Regulation Of Bcl-2 Expression Research Articles

Abstract 1035: Bcl2 is regulated by PKC and Akt and is associated with resistance to cisplatin in endometrial cancer cells.

Abstract Endometrial carcinomas often show resistance to cisplatin. Bcl2 has been shown to be associated with cisplatin resistance in different types of cancers. However, there is no information concerning its possible involvement in chemoresistance and its regulation in endometrial carcinomas. We have tested the ability of cisplatin to induce apoptosis in KLE, a resistant endometrial carcinoma cell line, and examined the putative role for Bcl2 in regulating drug sensitivity. We have investigated the impact of cisplatin treatment on Bcl2 family members and Xiap, an inhibitor of caspase activity. Cisplatin treatment upregulated Bcl2 protein expression in a time-dependant manner in KLE cells, but had no significant impact on the levels of Bax, Bcl-xL and Xiap proteins. As demonstrated by RT-PCR, Bcl2 mRNA was not upregulated in response to cisplatin, suggesting that cisplatin act on Bcl2 expression through a post-translational mechanism. In KLE cells, Bcl2 inhibition by HA14-1 led to increased apoptosis induced by cisplatin. It has been shown that Akt, MAPK and PKC pathways are involved in the regulation of Bcl2 under various conditions and in different cell types. Therefore, KLE cells were treated with inhibitors of MAPK (PD98059) and PKC (CalphostinC) prior to cisplatin treatment. Inhibition of PKC prevented cisplatin-dependant upregulation of Bcl2 protein, while MAPK inhibition had no impact. We previously showed that KLE cell line express high levels of phosphorylated Akt (pAkt) isoforms. Then, KLE cells stably expressing shRNA for all three Akt isoforms were established. Treatment of these cells with cisplatin prevented the increase of Bcl2 expression and led to increased apoptosis by cisplatin. These data suggest a role for Bcl2 in cisplatin resistance in endometrial carcinoma cells. Also, they indicate that PKC and Akt are involved in regulation of Bcl2 expression in this experimental model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1035.

Abstract LB-234: Bcl-2, a novel target of Bmi-1 (the stem cell associated factor), induced Wnt signaling: Implications on prostate cancer

Abstract In the current report we provide a novel finding about the regulation of Bcl-2 expression in prostate cancer (CaP) cells. Bcl-2 is known to be regulated by Sonic Hedgehog (SHH) signaling. Here, we provide evidence that human CaP cells exhibit SHH-independent Bcl-2 activation which is regulated by Bmi-1-induced Wnt signaling. Bmi-1 is a member of polycomb group gene family and is reported to be involved in stemness and self-renewal capability of cancer stem cells. We investigated the role of Bmi-1 in human CaP development and delineate its mechanism of action. On the basis of immunohistochemical analysis of prostatic specimens of 125 human CaP patients, we show that Bmi-1 protein levels are highly elevated in patients with advanced disease. To understand the mechanism of action of Bmi-1, we employed two-prong strategy. Firstly, Bmi-1 was knocked down in CaP cells (LNCaP, DU145 and PC-3) by employing siRNA technique. Bmi-1-silenced CaP cells exhibited decreased proliferative and clonogenic potential. Secondly, Bmi-1 was over-expressed in CaP cells by transfecting Bmi-1 overexpressing plasmid (pbabe-Bmi-1) in CaP cells. Bmi-1-overexpressing cells exhibited increased clonogenicity and rate of proliferation. We performed a micro-array analysis of Bmi-1-silenced cells and found that silencing of Bmi-1 caused a decrease in the Cyclin D1 (Wnt signaling target) and Bcl-2 (SHH signaling target), however an increase in p16 was observed. These studies were validated by performing immunoblot analysis. We generated a hypothesis that the Bmi-1 regulates the expression of Cyclin D1 and Bcl-2 by interacting with Wnt /SHH signaling in CaP cells. We show that despite blocking SHH signaling (by treating CaP cells with cyclopamine, a SHH signaling inhibitor), Bmi-1 induces the Bcl-2 expression in CaP cells suggesting the involvement of SHH-independent pathway in the regulation of Bcl-2 activation. We identified Wnt signaling as the pathway that regulates Bcl-2 independent of SHH signaling in CaP cells. We provide evidence that Bcl-2 promoter region of Bcl-2 gene has binding sites for Tcf transcriptional factor (Wnt signaling component). We found that Bmi-1 induces Wnt signaling in CaP cells. Taken together, these studies provide deep-insight into the mechanism of CaP cell proliferation (even after chemotherapy such as cyclopamine treatment). We suggest that Bmi-1 could be developed as a novel molecular and therapeutic target for human CaP treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-234.

