Regulation Of Bim Research Articles

Transcriptional and post-translational regulation of Bim is essential for TGF-β and TNF-α-induced apoptosis of gastric cancer cell

BackgroundTumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) are well known as central signaling molecules in natural antitumor mechanisms. However, some cancer cells are resistant to TNF-α or TGF-β-induced death signaling. Herein, we investigated synergistic activities of TGF-β and TNF-α and molecular mechanisms involved in apoptosis of gastric cancer cells. MethodsSNU620, a human gastric carcinoma cell line was tested for cell viability by treatment of TGF-β in combination with TNF-α. Cell apoptosis, proliferation, caspase activation and gene expression were tested using flow cytometry, Western blot, MTT assay, luciferase assay and real-time qRT-PCR analysis. Knockdown of target genes were performed using lentiviral shRNA system. ResultsTGF-β sensitizes SNU620 cells undergoing TNF-α-induced caspase-dependent apoptosis. TNF-α and TGF-β synergistically induced the degradation of poly(ADP–ribose) polymerase (PARP) and caspase cascade activation. We also confirmed that c-Jun NH2-terminal kinase (JNK) and Smad3 play critical roles in the apoptotic pathway. In addition, a pro-apoptotic protein Bim was critical for apoptosis and was regulated by TGF-β and TNF-α at the transcriptional and post-translational levels. Expression of Bim was induced at the transcriptional level by Smad3 while Bim protein stability was maintained by a JNK-mediated pathway. ConclusionBy understanding the synergistic activation of TGF-β and TNF-α in apoptosis, we may have a chance to identify good therapeutic approaches for the treatment of cancers that are resistant to death signals. General significanceOur results indicate that TGF-β and TNF-α act in concert to activate apoptosis in gastric cancer cell through crosstalk between Smad and JNK signaling pathways.

Melatonin induces transcriptional regulation of Bim by FoxO3a in HepG2 cells

Background:Melatonin induces apoptosis in many different cancer cell lines, including hepatocellular carcinoma cells. However, the responsible pathways have not been clearly elucidated. A member of the forkhead transcription factors' family, FoxO3a, has been implicated in the expression of the proapoptotic protein Bim (a Bcl-2-interacting mediator of cell death). In this study, we used human HepG2 liver cancer cells as an in vitro model to investigate whether melatonin treatment induces Bim through regulation by the transcription factor FoxO3a.Methods:Cytotoxicity of melatonin was compared in HepG2 hepatoblastoma cells and primary human hepatocytes. Proapoptotic Bim expression was analysed by reverse transcriptase–polymerase chain reaction and western blot. Reporter gene assays and chromatin immunoprecipitation assays were performed to analyse whether FoxO3a transactivates the Bim promoter. Small interfering RNA (siRNA) was used to study the role of FoxO3a in Bim expression. Immunofluorescence was performed to analyse FoxO3a localisation in HepG2 cells.Results:Melatonin treatment induces apoptosis in HepG2 cells, but not in primary human hepatocytes. The proapoptotic effect was mediated by increased expression of the BH3-only protein Bim. During melatonin treatment, we observed increased transcriptional activity of the forkhead-responsive element and could demonstrate that FoxO3a binds to a specific sequence within the Bim promoter. Furthermore, melatonin reduced phosphorylation of FoxO3a at Thr32 and Ser253, and induced its increased nuclear localisation. Moreover, silencing experiments with FoxO3a siRNA prevented Bim upregulation.Conclusion:This study shows that melatonin can induce apoptosis in HepG2 hepatocarcinoma cells through the upregulation of proapoptotic Bim mediated by nuclear translocation and activation of the transcription factor FoxO3a.

