Impaired Hematopoiesis in Paroxysmal Nocturnal Hemoglobinuria/Aplastic Anemia Is Not Associated With a Selective Proliferative Defect in the Glycosylphosphatidylinositol-Anchored Protein-Deficient Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) results from somatic mutations in the PIG-A gene, leading to poor presentation of glycosylphosphatidylinositol (GPI)-anchored surface proteins. PNH frequently occurs in association with suppressed hematopoiesis, including frank aplastic anemia (AA). The relationship between GPI-anchored protein expression and bone marrow (BM) failure is unknown. To assess the hematopoietic defect in PNH, the numbers of CD34+ cells, committed progenitors (primary colony-forming cells [CFCs]), and long-term culture-initiating cells (LTC-ICs; a stem cell surrogate) were measured in BM and peripheral blood (PB) of patients with PNH/AA syndrome or patients with predominantly hemolytic PNH. LTC-IC numbers were extrapolated from secondary CFC numbers after 5 weeks of culture, and clonogenicity of LTC-ICs was determined by limiting dilution assays. When compared with normal volunteers (n = 13), PNH patients (n = 14) showed a 4.7-fold decrease in CD34+ cells and an 8.2-fold decrease in CFCs. LTC-ICs in BM and in PB were decreased 7.3-fold and 50-fold, respectively. Purified CD34+ cells from PNH patients had markedly lower clonogenicity in both primary colony cultures and in the LTC-IC assays. As expected, GPI-anchored proteins were decreased on PB cells of PNH patients. On average, 23% of monocytes were deficient in CD14, and 47% of granulocytes and 58% of platelets lacked CD16 and CD55, respectively. In PNH BM, 27% of CD34+ cells showed abnormal GPI-anchored protein expression when assessed by CD59 expression. To directly measure the colony-forming ability of GPI-anchored protein-deficient CD34+ cells, we separated CD34+ cells from PNH patients for the GPI+ and GPI− phenotype; CD59 expression was chosen as a marker of the PNH phenotype based on high and homogeneous expression on fluorescent staining. CD34+CD59+ and CD34+CD59− cells from PNH/AA patients showed similarly impaired primary and secondary clonogeneic efficiency. The progeny derived from CD34+CD59− cells were both CD59− and CD55−. A very small population of CD34+CD59− cells was also detected in some normal volunteers; after sorting, these CD34+CD59− cells formed normal numbers of colonies, but their progeny showed lower CD59 levels. Our results are consistent with the existence of PIG-A–deficient clones in some normal individuals. In PNH/AA, progenitor and stem cells are decreased in number and function, but the proliferation in vitro is affected similarly in GPI-protein–deficient clones and in phenotypically normal cells. As measured in the in vitro assays, expansion of PIG-A– clones appears not be caused by an intrinsic growth advantage of cells with the PNH phenotype.

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Apoptosis Resistance of Blood Cells From Patients With Paroxysmal Nocturnal Hemoglobinuria, Aplastic Anemia, and Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v90.7.2716.2716_2716_2722 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2716-2722 ◽

Cited By ~ 2

Author(s):

Kentaro Horikawa ◽

Hideki Nakakuma ◽

Tatsuya Kawaguchi ◽

Norihiro Iwamoto ◽

Shoichi Nagakura ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Clinical Manifestations ◽

Interferon Γ ◽

Apoptosis Resistance ◽

Molecular Events ◽

Cd34 Cells ◽

Factor Α

Bone marrow (BM) hypoplasia is a major cause of death in paroxysmal nocturnal hemoglobinuria (PNH). However, little is known about the molecular events leading to the hypoplasia. Considering the close pathologic association between PNH and aplastic anemia (AA), it is suggested that a similar mechanism operates in the development of their BM failure. Recent reports have indicated apoptosis-mediated BM suppression in AA. It is thus conceivable that apoptosis also operates to cause BM hypoplasia in PNH. If this is the case, PNH clones need to survive apoptosis and show considerable expansion leading to clinical manifestations. We report here that granulocytes obtained from 11 patients with PNH were apparently less susceptible than those from 20 healthy individuals to both spontaneous apoptosis without any ligands and that induced by anti-FAS (CD95) antibody in vitro. The patients' BM CD34+ cells were also resistant to apoptosis induced by treatment with tumor necrosis factor-α, interferon-γ, and subsequently with anti-FAS antibody. In lymphocytes, the pathologic resistance was not discriminated from inherent resistance to apoptosis. Granulocytes from 13 patients with AA and 12 patients with myelodysplastic syndrome (MDS) exhibited similar resistance to apoptosis. CD34+ cells from MDS-BM also showed similar tendency. Thus, the comparative resistance to apoptosis supports the pathogenic implication of apoptosis in marrow injury of PNH and related stem cell disorders.