Abstract 2947: Knockdown of splicing factor SRp20 induces apoptosis in ovarian tumor cells

Abstract SRp20 is a member of the serine/arginine-rich (SR) protein family with multiple functions in RNA processing such as alternative splicing and polyadnylation. Our previous studies found that SRp20, together with another splicing factor, polypyrimidine tract-binding protein (PTB), is up-regulated in human ovarian tumors compared to normal ovarian surface epithelia (He, X et al, Clin Cancer Res, 10:4652). In order to determine whether overexpressed SRp20 is required for ovarian tumor cell growth, we studied the effects of SRp20 knockdown in several ovarian cancer cell lines. The knockdown of SRp20 expression was achieved by lentivirus-delivered, doxycycline (Dox)-induced siRNAs. We identified two effective siRNAs against SRp20, SRp20si1 and SRp20si2, which can suppress SRp20 expression by approximately 50% and 90%, respectively. In ovarian cancer cell lines A2780, SKOV3 and IGROV1, knockdown of SRp20 by either SRp20 siRNA caused substantial inhibition of cell growth and colony formation in soft agar. The degree of inhibition correlated with the extent of SRp20 suppression. Massive down-regulation of SRp20 expression by SRp20si2 led to almost complete loss of A2780's capability to form colonies in soft agar. Moreover, Hoechst 33342 staining revealed that the percentage of apoptotic cells was remarkably increased when SRp20 was knocked down ∼90% by SRp20si2. In contrast, moderate suppression (∼50%) of SRp20 by SRp20si1 did not trigger apoptosis. These results suggest that overexpression of SRp20 is required for ovarian cancer cell growth and survival. To determine which apoptotic pathway is activated by SRp20 knockdown, we examined the cleaved fragments of several caspases by immunoblotting and found that caspases 9, 3 and 7 but not caspases 2 and 8were cleaved in cells with SRp20 knocked down by SRp20si2. These results imply that the intrinsic apoptotic pathway is activated by massive SRp20 knockdown. In support of this, we also observed a substantial down-regulation of Bcl-2, a major anti-apoptotic regulator of the intrinsic apoptotic pathway, in the above cells. How SRp20 regulates Bcl-2 expression is unclear at present and currently under investigation. (Supported in part by CA40570 and CA138762 from NCI (to WTB), in part by OCRF (to XH), and in part by UIC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2947.

P35, the non-cyclin activator of Cdk5, protects podocytes against apoptosis in vitro and in vivo

Cyclin-dependent kinase-5 is widely expressed and predominantly regulated by the non-cyclin activator p35. Since we recently showed that expression of p35 in the kidney is restricted to podocytes, we examined here its function in mice in which p35 was genetically deleted. The mice did not exhibit kidney abnormalities during glomerular development or during adult life. Conditionally immortalized cultured podocytes, derived from these null mice, did not have any change in their morphology, differentiation, or proliferation. However, when these cultured podocytes were exposed to UV-C irradiation, serum depletion, puromycin aminonucleoside, or transforming growth factor-beta-1, they showed increased apoptosis compared to those from wild-type mice. Levels of Bcl-2 were decreased in these null podocytes but increased after transduction with human p35. Restoration of p35 or the ectopic expression of Bcl-2 reduced the susceptibility of p35-null podocytes to apoptosis. Experimental glomerulonephritis, characterized by podocyte apoptosis and subsequent crescent formation, was utilized to test these findings in vivo. Podocyte apoptosis was significantly increased in diseased p35-null compared with wild-type mice, accompanied by increased glomerulosclerosis and decreased renal function. Our study shows that p35 does not affect glomerulogenesis but controls podocyte survival following injury, in part, by regulating Bcl-2 expression.

