Proapoptotic Bid mediates the Atr-directed DNA damage response to replicative stress

Proapoptotic BH3 interacting domain death agonist (Bid), a BH3-only Bcl-2 family member, is situated at the interface between the DNA damage response and apoptosis, with roles in death receptor-induced apoptosis as well as cell cycle checkpoints following DNA damage.(1, 2, 3) In this study, we demonstrate that Bid functions at the level of the sensor complex in the Atm and Rad3-related (Atr)-directed DNA damage response. Bid is found with replication protein A (RPA) in nuclear foci and associates with the Atr/Atr-interacting protein (Atrip)/RPA complex following replicative stress. Furthermore, Bid-deficient cells show an impaired response to replicative stress manifest by reduced accumulation of Atr and Atrip on chromatin and at DNA damage foci, reduced recovery of DNA synthesis following replicative stress, and decreased checkpoint kinase 1 activation and RPA phosphorylation. These results establish a direct role for the BH3-only Bcl-2 family member, Bid, acting at the level of the damage sensor complex to amplify the Atr-directed cellular response to replicative DNA damage.     

Transient activation of hematopoietic stem and progenitor cells by IFNγ during acute bacterial infection

How hematopoietic stem cells (HSCs) respond to inflammatory signals during infections is not well understood. Our studies have used a murine model of ehrlichiosis, an emerging tick-born disease, to address how infection impacts hematopoietic function. Infection of C57BL/6 mice with the intracellular bacterium, Ehrlichia muris, results in anemia and thrombocytopenia, similar to what is observed in human ehrlichiosis patients. In the mouse, infection promotes myelopoiesis, a process that is critically dependent on interferon gamma (IFNγ) signaling. In the present study, we demonstrate that E. muris infection also drives the transient proliferation and expansion of bone marrow Lin-negative Sca-1(+) cKit(+) (LSK) cells, a population of progenitor cells that contains HSCs. Expansion of the LSK population in the bone marrow was associated with a loss of dormant, long-term repopulating HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation within that population. The reduced engraftment and myeloid bias of the infection-induced LSK cells was transient, and was most pronounced on day 8 post-infection. The infection-induced changes were accompanied by an expansion of more differentiated multipotent progenitor cells, and required IFNγ signaling. Thus, in response to inflammatory signals elicited during acute infection, HSCs can undergo a rapid, IFNγ-dependent, transient shift from dormancy to activity, ostensibly, to provide the host with additional or better-armed innate cells for host defense. Similar changes in hematopoietic function likely underlie many different infections of public health importance.     

Upregulation of nascent mitochondrial biogenesis in mouse hematopoietic stem cells parallels upregulation of CD34 and loss of pluripotency: A potential strategy for reducing oxidative risk in stem cells

Oxidative damage by reactive oxygen species generated in mitochondria is a potential cause of stem-cell dysregulation. Little is known about how hematopoietic stem cells mitigate/lessen this risk in the face of upregulated mitochondrial biogenesis/function necessary for the energy needs of differentiation and progenitor expansion. Here we report that upregulation of mitochondrial mass in mouse hematopoietic stem cells is closely linked to the appearance of CD34 on their surface, a marker indicating loss of long-term repopulating ability. These mitochondria have low membrane potential initially, but become active before exiting the primitive LSK compartment. Steady-state hematopoiesis perturbed by global expression of SDF-1/CXCL12 transgene causes a shift in ratios of these mitochondrialy-distinct LSK populations. Based on known effects of SDF-1 and signaling by it's receptor, CXCR4, along with finding primitive progenitors with high mitochondrial mass but low activity, we suggest a model of asymmetric self-renewing stem cell division that could lessen stem cell exposure to oxidative damage.     

The loss of the BH3-only Bcl-2 family member Bid delays T-cell leukemogenesis in Atm?/? mice

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.     

