Reduced C/EBPα Expression Favors Monopoiesis Over Granulopoiesis

Abstract Abstract 1230 C/EBPα is required for the formation of the granulocyte-monocyte progenitor; however, its role in subsequent lineage specification remains uncertain. We previously compared G-CSF with M-CSF signaling in murine marrow, finding that G-CSF more potently induces phosphorylation of SHP2 whereas M-CSF more potently activates ERK. Knockdown (KD) of SHP2 impaired granulopoiesis relative to monopoiesis and unexpectedly led to reduced C/EBPα RNA. A. Cantor and colleagues recently reported that SHP2 dephosphorylates Src kinase-modified RUNX1 and increases the ability of RUNX1 to induce megakaryopoiesis, and we demonstrated that in myeloid cells RUNX1 binds and activates the CEBPA gene, via its promoter and via a conserved 450 bp enhancer located at +37 kb containing 4 RUNX1-binding sites. Thus, G-CSF or other cytokines potentially direct SHP2 to activate RUNX1 which then increases C/EBPα transcription in myeloid progenitors to favor granulopoiesis. As a test of this model, we have now transduced lineage-depleted murine marrow cells, cultured in SCF, TPO, and Flt3L to minimize myeloid differentiation, with shRNAs targeting CEBPA or with a control lentiviral vector. Western blotting indicated 2-fold KD by shRNAs B9 or B11 after 2 days of transduction followed by 2 days of puromycin selection. As this analysis was done on a mixed population, greater KD may occur in rare progenitors. Upon transfer to IL3, IL6, and SCF to favor myeloid maturation, vector-transduced cells produced 4-fold more CFU-G than CFU-M in methylcellulose, whereas CEBPA KD resulted in 4- or 5-fold more CFU-M than CFU-G. FACS analysis of pooled CFUs demonstrated significant reduction of Mac-1+Gr-1+ granulocytes and an increase in Mac-1+Gr-1− monocytes, and examination of cell morphology after Wright's-Giemsa staining further confirmed a shift towards monopoiesis in response to reduced C/EBPα. Similar findings were obtained for cells placed in liquid culture. In addition, on day 3 of liquid culture Q-RTPCR analysis showed that CEBPA shRNAs induced 2- or 3-fold CEBPA RNA KD, reduced granulocytic MPO, NE, PR3, and GCSFR RNAs, increased monocytic MCSFR and CD14 RNAs, and increased expression of EGR1, IRF8, and c-Jun RNAs, encoding transcription factors that help direct monopoiesis. RUNX1 RNA was unchanged, and PU.1 RNA was reduced <2-fold. To determine the effect of CEBPA KD in vivo, transduced, puromycin-selected CD45.2+ marrow cells were transplanted into lethally irradiated, syngeneic CD45.1+ recipients together with CD45.1+ carrier cells. At day 28, FACS analysis indicated a shift towards monopoiesis amongst shRNA B9- or B11-transduced marrow cells, and CFU assay of sorted CD45.2+ cells demonstrated significantly reduced CFU-G with increased CFU-M. Besides affecting myeloid lineage determination, CEBPA KD increased the rate of cell proliferation in liquid culture, associated with blasts evident upon morphologic analysis and an increase in Mac-1−Gr-1− cells, which mainly consisted of c-Kit+Sca-1− myeloid progenitors. In addition, CEBPA KD by shRNA B9, which impaired granulopoiesis more effectively than shRNA B11, increased CFU replating to 8 generations, compared with 4 generations for control cells. Analysis on day 3 of liquid culture did not reveal an alteration in the proportion of cells in G1, S, or G2/M or increased apoptosis, and c-Myc and Bcl-2 RNA levels were unaffected. We propose that CEBPA KD not only strongly impairs granulopoiesis, but also modestly impedes monopoiesis to produce a transient accumulation of cells with blast morphology and the ability to proliferate. Overall, our findings suggest that relative to monopoiesis, granulopoiesis requires increased level or increased activity of C/EBPα. Offering a potential mechanism, we previously found that C/EBPα :AP-1 leucine zipper heterodimers are more abundant than C/EBPα homodimers during monopoiesis compared with granulopoiesis, in part due to c-Fos, c-Jun, and JunB induction, and that C/EBPα :AP-1 heterodimers can direct marrow monopoiesis. Perhaps higher levels of C/EBPα favor homodimer formation, with reduced levels still able to heterodimerize with AP-1 proteins. ERK modification of C/EBPα on S21, as occurs downstream of M-CSF, reduces its ability to direct granulopoiesis; phospho-C/EBPα (S21) potentially retains the ability to direct AP-1 proteins to hybrid DNA elements recently identified in numerous monocytic enhancers. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.2504.2504 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2504-2504

Author(s):

Russell Garrett ◽

Gerd Bungartz ◽

Alevtina Domashenko ◽

Stephen G. Emerson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Poly I:C ◽

Hematopoietic Stem ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Canonical NF-κB Signalling Is a Potential Target in FLT3/ITD AML.

