Hematopoietic Cells from Gadd45a, Gadd45b Deficent Mice Exhibit Altered Myelopoiesis and Higher Apoptosis after Cytokine Treatment.

Abstract Growth arrest and DNA damage, Gadd45 gene family members are rapidly induced by genotoxic agents as well as by apoptosis and differentiation inducing cytokines. Their role in hemetopoiesis, wherein proliferation, differentiation and apoptosis integrate to maintain cellular homeostasis, is not clear. Using bone marrow cells from gadd45a or gadd45b deficient and wild type littermate mice we have investigated the role of Gadd45 proteins in cytokine induced myeloid cell differentiation in vitro. Bone marrow cells obtained from either gadd45a or gadd45b deficient mice displayed compromised cytokines (IL3, GM-CSF, M-CSF or G-CSF) induced myelopoiesis, resulting in a quantitatively decreased population of mature myeloid cells. Immuno-phenotyping with antibodies to cell surface molecules associated with myeloid cell maturation confirmed impaired myeloid cell maturation in Gadd45a or b deficient bone marrow cells treated with the above cytokines. Analysis of apoptosis by annexin-V and PI staining followed by FACS analysis showed a substantially higher apoptosis in Gadd45a−/− as well as gadd45b−/− cells compared to wild type cells after treatment with M-CSF or G-CSF. Gadd45a−/− as well as gadd45b−/− bone marrow cells were found to be less clonogenic in methylcellulose medium. Morphologically compact and round colonies consisting of immature myeloid cells prevailed over dispersed- colonies consisting of mature myeloid cells in gadd45- deficient cells cultured in methyl cellulose containing IL-3. Furthermore, colony re-plating assay showed better self-renewal abilities in gadd45a−/− as well as gadd45b−/− progenitors, compared to wild type progenitor cells. Altered myelopoiesis in gadd45 a or b deficient mice was further confirmed in vivo by intra-peritoneal administration of sodium casienate - a known inducer of inflammatory response and myelopoiesis in mice bone marrow. Sodium casienate failed to enhance myelopoiesis in gadd45a or gadd45b deficient mice bone marrow, while wild type littermate mice showed a rapid induction of myelopoiesis. Simultaneously peritoneal exudates collected from gadd45 deficient mice consisted of 2–3 fold less myeloid cells compared to age matched wild type control mice after sodium casienate treatment. Gadd45a−/− or gadd45b−/− mice showed a slow recovery after myelo-suppressive effect of antimetabolite 5-Fluorouracil, which further confirmed that gadd45 deficiency leads to delayed myelopoiesis in mouse. Mechanistic aspects of Gadd45 deficiency, which results in impaired myelopoiesis are under investigation.

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Stat3β Promotes Basal Granulopoiesis and Inhibits Emergency Granulopoiesis, While Stat3α Inhibits Basal Granulopoiesis and Promotes Emergency Granulopoiesis

Blood ◽

10.1182/blood.v112.11.3871.3871 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3871-3871

Author(s):

Michele Redell ◽

S. Wen-Wen Chen ◽

Marcos J. Ruiz ◽

David J. Tweardy

Keyword(s):

