ABSTRACT
Foxp3⺠regulatory T (Treg) cells are a crucial immunosuppressive population of CD4⺠T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.
Subject(s)
Apoptosis/immunology , Forkhead Transcription Factors/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Animals , Apoptosis/genetics , Cell Survival/genetics , Cell Survival/immunology , Female , Forkhead Transcription Factors/genetics , Gene Deletion , Homeostasis/immunology , Interleukin-2/metabolism , Lymphocyte Count , Male , Mice , Mice, Knockout , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins c-bcl-2/genetics , Signal TransductionABSTRACT
The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.
Subject(s)
Carrier Proteins/metabolism , Cell Death , Embryonic Development , Hematopoiesis , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Caspase 8/genetics , Caspase 8/metabolism , Cell Death/genetics , Embryo Loss/genetics , Embryonic Development/genetics , Endothelial Cells/cytology , Female , Hematopoiesis/genetics , Mice , Mice, Inbred C57BL , Protein Domains , Protein Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Receptors, Tumor Necrosis Factor, Type I/metabolism , Signal Transduction , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/geneticsABSTRACT
Due to the myriad interactions between prosurvival and proapoptotic members of the Bcl-2 family of proteins, establishing the mechanisms that regulate the intrinsic apoptotic pathway has proven challenging. Mechanistic insights have primarily been gleaned from in vitro studies because genetic approaches in mammals that produce unambiguous data are difficult to design. Here we describe a mutation in mouse and human Bak that specifically disrupts its interaction with the prosurvival protein Bcl-xL Substitution of Glu75 in mBak (hBAK Q77) for leucine does not affect the three-dimensional structure of Bak or killing activity but reduces its affinity for Bcl-xL via loss of a single hydrogen bond. Using this mutant, we investigated the requirement for physical restraint of Bak by Bcl-xL in apoptotic regulation. In vitro, Bak(Q75L) cells were significantly more sensitive to various apoptotic stimuli. In vivo, loss of Bcl-xL binding to Bak led to significant defects in T-cell and blood platelet survival. Thus, we provide the first definitive in vivo evidence that prosurvival proteins maintain cellular viability by interacting with and inhibiting Bak.
Subject(s)
Apoptosis/genetics , Blood Platelets/cytology , T-Lymphocytes/cytology , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-X Protein/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Blood Platelets/drug effects , Blood Platelets/metabolism , Cell Line , Cell Survival/genetics , Humans , Mice , Mice, Inbred C57BL , Mutation , Protein Binding , Protein Conformation , Protein Domains/genetics , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , bcl-2 Homologous Antagonist-Killer Protein/geneticsABSTRACT
The role of RNA-binding proteins of the CCCH-containing family in regulating proinflammatory cytokine production and inflammation is increasingly recognized. We have identified ZC3H12C (Regnase-3) as a potential post-transcriptional regulator of tumor necrosis factor expression and have investigated its role in vivo by generating Zc3h12c-deficient mice that express green fluorescent protein instead of ZC3H12C. Zc3h12c-deficient mice develop hypertrophic lymph nodes. In the immune system, ZC3H12C expression is mostly restricted to the dendritic cell (DC) populations, and we show that DC-restricted ZC3H12C depletion is sufficient to cause lymphadenopathy. ZC3H12C can regulate Tnf messenger RNA stability via its RNase activity in vitro, and we confirmed the role of Tnf in the development of lymphadenopathy. Finally, we found that loss of ZC3H12C did not impact the outcome of skin inflammation in the imiquimod-induced murine model of psoriasis, despite Zc3h12c being identified as a risk factor for psoriasis susceptibility in several genome-wide association studies. Our data suggest a role for ZC3H12C in DC-driven skin homeostasis.
Subject(s)
Lymphadenopathy , Psoriasis , Animals , Dendritic Cells , Genome-Wide Association Study , Inflammation/pathology , Lymph Nodes/pathology , Lymphadenopathy/pathology , Mice , Mice, Inbred C57BL , Skin/pathologyABSTRACT
STAT5 and interleukin 7 (IL-7) signaling are thought to control B lymphopoiesis by regulating the expression of key transcription factors and by activating variable (V(H)) gene segments at the immunoglobulin heavy-chain (Igh) locus. Using conditional mutagenesis to delete the gene encoding the transcription factor STAT5, we demonstrate that the development of pro-B cells was restored by transgenic expression of the prosurvival protein Bcl-2, which compensated for loss of the antiapoptotic protein Mcl-1. Expression of the genes encoding the B cell-specification factor EBF1 and the B cell-commitment protein Pax5 as well as V(H) gene recombination were normal in STAT5- or IL-7 receptor alpha-chain (IL-7Ralpha)-deficient pro-B cells rescued by Bcl-2. STAT5-expressing pro-B cells contained little or no active chromatin at most V(H) genes. In contrast, rearrangements of the immunoglobulin-kappa light-chain locus (Igk) were more abundant in STAT5- or IL-7Ralpha-deficient pro-B cells. Hence, STAT5 and IL-7 signaling control cell survival and the developmental ordering of immunoglobulin gene rearrangements by suppressing premature Igk recombination in pro-B cells.
