Bcl-x Promoter Research Articles

Differential Regulation of Bcl-xL Gene Expression by Corticosterone, Progesterone, and Retinoic Acid.

Corticosterone (CT), progesterone (PG), and retinoic acid (RA) are capable of inhibiting Doxorubicin (Dox) from inducing apoptosis in rat cardiomyocytes. Mechanistically, CT, PG, and RA induce increases of Bcl-xL protein and mRNA, and activate a 3.2kb bcl-x gene promoter. CT and RA, but not PG, induced the activity of a 0.9kb bcl-x promoter, containing sequences for AP-1 and NF-kB binding. RA, but not CT or PG, induced NF-kB activation. CT, but not PG or RA, induced AP-1 activation, and induction of the 0.9kb bcl-x reporter by CT was inhibited by dominant negative c-Jun TAM-67 or removal of AP-1 binding site. Therefore, although CT, PG, and RA all induce Bcl-xL mRNA and protein, three independent mechanisms are in operation: while CT induces Bcl-xL via AP-1 transcription factor, and RA induces NF-kB activation and bcl-x promoter activity, PG induces Bcl-xL via a mechanism independent of NF-kB or AP-1.

Role of 3′-5′-cyclic adenosine monophosphate on the epidermal growth factor dependent survival in mammary epithelial cells

Epidermal growth factor (EGF) has been suggested to play a key role in the maintenance of epithelial cell survival during lactation. Previously, we demonstrated that EGF dependent activation of PI3K pathway prevents apoptosis in confluent murine HC11 cells cultured under low nutrient conditions. The EGF protective effect is associated with increased levels of the antiapoptotic protein Bcl-XL. Here, we identify the EGF-dependent mechanism involved in cell survival that converges in the regulation of bcl-X expression by activated CREB. EGF induces Bcl-XL expression through activation of a unique bcl-X promoter, the P1; being not only the PI3K/AKT signaling pathway but also the increase in cAMP levels and the concomitant PKA/CREB activation necessary for both bcl-XL upregulation and apoptosis avoidance. Results presented in this work suggest the existence of a novel connection between the EGF receptor and the adenylate cyclase that would have an impact in preventing apoptosis under low nutrient conditions.

Deregulation of Apoptotic Factors Bcl-xL and Bax Confers Apoptotic Resistance to Myeloid-derived Suppressor Cells and Contributes to Their Persistence in Cancer

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that accumulate in response to tumor progression. Compelling data from mouse models and human cancer patients showed that tumor-induced inflammatory mediators induce MDSC differentiation. However, the mechanisms underlying MDSC persistence is largely unknown. Here, we demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis as compared with myeloid cells with the same phenotypes from tumor-free mice. Consistent with the decreased apoptosis, cell surface Fas receptor decreased significantly in tumor-induced MDSCs. Screening for changes of key apoptosis mediators downstream the Fas receptor revealed that expression levels of IRF8 and Bax are diminished, whereas expression of Bcl-xL is increased in tumor-induced MDSCs. We further determined that IRF8 binds directly to Bax and Bcl-x promoter in primary myeloid cells in vivo, and IRF8-deficient MDSC-like cells also exhibit increased Bcl-xL and decreased Bax expression. Analysis of CD69 and CD25 levels revealed that cytotoxic T lymphocytes (CTLs) are partially activated in tumor-bearing hosts. Strikingly, FasL but not perforin and granzymes were selectively activated in CTLs in the tumor-bearing host. ABT-737 significantly increased the sensitivity of MDSCs to Fas-mediated apoptosis in vitro. More importantly, ABT-737 therapy increased MDSC spontaneous apoptosis and decreased MDSC accumulation in tumor-bearing mice. Our data thus determined that MDSCs use down-regulation of IRF8 to alter Bax and Bcl-xL expression to deregulate the Fas-mediated apoptosis pathway to evade elimination by host CTLs. Therefore, targeting Bcl-xL is potentially effective in suppression of MDSC persistence in cancer therapy.