Bone Marrow ◽

B Lymphocyte ◽

Inflammatory Responses ◽

Bone Marrow Cells ◽

Myeloid Cells ◽

Lymphoid Cells ◽

Transactivation Domain ◽

Wild Type ◽

Alternative Mrna Splicing ◽

Deficient Mice

Abstract Signal transducer and activator of transcription 3 (Stat3) is a key signaling intermediate that is activated by several cytokines that regulate hematopoiesis, including granulocyte-colony stimulating factor (G-CSF), interleukin 6, and stem cell factor (SCF). Studies using mice with Stat3 deletion targeted to hematopoietic cells have shown that Stat3 negatively regulates basal granulopoiesis but positively regulates emergency granulopoiesis. Stat3 also has been reported to promote B lymphocyte differentiation. Defining the hematopoietic functions of Stat3 is further complicated by the existence of two isoforms: full-length Stat3α (p92), and truncated Stat3β (p83). Stat3β is derived from alternative mRNA splicing resulting in replacement of the C-terminal transactivation domain with 7 unique amino acids (CT7), which have been demonstrated to confer markedly prolonged nuclear retention. Homozygous Stat3α-deficient mice are not viable, whereas Stat3β-deficient mice survive to adulthood and are fertile, but have increased inflammatory responses compared to wild-type mice. We compared basal granulopoiesis and lymphopoiesis, as well as emergency granulopoiesis, in homozygous Stat3β-deficient mice (βΔ/βΔ), which express only Stat3α, vs. their wild-type (+/+) littermates. We found that βΔ/βΔ mice were significantly leukopenic (2880 ± 1260/ml v. 4600 ± 1670/ml; p<0.05), with lower absolute neutrophil counts (ANC, 360 ± 180/ml v. 800 ± 380/ml, p<0.05) and B lymphocyte counts (780 ± 470/ml v. 1830 ± 1260/ml, p<0.05), compared to +/+ mice. Within the circulating B-lymphocyte population, the mature B220hi/IgM− cells were most dramatically reduced (170 ± 70/ml v. 480 ± 350/ml, p<0.05). Percentages of myeloid and lymphoid cells in the spleen and bone marrow were not significantly different between βΔ/βΔ and +/+ mice. Bone marrow from βΔ/βΔ mice generated significantly fewer myeloid colonies (CFU-GM) compared to wild-type marrow (28 ± 9 v. 42 ± 8 colonies per 20,000 cells, p<0.05). Additionally, βΔ/βΔ lineage-depleted bone marrow cells cultured in G-CSF and SCF produced significantly fewer CD11b+/Gr1+ myeloid cells compared to +/+ cells (52.8 ± 6.5% v. 68.3 ± 2.6%, p<0.05). In contrast, bone marrow from βΔ/βΔ and +/+ mice produced equal numbers of pro-B colonies in CFU assays containing the lymphopoietic cytokine IL-7. Finally, as a test of emergency granulopoiesis, we administered a single dose of G-CSF (250 μg/kg subcutaneously) or an equal volume of PBS, and 24 hr later measured the ANC, percentage of CD11b+/Gr1+ myeloid cells in the bone marrow, and CFU-GM generation. Mice of both genotypes responded to G-CSF stimulation with increases in ANC, percent of myeloid cells within the marrow, and CFU-GM. Bone marrow from βΔ/βΔ mice showed a larger G-CSF-induced increase in CFU-GM (PBS: 22 ± 5 v. G-CSF: 39 ± 1, p<0.05) compared to +/+ marrow (PBS: 24 ± 14 v. G-CSF: 31 ± 14, NS). Thus, Stat3β positively regulates basal granulopoiesis in the bone marrow, and may negatively regulate emergency granulopoiesis. This pattern is the opposite of that seen with deletion of both Stat3 isoforms, indicating that Stat3α’s function is to negatively regulate basal granulopoiesis and positively regulate emergency granulopoiesis. Stat3β also positively regulates circulating B lymphocyte numbers, via a mechanism other than B lymphocyte production in the bone marrow.

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Myeloid Progenitor Cells from Gadd45a and Gadd45b-Deficient Mice Are Sensitized to Genotoxic-Stress Induced Apoptosis.

Blood ◽

10.1182/blood.v104.11.4239.4239 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4239-4239

Author(s):

Mamta Gupta ◽

Shiv K. Gupta ◽

Arthur G. Balliet ◽

Barbara Hoffman ◽

Dan A. Lieberman

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Genotoxic Stress ◽

Wild Type ◽

Myeloid Progenitor ◽

Myeloid Progenitor Cells ◽

Induced Apoptosis ◽

Deficient Mice ◽

Marrow Cells

Abstract GADD45 (Growth arrest and DNA damge) regulates cell growth following exposure to diverse stimuli. It has been shown that, mice lacking the gadd45a gene exhibit genomic instability and increased carcinogenesis, but the exact role of the gadd45 family genes still remains unclear. In this study we have aimed at determining the effect of gadd45a or gadd45b deficiency on the response of bone marrow derived myeloid cells to genotoxic stress agents by using gadd45a or gadd45b null mice. We have found that myeloid progenitor cells from gadd45a or gadd45b-null mice are more sensitive to ultraviolet-radiation (UV), VP-16 or daunorubicin induced apoptosis. Introduction of wild-type gadd45 into gadd45-deficient bone marrow cells restored the wild-type apoptotic phenotype. In-vitro colony formation following stress responses has shown that bone marrow cells from gadd45a or gadd45b-deficient mice have a decreased ability to form haematopoetic colonies. Gadd45a or gadd45b-deficient bone marrow cells also displayed defective G2/M cell cycle checkpoint following exposure to either UV and V-16 but were still able to undergo G2/M arrest following exposure to daunorubicin, indicating the existence of different G2/M checkpoints in response to these anticancer agents. Taken together these findings identify gadd45a or gadd45b as anti-apoptotic gene(s), and suggests that the absence of gadd45a or gadd45b results in higher susceptibility of haematopoetic cells to UV radiation and certain anticancer drugs.