B7-H1 limits the entry of effector CD8+ T cells to the memory pool by upregulating Bim

Protective T‑cell immunity against cancer and infections is dependent on the generation of a durable effector and memory T‑cell pool. Studies from cancer and chronic infections reveal that B7-H1 (PD-L1) engagement with its receptor PD-1 promotes apoptosis of effector T cells. It is not clear how B7-H1 regulates T‑cell apoptosis and the subsequent impact of B7-H1 on the generation of memory T cells. In immunized B7-H1-deficient mice, we detected an increased expansion of effector CD8+ T cells and a delayed T‑cell contraction followed by the emergence of a protective CD8+ T‑cell memory capable of completely rejecting tumor metastases in the lung. Intracellular staining revealed that antigen-primed CD8+ T cells in B7-H1-deficient mice express lower levels of the pro-apoptotic molecule Bim. The engagement of activated CD8+ T cells by a plate-bound B7-H1 fusion protein led to the upregulation of Bim and increased cell death. Assays based on blocking antibodies determined that both PD-1 and CD80 are involved in the B7-H1-mediated regulation of Bim in activated CD8+ T cells. Our results suggest that B7-H1 may negatively regulate CD8+ T‑cell memory by enhancing the depletion of effector CD8+ T cells through the upregulation of Bim. Our findings may provide a new strategy for targeting B7-H1 signaling in effector CD8+ T cells to achieve protective antitumor memory responses.

Mechanisms and clinical significance of BIM phosphorylation in chronic lymphocytic leukemia

AbstractB-cell receptor and microenvironment-derived signals promote accumulation of chronic lymphocytic leukemia (CLL) cells through increased proliferation and/or decreased apoptosis. In this study, we investigated the regulation of BIM, a proapoptotic BCL2-related protein, which is tightly regulated by phosphorylation. Surface IgM stimulation increased phosphorylation of 2 BIM isoforms, BIMEL and BIML, in a subset of CLL samples. In contrast, in normal B cells, anti-IgM triggered selective phosphorylation of BIMEL only. In CLL, anti-IgM–induced BIM phosphorylation correlated with unmutated IGHV gene status and with progressive disease. Strikingly, it was also associated with progressive disease within the mutated IGHV gene subset. BIM phosphorylation was dependent on MEK1/2 kinase activity, and we identified BIMEL serine 69, previously linked to pro-survival responses, as the major site of phosphorylation in CLL and in Ramos cells. BIMEL/BIML phosphorylation was associated with release of the pro-survival protein MCL1. Coculture of CLL cells with HK cells, a model of the CLL microenvironment, promoted CLL cell survival and was associated with MEK1/2 activation and BIMEL phosphorylation. Hence, BIM phosphorylation appears to play a key role in apoptosis regulation in CLL cells, potentially coordinating antigen and microenvironment-derived survival signals. Antigen-mediated effects on BIM may be an important determinant of clinical behavior.

Regulation of pathogenic effector/memory T helper 1 lymphocyte survival by microRNA

Backgroundand objectivesAuto-antigen specific effector memory T helper type 1 lymphocytes (Th1) are critically involved in the development and maintenance of chronic inflammation in autoimmune diseases. These cells reside at inflamed...

Alternative splicing of Bim and Erk-mediated BimEL phosphorylation are dispensable for hematopoietic homeostasis in vivo

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim(EL), Bim(L) and Bim(S)) are generated by alternative splicing. Bim(EL), the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim(EL) through the DEF2 domain of Bim(EL) and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim(EL) degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim(EL) and Bim(L) are interchangeable, and that Bim(S) is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim(EL).

B cell autonomous function of Bim in B cell apoptosis and tolerance (159.10)

