scholarly journals A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1446-1454
Author(s):  
N Katayama ◽  
M Nishikawa ◽  
F Komada ◽  
N Minami ◽  
S Shirakawa
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Hematopoietic Progenitor Cells ◽  
Dependent Manner ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Gm Csf ◽  
Erythroid Progenitor Cells ◽  
Dependent Inhibition ◽  
Dose Dependent

A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

scholarly journals A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1446-1454 ◽  
Author(s):  
N Katayama ◽  
M Nishikawa ◽  
F Komada ◽  
N Minami ◽  
S Shirakawa
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Hematopoietic Progenitor Cells ◽  
Dependent Manner ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Gm Csf ◽  
Erythroid Progenitor Cells ◽  
Dependent Inhibition ◽  
Dose Dependent

Abstract A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

scholarly journals DOT1L methyltransferase regulates the calcium influx in erythroid progenitor cells in response to erythropoietin

2020 ◽  
Author(s):  
Yi Feng ◽  
Shaon Borosha ◽  
Anamika Ratri ◽  
Sami M. Housami ◽  
V. Praveen Chakravarthi ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Transient Receptor Potential ◽  
Calcium Influx ◽  
Receptor Potential ◽  
Proximal Region ◽  
Yolk Sac ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

ABSTRACTErythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of hematopoietic progenitor cells. An important downstream response of EPO signaling is calcium influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in c-KIT positive erythroid progenitor cells is regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development, including early erythropoiesis. We previously reported that Dot1L knockout (Dot1L-KO) hematopoietic progenitors in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we have detected a marked downregulation of Trpc6 gene expression in Dot1L-KO progenitor cells in the yolk sac compared to wildtype. However, the expression of Trpc2, the positive regulator of Ca2+influx, remained unchanged. The promoter and the proximal region of the Trpc6 gene loci exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. As the loss of DOT1L affects the expression of TRPC6, which inhibits Ca2+influx by TRPC2, Dot1L-KO progenitor cells in the yolk sac exhibit accelerated and sustained high levels of Ca2+influx. Such heightened Ca2+ levels might have detrimental effects on the development of hematopoietic progenitor cells in response to erythropoietin.

DPP4 (CD26) Truncation Of GM-CSF and IL-3 Alters Their Signaling and Functional Activity In Normal and Leukemic Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1206-1206 ◽  
Author(s):  
Heather A. O'Leary ◽  
Charlie Mantel ◽  
Xianyin Lai ◽  
Scott Cooper ◽  
Giao Hangoc ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Receptor Binding ◽  
Functional Activity ◽  
Colony Formation ◽  
Full Length ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Penultimate Proline