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ATRA and the RARα Specific Agonist, NRX195183, Have Opposing Effects on the In Vitro Clonogenicity of Pre-Leukemic Murine AML1-ETO Bone Marrow Cells

Blood ◽

10.1182/blood.v116.21.999.999 ◽

2010 ◽

Vol 116 (21) ◽

pp. 999-999

Author(s):

Lynette C.Y. Chee ◽

Jean Hendy ◽

Louise Purton ◽

Grant A. McArthur

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Myeloid Cells ◽

Myeloid Cell ◽

Sustained Remission ◽

Self Renewal ◽

Cells Cultured ◽

Marrow Cells

Abstract Abstract 999 All-trans retinoic acid (ATRA) is used successfully to treat acute promyelocytic leukemia (APML), however, to date it has not shown promise in treating other AML subtypes. ATRA has been shown to enhance hematopoietic stem cell (HSC) self-renewal (requiring RARγ activation) but promotes differentiation of myeloid progenitors likely through RARα activation. We hypothesized that (1) the lack of success of ATRA in treating other AML subtypes may be due to the potential ability of ATRA to enhance self-renewal of the leukemic stem cell and (2) the use of a specific RARα agonist may have more promise in enhancing AML differentiation. We therefore compared the effects of pharmacological levels (1μM) of ATRA and an RARα-specific agonist, NRX195183, on bone marrow cells harvested from a Cre-inducible conditional AML1-ETO (AE) knock-in murine model. AE cells cultured for 2 weeks with ATRA showed significant reductions in the proportions of mature myeloid cells (Gr1brightCD11b+) by fluorescence activated cell sorting (FACS) (DMSO: 14.2±4.3%, ATRA: 4.0±1.6%, p=0.04, n=4). By 4 weeks of culture, ATRA-treated AE cells had increased blast and reduced maturing myeloid cell proportions (Blasts %: DMSO 70.2 ± 3.0, ATRA 95.3 ± 1.2, p=0.08; Intermediate %: DMSO 14.3 ± 2.6, ATRA 3.8 ± 1.0, p=0.01; Neutrophils %: DMSO 2.3± 1.0, ATRA 0.3 ± 0.2, p=0.07, n=6). Furthermore, ATRA potentiated the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells: DMSO 505.8±337.0, ATRA 4394±388.9, p=0.001; n=6). In contrast, AE cells cultured for 2 weeks with NRX195183 showed significant increases in the proportions of mature myeloid cells by FACS (DMSO: 15.8±3.5%, NRX195183 26.7±3.0%, p=0.03; n=5). By 4 weeks of culture, NRX195183-treated AE cells had decreased blast and increased maturing myeloid cell proportions (Blasts %: DMSO 82.4±3.0, NRX195183 58.8±9.1, p=0.03; Intermediate %: DMSO 14.5±2.5, NRX195183 29.0±6.8, p=0.07; Neutrophils %: DMSO 1.6±0.8, NRX195183 8.2±4.7 p=ns; DMSO n=8, NRX195183 n=5). Moreover, NRX195183 reduced the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells DMSO 554.8±252.6, NRX195183 82.6±61.6, p=0.05; n=8). Short-term in vivo transplants of fetal liver cells overexpressing the truncated AE gene, AE9a, into sublethally irradiated recipients revealed similar findings in the NRX195183-treated mice with a decrease in blasts and an increase in mature neutrophils in the peripheral blood on morphological analysis after 4 weeks of treatment (Blasts x106/ml: DMSO 3.1±1.0, NRX195183 0.9±0.3, p=0.08; Neutrophils x106/ml: DMSO 0.5±0.1, NRX195183 0.8±0.1, p=0.04; DMSO n=16, NRX195183 n=11). Taken together, these findings support a model whereby ATRA promotes self-renewal of leukemic blasts whilst NRX195183 has the opposing effect. To understand the mechanism by which ATRA promotes self-renewal in AE cells, we performed genome-wide gene expression analyses on the ATRA- versus control-treated AE cells. This revealed 16 differentially upregulated genes after 24 hours of treatment. Using Ingenuity Pathway Analysis, the top scoring network in the ATRA-treated AE cells was cell-to-cell signalling and interaction (p=1.1E-7-2.4E-3), lipid metabolism (p=2.3E-7-2.0E-3) and small molecule biochemistry (p=2.3E-7-2.1E-3); SERPINE1 and BMP2 were the genes with the highest connectivity within the network interacting with molecules known for their roles in tumorigenesis, including AKT, NF-kβ complex and TGFβ1. SERPINE1 upregulation has been shown to be RARα-mediated whilst BMP2 has been shown to be a RARγ-regulated gene. Interestingly, the specific RARγ agonist, NRX204723, had no effect on the clonogenic potential of these AE progenitors thus raising the hypothesis that both RARα and RARγ activation are required to promote self-renewal of the AE progenitors. Further studies using both RARα/RARγ agonists are warranted to assess if the ATRA effects on AE cells are phenocopied. Collectively, these findings reveal the contrasting roles of specific RARα activation in promoting loss of self-renewal ability and enhancing differentiation in the AE cells whilst ATRA promotes clonogenicity of these cells. This has potential significant implications in AML treatment as specific RARα agonists may be beneficial in improving the efficacy of current treatment modalities to achieve sustained remission in other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