Abstract Splenic immature B cells traverse through the early transitional (T1) and late transitional (T2) stages to produce the mature B cell repertoire. BCR specificity-based selection ensures elimination of pathogenic B cells by apoptosis, receptor editing or anergy. These processes are tightly regulated by anti- and pro-apoptotic members of the Bcl-2 family. Conventional gene deletion of the pro-apoptotic member Bim lead to prolonged life-span of autoreactive B cells, defective anergy and production of autoantibodies. Whether these pathophysiological conditions arise as a result of B cell autonomous function of Bim remains unclear. We have found that T1 cells express higher levels of Bim and display increased susceptibility to BCR-induced apoptosis than more mature B cells. These properties are shared by BCR signaling defective Bruton’s tyrosine kinase (Btk) deficient B cells. We investigated the B cell-intrinsic role of Bim in the regulation of B cell maturation and survival in mice carrying floxed Bim and CD19-Cre in Btk-sufficient or deficient background. B lineage-specific deletion of Bim increased the B cell compartment through selective expansion of mature B cells and significantly restored mature B cells in Btk-mutant mice. These studies demonstrate a B cell autonomous role for Bim in BCR-dependent regulation of B cell homeostasis. Analysis of these mice in the context of BCR transgene will reveal the B cell autonomous function of Bim in tolerance and autoimmunity.

TDP-43-induced Death Is Associated with Altered Regulation of BIM and Bcl-xL and Attenuated by Caspase-mediated TDP-43 Cleavage

Abnormal aggregates of transactive response DNA-binding protein-43 (TDP-43) and its hyperphosphorylated and N-terminal truncated C-terminal fragments (CTFs) are deposited as major components of ubiquitinated inclusions in most cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). The mechanism underlying the contribution of TDP-43 to the pathogenesis of these neurodegenerative diseases remains unknown. In this study, we found that a 2-5-fold increase in TDP-43 expression over the endogenous level induced death of NSC34 motor neuronal cells and primary cortical neurons. TDP-43-induced death is associated with up-regulation of Bim expression and down-regulation of Bcl-xL expression. siRNA-mediated reduction of Bim expression attenuates TDP-43-induced death. Accumulated evidence indicates that caspases are activated in neurons of ALS and FTLD-U patients, and activated caspase-mediated cleavage of TDP-43 generates CTFs of TDP-43. Here, we further found that the ER (endoplasmic reticulum) stress- or staurosporine-mediated activation of caspases leads to cleavage of TDP-43 at Asp(89) and Asp(169), generating CTF35 (TDP-43-(90-414)) and CTF27 (TDP-43-(170-414)) in cultured neuronal cells. In contrast to TDP-43, CTF27 is unable to induce death while it forms aggregates. CTF35 was weaker than full-length TDP-43 in inducing death. A cleavage-resistant mutant of TDP-43 (TDP-43-D89E/D169E) showed stronger death-inducing activity than wild-type TDP-43. These results suggest that disease-related activation of caspases may attenuate TDP-43-induced toxicity by promoting TDP-43 cleavage.

Egr-1 TransactivatesBimGene Expression to Promote Neuronal Apoptosis

The proapoptotic BH3-only protein Bim is a crucial regulator of neuronal apoptosis. Previous studies have indicated the involvement of the c-Jun, FOXO1/3a, and B/C-Myb transcription factors in the regulation of Bim during neuronal apoptosis. However, the mechanism underlying the transcriptional regulation of Bim in activity deprivation-induced neuronal apoptosis has remained unclear. The present study demonstrates that early growth response 1 (Egr-1), rather than c-Jun, FOXO1/3a, or B/C-Myb, directly transactivates Bim gene expression to mediate apoptosis of rat cerebellar granule neurons. We showed that Egr-1 was sufficient and necessary for neuronal apoptosis. Suppression of Egr-1 activity using dominant-negative mutant or knockdown of Egr-1 using small interfering RNAs led to a decrease in Bim expression, whereas overexpression of Egr-1 resulted in induction of Bim. Deletion and site-directed mutagenesis of the Bim promoter revealed that Bim transcriptional activation depends primarily on a putative Egr-binding sequence between nucleotides -56 and -47 upstream of the start site. We also showed that Egr-1 binding to this sequence increased in response to activity deprivation in vitro and in vivo. Moreover, inhibition of Egr-1 binding to the Bim promoter, by mithramycin A and chromomycin A3, reduced the activity deprivation-induced increases in Bim promoter activity and mRNA and protein levels and protected neurons from apoptosis, further supporting the Egr-1-mediated transactivation of Bim. Additionally, Bim overcame the Egr-1 knockdown-mediated inhibition of apoptosis, whereas Bim knockdown impaired the increase in apoptosis induced by Egr-1. These findings establish Bim as an Egr-1 target gene in neurons, uncovering a novel Egr-1/Bim pathway by which activity deprivation induces neuronal apoptosis.