Abstract 2285: Stat3 modulates heregulin (HRG)-induced progesterone receptor (PR) transcriptional activation

Abstract The signal transducer and activator of transcription (Stat) family of proteins was found to be involved in crosstalks with both steroid hormones and type I receptor tyrosine kinases (RTKs) signaling pathways. We have previously demonstrated that HRG, a ligand of RTKs, transactivates PR both in C4HD cells from an experimental model of hormonal carcinogenesis in which MPA induced mammary adenocarcinomas in Balb/c mice, and in the human breast cancer cell line T47D (Mol Cell Biol. 2003 Feb;23(3):1095-111). We have also shown that HRG induces Stat3 tyrosine phosphorylation and transcriptional activation by a mechanism that requires PR signaling (Mol Cell Biol. 2009 Mar;29(5):1249-65). In the present work we explored whether Stat3 acting as a coactivator, could modulate ligand-independent activation of PR by HRG. Assessment of the expression of the progestin-regulated gene bcl-X showed that HRG treatment of C4HD cells resulted in an increase in bcl-X protein levels. This effect was completely abolished when Stat3 expression was silenced using siRNAs. HRG treatment of cells transfected with a luciferase reporter plasmid under the control of the murine bcl-X promoter which contains two progesterone response elements (PREs), resulted in an increase in luciferase activity. HRG had no effect on PR transcriptional activation when cells were transfected with a mutant vector containing a deletion spanning both PREs in bcl-X promoter. We assessed the specific association of Stat3 and PR to the PRE region of bcl-X promoter in the context of living cells by performing Chromatin Immunoprecipitation (chIP) Assays. We found that HRG treatment of primary cultures of C4HD cells induced PR and Stat3 recruitment to the bcl-X promoter. HRG also induced PR and Stat3 occupancy of the stably integrated MMTV promoter in T47D-Cat0 breast cancer cells. By performing sequential chIP assays we showed that HRG induced simultaneous PR and Stat3 occupancy of the bcl-X promoter region. We explored Stat3 role in the non-classical mechanism of action of PR, where PR is recruited to p21 promoter indirectly through interaction with Sp1 transcription factor. Interestingly, when cells were treated with HRG, we detected Stat3 binding to the Sp1 binding sites of the PR-regulated p21 promoter, together with Sp1 and PR. These results provide the first evidence that Stat3 modulates ligand-independent activation of PR by HRG in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2285. doi:10.1158/1538-7445.AM2011-2285

Novel role of signal transducer and activator of transcription 3 as a progesterone receptor coactivator in breast cancer

Interactions between progesterone receptor (PR) and signal transducer and activator of transcription 3 (Stat3)-mediated signaling pathways have already been described. In the present study, we explored the capacity of Stat3 to functionally interact with progesterone receptor (PR) and modulate PR transcriptional activation in breast cancer cells. We found that the synthetic progestin medroxyprogesterone acetate (MPA) induced the association of a PR/Stat3 complex in which Stat3 acts as a coactivator of PR. We demonstrated that Stat3 activation is required for MPA modulation of the endogenous genes bcl-X and p21 CIP1 which are involved in MPA-induced cell cycle regulation. Stat3 activity as a coactivator of PR was observed in both the classical and nonclassical ligand activated-PR transcriptional mechanisms, since the effects described were identified in the bcl-X promoter which contains a progesterone responsive element and in the p21 CIP1 promoter which carries Sp1 binding sites where PR is recruited via the transcription factor Sp1. The data herein presented identifies a potential therapeutic intervention for PR-positive breast tumors consisting of targeting Stat3 function or PR/Stat3 interaction which will result in the inhibition of PR function.

