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Hematopoiesis is a dynamic system that requires balanced cell division, differentiation, and death. The 2 major modes of programmed cell death, apoptosis and necroptosis, share molecular machinery but diverge in outcome with important implications for the microenvironment; apoptotic cells are removed in an immune silent process, whereas necroptotic cells leak cellular contents that incite inflammation. Given the importance of cytokine-directed cues for hematopoietic cell survival and differentiation, the impact on hematopoietic homeostasis of biasing cell death fate to necroptosis is substantial and poorly understood. Here, we present a mouse model with increased bone marrow necroptosis. Deletion of the proapoptotic Bcl-2 family members Bax and Bak inhibits bone marrow apoptosis. Further deletion of the BH3-only member Bid (to generate triple-knockout [TKO] mice) leads to unrestrained bone marrow necroptosis driven by increased Rip1 kinase (Ripk1). TKO mice display loss of progenitor cells, leading to increased cytokine production and increased stem cell proliferation and exhaustion and culminating in bone marrow failure. Genetically restoring Ripk1 to wild-type levels restores peripheral red cell counts as well as normal cytokine production. TKO bone marrow is hypercellular with abnormal differentiation, resembling the human disorder myelodysplastic syndrome (MDS), and we demonstrate increased necroptosis in MDS bone marrow. Finally, we show that Bid impacts necroptotic signaling through modulation of caspase-8-mediated Ripk1 degradation. Thus, we demonstrate that dysregulated necroptosis in hematopoiesis promotes bone marrow progenitor cell death that incites inflammation, impairs hematopoietic stem cells, and recapitulates the salient features of the bone marrow failure disorder MDS.
© 2019 by The American Society of Hematology.
Bcl-2 family proteins reorganize mitochondrial membranes during apoptosis, to form pores and rearrange cristae. In vitro and in vivo analysis integrated with human genetics reveals a novel homeostatic mitochondrial function for Bcl-2 family protein Bid. Loss of full-length Bid results in apoptosis-independent, irregular cristae with decreased respiration. mice display stress-induced myocardial dysfunction and damage. A gene-based approach applied to a biobank, validated in two independent GWAS studies, reveals that decreased genetically determined BID expression associates with myocardial infarction (MI) susceptibility. Patients in the bottom 5% of the expression distribution exhibit >4 fold increased MI risk. Carrier status with nonsynonymous variation in Bid's membrane binding domain, Bid, associates with MI predisposition. Furthermore, Bid but not Bid associates with Mcl-1, previously implicated in cristae stability; decreased MCL-1 expression associates with MI. Our results identify a role for Bid in homeostatic mitochondrial cristae reorganization, that we link to human cardiac disease.
© 2018, Salisbury-Ruf et al.
Apoptosis is programmed cell death triggered by activation of death receptors or cellular stress. Activation of caspases is the hallmark of apoptosis. Arrestins are best known for their role in homologous desensitization of G protein-coupled receptors (GPCRs). Arrestins quench G protein activation by binding to activated phosphorylated GPCRs. Recently, arrestins have been shown to regulate multiple signalling pathways in G protein-independent manner via scaffolding signalling proteins. Here we demonstrate that arrestin-2 isoform is cleaved by caspases during apoptosis induced via death receptor activation or by DNA damage at evolutionarily conserved sites in the C-terminus. Caspase-generated arrestin-2-(1-380) fragment translocates to mitochondria increasing cytochrome C release, which is the key checkpoint in cell death. Cells lacking arrestin-2 are significantly more resistant to apoptosis. The expression of wild-type arrestin-2 or its cleavage product arrestin-2-(1-380), but not of its caspase-resistant mutant, restores cell sensitivity to apoptotic stimuli. Arrestin-2-(1-380) action depends on tBID: at physiological concentrations, arrestin-2-(1-380) directly binds tBID and doubles tBID-induced cytochrome C release from isolated mitochondria. Arrestin-2-(1-380) does not facilitate apoptosis in BID knockout cells, whereas its ability to increase caspase-3 activity and facilitate cytochrome C release is rescued when BID expression is restored. Thus, arrestin-2-(1-380) cooperates with another product of caspase activity, tBID, and their concerted action significantly contributes to cell death.