Blood ◽

10.1182/blood.v120.21.2447.2447 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2447-2447 ◽

Cited By ~ 2

Author(s):

Jing Zhang ◽

Li Li ◽

Alan D. Friedman ◽

Donald Small ◽

Ido Paz-Priel

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Myeloproliferative Neoplasm ◽

Initial Response ◽

Wild Type ◽

Normal Numbers ◽

Toxic Dose ◽

Marrow Cells

Abstract Abstract 2447 Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) receptor is common in acute myeloid leukemia (AML) and is associated with a dismal outcome. Despite initial response, FLT3/ITD AMLs often relapse early, suggesting a residual population of resistant leukemia stem cells (LSC). Clinically, FLT3 inhibitors asmonotherapy have yet to improve outcome significantly and therefore, targeting additional pro-survival pathways may be necessary for this group of AML patients. Mice genetically egineered to express a hemizygous FLT3/ITD mutation develop a progressive, fatal, myeloproliferative neoplasm. Lin−cells isolated from the bone marrow of FLT3/ITD or control mice were subjected to gel shift analysis using a radio-labeled NF-kB binding site. This analysis demonstrated high levels of nuclear activation of NF-kB in the FLT3/ITD-expressing cells, suggesting its activation downstream of mutant FLT3 signaling. MV4–11 is a human AML-derived cell line harboring a homozygous FLT3/ITD mutation. Cells expressing high levels of aldehyde dehydrogenase (ALDH) have been shown to be enriched for LSC in primary AML samples and cell lines. High ALDH expressing MV4–11 cells were isolated using FACS and analyzed for NF-kB activation. Western blot analysis demonstrated preferential phosphorylation of NF-kB p65 by activated IKK on Ser536 in this subpopulation, compared with cells with low ALDH activity. These findings indicate activation of NF-kB in MV4–11 LSCs. We wanted to next test the requirement for NF-kB signaling in transformation by FLT3/ITD mutations. NF-kB p65 null mice die in utero. We therefore established C57BL/6 p65(flox/flox);Mx1-Cre mice. Intra-peritoneal injection of pIpC every other day for 7 doses efficiently deletes the RelA/p65 gene, resulting in expression of <1% of the corresponding RNA or protein. Despite effective excision of p65, the mice survive. Bone marrow cells harvested from control or p65(del/del) mice were transduced with a FLT3/ITD-expressing lentivirus and seeded in methylcellulose without cytokines. Equal transduction rate was verified by measurement of GFP expression by flow cytometry. Reproducibly, p65(del/del) marrow transduced with FLT3/ITD was ineffective in forming cytokine independent colonies, in contrast to wild-type marrow (5 +/− 0.6 vs. 55 +/− 6 colonies per 1E5 cells, P<0.001), and the few p65(del/del) colonies that resulted were smaller than those from p65 expressing wild-type marrow cells. Cells transduced with a lentiviral vector expressing GFP but not FLT3/ITD did not form colonies without cytokines, and p65(del/del) marrow formed normal numbers of colonies of normal size and distribution in the presence of IL-3, IL-6, and SCF. Sorafenib inhibits FLT3 signaling and kills MV4–11 cells with an IC50 of approximately 10 nM. Reproducibly, a sub-toxic dose of sorafenib (5 nM) combined with sub-toxic levels of the IKKb inhibitor IMD-0354 (400 nM) resulted in synergistic cell killing as indicated by the calculated combination index of 0.55. Currently, clinical efforts in FLT3/ITD leukemia concentrate on FLT3 inhibition alone. Our data suggest that canonical NF-kB may be an important pathway in FLT3/ITD AML and that simultaneously targeting FLT3 and NF-kB in this disease may be an effective approach. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Divisional Memory Drives Hematopoietic Stem Cell Functional Diversity

Blood ◽

10.1182/blood-2021-153296 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 20-20

Author(s):

