Differential Localization Of RAC1 and RAC2 Reflects Their Specific Functions In Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2892-2892
Author(s):  
Marta Ewa Capala ◽  
Francesco Bonardi ◽  
Henny Maat ◽  
Edo Vellenga ◽  
Jan Jacob Schuringa
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Hematopoietic Cells ◽  
Confocal Imaging ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Interaction Partners

Abstract Hematopoietic stem cells (HSCs) depend on the bone marrow niche to provide signals for their survival, quiescence and differentiation. Many of these microenvironmental signals converge on RAC GTPases. In the hematopoietic system, two members of the RAC family are expressed, RAC1 and RAC2. Although RAC1 and RAC2 share a very high sequence homology, specific functions of these proteins have been suggested. However, little has been revealed about the downstream effectors and molecular mechanisms. In this study, we used multiple approaches to gain insight into the molecular biology of RAC1 and RAC2 in normal and leukemic human HSCs. Firstly, GFP-tagged constructs of RAC1 and RAC2 were used to study localization of these proteins in CD34+/CD38-/Lin- HSCs. Time-lapse confocal imaging of living cells plated on stroma revealed that RAC1 was strongly enriched in the plasma membrane. In contrast, RAC2 localized predominantly in the cytoplasm of both resting and dividing HSCs, whereby localization changed dramatically when cells progressed from S to the G2 phase of the cell cycle. This very distinct localization pattern implied different functions of RAC1 and RAC2. Therefore, we specifically downregulated RAC1 and/or RAC2 to study the effects of their depletion in normal and BCR-ABL-transduced leukemic HSCs. In normal HSCs, simultaneous downregulation of RAC1 and RAC2 resulted in a modest but significant decrease in proliferation and progenitor frequencies in the long term stromal co-cultures. However, in BCR-ABL-transduced HSCs depletion of RAC2 alone, but not RAC1, was sufficient to induce a marked proliferative disadvantage, decreased progenitor frequency, reduced leukemic cobblestone formation and diminished replating capacity, indicative for reduced self-renewal. Consistently, the frequency of long-term culture initiating leukemic cells was markedly reduced upon RAC2 downregulation. To elucidate the mechanisms involved in the observed phenotypes, we employed an in vivo biotin labeling strategy of Avi-tagged RAC1 and RAC2 followed by pull down and mass-spectrometry to identify specific interaction partners of RAC1 and RAC2 in BCR-ABL-expressing hematopoietic cells. Several of the RAC1-specific interaction partners were annotated as plasma membrane proteins, involved in cell adhesion, cytoskeleton assembly and regulation of endocytosis. In contrast, RAC2-interacting proteins were cytoplasmic or mitochondria-associated, and involved in processes such as cell cycle progression and regulation of apoptosis. Consistently, the proportion of dividing cells was decreased in RAC2-depleted BCR-ABL leukemic cobblestones coinciding with an increased apoptosis. Finally, a marked decrease in mitochondrial membrane potential was observed upon RAC2 but not RAC1 downregulation pointing to mitochondrial dysfunction as the initiating event of the apoptotic response. Moreover, preliminary electron microscopy data suggest that this functional change may be paralleled by structural aberrations of mitochondria. Further functional characterization of RAC1 and RAC2-specific interactions is currently ongoing and will be discussed, but our data clearly indicate that distinct subcellular localization of RAC1 and RAC2 dictates their interaction with specific sets of proteins and consequently their specific functions in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

Differential Localization of RAC1 and RAC2 Reflects Their Specific Functions in Normal and Leukemic Human Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2302-2302
Author(s):  
Marta Ewa Capala ◽  
Henny Maat ◽  
Francesco Bonardi ◽  
Edo Vellenga ◽  
Jan Jacob Schuringa
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Specific Interaction ◽  
Hematopoietic Cells ◽  
Time Lapse ◽  
Confocal Imaging ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Interaction Partners

