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 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 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.
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ItemCombined loss of proapoptotic genes Bak or Bax with Bim synergizes to cause defects in hematopoiesis and in thymocyte apoptosis(ROCKEFELLER UNIV PRESS, 2005-06-20)The proapoptotic members of the Bcl-2 family can be subdivided into members that contain several Bcl-2 homology (BH) domains and those that contain only the BH3 domain. Although it is known that BH3-only proteins and the multi-BH domain proteins, Bak and Bax, are essential for programmed cell death, the overlapping role of these two subgroups has not been examined in vivo. To investigate this, we generated Bak/Bim and Bax/Bim double deficient mice. We found that although Bax-/-Bim-/-, but not Bak-/-Bim-/-, mice display webbed hind and front paws and malocclusion of the incisors, both groups of mice present with dysregulated hematopoiesis. Combined loss of Bak and Bim or Bax and Bim causes defects in myeloid and B-lymphoid development that are more severe than those found in the single knock-out mice. Bak-/-Bim-/- mice have a complement of thymocytes that resembles those in control mice, whereas Bax-/-Bim-/- mice are more similar to Bim-/- mice. However, thymocytes isolated from Bak-/-Bim-/- or Bax-/-Bim-/- mice are markedly more resistant to apoptotic stimuli mediated by the intrinsic pathway as compared with thymocytes from single-knockout mice. These data suggest an essential overlapping role for Bak or Bax and Bim in the intrinsic apoptotic pathway.
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ItemLoss of the pro-apoptotic BH3-only Bcl-2 family member Bim inhibits BCR stimulation-induced apoptosis and deletion of autoreactive B cells(ROCKEFELLER UNIV PRESS, 2003-10-06)During development, the stochastic process assembling the genes encoding antigen receptors invariably generates B and T lymphocytes that can recognize self-antigens. Several mechanisms have evolved to prevent the activation of these cells and the concomitant development of autoimmune disease. One such mechanism is the induction of apoptosis in developing or mature B cells by engagement of the B cell antigen receptor (BCR) in the absence of T cell help. Here we report that B lymphocytes lacking the pro-apoptotic Bcl-2 family member Bim are refractory to apoptosis induced by BCR ligation in vitro. The loss of Bim also inhibited deletion of autoreactive B cells in vivo in two transgenic systems of B cell tolerance. Bim loss prevented deletion of autoreactive B cells induced by soluble self-antigen and promoted accumulation of self-reactive B cells developing in the presence of membrane-bound self-antigen, although their numbers were considerably lower compared with antigen-free mice. Mechanistically, we determined that BCR ligation promoted interaction of Bim with Bcl-2, inhibiting its survival function. These findings demonstrate that Bim is a critical player in BCR-mediated apoptosis and in B lymphocyte deletion.
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ItemThe role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins(ROCKEFELLER UNIV PRESS, 2009-08-10)Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim's proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.