Pro-apoptotic Bid is required for the resolution of the effector phase of inflammatory arthritis

Rheumatoid arthritis is an autoimmune disease characterized by hyperplasia of the synovial lining and destruction of cartilage and bone. Recent studies have suggested that a lack of apoptosis contributes to the hyperplasia of the synovial lining and to the failure in eliminating autoreactive cells. Mice lacking Fas or Bim, two pro-apoptotic proteins that mediate the extrinsic and intrinsic death cascades, respectively, develop enhanced K/BxN serum transfer-induced arthritis. Since the pro-apoptotic protein Bid functions as an intermediate between the extrinsic and intrinsic apoptotic pathways, we examined the role that it plays in inflammatory arthritis. Mice deficient in Bid (Bid-/-) show a delay in the resolution of K/BxN serum transfer-induced arthritis. Bid-/- mice display increased inflammation, bone destruction, and pannus formation compared to wild-type mice. Furthermore, Bid-/- mice have elevated levels of CXC chemokine and IL-1β in serum, which are associated with more inflammatory cells throughout the arthritic joint. In addition, there are fewer apoptotic cells in the synovium of Bid-/- compared to Wt mice. These data suggest that extrinsic and intrinsic apoptotic pathways cooperate through Bid to limit development of inflammatory arthritis.     

FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress

To understand the role of FoxO family members in hematopoiesis, we conditionally deleted FoxO1, FoxO3, and FoxO4 in the adult hematopoietic system. FoxO-deficient mice exhibited myeloid lineage expansion, lymphoid developmental abnormalities, and a marked decrease of the lineage-negative Sca-1+, c-Kit+ (LSK) compartment that contains the short- and long-term hematopoietic stem cell (HSC) populations. FoxO-deficient bone marrow had defective long-term repopulating activity that correlated with increased cell cycling and apoptosis of HSC. Notably, there was a marked context-dependent increase in reactive oxygen species (ROS) in FoxO-deficient HSC compared with wild-type HSC that correlated with changes in expression of genes that regulate ROS. Furthermore, in vivo treatment with the antioxidative agent N-acetyl-L-cysteine resulted in reversion of the FoxO-deficient HSC phenotype. Thus, FoxO proteins play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment, a function that is required for its long-term regenerative potential.     

Specific overexpression of SIRT1 in mesenchymal stem cells rescues hematopoiesis niche in BMI1 knockout mice through promoting CXCL12 expression

Osteoblastic lineage cells (OBCs) are bone-building cells and essential component of hematopoietic niche, but mechanisms whereby bone-building and hematopoiesis-supportive activities of OBCs could be regulated simultaneously remain largely unknown. Here we found that B cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) was involved in such a co-regulatory mechanism. In this study, we first found that, accompanied with marked decline of osteogenic activity, the hematopoietic niche in Bmi1 knockout (KO) mice was severely impaired and manifested as CXCL12 expression falls and LSK homing failure; however, intratibial injection with CXCL12 effectively facilitated LSK accumulation in bone marrow of Bmi1 KO mice. To try to rescue these defects in Bmi1 KO mice, we generated Bmi1^KO/Sirt1^Tg (KO-TG) double mutant mice with Sirt1 specific overexpression in mesenchymal progenitor cells (MPCs) in Bmi1 KO mice, and our data showed that KO-TG mice had significantly increased bone-building activity, elevated Cxcl12 expression by MPCs, increased LSK homing and expanded LSK pool in bone marrow compared to Bmi1 KO mice. Of note, similar improvements in KO-TG mice were observed in Bmi1 KO mice fed with dietary resveratrol, an established Sirt1 activator, comparing with KO control mice. Therefore, pharmacologic activation of Bmi1/Sirt1 signaling pathway could simultaneously promote bone-building and hematopoiesis-supportive activities of OBCs.     

Evi-1 is a critical regulator for hematopoietic stem cells and transformed leukemic cells

Evi-1 has been recognized as one of the dominant oncogenes associated with murine and human myeloid leukemia. Here, we show that hematopoietic stem cells (HSCs) in Evi-1-deficient embryos are severely reduced in number with defective proliferative and repopulating capacity. Selective ablation of Evi-1 in Tie2(+) cells mimics Evi-1 deficiency, suggesting that Evi-1 function is required in Tie2(+) hematopoietic stem/progenitors. Conditional deletion of Evi-1 in the adult hematopoietic system revealed that Evi-1-deficient bone marrow HSCs cannot maintain hematopoiesis and lose their repopulating ability. In contrast, Evi-1 is dispensable for blood cell lineage commitment. Evi-1(+/-) mice exhibit the intermediate phenotype for HSC activity, suggesting a gene dosage requirement for Evi-1. We further demonstrate that disruption of Evi-1 in transformed leukemic cells leads to significant loss of their proliferative activity both in vitro and in vivo. Thus, Evi-1 is a common and critical regulator essential for proliferation of embryonic/adult HSCs and transformed leukemic cells.     

