TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

Induction of Bim and Bid gene expression during accelerated apoptosis in severe sepsis

ABSTRACT: INTRODUCTION: In transgenic animal models of sepsis, members of the Bcl-2-family of proteins regulate lymphocyte apoptosis and survival of sepsis. This study investigates the gene regulation of pro- and anti-apoptotic members of the Bcl-2-family of proteins in patients with early stage severe sepsis. METHODS: In this prospective case-control study patients were recruited from three intensive care units in a university hospital. Sixteen patients were enrolled as soon as they fulfilled the criteria of severe sepsis. Ten critically ill but non-septic patients and eleven healthy volunteers served as controls. Blood samples were immediately obtained at inclusion. To confirm the presence of accelerated apoptosis in the patient groups, caspase-3 activation and phosphatidylserine (PS) externalization in CD4+, CD8+ and CD19+ lymphocyte subsets were assessed by flow cytometry. Specific mRNA's of Bcl-2 family members were quantified from whole blood by real-time polymerase chain reaction. To test for statistical significance, Kruskal-Wallis testing with Dunn's multiple comparison test for post hoc testing was performed. RESULTS: In all lymphocyte populations caspase-3 (p<0.05) was activated, which was reflected in an increased PS externalization (p<0.05). Accordingly, lymphocyte counts were decreased in early severe sepsis. In CD4+ T-cells (p<005) and in B-cells (p<0.001) the Bcl-2 protein was decreased in severe sepsis. Gene expression of the BH3-only Bim was massively upregulated as compared to critically ill patients (p<0.001) and 51.6 fold as compared to healthy controls (p<0.05). Bid was increased 12.9 fold compared to critically ill (p<0.001). In the group of the mitochondrial apoptosis-inducers, Bak was upregulated 5.6 fold, while the expression of Bax showed no significant variations. By contrast, the pro-survival members Bcl-2 and Bcl-xl were both downregulated in severe sepsis (p<0.001, p<0.05). CONCLUSIONS: In early severe sepsis a gene expression pattern with induction of the pro-apoptotic Bcl-2 family members Bim, Bid and Bak and a downregulation of the anti-apoptotic Bcl-2 and Bcl-xl was observed in peripheral blood. This constellation may affect cellular susceptibility to apoptosis and complex immune dysfunction in sepsis.

MEK/ERK-mediated phosphorylation of Bim is required to ensure survival of T and B lymphocytes during mitogenic stimulation

Survival and death of lymphocytes are regulated by the balance between pro- and antiapoptotic members of the Bcl-2 family; this is coordinated with the control of cell cycling and differentiation. Bim, a proapoptotic BH3-only member of the Bcl-2 family, can be regulated by MEK/ERK-mediated phosphorylation, which affects its binding to pro-survival Bcl-2 family members and its turnover. We investigated Bim modifications in mouse B and T lymphoid cells after exposure to apoptotic stimuli and during mitogenic activation. Treatment with ionomycin or cytokine withdrawal caused an elevation in Bim(EL), the most abundant Bim isoform. In contrast, in mitogenically stimulated T and B cells, Bim(EL) was rapidly phosphorylated, and its levels declined. Pharmacological inhibitors of MEK/ERK signaling prevented both of these changes in Bim, reduced proliferation, and triggered apoptosis of mitogen-stimulated T and B cells. Loss of Bim prevented this cell killing but did not restore cell cycling. These results show that during mitogenic stimulation of T and B lymphocytes MEK/ERK signaling is critical for two distinct processes, cell survival, mediated (at least in part) through phosphorylation and consequent inhibition of Bim, and cell cycling, which proceeds independently of Bim inactivation.

Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

Puma indirectly activates Bax to cause apoptosis in the absence of Bid or Bim

Bcl-2 family members regulate apoptosis in response to cytokine withdrawal and a broad range of cytotoxic stimuli. Pro-apoptotic Bcl-2 family members Bax and Bak are essential for apoptosis triggered by interleukin-3 (IL-3) withdrawal in myeloid cells. The BH3-only protein Puma is critical for initiation of IL-3 withdrawal-induced apoptosis, because IL-3-deprived Puma(-/-) cells show increased capacity to form colonies when IL-3 is restored. To investigate the mechanisms of Puma-induced apoptosis and the interactions between Puma and other Bcl-2 family members, we expressed Puma under an inducible promoter in cells lacking one or more Bcl-2 family members. Puma rapidly induced apoptosis in cells lacking the BH3-only proteins, Bid and Bim. Puma expression resulted in activation of Bax, but Puma killing was not dependent on Bax or Bak alone as Puma readily induced apoptosis in cells lacking either of these proteins, but could not kill cells deficient for both. Puma co-immunoprecipitated with the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1 but not with Bax or Bak. These data indicate that Puma functions, in the context of induced overexpression or IL-3 deprivation, primarily by binding and inactivating anti-apoptotic Bcl-2 family members.

