The Experts below are selected from a list of 207 Experts worldwide ranked by ideXlab platform
Stephen J. Brandt - One of the best experts on this subject based on the ideXlab platform.
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The TAL1/SCL transcription factor regulates cell cycle progression and proliferation in differentiating murine bone marrow monocyte precursors.
Molecular and cellular biology, 2010Co-Authors: Soumyadeep Dey, David J. Curtis, Stephen M. Jane, Stephen J. BrandtAbstract:Monocytopoiesis involves the stepwise differentiation in the bone marrow (BM) of common myeloid precursors (CMPs) to monocytes. The basic helix-loop-helix transcription factor TAL1/SCL plays a critical role in other hematopoietic lineages, and while it had been reported to be expressed by BM-derived macrophages, its role in Monocytopoiesis had not been elucidated. Using cell explant models of monocyte/macrophage (MM) differentiation, one originating with CMPs and the other from more committed precursors, we characterized the phenotypic and molecular consequences of inactivation of Tal1 expression ex vivo. While Tal1 knockout had minimal effects on cell survival and slightly accelerated terminal differentiation, it profoundly inhibited cell proliferation and decreased entry into and traversal of the G(1) and S phases. In conjunction, steady-state levels of p16(Ink4a) mRNA were increased and those of Gata2 mRNA decreased. Chromatin immunoprecipitation analysis demonstrated the association of Tal1 and E47, one of its E protein DNA-binding partners, with an E box-GATA sequence element in intron 4 of the Gata2 gene and with three E boxes upstream of p16(Ink4a). Finally, wild-type Tal1, but not a DNA binding-defective mutant, rescued the proliferative defect in Tal1-null MM precursors. These results document the importance of this transcription factor in cell cycle progression and proliferation during Monocytopoiesis and the requirement for direct DNA binding in these processes.
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Novel Role of the TAL1/SCL Transcription Factor in Murine Monocytopoiesis.
Blood, 2008Co-Authors: Soumyadeep Dey, David J. Curtis, Stephen M. Jane, Stephen J. BrandtAbstract:Abstract The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev., 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-ã-activated macrophages. To elucidate Tal1’sfunctions in murine Monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1fl/fl/lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1-null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1,PU.1, Csfr1, Msr1 (mouse scavenger receptor), Cd68, or Il6ra. Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining ofTal1-null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.
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novel role of the tal1 scl transcription factor in murine Monocytopoiesis
Blood, 2008Co-Authors: Soumyadeep Dey, David J. Curtis, Stephen M. Jane, Stephen J. BrandtAbstract:The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev., 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-a-activated macrophages. To elucidate Tal1’sfunctions in murine Monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1 fl/fl /lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1 -null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1 , PU.1 , Csfr1 , Msr1 (mouse scavenger receptor), Cd68 , or Il6ra . Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining of Tal1 -null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1 hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.
Ravi Shankar - One of the best experts on this subject based on the ideXlab platform.
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Murine hematopoietic stem cells and progenitors express adrenergic receptors.
Journal of neuroimmunology, 2007Co-Authors: Kuzhali Muthu, Richard L. Gamelli, Ravi Shankar, Sivaraman Iyer, Andrea Szilagyi, Stephen B JonesAbstract:Association between the nervous and immune system is well documented. Immune cells originate within the bone marrow that is innervated. Thermal injury induces adrenergic stimulation, augments Monocytopoiesis and alters the beta-adrenergic receptor (AR) profile of bone marrow monocyte committed progenitors. This provides an impetus to study AR expression in hematopoietic progenitors along myeloid lineage. Using FACS analysis and confocal microscopy, we report the expression of alpha1-, alpha2- and beta(2)-AR in enriched populations of ER-MP209(+) and ER-MP12(+) myeloid progenitors, CD117(+) and CD34(+) multi-potential progenitors and more importantly pluripotent stem cells suggesting a plausible role for catecholamine in hematopoietic development.
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Thermal injury and sepsis modulates beta-adrenergic receptors and cAMP responses in monocyte-committed bone marrow cells.