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Apoptosis Resistance of Blood Cells From Patients With Paroxysmal Nocturnal Hemoglobinuria, Aplastic Anemia, and Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v90.7.2716 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2716-2722 ◽

Cited By ~ 78

Author(s):

Kentaro Horikawa ◽

Hideki Nakakuma ◽

Tatsuya Kawaguchi ◽

Norihiro Iwamoto ◽

Shoichi Nagakura ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Clinical Manifestations ◽

Interferon Γ ◽

Apoptosis Resistance ◽

Molecular Events ◽

Cd34 Cells ◽

Factor Α

Abstract Bone marrow (BM) hypoplasia is a major cause of death in paroxysmal nocturnal hemoglobinuria (PNH). However, little is known about the molecular events leading to the hypoplasia. Considering the close pathologic association between PNH and aplastic anemia (AA), it is suggested that a similar mechanism operates in the development of their BM failure. Recent reports have indicated apoptosis-mediated BM suppression in AA. It is thus conceivable that apoptosis also operates to cause BM hypoplasia in PNH. If this is the case, PNH clones need to survive apoptosis and show considerable expansion leading to clinical manifestations. We report here that granulocytes obtained from 11 patients with PNH were apparently less susceptible than those from 20 healthy individuals to both spontaneous apoptosis without any ligands and that induced by anti-FAS (CD95) antibody in vitro. The patients' BM CD34+ cells were also resistant to apoptosis induced by treatment with tumor necrosis factor-α, interferon-γ, and subsequently with anti-FAS antibody. In lymphocytes, the pathologic resistance was not discriminated from inherent resistance to apoptosis. Granulocytes from 13 patients with AA and 12 patients with myelodysplastic syndrome (MDS) exhibited similar resistance to apoptosis. CD34+ cells from MDS-BM also showed similar tendency. Thus, the comparative resistance to apoptosis supports the pathogenic implication of apoptosis in marrow injury of PNH and related stem cell disorders.

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Pravastatin Promotes Endothelial Colony-Forming Cell Function, Angiogenic Signaling and Protein Expression In Vitro

Journal of Clinical Medicine ◽

10.3390/jcm10020183 ◽

2021 ◽

Vol 10 (2) ◽

pp. 183

Author(s):

Nadia Meyer ◽

Lars Brodowski ◽

Katja Richter ◽

Constantin S. von Kaisenberg ◽

Bianca Schröder-Heurich ◽

...

Keyword(s):