Abstract DPP4 (CD26) is a dipeptidyl peptidase that functions by enzymatically cleaving the penultimate proline, alanine or select other amino acids such as serine of proteins, resulting in functional alterations of the protein. We recently published that many cytokines, chemokines and growth factors have putative DPP4 truncations sites and that DPP4 specifically was able to truncate some colony stimulating factors such as GM-CSF and IL-3 with resultant blunting of their activity. However, the mechanism of action of the truncated factors is still unknown and requires further investigation. The expression, and activity, of DPP4 is relevant in normal and malignant hematopoiesis as we have data showing that CD34+ umbilical cord blood cells (UCB) as well as Acute Myelogenous Leukemia (AML) patient samples express active DPP4. Further, specific inhibition of DPP4 increases homing and engraftment of both human UCB and mouse bone marrow cells after transplantation in mice indicating the therapeutic potential of DPP4 activity altering compounds. Due to its potential importance in disease states, and their subsequent treatment, it is relevant to study how the activity of DPP4 alters the functions of the molecules it cleaves, and subsequently their interactions with each other. DPP4 can cleave the penultimate proline of GM-CSF and IL-3 resulting in truncated forms which have blunted colony stimulating factor activity for hematopoietic progenitor cells (HPC). Since GM-CSF and IL-3 receptors share a common receptor beta chain, we investigated if DPP4 truncation of GM-CSF (TGM) or IL-3 (T3) could inhibit the receptor binding and functional activity of the full length (FL) alternate compound (i.e TGM inhibition of FL3 activity or T3 inhibition of FLGM activity) in the factor dependent TF-1 cell line, UCB cells and in in vivo mouse studies. We determined using TF-1 and UCB that both T3 and TGM bound to the receptors with higher affinity than their FL forms and could blunt the receptor binding of the FLGM and FL3. Additionally, TGM and T3 decreased colony formation induced by either FLGM or FL3 in both TF-1, UCB, and primary AML patient cell samples. Strikingly, this inhibition of colony formation did not require a 1:1 ratio of the full length to truncated forms of these cytokines. Rather, approximately 4-10 fold less truncated molecules could be used to efficiently inhibit the colony formation activity of the full length form, even across molecules. In vivo injection of FL, T, or a mixture of FL/T or T/T factors into DPP4 activity knockout mice followed by colony assays showed the TGM and T3 suppresed the effect of FLGM or FL3 on progenitor cell numbers per femur and diminished cycling of hematopoietic progenitor cells as detected by high specificity tritiated thymidine kill assay. Proteomic analysis of the effects of full length and truncated factors (FLGM, FL3, TGM, T3) were performed with TF-1 cells where we detected differential protein regulation by the full length vs truncated factors. After 24 hour treatment with 10ng/ml of FLGM or TGM, TF-1 cells displayed statistically significant (p < .05) differences in 26 proteins of which 17 were higher in the FL vs the T, and 9 higher in the T vs FL treated groups. These proteins included, but were not limited to, cell cycle proteins such as CDK6, HDAC6, as well as signal transduction proteins and redox control proteins such as STAM1 and Glutaredoxin. Additionally, alterations in protein phosphphorylation were detected for TF-1 cells treated for 15 or 30 min with the full length vs truncated IL-3 and GM-CSF proteins. Interestingly, the protein expression or phosphorylation levels were not always decreased by the truncated protein compared to the full length. In some cases, the truncated molecules induced an increase in the protein expression or phosphorylation. These data suggest interesting roles for full length and truncated GM-CSF and IL-3 in both normal and malignant hematopoiesis. Further investigation into the regulation of DPP4, and the roles that full length and truncated factors play during normal and malignant hematopoiesis, are important and will allow for a better understanding of the signficance of DPP4 activity during steady state, stressed, and disease hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Interleukin 6 is a permissive factor for monocytic colony formation by human hematopoietic progenitor cells.

1992 ◽  
Vol 175 (4) ◽  
pp. 1151-1154 ◽  
Author(s):  
J H Jansen ◽  
J C Kluin-Nelemans ◽  
J Van Damme ◽  
G J Wientjens ◽  
R Willemze ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Progenitor Cells ◽  
Interleukin 6 ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Gm Csf ◽  
Marrow Cells

Since monocytes and macrophages that arise during the culture of bone marrow progenitor cells are potential sources of interleukin 6 (IL-6), we investigated whether auto- or paracrine production of this factor is involved in colony formation by normal hematopoietic progenitor cells. We added a polyclonal anti-IL-6 antiserum and a monoclonal anti-IL-6 antibody to cultures of monocyte- and T cell-depleted bone marrow cells. Colony formation was stimulated with granulocyte/monocyte-colony-stimulating factor (GM-CSF), monocyte-CSF, or IL-3. Addition of anti-IL-6 antibody resulted in decreased numbers of monocytic colonies to 40-50% of control values, whereas the numbers of granulocytic colonies were not altered. The inhibitory effect was preserved in cultures of CD34(+)-enriched bone marrow cells. As a second approach, we added a monoclonal antibody directed against the IL-6 receptor to cultures of monocyte- and T cell-depleted bone marrow cells. This antibody almost completely inhibited the growth of monocytic colonies, again without decreasing the number of granulocytic colonies. Finally, the importance of IL-6 in monocytopoiesis was demonstrated in serum-deprived bone marrow cultures: addition of exogenous IL-6 to cultures stimulated with GM-CSF resulted in increased numbers of monocytic colonies. Our results indicate that the permissive presence of IL-6 is required for optimal monocytic colony formation by bone marrow progenitor cells.

