Absence Of Bim Research Articles

BH3-only protein expression determines hepatocellular carcinoma response to sorafenib-based treatment

Hepatocellular carcinoma (HCC) represents a global health challenge with limited therapeutic options. Anti-angiogenic immune checkpoint inhibitor-based combination therapy has been introduced for progressed HCC, but improves survival only in a subset of HCC patients. Tyrosine-kinase inhibitors (TKI) such as sorafenib represent an alternative treatment option but have only modest efficacy. Using different HCC cell lines and HCC tissues from various patients reflecting HCC heterogeneity, we investigated whether the sorafenib response could be enhanced by combination with pro-apoptotic agents, such as TNF-related apoptosis-inducing ligand (TRAIL) or the BH3-mimetic ABT-737, which target the death receptor and mitochondrial pathway of apoptosis, respectively. We found that both agents could enhance sorafenib-induced cell death which was, however, dependent on specific BH3-only proteins. TRAIL augmented sorafenib-induced cell death only in NOXA-expressing HCC cells, whereas ABT-737 enhanced the sorafenib response also in NOXA-deficient cells. ABT-737, however, failed to augment sorafenib cytotoxicity in the absence of BIM, even when NOXA was strongly expressed. In the presence of NOXA, BIM-deficient HCC cells could be in turn strongly sensitized for cell death induction by the combination of sorafenib with TRAIL. Accordingly, HCC tissues sensitive to apoptosis induction by sorafenib and TRAIL revealed enhanced NOXA expression compared to HCC tissues resistant to this treatment combination. Thus, our results suggest that BH3-only protein expression determines the treatment response of HCC to different sorafenib-based drug combinations. Individual profiling of BH3-only protein expression might therefore assist patient stratification to certain TKI-based HCC therapies.

MiR17~92 restrains pro-apoptotic BIM to ensure survival of haematopoietic stem and progenitor cells.

The miR17~92 cluster plays important roles in haematopoiesis. However, it is not clear at what stage of differentiation and through which targets miR17~92 exerts this function. Therefore, we generated miR17~92fl/fl; RosaCreERT2 mice for inducible deletion of miR17~92 in haematopoietic cells. Bone marrow reconstitution experiments revealed that miR17~92-deleted cells were not capable to contribute to mature haematopoietic lineages, which was due to defects in haematopoietic stem/progenitor cells (HSPCs). To identify the critical factor targeted by miR17~92 we performed gene expression analysis in HSPCs, demonstrating that mRNA levels of pro-apoptotic Bim inversely correlated with the expression of the miR17~92 cluster. Strikingly, loss of pro-apoptotic BIM completely prevented the loss of HSPCs caused by deletion of miR17~92. The BIM/miR17~92 interaction is conserved in human CD34+ HSPCs, as miR17~92 inhibition or blockade of its binding to the BIM 3'UTR reduced the survival and growth of these cells. Despite the prediction that miR17~92 functions by impacting a plethora of different targets, the absence of BIM alone is sufficient to prevent all defects caused by deletion of miR17~92 in haematopoietic cells.

Bcl-2 Is a Therapeutic Target for Hypodiploid B-Lineage Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. The highest rates of treatment failure occur in specific genetic subsets of ALL, including hypodiploid B-cell ALL (B-ALL), for which effective alternative therapies to current intensive chemotherapy treatments have yet to be developed. Here, we integrated biochemical and genomic profiling with functional drug assays to select effective agents with therapeutic potential against hypodiploid B-ALL. ABT-199, a selective Bcl-2 inhibitor, was effective in reducing leukemic burden in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. Daily oral treatment with ABT-199 significantly increased survival in xenografted mice. The unexpected efficacy of ABT-199 observed in hypodiploid leukemias lacking BIM expression (the major reported mediator of ABT-199-induced apoptosis) led us to investigate the mechanism of action of ABT-199 in the absence of BIM. Treatment with ABT-199 elicited responses in a dose-dependent manner, from cell-cycle arrest at low nanomolar concentrations to cell death at concentrations above 100 nmol/L. Collectively, these results demonstrate the efficacy of Bcl-2 inhibition and potential therapeutic strategy in hypodiploid B-ALL. SIGNIFICANCE: These results demonstrate the efficacy of ABT-199 in vivo and provide encouraging preclinical data of Bcl-2 as a potential target for the treatment of hypodiploid B-ALL.