Cardiovascular Diseases ◽

Growth Factor ◽

Endothelial Dysfunction ◽

Protein Expression ◽

Tube Formation ◽

In Vitro Assays ◽

Heme Oxygenase 1 ◽

Expression Levels ◽

Functional Capacities

Endothelial dysfunction is a primary feature of several cardiovascular diseases. Endothelial colony-forming cells (ECFCs) represent a highly proliferative subtype of endothelial progenitor cells (EPCs), which are involved in neovascularization and vascular repair. Statins are known to improve the outcome of cardiovascular diseases via pleiotropic effects. We hypothesized that treatment with the 3-hydroxy-3-methyl-glutaryl–coenzyme A (HMG-CoA) reductase inhibitor pravastatin increases ECFCs’ functional capacities and regulates the expression of proteins which modulate endothelial health in a favourable manner. Umbilical cord blood derived ECFCs were incubated with different concentrations of pravastatin with or without mevalonate, a key intermediate in cholesterol synthesis. Functional capacities such as migration, proliferation and tube formation were addressed in corresponding in vitro assays. mRNA and protein levels or phosphorylation of protein kinase B (AKT), endothelial nitric oxide synthase (eNOS), heme oxygenase-1 (HO-1), vascular endothelial growth factor A (VEGF-A), placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (Eng) were analyzed by real time PCR or immunoblot, respectively. Proliferation, migration and tube formation of ECFCs were enhanced after pravastatin treatment, and AKT- and eNOS-phosphorylation were augmented. Further, expression levels of HO-1, VEGF-A and PlGF were increased, whereas expression levels of sFlt-1 and Eng were decreased. Pravastatin induced effects were reversible by the addition of mevalonate. Pravastatin induces beneficial effects on ECFC function, angiogenic signaling and protein expression. These effects may contribute to understand the pleiotropic function of statins as well as to provide a promising option to improve ECFCs’ condition in cell therapy in order to ameliorate endothelial dysfunction.

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A genomic and proteomic analysis of surface proteins in bovine-adapted lineages of Staphylococcus aureus

Access Microbiology ◽

10.1099/acmi.ac2020.po0394 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

Shauna D. Drumm ◽

Rebecca Owens ◽

Jennifer Mitchell ◽

Orla M. Keane

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Surface Proteins ◽

Host Cells ◽

Mass Spectrometry Analysis ◽

In Vitro Assays ◽

Immune Recognition ◽

Independent Manner ◽

Genes Encoding

In Ireland, Staphylococcus aureus is the most common cause of intramammary infection (IMI) in cattle with the bovine-adapted lineages CC151 and CC97 most commonly found. Surface proteins play a major role in establishing and maintaining the infection. A previous study revealed that a strain from the CC151 lineage showed significant decay in genes encoding predicted surface proteins. Twenty-three S. aureus strains, twelve belonging to CC151 and eleven belonging to CC97, isolated from clinical IMI, were sequenced and genes encoding cell wall anchored (CWA) proteins predicted. Analysis showed that a minority of genes encoding putative CWA proteins were intact in the CC151 strains compared to CC97. Of the 26 known CWA proteins in S. aureus, the CC151 strains only encoded 10 intact genes while CC97 encoded on average 18 genes. Also within the CC97 lineage, the repertoire of genes varied depending on individual strains, with strains encoding between 17-20 intact genes. Although CC151 is reported to internalize within bovine host cells, it does so in a fibronectin-binding protein (FnBPA and FnBPB) independent manner. In-vitro assays were performed and results showed that strains from CC151, and surprisingly also CC97, weakly bound bovine fibronectin and that the FnBPs were poorly expressed in both these lineages. Mass spectrometry analysis of cell wall extracts revealed that SdrE and AdsA were the most highly expressed CWA proteins in both lineages. These results demonstrate significant differences between CC151 and CC97 in their repertoire of genes encoding CWA proteins, which may impact immune recognition of these strains and their interactions with host cells.

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Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma

Blood ◽

10.1182/blood.v77.8.1717.1717 ◽

1991 ◽

Vol 77 (8) ◽

pp. 1717-1722 ◽

Cited By ~ 340

Author(s):

RJ Berenson ◽

WI Bensinger ◽

RS Hill ◽

RG Andrews ◽

J Garcia-Lopez ◽

...

Keyword(s):