Growth Factor Stimulation Induces Cell Survival by c-Jun·ATF2-dependent Activation of Bcl-XL

Growth factor stimulation induces c-Jun-dependent survival of primary endothelial cells. However, the mechanism of c-Jun anti-apoptotic activity has not been identified. We here demonstrate that in response to growth factor treatment, primary human endothelial cells as well as mouse fibroblasts respond with an increased expression of c-Jun that forms a complex with ATF2. This complex activates the expression of the anti-apoptotic protein Bcl-X(L). By site-directed mutagenesis experiments, we identified two AP-1-binding sites located within the proximal promoter of the Bcl-X gene. Site-directed mutagenesis demonstrated that these AP-1 sites are required for the transcriptional activation of the promoter. Chromatin immunoprecipitation experiments show that in response to growth factor treatment, the heterodimer c-Jun.ATF2 binds to these functional AP-1 sites. Silencing of either c-Jun or ATF2 demonstrated that both nuclear factors are required for the activation of the proximal Bcl-X promoter. Taken together, our experiments provide evidence that growth factor-independent signaling pathways converge in the formation of an active c-Jun.AFT2 dimer, which induces the expression of the anti-apoptotic factor Bcl-X(L) that mediates a pro-survival response.

Oncoproteomic Analysis Reveals Co-Upregulation of RELA and STAT5 in Carboplatin Resistant Ovarian Carcinoma

BackgroundOvarian cancer is one of the most lethal types of female malignancy. Although most patients are initially responsive to platinum-based chemotherapy, almost all develop recurrent chemoresistant tumors and succumb to their diseases. Elucidating the pathogenesis underlying drug resistance is fundamental to the development of new therapeutics, leading to improved clinical outcomes in these patients.Methods and FindingsWe compared the proteomes of paired primary and recurrent post-chemotherapy ovarian high-grade serous carcinomas from nine ovarian cancer patients using CIEF/Nano-RPLC coupled with ESI-Tandem MS. As compared to their primary tumors, more than half of the recurrent tumors expressed higher levels of several proteins including CP, FN1, SYK, CD97, AIF1, WNK1, SERPINA3, APOD, URP2, STAT5B and RELA (NF-κB p65), which were also validated by quantitative RT-PCR. Based on shRNA screening for the upregulated genes in in vitro carboplatin-resistant cells, we found that simultaneous knockdown of RELA and STAT5B was most effective in sensitizing tumor cells for carboplatin treatment. Similarly, the NF-κB inhibitor, BMS-345541, and the STAT5 inhibitor, Dasatinib, significantly enhanced cell sensitivity to carboplatin. Moreover, both RELA and STAT5 are known to bind to the promoter region of Bcl-X, regulating its promoter activity. In this regard, augmented Bcl-xL expression was detected in carboplatin-resistant cells. Combined ectopic expression of RELA and STAT5B enhanced Bcl-xL promoter activity while treatment with BMS-345541 and Dasatinib decreased it. Chromatin immunoprecipitation of the Bcl-X promoter region using a STAT5 antibody showed induction of RELA and STAT5 DNA-binding segments both in naïve cells treated with a high concentration of carboplatin as well as in carboplatin-resistant cells.ConclusionsProteomic analysis identified RELA and STAT5 as two major proteins associated with carboplatin resistance in ovarian tumors. Our results further showed that NF-κB and STAT5 inhibitor could sensitize carboplatin-resistant cells and suggest that such inhibitors can be used to benefit patients with carboplatin-resistant recurrent ovarian cancer.

Overexpression of GATA1 Confers Chemotherapy Resistance in Pediatric Acute Megakaryocytic Leukemia.

