Kras Plays An Important Role In Generating Differentiated Blood Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2451-2451 ◽  
Author(s):  
Alisa Damnernsawad ◽  
Guangyao Kong ◽  
Yangang Liu ◽  
Yuan-I Chang ◽  
Jingfang Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Conditional Knockout ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Background Kras is a small GTPase essential for mouse embryonic development. Although Kras-/- fetal liver cells reconstitute recipient mice indistinguishably from wild-type cells, chimeric mice generated from injection of Kras-/- embryonic stem cells into wild-type blastocysts show little contribution of knockout cells to hematopoietic tissues even when these cells contribute to all the other tissues to a high degree. These results suggest that Kras plays an important role in adult hematopoiesis. However, early embryonic lethality of Kras-/- mice prevents further investigation of Kras functions in adulthood. To overcome this problem, we generated Kras conditional knockout mice (Krasfl/fl), which allow the deletion of Kras by the Cre recombinase in desired tissues and at desired developmental stages. Method We used two transgenic Cre lines, Mx1-Cre and Vav-Cre, to knockout Kras in adult hematopoietic system. The Mx1 promoter is induced by interferon signaling, which can be triggered by injections of polyinosinic-polycytidylic acid (pI-pC). The Vav promoter drives Cre expression specifically in fetal liver hematopoietic cells since E11.5 as well as in adult hematopoietic tissues. Both Cre lines efficiently deleted Kras expression in above 95% of hematopoietic cells as judged by single hematopoietic stem cell (HSC) genotyping. Results obtained from these two different Cre lines were essentially same. Results We found that the frequency and absolute number of Kras-/- HSCs, multipotent progenitors (MPPs), LSK (Lin- Sca1+ cKit+) cells, myeloid progenitors and common lymphoid progenitors are comparable to wild-type control cells. Consistent with this observation, cytokine signaling in Kras-/- hematopoietic stem/progenitor cells (HSPCs, Lin- cKit+) is indistinguishable from control HSPCs. In contrast, the percentage of CD19+ B-cells is moderately but significantly reduced in Kras-/- spleens and concomitantly cytokine-evoked ERK1/2 activation is greatly reduced in differentiated blood cells. To determine whether Kras plays an important role in regulating HSC functions, we performed a competitive bone marrow reconstitution assay using CD45.2+ control or Kras-/- bone marrow cells mixed together at ratios 1:1 and 3:1 with congeneic competitor cells (CD45.1+ bone marrow cells). Kras-/- bone marrow cells show significantly reduced long-term reconstitution in recipient mice compared to control cells (10% vs 45%). The reduced reconstitution is persistent in the secondary and tertiary recipients. However, detailed analysis in primary and secondary recipients revealed that the frequency of Kras-/- HSCs and MPPs is comparable to that of control cells and Kras-/- progenitor cells are also largely normal, indicating that Kras is dispensable for adult HSC functions but might play an important role in generating differentiated blood cells. The reduced generation of myeloid cells is further validated in an in vitro culture assay, in which we quantitatively measured the myeloid cell production from Lin- progenitor cells. Conclusions Our results indicate that loss-of-Kras could be compensated by other Ras isoforms in adult HSCs. However, in mature blood cells, Kras deficiency results in greatly reduced cytokine-evoked ERK1/2 activation. Under a stressed condition (e.g. competitive bone marrow transplantation), the generation of Kras-/- blood cells is defective. Taken together, our study reveals a novel and unique function of Kras in regulating adult hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 941-941
Author(s):  
Brian Wadugu ◽  
Amanda Heard ◽  
Joseph Bradley ◽  
Matthew Ndonwi ◽  
Jin J Shao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Splicing ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Mutant Cells ◽  
Marrow Cells

Abstract Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 857-857
Author(s):  
Gregor B. Adams ◽  
Ian R. Alley ◽  
Karissa T. Chabner ◽  
Ung-il Chung ◽  
Emily S. Marsters ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Conditional Knockout ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

scholarly journals Mutant ASXL1 Disrupts Paraspeckle Formation through Aberrant Interaction with Nono in Hematopoietic Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2514-2514
Author(s):  
Yamamoto Keita ◽  
Susumu Goyama ◽  
Shuhei Asada ◽  
Takeshi Fujino ◽  
Tomofusa Fukuyama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alternative Splicing ◽  
Rna Splicing ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Truncated Form ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Marrow Cells