James Bartram ◽

Baobao (Annie) Song ◽

Juying Xu ◽

Nathan Salomonis ◽

H. Leighton Grimes ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Functional Decline ◽

Functional Heterogeneity ◽

Hematopoietic Stem ◽

Donor Cells ◽

Marrow Cells

Abstract Hematopoietic stem cells are endowed with high regenerative potential but their actual self-renewal capacity is limited. Studies using the H2B-retention labeling system show HSC functional decline at each round of division (Qiu, Stem Cell Reports 2014). We have shown that mitochondria drive HSC functional decline with division history after transplantation (Cell Stem Cell 2020). Here we examined the link between mitochondrial metabolism, in vivo division at steady state, and HSC functions using the GFP label-Histone 2B (GFP-H2B) mouse model driven by a doxycycline-inducible promoter. Five months after doxycycline removal, mitochondrial membrane potential (MMP) was examined using TMRE in HSC with varying GFP intensity. HSC were separated into an H2B-labeled retention population and an H2B-labeled population. Interestingly, within the H2B-labeled retention population, HSC could be further subdivided into GFP high, medium, and low. MMP increased in a stepwise fashion with GFP dilution in HSC. We noted the presence of 2 TMRE peaks within each GFP high and medium populations leading to 5 populations: GFP-high;MMP-low (G1), GFP-high;MMP-high (G2), GFP-medium;MMP-low (G3), GFP-medium;MMP-high (G4), GFP-low;MMP-high (G5). We examined the repopulation activity of each population in a serial competitive transplant assay. G1 and G2 maintained higher peripheral blood chimerism up to 24 weeks post-transplant than G3 and G4. G5 did not engraft at all. However, only G1 reconstituted high frequency of HSC in primary recipients. In secondary recipients, G1, G2, G3 but not G4 gave rise to positive engraftment. Interestingly, G1 and G2 grafts showed myeloid/lymphoid balanced engraftment whereas the G3 graft was myeloid-bias, suggesting that myeloid skewing can be acquired upon HSC division. We further examined lineage fate maps of bone marrow cells derived from G1 or G3 population in vivo, using single cell RNA sequencing, 10X genomics. Surprisingly, G3-derived bone marrow cells displayed a distinct myeloid cell trajectory from G1-derived bone marrow cells, in which G3 gave rise to increased immature neutrophils but fewer myeloid precursors. Remarkably, each lineage population derived from G3 donor cells had different gene expression signatures than those derived from G1 donor cells. Therefore, HSC that have divided in vivo in the same bone marrow microenvironment are intrinsically and molecularly different such that not only do they exhibit lineage potential differences but they also produce progeny that are transcriptionally different. These findings imply that cellular division rewires HSC and that this rewiring is passed down to their fully differentiated progeny. When G1 and G3 single HSC were cultured in-vitro, G1 had a slower entry into cell-cycle which has been associated with increased stemness. Additionally, when single HSC from G1 and G3 were assessed for their multipotency in a lineage differentiation assay, G1 HSC had a higher propensity to produce all four myeloid lineages (megakaryocytes, neutrophils, macrophages, and erythroid), further supporting increased stemness in G1 compared to G3 HSC. Finally, HSC from G1, G2, G3 and G4 populations carried mitochondria that were morphologically different, and express distinct levels of Sca-1, CD34 and EPCR, with Sca-1 high, CD34-, EPCR+ cells more enriched in G1. In summary, this study suggests that HSC transition into distinct metabolic and functional states with division history that may contribute to HSC diversity and functional heterogeneity. It also suggests the existence of a cell-autonomous mechanism that confers HSC divisional memory to actively drive HSC functional heterogeneity and aging. Disclosures No relevant conflicts of interest to declare.

Download Full-text

A Novel RHCE*ce Variant Allele Is Highly Frequent in Chinese D-Negative Individuals and Results in C-Expression

Blood ◽

10.1182/blood.v124.21.2892.2892 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2892-2892

Author(s):

Tamara C Stegmann ◽

Y L Ji ◽

Lonneke Haer-Wigman ◽

L Wei ◽

Y Zhao ◽

...

Keyword(s):

Conflicts Of Interest ◽

Lentiviral Vector ◽

Variant Allele ◽

The Novel ◽

Facs Analysis ◽

Exon 2 ◽

Chinese Populations ◽

New Variant ◽

Novel Variant ◽

Dependent Probe

Abstract Background: The RHCE allele is highly polymorphic and many variants have been described, especially in individuals of African origin. Donors carrying these variants can be falsely typed and elicit transfusion reactions, and patients carrying such a variant may be at risk to develop allo-antibodies in response to mismatched transfusions. Not much is known about the frequency of RHCE variants in Chinese populations, whereas in China genotyping assays are increasingly applied for typing of blood donors and patients. Methods: Standard column agglutination was used to serologically type for C/c and E/e expression in 200 serologically D-negative and 200 serologically D-positive Chinese donors. The RH Multiplex Ligation-dependent Probe Amplification (MLPA) genotyping assay was used for genotyping the RHCE status (Transfusion 2013;53:1559). In donors with discrepant results of genotyping and phenotyping all 10 exons of RHCE were amplified and directly sequenced. A lentivirus containing the novel RHce variant was created to transduce human erythroblasts cultured from peripheral blood from 3 ccDee and 3 CCDee donors as previously described (Haematologica 2010;95:1594). FACS analysis was used to assess the c- and C-expression caused by the variant. Results: In 6 out of 200 Chinese D-negative donors the results of the RH-MLPA indicated the presence of only one copy of exon 2 of the CE*c-allele, and no copy of exon 2 of the CE*C-allele, whereas these donors were serologically typed as Cc. Sequencing of all 10 RHCE exons revealed a novel RHCE*ce allele defined by 308C>T (p.103Pro>Leu) mutation next to a normal RHCE*ce allele. The variant allele was not found in the 200 Chinese D-positive donors, indicating the linkage of this new variant RHCE*ce allele with the D-negative haplotype. Wild type Rhce cDNA was mutated to create the RHce*308C>T mutation and subsequently cloned into a lentiviral vector. Transduction of human ccDee erythroblasts with this vector resulted in C expression, whereas virtually no c-expression was induced by transduction of human CCDee erythroblasts as assessed by FACS analysis with the monoclonals MS33, MS35 and MS42 to detect c expression and monoclonals MS24 and MS273 to detect C. Discussion: A new RHCE variant (RHCE*ce308T) is identified, which is present in 3% of D-negative Chinese individuals. The 308C>T mutation in the triplet encoding the 103Pro results in the expression of 103Leu. Position 103 is one of the 4 aminoacid differences between the c- and C-carrying polypeptides (Pro and Ser, respectively). The Pro>Leu mutation in the novel variant leads to C-expression and loss or strongly diminished c-expression. Most Rhc-genotyping assays target the c-specific-307C nucleotide and most RhC-genotyping assays target the C-specific-intron 2, which are respectively present and absent in this variant allele. Therefore, when individuals carrying this allele are genotyped, the predictive phenotype will be falsely C-negative. Conclusion: RHC-genotyping assays applied in Chinese populations, should be adapted to recognize the presence of this new RHCE*ce308T allele to prevent C-mismatched transfusions. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Retroviral Mediated Transfer of MLL Fusion Genes into Human CD34+ Cord Blood Cells Supports the Establishment of Long-Term Myeloid Cultures with Growth Rates, Immunophenotypic and Transcriptional Features Distinct from Those of AML1-ETO Transduced Cultures.