Journal of neuroimmunology, 2005Co-Authors: Kuzhali Muthu, Ravi Shankar, Jiangping Deng, Fred Romano, Richard Gamelli, Stephen B JonesAbstract:We have previously reported that adrenergic stimulation enhances Monocytopoiesis following experimental burn injury and sepsis (BI/S). In the present work we measured beta-adrenergic receptor number and affinity in bone marrow committed monocyte progenitor cells (CD59(+)) following BI/S. We find that BI/S treatment significantly decreased monocyte progenitor cell beta-adrenergic receptors but significantly increased receptor binding affinity and isoproterenol-stimulated cAMP production. CD14 expression in macrophages derived in vitro from CD59(+) cells following BI/S was significantly increased by epinephrine and this change was blocked by beta(2)-adrenergic receptor antagonist. PCR analysis suggests the presence of beta(2)- but not beta(1)-adrenergic receptors. Enhanced adrenergic receptor signaling in CD59(+) bone marrow cells following BI/S may be important in macrophage development.
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Myeloid commitment shifts toward Monocytopoiesis after thermal injury and sepsis
Annals of surgery, 2001Co-Authors: Steve Santangelo, Richard L. Gamelli, Ravi ShankarAbstract:Activation of monocytes and macrophages and the resultant overproduction of proinflammatory cytokines are considered central to the pathology of sepsis. 1 The correlation between the severity of sepsis and the overproduction of the cytokines, such as tumor necrosis factor-α, interleukin-1, and interleukin-6, is well documented, so much so that several clinical trials were initiated to verify whether negating their levels in patients with sepsis would lead to improvements in survival. 2,3 Although unequivocal evidence for the efficacy of these treatments is still forthcoming, thus far the results from these multicenter trials indicate that these treatment modalities do not result in a significant survival advantage in patients with sepsis. 2,3 Although much is known about the potential role of monocyte and macrophage activation in the pathogenesis of sepsis, few studies have focused on the production of monocytes by the bone marrow under the same conditions. To understand the role of monocytes and macrophages in sepsis and severe injury states, both activation and rate of production are equally important. The origin of monocytes and macrophages is the bone marrow, wherein the monocytic progenitors give rise to monocytes, which differentiate in tissues to become macrophages. 4 In the bone marrow, Monocytopoiesis is coordinated through a series of tightly coupled proliferation and differentiation signals that allow the maturation of the pluripotent stem cells into monocytes. 5 The stem cells give rise to the bipotential progenitor colony-forming unit granulocyte–macrophage (CFU-GM), which can differentiate into either granulocytes or monocytes. 6 The development of monocytes from CFU-GM is predominantly under the influence of macrophage colony-stimulating factor (M-CSF). 7 Studies with op/op mice, which have a null mutation at the M-CSF locus, and granulocyte/macrophage colony-stimulating factor (GM-CSF) knockout mice clearly establish M-CSF as the essential growth factor for monocyte production. 8–10 Trauma and thermal injury have been shown to be associated with increased production of bone marrow CFU-GM in animal models. 11,12 Further, similar increases in CFU-GM have also been demonstrated in patients with various infections and peritonitis. 13 We have previously demonstrated an arrest in granulocyte lineage maturation and severe neutropenia in burn sepsis. 14 Whether monocyte production is also similarly downregulated or whether CFU-GM is preferentially directed toward the monocytic lineage in thermal injury and sepsis remains an open question. In addition, evidence suggesting the potential mechanism that regulates the development of progenitors along the monocyte lineage under these conditions is also lacking. In the present study, we performed experiments designed to test the status of bone marrow Monocytopoiesis in a murine thermal injury and sepsis model. To achieve this goal, we need reliable cell surface markers whose expression pattern will allow us to determine the progression of monocyte progenitor development. Recently, the antibodies to cell surface markers ER-MP12 and ER-MP20 have been used for the identification of myeloid committed progenitors and murine monocyte/macrophage development. 15–17 Through the expression pattern of these monocyte lineage-specific developmental antigens and clonogenic assays, we demonstrated that Monocytopoiesis is augmented by thermal injury and sepsis. We present concrete evidence that the increase in Monocytopoiesis is accompanied by a parallel increase in the expression of macrophage colony-stimulating factor receptor (M-CSFR), encoded by c-fms, thus providing a potential mechanism for our observations.
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Prostaglandin E2 receptor antagonist (SC-19220) treatment restores the balance to bone marrow myelopoiesis after burn sepsis.