Paraspeckles are sub-nuclear structures found in the interchromatin space of mammalian cells. The core paraspeckle components include a lncRNA NEAT1 and members of the DBHS family of proteins: NONO, SFPQ, and PSPC1. Paraspeckles and their components play diverse roles in gene regulatory networks, including transcription, alternative RNA splicing, nuclear retention of RNA, and DNA repair. Although a previous study showed the presence of paraspeckles in hematopoietic stem and progenitor cells (HSPCs), their roles in normal and malignant hematopoiesis remain largely unknown. ASXL1 regulates gene expression through interactions with multiple epigenetic regulators. Somatic mutations in ASXL1 gene occur frequently in myeloid neoplasms. We previously generated a hematopoietic lineage-specific conditional knockin (KI) mouse of a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), and showed that ASXL1-MT inhibited repopulating capability of HSPCs. We performed deep RNA sequencing using HSPCs from ASXL1-MT-KI mice, and found aberrant alternative splicing in multiple genes involved in hematopoiesis. The altered splicing in ASXL1-MT-KI HSPCs included abnormal exon skipping or retention in Runx1, Traf6, Atm, and Dnmt3b. These findings, together with a previous report showing that ASXL1 mutations affect alternative splicing in U937 cells, strongly indicate the involvement of ASXL1 in RNA splicing machinery. Because a previous interactome analysis suggested the association between NONO and ASXL1, we hypothesized that ASXL1 may play a role in RNA maturation processes through interactions with paraspeckle proteins. To test this hypothesis, we examined physical and functional interactions between paraspeckle components and ASXL1. We found that both wild-type and mutant ASXL1 interact with NONO and SFPQ in 293T cells. Interestingly, protein and RNA immunoprecipitation (RIP) analyses revealed that coexpression of wild-type ASXL1, but not mutant ASXL1, enhanced interactions between NONO and histone H3 as well as NONO and NEAT1. These results suggest that ASXL1 acts as a scaffolding protein that assembles paraspeckle proteins and histones to promote transcription and RNA processing. Importantly, mutant ASXL1 loses this function. Next, we assessed subcellular localization of Nono in HSPCs from control and ASXL1-MT-KI mice. We observed predominant cytoplasmic expression of Nono in ASXL1-MT KI HSPCs, while Nono mainly localized in the nucleus in control cells (Figure 1). In addition, expression of NEAT1_2 isoform, which is essential for paraspeckle formation and maintenance, was substantially downregulated in ASXL1-MT-KI HSPCs. Consistent with these observations, RNA FISH against NEAT1 and immunofluorescence against NONO revealed disrupted paraspeckle formation in ASXL1-MT-KI HSPCs. These data suggest that ASXL1-MT promotes nuclear export of Nono, which results in disruption of paraspeckles in HSPCs. NONO has nuclear localization signal (NLS) at its C-terminus, and it was previously shown that a cytoplasmic C-truncated form of NONO induced senescence in fibroblasts. To assess the effect of forced expression of the cytoplasmic NONO in hematopoietic cells, we transduced vector or a NONO mutant lacking the NLS domain (NONO-ΔNLS) into c-Kit+ bone marrow cells, and transplanted these cells into recipient mice. NONO-ΔNLS induced overproduction of reactive oxygen species (ROS) and reduced engraftment of bone marrow progenitors as ASXL1-MT did. We then assessed the effect of Nono depletion in ASXL1-MT-KI HSPCs using CRISPR/Cas9 system. We crossed ASXL1-MT-KI mice with Rosa26-LSL-Cas9-KI mice, and c-Kit+ bone marrow cells derived from these mice were transduced with a non-targeting or Nono-targeting sgRNAs. This experiment revealed that Nono depletion reverted the impaired repopulation of ASXL1-MT-KI HSPCs after transplantation. Taken together, these data indicate that the cytoplasmic localization of Nono induced by ASXL1-MT has the negative impact on HSPC function. In summary, this study reveals a novel link between an epigenetic regulator ASXL1 and paraspeckle formation. The aberrant interaction between mutant ASXL1 and NONO results in NONO mislocalization, paraspeckle disruption and HSPC dysfunction. Our findings also suggest potentially important roles for paraspeckles to maintain normal hematopoiesis. Disclosures Ogawa: Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; Asahi Genomics: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; Kan Research Laboratory, Inc.: Consultancy.