Abstract Abstract 2302 Hematopoietic stem cells (HSCs) depend on the bone marrow niche to provide signals for their survival, quiescence and differentiation. Many of these microenvironmental signals converge on RAC GTPases. In the hematopoietic system, two members of the RAC family are expressed, RAC1 and RAC2. Although RAC1 and RAC2 share a very high sequence homology, specific functions of these proteins have been suggested. However, little has been revealed about the downstream effectors and molecular mechanisms. In this study, we used multiple approaches to gain insight into the molecular biology of RAC1 and RAC2 in normal and leukemic human HSCs. Firstly, GFP-tagged constructs of RAC1 and RAC2 were used to study localization of these proteins in CD34+/CD38−/Lin− HSCs. Time-lapse confocal imaging of living cells plated on stroma revealed that RAC1 was strongly enriched in the plasma membrane. In contrast, RAC2 localized predominantly in the cytoplasm of both resting and dividing HSCs, whereby localization changed dramatically when cells progressed from S to the G2 phase of the cell cycle. This very distinct localization pattern implied different functions of RAC1 and RAC2. Therefore, we specifically downregulated RAC1 and/or RAC2 to study the effects of their depletion in normal and BCR-ABL-transduced leukemic HSCs. In normal HSCs, simultaneous downregulation of RAC1 and RAC2 resulted in a modest but significant decrease in proliferation and progenitor frequencies in the long term stromal co-cultures. However, in BCR-ABL-transduced HSCs depletion of RAC2 alone, but not RAC1, was sufficient to induce a marked proliferative disadvantage, decreased progenitor frequency, reduced leukemic cobblestone formation and diminished replating capacity. To elucidate the mechanisms involved in the observed phenotypes, we employed an in vivo biotin labeling strategy of Avi-tagged RAC1 and RAC2 followed by pull down and mass-spectrometry to identify specific interaction partners of RAC1 and RAC2 in BCR-ABL-expressing hematopoietic cells. Several of the RAC1-specific interaction partners were annotated as plasma membrane proteins, involved in cell adhesion, cytoskeleton assembly and regulation of endocytosis. In contrast, RAC2-interacting proteins were cytoplasmic and involved in processes such as cell cycle progression, mitosis and regulation of apoptosis. Consistently with these findings, confocal time-lapse imaging of living hematopoietic cells revealed that pharmacological inhibition of RAC2 activity resulted in greatly decreased frequency of cell division. Moreover, the average division time was significantly extended upon RAC2 inhibition. Further functional characterization of RAC1 and RAC2-specific interactions is currently ongoing and will be discussed, but our data clearly indicate that distinct subcellular localization of RAC1 and RAC2 dictates their interaction with specific sets of proteins and consequently their specific functions in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals GATA2 Enhancer Modules Governing Hematopoietic Regeneration

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-16
Author(s):  
Alexandra A Soukup ◽  
Daniel R Matson ◽  
Kirby D Johnson ◽  
Emery H Bresnick
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Steady State ◽  
Hematopoietic Cells ◽  
Hematopoietic Cell ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
E Box

