Medical Biology - Research Publications
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ItemNo Preview AvailableIn vitro and in vivo assays for osteoclast apoptosis(BIOMED CENTRAL LTD, 2005-05-09)Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone.
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ItemNo Preview AvailableCAML regulates Bim-dependent thymocyte death(NATURE PUBLISHING GROUP, 2010-10)Appropriate control of apoptosis during T lymphocyte differentiation is critical for destruction of T cells bearing potentially autoreactive or useless immuno-receptors and for survival of those T cells bearing antigen receptors that may recognize foreign proteins. Despite the well-established importance of thymocyte survival, the exact signals regulating thymocyte apoptosis have not been fully elucidated. Here, we show that thymocytes lacking the endoplasmic reticulum protein calcium-modulating cyclophilin ligand (CAML) failed to undergo normal T-cell development and exhibited dramatically increased rates of apoptosis. In vitro, CAML-deficient thymocytes accumulated high levels of reactive oxygen species (ROS) and underwent abnormally accelerated death in response to several cytotoxic stimuli, including treatment with etoposide, cytokine deprivation, or Fas ligation. Although neither p53 deletion nor loss of Fas rescued the survival and continued development of CAML-deficient thymocytes, removal of the pro-apoptotic BH3-only Bcl-2 family member Bim significantly restored their survival. This work reveals CAML to be a critically important regulator of ROS- and Bim-dependent thymocyte death.
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ItemNo Preview AvailableIndividual and overlapping roles of BH3-only proteins Bim and Bad in apoptosis of lymphocytes and platelets and in suppression of thymic lymphoma development(NATURE PUBLISHING GROUP, 2010-10)BH3-only proteins, such as Bim and Bad, contribute to tissue homeostasis by initiating apoptosis in a cell type- and stimulus-specific manner. Loss of Bim provokes lymphocyte accumulation in vivo and renders lymphocytes more resistant to diverse apoptotic stimuli and Bad has been implicated in the apoptosis of haematopoietic cells upon cytokine deprivation. To investigate whether their biological roles in apoptosis overlap, we generated mice lacking both Bim and Bad and compared their haematopoietic phenotype with that of the single-knockout and wild-type (wt) animals. Unexpectedly, bad(-/-) mice had excess platelets due to prolonged platelet life-span. The bim(-/-)bad(-/-) mice were anatomically normal and fertile. Their haematopoietic phenotype resembled that of bim(-/-) mice but lymphocytes were slightly more elevated in their lymph nodes. Although resting B and T lymphocytes from bim(-/-)bad(-/-) and bim(-/-) animals displayed similar resistance to diverse apoptotic stimuli, mitogen activated bim(-/-)bad(-/-) B cells were more refractory to cytokine deprivation. Moreover, combined loss of Bim and Bad-enhanced survival of thymocytes after DNA damage and accelerated development of γ-irradiation-induced thymic lymphoma. Unexpectedly, their cooperation in the thymus depended upon thymocyte-stromal interaction. Collectively, these results show that Bim and Bad can cooperate in the apoptosis of thymocytes and activated B lymphocytes and in the suppression of thymic lymphoma development.
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ItemNo Preview AvailableMembrane-bound Fas ligand only is essential for Fas-induced apoptosis(NATURE PUBLISHING GROUP, 2009-10-01)Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasL(Deltas/Deltas)) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasL(Deltam/Deltam)) could not kill cells through Fas activation. FasL(Deltam/Deltam) mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasL(gld/gld) mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasL(Deltam/Deltam) mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasL(gld/gld) mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.
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ItemLoss of the BH3-only protein Bmf impairs B cell homeostasis and accelerates γ irradiation-induced thymic lymphoma development(ROCKEFELLER UNIV PRESS, 2008-03-17)Members of the Bcl-2 protein family play crucial roles in the maintenance of tissue homeostasis by regulating apoptosis in response to developmental cues or exogenous stress. Proapoptotic BH3-only members of the Bcl-2 family are essential for initiation of cell death, and they function by activating the proapoptotic Bcl-2 family members Bax and/or Bak, either directly or indirectly through binding to prosurvival Bcl-2 family members. Bax and Bak then elicit the downstream events in apoptosis signaling. Mammals have at least eight BH3-only proteins and they are activated in a stimulus-specific, as well as a cell type-specific, manner. We have generated mice lacking the BH3-only protein Bcl-2-modifying factor (Bmf) to investigate its role in cell death signaling. Our studies reveal that Bmf is dispensable for embryonic development and certain forms of stress-induced apoptosis, including loss of cell attachment (anoikis) or UV irradiation. Remarkably, loss of Bmf protected lymphocytes against apoptosis induced by glucocorticoids or histone deacetylase inhibition. Moreover, bmf(-/-) mice develop a B cell-restricted lymphadenopathy caused by the abnormal resistance of these cells to a range of apoptotic stimuli. Finally, Bmf-deficiency accelerated the development of gamma irradiation-induced thymic lymphomas. Our results demonstrate that Bmf plays a critical role in apoptosis signaling and can function as a tumor suppressor.
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ItemGefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics(PUBLIC LIBRARY SCIENCE, 2007-10)BACKGROUND: The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms. METHODS AND FINDINGS: We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called "mitochondrial") apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK-ERK1/2 (mitogen-activated protein kinase kinase-extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737. CONCLUSIONS: Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR-MEK-ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.