Grb10 interacts with Bim L and inhibits apoptosis

Bim is a proapoptotic member of the Bcl-2 family and is primarily involved in the regulation of the intrinsic apoptotic pathway. However, the detail of regulation of Bim's proapoptotic activity has not been clarified yet. Using Bim L as bait, we screened a human fetal cDNA library for interacting proteins and identified Grb10 as an interactor. This interaction was verified by co-immunoprecipitation and intracellular co-localization studies. The potential segment of Bim L that binds Grb10 was identified via a yeast mating test. Grb10 interacted with the DBD (dynein binding domain) of Bim and inhibited apoptosis triggered by overexpression of DBD containing Bim isoforms. The putative phosphorylation sites on DBD of Bim play a role for the anti-proapoptotic activity of Grb10. Our results suggest that Grb10 interacts with Bim L and inhibits its proapoptotic activity in a phosphorylation-dependant manner.

Identification of proapoptotic Bim as a tumor suppressor in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs

AbstractSystemic mastocytosis (SM) is a myeloid neoplasm involving mast cells (MCs) and their progenitors. In most cases, neoplastic cells display the D816V-mutated variant of KIT. KIT D816V exhibits constitutive tyrosine kinase (TK) activity and has been implicated in increased survival and growth of neoplastic MCs. Recent data suggest that the proapoptotic BH3-only death regulator Bim plays a role as a tumor suppressor in various myeloid neoplasms. We found that KIT D816V suppresses expression of Bim in Ba/F3 cells. The KIT D816–induced down-regulation of Bim was rescued by the KIT-targeting drug PKC412/midostaurin. Both PKC412 and the proteasome-inhibitor bortezomib were found to decrease growth and promote expression of Bim in MC leukemia cell lines HMC-1.1 (D816V negative) and HMC-1.2 (D816V positive). Both drugs were also found to counteract growth of primary neoplastic MCs. Furthermore, midostaurin was found to cooperate with bortezomib and with the BH3-mimetic obatoclax in producing growth inhibition in both HMC-1 subclones. Finally, a Bim-specific siRNA was found to rescue HMC-1 cells from PKC412-induced cell death. Our data show that KIT D816V suppresses expression of proapoptotic Bim in neoplastic MCs. Targeting of Bcl-2 family members by drugs promoting Bim (re)-expression, or by BH3-mimetics such as obatoclax, may be an attractive therapy concept in SM.

Overactivation of the MEK/ERK Pathway in Liver Tumor Cells Confers Resistance to TGF-β–Induced Cell Death through Impairing Up-regulation of the NADPH Oxidase NOX4

Transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes, being considered a liver tumor suppressor. However, many human hepatocellular carcinoma (HCC) cells escape from its proapoptotic effects, gaining response to this cytokine in terms of malignancy. We have recently reported that the apoptosis induced by TGF-beta in hepatocytes requires up-regulation of the NADPH oxidase NOX4, which mediates reactive oxygen species (ROS) production. TGF-beta-induced NOX4 expression is inhibited by antiapoptotic signals, such as the phosphatydilinositol-3-phosphate kinase or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. The aim of the present work was to analyze whether resistance to TGF-beta-induced apoptosis in HCC cells is related to the impairment of NOX4 up-regulation due to overactivation of survival signals. Results indicate that inhibition of the MAPK/ERK kinase (MEK)/ERK pathway in HepG2 cells, which are refractory to the proapoptotic effects of TGF-beta, sensitizes them to cell death through a mitochondrial-dependent mechanism, coincident with increased levels of BIM and BMF, decreased levels of BCL-XL and MCL1, and BAX/BAK activation. Regulation of BMF, BCL-XL, and MCL1 occurs at the mRNA level, whereas BIM regulation occurs post-transcriptionally. ROS production and glutathione depletion are only observed in cells treated with TGF-beta and PD98059, which correlates with NOX4 up-regulation. Targeting knockdown of NOX4 impairs ROS increase and all the mitochondrial-dependent apoptotic features by a mechanism that is upstream from the regulation of BIM, BMF, BCL-XL, and MCL1 levels. In conclusion, overactivation of the MEK/ERK pathway in liver tumor cells confers resistance to TGF-beta-induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis.

