PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs

Abstract Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.2629.2629 ◽

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.

Download Full-text

Non-Canonical NF-Kb Signaling Regulates Hematopoietic Stem Cell Self-Renewal and Microenvironment Interactions

Blood ◽

10.1182/blood.v118.21.859.859 ◽

2011 ◽

Vol 118 (21) ◽

pp. 859-859 ◽

Cited By ~ 1

Author(s):

Chen Zhao ◽

Yan Xiu ◽

John M Ashton ◽

Lianping Xing ◽

Yoshikazu Morita ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Wild Type ◽

Double Knockout ◽

Self Renewal ◽

Hematopoietic Stem ◽

Physiological Processes ◽

Marrow Cells

Abstract Abstract 859 RelB and NF-kB2 are the main effectors of NF-kB non-canonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-kB2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Further, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-kB2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Prostaglandin E2 restrains macrophage maturation via E prostanoid receptor 2/protein kinase A signaling

Blood ◽

10.1182/blood-2011-08-374207 ◽

2012 ◽

Vol 119 (10) ◽

pp. 2358-2367 ◽

Cited By ~ 40

Author(s):

Zbigniew Zasłona ◽

Carlos H. Serezani ◽

Katsuhide Okunishi ◽

David M. Aronoff ◽

Marc Peters-Golden

Keyword(s):

Bone Marrow ◽

Protein Kinase ◽

Prostaglandin E2 ◽

Protein Kinase A ◽

Bone Marrow Cells ◽

Lipid Mediator ◽

Wild Type ◽

Kinase A ◽

Marrow Cells

Abstract Prostaglandin E2 (PGE2) is a lipid mediator that acts by ligating 4 distinct G protein–coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE2 in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE2 receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2−/−) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2−/− mice exhibited a higher percentage of F4/80high/CD11bhigh cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE2 analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2−/− mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE2/EP2/protein kinase A signaling in the suppression of macrophage maturation.

Download Full-text

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

Blood ◽

10.1182/blood-2018-99-110836 ◽

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.

Download Full-text

miR-21 Is a Transcriptional Target of Growth Factor Independent -1; Implications for Transcriptional Regulation of microRNA.

Blood ◽

10.1182/blood.v108.11.1178.1178 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1178-1178

Author(s):

Chinavenmeni S. Velu ◽

Anil G. Jegga ◽

Vivek Kaimal ◽

Bruce Aronow ◽

H. Leighton Grimes

Keyword(s):

Bone Marrow ◽

Binding Sites ◽

Bone Marrow Cells ◽

Regulatory Sequences ◽

Cis Elements ◽

Wild Type ◽

Expression Array ◽

Hematopoietic Stem ◽

Microrna Genes ◽

Marrow Cells

Abstract Hematopoiesis is a tightly regulated multistage process, wherein pluripotent self-renewing stem cells gives rise to all blood cell lineages. Growth factor independent -1 (Gfi1) is a transcription factor that relies on corepressor proteins such as G9a to repress transcription of target genes. Gfi1 is required for hematopoietic stem cell self-renewal and subsequent granulocytic lineage development. Recent studies have identified microRNAs (miRs) as 21–23 nucleotide non-coding RNA that regulate gene expression. It is currently unclear which microRNAs control hematopoiesis. The deregulated Gfi1 null environment provides a unique setting to screen for miRNA that may control hematopoiesis. Therefore, we performed microRNA gene expression array analyses on RNA extracted from total bone marrow cells of Gfi1 null and wild type littermate mice. Our results demonstrate that several miR genes are differentially expressed in Gfi1 knock-out mice; however, we have focused further analyses on miR-21, which was consistently upregulated in Gfi1 null cells. Thus, miR-21 provides a potential direct target for Gfi1. Moreover, miR-21 was previously shown to regulate apoptosis in neuronal cell lines. As demonstrated by both expression array and Taqman, the relative expression of the mature miR-21 in Gfi1 null bone marrow cells is more than 7 fold higher than wild type Gfi1 littermate bone marrow. Because loss of Gfi1 alters hematopoiesis, we normalized the cells under analysis by lineage marker depletion. Still, in comparison to similar Lin-populations from wild type littermates, the expression of miR-21 was consistently higher in Gfi1 null Lin-bone marrow cells. We next attempted to determine whether miR-21 is directly regulated by Gfi1; however, bioinformatic analyses of microRNA regulatory sequences are currently underdeveloped. We therefore adopted a systematic gene-centric pipeline approach and adapted a web-accessible database resource termed Genome TraFac (http://genometrafac.cchmc.org) to microRNA orthologs that are conserved between human and murine genomes. The new “miRBase” analyzed for the occurrence of conserved individual cis-elements and compositionally similar cis-culsters to identify validated transcription factor targets within promoter and distal genomic regulatory regions of microRNA genes. Interestingly, many microRNA genes contain conserved regulatory sequences within transcribed precursor sequences, not in the predicted promoter regions. Within the miR-21 gene, two putative Gfi1 binding sites were identified within transcribed precursor sequences. To determine the accuracy of our bioinformatic analyses, we performed chromatin immunoprecipitation (ChIP) with anti-Gfi1 and an anti-G9A antisera. Our data reveal Gfi1, along with its co-factor G9A, is bound to the predicted region (but not control regions lacking predicted Gfi1 binding sites). Notably, electrophoretic mobility shift assay (EMSA) indicated that in vitro transcribed and translated Gfi1 binds strongly to one of the two putative Gfi1 binding sites in the miR-21 gene. These data suggest that the level of miR-21 is regulated directly by Gfi1, and that microRNA genes are commonly regulated by conserved cis-elements within transcribed precursor sequences. The role of miR-21 in controlling differentiation/proliferation in hematopoietic stem cells is currently under investigation.