Breast Cancer ◽

Stage Iv ◽

In Vitro Assays ◽

Hematopoietic Progenitors ◽

Cd34 Antigen ◽

Stage Iv Breast Cancer ◽

Cd34 Cells ◽

Selection Of ◽

Marrow Cells

Abstract The CD34 antigen is expressed by 1% to 4% of human and baboon marrow cells, including virtually all hematopoietic progenitors detectable by in vitro assays. Previous work from our laboratory has shown that CD34+ marrow cells can engraft lethally irradiated baboons. Because the CD34 antigen has not been detected on most solid tumors, positive selection of CD34+ cells may be used to provide marrow cells capable of engraftment, but depleted of tumor cells. In seven patients with stage IV breast cancer and two patients with stage IV neuroblastoma, 2.5 to 17.5 x 10(9) marrow cells were separated by immunoadsorption with the anti-CD34 antibody 12–8 and 50 to 260 x 10(6) positively selected cells were recovered that were 64 +/- 16% (range 35% to 92%) CD34+. The patients received 1.0 to 5.2 x 10(6) CD34-enriched cells/kg after marrow ablative therapy. Six patients engrafted, achieving granulocyte counts of greater than 500/mm3 at 34 +/- 10 (range 21 to 47) days and platelets counts of greater than 20,000/mm3 at 46 +/- 14 (range 28 to 66) days posttransplant. Five of these patients showed durable engraftment until the time of death 82 to 386 days posttransplant. One patient failed to sustain engraftment associated with metastatic marrow disease. Three patients died at days 14, 14, and 17 posttransplant, two of whom had evidence of early engraftment. These studies suggest that CD34+ marrow cells are capable of reconstituting hematopoiesis in humans.

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Aplastic anemia and paroxysmal nocturnal hemoglobinuria: search for a pathogenetic link

Blood ◽

10.1182/blood.v85.5.1354.bloodjournal8551354 ◽

1995 ◽

Vol 85 (5) ◽

pp. 1354-1363 ◽

Cited By ~ 76

Author(s):

A Griscelli-Bennaceur ◽

E Gluckman ◽

ML Scrobohaci ◽

P Jonveaux ◽

T Vu ◽

...

Keyword(s):

Protein Expression ◽

Aplastic Anemia ◽

Paroxysmal Nocturnal Hemoglobinuria ◽

Antithrombin Iii ◽

Clinical Symptoms ◽

Flow Cytometric Analysis ◽

Normal Subjects ◽

Initial Study ◽

Pnh Clone

The association of paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA) raises the yet unresolved questions as to whether these two disorders are different forms of the same disease. We compared two groups of patients with respect to cytogenetic features, glycosylphosphatidylinositol (GPI)-linked protein expression, protein C/protein S/thrombomodulin/antithrombin III activity, and PIG-A gene expression. The first group consisted of eight patients with PNH (defined as positive Ham and sucrose tests at diagnosis), and the second, 37 patients with AA. Twelve patients with AA later developed a PNH clone. Monoclonal antibodies used to study GPI-linked protein expression (CD14 [on monocytes], CD16 [on neutrophils], CD48 [on lymphocytes and monocytes], CD67 [on neutrophils and eosinophils], and, more recently, CD55, CD58, and CD59 [on erythrocytes]) were also tested on a cohort of 20 normal subjects and five patients with constitutional AA. Ham and sucrose tests were performed on the same day as flow- cytometric analysis. Six of 12 patients with AA, who secondarily developed a PNH clone, had clinical symptoms, while all eight patients with PNH had pancytopenia and/or thrombosis and/or hemolytic anemia. Cytogenetic features were normal in all but two patients. Proteins C and S, thrombomodulin, and antithrombin III levels were within the normal range in patients with PNH and in those with AA (with or without a PNH clone). In patients with PNH, CD16 and CD67 expression were deficient in 78% to 98% of the cells and CD14 in 76% to 100%. By comparison, a GPI-linked defect was detected in 13 patients with AA, affecting a mean of 32% and 33% of CD16/CD67 and CD14 cell populations, respectively. Two of three tested patients with PNH and 1 of 12 patients with AA had a defect in the CD48 lymphocyte population. In a follow-up study of our patient cohort, we used the GPI-linked molecules on granulocytes and monocytes investigated earlier and added the study of CD55, CD58, and CD59 on erythrocytes. Two patients with PNH and 14 with AA were studied for 6 to 13 months after the initial study. Among patients with AA, four in whom no GPI-anchoring defect was detected in the first study had no defect in follow-up studies of all blood-cell subsets (including erythrocytes). Analysis of granulocytes, monocytes, and erythrocytes was performed in 7 of 13 AA patients in whom affected monocytes and granulocytes were previously detected. A GPI-anchoring defect was detected on erythrocytes in five of six.(ABSTRACT TRUNCATED AT 400 WORDS)

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Comparative in-Vitro Evaluation of the Myeloid Toxicity of Pentostatin and the Novel PNP-Inhibitor Forodesine.