Ubc9 promotes breast cell invasion and metastasis in a sumoylation-independent manner.

Ubc9 is an E2 conjugating enzyme that transfers the activated SUMO (small ubiquitin-related modifier) to protein substrates, and thus it plays a critical role in sumoylation-mediated cellular pathways. We have previously reported that Ubc9 promotes tumor growth in the xenograft mouse model using breast cancer cell line MCF-7 in part through regulation of Bcl-2 expression. In this study, we show that ectopic expression of wild type Ubc9 (Ubc9-WT) promotes cell invasion and metastasis. Surprisingly, the dominant negative mutant Ubc9 (Ubc9-DN) also causes the same phenotype, indicating that the ability of Ubc9 to promote invasion and metastasis is distinct from its ability to conjugate SUMO to protein substrates. Of considerable interest, several microRNAs such as miR-224 are regulated by Ubc9. While ectopic expression of Ubc9 causes downregulation of miR-224, and suppression of Ubc9 by Ubc9-siRNAs leads to its upregulation. We further show that miR-224 can inhibit cell invasion and directly targets CDC42 and CXCR4, and that suppression of CDC42 and CXCR4 by RNAi causes inhibition of Ubc9-mediated invasion. Together, these results demonstrate a molecular link between Ubc9 and the metastasis genes such as CDC42 and CXCR4, and thus provide new insight into the mechanism by which Ubc9 promotes tumor invasion and metastasis.

Lyl1 and Scl Regulate Bcl2 Expression and Control Apoptosis at Different Stages of Hematopoietic Development.

Abstract 569The Lymphoblastic Leukemia gene 1 (Lyl1) encodes a basic-helix-loop-helix (bHLH) transcription factor that is involved in T cell acute lymphoblastic leukemia via chromosomal translocations. Lyl1 has an almost identical bHLH motif to Scl, a known master regulator of hematopoiesis. In our previous work, we used a double knock-out mouse model to demonstrate functional redundancy between Lyl1 and Scl in adult hematopoietic stem cells (HSCs), where in the absence of both genes in adult mice, HSCs and progenitors undergo rapid apoptosis. Despite their common and overlapping functions, the two genes appear to also function at different aspects of hematopoietic development. Lyl1 knock-out mice exhibit a unique defect in B-cell development. This phenotype is recapitulated in vitro, where Lyl1 progenitors grown on a layer of OP9 cells and allowed to differentiate with the addition of cytokines fail to differentiate to B cells as well as wild type progenitors. Rescue assays, where Lyl1 was transduced using a retrovirus into Lyl1 knock-out progenitors and transplanted into lethally irradiated recipients, showed complete rescue of the B cell lineage. Forced expression of Scl into Lyl1 knock-out progenitors failed to rescue, giving rise to only about 5% of peripheral blood B cells. This demonstrates the distinct functions of the two genes in differentiated cells types, despite their functional redundancy in the HSCs. We further sought to dissect the functions and differences between Lyl1 and Scl based on their target genes. Using bioinformatics analysis and a literature search, we identified a number of genes that were candidates for Lyl1 transcriptional regulation; these interactions with Lyl1 and Scl were confirmed by chromatin immunoprecipitation. We found that Bcl2, and genes belonging to this family, are direct target genes of both Lyl1 and Scl in progenitor cells, and that they are downregulated in Lyl1 and Scl single knock-out mouse models. Since Bcl2 is critical for B cell survival, we hypothesized that the B cell deficiency in Lyl1 knock-out mice could be partly due to a loss of Bcl2. We performed rescue assays where Bcl2 was transduced using a retrovirus into Lyl1 knock-out progenitors which were transplanted into lethally irradiated recipient mice. Bcl2 overexpression rescued the B-cell defect in Lyl1 knock-out bone marrow. Since Bcl2 was identified as a target gene for Scl as well, we tested whether Bcl2 could also rescue the rapid apoptosis phenotype of the Lyl1/Scl double knock-out progenitors. Forced expression of Bcl2 in these cells, followed by deletion of the floxed Scl allele, resulted in a delayed onset of apoptosis, but incomplete rescue of the progenitor cell death. These findings distinguish the activities of Lyl1 and Scl in B cells and demonstrate a possible mechanism for their functions, through in part by their activation of the antiapoptotic gene Bcl2. Disclosures:No relevant conflicts of interest to declare.

