Medical Biology - Research Publications
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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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ItemFas ligand, Bcl-2, granulocyte colony-stimulating factor, and p38 mitogen-activated protein kinase: Regulators of distinct cell death and survival pathways in granulocytes(ROCKEFELLER UNIV PRESS, 2000-09-04)The short life span of granulocytes, which limits many inflammatory responses, is thought to be influenced by the Bcl-2 protein family, death receptors such as CD95 (Fas/APO-1), stress-activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), and proinflammatory cytokines like granulocyte colony-stimulating factor (G-CSF). To clarify the roles of these various regulators in granulocyte survival, we have investigated the spontaneous apoptosis of granulocytes in culture and that induced by Fas ligand or chemotherapeutic drugs, using cells from normal, CD95-deficient lpr, or vav-bcl-2 transgenic mice. CD95-induced apoptosis, which required receptor aggregation by recombinant Fas ligand or the membrane-bound ligand, was unaffected by G-CSF treatment or Bcl-2 overexpression. Conversely, spontaneous and drug-induced apoptosis occurred normally in lpr granulocytes but were suppressed by G-CSF treatment or Bcl-2 overexpression. Although activation of p38 MAPK has been implicated in granulocyte death, their apoptosis actually was markedly accelerated by specific inhibitors of this kinase. These results suggest that G-CSF promotes granulocyte survival largely through the Bcl-2-controlled pathway, whereas CD95 regulates a distinct pathway to apoptosis that is not required for either their spontaneous or drug-induced death. Moreover, p38 MAPK signaling contributes to granulocyte survival rather than their apoptosis.
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ItemB lymphocytes differentially use the Rel and nuclear factor κB1 (NF-κB1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells(ROCKEFELLER UNIV PRESS, 1998-03-02)Rel and nuclear factor (NF)-kappaB1, two members of the Rel/NF-kappaB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-kappaB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1-/- mice, the level of apoptosis in cultures of quiescent nfkb1-/-, but not c-rel-/-, B cells is higher. The failure of c-rel-/- or nfkb1-/- B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel-/- and nfkb1-/- B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-kappaB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-kappaB-dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel-/- B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-kappaB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-kappaB factors to control cell cycle progression and prevent apoptosis.
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ItemPro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-xL(ROCKEFELLER UNIV PRESS, 2000-05-01)How Bcl-2 and its pro-survival relatives prevent activation of the caspases that mediate apoptosis is unknown, but they appear to act through the caspase activator apoptosis protease-activating factor 1 (Apaf-1). According to the apoptosome model, the Bcl-2-like proteins preclude Apaf-1 activity by sequestering the protein. To explore Apaf-1 function and to test this model, we generated monoclonal antibodies to Apaf-1 and used them to determine its localization within diverse cells by subcellular fractionation and confocal laser scanning microscopy. Whereas Bcl-2 and Bcl-x(L) were prominent on organelle membranes, endogenous Apaf-1 was cytosolic and did not colocalize with them, even when these pro-survival proteins were overexpressed or after apoptosis was induced. Immunogold electron microscopy confirmed that Apaf-1 was dispersed in the cytoplasm and not on mitochondria or other organelles. After the death stimuli, Bcl-2 and Bcl-x(L) precluded the release of the Apaf-1 cofactor cytochrome c from mitochondria and the formation of larger Apaf-1 complexes, which are steps that presage apoptosis. However, neither Bcl-2 nor Bcl-x(L) could prevent the in vitro activation of Apaf-1 induced by the addition of exogenous cytochrome c. Hence, rather than sequestering Apaf-1 as proposed by the apoptosome model, Bcl-2-like proteins probably regulate Apaf-1 indirectly by controlling upstream events critical for its activation.
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Itembcl-2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells(ROCKEFELLER UNIV PRESS, 2000-02-07)Immunization with T cell-dependent antigens generates long-lived memory B cells and antibody-forming cells (AFCs). Both populations originate in germinal centers and, predominantly, produce antibodies with high affinity for antigen. The means by which germinal center B cells are recruited into these populations remains unclear. We have examined affinity maturation of antigen-specific B cells in mice expressing the cell death inhibitor bcl-2 as a transgene. Such mice had reduced apoptosis in germinal centers and an excessive number of memory B cells with a low frequency of V gene somatic mutation, including those mutations encoding amino acid exchanges known to enhance affinity. Despite the frequency of AFCs being increased in bcl-2-transgenic mice, the fraction secreting high-affinity antibody in the bone marrow at day 42 remained unchanged compared with controls. The inability of BCL-2 to alter selection of bone marrow AFCs is consistent with these cells being selected within the germinal center on the basis of their affinity being above some threshold rather than their survival being due to a selective competition for an antigen-based signal. Continuous competition for antigen does, however, explain formation of the memory compartment.