Multicellular organisms maintain genomic integrity and resist tumorigenesis through a tightly regulated DNA damage response (DDR) that prevents propagation of deleterious mutations either through DNA repair or programmed cell death. An impaired DDR leads to tumorigenesis that is accelerated when programmed cell death is prevented. Loss of the ATM (ataxia telangiectasia mutated)-mediated DDR in mice results in T-cell leukemia driven by accumulation of DNA damage accrued during normal T-cell development. Pro-apoptotic BH3-only Bid is a substrate of Atm, and Bid phosphorylation is required for proper cell cycle checkpoint control and regulation of hematopoietic function. In this report, we demonstrate that, surprisingly, loss of Bid increases the latency of leukemogenesis in Atm-/- mice. Bid-/-Atm-/- mice display impaired checkpoint control and increased cell death of DN3 thymocytes. Loss of Bid thus inhibits T-cell tumorigenesis by increasing clearance of damaged cells, and preventing propagation of deleterious mutations.
The BH3-only Bid protein is a critical sentinel of cellular stress in the liver and the hematopoietic system. Bid's initial 'claim to fame' came from its ability-as a caspase-truncated product-to trigger the mitochondrial apoptotic program following death receptor activation. Today we know that Bid can response to multiple types of proteases, which are activated under different conditions such as T-cell activation, ischemical reperfusion injury and lysosomal injury. Activation of the mitochondrial apoptotic program by Bid-via its recently identified receptor mitochondrial carrier homolog 2-involves multiple mechanisms, including release of cytochrome c and second mitochondria-derived activator of caspase (Smac), alteration of mitochondrial cristae organization, generation of reactive oxygen species and engagement of the permeability transition pore. Bid is also emerging-in its full-length form-as a pivotal sentinel of DNA damage in the bone marrow regulated by the ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR) kinases. The ATM/ATR-Bid pathway is critically involved in preserving the quiescence and survival of hematopoietic stem cells both in the absence and presence of external stress, and a large part of this review will be dedicated to recent advances in this area of research.
Hematopoietic stem cells (HSCs) possess long-term self-renewal capacity and multipotent differentiative capacity, to maintain the hematopoietic system. Long-term hematopoietic homeostasis requires effective control of genotoxic damage to maintain HSC function and prevent propagation of deleterious mutations. Here we investigate the role of the BH3-only Bcl-2 family member Bid in the response of murine hematopoietic cells to long-term replicative stress induced by hydroxyurea (HU). The PI3-like serine/threonine kinase, ATR, initiates the DNA damage response (DDR) to replicative stress. The pro-apoptotic Bcl-2 family member, Bid, facilitates this response to replicative stress in hematopoietic cells, but the in vivo role of this DDR function of Bid has not been defined. Surprisingly, we demonstrate that long-term HU treatment expands wild-type myeloid progenitor cells (MPCs) and HSC-enriched Lin(-)Sca1(+)Kit(+) (LSK) cells to maintain bone marrow function as measured by long-term competitive repopulating ability. Bid-/- MPCs demonstrate increased sensitivity to HU and are depleted. Bid-/- LSK cells demonstrate increased mobilization manifest by increased Bromodeoxyuridine (BrdU) incorporation. Bid-/- MPCs and LSK cells are relatively depleted, however, and bone marrow from Bid-/- mice demonstrates decreased long-term competitive repopulating ability in both primary and secondary transplants. We thus describe a survival function of Bid in hematopoiesis in the setting of chronic replicative stress.
Proapoptotic BH3-interacting death domain agonist (BID) regulates apoptosis and the DNA damage response. Following replicative stress, BID associates with proteins of the DNA damage sensor complex, including replication protein A (RPA), ataxia telangiectasia and Rad3 related (ATR), and ATR-interacting protein (ATRIP), and facilitates an efficient DNA damage response. We have found that BID stimulates the association of RPA with components of the DNA damage sensor complex through interaction with the basic cleft of the N-terminal domain of the RPA70 subunit. Disruption of the BID-RPA interaction impairs the association of ATR-ATRIP with chromatin as well as ATR function, as measured by CHK1 activation and recovery of DNA replication following hydroxyurea (HU). We further demonstrate that the association of BID with RPA stimulates the association of ATR-ATRIP to the DNA damage sensor complex. We propose a model in which BID associates with RPA and stimulates the recruitment and/or stabilization of ATR-ATRIP to the DNA damage sensor complex.