Multimodal Bcl-2 Sub-Populations in CLL.

Abstract 4391 IntroductionThe Recently described NZB miRNA15a/16-1 mutation is syntenic to 13q14 deletion in CLL. The mir15a/16-1 complex is thought to regulate bcl-2 expression. Therefore the level of bcl-2 expression was examined more closely as it might be used to differentiate mono-allelic deletion from the diploid CLL clone. Patients and MethodsMulticolor flow-cytometry using a panel of anti-bcl-2 FITC (clone 124, DAKO Cytomation, Glostrup, Denmark) and CD19, CD20, CD5, CD23, CD38, kappa, lambda, and CD45 was used. Bcl-2 expression was evaluated using the conventional bcl-2 index (Mean fluorescence intensity (MFI) B cell/ MFI T cells) in 25 untreated CLL patients, and 10 normal donor controls. A modified ratio based on normal remaining (NR) polyclonal B cells was also used (MFI clone/MFI NR) with a new gating strategy for bcl-2 expression analysis. ResultsThe mean of the conventional bcl-2 index is significantly higher in CLL patients [1.52 ± 0.53(SD)] compared to the control [0.5 ± 0.19(SD)], p<0.001 Student‘s t test. When comparing the mean of conventional bcl-2 index (clone/T cell) versus the mean of the modified bcl-2 index (clone/NR) [1.78 ±0.61 (SD)] in only CLL patients there was no significant difference. On the other hand, the rank order analysis of the conventional and modified bcl-2 index showed an r=0.7, p< 0.001 indicating that both methods give similar results. In addition to the high correlation between the conventional and the modified ratio, the modified ratio was multimodal in distribution. ConclusionsBcl-2 expression varies largely among the lymphocyte sub-sets. The choice of the polyclonal B cells allows better segregation of multimodal bcl-2 expression. Since bcl-2 mRNA is a validated mir15a/16 target, increased bcl-2 expression maybe a direct result of and a surrogate marker for mir15a/16 levels and/or the 13q14 status. There are three possibilities: no deletion (diploid), heterozygous deletion, or homozygous deletion. This needs to be verified by fluorescence in situ hybridization (FISH) analysis of sorted CLL B cells based on bcl-2 expression. Disclosures:No relevant conflicts of interest to declare.

Sigma-1 Receptors Regulate Bcl-2 Expression by Reactive Oxygen Species-Dependent Transcriptional Regulation of Nuclear Factor κB

The expression of Bcl-2, the major antiapoptotic member of the Bcl-2 family, is under complex controls of several factors, including reactive oxygen species (ROS). The sigma-1 receptor (Sig-1R), which was recently identified as a novel molecular chaperone at the mitochondria-associated endoplasmic reticulum membrane (MAM), has been shown to exert robust cellular protective actions. However, mechanisms underlying the antiapoptotic action of the Sig-1R remain to be clarified. Here, we found that the Sig-1R promotes cellular survival by regulating the Bcl-2 expression in Chinese hamster ovary cells. Although both Sig-1Rs and Bcl-2 are highly enriched at the MAM, Sig-1Rs neither associate physically with Bcl-2 nor regulate stability of Bcl-2 proteins. However, Sig-1Rs tonically regulate the expression of Bcl-2 proteins. Knockdown of Sig-1Rs down-regulates whereas overexpression of Sig-1Rs up-regulates bcl-2 mRNA, indicating that the Sig-1R transcriptionally regulates the expression of Bcl-2. The effect of Sig-1R small interfering RNA down-regulating Bcl-2 was blocked by ROS scavengers and by the inhibitor of the ROS-inducible transcription factor nuclear factor kappaB (NF-kappaB). Knockdown of Sig-1Rs up-regulates p105, the precursor of NF-kappaB, while concomitantly decreasing inhibitor of nuclear factor-kappaBalpha. Sig-1R knockdown also accelerates the conversion of p105 to the active form p50. Lastly, we showed that knockdown of Sig-1Rs potentiates H(2)O(2)-induced apoptosis; the action is blocked by either the NF-kappaB inhibitor oridonin or overexpression of Bcl-2. Thus, these findings suggest that Sig-1Rs promote cell survival, at least in part, by transcriptionally regulating Bcl-2 expression via the ROS/NF-kappaB pathway.