Blood ◽

10.1182/blood.v106.11.1610.1610 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1610-1610

Author(s):

Jorge F. DiMartino ◽

Catherine Rettig ◽

Mark Wunderlich ◽

James C. Mulloy

Keyword(s):

Transcription Factors ◽

Cord Blood ◽

Leucine Zipper ◽

Antigen Expression ◽

Fusion Genes ◽

Human Cord Blood ◽

Myeloid Progenitors

Abstract Translocations involving the MLL gene, primarily t(9;11) and t(10;11) together with rearrangements affecting the core binding factor (CBF) genes, t(8;21) and inv(16) comprise the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). Although all of these rearrangements generate chimeric transcription factors (MLL-AF9, MLL-AF10, AML1-ETO and CBFβ-MYH11) clinicopathologic features and transcriptional profiles clearly distinguish MLL-rearranged from CBF-rearranged AML. To understand how these distinct subgroups of AML arise, we have developed a model for studying the effects of MLL and CBF fusion proteins on the growth, survival and differentiation human myeloid progenitors in vitro. Using retroviral mediated gene transfer, we transduced CD34 selected normal human cord blood (CB) cells with vector (MIEG3) alone or with vectors expressing MLL-AF9, MLL-AF10 or AML1-ETO fusion genes. Whereas CB transduced with MIEG3 proliferated in liquid culture for 6 to 8 weeks, MLL-AF9, MLL-AF10 and AML1-ETO transduced cells have continued to proliferate continuously in culture for more than 16 weeks without any sign of crisis. At any point after transduction, CB expressing MLL-AF9 or MLL-AF10 exhibited a faster rate of growth as compared with AML1-ETO or MIEG3 transduced CB. This difference in growth rate was associated with a reduced frequency of spontaneous apoptosis by annexin staining in the MLL cultures, as compared with the AML1-ETO cultures, but no difference in the fraction of cells in S-phase. MLL-AF9 and MLL-AF10 transduced CB also exhibited evidence of early myeloid maturation arrest based on morphology and surface antigen expression. However, while AML1-ETO transduced cells continue to express CD34 throughout their time in culture, MLL cultures lose expression of this stem cell-associated antigen and acquire expression of c-Kit and CD33, neither of which is expressed in AML1-ETO cultures. Also, unlike AML1-ETO transduced cells, CB transduced with with MLL fusions retain serial clonogenicity for 3 or more rounds of plating in methylcellulose assays. We used quantitative realtime RT-PCR to measure expression of 3 genes that are differentially expressed in patients with MLL or CBF gene fusions based on published microarray data. While expression of SPARC increased over time in MIEG3 cultures or remained stable in CB transduced with AML1-ETO, it decreased to nearly undetectable levels in MLL-AF9 transduced cultures. In contrast, expression of both BMI-1 and HOXA9 increased in the MLL-AF9 cultures and decreased in the MIEG3 and AML1-ETO cultures. The transcriptional changes in our long-term cultures mirror the gene expression differences that have been observed in AML associated with MLL or CBF fusions and suggest that this will be a useful model to study how chimeric transcription factors contribute to myeloid leukemogenesis. Interestingly, CB transduced with a mutated MLL-AF10 (MA10ΔLZ) lacking the leucine zipper domain required for transformation of primary murine myeloid progenitors did not differ, in terms of growth or differentation, from MIEG3 transduced cells. This suggests that the effects of MLL-AF9 and MLL-AF10 on normal CB may reflect early events in myeloid leukemogenesis. The in vivo leukemogenic potential of MLL fusion transduced CB is currently being evaluated in NOD/SCID mice.