The Cell Cycle in Hematopoietic Stem Cells, Lin−/C-kit+/Sca1+ Fraction, Is Regulated by Connexin 32, Specifically Expressed in the Stem Cell Compartment

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4775-4775
Author(s):  
Yoko Hirabayashi ◽  
Byung-Il Yoon ◽  
Isao Tsuboi ◽  
Yan Huo ◽  
Yukio Kodama ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
In The Wild ◽  
Marrow Cells

Abstract Connexin (Cx) functions in the organization of cell-cell communication via gap junctions in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control, cellular differentiation, apoptosis and the synchronization of electrotonic and metabolic functions. As Cxs are essential molecules for multicellular organisms, Cxs that organize cell-cell communication within the hematopoietic progenitor cell compartment are surmised to be present in bone marrow tissue. Recently, we first found that Cx32 is only Cx molecule expressed in the bone marrow in wild-type mice by means of comparison with Cx32-knockout (KO) mice, studied by a reverse biological approach. Cx32 is specifically expressed in primitive hematopoietic stem/progenitor cells, i.e., the lineage marker-negative (Lin−)/c-kit positive (c-kit+)/stem cell antigen-1-positive (Sca1+) (=LKS) fraction, and likely playing a role of restoration of stem/progenitor cell-quiescence, thereby preventing primitive stem cells from exhaustion. In this study, we present results on cell cycle analyses with respect to the function of Cx32; one for colony-forming progenitors by the method evaluating the cycling progenitor cells using incorporation of bromodeoxyuridine (BrdUrd) followed by ultraviolet-light cytocide and the other for primitive progenitor cells using a cell sorter with bioactive AT-rich DNA-binding dye Hoechst 33342. In the colonization assay on CFU-S-13 (primitive hematopoietic progenitor cells), the incorporation of BrdUrd starts from a higher percentage with rapid increase in Cx32-KO mice, suggesting suppression of cell cycle in these primitive hematopoietic progenitor cells with Cx32-mediated cell-cycle regulation in the wild-type steady state. This suppression may be attenuated in CFU-S-9, a differentiated progenitor cell compartment. The progenitor cells assayed by in vitro colonization on CFU-GM also showed accelerated cell cycle in the Cx32-KO mice. Following the incorporation of Hoechst 33342, the lineagedepleted bone marrow cells were analyzed by flow cytometry. The population sizes of the LKS fraction obtained were 0.052% in the Cx32-KO bone marrow cells and 0.035% in the wild-type bone marrow cells (p=0.0458<0.05). The lineage-depleted bone marrow cells were analyzed their cell-cycle patterns by flow cytometry, and the G0/G1 was calculated for the LKS fractions in both, the Cx32-KO mice and wild-type mice. The percentage of G0/G1 calculated for the LKS fractions were significantly lower in the Cx32-KO mice than those in wild-type mice (60.6% vs. 87.9% for Cx32-KO vs. wild-type; p=0.001). The results suggest that Cx32 may have suppressive functions on the hematopoietic stem cell compartment, the LKS fraction, under the physiological function of Cx32. The Cx32 in the wild-type mice is, thus considered to be expressed in the primitive hematopoietic stem/progenitor cells to prevent from their exhaustion.

C-Cbl Regulates Interaction of Immature Hematopoietic Cells with the Bone Marrow Microenvironment by Rac GTPase-Mediated Cytoskeletal Signals.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3643-3643
Author(s):  
Eisuke Uehara ◽  
Takahiro Suzuki ◽  
Hiroshi Okabe ◽  
Masuzu Ueda ◽  
Tadashi Nagai ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Bone Marrow Microenvironment ◽  
Negative Regulator ◽  
Wild Type ◽  
Rac Gtpase ◽  
Hematopoietic Stem ◽  
Deficient Mice ◽  
Marrow Cells