Subject(s)
B-Lymphocytes/cytology , Cell Differentiation/genetics , Lymphoid Progenitor Cells/cytology , Lymphopoiesis/genetics , STAT5 Transcription Factor/genetics , Signal Transduction/immunology , Animals , B-Lymphocytes/immunology , Cell Differentiation/immunology , Cell Survival/genetics , Cell Survival/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Regulation/immunology , Gene Rearrangement/genetics , Gene Rearrangement/immunology , Genes, Immunoglobulin/genetics , Genes, Immunoglobulin/immunology , Interleukin-7/genetics , Interleukin-7/immunology , Lymphoid Progenitor Cells/immunology , Lymphopoiesis/immunology , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , STAT5 Transcription Factor/immunologyABSTRACT
The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.
Subject(s)
Lymphoma/genetics , Lymphoma/therapy , Mutation , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Proto-Oncogene Proteins c-myc/metabolism , Tumor Suppressor Protein p53/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Cell Survival/genetics , Gene Expression Regulation, Neoplastic , Humans , Lymphoma/pathology , Mice , Mice, Inbred C57BL , Protein Binding , Proto-Oncogene Proteins c-myc/geneticsABSTRACT
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 T cells 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.
Subject(s)
Apoptosis Regulatory Proteins/immunology , Autoantigens/immunology , Autoimmunity/immunology , Membrane Proteins/immunology , Proto-Oncogene Proteins/immunology , Thymocytes/immunology , Tumor Suppressor Proteins/immunology , Animals , Apoptosis/genetics , Apoptosis/immunology , Apoptosis Regulatory Proteins/genetics , Autoimmunity/genetics , Bcl-2-Like Protein 11 , Cell Differentiation/genetics , Cell Differentiation/immunology , Female , Flow Cytometry , Male , Membrane Proteins/genetics , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/immunology , Self Tolerance/genetics , Self Tolerance/immunology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Thymocytes/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/immunology , Tumor Suppressor Proteins/geneticsABSTRACT
Trp63, a transcription factor related to the tumor suppressor p53, is activated by diverse stimuli and can initiate a range of cellular responses. TAp63 is the predominant Trp53 family member in primordial follicle oocyte nuclei and is essential for their apoptosis triggered by DNA damage in vivo. After γ-irradiation, induction of the proapoptotic BH3-only members Puma and Noxa was observed in primordial follicle oocytes from WT and Trp53(-/-) mice but not in those from TAp63-deficient mice. Primordial follicle oocytes from mice lacking Puma or both Puma and Noxa were protected from γ-irradiation-induced apoptosis and, remarkably, could produce healthy offspring. Hence, PUMA and NOXA are critical for DNA damage-induced, TAp63-mediated primordial follicle oocyte apoptosis. Thus, blockade of PUMA may protect fertility during cancer therapy and prevent premature menopause, improving women's health.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis/genetics , DNA Damage , Fertility/genetics , Oocytes/metabolism , Tumor Suppressor Proteins/genetics , Animals , Apoptosis/radiation effects , Apoptosis Regulatory Proteins/metabolism , Female , Gamma Rays , Gene Expression/radiation effects , Immunohistochemistry , In Situ Hybridization , Male , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Oocytes/cytology , Oocytes/radiation effects , Phosphoproteins/genetics , Phosphoproteins/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Acute myeloid leukemia (AML) frequently relapses after initial treatment. Drug resistance in AML has been attributed to high levels of the anti-apoptotic Bcl-2 family members Bcl-x(L) and Mcl-1. Here we report that removal of Mcl-1, but not loss or pharmacological blockade of Bcl-x(L), Bcl-2, or Bcl-w, caused the death of transformed AML and could cure disease in AML-afflicted mice. Enforced expression of selective inhibitors of prosurvival Bcl-2 family members revealed that Mcl-1 is critical for survival of human AML cells. Thus, targeting of Mcl-1 or regulators of its expression may be a useful strategy for the treatment of AML.
