenous apoptotic inhibitor by interfering with the CARD-CARD interaction between Apaf-1 (apoptotic protease activating factor-1) and procaspase-9. In addition, Casp9-gamma does not enhance NF-kappaB activation in transfected 293T cells, conflicting with previous evidence that the isolated CARD of caspase-9 activates NF-kappaB in ND7 cells. This suggests that the procaspase-9-mediated NF-kappaB activation in response to cellular stresses is cell type-specific through an unidentified mechanism.     

Human alphaA- and alphaB-crystallins bind to Bax and Bcl-X(S) to sequester their translocation during staurosporine-induced apoptosis

AlphaA- and alphaB-crystallins are distinct antiapoptotic regulators. Regarding the antiapoptotic mechanisms, we have recently demonstrated that alphaB-crystallin interacts with the procaspase-3 and partially processed procaspase-3 to repress caspase-3 activation. Here, we demonstrate that human alphaA- and alphaB-crystallins prevent staurosporine-induced apoptosis through interactions with members of the Bcl-2 family. Using GST pulldown assays and coimmunoprecipitations, we demonstrated that alpha-crystallins bind to Bax and Bcl-X(S) both in vitro and in vivo. Human alphaA- and alphaB-crystallins display similar affinity to both proapoptotic regulators, and so are true with their antiapoptotic ability tested in human lens epithelial cells, human retina pigment epithelial cells (ARPE-19) and rat embryonic myocardium cells (H9c2) under treatment of staurosporine, etoposide or sorbitol. Two prominent mutants, R116C in alphaA-crystallin and R120G, in alphaB-crystallin display much weaker affinity to Bax and Bcl-X(S). Through the interaction, alpha-crystallins prevent the translocation of Bax and Bcl-X(S) from cytosol into mitochondria during staurosporine-induced apoptosis. As a result, alpha-crystallins preserve the integrity of mitochondria, restrict release of cytochrome c, repress activation of caspase-3 and block degradation of PARP. Thus, our results demonstrate a novel antiapoptotic mechanism for alpha-crystallins.     

Exonic splicing enhancer-dependent selection of the bovine papillomavirus type 1 nucleotide 3225 3' splice site can be rescued in a cell lacking splicing factor ASF/SF2 through activation of the phosphatidylinositol 3-kinase/Akt pathway

Bovine papillomavirus type 1 (BPV-1) late pre-mRNAs are spliced in keratinocytes in a differentiation-specific manner: the late leader 5' splice site alternatively splices to a proximal 3' splice site (at nucleotide 3225) to express L2 or to a distal 3' splice site (at nucleotide 3605) to express L1. Two exonic splicing enhancers, each containing two ASF/SF2 (alternative splicing factor/splicing factor 2) binding sites, are located between the two 3' splice sites and have been identified as regulating alternative 3' splice site usage. The present report demonstrates for the first time that ASF/SF2 is required under physiological conditions for the expression of BPV-1 late RNAs and for selection of the proximal 3' splice site for BPV-1 RNA splicing in DT40-ASF cells, a genetically engineered chicken B-cell line that expresses only human ASF/SF2 controlled by a tetracycline-repressible promoter. Depletion of ASF/SF2 from the cells by tetracycline greatly decreased viral RNA expression and RNA splicing at the proximal 3' splice site while increasing use of the distal 3' splice site in the remaining viral RNAs. Activation of cells lacking ASF/SF2 through anti-immunoglobulin M-B-cell receptor cross-linking rescued viral RNA expression and splicing at the proximal 3' splice site and enhanced Akt phosphorylation and expression of the phosphorylated serine/arginine-rich (SR) proteins SRp30s (especially SC35) and SRp40. Treatment with wortmannin, a specific phosphatidylinositol 3-kinase/Akt kinase inhibitor, completely blocked the activation-induced activities. ASF/SF2 thus plays an important role in viral RNA expression and splicing at the proximal 3' splice site, but activation-rescued viral RNA expression and splicing in ASF/SF2-depleted cells is mediated through the phosphatidylinositol 3-kinase/Akt pathway and is associated with the enhanced expression of other SR proteins.     

bFGF inhibits the activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells during neuronal differentiation.

P19 embryonal carcinoma (EC) cells undergo apoptosis during neuronal differentiation induced by all-trans retinoic acid (RA). Caspase-3-like proteases are activated and involved in the apoptosis of P19 EC cells during neuronal differentiation.1 Recently it has been shown that growth factor signals protect against apoptosis by phosphorylation of Bad. Phosphorylated Bad, an apoptotic member of the Bcl-2 family, cannot bind to Bcl-xL and results in Bcl-xL homodimer formation and subsequent antiapoptotic activity. In the present study, we demonstrate that this system is used generally to protect against apoptosis during neuronal differentiation. Bcl-xL inhibited the activation of caspase-3-like proteases. Basic fibroblast growth factor (bFGF) inhibited more than 90% of the caspase-3-like activity, inhibited processing of caspase-3 into its active form, and inhibited DNA fragmentation. bFGF activated phosphatidyl-inositol-3-kinase (PI3K) and stimulated the phosphorylation of Bad. Phosphorylation was inhibited by wortmannin, an inhibitor of PI3K and its downstream target Akt. Thus, Bad is a target of the FGF receptor-mediated signals involved in the protection against activation of caspase-3.     