Abstract Abstract 3643 Poster Board III-579 <Background and purpose> c-Cbl is a ubiquitin E3 ligase and functions as a negative regulator for signals induced by various activated tyrosine kinases, by promoting ubiquitination and proteasomal degradation of these kinases. This inhibitory action is mainly mediated by the tyrosine kinase binding (TKB) and RING finger domains located in the NH3-terminal part of the molecule and these domains are evolutionally well-conserved from nematodes. But mammalian c-Cbl has a stretched structure toward the COOH-terminal end with putative tyrosine residues that can interact with p85 subunit of PI3-kinase, and several reports have denoted that this interaction may be important for cytoskeletal regulation. Recently, mutations of the c-cbl gene have been reported in hematopoietic malignancies, and regulation of hematopoietic stem/progenitor cells (HSPCs) by Cbl is attracting attention. Since it is well-known that cytoskeletal dysregulation is often associated with malignant transformation, in this study, we investigated cytoskeletal regulatory mechanisms mediated by Cbl in hematopoietic cells, using Cbl deficient mice. <Methods and results> To examine the migratory capacity of the HSPCs, lineage negative (Lin(-)) bone marrow cells were set in the Boyden Chamber assay for SDF-1 and fibronectin (FN) were performed. We found that Cbl deficient Lin(-) cells showed significantly decreased migration to these chemoattractants; the migration capacity of the Cbl deficient cells was one eighth for SDF-1 (p = 0.01) and one third for FN (p = 0.007), respectively, compared with the wild-type counterparts. Then, to evaluate in vivo homing ability to the bone marrow microenvironment, transplantation assays were performed. We transplanted 2.5 × 10e6 of Lin(-) HSPCs from Cbl deficient or wild-type Ly5.2 mice into sublethally irradiated wild-type Ly5.1 mice. Three hours after transplantation, chimerism of the transplanted Ly5.2 cells in the bone marrow was examined, and we found that the number of transplanted cells was significantly smaller in Cbl deficient cells, showing that homing capacity of HSPCs in Cbl deficient mice was impaired. Moreover, when we administered G-CSF to Cbl deficient mice, a significantly larger number of Lin(-) Sca1(+) c-Kit(+) cells were mobilized from the bone marrow (p = 0.01), indicating that in Cbl deficient mice, mobilization of HSPCs by G-CSF was also affected. In bone marrow cells or Lin(-) HPSCs of Cbl deficient mice, activity of Rac, a member of small G-protein GTPases, was significantly decreased (p = 0.002). These data ware supported by the experiments with embryonic fibroblast; Rac activities induced by FN stimulation were weaker in Cbl deficient fibroblasts than wild-type ones, and Cbl deficient fibroblasts showed impaired actin rearrangement. <Discussion> We found that Cbl deficient HSPCs showed impaired migration activities to chemoattractants and altered homing and mobilization to and from the bone marrow. Furthermore, Cbl deficient cells had impaired activation of Rac. Currently, little is known about the relationship between Cbl-mediated signals and cytoskeletal regulator Rac, and in this study, we found that Cbl is a positive regulator of Rac activity in the bone marrow hematopoietic cells. Rac activity is reported to be necessary for homing and retention of HSPCs in the bone marrow microenvironment, and our results indicate that interaction of HSPCs with the bone marrow microenvironment, e.g. trafficking of these cells, is regulated by Cbl via Rac GTPase signals. Disclosures: No relevant conflicts of interest to declare.

Inhibitory Receptor, gp49B1, Is Co-Expressed with c-Kit and Regulates Hematopoiesis during Development

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4751-4751
Author(s):  
Mi Deng ◽  
Zhigang Lu ◽  
Jaehyup Kim ◽  
Chengcheng Zhang
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Deficient Mouse ◽  
Hematopoietic Progenitor ◽  
Post Transplantation ◽  
Hematopoietic Stem