Epstein-Barr Virus Latency in B Cells Leads to Epigenetic Repression and CpG Methylation of the Tumour Suppressor Gene Bim

In human B cells infected with Epstein-Barr virus (EBV), latency-associated virus gene products inhibit expression of the pro-apoptotic Bcl-2-family member Bim and enhance cell survival. This involves the activities of the EBV nuclear proteins EBNA3A and EBNA3C and appears to be predominantly directed at regulating Bim mRNA synthesis, although post-transcriptional regulation of Bim has been reported. Here we show that protein and RNA stability make little or no contribution to the EBV-associated repression of Bim in latently infected B cells. However, treatment of cells with inhibitors of histone deacetylase (HDAC) and DNA methyltransferase (DNMT) enzymes indicated that epigenetic mechanisms are involved in the down-regulation of Bim. This was initially confirmed by chromatin immunoprecipitation analysis of histone acetylation levels on the Bim promoter. Consistent with this, methylation-specific PCR (MSP) and bisulphite sequencing of regions within the large CpG island located at the 5′ end of Bim revealed significant methylation of CpG dinucleotides in all EBV-positive, but not EBV-negative B cells examined. Genomic DNA samples exhibiting methylation of the Bim promoter included extracts from a series of explanted EBV-positive Burkitt's lymphoma (BL) biopsies. Subsequent analyses of the histone modification H3K27-Me3 (trimethylation of histone H3 lysine 27) and CpG methylation at loci throughout the Bim promoter suggest that in EBV-positive B cells repression of Bim is initially associated with this repressive epigenetic histone mark gradually followed by DNA methylation at CpG dinucleotides. We conclude that latent EBV initiates a chain of events that leads to epigenetic repression of the tumour suppressor gene Bim in infected B cells and their progeny. This reprogramming of B cells could have important implications for our understanding of EBV persistence and the pathogenesis of EBV-associated disease, in particular BL.

The imbalance between Survivin and Bim mediates tumour growth and correlates with poor survival in patients with multiple myeloma

Survivin is selectively expressed in most of common human cancers and is now viewed as a potent modulator of the cell death/proliferation balance in tumour cells. We previously found that myeloma cells expressed high levels of Survivin protein in correlation with disease progression and that Survivin knock-down by RNA interference decreased myeloma cell growth. We now demonstrate that Survivin overexpression promotes the proliferation and survival of human myeloma cells both in vitro and in vivo in the absence of their major growth factor, interleukin 6. Of particular interest, this effect correlates with the down regulation of Bim, a critical BH3-only cell death activator during cytokine deprivation, mainly at transcriptional level. The tight link between Survivin and Bim expression, reported for the first time here in myeloma cells and in other cell lines, is further confirmed in a panel of newly diagnosed patients with myeloma, and BIRC5 is validated as a gene significantly associated with short survival in these patients. Altogether, our findings provide evidence that Survivin directly contributes to malignant progression of myeloma and strongly suggest that targeting Survivin may disrupt the delicate balance controlling cell survival and proliferation, opening new avenues for the therapy of this still difficult-to-treat cancer.