Download Full-text

C/EBPα:AP-1 Leucine Zipper Heterodimers Bind Novel DNA Elements, Activate the PU.1 Promoter, and Direct Monocyte Lineage Commitment More Potently Than C/EBPα Homodimers or AP-1.

Blood ◽

10.1182/blood.v110.11.1232.1232 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1232-1232

Author(s):

Alan D. Friedman ◽

Dong Hong Cai ◽

Dehua Wang ◽

Jeffrey Keefer ◽

Christine Yeamans ◽

...

Keyword(s):

Leucine Zipper ◽

Biological Effects ◽

Myeloid Cell ◽

Salt Bridge ◽

Lineage Commitment ◽

Myeloid Cell Line ◽

Dna Elements ◽

Endogenous Proteins ◽

Bzip Proteins ◽

Myeloid Progenitors

Abstract The basic-region leucine zipper (BR-LZ or bZIP) transcription factors dimerize via their LZ domains to position the adjacent BRs for DNA-binding. Members of the C/EBP, AP-1, and CREB/ATF bZIP subfamilies form homodimeric or heterodimeric complexes with other members of the same subset and bind specific DNA motifs. Here we demonstrate that C/EBPα also zippers with AP-1 proteins and that this interaction allows contact with novel DNA elements and induction of monocyte lineage commitment in myeloid progenitors. A leucine zipper swap:gel shift assay demonstrates that C/EBPα zippers with c-Jun, JunB, or c-Fos, but not with c-Maf or MafB. In addition, C/EBPα co-ips c-Jun, JunB, or c-Fos, either expressed in 293T cells or as endogenous proteins in a myeloid cell line. Affinity of C/EBPα for AP-1 proteins appears weaker in this assay than affinity of C/EBPα for the related C/EBPβ, suggesting that interaction of C/EBP and AP-1 proteins is not primarily cross-inhibitory but instead generates heterodimers capable of binding novel DNA elements and inducing unique biological effects. To evaluate activities of specific homodimers or heterodimers we utilized LZs with acid (LZE) or basic (LZK) residues in their salt bridge positions. This approach is advantageous over efforts to fuse bZIP proteins together as zippering of tethered proteins may be sterically hindered and disruptive to the remainder of the protein. We find that C/EBPαLZE:C/EBPαLZK preferentially binds a C/EBP site, c-JunLZE:c-FosLZK an AP-1 site, and C/EBPαLZE:c-JunLZK a hybrid element identified as 5′-TTGCGTCAT by oligonucleotide selection. In contrast, c-Jun homodimers selected the consensus site 5′-TGACGTCAT, and C/EBPα homodimers selected 5′-NNGNNGCAAC. Note that 5′-TTGC in the selected hybrid site corresponds to 5′-GCAA in the C/EBPα site. In lineage-negative murine myeloid progenitors, either C/EBPα-ER or c-Jun-ER potently induce monocyte as compared to granulocyte development. Using LZE:LZK proteins linked to the ER ligand-binding domain, we find that C/EBPα:c-Jun or C/EBPα:c-Fos LZE:LZK heterodimers induce monocyte lineage commitment with markedly increased potency compared with C/EBPα or c-Jun homodimers or c-Jun:c-Fos heterodimers, demonstrating a positive functional consequence of C/EBP:AP-1 bZIP subfamily interaction. We propose that C/EBPα induction of monopoiesis requires zippering with endogenous c-Jun and that conversely induction of monopoiesis by c-Jun requires zippering with endogenous C/EBPα. Finally, using Ba/F3 lines stably expressing LZE:LZK ER fusion proteins, we find that C/EBPα:c-Jun binds the endogenous PU.1 promoter as assessed by chromatin immunoprecipitation and that exposure of cells expressing C/EBPαLZK-ER and c-JunLZE-ER to estradiol induces PU.1 mRNA expression as assessed by quantitative RT-PCR, whereas C/EBPαLZK-ER in combination with C/EBPαLZE-ER did not induce PU.1 mRNA in Ba/F3 cells. In addition, both endogenous C/EBPα and c-Jun were detected by the ChIP assay in the vicinity of the PU.1 promoter in 32Dcl3 myeloid cells. Thus, induction of PU.1 may contribute to stimulation of monopoiesis by C/EBPα:c-Jun heterodimers. In addition, as C/EBP and AP-1 proteins are widely expressed, zippering between these bZIP subfamilies may have broad biologic relevance. To gain insight into the spectrum of genes activated specifically by this complex, we have initiated microarray studies using our Ba/F3 cell lines.

Download Full-text

C/EBPα:C/EBPα and C/EBPα:Jun Leucine Zipper Complexes Are Detectable in Myeloid Cells Via Binding to Distinct DNA Elements.