Download Full-text

Protective Role of Connexin 32 in Experimental Leukemogenesis.

Blood ◽

10.1182/blood.v110.11.2239.2239 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2239-2239

Author(s):

Yoko Hirabayashi ◽

Byung-Il Yoon ◽

Isao Tsuboi ◽

Yan Huo ◽

Yukio Kodama ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Bone Marrow Cells ◽

Pcr Analysis ◽

Wild Type ◽

Hematopoietic Stem ◽

Immunomagnetic Bead ◽

Competitive Assay ◽

Marrow Cells

Abstract Connexin (Cx) functions in the organization of cell-cell communication in multicellular organisms. Gap junctions have been implicated in the homeostatic regulation of various cellular functions, including growth control and differentiation, apoptosis, and the synchronization of electrotonic and metabolic functions. Primitive hemopoietic progenitor cells form a multicellular system, but a previous report describes that Cx32 is not expressed in the bone marrow. Thus, a question arises as to why Cx molecules are not detected in the hematopoietic tissue other than stromal cells. Based on our preliminary study that suggested a potential impairment of hematopoiesis in Cx32-knockout (KO) mice, the objectives of the present study were to determine whether Cx32 functions in the bone marrow during steady-state hematopoiesis and further to examine its possible protective roles during regeneration after chemical abrasions and during leukemogenesis after the administration of a genotoxic chemical, methyl nitrosourea (MNU). As results, the Cx32 molecule functioning in the hematopoietic stem cell (HSC) compartment during steady-state hematopoiesis was observed for the first time; the expression of Cx32 at the mRNA level determined by PCR analysis and that at the protein level determined using an anti-Cx32 antibody were observed only in the lin−c-kit+ HSC fraction using a combination of immunobead-density gradient and immunomagnetic-bead separation. Hematopoiesis was impaired in the absence of Cx32; it was delayed during regeneration after chemical abrasion with 5-fluorouracil at 150 mg/kg body weight in Cx32-KO mice. Cx32-KO mice also showed increased leukemogenicity compared with wild-type mice after MNU injection; furthermore, in a competitive assay for leukemogenicity in mice that had been lethally irradiated and repopulated with a mixed population of equal amount of bone marrow cells from Cx32-KO mice and wild-type mice, the resulting leukemias were originated predominantly from Cx32-KO bone marrow cells. The present competitive assay clearly showed that Cx32-KO bone marrow cells have a higher risk of becoming leukemogenic. The above-mentioned findings in this study imply that Cxs play an essential role in maintaining the steady-state hematopoiesis and suppressing the neoplastic change. In summary, the role of Cx32 in hematopoiesis was not previously recognized and Cx32 was expressed only in HSCs and their progenitors. The results indicate that Cx32 in wild-type mice protects HSCs from chemical abrasion and leukemogenic impacts. Our results indicate that the risk of developing leukemia in patients with X-chromosome-linked Cx32 deficiency, called Charcot-Marie-Tooth syndrome, may not be incidental.