Abstract 2039Poster Board II-16Acute megakaryocytic leukemia (AMkL; M7) is a biologically heterogeneous form of AML, representing ∼10% of pediatric and 1-2% of adult AML cases. AMkL is the most common AML subtype of children with Down syndrome (DS). DS children with AMkL have an excellent prognosis with EFS rates of 80-100% when treated with ara-C/anthracycline-based protocols, in contrast to the <30% EFS rates of non-DS children with AMkL. This also contrasts to the ∼50% EFS rates of non-DS children with AML overall, indicating that AMkL is an extremely poor risk group amongst non-DS children with AML despite the use of intensive chemotherapy-based protocols. These clinical data make a compelling argument that new therapies are essential to improve the treatment outcome of this aggressive disease. Acquired somatic mutations of the transcription factor gene, GATA1 (localized to Xp11.23), have been detected uniformly in nearly all DS AMkL cases, but not in non-DS AML and non-AMkL DS leukemia cases. The net effect of GATA1 mutations is an introduction of early stop codons and synthesis of a shorter GATA1 protein (designated GATA1s) that has altered transactivation activity, potentially contributing to the uncontrolled proliferation of immature megakaryocytes. It is conceivable that the altered GATA1 function between DS and non-DS AMkL may account for differential expression of GATA1 target genes in these two groups of patients. On the other hand, overexpression of GATA1 in megakaryoblasts from non-DS children with AMkL compared to myeloblasts from non-DS children with other subtypes of AML may contribute to differences in chemotherapy sensitivity via regulation of GATA1 target genes. We previously reported that GATA1 mutations in DS AMkL are associated with decreased expression of cytidine deaminase (encodes an enzyme which can convert ara-C to ara-U, the inactive form of the drug), thus contributing to the enhanced ara-C sensitivity of DS AMkL blasts. Further, when GATA1 was ectopically expressed in a DS AMkL cell line, CMK, it caused significantly increased resistance to ara-C. In the present study, we confirmed overexpression of GATA1 in non-DS AMkL blasts compared to non-DS AML blasts by real-time RT-PCR quantitation of GATA1 transcripts in our cohort of patient samples. shRNA knockdown of GATA1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivities to ara-C and daunorubicin, the two main drugs used for AML treatment, and significantly increased basal level apoptosis. This was accompanied by significantly decreased Bcl-xL transcript and protein levels in the GATA1 shRNA knockdown clones compared to a shRNA negative control. Binding of GATA1 to the two GATA elements in Bcl-x promoter and transactivation of Bcl-x promoter activity by GATA1 was demonstrated by ChIP assays and luciferase reporter assays, respectively, in Meg-01 cells. In our cohort of non-DS AMkL and AML patient samples, significant overexpression of Bcl-xL in non-DS AMkL compared to non-DS AML cases and a significant correlation between Bcl-xL and GATA1 transcripts were detected. Besides Bcl-xL, additional GATA1 targets (e.g. TNF) related to apoptosis were also identified by gene expression and ChIP-on-ChIP microarray analyses. Interestingly, our microarray data also suggest that GATA1 may have an impact on PI3-kinase/Akt pathway through modulating directly or indirectly a group of genes within the pathway. Western blotting revealed increased phosphorylation of Akt in the GATA1 knockdown clones compared to the negative control cells. Previous studies reported that histone deacetylase inhibitors (HDACIs) treatment causes hyperacetylation and subsequent degradation of GATA1, suggesting that these agents may be effective in targeting GATA1 in AMkL. Treatment of Meg-01 cells with an HDACI, valproic acid (VPA), resulted in decreased protein levels for GATA1 and Bcl-xL and increased phosphorylation of Akt. Co-treatment of Meg-01 cells with VPA and ara-C resulted in synergistic induction of apoptosis and activation of caspase-3. This drug synergy was amplified when a non-toxic dose of the PI3-kinase inhibitor LY294002 was added. Our results demonstrate that GATA1 causes resistance to chemotherapy in non-DS AMkL by promoting AMkL blast survival through regulating its target genes. Treatment of AMkL may be improved by integrating HDACI and PI3-kinase or Akt inhibitors into the chemotherapy of this disease. Disclosures:No relevant conflicts of interest to declare.