G protein-coupled receptor kinase 5, overexpressed in the α-synuclein up-regulation model of Parkinson's disease, regulates bcl-2 expression

G protein-coupled receptor kinase 5 (GRK5) has been reported to accumulate in Lewy bodies (LBs), a histological hallmark of Parkinson's disease. Recent findings propose that GRK5 might function in Parkinson's disease via phosphorylation of α-synuclein, a major component of LBs. In this study, the changes of the expression levels of GRK5 and its possible effects in Parkinson's disease were evaluated in cell lines and transgenic mice model of α-synuclein overexpression. Both the expression levels of cytoplasmic and nuclear distributed GRK5 were induced an increase via α-synuclein overexpression in vivo and in vitro. The observations that the levels of α-synuclein phosphorylated at Ser-129 (pS129-α-synuclein) remain unchanged despite the downregulation of GRK5 by short hairpin ribonucleic acid (shRNA) transfection suggest that GRK5 is not the sole kinase involved in phosphorylating α-synuclein in Parkinson's disease. In addition, the findings that nuclear accumulation of GRK5 inhibits bcl-2 transcription and expression, at least in part by enhancing histone deacetylase (HDAC) activity, show an unexpected role for nuclear GRK5 in the regulation of an apoptosis-related gene. The present study suggests that GRK5 may be extensively involved in the mechanism of Parkinson's disease.

Regulation of Bcl-2 Expression by HuR in HL60 Leukemia Cells and A431 Carcinoma Cells

Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3'-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability.

CREB, a possible upstream regulator of Bcl-2 in trichosanthin-induced HeLa cell apoptosis

Our previous reports indicated that cyclic AMP response element-binding (CREB) protein was involved in the regulation of Bcl-2 expression in apoptotic HeLa cells induced by trichosanthin (TCS). Here we presented that blockade the binding site of CREB to Bcl-2 by a CRE decoy oligonucleotide abrogated the TCS-decreased Bcl-2 expression. Furthermore, overexpression of phosphorylated CREB (p-CREB) in cells transfected with p-CREB/GFP fusion construct resulted in an increase of Bcl-2 protein content, however, this increase was attenuated by TCS treatment. Therefore, this data supports the hypothesis that CREB is a possible upstream regulator of Bcl-2 in apoptotic HeLa cells induced by TCS. The study provides new insights into understanding the mechanism of TCS in the treatment of cervical cancer.

Role of Foxa1 in regulation of bcl2 expression during oxidative-stress-induced apoptosis in A549 type II pneumocytes

Forkhead box protein A1 (Foxa1) is an evolutionarily conserved winged helix transcription factor that was traditionally considered to be involved in embryonic development and cell differentiation. However, little is known about the role of Foxa1 in oxidative-stress-induced apoptosis. In this study, hydrogen peroxide (H(2)O(2))-induced apoptosis, upregulation of Foxa1, and the role of Foxa1 in the regulation of bcl2 gene expression were studied in A549 type II pneumocytes. H(2)O(2) upregulated Foxa1 mRNA and protein in a time- and dose-dependent manner. Overexpression of Foxa1 promoted apoptosis, whereas Foxa1 deficiency, induced by antisense oligonucleotides, decreased A549 cell apoptosis induced by H(2)O(2), as shown by flow cytometry. Moreover, Foxa1 overexpression decreased the expression of bcl2, while Foxa1 depletion increased the expression of bcl2. Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that Foxa1 bound to bcl2 promoter, and H(2)O(2) promoted its DNA binding activity. Luciferase reporter showed that Foxa1 also decreased the transcription activity of bcl2 promoter under normal conditions and oxidative stress. These results indicate that Foxa1 plays a pro-apoptotic role by inhibiting the expression of anti-apoptotic gene bcl2.