Neurotensin receptor-2 and -3 are crucial for the anti-apoptotic effect of neurotensin on pancreatic β-TC3 cells

The neuropeptide neurotensin (NT) has been recently shown to protect pancreatic beta cells from toxic agents-induced apoptosis through interaction with the NT receptor-2 (NTSR2) and activation of the phosphatidyl inositol-3 kinase pathway. However, expression of the NT receptor-3/sortilin (NTSR3) in the mouse pancreatic beta cell line β-TC3 led us to investigate its possible functional role in these cells. By using siRNA, immunoprecipitation, co-localization and caspase-3 assays, we provide evidence for a functional endogenous interaction between NTSR2 and NTSR3. Expression of both receptors is necessary for the protective action of NT on staurosporine-induced caspase-3 activity in β-TC3 cells. Moreover, NTSR2 and NTSR3 co-immunoprecipitate and are co-localized at the plasma membrane. Thus, the NT response in beta cells is controlled by the formation of a functional complex between NTSR2 and NTSR3.     

Antiapoptotic Activity of the Herpesvirus Saimiri-Encoded Bcl-2 Homolog: Stabilization of Mitochondria and Inhibition of Caspase-3-Like Activity

Viruses have evolved different strategies to interfere with host cell apoptosis. Herpesvirus saimiri (HVS) and other lymphotropic herpesviruses code for proteins that are homologous to the cellular antiapoptotic Bcl-2. In this study HVS-Bcl-2 was stably expressed in the human leukemia cell line Jurkat and in the murine T-cell hybridoma DO to assess its antiapoptotic spectrum and to gain further insight into its mode of action. HVS- Bcl-2 prevented apoptosis that occurs as a result of a disturbance of intracellular homeostasis by, for example, DNA damage or menadione, which gives rise to oxygen radicals. In Jurkat cells, HVS-Bcl-2 also inhibited apoptosis mediated by the death receptor CD95. In DO cells, HVS-Bcl-2 did not interfere with CD95-mediated apoptosis but blocked dexamethasone-induced cell death. Mitochondrial damage is a central coordinating event in apoptosis induced by different stimuli. To assess the integrity of mitochondria, we used rhodamine 123, which is released upon disturbance of the mitochondrial membrane potential, and determined the release of cytochrome c into the cytosol. Both signs of mitochondrial damage were prevented by HVS-Bcl-2. This viral protein also inhibited the generation of caspase-3-like DEVDase activity and blocked the cleavage of poly(ADP-ribose) polymerase, a natural substrate of caspase-3-like proteases. In conclusion, HVS-Bcl-2 protects against a great variety of apoptotic stimuli, stabilizes mitochondria, and acts upstream of the generation of caspase-3-like activity.     

Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

Retinal progenitor cells （RPCs） are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid （RA） is a powerful growth/differentiation factor that generally causes growth inhibition, differentiation and/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affecting RPC proliferation. The enhanced cell survival was accompanied by a significant upregulation of the expression of protein kinase A （PKA） and several protein kinase C （PKC） isoforms. Treatment of cells grown in RA-free media with 8-bromoadenosine3＇,5＇-cyclic monophosphate, a known activator of PKA, resulted in an anti-apoptotic effect similar to that caused by RA; whereas the PKA inhibitor N-[2-（p-bromocinnamylamino）ethyl]-5-isoquinolinesul- fonamide dihydrochloride led to a significant （-32%） increase in apoptosis. In contrast, treatment of RPCs with any of two PKC selective inhibitors, 2,2＇,3,3＇,4,4＇-hexahydroxy-1,1 ＇-biphenyl-6,6＇-dimethanol dimethyl ether and bisindolylmaleimide XI, led to diminished apoptosis; while a PKC activator, phorbol 12-myristate 13-acetate, increased apoptosis. These and other data suggest that the effect of RA on RPC survival is mostly due to the increased anti-apoptotic activity elicited by PKA, which might in turn be antagonized by PKC. Such a mechanism is a new example of tight regulation of important biological processes triggered by RA. Although the detailed mechanisms remain to be elucidated, we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changed expression of p53 or bcl-2, and appears to be independent of 15-amyloid, Fas ligand, TNF-α, ganglioside GM1 and ceramide C 16-induced apoptotic pathways.     