Subject(s)
Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Antineoplastic Agents, Hormonal/pharmacology , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival , Gene Deletion , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Inbred C57BL , Myeloid Cell Leukemia Sequence 1 Protein , Tamoxifen/pharmacology , Tumor Cells, CulturedABSTRACT
BPSM1 (Bone phenotype spontaneous mutant 1) mice develop severe polyarthritis and heart valve disease as a result of a spontaneous mutation in the Tnf gene. In these mice, the insertion of a retrotransposon in the 3' untranslated region of Tnf causes a large increase in the expression of the cytokine. We have found that these mice also develop inducible bronchus-associated lymphoid tissue (iBALT), as well as nodular lymphoid hyperplasia (NLH) in the bone marrow. Loss of TNFR1 prevents the development of both types of follicles, but deficiency of TNFR1 in the hematopoietic compartment only prevents the iBALT and not the NLH phenotype. We show that the development of arthritis and heart valve disease does not depend on the presence of the tertiary lymphoid tissues. Interestingly, while loss of IL-17 or IL-23 limits iBALT and NLH development to some extent, it has no effect on polyarthritis or heart valve disease in BPSM1 mice.
Subject(s)
Lymphoid Tissue/pathology , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Bone Marrow/pathology , Hyperplasia , Interleukin-17/genetics , Interleukin-17/metabolism , Interleukin-23/genetics , Interleukin-23/metabolism , Lymphoid Tissue/metabolism , Mice , Tumor Necrosis Factor-alpha/geneticsABSTRACT
FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1+ (NRP1+ ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1- Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.
Subject(s)
CARD Signaling Adaptor Proteins/deficiency , Dermatitis, Atopic/immunology , Homeostasis/immunology , Signal Transduction/immunology , T-Lymphocytes, Regulatory/immunology , Animals , CARD Signaling Adaptor Proteins/genetics , CARD Signaling Adaptor Proteins/immunology , Dermatitis, Atopic/genetics , Disease Models, Animal , Ethylnitrosourea/toxicity , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Homeostasis/genetics , Humans , Introns/drug effects , Introns/genetics , Introns/immunology , Loss of Function Mutation/drug effects , Loss of Function Mutation/immunology , Mice , Mice, Transgenic , Mutagenesis/immunology , Mutagens/toxicity , Neuropilin-1/immunology , Neuropilin-1/metabolism , Polymorphism, Single Nucleotide/drug effects , Polymorphism, Single Nucleotide/immunology , Signal Transduction/genetics , T-Lymphocytes, Regulatory/metabolismABSTRACT
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.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , BH3 Interacting Domain Death Agonist Protein/metabolism , Caspase 8/metabolism , Chemical and Drug Induced Liver Injury , Hepatocytes/pathology , Membrane Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Bcl-2-Like Protein 11 , Mice , Mice, Inbred C57BL , Mice, Knockout , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are chronic inflammatory diseases that together affect 2-3% of the population. RA and AS predominantly involve joints, but heart disease is also a common feature in RA and AS patients. Here we have studied a new spontaneous mutation that causes severe polyarthritis in bone phenotype spontaneous mutation 1 (BPSM1) mice. In addition to joint destruction, mutant mice also develop aortic root aneurism and aorto-mitral valve disease that can be fatal depending on the genetic background. The cause of the disease is the spontaneous insertion of a retrotransposon into the 3' untranslated region (3'UTR) of the tumor necrosis factor (TNF), which triggers its strong overexpression in myeloid cells. We found that several members of a family of RNA-binding, CCCH-containing zinc-finger proteins control TNF expression through its 3'UTR, and we identified a previously unidentified regulatory element in the UTR. The disease in BPSM1 mice is independent of the adaptive immune system and does not appear to involve inflammatory cytokines other than TNF. To our knowledge, this is the first animal model showing both polyarthritis and heart disease as a direct result of TNF deregulation. These results emphasize the therapeutic potential of anti-TNF drugs for the treatment of heart valve disease and identify potential therapeutic targets to control TNF expression and inflammation.