Increased lymphocyte apoptosis in mouse models of colitis upon ABT-737 treatment is dependent upon BIM expression.

Exaggerated activation of lymphocytes contributes to the pathogenesis of inflammatory bowel disease (IBD). Medical therapies are linked to the BCL-2 family-mediated apoptosis. Imbalance in BCL-2 family proteins may cause failure in therapeutic responses. We investigated the role of BCL-2 inhibitor ABT-737 for lymphocyte apoptosis in mice under inflammatory conditions. B.6129P2-interleukin (IL)-10(tm1Cgn) /J (IL-10(-/-) ) weighing 25-30 g with ongoing colitis were used. Fifty mg/kg/day ABT-737 was injected intraperitoneally (i.p.). Haematological analyses were performed with an ADVIA 2120 flow cytometer and mass cytometry with a CyTOF 2. Following i.p. administration, ABT-737 was detected in both spontaneous and acute colitis in peripheral blood (PBL) and colon tissue. Treatment led to lymphopenia. CD4(+) CD44(+) CD62L(+) central memory and CD8(+) , CD44(+) CD62L(-) central memory T cells were decreased in PBL upon ABT-737 compared to vehicle-receiving controls. Increased apoptosis upon ABT-737 was determined in blood lymphocytes, splenocytes and Peyer's patches and was accompanied by a decrease in TNF and IL-1B. ABT-737 positively altered the colonic mucosa and ameliorated inflammation, as shown by colonoscopy, histology and colon length. A decreased BIM/BCL-2 ratio or absence of BIM in both Bim(-) (/) (-) and Il10(-) (/) (-) × Bim(-) (/) (-) impeded the protective effect of ABT-737. The BIM/BCL-2 ratio decreased with age and during the course of treatment. Thus, long-term treatment resulted in adapted TNF levels and macroscopic mucosal damage. ABT-737 was efficacious in diminishing lymphocytes and ameliorating colitis in a BIM-dependent manner. Regulation of inappropriate survival of lymphocytes by ABT-737 may provide a therapeutic strategy in IBD.

Dexamethasone Synergizes with ABT-199 through the Induction of Bim and Bcl-2 Dependence in Myeloma