Protective effects of docosahexaenoic acid in staurosporine-induced apoptosis: involvement of phosphatidylinositol-3 kinase pathway

Docosahexaenoic acid (22:6n-3, DHA) is highly enriched in neuronal membranes and is considered to be essential for proper brain function. We have previously demonstrated in Neuro 2A cells that DHA as a membrane component protects cells from apoptotic death induced by serum deprivation (Kim et al. 2000). In the present study we demonstrate that staurosporine (ST) induces apoptosis in Neuro 2A cells and DHA enrichment prior to the ST treatment significantly inhibits the apoptotic cell death, as evidenced by the reduction of caspase-3 activity, cleavage of pro-caspase-3 to active caspase-3, DNA strand-breaking and laddering. Enrichment of cells with other fatty acids such as oleic and arachidonic acids did not exert such an effect, indicating that the antiapoptotic effect was specific to DHA enrichment. Among the several protein kinase inhibitors, only phosphatidylinositol 3-kinase (PI3-K) inhibitors, wortmanin, and LY-294002 abolished the protective effect of DHA in ST-induced apoptosis. Concurrently, ST-treatment significantly decreased the phosphorylation status of Akt at Ser-473 and Thr-308 as well as Akt activity, and this reduction was partially prevented by DHA enrichment. The extent of the antiapoptotic effect of DHA correlated with a time-dependent increase in the phosphatidylserine (PS) content upon DHA enrichment. When cells were enriched with DHA in serine-free medium, the PS increase diminished and the DHA effect on caspase-3 activation as well as Akt phosphorylation in ST-induced apoptosis was no longer apparent, suggesting that DHA's role in accumulating membrane PS is an important component for the observed protection. In summary, DHA enrichment uniquely protects ST-induced apoptosis in a PS- and PI3-K-dependent manner. From these data, we suggest that the antiapoptotic effect of DHA is mediated at least in part through the PI3-K/Akt pathway, facilitated by DHA-induced PS accumulation.     

Role of the mitochondrial signaling pathway in murine coronavirus-induced oligodendrocyte apoptosis

A previous study demonstrated that infection of rat oligodendrocytes by mouse hepatitis virus (MHV) resulted in apoptosis, which is caspase dependent (Y. Liu, Y. Cai, and X. Zhang, J. Virol. 77:11952-11963, 2003). Here we determined the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis. We found that caspase-9 activity was 12-fold higher in virus-infected cells than in mock-infected cells at 24 h postinfection (p.i.). Pretreatment of cells with a caspase-9 inhibitor completely blocked caspase-9 activation and partially inhibited the apoptosis mediated by MHV infection. Analyses of cytochrome c release further revealed an activation of the mitochondrial apoptotic pathway. Stable overexpression of the two antiapoptotic proteins Bcl-2 and Bcl-xL significantly, though only partially, blocked apoptosis, suggesting that activation of the mitochondrial pathway is partially responsible for the apoptosis. To identify upstream signals, we determined caspase-8 activity, cleavage of Bid, and expression of Bax and Bad by Western blotting. We found a drastic increase in caspase-8 activity and cleavage of Bid at 24 h p.i. in virus-infected cells, suggesting that Bid may serve as a messenger to relay the signals from caspase-8 to mitochondria. However, treatment with a caspase-8 inhibitor only slightly blocked cytochrome c release from the mitochondria. Furthermore, we found that Bax but not Bad was significantly increased at 12 h p.i. in cells infected with both live and UV-inactivated viruses and that Bax activation was partially blocked by treatment with the caspase-8 inhibitor. These results thus establish the involvement of the mitochondrial pathway in MHV-induced oligodendrocyte apoptosis.     

Down-regulation of CIBZ, a novel substrate of caspase-3, induces apoptosis

We previously identified and characterized a murine BTB domain-containing protein, CIBZ (ZBTB38 in human), that interacts with CtBP and binds to methylated CpGs. However, its physiological function remained unknown. As CtBP is reportedly involved in p53-independent programmed cell death, we examine here whether CIBZ is associated with apoptosis. We found that CIBZ was highly expressed in proliferating C2C12 cells but that its expression levels decreased upon induction of apoptosis by serum starvation. Knockdown of CIBZ by small interfering RNA in C2C12 cells induced apoptosis, as determined by an increase of annexin V/propidium iodide labeling, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. CIBZ inhibition also activated caspase-7 and caspase-9, suggesting that CIBZ-associated apoptosis occurs through the mitochondrial pathway. Notably, knockdown of CIBZ in p53(-/-) mouse embryonic fibroblast cells also activated caspase-3 and cleavage of poly(ADP-ribose) polymerase, indicating that CIBZ-associated apoptosis is mediated by a p53-independent pathway; however, because both common and distinct targets are regulated by CIBZ- and CtBP-associated apoptosis, we conclude that more than one pathway is involved. Finally, using mutagenesis and an in vitro caspase cleavage assay, we show that CIBZ is a novel substrate of caspase-3 and identify two caspase-3 recognition sites. These findings indicate, collectively, that CIBZ plays an important role by participating in the negative regulation of apoptosis in murine cells.