ERK/BCL-2 Pathway in the Resistance of Pancreatic Cancer to Anoikis

Anoikis is a special type of programmed cell death after loss of cell-cell and cell-extracellular matrix interactions. Resistance to anoikis is likely involved in the process of metastasis, specifically during the tumor cell migration through lymph or vascular channels. We have previously shown that BCL-2 confers resistance to other forms of programmed cell death (i.e., apoptosis); furthermore, the extracellular signaling-regulated kinase (ERK) signaling pathway regulates BCL-2 expression. We therefore tested the hypothesis that pancreatic cancer cell lines are resistant to anoikis and this resistance is due to activation of ERK1/2 and subsequent overexpression of BCL-2. Pancreatic cancer cell lines (MIA-PaCa-2 and BxPC-3) were examined for cell death following loss of adherence to extracellular matrix. Subclones of the MIA-PaCa-2 cell line (either selected in vivo for increased metastatic potential [MIA-LM2] or overexpressing BCL-2 [MIA-BCL2]) were also examined for induction of anoikis following loss of extracellular matrix adherence. Finally, the effect of the ERK inhibitor (PD98059) on BCL-2 expression and induction of anoikis was examined. Under conditions of loss of cell-extracellular matrix interaction, pancreatic cancer cells undergo varying amounts of anoikis. Basal levels of activated ERK and BCL-2 paralleled the sensitivity to induction of anoikis. The highly metastatic cell line, MIA-LM2, was more resistant to anoikis than the parental cell line. Inhibition of ERK down-regulated BCL-2 and was associated with restoration of sensitivity to anoikis. Activation of a signaling pathway from ERK to overexpression of BCL-2 may confer resistance to anoikis, a critical step in the development of metastasis. Targeting the ERK/BCL-2 pathway may lead to sensitization of pancreatic cancer to anoikis, thereby decreasing the ability of these cells to metastasize.

Effects of lithium and deafferentation on expression of glycogen synthase kinase-3β, NFκB, β-catenin and pCreb in the chick cochlear nucleus

The avian brainstem serves as a useful model to answer the question of how afferent activity influences the viability of target neurons. Approximately 20–30% of neurons in the avian cochlear nucleus, nucleus magnocellularis (NM) die following deafferentation (i.e., deafness produced by cochlea removal). Interestingly, Bcl-2 mRNA (but not protein) is upregulated in 20–30% of NM neurons following deafferentation. We have recently shown that chronic treatments of lithium upregulates the neuroprotective protein Bcl-2 and increases neuronal survival following deafferentation. The pathways leading to the upregulation of Bcl-2 expression following these two manipulations are unknown. The present experiments examine changes in glycogen synthase kinase-3 beta (Gsk-3β), and transcription factors nuclear factor κB (NFκB), β-catenin, and pCreb following lithium administration and following deafferentation. These molecules are known to be influenced by lithium and to regulate Bcl-2 expression in other model systems. Lithium decreased immunolabeling for Gsk-3β and increased expression for all three transcription factors. Deafferentation, however, did not alter Gsk-3β or NFκB, resulted in lower β-catenin expression, but did increase pCreb immunoreactivity. While it is possible that pCreb is a common link in the regulation of Bcl-2 following these two manipulations, the timing and distribution of pCreb labeling suggests that it is not the sole determinant of Bcl-2 upregulation following deafferentation. It is likely that the regulation of Bcl-2 gene expression by lithium and by deafferentation involves different molecular pathways.

Macrophage Migration Inhibitory Factor Induces B Cell Survival by Activation of a CD74-CD44 Receptor Complex

Macrophage migration inhibitory factor (MIF) is an upstream activator of innate immunity that regulates subsequent adaptive responses. It was previously shown that in macrophages, MIF binds to a complex of CD74 and CD44, resulting in initiation of a signaling pathway. In the current study, we investigated the role of MIF in B cell survival. We show that in B lymphocytes, MIF initiates a signaling cascade that involves Syk and Akt, leading to NF-kappaB activation, proliferation, and survival in a CD74- and CD44-dependent manner. Thus, MIF regulates the adaptive immune response by maintaining the mature B cell population.