The transcription factor GATA2 is essential for the generation and function of hematopoietic stem and progenitor cells (HSPCs), erythroid precursors, and endothelial cells. A conserved intronic GATA2 enhancer, 9.8 kb downstream of the transcriptional start site (+9.5 in the mouse), is mutated in patients with GATA2 deficiency syndrome. Patient mutations within this region include a c.1017+512del28 deletion, removing E-box and GATA motifs, c.1017+532T>A that disrupts the E-box, and, most frequently, C>T in a 3' Ets motif (c.1017+572C>T) (Soukup and Bresnick, 2020). Homozygous mutation of the Ets motif in mice allows normal developmental and steady-state hematopoiesis but impairs hematopoietic regeneration (Soukup et al., 2019). In addition to HSPCs, GATA2 is expressed in non-hematopoietic cells in the bone marrow niche, e.g. endothelial cells and neurons (Katsumura et al., 2017). As the +9.5(Ets) mutation is not hematopoietic cell-specific, we asked whether regenerative defects of +9.5(Ets)-/- mice reflect disruption of cell-intrinsic or -extrinsic activities. In a competitive transplant assay, +9.5(Ets)-/- HSPCs were 3-fold less effective at long-term reconstitution than WT, and mechanistic studies indicated that the motif functions in hematopoietic cells to promote regeneration (Soukup et al., 2019). We conducted a reciprocal transplant of WT HSPCs into irradiated WT or +9.5(Ets)-/- recipients and quantified reconstitution by peripheral blood counts 4, 8, 12, and 16 weeks post-transplant. This analysis revealed no significant differences between WT and mutant recipients. At week 16, donor-derived leukocytes were 92% (+9.5(Ets)-/- recipients) and 96% (WT recipients) of total; the contribution did not differ significantly at any time. After 16 weeks, animals were sacrificed and HSPCs analyzed, confirming no significant alterations in mutant recipients. These results rigorously establish the mutant microenvironment as competent to support WT HSPC functions, emphasizing the critical hematopoietic cell-intrinsic activity of the +9.5 Ets motif. As the +9.5 Ets motif promotes regenerative hematopoiesis, and the +9.5 E-box;GATA is essential for developmental hematopoiesis, we devised a strategy to leverage these activities to innovate new models for GATA2 function in adult HSPCs. We generated compound heterozygous (CH) mice containing a mutant E-box;GATA sequence on one allele and a mutant Ets motif on the other allele. CH mice survived past weaning, with adults exhibiting significant steady-state defects, including a 4.4-fold decrease in GATA2hi megakaryocytes (p < 0.0001) and 20% decrease (p = 0.02) in platelets. To test whether the CH mutations compromise regeneration, we quantified HSPC populations in bone marrow from mice treated with vehicle or 5-fluorouracil (FU) 9- and 10- days post treatment. Steady-state HSC (Lin−Sca1+Kit+CD48-CD150+) levels were unaltered in CH animals. Days 9 and 10 post-FU treatment, WT HSC levels increased 17- (p = 0.0006) and 18-fold (p = 0.0007) relative to vehicle-treated animals. CH HSCs did not expand and were <10% of the steady-state level. 7 days post-FU treatment, Gata2 expression increased 1.9-fold in WT HSCs (p = 0.029); this response was abrogated in CHs. We asked if CH HSCs were capable of reconstitution in a competitive transplant assay. Four weeks post-transplant, CH progeny were 40-fold lower than WT (p < 0.0001). At 8-, 12-, and 16-weeks post-transplant, CH contribution was reduced 90-, 266-, and 280-fold, respectively. Defects persisted upon secondary transplantation, demonstrating that the defects cannot be restored by passage through a WT microenvironment. Thus, CH and +9.5(Ets)-/- mice share phenotypes, but CH mutations more severely impair regeneration and long-term reconstituting activity. This supports a paradigm in which the Ets motif and additional +9.5 sequences are critical for regeneration. This study revealed molecular determinants for steady-state and regenerative enhancer functions to enable discovery of +9.5-like enhancers with common operating mechanisms. We predict that such enhancers reside at a GATA2-regulated gene cohort, including genes that will reveal new mechanisms in hematopoiesis. As CH mice are poised for hematopoietic collapse, but can be propagated as relatively normal adults, studies are underway with this unique model to identify triggers of bone marrow failure and leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2829-2837 ◽  
Author(s):  
Stephen J. Szilvassy ◽  
Todd E. Meyerrose ◽  
Barry Grimes
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Clinical Utility ◽  
Cell Function ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Short Term ◽  
Hematopoietic Stem