Nerve growth factor blocks thapsigargin‐induced apoptosis at the level of the mitochondrion viaregulation of Bim

This study examined how the neurotrophin, nerve growth factor (NGF), protects PC12 cells against endoplasmic reticulum (ER) stress-induced apoptosis. ER stress was induced using thapsigargin (TG) that inhibits the sarcoplasmic/ER Ca2+-ATPase pump (SERCA) and depletes ER Ca2+ stores. NGF pre-treatment inhibited translocation of Bax to the mitochondria, loss of mitochondrial transmembrane potential, cytochrome c release, activation of caspases (−3, −7 and −9) and apoptosis induction by TG. Notably, TG also caused a marked induction of Bimel mRNA and protein, and knockdown of Bim with siRNA protected cells against TG-induced apoptosis. NGF delayed the induction and increased the phosphorylation of Bimel. NGF-mediated protection was dependent on phosphatidylinositol-3 kinase (PI3K) signalling since all above apoptotic events, including expression and phosphorylation status of Bimel protein, could be reverted by the PI3K inhibitor LY294002. In contrast, NGF had no effect on the TG-mediated induction of the unfolded protein response (increased expression of Grp78, GADD34, splicing of XBP1 mRNA) or ER stress-associated pro-apoptotic responses (induction of C/EBP homologous protein [CHOP], induction and processing of caspase-12). These data indicate that NGF-mediated protection against ER stress-induced apoptosis occurs at the level of the mitochondria by regulating induction and activation of Bim and mitochondrial translocation of Bax.

Transcriptional Regulation of Bim by FOXO3a and Akt Mediates Scleroderma Serum–Induced Apoptosis in Endothelial Progenitor Cells

Endothelial progenitor cells (EPCs) contribute to vascular regeneration/repair and thus may protect against scleroderma vasculopathy. We aimed to determine whether circulating EPCs were reduced in scleroderma, whether scleroderma sera could induce EPC apoptosis, and, if so, what the underlying apoptotic signaling pathway was. Circulating EPC levels were quantified in 54 patients with scleroderma and 18 healthy control subjects by colony-forming unit assay and flow cytometry, which revealed markedly decreased EPC levels in scleroderma patients relative to healthy subjects. Substantial apoptosis was detected in EPCs after culturing in the presence of scleroderma sera compared with normal sera. Intriguingly, depletion of the IgG fraction from scleroderma sera completely abolished the apoptotic effects. Furthermore, scleroderma sera inhibited the activation/phosphorylation of Akt, which in turn suppressed the phosphorylation and degradation of forkhead transcription factor FKHRL1 (FOXO3a), resulting in the upregulation of apoptotic protein Bim. siRNA-mediated FOXO3a and Bim knockdown substantially reduced scleroderma serum-induced EPC apoptosis. Importantly, Bim expression and baseline apoptosis were increased in EPCs freshly isolated from scleroderma patients relative to that obtained from healthy subjects. Scleroderma serum-induced EPC apoptosis is mediated chiefly by the Akt-FOXO3a-Bim pathway, which may account, at least in part, for the decreased circulating EPC levels in scleroderma patients.

Withaferin A Causes FOXO3a- and Bim-Dependent Apoptosis and Inhibits Growth of Human Breast Cancer Cells In vivo

Withaferin A (WA) is derived from the medicinal plant Withania somnifera, which has been safely used for centuries in Indian Ayurvedic medicine for treatment of different ailments. We now show, for the first time, that WA exhibits significant activity against human breast cancer cells in culture and in vivo. The WA treatment decreased viability of MCF-7 (estrogen-responsive) and MDA-MB-231 (estrogen-independent) human breast cancer cells in a concentration-dependent manner. The WA-mediated suppression of breast cancer cell viability correlated with apoptosis induction characterized by DNA condensation, cytoplasmic histone-associated DNA fragmentation, and cleavage of poly-(ADP-ribose)-polymerase. On the other hand, a spontaneously immortalized normal mammary epithelial cell line (MCF-10A) was relatively more resistant to WA-induced apoptosis compared with breast cancer cells. The WA-mediated apoptosis was accompanied by induction of Bim-s and Bim-L in MCF-7 cells and induction of Bim-s and Bim-EL isoforms in MDA-MB-231 cells. The cytoplasmic histone-associated DNA fragmentation resulting from WA exposure was significantly attenuated by knockdown of protein levels of Bim and its transcriptional regulator FOXO3a in both cell lines. Moreover, FOXO3a knockdown conferred marked protection against WA-mediated induction of Bim-s expression. The growth of MDA-MB-231 cells implanted in female nude mice was significantly retarded by 5 weekly i.p. injections of 4 mg WA/kg body weight. The tumors from WA-treated mice exhibited reduced cell proliferation and increased apoptosis compared with tumors from control mice. These results point toward an important role of FOXO3a and Bim in regulation of WA-mediated apoptosis in human breast cancer cells.