Download Full-text

R-Ras Regulates Hematopoietic Stem/Progenitor Cell Adhesion, Migration, and Mobilization through Rac GTPase-Mediated Signals.

Blood ◽

10.1182/blood.v110.11.221.221 ◽

2007 ◽

Vol 110 (11) ◽

pp. 221-221

Author(s):

Xun Shang ◽

Lina Li ◽

Jose Concelas ◽

Fukun Guo ◽

Deidre Daria ◽

...

Keyword(s):

Bone Marrow ◽

Cell Adhesion ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Wild Type ◽

Rac Gtpase ◽

Hematopoietic Stem ◽

And Migration ◽

Marrow Cells

Abstract Hematopoietic stem/progenitor cells (HSPCs) are maintained by strictly regulated signals in the bone marrow microenvironment. One challenge in understanding the complex mode of HSPC regulation is to link intracellular signal components with extracellular stimuli. R-Ras is a member of the Ras family small GTPases. Previous mouse genetic studies suggest that R-Ras mRNA is primarily expressed in endothelial cells and R-Ras is involved in vascular angiogenesis. In clonal cell lines, although dominant mutant overexpression studies suggest a possible role of R-Ras in regulating cell adhesion and spreading, proliferation and/or differentiation in a cell-type dependent manner, it remains controversial whether R-Ras activity may promote or inhibit cell adhesion and migration. Here, in a mouse knockout model, we have examined the role of R-Ras in HSPC regulation by a combined in vivo and in vitro approach. Firstly, we found that R-Ras is expressed in the Lin− low density bone marrow cells of wild-type mice, and R-Ras activity in the cells is downregulated by cytokines and chemokines such as SCF and SDF-1a (∼ 20% and 40% of unstimulated control, respectively). Secondly, R-Ras deficiency did not significantly affect peripheral blood CBC, nor alter the frequency or distribution of long-term and short-term hematopoietic stem cells (defined by IL7Ra−Lin−Sca-1+c-Kit+CD34− and IL7Ra−Lin−Sca-1+c-Kit+CD34+ genotypes, respectively) in the bone marrow, peripheral blood and spleen. Competitive repopulation experiments using the wild-type and R-Ras−/− bone marrow cells at 1:1 ratio in lethally irradiated recipient mice showed no significant difference of blood cells of the two genotypes in the recipients up to 6 months post-transplantation. R-Ras−/− bone marrow cells did not show a detectable difference in colony forming unit activities assayed in the presence of various combinations of SCF, TPO, EPO, IL3, G-CSF and serum, compared with the matching wild-type cells. Thirdly, upon challenge with G-CSF, a HSPC mobilizing agent, R-Ras−/− mice demonstrated a markedly enhanced ability to mobilize HSPCs from bone marrow to peripheral blood as revealed by genotypic and colony-forming unit analyses (WT: 150 vs. KO: 320 per 200uL blood, p=0.018), and R-Ras−/− HSPCs exhibit significantly decreased homing activity (WT: 4.3% vs. KO: 2.8%, p<0.001). Fourthly, isolated R-Ras−/− HSPCs displayed a constitutively assembled cortical actin cytoskeleton structure in the absence of cytokine or chemokine stimulation, similar to that of activated wild-type HSPCs. The R-Ras−/− HSPCs were defective in adhesion of cobblestone area-forming cells to a bone marrow-derived stroma cell line (FBMD-1) and in adhesion to fibronectin CH296 fragment, and showed a drastically increased ability to migrate toward a SDF-1a gradient (WT: 16% vs. KO: 38%, p<0.001). These data point to a HSPC-intrinsic role of R-Ras in adhesion and migration. Finally, the functional changes of R-Ras−/− cells were associated with a ∼3 fold increase in Rac-GTP species and constitutively elevated Rac downstream signals of phsopho-PAK1 and phospho-myosin light chain. Partial inhibition of Rac activity by NSC23766, a Rac GTPase-specific inhibitor, readily reversed the migration phenotype under SDF-1a stimulation. Taken together, these studies demonstrate that R-Ras is a critical signal regulator for HSPC adhesion, homing, migration, and mobilization through a mechanism involving Rac GTPase-regulated cytoskeleton and adhesion machinery.