Blood ◽

10.1182/blood.v114.22.1458.1458 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1458-1458

Author(s):

SunHwa Hong ◽

Alan D. Friedman

Keyword(s):

Cell Lines ◽

Leucine Zipper ◽

Conflicts Of Interest ◽

Myeloid Cells ◽

Myeloid Cell ◽

Monocytic Differentiation ◽

New Findings ◽

Dna Elements ◽

Gel Shift

Abstract Abstract 1458 Poster Board I-481 Mice lacking C/EBPα have reduced granulocyte-monocyte progenitors, and C/EBPα plays a role in the maturation of GMP along the granulocyte or monocyte lineages. C/EBPα is a member of the bZIP family of transcription factors, dimerizing with itself or other C/EBP proteins via its leucine zipper (LZ) domain to bind palindromic DNA elements (e.g. ATTGCGCAAT, designated αα sites) via the adjacent basic region (BR). AP-1 proteins such as c-Fos and c-Jun represent a distinct bZIP subfamily that heterodimerize amongst themselves to bind AP-1 sites (e.g. TGACTCA, designated JJ sites) to also mediate monocytic differentiation in hematopoietic cell lines or transduced marrow cells. In addition, we previously demonstrated that C/EBPα or C/EBPβ can zipper with c-Jun, JunB, or c-Fos (Cai et al 2008). To accomplish this we employed hybrid proteins in which, for example, the c-Jun LZ was swapped in place of the C/EBPα LZ and binding to a C/EBP site was then assessed after expression in 293T cells. We also provided data suggesting that C/EBPα:c-Jun heterodimers bind preferentially to hybrid αJ sites (e.g. TGACGCAAT). To accomplish this we forced heterodimerization by replacing the LZ in C/EBPα with an acidic zipper containing multiple glutamic acid residues (LZE) and replaced the LZ in c-Jun with a basic zipper containing multiple lysines (LZK). We have now extended these initial findings in two ways. First, we demonstrate that the intact proteins, not only those containing artificial acid or basic leucine zippers, maintain binding site specificity. In particular, when expressed in 293T cells or when generated by coupled in vitro transcription/translation, C/EBPα alone preferentially binds an αα site whereas C/EBPα co-expressed with c-Jun prefers the αJ site. Neither C/EBPα alone nor C/EBPα:c-Jun bound a JJ site. Consistent with the weak affinity of C/EBPα for c-Jun, formation of sufficient C/EBPα:c-Jun heterodimers to allow detection of preferential binding to the αJ site required a 4:1 c-Jun:C/EBPα ratio, with input proteins normalized via a shared N-terminal myc tag. Super-shift assays confirmed that a single gel shift band contains both proteins under these conditions. Co-expression of c-Jun with c-Fos yielded complexes that bound JJ sites, but interestingly c-Jun:c-Fos bound αJ sites with even greater affinity. Second, we have used biotinylated αα and αJ oligonucleotides to demonstrate the presence of both endogenous C/EBPα:C/EBPα and C/EBPα:c-Jun complexes in myeloid cell lines. When 32Dcl3 cells are placed in G-CSF for 24 hrs to stimulate granulopoiesis, Western blotting indicates that C/EBPα levels are induced while c-Jun levels remain constant, and binding of C/EBPα to the αα biotin-oligo increases, likely reflecting the presence of endogenous C:EBPα homodimers, as no interaction of c-Jun with the αα probe was detected. Both endogenous C/EBPα and c-Jun interact the αJ biotin-oligo, suggesting binding by an endogenous C/EBPα:c-Jun complex, and binding of each of these proteins to this probe also increases upon G-CSF induction of C/EBPα. Further support for the presence of an endogenous C/EBPα:c-Jun complex comes from the observation that interaction of C/EBPα with the αJ oligo is several-fold stronger than with the αα oligo, before or after G-CSF addition, whereas gel shift assay results described above indicate that C/EBPα homodimers prefer the αα probe. We previously found that C/EBPαLZK:C/EBPαLZE homodimers favor granulopoiesis whereas C/EBPαLZK:c-JunLZE heterodimers favor monopoiesis of marrow progenitors (Cai et al 2008). In summary, new data indicate that when c-Jun is present in excess, co-expressed C/EBPα and c-Jun preferentially binds hybrid αJ site, and C/EBPα:c-Jun complexes are detected endogenously in myeloid cells. These new findings provide biochemical support for the idea that C/EBPα:AP-1 heterodimers bind novel DNA elements to help mediate monopoiesis, with C/EBPα:C/EBPα homodimers potentially favoring granulopoiesis. Our future focus will be to further define the cellular levels of endogenous C/EBP, AP-1, and C/EBP:AP-1 complexes in immature and mature monocytic and granulocytic cells and to elucidate the spectrum of genes these complexes bind and regulate during myelopoiesis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Inhibition of Let-7 Processing in Adult Murine Hematopoietic Stem Cells Induces a Fetal-Like High Self-Renewal Pattern in Their Progeny