Subject(s)
3' Untranslated Regions/genetics , Arthritis/genetics , Heart Valve Diseases/genetics , Mutagenesis, Insertional/genetics , Mutation/genetics , Retroelements/genetics , Tumor Necrosis Factor-alpha/genetics , Aneurysm/pathology , Animals , Aortic Valve Insufficiency/complications , Aortic Valve Insufficiency/diagnostic imaging , Aortic Valve Insufficiency/pathology , Arthritis/blood , Arthritis/diagnostic imaging , Arthritis/pathology , Base Sequence , Bone Marrow Transplantation , Chemokines/blood , Chronic Disease , Disease Models, Animal , Fibrosis , Heart Valve Diseases/blood , Heart Valve Diseases/diagnostic imaging , Heart Valve Diseases/pathology , Humans , Inflammation/pathology , Joints/pathology , Mice, Mutant Strains , Mitral Valve/pathology , Molecular Sequence Data , RNA Stability/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Short Interspersed Nucleotide Elements/genetics , Tumor Necrosis Factor-alpha/metabolism , Ultrasonography , X-Ray Microtomography , Zinc Fingers/geneticsABSTRACT
Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.
Subject(s)
Apoptosis , Dendritic Cells/cytology , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Cell Separation , Cell Survival , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Signal Transduction , Spleen/immunology , Spleen/metabolism , T-Lymphocytes/cytology , Transgenes , bcl-X Protein/metabolismABSTRACT
The B-cell CLL/lymphoma 2 (Bcl2) relative Myeloid cell leukemia sequence 1 (Mcl1) is essential for cell survival during development and for tissue homeostasis throughout life. Unlike Bcl2, Mcl1 turns over rapidly, but the physiological significance of its turnover has been unclear. We have gained insight into the roles of Mcl1 turnover in vivo by analyzing mice harboring a modified allele of Mcl1 that serendipitously proved to encode an abnormally stabilized form of Mcl1 due to a 13-aa N-terminal extension. Although the mice developed normally and appeared unremarkable, the homozygous males unexpectedly proved infertile due to defective spermatogenesis, which was evoked by enhanced Mcl1 prosurvival activity. Under unstressed conditions, the modified Mcl1 is present at levels comparable to the native protein, but it is markedly stabilized in cells subjected to stresses, such as protein synthesis inhibition or UV irradiation. Strikingly, the modified Mcl1 allele could genetically complement the loss of Bcl2, because introduction of even a single allele significantly ameliorated the severe polycystic kidney disease and consequent runting caused by Bcl2 loss. Significantly, the development of c-MYC-induced acute myeloid leukemia was also accelerated in mice harboring that Mcl1 allele. Our collective findings reveal that, under certain circumstances, the N terminus of Mcl1 regulates its degradation; that some cell types require degradation of Mcl1 to induce apoptosis; and, most importantly, that rapid turnover of Mcl1 can serve as a tumor-suppressive mechanism.
Subject(s)
Apoptosis , Cell Transformation, Neoplastic/genetics , Infertility, Male/genetics , Myeloid Cell Leukemia Sequence 1 Protein/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Alleles , Animals , Cell Death , Cell Survival , Female , Fibroblasts/metabolism , Flow Cytometry , Gene Expression Regulation , HEK293 Cells , Humans , Infertility, Male/metabolism , Leukemia, Myeloid, Acute/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Polycystic Kidney Diseases/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-myc/metabolism , Spermatogenesis , Testis/pathology , Time Factors , Ultraviolet RaysABSTRACT
BH3 domain-only proteins (BH3-only) proteins are members of the Bcl-2 family that play crucial roles in embryogenesis and the maintenance of tissue homeostasis by triggering apoptotic cell death. The BH3-only protein Bim is critical for developmental apoptosis of lymphocytes, securing establishment of tolerance and for the termination of immune responses. Bim is believed to act in concert with other BH3-only proteins or members of the tumor necrosis factor receptor family in getting rid of unwanted cells. Bmf, a related BH3-only protein, was shown to play a role in B-cell homeostasis and to mediate cell death in response to certain apoptotic triggers, including glucocorticoid, histone deacetylase inhibitors, and overexpression of the c-Myc proto-oncogene. Here we show that Bim and Bmf have overlapping functions during mouse development and coregulate lymphocyte homeostasis and apoptosis in a nonredundant manner. Double deficiency of Bim and Bmf caused more B lymphadenopathy than loss of either BH3-only protein alone, and this was associated with autoimmune glomerulonephritis and a range of malignancies in aged mice. Thus, our results demonstrate that Bim and Bmf act in concert to prevent autoimmunity and malignant disease, strengthening the rational for the development of BH3-only protein mimicking therapeutics for the treatment of such disorders.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Apoptosis Regulatory Proteins/genetics , Cell Death , Homeostasis , Lymphocytes/cytology , Membrane Proteins/genetics , Proto-Oncogene Proteins/genetics , Adaptor Proteins, Signal Transducing/physiology , Animals , Apoptosis , Apoptosis Regulatory Proteins/physiology , Autoantibodies/immunology , Autoimmunity , Bcl-2-Like Protein 11 , Cell Separation , Female , Flow Cytometry , Genotype , Immune System , Immunoglobulins/immunology , Male , Membrane Proteins/physiology , Mice , Mice, Transgenic , Phenotype , Protein Structure, Tertiary , Proto-Oncogene Proteins/physiology , Thymocytes/cytologyABSTRACT