LSK derived LSK- cells have a high apoptotic rate related to survival regulation of hematopoietic and leukemic stem cells

A balanced pool of hematopoietic stem cells (HSCs) in bone marrow is tightly regulated, and this regulation is disturbed in hematopoietic malignancies such as chronic myeloid leukemia (CML). The underlying mechanisms are largely unknown. Here we show that the Lin(-)Sca-1(+)c-Kit(-) (LSK(-)) cell population derived from HSC-containing Lin(-)Sca-1(+)c-Kit(+) (LSK) cells has significantly higher numbers of apoptotic cells. Depletion of LSK cells by radiation or the cytotoxic chemical 5-fluorouracil results in an expansion of the LSK(-) population. In contrast, the LSK(-) population is reduced in CML mice, and depletion of leukemia stem cells (LSCs; BCR-ABL-expressing HSCs) by deleting Alox5 or by inhibiting heat shock protein 90 causes an increase in this LSK(-) population. The transition of LSK to LSK(-) cells is controlled by the Icsbp gene and its downstream gene Lyn, and regulation of this cellular transition is critical for the survival of normal LSK cells and LSCs. These results indicate a potential function of the LSK(-) cells in the regulation of LSK cells and LSCs.     

Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis

Here we report that in staurosporine-induced apoptosis of HeLa cells, Bid, a BH3 domain containing protein, translocates from the cytosol to mitochondria. This event is associated with a change in conformation of Bax which leads to the unmasking of its NH2-terminal domain and is accompanied by the release of cytochrome c from mitochondria. A similar finding is reported for cerebellar granule cells undergoing apoptosis induced by serum and potassium deprivation. The Bax-conformational change is prevented by Bcl-2 and Bcl-xL but not by caspase inhibitors. Using isolated mitochondria and various BH3 mutants of Bid, we demonstrate that direct binding of Bid to Bax is a prerequisite for Bax structural change and cytochrome c release. Bcl-xL can inhibit the effect of Bid by interacting directly with Bax. Moreover, using mitochondria from Bax-deficient tumor cell lines, we show that Bid- induced release of cytochrome c is negligible when Bid is added alone, but dramatically increased when Bid and Bax are added together. Taken together, our results suggest that, during certain types of apoptosis, Bid translocates to mitochondria and binds to Bax, leading to a change in conformation of Bax and to cytochrome c release from mitochondria.     

Molecular and functional properties of lung SP cells

Previous analysis of lung injury and repair has provided evidence for region-specific stem cells that maintain proximal and distal epithelial compartments. However, redundant expression of lineage markers by cells at several levels of the stem cell hierarchy has complicated phenotypic and functional characterization of clonogenic airway cells. Based on the demonstration that rapid efflux of the DNA dye Hoechst 33342 can be used to prospectively purify long-term repopulating hematopoietic stem cells, we hypothesized that lung cells with similar biochemical properties would be enriched for clonogenic progenitors. We demonstrate that Hoechst-dim side population (SP) cells isolated from proximal and distal compartments of the mouse lung were relatively small and agranular, exhibited low red and green autofluorescence, and that the SP fraction was highly enriched in clonogenic cells. Quantitative RT-PCR indicated that vimentin mRNA was enriched and that epithelial markers were depleted in these preparations of SP cells. Bleomycin exposure was associated with decreased clonogenicity among alveolar SP and suggested that SP cell function was compromised under profibrotic conditions. We conclude that the SP phenotype is common to clonogenic cells at multiple airway locations and suggest that Hoechst efflux is a property of cells expressing a wound-repair phenotype.     

Stable Cellular Senescence Is Associated with Persistent DDR Activation

The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.     