Multiple myeloma is a plasma cell malignancy, with a 10-year survival rate of approximately 25%. While this survival rate is largely attributed to the FDA approval of new therapies for the disease, such as IMiDs and proteasome inhibitors, eventual relapse and drug resistance is the reality facing the majority of myeloma patients. Subsequently, the quest for new drugs and combination therapies is ongoing.Novel agents targeting the Bcl-2 family of cell survival regulators are promising avenues of research. We have previously reported on a method of predicting sensitivity of myeloma cell lines and patient samples to the Bcl-2/xL inhibitor ABT-737, based on the binding pattern of pro-apoptotic protein Bim to anti-apoptotic proteins Mcl-1, Bcl-xL, and Bcl-2. In Mcl-1-dependent cells, Bim is primarily associated with Mcl-1, and insensitive to ABT-737. Alternatively, in cells that are co-dependent on Mcl-1 and Bcl-2/xL for survival, Bim is either predominantly associated with Bcl-2/xL or when it is released from Bcl-2/xL it cannot bind to Mcl-1 because of the presence of the Mcl-1 inhibitor Noxa, rendering the cells sensitive to ABT-737. Unfortunately, ABT-737, or the clinical compound Navitoclax, is not a viable treatment for myeloma, due to its potential for causing thrombocytopenia. Therefore we chose to investigate the sensitivity of myeloma cell lines and freshly isolated patient samples to ABT-199.ABT-199, a specific inhibitor of anti-apoptotic protein Bcl-2, is currently in phase I clinical trials for multiple myeloma. We have previously reported on its preclinical efficacy as a single agent in myeloma as well as in combination with other commonly used therapeutics. While ABT-199 was ineffective as a single agent in the cell lines we tested, combining it with dexamethasone significantly decreased the IC50s (Fold change: 8226 – 2.6, MM.1s – 43.5, OPM2 – 14.1, KMS11 – 23.3, KMS18 – 4.8). Here we report on the mechanism by which dexamethasone increases sensitivity to ABT-199, as well as the effectiveness of this combination in freshly isolated patient samples.We performed real-time PCR analysis to determine changes in the expression of the Bcl-2 family of proteins following treatment with ABT-199, dexamethasone, and the combination. Minimal changes in expression were seen with ABT-199 treatment; however, dexamethasone treatment greatly induced the expression of both Bcl-2 and Bim, which was also seen in the combination treatment, along with a decrease in Bcl-xL expression. Next we performed CoIP studies to examine how these changes in expression affected the binding pattern of Bim to the anti-apoptotic proteins. As expected, we found that upon treatment with ABT-199, the small amount of Bim previously bound to Bcl-2 was released. Alternatively, when cells were treated with dexamethasone alone, the amount of Bim bound to Bcl-2 increased, likely due to the significant increase in expression of both proteins. When cells were subjected to both drugs simultaneously, there was a decrease in the amount of Bim bound to Bcl-xL, as well as an absence of Bim on Bcl-2. Thus, co-treatment brings about a scenario whereby ABT-199 releases Bim bound to Bcl-2, and at the same time, prevents the Bim induced by dexamethasone from binding to Bcl-2. These changes, coupled with a decrease in amount of Bcl-xL available to bind Bim, result in the induction of apoptosis.To further verify this drug synergy, we treated ficoll-isolated buffy coat cells from 10 myeloma patients with ABT-199, either alone or in combination with dexamethasone. Samples from 4 of the patients were sensitive to ABT-199 alone, thus combination had little effect. For the other 6 patient samples, the addition of dexamethasone to ABT-199 significantly decreased the IC50 over ABT-199 alone (Fold Change: MM61 – 7.3, MM62 – 17.5, MM63 – 20.1, MM64 – 2.2, MM65 – 1.8, MM66 – 8.7).Previous studies have reported that the t(11;14) subset of multiple myeloma is highly sensitive to ABT-199. Five of the 10 patient samples we tested were positive for the t(11;14), and all but one, MM61, were sensitive to ABT-199 alone. However, MM61 was sensitive to the combination treatment. Taken together, our data demonstrate that the addition of dexamethasone expands the potential of ABT-199 to a broader set of patients who would otherwise likely be resistant to monotherapy. DisclosuresKaufman:Millennium: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Onyx: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Spectrum: Consultancy, Honoraria; Merck: Research Funding. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding.

Id2 influences differentiation of killer cell lectin-like receptor G1(hi) short-lived CD8+ effector T cells.

CD8(+) T cells play a crucial role in the clearance of intracellular pathogens through the generation of cytotoxic effector cells that eliminate infected cells and long-lived memory cells that provide enhanced protection against reinfection. We have previously shown that the inhibitor of E protein transcription factors, Id2, is necessary for accumulation of effector and memory CD8(+) T cells during infection. In this study, we show that CD8(+) T cells lacking Id2 did not generate a robust terminally differentiated killer cell lectin-like receptor G1 (KLRG1)(hi) effector population, but displayed a cell-surface phenotype and cytokine profile consistent with memory precursors, raising the question as to whether loss of Id2 impairs the differentiation and/or survival of effector memory cells. We found that deletion of Bim rescued Id2-deficient CD8(+) cell survival during infection. However, the dramatic reduction in KLRG1(hi) cells caused by loss of Id2 remained in the absence of Bim, such that Id2/Bim double-deficient cells form an exclusively KLRG1(lo)CD127(hi) memory precursor population. Thus, we describe a role for Id2 in both the survival and differentiation of normal CD8(+) effector and memory populations.