A Protein Kinase Cε-Anti-apoptotic Kinase Signaling Complex Protects Human Vascular Endothelial Cells against Apoptosis through Induction of Bcl-2

Endothelial cell apoptosis is associated with vascular injury and predisposes to atherogenesis. Endothelial cells express anti-apoptotic genes including Bcl-2, Bcl-XL and survivin, which also contribute to angiogenesis and vascular remodeling. We report a central role for protein kinase Cepsilon (PKCepsilon) in the regulation of Bcl-2 expression and cytoprotection of human vascular endothelium against apoptosis. Using myristoylated inhibitory peptides, a predominant role for PKCepsilon in vascular endothelial growth factor-mediated endothelial resistance to apoptosis was revealed. Immunoblotting of endothelial cells infected with an adenovirus expressing a constitutively active form of PKCepsilon (Adv-PKCepsilon-CA) or control Adv-beta-galactosidase demonstrated a 3-fold, PKCepsilon-dependent increase in Bcl-2 expression, with no significant change in Bcl-XL, Bad, Bak, or Bax. The induction of Bcl-2 inhibited apoptosis induced by serum starvation or etoposide, and PKCepsilon activation attenuated etoposide-induced caspase-3 cleavage. The functional role of Bcl-2 was confirmed with Bcl-2 antagonist HA-14-1. Inhibition of phosphoinositide 3-kinase attenuated vascular endothelial growth factor-induced protection against apoptosis, and this was rescued by overexpression of constitutively active PKCepsilon, suggesting PKCepsilon acts downstream of phosphoinositide 3-kinase. Co-immunoprecipitation studies demonstrated a physical interaction between PKCepsilon and Akt, which resulted in formation of a signaling complex, leading to optimal induction of Bcl-2. This study reveals a pivotal role for PKCepsilon in endothelial cell cytoprotection against apoptosis. We demonstrate that PKCepsilon forms a signaling complex and acts co-operatively with Akt to protect human vascular endothelial cells against apoptosis through induction of the anti-apoptotic protein Bcl-2 and inhibition of caspase-3 cleavage.

CREB-mediated Bcl-2 expression in trichosanthin-induced Hela cell apoptosis

Bcl-2 plays a pivotal role in the control of cell death and is down-regulated in trichosanthin (TCS)-induced cell apoptosis. Because Bcl-2 expression is regulated by the transcription factor cyclic AMP response element-binding protein (CREB), we investigated the role of CREB activation in TCS-induced Hela cells apoptosis. Our results showed that TCS-caused Hela cell apoptosis was accompanied by the decrease of Bcl-2 and phosphorylated CREB protein levels. Interesting, this inhibitive effect can be abolished by the combined treatment of TCS/cAMP agonists. Furthermore, TCS-mediated Bcl-2 protein was abrogated by the suppression of CREB expression with antisense treatment, and blocking the interaction between CREB-binding protein and the Bcl-2 cyclic AMP-responsive element (CRE) by a CRE decoy oligonucleotide. Therefore, these data support the hypothesis that CREB plays a critical role in the regulation of Bcl-2 expression in TCS-induced Hela cell death.

Calcitonin Gene-Related Peptide (CGRP) Inhibits Apoptosis by Differential Phosphorylation of BAD Serines 112, 136 and 155 (84.12)

Abstract Calcitonin gene-related peptide (CGRP) is a sensory neuropeptide that inhibits early B cell differentiation. It enhances B cell survival by upregulating Pim-1, a S/T kinase that regulates Bcl-2 expression, and by shifting expression of Bcl-2 family members to anti-apoptotic members. Studies were done to further characterize CGRP’s effect on Bad phoshorylation. Inactive Bad (a pro-apoptotic Bcl-2 family member) is phosphorylated at ser112, ser136, and ser155 by different mechanisms. Bad ser112is a Pim-1 target. Dephosphorylated Bad forms heterodimers with Bcl-XL and Bcl-2, blocking their anti-apoptotic effects. Studies were done to determine if CGRP influences Bad phosphorylation. 70Z/3 cells were grown in low serum and treated with CGRP. Bad phosphorylation at ser112, ser136 and ser155 was measured by western blot. Serum starvation decreased Bad phosphorylation at 4 hrs and completely depletes phospho-Bad at 24 hours. CGRP upregulated phospho-ser112 and -ser155, peaking at 8–12 hrs and returning to untreated levels at 24 hrs. The results show that CGRP induced Bad phosphorylation at ser112 and ser155 is correlated with increased Pim-1 expression. In contrast, CGRP had no effect on ser136phosphorylation. This study provides further information on cellular and molecular mechanisms underlying CGRP’s protective effect in immature B cells.