MicroRNAs and lymphocyte homeostasis: Dangerous eggs in a single basket

MicroRNAs and lymphocyte homeostasis: Dangerous eggs in a single basket

Bim, Bak, and Bax Regulate Osteoblast Survival

Osteoblasts depend on a constant supply of prosurvival signals from their microenvironment. When trophic factors become limited by injury or disease, cells undergo apoptosis. This study establishes the regulation and function of Bim, Bak, and Bax in this response. MBA-15.4 murine osteoblasts and primary human bone marrow stromal cells (hBMSCs) were subjected to growth factor depletion by serum starvation (1% FCS or serum withdrawal). Protein phosphorylation, activation, or expression was quantified by Western blotting and gene expression by real-time PCR. Regulation of apoptosis in response to serum depletion was determined using siRNA specific for Bim, Bak, or Bax, followed by TUNEL staining. Statistical significance was determined by one-way ANOVA after multiple experimental repeats. Serum depletion strongly induced expression of the proapoptotic protein Bim in both hBMSC and MBA-15.4 osteoblasts. Detailed analysis of the mouse line showed that both mRNA and protein levels rose from 2 h to peak between 16 and 24 h, in conjunction with activation of caspase 3 and rising levels of apoptosis. Both actinomycin D and cycloheximide prevented this increase in Bim, indicating transcriptional regulation. Serum deprivation caused immediate and sustained decreases in phosphorylation of prosurvival kinases, ERK and PKB, preceding upregulation of Bim. Pathway inhibitors, U0126 or LY294002, strongly increased both Bim mRNA and protein, confirming that both kinases regulate Bim. These inhibitors also induced osteoblast apoptosis within 24-72 h. JC-1 tracer detected mitochondrial potential disruption after serum deprivation, indicating involvement of the intrinsic pathway. Moreover, activation-associated conformational changes were detected in the channel-formers, Bax and Bak. Selective knockdown of Bim or Bak by siRNA protected osteoblasts from serum depletion-induced apoptosis by 50%, whereas knockdown of Bax alone or Bak and Bax together reduced apoptosis by 90%. Our data indicate that Bim, Bak, and Bax actively mediate osteoblast apoptosis induced by trophic factor withdrawal. The complex upstream regulation of Bim may provide targets for therapeutic enhancement of osteoblast viability.

Rapid Culling of the CD4+ T Cell Repertoire in the Transition from Effector to Memory

Requirements for CD4+ T cell memory differentiation were analyzed with adoptively transferred SMARTA T cell receptor (TCR) transgenic cells specific for alymphocytic choriomeningitis virus (LCMV) epitope. LCMV-induced effector and memory differentiation of SMARTA cells mimicked the endogenous CD4+ T cell response. In contrast, infection with a recombinant Listeria expressing the LCMV epitope, although resulting initially in massive SMARTA expansion, led to loss of effector function and rapid cell death characterized by high expression of the apoptosis regulator Bim. Defective memory differentiation was seen after stimulation of naive but not memory SMARTA cells, was independent of precursor frequency, and correlated with a lower TCR avidity compared to endogenous responders. In addition, long-lived endogenous CD4+ memory T cells skewed to a higher functional avidity over time. These results support a model in which CD4+ T cell memory differentiation and longevity depend on the strength of the TCR signal during the primary response.