The Journal of trauma, 2000Co-Authors: Steve Santangelo, Richard L. Gamelli, Margo Shoup, Ravi ShankarAbstract:Although prostaglandin E2 (PGE2) has been shown to be immunosuppressive, its role in the development of specific bone marrow myeloid lineages after thermal injury and sepsis has yet to be elucidated. The purpose of this study was to demonstrate that alterations in bone marrow progenitor proliferation favoring Monocytopoiesis in burn sepsis can be restored by blocking the cellular interactions of PGE2. A murine model of burn sepsis with and without treatment with SC-19220, a PGE2 receptor antagonist, was used to determine peripheral monocyte and neutrophil counts as well as the colony forming potential of colony-stimulating factor responsive bone marrow progenitors. Burn sepsis augmented the growth of the early colony-forming unit granulocyte-macrophage and monocyte progenitors and the number of circulating monocytes, whereas granulocyte progenitors and circulating neutrophils demonstrated an opposite response. Treatment with SC-19220 nearly reversed these alterations. These data indicate that abrogating PGE2's actions during burn sepsis can restore the balance in bone marrow granulocyte and monocyte production, further consolidating the pivotal role PGE2 plays in the pathogenesis of burn sepsis.
Soumyadeep Dey - One of the best experts on this subject based on the ideXlab platform.
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The TAL1/SCL transcription factor regulates cell cycle progression and proliferation in differentiating murine bone marrow monocyte precursors.
Molecular and cellular biology, 2010Co-Authors: Soumyadeep Dey, David J. Curtis, Stephen M. Jane, Stephen J. BrandtAbstract:Monocytopoiesis involves the stepwise differentiation in the bone marrow (BM) of common myeloid precursors (CMPs) to monocytes. The basic helix-loop-helix transcription factor TAL1/SCL plays a critical role in other hematopoietic lineages, and while it had been reported to be expressed by BM-derived macrophages, its role in Monocytopoiesis had not been elucidated. Using cell explant models of monocyte/macrophage (MM) differentiation, one originating with CMPs and the other from more committed precursors, we characterized the phenotypic and molecular consequences of inactivation of Tal1 expression ex vivo. While Tal1 knockout had minimal effects on cell survival and slightly accelerated terminal differentiation, it profoundly inhibited cell proliferation and decreased entry into and traversal of the G(1) and S phases. In conjunction, steady-state levels of p16(Ink4a) mRNA were increased and those of Gata2 mRNA decreased. Chromatin immunoprecipitation analysis demonstrated the association of Tal1 and E47, one of its E protein DNA-binding partners, with an E box-GATA sequence element in intron 4 of the Gata2 gene and with three E boxes upstream of p16(Ink4a). Finally, wild-type Tal1, but not a DNA binding-defective mutant, rescued the proliferative defect in Tal1-null MM precursors. These results document the importance of this transcription factor in cell cycle progression and proliferation during Monocytopoiesis and the requirement for direct DNA binding in these processes.
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Novel Role of the TAL1/SCL Transcription Factor in Murine Monocytopoiesis.
Blood, 2008Co-Authors: Soumyadeep Dey, David J. Curtis, Stephen M. Jane, Stephen J. BrandtAbstract:Abstract The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev., 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-ã-activated macrophages. To elucidate Tal1’sfunctions in murine Monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1fl/fl/lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1-null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1,PU.1, Csfr1, Msr1 (mouse scavenger receptor), Cd68, or Il6ra. Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining ofTal1-null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.
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novel role of the tal1 scl transcription factor in murine Monocytopoiesis
Blood, 2008Co-Authors: Soumyadeep Dey, David J. Curtis, Stephen M. Jane, Stephen J. BrandtAbstract:The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev., 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-a-activated macrophages. To elucidate Tal1’sfunctions in murine Monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1 fl/fl /lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1 -null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1 , PU.1 , Csfr1 , Msr1 (mouse scavenger receptor), Cd68 , or Il6ra . Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining of Tal1 -null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1 hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.
Cesare Peschle - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Monocytopoiesis by microRNAs.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Laura Fontana, Antonio Sorrentino, Cesare PeschleAbstract:MicroRNAs (miRNAs or miRs) are approximately 22 nt single-stranded noncoding RNAs that control gene expression in eukaryotes. miRNAs play an essential role in all basic cellular processes including cell development, proliferation, differentiation, and apoptosis. Importantly, miRNAs regulate hematopoietic progenitor cells differentiation toward the different hematopoietic lineages. This occurs through the regulation of key factors involved in hematopoiesis (e.g., transcription factors, growth factor receptors). We, hereby, describe how to investigate the role of miRNAs in Monocytopoiesis.