Download Full-text

Deregulation of MicroRNA Underlies Severe Congenital Neutropenia Pathogenesis

Blood ◽

10.1182/blood.v112.11.313.313 ◽

2008 ◽

Vol 112 (11) ◽

pp. 313-313

Author(s):

ChinavenMeni S. Velu ◽

Avinash M Baktula ◽

Tristan Bourdeau ◽

H. Leighton Grimes

Keyword(s):

Bone Marrow ◽

Zinc Finger Protein ◽

Bone Marrow Cells ◽

Flow Cytometric Analysis ◽

Leukemia Cell ◽

Severe Congenital Neutropenia ◽

Wild Type ◽

Congenital Neutropenia ◽

Hematopoietic Stem ◽

Marrow Cells

Abstract The zinc finger protein Growth factor independent-1 (Gfi1) is a transcriptional repressor that regulates hematopoietic stem cell (HSC) maintenance and granulocytic lineage differentiation. Humans with severe congenital neutropenia (SCN) display mutations in GFI1 (encoding GFI1N382S) which generate dominant negative acting proteins. GFI1N382S proteins sequester limiting cofactors to deregulate a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs and suggest that that transcriptional control of microRNA genes is critical for GFI1N382S-associated SCN phenotypes. First, the expression of Gfi1 and miR21 and miR-196 is reciprocal: 1) in wild type and Gfi1−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), 3) during treatment-induced differentiation of human myeloid leukemia cell lines, and 4) upon conditional deletion of Gfi1 in primary sorted murine CMP and GMP. Biochemical analyses reveal that miR21 and miR-196 are direct transcriptional targets of Gfi1. Subsequently, forced expression of wild type Gfi1 rescues expression of microRNA in Gfi1−/− Lin- bone marrow cells, while forced expression of Gfi1N382S in wild type Lin- bone marrow cells significantly deregulates miR-21 and miR-196 expression. Similarly, we demonstrate elevated miR21 and miR196b levels in CD34+ cells from a GFI1N382S SCN patient. Flow cytometric analysis and colony assays reveal that the overexpression or knockdown of either miR induces changes in myeloid development, but that co-expression of both miR (as seen in Gfi1−/− mice and GFI1N382S SCN patients) completely blocks G-CSF-induced granulopoiesis. These data provide a molecular understanding of SCN disease pathogenesis.

Download Full-text

Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response.

Blood ◽

10.1182/blood.v114.22.1492.1492 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1492-1492

Author(s):

Akil Merchant ◽

Anju Singh ◽

Giselle Joseph ◽

Qiuju Wang ◽

Ping Zhang ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Bone Marrow ◽

Transcription Factor ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Cytokine Signaling ◽

Wild Type ◽

Increased Sensitivity ◽

Marrow Cells

Abstract Abstract 1492 Poster Board I-515 Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2null mice and found increased total bone marrow cellularity, cKit+Sca1+Lin− (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34−KSL) compared to wild type mice (p<0.05). Transplantation of equal numbers of KSL cells from Nrf2wt and Nrf2null resulted in a five-fold decrease in peripheral blood chimerism from Nrf2null derived cells at 16 weeks (15% wild type vs. 3% null, p<0.05). Unlike other models of deficiencies in genes associated with ROS handling, such as ATM or the FoxO family of transcription factors, basal ROS levels were not elevated in Nrf2null HSC. However, Nrf2null bone marrow cells demonstrated increased sensitivity to induced oxidative stress and in vitro treatment with H2O2 resulted in a 2 fold decrease in colony formation in methylcellulose. We also examined the in vivo sensitivity of Nrf2null cells to oxidative stress by irradiating (400 rads) stably chimeric mice 20 weeks following transplantation with either Nrf2wt or Nrf2null HSC. Mice receiving Nrf2null HSC demonstrated a 50% decrease in peripheral blood chimerism at 4 months following radiation compared to no change in Nrf2wt recipients (p<0.05) confirming that loss of Nrf2 leads to increased sensitivity to oxidative stress. Microarray gene expression analysis from Nrf2wt and Nrf2null mice revealed down regulation of the G-CSF cytokine receptor in Nrf2null HSC and suggested that defective cytokine signaling may contribute to the HSC dysfunction seen in Nrf2null bone marrow cells. To test this hypothesis, we attempted to rescue the function of Nrf2null HSC by treating mice with exogenous G-CSF. Nrf2wt and Nrf2null mice were treated with one week of daily G-CSF and then HSC were harvested and transplanted. In contrast to the defects in engraftment of untreated Nrf2null HSC, there was no significant difference in peripheral blood chimerism following transplantation of G-CSF treated Nrf2wt or Nrf2null HSC, thus demonstrating that G-CSF treatment could rescue the HSC defect in mutant mice. In conclusion, the Nrf2 transcription factor appears to be a novel and essential regulator of normal HSC function through the modulation of oxidative stress response and cytokine signaling. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Loss of Sos1 Inhibits Oncogenic Kras-Induced Hyperactivation of Wild-Type Ras during Leukemogenesis