Wnt5a Does Not Support Hematopoiesis in Stroma-Free,Serum-Free Cultures

Previously we reported that Wnt5a is highly expressed in the murine urogenital ridge-derived UG26-1B6 cells but not embryonic liver-derived EL08-1D2 cells. Mouse long-term repopulating hematopoietic stem cells (LTR-HSC) were maintained in non-contact UG26-1B6 cultures but not EL08-1D2 non-contact cultures, unless Wnt5a was also added to the cultures, suggesting a role for Wnt5a in the in vitro maintenance of LTR-HSC. Here, we investigated if the effect of Wnt5a on adult LTR-HSC activity is HSC-autonomous. To test the effect of Wnt5a on maintenance of LTR-HSC, we performed limiting dilution competitive transplantation assays of murine Lin-Sca1⁺ c-kit⁺ (LSK) cells cultured for 5 days with TPO and SCF with and without Wnt5a. The effect of Wnt5a on the generation of colony forming units (CFU) and the homing ability of LSK progeny was also tested. No effects were found of Wnt5a on total cell expansion, the number of CFU, or homing ability of day 5 LSK progeny. Furthermore, addition of Wnt5a did not improve, but may have impeded maintenance of LTR-HSC. In conclusion, our data indicate that Wnt5a does not enhance the maintenance and expansion of adult murine LTR-HSCs or committed progenitors cultured in vitro in serum- and stroma-free conditions.     

Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6

Parathyroid hormone (PTH) stimulates hematopoietic cells through mechanisms of action that remain elusive. Interleukin-6 (IL-6) is upregulated by PTH and stimulates hematopoiesis. The purpose of this investigation was to identify actions of PTH and IL-6 in hematopoietic cell expansion. Bone marrow cultures from C57B6 mice were treated with fms-like tyrosine kinase-3 ligand (Flt-3L), PTH, Flt-3L plus PTH, or vehicle control. Flt-3L alone increased adherent and non-adherent cells. PTH did not directly impact hematopoietic or osteoclastic cells but acted in concert with Flt-3L to further increase cell numbers. Flt-3L alone stimulated proliferation, while PTH combined with Flt-3L decreased apoptosis. Flt-3L increased blasts early in culture, and later increased CD45(+) and CD11b(+) cells. In parallel experiments, IL-6 acted additively with Flt-3L to increase cell numbers and IL-6-deficient bone marrow cultures (compared to wildtype controls) but failed to amplify in response to Flt-3L and PTH, suggesting that IL-6 mediated the PTH effect. In vivo, PTH increased Lin(-) Sca-1(+)c-Kit(+) (LSK) hematopoietic progenitor cells after PTH treatment in wildtype mice, but failed to increase LSKs in IL-6-deficient mice. In conclusion, PTH acts with Flt-3L to maintain hematopoietic cells by limiting apoptosis. IL-6 is a critical mediator of bone marrow cell expansion and is responsible for PTH actions in hematopoietic cell expansion.     

Kit regulates maintenance of quiescent hematopoietic stem cells

Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (Kit(W41/W41)) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult Kit(W41/W41) mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin(-)Sca-1(+)Kit(high) (LSK)CD34(-)Flt3(-) long-term HSCs by 12 wk of age, whereas LSKCD34(+)Flt3(-) short-term HSCs and LSKCD34(+)Flt3(+) multipotent progenitors were less affected. Whereas homing and initial reconstitution of Kit(W41/W41) bone marrow cells in myeloablated recipients were close to normal, self-renewing Kit(W41/W41) HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation Kit(W41/W41) HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis.     

Granulocyte colony stimulating factor expands hematopoietic stem cells within the central but not endosteal bone marrow region

Granulocyte colony stimulating factor (G-CSF) is clinically well established for the mobilization of hematopoietic stem cells (HSC). Extensive data on the underlying mechanism of G-CSF induced mobilization is available; however, little is known regarding the functional effect of G-CSF on HSC within the bone marrow (BM). In this study we analyzed the proportion and number of murine HSC in the endosteal and central bone marrow regions after 4 days of G-CSF administration. We demonstrate that the number of HSC, defined as CD150(+)CD48(-)LSK cells (LSKSLAM cells), increased within the central BM region in response to G-CSF, but not within the endosteal BM region. In addition the level of CD150 and CD48 expression also increased on cells isolated from both regions. We further showed that G-CSF mobilized proportionally fewer LSKSLAM compared to LSK cells, mobilized LSKSLAM had colony forming potential and the presence of these cells can be used as a measure for mobilization efficiency. Together we provide evidence that HSC in the BM respond differently to G-CSF and this is dependent on their location. These findings will be valuable in developing new agents which specifically mobilize HSC from the endosteal BM region, which we have previously demonstrated to have significantly greater hematopoietic potential compared to their phenotypically identical counterparts located in other regions of the BM.     

Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax

Following caspase-8 mediated cleavage, a carboxyl-terminal fragment of the BH3 domain-only Bcl-2 family member Bid transmits the apoptotic signal from death receptors to mitochondria. In a screen for possible regulators of Bid, we defined Bfl-1/A1 as a potent Bid interacting protein. Bfl-1 is an anti-apoptotic Bcl-2 family member, whose preferential expression in hematopoietic cells and endothelium is controlled by inflammatory stimuli. Its mechanism of action is unknown. We find that Bfl-1 associates with both full-length Bid and truncated (t)Bid, via the Bid BH3 domain. Cellular expression of Bfl-1 confers protection against CD95- and Trail receptor-induced cytochrome c release. In vitro assays, using purified mitochondria and recombinant proteins, demonstrate that Bfl-1 binds full-length Bid, but does not interfere with its processing by caspase-8, or with its mitochondrial association. Confocal microscopy supports that Bfl-1, which at least in part constitutively localizes to mitochondria, does not impede tBid translocation. However, Bfl-1 remains tightly and selectively bound to tBid and blocks collaboration between tBid and Bax or Bak in the plane of the mitochondrial membrane, thereby preventing mitochondrial apoptotic activation. Lack of demonstrable interaction between Bfl-1 and Bak or Bax in the mitochondrial membrane suggests that Bfl-1 generally prevents the formation of a pro-apoptotic complex by sequestering BH3 domain-only proteins.     

Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation

The in vivo regulation of hematopoietic stem cell (HSC) function is poorly understood. Here, we show that hematopoietic repopulation can be augmented by administration of a glycogen synthase kinase-3 (GSK-3) inhibitor to recipient mice transplanted with mouse or human HSCs. GSK-3 inhibitor treatment improved neutrophil and megakaryocyte recovery, recipient survival and resulted in enhanced sustained long-term repopulation. The output of primitive Lin(-)c-Kit(+)Sca-1(+) cells and progenitors from HSCs increased upon GSK-3 inhibitor treatment without altering secondary repopulating ability, suggesting that the HSC pool is maintained while overall hematopoietic reconstitution is increased. GSK-3 inhibitors were found to modulate gene targets of Wnt, Hedgehog and Notch pathways in cells comprising the primitive hematopoietic compartment without affecting mature cells. Our study establishes GSK-3 as a specific in vivo modulator of HSC activity, and suggests that administration of GSK-3 inhibitors may provide a clinical means to directly enhance the repopulating capacity of transplanted HSCs.     

Parvovirus infection suppresses long-term repopulating hematopoietic stem cells

The functional disturbance of self-renewing and multipotent hematopoietic stem cells (HSCs) in viral diseases is poorly understood. In this report, we have assessed the susceptibility of mouse HSCs to strain i of the autonomous parvovirus minute virus of mice (MVMi) in vitro and during persistent infection of an immunodeficient host. Purified 5FU(r) Lin(-) Sca-1(+) primitive hematopoietic precursors were permissive for MVMi genome replication and the expression of viral gene products. The lymphoid and myeloid repopulating capacity of bone marrow (BM) cells was significantly impaired after in vitro infection, although the degree of functional effect proportionally decreased with the posttransplantation time. This indicated that MVMi targets the heterogeneous compartment of repopulating cells with differential affinity and suggests that the virus may persist in some primitive HSCs in the quiescent stage, killing those eventually recruited for proliferative activity. Immunodeficient SCID mice oronasally infected with MVMi were cured of the characteristic virus-induced lethal leukopenia by transplantation of immunocompetent BM grafts. However, two double-stranded viral DNA species, probably uncommon replicative intermediates, remained in the marrow of every transplanted mouse months after infectious virus clearance. Genetic analysis of the rescued mice showed that the infection ensured a stable engraftment of donor hematopoiesis by markedly depleting the pool of endogenous HSCs. The MVMi-induced suppression of HSC functions illustrates the accessibility of this compartment to infection during a natural viral hematological disease. These results may provide clues to understanding delayed hematopoietic syndromes associated with persistent viral infections and to prospective gene delivery to HSCs in vivo.