Induction of Bim limits cytokine-mediated prolonged survival of neutrophils

Under inflammatory conditions, neutrophil apoptosis is delayed due to survival-factor exposure, a mechanism that prevents the resolution of inflammation. One important proinflammatory cytokine involved in the regulation of neutrophil survival/activation is granulocyte-macrophage colony-stimulating factor (GM-CSF). Although GM-CSF mediates antiapoptotic effects in neutrophils, it does not prevent apoptosis, and the survival effect is both time dependent and limited. Here, we identified the proapoptotic Bcl-2 family member Bim as an important lifespan limiting molecule in neutrophils, particularly under conditions of survival factor exposure. Strikingly, GM-CSF induced Bim expression in both human and mouse neutrophils that was blocked by pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). Increased Bim expression was also seen in human immature bone marrow neutrophils as well as in blood neutrophils from septic shock patients; both cell populations are known to be exposed to GM-CSF under in vivo conditions. The functional role of Bim was investigated using Bim-deficient mouse neutrophils in the presence and absence of the survival cytokines interleukin (IL)-3 and GM-CSF. Lack of Bim expression resulted in a much higher efficacy of the survival cytokines to block neutrophil apoptosis. Taken together, these data demonstrate a functional role for Bim in the regulation of neutrophil apoptosis and suggest that GM-CSF and other neutrophil hematopoietins initiate a proapoptotic counterregulation that involves upregulation of Bim.

Foxo-mediated Bim transcription is dispensable for the apoptosis of hematopoietic cells that is mediated by this BH3-only protein

The BH3-only protein Bim is a critical initiator of apoptosis in hematopoietic cells. Bim is upregulated in response to growth factor withdrawal and in vitro studies have implicated the transcription factor Foxo3a as a critical inducer. To test the importance of this regulation in vivo, we generated mice with mutated Foxo-binding sites within the Bim promoters (Bim(ΔFoxo/ΔFoxo)). Contrary to Bim-deficient mice, Bim(ΔFoxo/ΔFoxo) mice had a normal hematopoietic system. Moreover, cytokine-dependent haematopoietic cells from Bim(ΔFoxo/ΔFoxo) and wt mice died at similar rates. These results indicate that regulation of Bim by Foxo transcription factors is not critical for the killing of hematopoietic cells.

Thiazolides promote apoptosis in colorectal tumor cells via MAP kinase-induced Bim and Puma activation

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-xL leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of anti-tumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.

F. Bimḳo : der Dramaturg un Reʾalisṭ

Aharon Beḳerman

BIM MEDIATES IMATINIB-INDUCED APOPTOSIS OF GASTROINTESTINAL STROMAL TUMORS: TRANSLATIONAL IMPLICATIONS

Gastrointestinal stromal tumors (GISTs) are oncogene-addicted cancers driven by activating mutations in the genes encoding receptor tyrosine kinases KIT and PDGFR-α. Imatinib mesylate, a specific inhibitor of KIT and PDGFR-α signaling, delays progression of GIST, but is incapable of achieving cure. Thus, most patients who initially respond to imatinib therapy eventually experience tumor progression, and have limited therapeutic options thereafter. To address imatinib-resistance and tumor progression, these studies sought to understand the molecular mechanisms that regulate apoptosis in GIST, and evaluate combination therapies that kill GISTs cells via complementary, but independent, mechanisms. BIM (Bcl-2 interacting mediator of apoptosis), a pro-apoptotic member of the Bcl-2 family, effects apoptosis in oncogene-addicted malignancies treated with targeted therapies, and was recently shown to mediate imatinib-induced apoptosis in GIST. This dissertation examined the molecular mechanism of BIM upregulation and its cytotoxic effect in GIST cells harboring clinically-representative KIT mutations. Additionally, imatinib-induced alterations in BIM and pro-survival Bcl-2 proteins were studied in specimens from patients with GIST, and correlated to apoptosis, FDG-PET response, and survival. Further, the intrinsic pathway of apoptosis was targeted therapeutically in GIST cells with the Bcl-2 inhibitor ABT-737. These studies show that BIM is upregulated in GIST cells and patient tumors after imatinib exposure, and correlates with induction of apoptosis, response by FDG-PET, and disease-free survival. These studies contribute to the mechanistic understanding of imatinib-induced apoptosis in clinically-relevant models of GIST, and may facilitate prediction of resistance and disease progression in patients. Further, combining inhibition of KIT and Bcl-2 induces apoptosis synergistically and overcomes imatinib-resistance in GIST cells. Given that imatinib-resistance and GIST progression may reflect inadequate BIM-mediated inhibition of pro-survival Bcl-2 proteins, the preclinical evidence presented here suggests that direct engagement of apoptosis may be an effective approach to enhance the cytotoxicity of imatinib and overcome resistance.

Reseña del libro : Memorias del BIM : Biografías. Las víctimas de la Fuerza de Tareas 5 en La Plata, Berisso y Ensenada de Ana Julia Ramírez y Margarita Merbilhaá (editoras)

Fil: Codaro, Laura. Universidad Nacional de La Plata. Facultad de Humanidades y Ciencias de la Educación; Argentina.

Reseña del libro : Memorias del BIM : Biografías. Las víctimas de la Fuerza de Tareas 5 en La Plata, Berisso y Ensenada de Ana Julia Ramírez y Margarita Merbilhaá (editoras)

Fil: Codaro, Laura. Universidad Nacional de La Plata. Facultad de Humanidades y Ciencias de la Educación; Argentina.

Reseña del libro : Memorias del BIM : Biografías. Las víctimas de la Fuerza de Tareas 5 en La Plata, Berisso y Ensenada de Ana Julia Ramírez y Margarita Merbilhaá (editoras)

Fil: Codaro, Laura. Universidad Nacional de La Plata. Facultad de Humanidades y Ciencias de la Educación; Argentina.