Blood ◽

10.1182/blood.v118.21.45.45 ◽

2011 ◽

Vol 118 (21) ◽

pp. 45-45 ◽

Cited By ~ 1

Author(s):

Michael R. Copley ◽

David G. Kent ◽

Claudia Benz ◽

Stefan Wohrer ◽

Keegan M. Rowe ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Mirna Biogenesis ◽

Self Renewal ◽

Hematopoietic Stem ◽

Control Virus ◽

Elevated Expression

Abstract Abstract 45 Fetal hematopoietic stem cells (HSCs) in mice differ from their adult counterparts in a number of key properties. These include a higher cycling activity, an ability to more rapidly reconstitute the HSC compartment of irradiated recipient mice, a higher output of myeloid as compared to lymphoid progeny, and a greater sensitivity to the self-renewal promoting activity of Steel factor. We have previously shown that most of these features of fetal HSCs are sustained until 3 weeks after birth at which time they are rapidly (within 1 week), completely and permanently replaced with the corresponding properties of adult HSCs. A candidate regulator of this transition, Hmga2, was identified based on its greater expression in highly purified fetal versus adult HSCs (CD45+EPCR+CD48−CD150+; E-SLAM cells) with persistence of this difference in the matching lineage-negative (lin−) compartments. Experiments in which Hmga2 was overexpressed by lentiviral transduction of purified adult HSCs which were then transplanted into irradiated mice provided evidence that this chromatin remodeling factor can activate a fetal-like HSC program in these cells; i.e., more rapidly reconstitute the HSC compartment (increased self-renewal response) and produce clones with a higher proportion of myeloid cells. Based on the known ability of the let-7 family of microRNAs (miRNAs) to target Hmga2 transcripts resulting in their degradation and/or translational repression, we next hypothesized that let-7 miRNAs might be involved in controlling HSC developmental programs. A comparison of the levels of expression of 6 members of the let-7 family in purified fetal and adult HSCs, as well as in lin− hematopoietic cells, showed that transcripts for all of these are higher in the adult subsets, although this difference was significant only for let-7b (p<0.05). Since Lin28 is a natural inhibitor of let-7 miRNA biogenesis we proposed that overexpression of this protein might be used to simultaneously inhibit all let-7 miRNA species and therefore modulate let-7-mediated effects in HSCs. Transduction of BA/F3 cells with a Lin28-YFP lentiviral vector led to an elevated expression of Lin28 and a significant decrease in multiple let-7 miRNAs. To investigate the influence of Lin28 overexpression on adult HSC self-renewal activity in vivo, we used the same Lin28 lentiviral vector (or a control YFP vector) to transduce highly purified HSCs (40 E-SLAM cells, i.e. ∼20 HSCs/group/experiment, 3 experiments) in a 3–4-hour exposure protocol and then transplanted all of the cells directly into irradiated mice (total of 3–4 mice/group). The number of HSCs regenerated 6 weeks later was subsequently measured by performing limiting-dilution transplants in secondary mice (total of 12–16 secondary mice/group/experiment). Interestingly, analysis of the secondary recipients showed that the Lin28-overexpressing adult HSCs had expanded in the primary recipients ∼6-fold more than the control-virus transduced HSCs (p<0.001). These findings support our thesis that alterations in let-7 miRNA levels play a key role in regulating the developmental switch from fetal to adult HSCs programs that occurs between 3 and 4 weeks after birth in mice. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Innate Immune System Favors Emergency Monopoiesis at the Expense of DC Differentiation to Control Bacterial Infections

Blood ◽

10.1182/blood.v124.21.2722.2722 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2722-2722

Author(s):

Kristin Bieber ◽

Karina A. Pasquevich ◽

Manina Günter ◽

Matthias Grauer ◽

Oliver Pötz ◽

...

Keyword(s):