Loss of long-term hematopoietic stem cell function in vitro is associated with cell cycle progression. To determine whether cytokine-induced proliferation also limits the rate of short-term engraftment and potential clinical utility of ex vivo expanded hematopoietic cells, murine Sca-1+c-kit+Lin− cells were cultured in interleukin-6 (IL-6), IL-11, granulocyte colony-stimulating factor (G-CSF), stem cell factor, flk-2 ligand, and thrombopoietin for 7 days. Cells amplified 2000-fold were then stained with Hoechst 33342, separated into G0/G1 (72% ± 3%) or S/G2/M (27% ± 3%) fractions by flow sorting, and injected into lethally irradiated mice. Although long-term (more than 6 months) engraftment of lymphoid and myeloid lineages was greater in primary and secondary recipients of expanded cells residing in G0/G1 at the time of transplantation, there were no noted differences in the short-term (less than 6 weeks) recovery kinetics of circulating blood cells. When hematopoietic cells were expanded in cultures containing the tetrapeptide stem cell inhibitor N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) to reduce progenitor cycling prior to transplantation, again there were no differences observed in short-term reconstitution by inhibited or uninhibited cells. Interestingly, AcSDKP significantly accelerated engraftment by expanded hematopoietic cells when administered in vivo at the time of transplantation. Leukocytes recovered to 20% of normal levels approximately 1 week faster, and thrombocytopenia was largely abrogated in AcSDKP-treated versus untreated mice. Therefore, while AcSDKP can accelerate the engraftment of ex vivo expanded hematopoietic progenitors, which suggests a relatively simple approach to improve their clinical utility, its effects appear unrelated to cell cycle arrest.

IRF8 Regulates Cell Cycle of Hematopoietic Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2353-2353
Author(s):  
Qingsong Qiu ◽  
Ping Liu ◽  
Xuemei Zhao ◽  
Chun Zhang ◽  
Donghe Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Innate Immune ◽  
Myelogenous Leukemia ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract IRF8 is expressed predominately in hematopoietic cells as a transcription factor and regulator of innate immune receptors signaling. It plays a critical role in the development of innate immune and adaptive immune cells, including dendritic cells, monocytes, eosinophils, basophils, B and T lymphocytes. It also functions as a tumor suppressor, as IRF8 deficient mice manifest a chronic myelogenous leukemia (CML)-like syndrome. In addition to various lineages of hematopoietic cells, we have found that IRF8 is expressed in hematopoietic stem cells (HSCs). However, the function of IRF8 in HSCs was unknown. In this study we investigated the role of IRF8 in regulating HSCs. We found that the number of long-term (LT)-HSCs (Lin- Sca1+ c-Kit+ CD48- CD150+) is significantly reduced in IRF8 knockout mice (IRF8-/-), comparing to the wild-type (WT) controls. Long-term reconstitution assays showed that IRF8-/- LT-HSC's repopulation capability is severely impaired compared to equal amount of WT mouse LT-HSCs. The effect of IRF8 depletion on HSC's self-renewal capacity is unlikely due to the influence of the CML-like syndrome, since the disease is not transplantable and only seen in the primary mice. In addition, the number of LT-HSCs is also decreased in E14.5 fetal liver of IRF8-/- mice, when the myeloproliferative disorder has not been manifested. A cell cycle analysis showed that the number of LT-HSCs in S, G2 or M phase is greatly reduced in IRF8-/- mice comparing to that in WT mice. Transcription profiling analysis of LT-HSCs revealed that the expression of key regulators of cytokine/growth factor signaling and factors controlling HSC self-renewal are downregulated in IRF8-/- mice comparing to that in WT mice. These results indicate that IRF8 plays a critical role in regulating cell cycle entry of HSCs. This function of IRF8 may play an important role in activating HSCs to enhance immunity and innate immunity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

scholarly journals Activation of the receptor tyrosine kinase RET improves long-term hematopoietic stem cell outgrowth and potency

Blood ◽  
2020 ◽  
Vol 136 (22) ◽  
pp. 2535-2547 ◽  
Author(s):  
W. Grey ◽  
R. Chauhan ◽  
M. Piganeau ◽  
H. Huerga Encabo ◽  
M. Garcia-Albornoz ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Culture Conditions ◽  
Cellular Growth ◽  
Great Promise ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Intracellular Signaling Pathway ◽  
Wide Range

Abstract Expansion of human hematopoietic stem cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche; how to apply this process to HSC maintenance and expansion has yet to be explored. We show a role for the GFL receptor, RET, at the cell surface of HSCs in mediating sustained cellular growth, resistance to stress, and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/coreceptor complex, glial-derived neurotrophic factor and its coreceptor, exhibit improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally, we show that RET drives a multifaceted intracellular signaling pathway, including key signaling intermediates protein kinase B, extracellular signal-regulated kinase 1/2, NF-κB, and p53, responsible for a wide range of cellular and genetic responses that improve cell growth and survival under culture conditions.

Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients

Blood ◽  
2002 ◽  
Vol 99 (5) ◽  
pp. 1585-1593 ◽  
Author(s):  
Anna Jetmore ◽  
P. Artur Plett ◽  
Xia Tong ◽  
Frances M. Wolber ◽  
Robert Breese ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Hematopoietic Cells ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells ◽  
G1 Cells ◽  
Active Proliferation

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34+ cells, or their G0(G0CD34+) or G1(G1CD34+) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34+ cells harbored similar numbers of CD34+ and CD34− cells, indicating that CD34+ cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G2+M than in recipients of G0/G1 cells (34.6% ± 5.9% and 17.1% ± 6.3%, respectively; P < .01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.

scholarly journals Transcriptional signature of prion-induced neurotoxicity in a Drosophila model of transmissible mammalian prion disease

2020 ◽  
Vol 477 (4) ◽  
pp. 833-852
Author(s):  
Alana M. Thackray ◽  
Brian Lam ◽  
Anisa Shahira Binti Ab Razak ◽  
Giles Yeo ◽  
Raymond Bujdoso
Keyword(s):  
Cell Cycle ◽  
Plasma Membrane ◽  
Prion Disease ◽  
Molecular Mechanisms ◽  
Control Strategies ◽  
Prion Diseases ◽  
Transcriptional Signature ◽  
Natural Hosts ◽  
Regulation Of Cell Cycle ◽  
Transgenic Drosophila

Prion diseases are fatal transmissible neurodegenerative conditions of humans and animals that arise through neurotoxicity induced by PrP misfolding. The cellular and molecular mechanisms of prion-induced neurotoxicity remain undefined. Understanding these processes will underpin therapeutic and control strategies for human and animal prion diseases, respectively. Prion diseases are difficult to study in their natural hosts and require the use of tractable animal models. Here we used RNA-Seq-based transcriptome analysis of prion-exposed Drosophila to probe the mechanism of prion-induced neurotoxicity. Adult Drosophila transgenic for pan neuronal expression of ovine PrP targeted to the plasma membrane exhibit a neurotoxic phenotype evidenced by decreased locomotor activity after exposure to ovine prions at the larval stage. Pathway analysis and quantitative PCR of genes differentially expressed in prion-infected Drosophila revealed up-regulation of cell cycle activity and DNA damage response, followed by down-regulation of eIF2 and mTOR signalling. Mitochondrial dysfunction was identified as the principal toxicity pathway in prion-exposed PrP transgenic Drosophila. The transcriptomic changes we observed were specific to PrP targeted to the plasma membrane since these prion-induced gene expression changes were not evident in similarly treated Drosophila transgenic for cytosolic pan neuronal PrP expression, or in non-transgenic control flies. Collectively, our data indicate that aberrant cell cycle activity, repression of protein synthesis and altered mitochondrial function are key events involved in prion-induced neurotoxicity, and correlate with those identified in mammalian hosts undergoing prion disease. These studies highlight the use of PrP transgenic Drosophila as a genetically well-defined tractable host to study mammalian prion biology.

scholarly journals CXCR4 is required for the quiescence of primitive hematopoietic cells

2008 ◽  
Vol 205 (4) ◽  
pp. 777-783 ◽  
Author(s):  
Yuchun Nie ◽  
Yoon-Chi Han ◽  
Yong-Rui Zou
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Regulatory Factor ◽  
Cell Compartment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Stem Cell Compartment

The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4−/− HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.