Blood ◽

10.1182/blood.v114.22.3766.3766 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3766-3766

Author(s):

Mario Schubert ◽

Christian Wallenwein ◽

Larissa Pietsch ◽

Dan Ran ◽

Isabel Taubert ◽

...

Keyword(s):

Primary Cultures ◽

Flow Cytometric Analysis ◽

Chemotherapeutic Agents ◽

Dose Effect ◽

In Vitro Assays ◽

In Vitro Toxicity ◽

The Novel ◽

Xtt Assay ◽

Cd34 Cells

Abstract Abstract 3766 Poster Board III-702 Inhibitors of the purine metabolism show promising results in the treatment of lymphatic malignancies due to their suppressive effects on lymphogenesis. Their first representative, Pentostatin (Pento), an inhibitor of the deoxyadenosine deaminase, has been in clinical use for several decades. However, early clinical trials with higher dose ranges of the drug reported unforseen severe myelotoxic effects. Recently, Forodesine (Foro), a novel inhibitor of the nucleoside phosphorylase (PNP) has been introduced and is currently deployed in clinical phase I/II trials for the treatment of acute lymphatic leukemia (ALL). In order to systematically evaluate the myelotoxic effects of Pento and Foro, we have now examined their influence on the proliferation and differentiation of primitive and lineage committed hematopoietic progenitor cells (HPCs). In vitro dose/effect-curves for Foro, Pento, and Cytarabine (AraC) were generated for the leukemic cell line jurkat by 48 hours of co-incubation with the compounds. Adequate cytotoxic effects, measured in the XTT assay and by flow cytometric analysis, were observed in clinically relevant dose ranges. For the following studies, an equivalent IC60 dose of each chemotherapeutic agent was selected and CD34+ HPCs from either bone marrow, mobilized peripheral blood, or umbilical cord blood were incubated with the compounds for 48 hours. Subsequently, the rate of vital cells was determined by flow cytometry after stainig with Annexin-V and Propidium Iodide. Compared to the untreated control, the lowest amount of vital CD34+ cells was found in AraC-treated samples (30%); Foro and Pento yielded more vital cells (66% vs 61%). The combination of Foro and Pento unexpectedly had the least toxic effect on CD34+ cells (72%; n=5; p<0.05). Cells from those primary cultures were harvested and short- and long term in vitro assays for colony forming units were performed to evaluate the compounds' toxicity on primitive and lineage committed HPCs. The frequency of primitive myeloic progenitors (LTC-IC) was 2.3% in the untreated samples and diminished after treatment with AraC (1.2%) and Pento (1.9%) but surprisingly significantly increased after Foro-treatment (2.7%); the combination of Foro and Pento resulted in a LTC-IC frequency of 2.3% (p<0.01; n=5) suggesting that Foro may have attenuated the myelotoxicity of Pento. Similar effects of Foro were also observed in the short term colony forming assays where Foro seemed to have a protective effect on multipotent GEMM-progenitors: colony count increased 1.3-fold in comparison to the control; AraC yielded only 0.1-fold, Pento 0.8-fold and the combination of Pento and Foro reached 0.9-fold of the control (p<0.05; n=15). In summary, the novel PNP-inhibitor Forodesine has not only proven to have a low in vitro toxicity on lineage committed HPCs but, surprisingly, the frequency of primitive myeloic progenitors (LTC-IC) increased; clinical studies should therefore be performed to evaluate whether Forodesine, while adding to the therapeutic efficiency, may attenuate adverse effects in combination with other chemotherapeutic agents, such as Pentostatin. Disclosures: Schubert: Mundipharma Int. LTD: Research Funding.

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MicroRNA Expression and Regulation of Hematopoiesis in CD34+ Cells: A Bioinformatic Circuit Diagram of the Hematopoietic Differentiation Control.

Blood ◽

10.1182/blood.v108.11.1334.1334 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1334-1334

Author(s):

Robert W. Georgantas ◽

Richard Hildreth ◽

Jonathan Alder ◽

Carlo M. Croce ◽

George A. Calin ◽

...