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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.

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Gadd45a and Gadd45b Protect Haematopoetic Cells from Ultraviolet Radiation Induced Apoptosis Via Distinct Signaling Pathways.

Blood ◽

10.1182/blood.v104.11.1258.1258 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1258-1258

Author(s):

Mamta Gupta ◽

Shiv K. Gupta ◽

Barbara Hoffman ◽

Dan A. Liebermann

Keyword(s):

Bone Marrow ◽

Ultraviolet Radiation ◽

Cell Survival ◽

Signaling Pathways ◽

Bone Marrow Cells ◽

Myeloid Cells ◽

Genotoxic Stress ◽

Induced Apoptosis ◽

Deficient Mice ◽

Marrow Cells

Abstract Gadd45 expression, which is stress inducible, has been associated with growth arrest, but the exact role of gadd45 family genes in apoptosis still remains unclear. We have found that myeloid progenitor cells from gadd45a and gadd45b-deficient mice are more sensitive to ultra-violet radiation, VP-16 or daunorubicin induced apoptosis. indicating that gadd45a or gadd45b protect haematopoetic cells from DNA damaging agents. To determine, how gadd45a or gadd45b proteins exert their anti-apoptotic function, bone marrow cells from wild-type and gadd45a or gadd45b deficient mice were exposed to ultraviolet radiation (UV) and analyzed for expression of stress responsive kinases, including JNK and p38. It was observed that P38 and JNK were activated in wt bone marrow cells in response to UV but not in bone marrow cells defecient in gadd45a. Also, the transcription factor NF-kB was activated in wt bone marrow cells, but not in gadd45a−/− cells. The pharmacological inhibitor SB203580 specific for p38, increased apoptosis in reponse to UV, indicating that p38 is implicated in signaling myeloid cell survival. SB203580 was observed also to inhibit the expression of certain NF-kB target genes, including cIAP-1, c-IAP-2, bcl-2 and bcl-xl, in gadd45a+/+ cells but not in gadd45a deficient bone marrow cells. Taken together this data provides first evidence for the role gadd45a plays in the control of hematopoietic cell survival in response to UV, via modulation of P38 MAPK and NF-kB signaling pathways. Unlike in gadd45a−/− bone marrow cells, p38 activation appeared not to be impaired in gadd45b−/− cells, indicating that gadd45b is not involved in p38 activation in myeloid cells. However, UV induced JNK activation was sustained in gadd45b−/− myeloid cells compared to wt cells, indicating that gadd45b is a negative modulator of UV induced JNK signaling in myeloid cells. UV induced activation of MKK4 an upstream regulator of JNK also was impaired in gadd45b−/−. NF-kB was also found activated in wt cells, but not in gadd45b−/− cells. This data indicates that in bone marrow cells exposed to UV, NF-kB induced expression of Gadd45b plays a protective role against UV induced apoptosis via inhibition of MKK4 kinase which in turn results in suppression of JNK activity. Taken together this data provides evidence that Gadd45a and Gadd45b protect haematopoetic cells from genotoxic-stress induced apoptosis via distinct signaling pathways.

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Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transplantation factor NGFI-A (EGR1).