Modulation of Vascular Cell Function by Bim Expression

Apoptosis of vascular cells, including pericytes and endothelial cells, contributes to disease pathogenesis in which vascular rarefaction plays a central role. Bim is a proapoptotic protein that modulates not only apoptosis but also cellular functions such as migration and extracellular matrix (ECM) protein expression. Endothelial cells and pericytes each make a unique contribution to vascular formation and function although the details require further delineation. Here we set out to determine the cell autonomous impact of Bim expression on retinal endothelial cell and pericyte function using cells prepared from Bim deficient (Bim−/−) mice. Bim−/− endothelial cells displayed an increased production of ECM proteins, proliferation, migration, adhesion, and VEGF expression but, a decreased eNOS expression and nitric oxide production. In contrast, pericyte proliferation decreased in the absence of Bim while migration, adhesion, and VEGF expression were increased. In addition, we demonstrated that the coculturing of either wild-type or Bim−/− endothelial cells with Bim−/− pericytes diminished their capillary morphogenesis. Thus, our data further emphasizes the importance of vascular cell autonomous regulatory mechanisms in modulation of vascular function.

Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3‐only proteins Bim and Bmf

Anti-apoptotic Bcl-2 family members are critical for the regulation of haematopoietic stem and progenitor cell (HSPC) survival. Little is known about the role of their pro-apoptotic antagonists, i.e. ‘BH3-only’ proteins, in this cell compartment. Based on the analysis of cytokine deprivation-induced changes in mRNA expression levels of Bcl-2 family proteins, we determined the consequences of BH3-only protein depletion on HSPC survival in culture and, for selected candidates, on engraftment in vivo. Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution. HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment. Moreover, in the absence of Bim, significantly lower numbers of transplanted HSPCs were able to fully engraft radio-depleted recipients. Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34+ cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.

Deficiency in Bim, Bid and Bbc3 (Puma) do not prevent axonal injury induced death

Dear Editor, BAX activation is a critical step in the apoptotic pathway in neurons. BAX activation is multistep process that can be driven by a subset of other pro-apoptotic Bcl-2 family members, specifically the BH3-only proteins BIM, BBC3 (PUMA), and BID.1 In fact BAX activation appears to require at least one of these pro-apoptotic chaperones during development and after many apoptotic insults.2 Retinal ganglion cells (RGCs) die by a BAX-dependent mechanism in development and after axonal injury,3 a key insult in many neurodegenerations. During development BAX-dependent death of RGCs requires BBC3 (Harder and Libby4 and Supplementary Figure 1A); however, the molecules controlling BAX activation after axonal injury are unknown. We have previously shown that single deficiency in Bim or Bbc3 lessen early RGC death after axonal injury.4, 5 However, by 5 days after injury, RGC death is no longer different in Bbc3-deficient mice and there is a significant number of apoptotic RGCs in Bim-deficient mice (Supplementary Figure 1B). These data show that both BIM and BBC3 are involved in BAX activation in axonally injured RGCs, but are not solely responsible for RGC death. As BIM and BBC3 appear to regulate BAX activation in axonally injured RGCs, we tested whether deficiency in both Bim and Bbc3 phenocopied Bax deficiency and prevented RGC loss after axonal injury. The amount of protection in Bim Bbc3 double knockouts was additive and significant compared with wildtype and single mutants as judged by the number of RGCs surviving 21 days after injury (Supplementary Figure 1C). However, apoptotic cell death persisted in the Bim Bbc3 double mutants after axonal injury, which led to significant RGC loss over time (P<0.001; Supplementary Figure 1C). BID has been implicated in neuronal death after many types of insults;2 therefore, it is possible that it is contributing to RGC death in the absence of BIM and BBC3. Bid deficiency alone did not alter the rate or amount of RGC death after injury (Supplementary Figures 1B and C). Bid deficiency was also not additive in the context of Bim deficiency or Bbc3 deficiency (Supplementary Figures 1B and C). In order to test whether removing the major direct activators of BAX was necessary to prevent RGC loss, mice deficient in Bim, Bbc3, and Bid were generated. Triple deficiency did not prevent RGC death after axonal injury and did not increase the long term survival of RGCs compared with the protection of Bim Bbc3 combined deficiency (64±3 and 61±4% of control, respectively; Supplementary Figure 1C). However, triple deficiency did reduce the rate of RGC death compared with Bim Bbc3 double-deficient animals suggesting that BID can have a role in RGC death after axonal injury (Supplementary Figure 1B). Thus, in RGCs axonal injury induces cell death independently of BIM, BBC3, and BID despite the requirement for BAX. RGC death in the absence of BIM, BBC3, and BID is consistent with observations suggesting that BAX (and BAK) could be activated independently of these BH3-only proteins.6 BH3-only proteins with a lower affinity for BAX have been implicated in neuronal death, such as HRK, BMF, and NOXA after neuronal trauma.7 These molecules may be less efficient at killing cells due to their primary function of antagonizing pro-survival Bcl-2 family members. Interestingly, lowering levels of pro-survival Bcl-2 family members alone can lead to apoptosis.8 Necroptotic cell death is also dependent on BAX9 and observed in injured neurons, but may not require BIM, BBC3, and BID. Finally, it is possible that BAX activation could be occurring independently of the Bcl-2 family (e.g. Chipuk et al.10). Therefore, further defining the mechanism of BAX activation may be important in understanding cell death in neuronal degenerations or after neuronal trauma.