Keyword(s):

Protein Expression ◽

Expression Profiles ◽

In Vivo Studies ◽

Luciferase Reporter ◽

Hematopoietic Differentiation ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract MicroRNAs (miRs) are a recently realized class of epigenetic elements which block translation of mRNA to protein. MicroRNAs have been shown to control cellular metabolism, apoptosis, differentiation and development in numerous organisms including drosophila, rat, mouse, and humans. Recently, miRs have been implicated in the control of hematopoiesis. Importantly, both aberrant expression and deletion of miRs are have been associated with the development of various cancers. In a previous study, we determined the gene expression profiles of HSC-enriched, HPC-enriched, and total CD34+ cells from human PBSC, BM, and CB. One rather surprising finding from this study was that virtually all of “hematopoietic important” genes were expressed at virtually identical levels within all populations examined. One of our hypotheses to explain this phenomena was that miRs may control differentiation by controlling protein expression from these “hematopoietic” RNAs. To examine the possible role of miRs in normal hematopoiesis and their relation to the HSPC transcriptome, we used mir-miroarrays to determine the miR expression profile of primary normal human mobilized blood and bone marrow CD34+ hematopoietic stem-progenitor cells (HSPCs). We have combined this miR data with (1) our extensive mRNA expression data obtained previously for CD34+ HSPCs, CD34+/CD38−/Lin- stem cell-enriched, CD34+/CD38+/Lin+ progenitor-enriched populations, and total CD34+ HSPC (Georgantas, Cancer Research 64:4434) and (2) miR target predictions from various published algorithms. Combining these datasets into one integrated database allowed us to bioinformaticly examine the global interaction of HSPC mRNAs and miRs during hematopoiesis. The 3′UTR sequences from many of these “hematopoietic” mRNA were cloned behind a luciferase reporter. K562 cells were transfected with these luc-3′UTR constructs, confirmating that expression of many important hematopoietic proteins are controlled by miRs. Based on our bioinformatic and protein expression studies, we present a global in silico model by which microRNAs control and direct hematopoietic differentiation. Actual in vitro and in vivo studies addressing the action of specific miRs in hematopoietic differentiation are presented in separate abstracts.

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A Novel Role for Thrombin in Hematopoietic Stem/Progenitor Cell Homing.

Blood ◽

10.1182/blood.v108.11.3374.3374 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3374-3374

Author(s):

Neeta Shirvaikar ◽

Ali Jalili ◽

Mariusz Z. Ratajczak ◽

Anna Janowska-Wieczorek

Keyword(s):

Protein Expression ◽

Lipid Rafts ◽

Leading Edge ◽

Membrane Lipid ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

And Migration ◽

Stimulation Of

Abstract Thrombin, an important serine protease, not only plays a pivotal role in platelet aggregation and coagulation, but also through activation of its receptor, seven transmembrane, G-protein-coupled receptor PAR-1, elicits numerous cellular responses in platelets and endothelial cells such as induction of adhesion molecules, production of chemokines, activation of matrix metalloproteinase (MMP)-2, cytoskeletal reorganization and migration. Thrombin is also one of the inflammatory molecules elevated during G-CSF mobilization of hematopoietic stem/progenitor cells (HSPC) and their collection by leukapheresis. We recently reported that components of leukapheresis products including thrombin enhance in vitro chemotaxis of CD34+ cells towards an SDF-1 gradient and in vivo homing to bone marrow (BM) niches in a murine model (Blood2005; 105:40). In this study we investigated whether thrombin enhances the homing-related responses of human HSPC (CD34+ cells) through MMPs, especially membrane-type (MT)1-MMP which is known to be localized on the leading edge of migrating cells and both activates latent proMMPs (MMP-2, -9) and itself has strong pericellular proteolytic activity. We found that stimulation of CD34+ cells with thrombin upregulates mRNA for MT1-MMP and MMP-9 as well as MT1-MMP protein expression (Western blot, flow cytometry) and proMMP-2 and proMMP-9 secretion (zymography). Thrombin was also found to (i) prime trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP, and (ii) activate MMP-2 in of co-cultures of CD34+ cells with stromal cells (BM fibroblasts and HUVEC) which secrete proMMP-2. We also found that SDF-1 upregulates mRNA and protein expression of MT1-MMP. Moreover, using confocal microscopy we demonstrate for the first time that in CD34+ cells, PAR-1, like CXCR4, is localized in the GM1 fraction of lipid rafts and stimulation of these cells with thrombin as well as SDF-1 increases incorporation of MT1-MMP into membrane lipid rafts. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibits MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards SDF-1. Thus we conclude that thrombin, through PAR-1 signalling and the SDF-1-CXCR4 axis, upregulates the incorporation of MT1-MMP into membrane lipid rafts and the interaction of these axes enhances the homing-related responses of HSPC towards SDF-1.