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4566 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4566-4572 ◽

Cited By ~ 67

Author(s):

S L Lee ◽

Y Wang ◽

J Milbrandt

Keyword(s):

Bone Marrow ◽

Cell Differentiation ◽

Zinc Finger ◽

Bone Marrow Cells ◽

Myeloid Cell ◽

Hematopoietic Progenitor Cell ◽

Wild Type ◽

Granulocytic Differentiation ◽

Macrophage Differentiation ◽

Marrow Cells

The zinc finger protein NGFI-A (also called EGR1, Krox24, or zif268) is a candidate regulator of myeloid cell differentiation. Evidence supporting this hypothesis is twofold. First, NGFI-A antisense oligonucleotides prevent macrophage differentiation in HL-60 and U937 myeloid leukemia cell lines and in normal bone marrow cells. Second, enforced expression of NGFI-A blocks granulocytic differentiation and promotes macrophage differentiation in HL-60 cells and in the hematopoietic progenitor cell line 32D. We sought to determine the effect of NGFI-A deficiency on macrophage differentiation and function in vivo by examining native bone marrow cells from mice homozygous for a disrupted allele of NGFI-A derived from gene-targeted ES cells. Macrophages were observed in peripheral blood and several tissues, indicating that NGFI-A was not required for the formation of a variety of macrophage compartments. No differences in myeloid cell differentiation were observed between wild-type and NGFI-A-/- bone marrow cells cultured in the presence of macrophage, granulocyte-macrophage, or granulocyte colony-stimulating factor (M-CSF, GM-CSF, or G-CSF). Activation of NGFI-A-/- macrophages was comparable to that of wild-type macrophages as determined by nitric oxide production and increased cell surface expression of class II major histocompatibility complex molecules. Moreover, NGFI-A-/- mice showed no increased mortality or bacteria] burden when challenged with Listeria monocytogenes. Together, these results indicate that NGFI-A is not required for macrophage differentiation or activation.

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Expression of V617F JAK2 in Mice Leads to MPD Mimicking Human ET, Idiopahtic Myelofibrosis, and PV.

Blood ◽

10.1182/blood.v110.11.2531.2531 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2531-2531

Author(s):

Kotaro Shide ◽

Haruko K. Shimoda ◽

Takashi Kumano ◽

Kennosuke Karube ◽

Takuro Kameda ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Myeloid Cell ◽

Jak2 V617f ◽

Myeloproliferative Disorders ◽

Single Mutation ◽

Wild Type ◽

Idiopathic Myelofibrosis ◽

Marrow Cells

Abstract An acquired JAK2 V617F mutation has been detected in up to 90% of patients with polycythemia vera (PV) and in a sizeable proportion of patients with other myeloproliferative disorders such as essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). However, how a single mutation may be responsible for such different clinical phenotypes is unknown. Mice transplanted with bone marrow cells in which V617F JAK2 was retrovirally expressed developed PV-like features, but not ET or IMF. To address the contribution of this mutation to the pathogenesis of these three MPDs, we generated transgenic mice expressing V617F JAK2 driven by the murine H2Kb promoter. We established two lines. The expression of V617F JAK2 mRNA in bone marrow cells was 0.45 and 1.35 that of endogenous wild-type JAK2 in the two lines. One line showed leukocytosis after 4 months of age, with a predominance of granulocytes. Among 43 mice, examined after 3 months of age, 8 (19%) showed polycythemia and 14 (33%) showed thrombocythemia. Two polycythemia cases also showed thromobocytosis. The other line showed extreme leukocytosis and thromobocytosis at one month of age. The leukocytosis progressed as the animals aged, but the thrombocytosis tended to resolve at 8 months. They showed anemia that means Hb value from 9 to 10 g/dL at one month old. Myeloid cells and megakaryocytes were predominant in the bone marrow of these animals, and splenomegaly with myeloid cell and megakaryocyte invasion was observed. We conclude that in vivo expression of V617F JAK2 results in ET-like, IMF-like, and PV-like disease.

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VEGF and AMD3100-Induced Rapid Mobilization of C-Kit/Sca-1 Positive Murine Bone Marrow Cells and of Gr-1+/CD-11b+ Myeloid Cells Is Impaired in Urokinase (uPA) and Urokinase Receptor (uPAR) Deficient Mice.