Up-Regulation of Bcl-xl by Hepatocyte Growth Factor in Human Mesothelioma Cells Involves ETS Transcription Factors

Bcl-xl and the hepatocyte growth factor (HGF) receptor c-Met are both highly expressed in mesotheliomas, where they protect cells from apoptosis and can confer resistance to conventional therapeutic agents. In our current study, we investigate a model for the transcriptional control of Bcl-xl that involves ETS transcription factors and the HGF/Met axis. In addition, the effects of activated c-Met on the phosphorylation of the ETS family transcriptional factors were examined. The transient expression of ETS-2 and PU.1 cDNAs in mesothelioma cell lines resulted in an increase in the promoter activity of Bcl-xl and consequently in its mRNA and protein expression levels, whereas the transcriptional repressor Tel suppressed Bcl-xl transcription. The activation of the HGF/Met axis led to rapid phosphorylation of ETS family transcription factors in mesothelioma cells through the mitogen-activated protein kinase pathway and via nuclear accumulation of ETS-2 and PU.1. A chromatin immunoprecipitation assay further demonstrated that the activation of c-Met enhanced the binding of ETS transcriptional factors to the Bcl-x promoter. Finally, we determined the Bcl-xl and phosphorylated c-Met expression levels in mesothelioma patient samples; these data suggest a strong correlation between Bcl-xl and phosphorylated c-Met levels. Taken together, these findings support a role for c-Met as an inhibitor of apoptosis and an activator of Bcl-xl.

STAT5 Mediates Antiapoptotic Effects of Methylprednisolone on Oligodendrocytes

Methylprednisolone (MP), a synthetic glucocorticoid agonist, is widely used for the clinical therapy of white matter diseases in the nervous system, such as spinal cord injury and multiple sclerosis. In addition to its potent anti-inflammatory and antioxidant properties, we recently discovered a selective antiapoptotic effect of MP on oligodendrocytes via the activation of the glucocorticoid receptor (GR) and the upregulation of bcl-X(L), a splicing isoform of the bcl-x gene. Based on published findings of the functional interactions between GR and STAT5, a transcription factor from the family of signal transducers and activators of transcription (STAT), we examined whether the glucocorticoid signaling pathway interacts with STAT5 to upregulate bcl-X(L) and protect oligodendrocytes. We show herein that (1) the GR and STAT5 complex is present on the STAT5-binding site of the bcl-x promoter region in oligodendrocytes; (2) the overexpression of an activated form of STAT5 prevents alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced oligodendrocyte cell death; and (3) this prevention is lost when the STAT5 gene is knocked down. Thus, our results provide one molecular mechanism underlying the postinjury protective effects of oligodendrocytes by stress hormones.

Androgen receptor‐dependent regulation of Bcl‐xL expression: Implication in prostate cancer progression

Recently we reported that silencing the androgen receptor (AR) gene reduced Bcl-xL expression that was associated with a profound apoptotic cell death in prostate cancer cells. In this study we further investigated AR-regulated Bcl-xL expression. Prostate cancer cell line LNCaP and its sublines, LNCaP/PURO and LNCaP/Bclxl, were used for cell proliferation assay and xenograft experiments in nude mice. Luciferase gene reporters driven by mouse or human bcl-x gene promoter were used to determine androgen regulation of Bcl-xL expression. RT-PCR and Western blot assays were conducted to assess Bcl-xL gene expression. Chromatin immunoprecipitation assay was performed to determine AR interaction with Bcl-xL promoter. Bcl-xL-induced alteration of gene expression was examined using cDNA microarray assay. In cultured prostate cancer LNCaP cells, androgen treatment significantly increased Bcl-xL expression at mRNA and protein levels via an AR-dependent mechanism. Promoter analyses demonstrated that the AR mediated androgen-stimulated bcl-x promoter activation and that the AR interacted with bcl-x promoter. Enforced expression of Bcl-xL gene dramatically increased cell proliferation in vitro and promoted xenograft tumor growth in vivo. Genome-wide gene profiling analysis revealed that Bcl-xL expression was significantly higher in metastatic and castration-resistant diseases compared to normal prostate tissues or primary cancers. Bcl-xL overexpression significantly increased the expression of cyclin D2, which might be responsible for Bcl-xL-induced cell proliferation and tumor growth. Taken together, our data strongly suggest that androgen stimulates Bcl-xL expression via the AR and that increased Bcl-xL expression plays a versatile role in castration-resistant progression of prostate cancer.