Immune System ◽

Bacterial Infection ◽

Bacterial Infections ◽

Conflicts Of Interest ◽

Bacterial Load ◽

Innate Immune ◽

General Response ◽

The Impact ◽

Myeloid Progenitors

Abstract Dendritic cells (DCs) are critical in host defense against infection, bridging the innate and adaptive immune system. Patients with sepsis display reduced circulating and splenic DCs and impaired DC function that may contribute to prolonged immune suppression and exacerbation of infection. However, the mechanisms of pathogen-induced DC depletion remain poorly understood. Here, a mouse model of systemic bacterial infection was employed to analyze the impact of different bacterial pathogens on DC development in vivo. We found that the numbers of bone marrow (BM) hematopoietic progenitors committed to the DC lineages were reduced following systemic infection with different Gram-positive and Gram-negative bacteria. In parallel, a TLR4-dependent increase of committed monocyte progenitors in the BM as well as mature monocytes in the spleen was observed. In line, adoptively transferred FLT3+ myeloid progenitors (MPs) developed preferentially to monocytes at the expense of DCs in infected animals. Analyses performed on mixed BM chimeras suggested that both the reduction of DC progenitors and the induction of monopoiesis following infection were dependent on extrinsic TLR4 signaling driving the secretion of IFN-g regulated chemokines. Consistently, these effects were completely abrogated by suppression of IFN-g signaling. Elevated monocyte numbers in the spleen triggered by infection were due to a CCR2-dependent egress from the BM. In CCR2-deficient mice, in which monocytosis reportedly is abrogated, we observed a significantly increased bacterial load in the spleen and a reduced survival rate, highlighting the importance of monocytes for bacterial clearance. Together, our data provide evidence for a general response of myeloid progenitors upon bacterial infection to enhance monocyte production, thereby increasing the availability of innate immune cells as a first line of defense against invading pathogens. Concomitantly the development of DCs is impaired, which may be responsible for transient immunosuppression in e.g. bacterial sepsis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Efficiency of Homing and Engraftment Is Higher in VEGFR-3+CD34+CD38- Cells Than in VEGFR-3-CD34+CD38- Cells in Leukemic Patients

Blood ◽

10.1182/blood.v124.21.4782.4782 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4782-4782

Author(s):

Heeje Kim ◽

Ji-Yoon Lee ◽

Sohye Park ◽

Jae-Ho Yoon ◽

Woo-Sung Min ◽

...

Keyword(s):

Stem Cells ◽

Conflicts Of Interest ◽

Annexin V ◽

Leukemic Cell ◽

Phenotypic Characterization ◽

Facs Analysis ◽

Hematopoietic Stem ◽

Tumor Microenvironments ◽

Cd34 Cells

Abstract Surviving leukemic stem cells (LSCs) after chemotherapy lead to relapses in acute myeloid leukemia (AML). Because LSCs will not be eradicated after standard chemotherapy, inhibiting the propagation of AML cells by LSCs is a major part in AML treatment. Although CD34+CD38- cells in leukemia are representative LSCs, it is still difficult to distinguish them apart from normal hematopoietic stem cells (HSCs). So far, many studies have shown rapid advances in phenotypic characterization. However, heterogeneous diversity in AML patients may not allow effective eradication of LSCs. Vascular endothelial growth factor receptors (VEGFR)-3 is expressed in AML blasts and in the bone marrow (BM) environment including sinusoidal vessels. In particular, VEGFR-3 is strongly correlated with poor prognosis, leukemic cell proliferation and survival in AML. Based on previous reports, we were able to hypothesize that VEGFR-3 is expressed in LSCs and functions as a LSC marker. Here, we observed high expressions of VEGFR-3 on CD34+CD38− cells in AML patients who received chemotherapy and further showed low homing and engraftment capacity of CD45dim blast cells in the BM by a VEGFR-3 antagonist. In order to determine the expression of VEGFR-3 on LSCs, data was collected from 64 AML patients, 12 of whom were after complete remission (CR), as well as from 14 healthy volunteers. MNCs were first isolated and were then subjected to FACS analysis and immunocytochemistry. NOD-Scid IL2RγNull mice were used for homing efficiency and engraftment in vivo. MAZ51 was used as a VEGFR-3 antagonist. FACS analysis showed that VEGFR-3 was increased on CD34+CD38− LSC cells. (Normal vs. AML vs. CR, VEGFR-3 on CD34+CD38- cells: 8.33 ± 4.37% vs. 25.62 ± 2.46% vs. 23.46 ± 5.47%, P < 0.05). Similarly, immunocytochemistry clearly displayed the co-expression of VEGFR-3 on isolated CD34+CD38− LSC cells, suggesting the possibility of it as a LSC marker. We checked the ability of LSCs to use colony forming units assay. VEGFR-3+CD34+ cells showed unarguably enhanced colony forming ability compared to that of VEGFR-3-CD34+ cells from patients. To test whether VEGFR-3-CD34+ cells are not on apoptotic procedure, annexin-V and proliferation assay with Ki67 were performed, and there was no difference in apoptotic and proliferative movement in both cells. Intending to determine homing efficiency and engraftment, CD34 and CD45 markers were used in the BM and it was discovered that sorted VEGFR-3+CD34+CD38- cells showed significantly increased homing and engraftment efficiency compared to those of VEGFR-3-CD34+CD38- cells (by FACS, homing capacity: 1.93 ± 0.19% vs. 0.13 ± 0.06%, P = 0.02; engraftment: 26.4 ± 9.42% vs. 5.30 ± 2.31%, P < 0.05), implying that VEGFR-3 can serve as a marker for LSCs. We demonstrated that VEGFR-3 was highly expressed and enriched on CD34+CD38- cells in CR status as well as in the initial diagnosis of AML. Therefore, the targeting of VEGFR-3 may diminish LSC function in human AML. These findings could suggest some clues to develop therapeutic strategies targeting VEGFR-3+ LSCs with favorable tumor microenvironments. Disclosures No relevant conflicts of interest to declare.

Download Full-text