Abstract During development, hematopoietic stem cells (HSCs) undergo dramatic expansion in fetal liver, and then migrate to spleen and bone marrow afterwards. Mouse HSCs are enriched in lineage- Sca-1+ c-Kit+ (LSK) cells and defined by their ability to reconstitute the hematopoietic system of lethally irradiated recipients. Although c-Kit is required for HSCs function, heterogeneous c-Kit expression represents functionally distinct subsets of HSCs and progenitor cells. Recently, we demonstrated the one of inhibitory leukocyte immunoglobulin-like receptors, LILRB2, and its mouse homolog, PIRB, are expressed in HSCs. Besides PIRB, the gp49B1 is the only other member of mouse LILRB family. The function of gp49B1 in hematopoiesis is not known. Here we demonstrated that gp49B1 is not expressed by LSK cells of adult mice but is expressed on neonatal bone marrow and spleen LSK cells. Two distinct populations of neonatal LSK cells can be identified based on c-Kit expression. In neonatal bone marrow, 96% of c-Kithi LSK cells are gp49B1+ whereas only 3% of c-Kitlo LSK cells express gp49B1. Similarly, 99% of c-Kithi but only 9% of c-Kitlo LSK cells are gp49B1+ in neonatal spleen. The gp49B1+ LSK cells showed 4.2-folds higher expression level of c-Kit than that of the gp49B1- LSK cells. Because c-Kit is required for hematopoietic progenitor or HSC (HSPC) function, we sought to test whether gp49B1 has regulatory effects on HSC activity. Neonatal splenic gp49B1- LSK cells produced 26-folds more colonies than gp49+ LSK cells after 7 day in methylcellulose media. To compare their reconstitution abilities, we injected 1,000 sorted neonatal splenic gp49B1+ or gp49B1- LSK cells into lethally irradiated 8 weeks-old C57BL6 mice. All mice transplanted by gp49B1+ LSK cells died within 2 weeks post-transplantation, whereas all gp49B1- LSK cells transplanted survived. These results suggest gp49B1- LSK cells, which have less c-Kit expression, are enriched for HSC activity. To further confirm it, 500 sorted gp49B1+ or gp49B1- LSK cells (CD45.2+) were transplanted with 100,000 competitor bone marrow cells (CD45.1+) into lethally irradiated congenic recipients (CD45.1+). Mice transplanted with gp49B1- LSK cells exhibited increasing peripheral blood donor CD45 chimerism levels from 3 to 18 weeks after transplant (14.7%~68.2%); but gp49B1+ LSK cells transplanted mice only have modest chimerism levels (<2%; exception of 1 out of 7 mice has 13% at 18 weeks). Interestingly, neonatal splenic gp49B1+ LSK cells exhibited a lineage bias compared to gp49B1- LSK cells after transplantation (B cell: 2% vs. 30.1%, p<0.01; T cell: 61.3% vs. 17.4%, p<0.05; and Myeloid cell: 42.8% vs. 60.2%, p=0.32). Consistently, c-Kithi LSK cells of which over 96% are gp49B1+ showed much less HSPC activities comparing with c-Kitlo LSK cells in colony formation (40-folds less), non-competitive transplantation (all died in 2 weeks vs. all survived after transplantation), and competitive transplantation (donor CD45 chimerism: 0.04~0.34% vs. 5.8~33.6%, from 6 to 20 weeks). We continued to study the function of gp49B1's function using gp49b1 deficient mice. We found that c-Kithi LSK cells were increased in gp49B1-deficient mouse (0.18% vs. 0.14%), whereas KO c-Kithi LSK% decreased (0.09% vs. 0.14%). The c-Kithi LSK cells of gp49B1-deficient mouse also exhibited low repopulation potential. While the same number of WT and KO LSK cells had comparable repopulation abilities, five hundred gp49b1-deficient c-Kitlo LSK cells exhibited a greater reconstitution capacity (65.3% vs. 33.6%) than wild-type c-Kitlo LSK cells. These results suggest that gp49B1 may regulate the repopulation of primitive neonatal hematopoietic cells. Together, our results demonstrate that the gp49B1 is co-expressed with high level of c-Kit in hematopoietic progenitor cells of neonatal mouse, and it regulates maturation, repopulation, and differentiation of hematopoietic cells during development. Disclosures No relevant conflicts of interest to declare.

scholarly journals Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2148-2159 ◽  
Author(s):  
Harshal H. Nandurkar ◽  
Lorraine Robb ◽  
David Tarlinton ◽  
Louise Barnett ◽  
Frank Köntgen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
White Blood Cells ◽  
Null Mutation ◽  
Wild Type ◽  
Normal Numbers ◽  
Interleukin 11 ◽  
Marrow Cells

Abstract Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

Phenotypic correction of Fanconi anemia group C knockout mice

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 700-704 ◽  
Author(s):  
Kimberly A. Gush ◽  
Kai-Ling Fu ◽  
Markus Grompe ◽  
Christopher E. Walsh
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mitomycin C ◽  
Fanconi Anemia ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Genetic Disorder ◽  
Hematopoietic Stem ◽  
Marrow Cells