Selected Members of the Angiopoietin-Like Family of Human Proteins Enhance Survival and Replating Capacity of Immature Human Hematopoietic Progenitor Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3367-3367
Author(s):  
Hal E. Broxmeyer ◽  
Edward F. Srour ◽  
Scott Cooper ◽  
Carrie T. Wallace ◽  
Giao Hangoc ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Gm Csf ◽  
Cd34 Cells

Abstract Angiopoietin-like (ANGPTL) molecules are a family of secreted proteins which have characteristic structures of angiopoietins. This includes a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils (CC), a short linker peptide, and a globular fibrinogen-like domain (FLD). Zhang et. al. (Nat. Med., 12(2):240–245, 2006) reported that human ANGPTL-2, 3, 3CC, 5 and 7, but not ANGPTL4, enhanced ex-vivo expansion of highly enriched mouse bone marrow (BM) long term competitive repopulating hematopoietic stem cells in serum-free culture with SCF, TPO, IGF-2, and FGF-1. To the present, there have not been publications describing effects of human ANGPTL molecules on hematopoietic progenitor cells (HPC) or on human hematopoietic cells. Thus, we evaluated purified recombinant human ANGPTL-2CC, 3, 3CC, 3FLD, 4, 4CC, 5, 6 and 7 (AdipoGen, Inc, Seoul, Korea) for effects on proliferation and survival of HPC from human cord blood (CB). No endotoxin was detected in the ANGPTL molecule preparations (<0.1 EU/ug endotoxin per LAL method). None of the ANGPTL molecules at up to 500ng/ml stimulated HPC colony formation by themselves, or enhanced or inhibited HPC colony formation of low density (LD) or CD34+ human cord blood (CB) cells stimulated by GM-CSF, GM-CSF plus SCF, Epo plus SCF, or the combination of Epo, SCF, IL-3 and GM-CSF. However, ANGPTL-2CC, 3, and 3CC at 200 and 100, but not 10ng/ml significantly enhanced the survival of human LD and CD34+ HPC (CFU-GM, BFU-E, CFU-GEMM) subjected to delayed addition of growth factors (Epo, SCF, IL-3, GM-CSF). Survival is a measure of anti-apoptosis for the hematopoietic progenitor cells in this context. The other ANGPTL molecules were not active at up to 500ng/ml. The survival enhancing effects of ANGPTL-3 was neutralized by purified rabbit anti-ANGPTL-3 IgG, but not by anti-ANGPTL-4, -6, or -7. Replating of HPC colonies offers an estimate of the self-renewal capabilities of HPC. We found that ANGPTL-3, but not -4, -6, or -7 enhanced the replating capacity of single CFU-GEMM colonies by greater than 2 fold. Thus far, we have not detected significant effects of the ANGPTL molecules on ex-vivo expansion of human CB CD34+ cells, alone, or in combination with SCF, TPO, Flt3-ligand, with or without IL-3, after assessing output of HPC, % and numbers of CD34+ cells, or cell cycle status of produced cells. In summary, we have implicated a few members of the ANGPTL family of proteins in functional effects on human HPC survival and replating/”self-renewal” activity, effects requiring the CC domain of the ANGPTL molecules. This information may be of relevance to regulation of HPC, and of use for protocols to use these cells for transplantation.

scholarly journals Lymphokine requirement for the generation of natural killer cells from CD34+ hematopoietic progenitor cells

Blood ◽  
1995 ◽  
Vol 85 (12) ◽  
pp. 3538-3546 ◽  
Author(s):  
A Shibuya ◽  
K Nagayoshi ◽  
K Nakamura ◽  
H Nakauchi
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Progenitor Cells ◽  
Interleukin 2 ◽  
Hematopoietic Progenitor Cells ◽  
Dependent Manner ◽  
Hematopoietic Progenitor ◽  
A Cell ◽  
Dose Dependent ◽  
Adult Bone