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ItemPro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis(NATURE PUBLISHING GROUP, 2010-05)The process of apoptosis in immune cells like mast cells is essential to regain homeostasis after an inflammatory response. The intrinsic pathway of apoptosis is ultimately controlled by the pro-apoptotic Bcl-2 family members Bax and Bak, which upon activation oligomerize to cause increased permeabilization of the mitochondria outer membrane leading to cell death. We examined the role of Bax and Bak in cytokine deprivation-induced apoptosis in mast cells using connective tissue-like mast cells and mucosal-like mast cells derived from bax(-/-), bak(-/-) and bax(-/-)bak(-/-) mice. Although both Bax and Bak were expressed at readily detectable protein levels, we found a major role for Bax in mediating mast cell apoptosis induced by cytokine deprivation. We analyzed cell viability by propidium iodide exclusion and flow cytometry after deprivation of vital cytokines for each mast cell population. Upon cytokine withdrawal, bak(-/-) mast cells died at a similar rate as wild type, whereas bax(-/-) and bax(-/-)bak(-/-) mast cells were partially or completely resistant to apoptosis, respectively. The total resistance seen in bax(-/-)bak(-/-) mast cells is comparable with mast cells deficient of both pro-apoptotic Bim and Puma or mast cells overexpressing anti-apoptotic Bcl-2. These results show that Bax has a predominant and Bak a minor role in cytokine deprivation-induced apoptosis in both connective tissue-like and mucosal-like mast cells.
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ItemAMP kinase-mediated activation of the BH3-only protein Bim couples energy depletion to stress-induced apoptosis(ROCKEFELLER UNIV PRESS, 2010-04-05)Excitotoxicity after glutamate receptor overactivation induces disturbances in cellular ion gradients, resulting in necrosis or apoptosis. Excitotoxic necrosis is triggered by rapid, irreversible ATP depletion, whereas the ability to recover cellular bioenergetics is suggested to be necessary for the activation of excitotoxic apoptosis. In this study, we demonstrate that even a transient decrease in cellular bioenergetics and an associated activation of adenosine monophosphate-activated protein kinase (AMPK) is necessary for the activation of excitotoxic apoptosis. We show that the Bcl-2 homology domain 3 (BH3)-only protein Bim, a proapoptotic Bcl-2 family member, is activated in multiple excitotoxicity paradigms, mediates excitotoxic apoptosis, and inhibits delayed Ca(2+) deregulation, mitochondrial depolarization, and apoptosis-inducing factor translocation. We demonstrate that bim activation required the activation of AMPK and that prolonged AMPK activation is sufficient to induce bim gene expression and to trigger a bim-dependent cell death. Collectively, our data demonstrate that AMPK activation and the BH3-only protein Bim couple transient energy depletion to stress-induced neuronal apoptosis.
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ItemUltraviolet radiation triggers apoptosis of fibroblasts and skin keratinocytes mainly via the BH3-only protein Noxa(ROCKEFELLER UNIV PRESS, 2007-02-12)To identify the mechanisms of ultraviolet radiation (UVR)-induced cell death, for which the tumor suppressor p53 is essential, we have analyzed mouse embryonic fibroblasts (MEFs) and keratinocytes in mouse skin that have specific apoptotic pathways blocked genetically. Blocking the death receptor pathway provided no protection to MEFs, whereas UVR-induced apoptosis was potently inhibited by Bcl-2 overexpression, implicating the mitochondrial pathway. Indeed, Bcl-2 overexpression boosted cell survival more than p53 loss, revealing a p53-independent pathway controlled by the Bcl-2 family. Analysis of primary MEFs lacking individual members of its BH3-only subfamily identified major initiating roles for the p53 targets Noxa and Puma. In the transformed derivatives, where Puma, unexpectedly, was not induced by UVR, Noxa had the dominant role and Bim a minor role. Furthermore, loss of Noxa suppressed the formation of apoptotic keratinocytes in the skin of UV-irradiated mice. Collectively, these results demonstrate that UVR activates the Bcl-2-regulated apoptotic pathway predominantly through activation of Noxa and, depending on cellular context, Puma.
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ItemLoss of Bim increases T cell production and function in interleukin 7 receptor-deficient mice(ROCKEFELLER UNIV PRESS, 2004-11-01)Interleukin (IL)-7 receptor (R) signaling is essential for T and B lymphopoiesis by promoting proliferation, differentiation, and survival of cells. Mice lacking either IL-7 or the IL-7Ralpha chain have abnormally low numbers of immature as well as mature T and B lymphocytes. Transgenic expression of the apoptosis inhibitor Bcl-2 rescues T cell development and function in IL-7Ralpha-deficient mice, indicating that activation of a proapoptotic Bcl-2 family member causes death of immature and mature T cells. BH3-only proteins such as Bim, which are distant proapoptotic members of the Bcl-2 family, are essential initiators of programmed cell death and stress-induced apoptosis. We generated Bim/IL-7Ralpha double deficient mice and found that loss of Bim significantly increased thymocyte numbers, restored near normal numbers of mature T cells in the blood and spleen, and enhanced cytotoxic T cell responses to virus infection in IL-7Ralpha-/- mice. These results indicate that Bim cooperates with other proapoptotic proteins in the death of IL-7-deprived T cell progenitors in vivo, but is the major inducer of this pathway to apoptosis in mature T cells. This indicates that pharmacological inhibition of Bim function might be useful for boosting immune responses in immunodeficient patients.