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New G-CSF-Dependent Signaling Pathway and Its Role In Patients with Severe Congenital Neutropenia and Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.385.385 ◽

2010 ◽

Vol 116 (21) ◽

pp. 385-385

Author(s):

Julia Skokowa ◽

Elena Chirvon ◽

Lan Dan ◽

Kshama Gupta ◽

Ana Gigina ◽

...

Keyword(s):

Bone Marrow ◽

Protein Expression ◽

Cell Lines ◽

Myeloid Leukemia ◽

Gene Promoter ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Cd34 Cells ◽

Acute Myeloid

Abstract Abstract 385 We demonstrated that stimulation of hematopoietic CD34+ cells with granulocyte-colony stimulating factor (G-CSF) leads to phosphorylation of hematopoietic cell-specific Lyn substrate 1 (HCLS1) protein and HCLS1 interaction with G-CSF receptor-associated tyrosine kinases Lyn and Syk. Activated HCLS1 binds to lymphoid-enhancer factor 1 (LEF-1) protein, inducing LEF-1-dependent autoregulation of the LEF-1 gene promoter. Transcription factor LEF-1 is a downstream effector of Wnt signaling and a master regulator of myeloid differentiation. In patients with severe congenital neutropenia (CN), inherited mutations in the HCLS1-associated protein × 1 (HAX1) lead to profound defects in the G-CSF-triggered expression and phosphorylation of HCLS1 protein and subsequently, to reduced expression of LEF-1 and abrogated granulopoiesis. Using immunoprecipitation assay, we identified endogenous HAX1 protein in the complex with LEF-1 and HCLS1 proteins. HAX1 is known as a mitochondrial protein, however using cell fractionation experiments of different myeloid cell lines (NB4, HL60, THP-1) and of primary CD34+ cells we identified endogenous HAX1 protein in different intracellular compartments. Thus, HAX1 protein was expressed in mitochondria, cytoplasm, nucleus, cytosol and in membranous fraction. Expression levels of HAX1 protein varied between different cell lines and between maturation stage of the cells. In vitro inhibition of HCLS1 or HAX1 by shRNA severely disrupted granulocytic differentiation of CD34+ cells, due to a lack of LEF-1 and C/EBPa protein expression and subsequently abolished activation of LEF-1 and C/EBPa gene promoters. Intriguingly, LEF-1 in turn induced HCLS1 mRNA and protein expression by direct binding to the HCLS1 gene promoter and inhibition of LEF-1 in CD34+ cells leads to severe downregulation of HCLS1 protein levels. These findings demonstrated a reciprocal feed-back regulation between LEF-1 and HCLS1 proteins. Moreover, HCLS1-/- mice are neutropenic due to a lack of LEF-1 protein expression and defective in vitro G-CSF-triggered F-actin rearrangement in bone marrow myeloid progenitor cells, as compared to WT mice. At the same time, in bone marrow biopsies of 88.46 % of tested patients (46 out of 52 patients) with acute myeloid leukemia (AML), we found significantly elevated levels of HCLS1 protein. Inhibition of HCLS1 in the AML cell lines and primary blasts of AML patients resulted in reduced proliferation and increased apoptosis. Taken together, we described a novel G-CSFR downstream signaling pathway, with dose-dependent effects on myelopoiesis: diminished expression of HCLS1 and LEF-1 resulted in a “maturation arrest” of granulopoiesis and severe neutropenia, but hyperactivation of one of these proteins led to a hyperproliferation of myeloid progenitors and AML. Disclosures: No relevant conflicts of interest to declare.

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