Upregulation of SOCS-1 and Downregulation of miR-214 Correlate with Disease Course in Chronic Lymphocytic Leukemia.

SOCS-1, suppressor of cytokine signaling, is a negative regulator of the JAK2/STAT signal transduction pathway. This pathway, often deregulated in myeloproliferative disorders, has not yet been implicated in chronic lymphocytic leukemia (CLL). We demonstrated SOCS-1 upregulation and miR-214 downregulation with clinical response in a patient treated with cladribine. Similar changes were not seen in a treated patient with progressive disease. MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate expression of other genes by complementary pairing at the seed sequence in the 3′-UTR of the target mRNA. Some patients with CLL reportedly regulate bcl-2 expression by downregulating miR-15a and miR-16-1. Although no experimentally verified targets have been demonstrated for miR-214, complete inhibition of miR-214 by anti-sense inhibitors prevents apoptosis in Hela cells. Of the 44 predicted human miRNAs that target SOCS-1 (http://microrna.sanger.ac.uk), we determined the expression of 24 of these by real-time PCR using the Early Access Human Panel from Applied Biosystems. MiR-214 was the only downregulated miRNA after treatment. The other 23 miRNAs were either upregulated or unchanged. We demonstrated SOCS-1 protein upregulation by two fold after cladribine in the “responsive” patient without a concomitant increase in the transcript level as measured by real time PCR. SOCS-1 expression is often silenced by DNA promoter methylation in hematologic and solid tumor malignancies. We report another mechanism of regulation through miRNA translational repression. This suggests involvement of the JAK2/STAT pathway in B-cell survival in CLL.

Interaction of Vascular Endothelial Growth Factor 165 with Neuropilin-1 Protects Rheumatoid Synoviocytes from Apoptotic Death by Regulating Bcl-2 Expression and Bax Translocation

Rheumatoid arthritis (RA) synoviocytes are resistant to apoptosis and exhibit a transformed phenotype, which might be caused by chronic exposure to genotoxic stimuli including reactive oxygen species and growth factors. In this study, we investigated the role of vascular endothelial growth factor165 (VEGF165), a potent angiogenic factor, and its receptor in the apoptosis of synoviocytes. We demonstrated here that neuropilin-1, rather than fms-like tyrosine kinase-1 and kinase insert domain-containing receptor, is the major VEGF165 receptor in the fibroblast-like synoviocytes. Neuropilin-1 was highly expressed in the lining layer, infiltrating leukocytes, and endothelial cells of rheumatoid synovium. The production of VEGF165, a ligand for neuropilin, was significantly higher in the RA synoviocytes than in the osteoarthritis synoviocytes. The ligation of recombinant VEGF165 to its receptor prevented the apoptosis of synoviocytes induced by serum starvation or sodium nitroprusside (SNP). VEGF165 rapidly triggered phospho-Akt and phospho-ERK activity and then induced Bcl-2 expression in the rheumatoid synoviocytes. The Akt or ERK inhibitor cancelled the protective effect of VEGF165 on SNP-induced synoviocyte apoptosis. Moreover, VEGF165 blocks SNP-induced Bcl-2 down-regulation as well as SNP-induced Bax translocation from the cytosol to the mitochondria. The down-regulation of the neuropilin-1 transcripts by short interfering RNA caused spontaneous synoviocyte apoptosis, which was associated with both the decrease in Bcl-2 expression and the increase in Bax translocation to mitochondria. Collectively, our data suggest that the interaction of VEGF165 with neuropilin-1 is crucial to the survival of rheumatoid synoviocytes and provide important implications for the abnormal growth of synoviocytes and therapeutic intervention in RA.