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital anomalies, and a predisposition to malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of the FANCC gene (fancc −/− nullizygous mice) exhibit many of the characteristic features of FA and provide a valuable tool for testing novel therapeutic strategies. We have exploited the inherent hypersensitivity offancc −/− hematopoietic cells to assay for phenotypic correction following transfer of the FANCC complementary DNA (cDNA) into bone marrow cells. Murine fancc −/− bone marrow cells were transduced with the use of retrovirus carrying the humanfancc cDNA and injected into lethally irradiated recipients. Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to challenge the transplanted animals. Phenotypic correction was determined by assessment of peripheral blood counts. Mice that received cells transduced with virus carrying the wild-type gene maintained normal blood counts following MMC administration. All nullizygous control animals receiving MMC exhibited pancytopenia shortly before death. Clonogenic assay and polymerase chain reaction analysis confirmed gene transfer of progenitor cells. These results indicate that selective pressure promotes in vivo enrichment offancc-transduced hematopoietic stem/progenitor cells. In addition, MMC resistance coupled with detection of the transgene in secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.

Requirement for p85α Regulatory Subunit of Class IA PI3Kinase and Rac2 GTPase in Myeloproliferative Disease Driven by Activation Loop Mutant of KIT.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 89-89
Author(s):  
Veerendra Munugalavadla ◽  
Emily C. Sims ◽  
Stephen D. Lenz ◽  
Reuben Kapur
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Regulatory Subunit ◽  
Activation Loop ◽  
Myeloproliferative Disease ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Oncogenic activation-loop mutants of KIT, the receptor for stem cell factor (SCF), are commonly observed in acute myeloid leukemia (AML) and systemic mastocytosis (SM); however, unlike the KIT juxtamembrane mutants (found in patients with gastrointestinal stromal tumors [GISTs]), the activation-loop mutants are commonly insensitive to inhibition by tyrosine kinase inhibitors. Furthermore, little is known about the signaling pathways that contribute to oncogenic KIT-induced transformation in SM or AML. We demonstrate that expression of KITD814V (KIT activation-loop mutant) in primary hematopoietic stem and progenitor cells induces constitutive KIT autophosphorylation, promotes ligand-independent hyperproliferation, skews myeloid differentiation towards the granulocytic lineage, and promotes promiscuous cooperation with multiple cytokines, including G-CSF, M-CSF and IL-3. KITD814V expressing primary mast cells also demonstrated hyperproliferation in response to SCF, IL-3, IL-4 and IL-10. Biochemical analyses of KITD814V expressing cells revealed constitutively elevated levels of phosphatidylinositol-3-kinase (PI3K) and its downstream substrate, the Rho family GTPase Rac. Genetic disruption of p85a, the regulatory subunit of class IA PI-3Kinase, but not of p85β, or genetic disruption of the hematopoietic cell-specific Rho GTPase, Rac2, normalized KITD814V-induced ligand independent hyperproliferation in vitro. Additionally, deficiency of p85α or Rac2 corrected the promiscuous hyperproliferation observed in response to multiple cytokines in both KITD814V expressing stem/progenitor cells as well as mast cells in vitro. Although p85α is hyperphosphorylated and constitutively bound to KITD814V in bone marrow cells in vitro; its physiologic role in transformation in vivo is not known. To address this, we generated a new mouse model to study KITD814V induced transformation in myeloid cells as opposed to previously described models that primarily result in the generation of phenotypes resembling acute lymphocytic leukemia via this mutation. Our results show that transplantation of KITD814V expressing bone marrow cells from C57/BL6 strain of mice into syngeneic recipients results in a fatal myeloproliferative disease (MPD) characterized by leukocytosis, splenomegaly, disruption of the splenic architecture as well as myeloid cell infiltration in the lung and liver. Importantly, in this model, transplantation of KITD814V expressing p85α deficient bone marrow cells rescued the MPD phenotype, including splenomegaly, peripheral blood leukocytosis and the reduced life span associated with the transplantation of KITD814V expressing wildtype bone marrow cells. Treatment of KITD814V-expressing hematopoietic progenitors with either a Rac inhibitor (NC23766) or rapamycin showed a dose-dependent suppression in KITD814V induced growth. Taken together, our results describe the generation of a new murine transplant model to study KITD814V induced transformation and identify p85a and Rac2 as potential novel therapeutic target for the treatment of KITD814V-bearing diseases including SM and AML.

Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2629-2629
Author(s):  
Ying Zhao ◽  
Flora Ling ◽  
Hong-Cheng Wang ◽  
Xiao-Hong Sun
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Chronic Inflammation ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Inflammatory Conditions ◽  
The Impact ◽  
Lps Treatment ◽  
Marrow Cells

Abstract Abstract 2629 The overall objectives of this study are to investigate the impact of inflammatory conditions on hematopoietic stem cell (HSC) maintenance and to elucidate the underlying mechanisms. HSCs are exposed to a variety of inflammatory conditions through life. How these conditions influence the integrity of HSCs is a fundamental issue of clinical importance but it is poorly understood. Equally unknown is the molecular regulation of HSC maintenance during inflammatory. In this context, our focus is on the role of basic helix-loop-helix (bHLH) proteins, which include transcription activators such as E2A proteins and their inhibitors including Id proteins. We and others have shown that these regulators are involved in normal hematopoiesis such as stem cell function and lineage specific differentiation. Recently, we have obtained evidence to suggest that signaling through Toll-like receptors (TLRs), which is closely linked to inflammation, causes down-regulation of E2A function by stimulating Id1 expression. Therefore, we hypothesize that inflammatory conditions causes down-regulation of E protein function, which disturbs the quiescence of long-term (LT)-HSC, leading to stem cell exhaustion over time. To test this hypothesis, we induced chronic inflammation in wild type and Id1-/- mice by daily injection of 1 mg of LPS, i.p. for 30 days. Peripheral blood was collected on days 15 and 30 and levels of a panel of inflammatory cytokines were assayed using a Luminex multiplex kit. On day 15, dramatic increases were found in the levels of IL-10, IL-6, KC and TNFα but not IFN-γ, IL12-p70 and IL-1β. Interestingly, levels of IL-6 and TNFα were significantly lower in Id1-/- mice compared to wild type mice. By day 30 of LPS treatment, levels of these cytokines returned to the levels in animals without LPS injection. These results suggest that this chronic LPS treatment indeed elicited an inflammatory response that included transient elevation of inflammatory cytokines. Whether secretion of these cytokines has any direct effects on HSCs remains to be determined. To measure HSC activity in these LPS-treated mice, we performed serial bone marrow transplant assays. Lin−Sca-1+c-kit+ (LSK) stem/progenitor cells were isolated from wild type or Id1-/- mice treated with or without LPS. These cells were transplanted into lethally irradiated CD45.1+ recipients along with equal numbers of YFP-expressing LSK as competitors. Six weeks later, cohorts of mice were sacrificed and bone marrow cells were collected. Pooled whole bone marrow cells within each cohort were injected into lethally irradiated secondary recipients. Secondary recipients were sacrificed 8 and 16 weeks post transplant. For assessment of primary and secondary engraftment, bone marrow cells were examined for expression of donor and lineage specific markers. Robust engraftment was observed in primary or secondary recipients. Donor derived cells were then gated for YFP− and YFP+ cells, which separate cells originated from tester and competitor LSK, respectively. While YFP− and YFP+ cells engrafted equivalently in primary recipients transplanted with cells treated with or without LPS, LPS treatment of wild type mice caused a great disparity in secondary recipients. In contrast, HSC in Id1-/- mice did not appear to be affected by the same treatment even though HSCs in Id1 deficient mice are normally lower in numbers and activities as we previously reported. These results suggest that chronic inflammation diminishes the LT-stem cell activity and this may involve the up-regulation of Id1 expression. To investigate the underlying mechanism, we performed label retaining assays to examine the quiescence of LT-HSCs. We found that BrdU-labeling in HSCs was 2-fold lower in mice treated with LPS compared to the untreated controls, suggesting that treatment with LPS promoted the cycling of HSCs, thus impairing their stem cell function. Taken together, our study illustrates that chronic inflammation has a detrimental effect on LT-stem cell activity. Although HSCs have an enormous capability to repopulate the bone marrow by compensatory proliferation, pro-longed inflammation could eventually lead to stem cell exhaustion and seriously compromise hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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