We have established a cell culture system without stromal cells that allows the CD34+ hematopoietic progenitor cells (HPC) to differentiate into natural killer (NK) cells. CD34+Lin (CD3, CD16, CD56)-cells were purified using fluorescence-activated cell sorting from normal adult bone marrow (BM) and cultured for 28 days in medium supplemented with interleukin-2 (IL-2) and stem cell factor (SCF). NK (CD3-CD16-CD56+) cells were generated in a dose-dependent manner in response to SCF. NK cells originated from CD34+CD33+Lin- cells, but they were barely detectable in cultures of CD34+CD33-Lin- cells. However, on addition of IL-3, an induced differentiation of NK cells from CD34+CD33-Lin- cells was observed, although at a lower frequency. Supplementing of the cell cultures with SCF alone or both SCF and IL-3 for the first 7 days followed by IL-2 for the next 21 days is essential for production of NK cells from CD34+CD33+Lin- cells and from CD34+CD33-Lin- cells, respectively. These data provide direct evidence that NK cells arise from CD34+HPC and show the minimum lymphokine requirement for their differentiation.

scholarly journals Interleukin 13: novel role in direct regulation of proliferation and differentiation of primitive hematopoietic progenitor cells.

1994 ◽  
Vol 180 (1) ◽  
pp. 75-82 ◽  
Author(s):  
S E Jacobsen ◽  
C Okkenhaug ◽  
O P Veiby ◽  
D Caput ◽  
P Ferrara ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Biological Activities ◽  
Hematopoietic Progenitor Cells ◽  
Synergistic Activity ◽  
Hematopoietic Progenitor ◽  
Interleukin 13 ◽  
Gm Csf ◽  
Macrophage Colony

The recently cloned interleukin 13 (IL-13) shares most investigated biological activities on B lymphocytes and monocytes with IL-4. In this study we investigated for the first time the potential role of IL-13 in the regulation of the growth of hematopoietic progenitor cells. IL-13 enhanced stem cell factor (SCF)-induced proliferation of Lin-Sca-1+ bone marrow progenitor cells more potently than IL-4. The effect of IL-13 was purely synergistic, since IL-13 alone stimulated no colony formation. Single cell experiments suggested that the synergistic effect of IL-13 on Lin-Sca-1+ progenitors was directly mediated. In contrast, IL-13 had no synergistic activity on SCF-induced proliferation of the more mature Lin-Sca-1- progenitor cells. Thus, the cloning frequency in response to SCF + IL-13 was at least 20-fold higher in the Lin-Sca-1+ than the Lin-Sca-1- progenitor cell population. Furthermore, IL-13 but not IL-4 synergistically enhanced colony formation of Lin-Sca-1+ progenitors in response to granulocyte/macrophage colony-stimulating factor (GM-CSF) (threefold), whereas both IL-4 and IL-13 enhanced G-CSF-induced colony formation (threefold), and neither of the two significantly affected CSF-1 and IL-3-induced proliferation. Finally, whereas stimulation of Lin-Sca-1+ progenitors by SCF + G-CSF resulted in the formation of 90% granulocytes, the addition of IL-13 resulted in the production of macrophages exclusively. This novel effect on differentiation was directly mediated, shared with IL-4, and could not be observed on Lin-Sca-1- progenitor cells. Collectively, these findings indicate a novel role of IL-13 in early myelopoiesis, partially overlapping but also different from that of IL-4.

scholarly journals Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

1995 ◽  
Vol 15 (6) ◽  
pp. 3147-3153 ◽  
Author(s):  
G A Blobel ◽  
C A Sieff ◽  
S H Orkin
Keyword(s):  
Bone Marrow ◽  
Estrogen Receptor ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Erythroid Cell ◽  
High Dose ◽  
Dependent Manner ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

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