The BH3-Only Proteins Bim and Puma Cooperate to Impose Deletional Tolerance of Organ-Specific Antigens

Although the proapoptotic BH3-only protein, Bim, is required for deletion of autoreactive thymocytes, Bim-deficient mice do not succumb to extensive organ-specific autoimmune disease. To determine whether other BH3-only proteins safeguard tolerance in the absence of Bim, we screened mice lacking Bim as well as other BH3-only proteins. Most strains showed no additional defects; however, mice deficient for both Puma and Bim spontaneously developed autoimmunity in multiple organs, and their Tcells could transfer organ-specific autoimmunity. Puma- and Bim-double-deficient mice had a striking accumulation of mature, single-positive thymocytes, suggesting an additional defect in thymic deletion was the basis for disease. Transgenic mouse models of thymocyte deletion by peripheral neoantigens confirmed that the loss of Bim and Puma allowed increased numbers of autoreactive thymocytes to escape deletion. Our data show that Puma cooperates with Bim to impose a thymic-deletion checkpoint to peripheral self-antigens and cement the notion that defects in apoptosis alone are sufficient to cause autoimmune disease.

Potential synergism of Bim with p53 in mice with Myc‑induced lymphoma in a mouse lymphoma model

We report that Bim has an apoptotic function through Bax/mitochondrial apoptosis signaling in the p53-independent mode, which is somewhat additive to the effects of p53. Bim-deficient mouse embryonic fibroblast (MEF) cells were resistant to the apoptotic effects of Myc, while loss of Bim accelerated lymphoma development. Furthermore, Bim was overexpressed at the same frequency in Myc-initiated lymphomas, irrespective of p53 status, suggesting that Bim resides in a pathway separate from p53. Loss of Bim further augmented resistance to apoptosis in p53-/- MEFs. Mice with p53 knockdown exhibited exacerbated malignancies in the absence of Bim. The combined loss of these proteins promoted more severe spontaneous tumorigenesis. Thus, Myc-induced apoptotic signals through Bim and p53 must bifurcate to activate Bax, suggesting that the activation of Bim and p53 significantly contribute to apoptosis. Our results, therefore, establish that p53 and Bim are effective key initiators of apoptosis in lymphoma cells, particularly when combined.