High levels of the pro-inflammatory cytokine tumour necrosis factor (TNF) have been associated with many diseases including rheumatoid arthritis (RA), ankylosing spondylitis (AS), inflammatory bowel disease (IBD) and psoriasis. Although it has been clear for twenty-five years that TNF plays a major role in RA and AS, two major questions remain unanswered: (1) What mechanism underlies the loss of control of TNF levels in patients? (2) How does TNF exert its detrimental effects? Nonetheless, biological anti-TNF drugs have become the most successful treatment of these conditions with a third of patients entering remission, and the global market for biological TNF inhibitors is now estimated at around US$35 billions. However, their use is limited by their cost, the fact that they need to be injected, non-negligible side effects and the development of resistance due to the protein (thus antigenic) nature of these TNF inhibitors. It looks inevitable that new approaches to lower the amount of TNF should be considered. To do this, a better understanding of the regulation of TNF expression is necessary.
Subject(s)
3' Untranslated Regions/genetics , Gene Expression Regulation , Heart Valve Diseases/genetics , Regulatory Elements, Transcriptional/genetics , Tumor Necrosis Factors/genetics , Animals , Arthritis/genetics , Humans , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Mutant Strains , Models, Genetic , Mutagenesis, Insertional , Mutation , Retroelements/geneticsABSTRACT
Apoptosis plays a prominent role during ovarian development by eliminating large numbers of germ cells from the female germ line. However, the precise mechanisms and regulatory proteins involved in germ cell death are yet to be determined. In this study, we characterised the role of the pro-apoptotic BH3-only protein, BCL2-modifying factor (BMF), in germ cell apoptosis in embryonic and neonatal mouse ovaries. BMF protein was immunohistochemically localised to germ cells at embryonic days 15.5 (E15.5) and E17.5 and postnatal day 1 (PN1), coincident with entry into the meiotic prophase, but was undetectable at E13.5, and only present at low levels at PN3 and PN5. Consistent with this expression pattern, loss of BMF in female mice was associated with a decrease in apoptosis at E15.5 and E17.5. Furthermore, increased numbers of germ cells were found in ovaries from Bmf(-/-) mice compared with WT animals at E15.5 and PN1. However, germ cell numbers were comparable between Bmf(-/-) and WT ovaries at PN3, PN5 and PN10. Collectively, these data indicate that BMF mediates foetal oocyte loss and its action limits the maximal number of germ cells attained in the developing ovary, but does not influence the number of primordial follicles initially established in ovarian reserve.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Apoptosis , Embryo, Mammalian/pathology , Germ Cells/pathology , Oocytes/pathology , Oogenesis/physiology , Ovary/pathology , Animals , Blotting, Western , Cells, Cultured , Embryo, Mammalian/metabolism , Female , Fluorescent Antibody Technique , Germ Cells/metabolism , Immunoenzyme Techniques , Mice , Mice, Inbred C57BL , Mice, Knockout , Oocytes/metabolism , Ovary/metabolism , Proto-Oncogene Proteins c-bcl-2ABSTRACT
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.
Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis , Forkhead Transcription Factors/metabolism , Hematopoietic System/cytology , Membrane Proteins/genetics , Proto-Oncogene Proteins/genetics , Transcription, Genetic , Animals , Apoptosis/drug effects , Apoptosis/genetics , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/metabolism , Base Sequence , Bcl-2-Like Protein 11 , Binding Sites , Carcinogenesis/metabolism , Carcinogenesis/pathology , Cell Death/drug effects , Cytokines/pharmacology , Forkhead Box Protein O3 , Forkhead Transcription Factors/deficiency , HEK293 Cells , Hematopoiesis/drug effects , Hematopoietic System/drug effects , Hematopoietic System/metabolism , Humans , Lymphoma/pathology , Membrane Proteins/deficiency , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Mutation/genetics , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Binding/genetics , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Thymocytes/cytology , Thymocytes/drug effects , Thymocytes/metabolism , Transcription, Genetic/drug effectsABSTRACT
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.