C/EBPβ Isoforms Regulate Proliferation and Differentiation of Regenerating Hematopoietic Stem/Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3713-3713
Author(s):  
Atsushi Sato ◽  
Asumi Yokota ◽  
Yoshihiro Hayashi ◽  
Naoka Kamio ◽  
Satoshi Sagai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Flow Cytometric Analysis ◽  
Myeloid Cells ◽  
Stress Conditions ◽  
Hematopoietic Stem ◽  
N Terminus ◽  
Factor C ◽  
The Impact

Under stress or regenerative conditions, HSCs rapidly enter into cell cycle and are reprogrammed toward myeloid-biased hematopoiesis to meet the increasing demand of myeloid cells. We have previously shown that the transcription factor C/EBPβ plays critical roles at the level of HSPCs under stress conditions (Nat Immunol 2006, J Immunol 2012, Leukemia 2013 and Blood Adv 2019). However, underlying molecular mechanisms of action remain largely unknown. In this study, we have investigated the detailed function of C/EBPβ in regulation of HSPCs. We first evaluated the impact of C/EBPβ on the cell cycle status of LT-HSCs. To exclude the cell-extrinsic contribution of C/EBPβ, CD45.2+ BM cells from WT or Cebpb-/- mice were transplanted into lethally irradiated CD45.1+ WT mice, and these "BM-replaced" recipients were subjected to the following experiments. At steady state, the cell cycle statuses and the numbers of HSPCs did not significantly differ between the recipients of WT cells and those of Cebpb-/- cells. Immediately after 5-FU treatment, WT LT-HSCs entered the cell cycle, as revealed by the decreased percentage of cells in G0 phase and the increased percentage of cells in S/G2M phase. All these parameters of cell cycle acceleration were observed prior to the nadir of LT-HSCs induced by 5-FU and were significantly attenuated in Cebpb-/- LT-HSCs. Next, we assessed the numbers of LT-HSCs, KSL cells, and KL cells after 5-FU treatment. Following the nadir, the recovery of LT-HSCs preceded that of KSL and KL cells, suggesting the differentiation of LT-HSCs to KSL and KL cells. In the recipients of Cebpb-/- cells, the recovery of KSL and KL cells was delayed significantly. Collectively, cell cycle acceleration and subsequent differentiation of LT-HSCs under stress conditions were impaired in the absence of Cebpb. The Cebpb is a single exon gene, and three isoforms, namely, LAP*, LAP and LIP which lacks N-terminus, are translated from its unique mRNA. Due to their structural difference, they should have distinct functions. Here, we focused on expression and functions of these isoforms in regenerating HSPCs. To monitor expression of these isoforms in small numbers of HSCs, we devised a novel intracellular double staining method for flow cytometric analysis using two distinct anti-C/EBPβ antibodies. An antibody against the C-terminus of C/EBPβ recognized all three isoforms, while an antibody against the N-terminus of C/EBPβ only recognized LAP* and LAP. Thus, simultaneous staining with both antibodies should enable us to distinguish cells that dominantly expressed LIP (C-term+ N-term-) from those that expressed all three isoforms (C-term+ N-term+). Using this method, we monitored the expression patterns of these isoforms in LT-HSCs after 5-FU treatment. LT-HSCs initially became C-term single positive in response to 5-FU and subsequently changed to C- and N-term double positive, suggesting that LIP was upregulated prior to LAP/LAP* under stress conditions. These results suggest that phase-specific upregulation of LIP and LAP/LAP* is strongly associated with phase-specific functions of C/EBPβ in cell cycle activation and differentiation, respectively. Indeed, when EML cells, a mouse HSC line, were retrovirally transduced with LIP, the transduced cells were more proliferative and actively cycling than those transduced with the control vector, whereas proliferation and cell cycle were markedly suppressed in LAP*- and LAP-expressing EML cells. LIP-expressing cells remained undifferentiated, while LAP*- and LAP-expressing cells rapidly differentiated into CD11b+ myeloid cells and eventually stopped proliferating. In summary, our findings clearly suggest that sequential upregulation of C/EBPβ isoforms is critical for the regulation of HSCs under stress conditions. LIP amplifies the "reservoir" of HSPCs by accelerating the proliferation of HSCs during the early phase of regeneration, while LAP*/LAP induce their myeloid differentiation at a later phase. These findings should facilitate our understanding of the pathophysiology of infection, inflammation, and regenerating hematopoiesis in response to myeloablative chemotherapies or hematopoietic stem cell transplantation, all of which increase the hematopoietic demands. Disclosures Hirai: Kyowa Kirin: Research Funding.

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