We provide evidence for the first time, that two natural compounds ginsenoside Rh2 (G-Rh2) and betulinic acid (Bet A) synergistically induce apoptosis in human cervical adenocarcinoma (HeLa), human lung cancer A549, and human hepatoma HepG2 cells. G-Rh2 and Bet A cooperated to induce Bax traslocation to mitochondria and cytochrome c release. Co-treatment of G-Rh2 and Bet A resulted in enhanced cleavage of caspase-8 and Bid. Moreover, specific inhibition of caspase-8 by siRNA technology effectively reduced caspase-9 processing, poly (ADP-ribose) polymerase (PARP) cleavage, caspase-3 activation, and apoptosis in co-treated cells, which indicated that the caspase-8 feedback amplification pathway may have been involved in the apoptosis process. A previous study has shown that G-Rh2 induces cancer cell apoptosis via a Bcl-2 and/or Bcl-xL-independent mechanism, and Bet A induces apoptosis mainly through a mitochondrial pathway with tumor specificity. Since the antiapoptotic Bcl-2 and Bcl-xL are frequently overexpressed in human cancer cells, combined treatment with G-Rh2 and Bet A may be a novel strategy to enhance efficacy of anticancer therapy. © 2011 Wiley-Liss, Inc.
Copyright © 2011 Wiley-Liss, Inc.
Preservation of genome integrity via the DNA-damage response is critical to prevent disease. ATR (ataxia telangiectasia mutated- and Rad3-related) is essential for life and functions as a master regulator of the DNA-damage response, especially during DNA replication. ATR controls and co-ordinates DNA replication origin firing, replication fork stability, cell cycle checkpoints and DNA repair. Since its identification 15 years ago, a model of ATR activation and signalling has emerged that involves localization to sites of DNA damage and activation through protein-protein interactions. Recent research has added an increasingly detailed understanding of the canonical ATR pathway, and an appreciation that the canonical model does not fully capture the complexity of ATR regulation. In the present article, we review the ATR signalling process, focusing on mechanistic findings garnered from the identification of new ATR-interacting proteins and substrates. We discuss how to incorporate these new insights into a model of ATR regulation and point out the significant gaps in our understanding of this essential genome-maintenance pathway.
The Bcl-2-associated death promoter (BAD) protein, like many other BH3-only proteins, is known to promote apoptosis through the intrinsic mitochondrial pathway. Unlike the BH3-interacting domain death agonist (BID) protein, BAD cannot directly trigger apoptosis but, instead, lowers the threshold at which apoptosis is induced. In many mathematical models of apoptosis, BAD is neglected or abstracted. The work presented here considers the incorporation of BAD and its various modifications in a model of the tBID-induction of BAK (Bcl-2 homologous antagonist killer) or the tBID-induction of BAX (Bcl-2-associated X protein). Steady state equations are used to develop an explicit formula describing the total concentration level of tBID, guaranteed to trigger apoptosis, as a bilinear function of the total BAD concentration level and the total anti-apoptotic protein concentration level (usually Bcl-2 or Bcl-xL). In particular, the formula explains how the pro-apoptotic protein BAD lowers the threshold at which tBID induces BAK/BAX activation-reducing the level of total Bcl-2/Bcl-xL available to inhibit tBID signaling in the mitochondria. Attention is then turned to the experimental data surrounding BAD phosphorylation, a process known to inhibit the pro-apoptotic effects of BAD. To address this data, the phosphorylation process is modeled following two separate kinetics in which either free unbound BAD is the assumed substrate or Bcl-xL/Bcl-2-bound BAD is the assumed substrate. Bifurcation analysis and further analysis of the bilinear equation validate experiments, which suggest that BAD phosphorylation prevents irreversible BAK/BAX-mediated apoptosis, even when phosphorylation-induced dissociation of Bcl-xL/Bcl-2-bound BAD is blocked. It is also shown that a cooperative, even synergistic, removal of mitochondrial BAD is seen when both types of phosphorylation are assumed possible. The presented work, however, reveals that the balance between BAD phosphorylation and dephosphorylation modulates the degree to which BAD influences the signaling from tBID to BAK/BAX. Our model shows that both the mode(s) of phosphorylation and the BAD dephosphorylation rate become important factors in determining whether BAD influences the activation of the BAK/BAX signal or not. Such potential variations in the pro-apoptotic effects of BAD are used to explain some of the inconsistent experimental data surrounding BAD phosphorylation. Nonetheless, our model serves to evaluate BAD and its sensitizing effects on the tBID-induction of BAK/BAX and thus aid in predicting when the incorporation of BAD in an apoptosis signaling model is important and when it is not.
Copyright Â© 2010 Elsevier Ltd. All rights reserved.