Preferential control of induced regulatory T cell homeostasis via a Bim/Bcl-2 axis

Apoptosis has an essential role in controlling T cell homeostasis, especially during the contraction phase of an immune response. However, its contribution to the balance between effector and regulatory populations remains unclear. We found that Rag1−/− hosts repopulated with Bim−/− conventional CD4+ T cells (Tconv) resulted in a larger induced regulatory T cell (iTreg) population than mice given wild-type (WT) Tconv. This appears to be due to an increased survival advantage of iTregs compared with activated Tconv in the absence of Bim. Downregulation of Bcl-2 expression and upregulation of Bim expression were more dramatic in WT iTregs than activated Tconv in the absence of IL-2 in vitro. The iTregs generated following Tconv reconstitution of Rag1−/− hosts exhibited lower Bcl-2 expression and higher Bim/Bcl-2 ratio than Tconv, which indicates that iTregs were in an apoptosis-prone state in vivo. A significant proportion of the peripheral iTreg pool exhibits low Bcl-2 expression indicating increased sensitivity to apoptosis, which may be a general characteristic of certain Treg subpopulations. In summary, our data suggest that iTregs and Tconv differ in their sensitivity to apoptotic stimuli due to their altered ratio of Bim/Bcl-2 expression. Modulating the apoptosis pathway may provide novel therapeutic approaches to alter the balance between effector T cells and Tregs.

The Proapoptotic Bcl-2 Family Member Bim Plays a Central Role during the Development of Virus-Induced Hepatitis

The proapoptotic Bcl-2 homolog Bim was shown to control the apoptosis of both T cells and hepatocytes. This dual role of Bim might be particularly relevant for the development of viral hepatitis, in which both the sensitivity of hepatocytes to apoptosis stimuli and the persistence of cytotoxic T cells are essential factors for the outcome of the disease. The relevance of Bim in regulating survival of cytotoxic T cells or induction of hepatocyte death has only been investigated in separate systems, and their relative contributions to the pathogenesis of T cell-mediated hepatitis remain unclear. Using the highly dynamic model system of lymphocytic choriomeningitis virus-mediated hepatitis and bone marrow chimeras, we found that Bim has a dual role in the development of lymphocytic choriomeningitis virus-induced, T cell-mediated hepatitis. Although the absence of Bim in parenchymal cells led to markedly attenuated liver damage, loss of Bim in the lymphoid compartment moderately enhanced hepatitis. However, when both effects were combined in Bim(-/-) mice, the effect of Bim deficiency in the lymphoid compartment was overcompensated for by the reduced sensitivity of Bim(-/-) hepatocytes to T cell-induced apoptosis, resulting in the protection of Bim(-/-) mice from hepatitis.

Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens

Positive and negative selection of thymocytes in the thymus are critical for the development of a mature and self-tolerant T-cell repertoire. The proapoptotic Bcl-2 family member Bim is important for negative selection by inducing apoptosis in thymocytes receiving a strong signal through their antigen receptor. However, in the case of ubiquitous self-antigens (UbA), Bim is not required for the clonal deletion of self-reactive thymocytes, suggesting the existence of nonapoptotic clonal deletion mechanisms. Unlike UbA, clonal deletion to tissue-restricted antigens (TRAs) requires positive selection and CCR7-mediated migration to the medulla. This led us to hypothesize that Bim is required for the latter. To study the role of Bim in clonal deletion to TRA, we constructed bone marrow (BM) chimeras using OT-I Bim-deficient or -sufficient donor bone marrow and recipients that express membrane bound chicken ovalbumin under control of the rat insulin promoter (Rip-mOVA). We found that clonal deletion to TRA was completely abrogated in the absence of Bim and large numbers of mature OT-I CD8 T cells survived in the periphery. Despite the large numbers of autoreactive T cells, the chimeras did not develop diabetes and OT-I Bim-deficient T cells from these chimeras were functionally impaired. Collectively, these data provide unique evidence of a differential, thymocyte-intrinsic, molecular requirement downstream of the T-cell receptor (TCR) for clonal deletion to UbA versus TRA and highlight the profound ability of other tolerance mechanisms to control T-cell autoreactivity in the absence of thymic clonal deletion.