Human TIEG2/KLF11 induces oligodendroglial cell death by downregulation of Bcl-XL expression

TGF-beta-induced apoptosis is essential for embryonic development and mainteanance of adult tissues. Impairment of the apoptotic pathway, regulated by TGF-beta, plays a center role in tumorigenesis and manifestations of different diseases. TIEG2/KLF11 is a recently identified human TGF-beta-inducible zinc finger protein belonging to the family of Sp1/KLF-like transcription factors. In human and murine tissues it has been shown that TIEG1 and TIEG2 induce apoptosis and inhibit cell growth. Since TGF-beta and Tieg1 are able to induce apoptosis in the oligodendroglial cell line OLI-neu, we analysed the ability of TIEG2 to mimic the effects observed after treatment with TGF-beta and overexpression of Tieg1. Herein we report that TIEG2 induces Caspase3-dependent apoptosis in murine OLI-neu cells. Furthermore, we could demonstrate that TIEG2 decreases the levels of the anti-apoptotic protein Bcl-X(L) and inhibits transcription driven by the Bcl-X(L) promoter. These data suggest that TIEG2 serves as a downstream mediator of TGF-beta, bridging TGF-beta-dependent signaling to the intracellular pathway of apoptosis.

Regulation of Bcl-xL expression in lung vascular smooth muscle.

Pulmonary hypertension is characterized by thickened pulmonary arterial walls due to increased number of pulmonary artery smooth muscle cells (PASMC). Apoptosis of PASMC may play an important role in regulating the PASMC number and may be useful for reducing pulmonary vascular thickening. The present study examined the regulation of an anti-apoptotic protein Bcl-x(L). Bcl-x(L) expression was found to be increased in the pulmonary artery of chronic hypoxia-treated rats with pulmonary vascular remodeling. Adenovirus-mediated gene transfer of Bcl-x(L) indeed showed that this protein has anti-apoptotic activities in PASMC. Treatment of remodeled pulmonary artery with sodium nitroprusside (SNP) reduced Bcl-x(L) expression by targeting the bcl-x(L) promoter. The bcl-x(L) promoter contains two GATA elements, and SNP decreases the GATA-4 DNA-binding activity. Overexpression of GATA-4 attenuated the SNP-mediated suppression of Bcl-x(L) expression, providing direct evidence for the role of GATA-4 in Bcl-x(L) gene transcription. We established that SNP targets the 250 proximal region of the gata4 promoter and suppresses its gene transcription. Thus, inducers of pulmonary hypertension enhance anti-apoptotic Bcl-x(L) gene transcription, which can be suppressed by targeting gata4 gene transcription.

Prevention of Ischemic Neuronal Death by Intravenous Infusion of a Ginseng Saponin, Ginsenoside Rb1, That Upregulates Bcl-xL Expression

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb(1) (gRb(1)) (C(54)H(92)O(23), molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb(1)-induced expression of gene products responsible for neuronal death or survival, we showed that gRb(1) stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-x(L) in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb(1) treatment. Ginsenoside Rb(1) appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.