Blood ◽

10.1182/blood.v128.22.1543.1543 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1543-1543

Author(s):

Xiaona You ◽

Guangyao Kong ◽

Erik A. Ranheim ◽

Yun Zhou ◽

Jing Zhang

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Myeloid Cells ◽

Small Gtpase ◽

Wild Type ◽

Hematopoietic Stem ◽

Erk Activation ◽

Gm Csf ◽

Marrow Cells

Abstract As members of small GTPase super family, the functional output of Ras proteins depends on their GTP binding status, which is regulated by the interactions with guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Activating mutations in NRAS and KRAS isoforms are identified in various types of hematopoietic malignancies. Interestingly, the same oncogenic mutation (G12D) at the endogenous Kras locus displays much more potent leukemogenic activity than that at the endogenous Nras locus in vivo. Moreover, combined inhibition of MEK and ERK provides long-term disease-free survival in NrasG12D/G12D mice but had much less effect in KrasG12D/+ mice. During our investigation to understand the potent leukemogenic activity of oncogenic Kras, we found that in total bone marrow cells, oncogenic Kras, but not oncogenic Nras, induces hyperactivation of wild-type (WT) Hras and Nras. We hypothesize that the hyperactivated WT Ras significantly contributes to oncogenic Kras-mediated leukemogenesis and inhibition of this process might improve the sensitivity of oncogenic Kras cells towards combined therapy. Because Sos1, a RAS GEF, has been implicated in oncogenic Ras-mediated activation of WT Ras in human cancer cell lines, we investigated whether Sos1 plays an essential role in this process in vivo. We find that Sos1 is overexpressed in KrasG12D/+ bone marrow cells. Genetic deletion of Sos1 indeed significantly decreases the GTP-bound active form of WT Nras and Hras without affecting the activation status of oncogenic Kras. Consequently, Sos1 deficiency-mediated downregulation of ERK activation rescues oncogenic Kras mediated depletion of hematopoietic stem cells (HSCs). HSCs, multipotent progenitors (MPPs) and LSKs (Lin-Sca-1+c-Kit+) in KrasG12D/+;Sos1-/- mice are much more quiescent than those in KrasG12D/+ mice. Moreover, Sos1 deficiency significantly inhibits granulocyte-macrophage colony stimulating factor (GM-CSF) evoked ERK signaling in KrasG12D/+ myeloid progenitor and precursor cells. Consistent with these biochemical data, we show that myeloproliferative neoplasm (MPN) phenotypes are significantly alleviated in KrasG12D/+;Sos1-/- mice and these animals survived significantly longer than KrasG12D/+ mice. However, we find that in differentiated myeloid cells (e.g. neutrophils), loss of Sos1 does not affect GM-CSF-evoked ERK activation. This result is consistent with our previous finding that Ras-mediated ERK activation in differentiated myeloid cells is predominantly through Kras but not Hras or Nras. Together, our results demonstrate that Sos1 mediates oncogenic Kras-induced hyperactivation of WT Ras. Inhibition of Sos1 thus blocks this process and attenuates the leukemogenic activity of oncogenic Kras. In contrast, Sos1 deficiency does not affect the unique signaling mediated by oncogenic Kras itself. Therefore, we hypothesize that targeting Sos1 alone will not effectively treat KrasG12D-associated leukemias but it might increase the sensitivity of KrasG12D cells to other therapies, such as combined inhibition of MEK and JAK. We are currently testing this hypothesis in vivo. Disclosures No relevant conflicts of interest to declare.

Download Full-text