Bim is a component of the endosomal-lysosomal interface in Interleukin-7 dependent T cells (57.10)

Abstract Bim is a key protein in T cell homeostasis. Bim deficiency prevents activated T cell death, and protects splenic T cells in IL-7R/Bim double knockout mice through an undetermined mechanism. We propose a novel function for Bim in T cells involving lysosomal development in both apoptotic and non-apoptotic cells. Using IL-7 dependent T cell lines, Bim-containing and Bim-deficient (Bim-/-), we determined that the transcription of bim increased in the absence of IL-7 and the loss of Bim protected cells from apoptosis induced by IL-7 withdrawal. Treatment of Bim-containing cells with Bim siRNA confirmed these results. Bim-/- cells had increased endosomal and decreased lysosomal proteins, suggesting that Bim plays a role in endosomal to lysosomal trafficking and maturation. Subcellular organelle fractionation revealed that the BimL isoform associated with both endosomal and lysosomal fractions in non-apoptotic cells. However, during IL-7 withdrawal, the BimEL isoform associated with lysosomal fractions and less so with endosomal fractions. In fact, reduced co-localization of endosomes and lysosomes in the absence of Bim was observed by confocal microscopy, suggesting a defect in an endocytic compartment. Overexpression of the individual Bim isoforms restored acidic organelle distribution with BimL contributing the most in non-apoptotic cells. These results demonstrate that Bim has both apoptotic and non-apoptotic activities in T cells that affects lysosomal equilibrium.

BH3 Domains other than Bim and Bid Can Directly Activate Bax/Bak

Bcl-2 family proteins regulate a critical step in apoptosis referred to as mitochondrial outer membrane permeabilization (MOMP). Members of a subgroup of the Bcl-2 family, known as the BH3-only proteins, activate pro-apoptotic effectors (Bax and Bak) to initiate MOMP. They do so by neutralizing pro-survival Bcl-2 proteins and/or directly activating Bax/Bak. Bim and Bid are reported to be direct activators; however, here we show that BH3 peptides other than Bim and Bid exhibited various degrees of direct activation of the effector Bax or Bak, including Bmf and Noxa BH3s. In the absence of potent direct activators, such as Bim and Bid, we unmasked novel direct activator BH3 ligands capable of inducing effector-mediated cytochrome c release and liposome permeabilization, even when both Bcl-xL- and Mcl-1-type anti-apoptotic proteins were inhibited. The ability of these weaker direct activator BH3 peptides to cause MOMP correlated with that of the corresponding full-length proteins to induce apoptosis in the absence of Bim and Bid. We propose that, in certain contexts, direct activation by BH3-only proteins other than Bim and Bid may significantly contribute to MOMP and apoptosis.

A Major Role for Bim in Regulatory T Cell Homeostasis

We have previously shown that regulatory T cells (Treg) accumulate dramatically in aged animals and negatively impact the ability to control persistent infection. However, the mechanisms underlying the age-dependent accrual of Treg remain unclear. In this study, we show that Treg accumulation with age is progressive and likely not the result of increased thymic output, increased peripheral proliferation, or from enhanced peripheral conversion. Instead, we found that Treg from aged mice are more resistant to apoptosis than Treg from young mice. Although Treg from aged mice had increased expression of functional IL-7Rα, we found that IL-7R signaling was not required for maintenance of Treg in vivo. Notably, aged Treg exhibit decreased expression of the proapoptotic molecule Bim compared with Treg from young mice. Furthermore, in the absence of Bim, Treg accumulate rapidly, accounting for >25% of the CD4(+) T cell compartment by 6 mo of age. Additionally, accumulation of Treg in Bim-deficient mice occurred after the cells left the transitional recent thymic emigrant compartment. Mechanistically, we show that IL-2 drives preferential proliferation and accumulation of Bim(lo) Treg. Collectively, our data suggest that chronic stimulation by IL-2 leads to preferential expansion of Treg having low expression of Bim, which favors their survival and accumulation in aged hosts.

Opposing effects of bim and bcl-2 on lung endothelial cell migration.