The Tumor Necrosis Factor-like Weak Inducer of Apoptosis (TWEAK)-Fibroblast Growth Factor-inducible 14 (Fn14) Signaling System Regulates Glioma Cell Survival via NFκB Pathway Activation and BCL-XL/BCL-W Expression

The Fn14 gene encodes a type Ia transmembrane protein that belongs to the tumor necrosis factor receptor superfamily. We recently showed that fibroblast growth factor-inducible 14 (Fn14) is overexpressed in migrating glioma cells in vitro and in glioblastoma multiforme clinical specimens in vivo. To determine the biological role of Fn14 in brain cancer progression, we examined the activity of Fn14 as a potential mediator of cell survival. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-stimulated glioma cells had increased cellular resistance to cytotoxic therapy-induced apoptosis. Either TWEAK treatment or Fn14 overexpression in glioma cells resulted in the activation of NFkappaB and subsequently the translocation of NFkappaB from the cytoplasm to the nucleus. In addition, Fn14 activation induced BCL-XL and BCL-W mRNA and protein levels, and this effect was dependent upon NFkappaB transcriptional activity. Substitution of a putative NFkappaB binding site identified in the BCL-X promoter significantly decreased Fn14-induced transactivation. Furthermore Fn14-induced transactivation of the BCL-X promoter was also diminished by the super-repressor IkappaBalpha mutant, which specifically inhibits NFkappaB activity, and by mutations in the NFkappaB binding motif of the BCL-X promoter. Additionally small interfering RNA-mediated depletion of either BCL-XL or BCL-W antagonized the TWEAK protective effect on glioma cells. Our results suggest that NFkappaB-mediated up-regulation of BCL-XL and BCL-W expression in glioma cells increases cellular resistance to cytotoxic therapy-induced apoptosis. We propose that the Fn14 protein functions, in part, through the NFkappaB signaling pathway to up-regulate BCL-XL and BCL-W expression to foster malignant glioblastoma cell survival. Targeted therapy against Fn14 as an adjuvant to surgery may improve management of invasive glioma cells and advance the outcome of this devastating cancer.

Steroid Hormones Induce bcl-X Gene Expression through Direct Activation of Distal Promoter P4

Bcl-X exists in at least five different isoforms with complex effects on programmed cell death. Glucocorticoids and progestins control bcl-X expression and influence the ratio between bcl-X(L) (antiapoptotic isoform) and bcl-X(S) (proapoptotic isoform) in different tissues. The 5'-UTR region of the mouse bcl-X gene contains at least five different promoters, which exhibit a tissue-specific pattern of promoter usage. Several mRNAs with different 5'-leading exons can be generated upon promoter activation. Here we explore the potential of the various bcl-X gene promoters to be regulated by glucocorticoids or progestins. We found that the region located immediately upstream of promoter 4 (P4) contains two hormone response element (HRE)-like sequences at positions -3040 (HRE I) and -3001 (HRE II) relative to the translation initiation codon. These HRE-like sequences confer hormone responsiveness to a core promoter and bind glucocorticoid or progesterone receptors in vitro. Point mutations of both HREs that prevent steroid receptor binding also eliminate hormonal inducibility. In cells treated with glucocorticoids, the hormone receptor is recruited to the P4 region containing the HREs. Analysis of the products of the endogenous bcl-X in epithelial mammary cells showed that only transcripts originating from P4 increased upon hormone treatment. This observation correlates with the induction of the bcl-X(L) mRNA, suggesting that P4 is one of the bcl-X promoters responsible for the generation of this antiapoptotic isoform.

Glucocorticoids Inhibit Apoptosis during Fibrosarcoma Development by Transcriptionally Activating Bcl-xL