Blood ◽

10.1182/blood.v112.11.1384.1384 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1384-1384

Author(s):

Mohammad Dehghani ◽

Damla Olcaydu ◽

Pavel Uhrin ◽

Bernd Binder ◽

Johannes Breuss

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Myeloid Cells ◽

Urokinase Receptor ◽

Facs Analysis ◽

Β1 Integrins ◽

Deficient Mice ◽

Marrow Cells

Abstract Hematopoietic Progenitor Cells (HPC) can be mobilized from bone marrow into the circulation in response to a number of stimuli including G-CSF, AMD3100 (antagonist of CXCR4-DSF-1 axis) and vascular endothelial growth factor (VEGF). The main mechanism for mobilization of HPCs upon stimulation by classical “mobilizers”as G-CSF is thought to be through extracellular matrix proteolysis in the marrow. Urokinase is a serine protease present in the marrow and contributes to mobilization of stem cells upon binding to its receptor (uPAR) and activating plasminogen that leads to matrix degradation. Our previous data show that the effect of VEGF on endothelial cell migration is exerted through activation of the uPA/uPAR system and through co-internalization of β 1 integrins. Upon internalization of these receptors, cells detach from their underlying extra-cellular matrix (ECM) as well as from stromal cells. We hypothesize that the contribution of VEGF to HPC mobilization occurs through a similar mechanism. We also want to analyze the influence of uPA/uPAR deficiencies on mobilization of Gr-1+/CD-11b+ myeloid and c-kit +/Sca-1+ (SK)cells by VEGF and AMD3100 and compare it with G-CSF as a classical “mobilizer”. Wild type, uPA knockout and uPAR knockout mice in C57BL6 background were used for in vivo experiments. We collected peripheral blood before and 2 hours after i.p. injection of VEGF-E and AMD3100 and assessed the number of SK cells and myeloid cells by FACS analysis. We also administered G-CSF for 5 days and compared blood samples before and after the experiment. To evaluate the effect of VEGF on HPC integrin expression, femurs of the respective animals were incubated with VEGF in an ex vivo experimental model and β1 expression was assessed by FACS analysis. In vivo data demonstrated a significantly reduced responsiveness of uPA−/− mice to VEGF-E in the first 2 hours after the injection. This decreased responsiveness to VEGFis observed in uPAR−/− mice but to a lesser degree than in uPA−/− mice..(40 +/−16 % and21 +/− 20% respectively vs 65 +/− 24 % in wt, means and SD). Injection of urokinase together with VEGF to uPA−/− mice rescues the lack of mobilization of SK cells. Ex vivo stimulation of uPAR knockout femoral bone marrow cells with VEGF for 20 minutes provides evidence that the internalization of β1 integrins upon VEGF stimulation is uPAR dependent. VEGF can also increase in vivo the number of Gr-1+/CD-11b+ myeloid cells after 2 hours in wt mice (96 +/− 45%) but not in urokinase deficient or urokinase receptor deficient mice (7 +/− 11% and 21+/−33%, respectively). AMD3100 has a strong effect on mobilization of SK cells in wt animals within 2 hours (increase of 2.8+/−0.78 times) but cannot mobilize these cells in uPA and uPAR deficient mice to the same extend (0.8+/−0.65 times and 0.1+/−0.07 respectively). G-CSF injection for 5 days mobilizes Gr-1+/CD-11b+and SK cells in wt and knock out mice to a similar extent, indicating that the capacity to release these cells from the bone marrow is not affected by uPA or uPAR gene deficiency. Our results demonstrate a reduced mobilization of uPA−/− and uPAR−/− HPCs and myeloid cells in response to VEGF compared to wt mice. VEGF leads to internalization of the expression of β1 integrins on the surface of SK cells in wt but not in uPAR−/− mice. In addition, we could show that the uPA/uPAR system plays a role in AMD3100-dependent mobilization of these cells. These data indicate that the uPA – uPAR system plays a pivotal role in short-term but not long-term bone marrow HPC and PMN leukocyte mobilization.

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

Blood ◽

10.1182/blood.v114.22.3643.3643 ◽

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.

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Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

Biochemical Journal ◽

10.1042/bj3310733 ◽

1998 ◽

Vol 331 (3) ◽

pp. 733-742 ◽

Cited By ~ 8

Author(s):

Masafumi YOSHIMURA ◽

Yoshito IHARA ◽

Tetsuo NISHIURA ◽

Yu OKAJIMA ◽

Megumu OGAWA ◽

...

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

Stromal Cells ◽

Bone Marrow Cells ◽

Adherent Cell ◽

Wild Type ◽

Spleen Cells ◽

Bisecting Glcnac ◽

Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

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