Integration of cell adhesive, survival, and proliferative processes is essential for capillary morphogenesis of endothelial cells (EC) in vitro and vascular development and function in vivo. Unfortunately, the molecular and cellular mechanisms that impact these processes are poorly defined. Here we examined how lack of bim and/or bcl-2 expression impact lung EC function. The absence of bcl-2 or bim had a significant impact on EC adhesion and migration. Lack of bcl-2 expression decreased lung EC migration, whereas lack of bim expression increased migration compared with their wild-type counterparts. Decreased adhesion to fibronectin and vitronectin was observed in both bcl-2-/- and bim-/- lung EC, with bcl-2-/- EC having very little adhesion to either matrix protein. Capillary morphogenesis was greatly diminished in bcl-2-/- EC, which correlated with decreased lung alveolarization in vivo, an angiogenesis-dependent process. We also observed aberrant production of extracellular matrix proteins, eNOS expression, and nitric oxide production in bcl-2-/- lung EC, which could contribute to inability to undergo capillary morphogenesis. The changes in cell adhesion and migration noted in the absence of bim or bcl-2 were independent of their impact on apoptosis. We observed no significant affect on the steady-state rate of apoptosis of lung EC in the absence of bim or bcl-2. Thus, bcl-2 family members, bim and bcl-2, play a central role in modulation of EC proangiogenic properties, which goes beyond their role as simple mediators of mitochondrial homeostasis and apoptosis.

Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim sustains B lymphopoiesis in the absence of IL-7

IL-7 is pivotal for B cell development. Proteins of the Bcl-2 family are essential regulators of lymphocyte survival. Particularly, the pro-apoptotic BH3-only members Bim and Puma mediate lymphocyte apoptosis provoked by cytokine deprivation. Herein, we addressed whether the absence of Bim or Puma within the hematopoietic compartment could bypass the requirement for IL-7-driven B cell development in adult mice. We found that deficiency of Bim, but not Puma, partially rescued B cell development in the absence of IL-7. The numbers of both sIgM(-) and sIgM(+) B cells were markedly increased in the bone marrow of recipients lacking IL-7 upon reconstitution with Bim-deficient hematopoietic progenitors, compared with their control or Puma-deficient counterparts. The augmentation of B cell lymphopoiesis in the absence of Bim was reflected in the mature peripheral compartment by an increase in both the number of immature and mature B cells in the spleen and in the circulating IgM levels. Bim-deficient B cells were also increased in IL-7-sufficient recipients suggesting that peripheral B cells homeostasis is governed by a Bim-dependent and IL-7-independent mechanism. Our data highlight the role of Bim as a key regulator of cell survival during B lymphocyte development in vivo.

Polyglutamine-Expanded Androgen Receptor Truncation Fragments Activate a Bax-Dependent Apoptotic Cascade Mediated by DP5/Hrk

Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disorder caused by a polyglutamine (polyQ) repeat expansion in the androgen receptor (AR). PolyQ-AR neurotoxicity may involve generation of an N-terminal truncation fragment, as such peptides occur in SBMA patients and mouse models. To elucidate the basis of SBMA, we expressed N-terminal truncated AR in motor neuron-derived cells and primary cortical neurons. Accumulation of polyQ-AR truncation fragments in the cytosol resulted in neurodegeneration and apoptotic, caspase-dependent cell death. Using primary neurons from mice transgenic or deficient for apoptosis-related genes, we determined that polyQ-AR apoptotic activation is fully dependent on Bax. Jun N-terminal kinase (JNK) was required for apoptotic pathway activation through phosphorylation of c-Jun. Expression of polyQ-AR in DP5/Hrk null neurons yielded significant protection against apoptotic activation, but absence of Bim did not provide protection, apparently due to compensatory upregulation of DP5/Hrk or other BH3-only proteins. Misfolded AR protein in the cytosol thus initiates a cascade of events beginning with JNK and culminating in Bax-dependent, intrinsic pathway activation, mediated in part by DP5/Hrk. As apoptotic mediators are candidates for toxic fragment generation and other cellular processes linked to neuron dysfunction, delineation of the apoptotic activation pathway induced by polyQ-expanded AR may shed light on the pathogenic cascade in SBMA and other motor neuron diseases.