Glucocorticoids influence many physiological processes, and in particular apoptosis, often with opposite effects depending on the cell type examined. We found that during fibrosarcoma development there is a strong increase in apoptosis at the tumor stage, which is repressed by dexamethasone to levels observed in normal fibroblasts. The anti-apoptotic Bcl-2 family protein Bcl-x(L) is induced by dexamethasone at the transcriptional level at all stages of fibrosarcoma development. The ligand-activated glucocorticoid receptor (GR) activates the Bcl-x promoter in transient transfection experiments, and GR binds to specific Bcl-x promoter sequences in vitro and in vivo. Furthermore, a GR antagonist abolishes this effect, indicating that Bcl-x(L) induction is mediated by GR. Importantly, exogenous Bcl-x(L) inhibits apoptosis and caspase-3 activity in fibrosarcoma cells to levels found in dexamethasone-treated fibrosarcoma cells. We conclude that Bcl-x(L) is a key target mediating the anti-apoptotic effects of glucocorticoids during fibrosarcoma development. These observations provide further understanding of the molecular basis of glucocorticoid regulation of cell death during tumorigenesis.

The function of the bcl-xpromoter in erythroid progenitor cells

The protein Bcl-x(L) is essential for survival of erythroid progenitor cells, and it increases substantially during late erythrocyte differentiation due to an increase of mRNA. We mapped the transcription start sites of bcl-x mRNA in mouse and human erythroblasts, and we analyzed the function of the mouse bcl-x promoter by transient and stable transfection assays in a mouse erythroid cell line using plasmids containing the bcl-x promoter fused to a luciferase reporter gene. In mouse erythroblasts, a cluster of start sites at positions -664, -655, and -644 relative to the ATG initiation codon account for almost all transcripts. Human erythroblasts exhibit a start site at -654 that is homologous to the triplet in the mouse. A short sequence element in the mouse bcl-x promoter that includes nucleotides -1804 through -1734 was identified as very important for transcription. This element also showed strong enhancerlike activity in concert with the SV40 promoter in an enhancer test vector. Analyses of mutations indicated that 2 short sequences within the element, about 15 base pair apart, are necessary for full enhancer activity. Gel shift experiments with oligonucleotides representing these sequences revealed specific binding of nuclear proteins from erythroblasts. Some of these proteins are regulated during the late erythroid differentiation.

The Brn-3a POU family transcription factor stimulates p53 gene expression in human and mouse tumour cells

The Brn-3a POU family transcription factor is able to induce the expression of genes encoding anti-apoptotic proteins such as Bcl-2 and Bcl-x and protects neuronal cells from apoptosis. This effect is opposed by the pro-apoptotic p53 protein which completely inhibits the ability of Brn-3a to activate the Bcl-2 and Bcl-x promoters. Here we demonstrate that Brn-3a is able to stimulate p53 expression. Thus, in co-transfection experiments, Brn-3a activates the p53 promoter acting via a region from +22 to +67, located between the most proximal (+1) and the most distal (+105) transcriptional start sites. Similarly, reduction of Brn-3a expression using anti-sense constructs reduces endogenous p53 expression in human neuroblastoma or cervical carcinoma cell lines growing in vitro and as tumours in nude mice whilst increasing Brn-3a levels enhances p53 expression. These results suggest the existence of a negative feedback loop in which elevated Brn-3a expression induces the expression of p53 which, in turn, antagonises the anti-apoptotic activity of Brn-3a.

Bruton’s tyrosine kinase targets NF-κB to the bcl-x promoter via a mechanism involving phospholipase C-γ2 following B cell antigen receptor engagement

Disruption of Bruton’s tyrosine kinase (BTK) function leads to x-linked immunodeficiency ( xid) in mice. BTK-deficient ( btk −/−) B cells are defective for survival. Prior studies show that BTK is required for the induction of Bcl-x L following B cell antigen receptor (BCR) engagement. However, the mechanism underlying Bcl-x L induction in response to BCR ligation remains unresolved. We now demonstrate that BTK regulates bcl-x expression by transcriptional control in response to BCR engagement. BTK targets nuclear factor-κB (NF-κB) to activate the bcl-x promoter via a phospholipase C-γ2 (PLC-γ2)-dependent mechanism. Perturbation of the BTK/PLC-γ2/NF-κB signaling axis likely contributes to the defective expression of bcl-x and compromised survival of xid B cells.