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micrornas 17 5p 20a 106a control Monocytopoiesis through aml1 targeting and m csf receptor upregulation
Nature Cell Biology, 2007Co-Authors: Laura Fontana, Paolo Greco, Serena Racanicchi, Francesca Liuzzi, Elvira Pelosi, Ugo Testa, Ercole Brunetti, Francesco Grignani, Carlo M. Croce, Cesare PeschleAbstract:MicroRNAs 17-5p–20a–106a control Monocytopoiesis through AML1 targeting and M-CSF receptor upregulation
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MicroRNAs 17-5p–20a–106a control Monocytopoiesis through AML1 targeting and M-CSF receptor upregulation
Nature cell biology, 2007Co-Authors: Laura Fontana, Paolo Greco, Serena Racanicchi, Francesca Liuzzi, Elvira Pelosi, Ugo Testa, Ercole Brunetti, Francesco Grignani, Carlo M. Croce, Cesare PeschleAbstract:We investigated the role of microRNAs (miRNA) 17-5p, 20a and 106a in monocytic differentiation and maturation. In unilineage monocytic culture generated by haematopoietic progenitor cells these miRNAs are downregulated, whereas the transcription factor acute myeloid leukaemia-1 (AML1; also known as Runt-related transcription factor 1, Runx1) is upregulated at protein but not mRNA level. As miRNAs 17-5p, 20a and 106a bind the AML1 mRNA 3'UTR, their decline may unblock AML1 translation. Accordingly, transfection with miRNA 17-5p-20a-106a suppresses AML1 protein expression, leading to M-CSF receptor (M-CSFR) downregulation, enhanced blast proliferation and inhibition of monocytic differentiation and maturation. Treatment with anti-miRNA 17-5p, 20a and 106a causes opposite effects. Knockdown of AML1 or M-CSFR by short interfering RNA (siRNA) mimics the action of the miRNA 17-5p-20a-106a, confirming that these miRNAs target AML1, which promotes M-CSFR transcription. In addition, AML1 binds the miRNA 17-5p-92 and 106a-92 cluster promoters and transcriptionally inhibits the expression of miRNA 17-5p-20a-106a. These studies indicate that Monocytopoiesis is controlled by a circuitry involving sequentially miRNA 17-5p-20a-106a, AML1 and M-CSFR, whereby miRNA 17-5p-20a-106a function as a master gene complex interlinked with AML1 in a mutual negative feedback loop.
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MicroRNAs 17-5p/20a/106a Function as a Master Gene Complex Controlling Monocytopoiesis through AML1 Targeting.
Blood, 2006Co-Authors: Laura Fontana, Paolo Greco, Francesca Liuzzi, Elvira Pelosi, Ugo Testa, Ercole Brunetti, Carlo M. Croce, Cesare PeschleAbstract:Abstract MicroRNAs (miRs) are a novel regulatory class of non-coding, single-stranded RNAs of ~ 22 nucleotides, identified in plants and animals. MiRs repress protein expression at post-transcriptional level, mostly through base pairing to the 3′ untranslated region (UTR) of the target mRNA, thus leading to its degradation and/or reduced translation. MiRs have been shown to control basic biological functions, such as cell proliferation and differentiation. However, little is known on their role in hematopoiesis. Furthermore, in diverse types of cancer miRs may function as “oncomirs”. We investigated the role of miR-17-5p, -20a and -106a, located in the miR-17-5p-92 and miR-106a-92 clusters respectively, in monocytic-macrophage (Mo) differentiation-maturation. In unilineage Mo culture generated by cord blood (CB) hematopoietic progenitor cells (HPC), these miRs are gradually and markedly downmodulated, whereas AML1 is upmodulated at protein but not at mRNA level. The acute myeloid leukaemia-1 protein (AML1, also known as Runt-related transcription factor 1, Runx1) is the DNA-binding subunit of the hematopoietic transcription factor CBF, which plays a pivotal role in Mo differentiation-maturation. Luciferase assay shows that miR-17/20/106 bind the AML1 mRNA 3′UTR, implying that during Monocytopoiesis their decline unblocks AML1 translation. Mo culture transfection with miR-17/20/106 stimulates blast cell proliferation and inhibits differentiation-maturation: this is coupled with AML1 translation blockade, leading in turn to inhibition of M-CSF receptor (M-CSFR) expression. Equivalent results were observed in Mo clonogenic culture of HPCs. Knockdown of endogenous miR-17/20/106 by anti-miR oligonucleotide transfection causes opposite effects. Altoghether, our studies indicate that miR-17/20/106 downmodulation during Monocytopoiesis “unblocks” the translation of AML1, which in turn transactivates the M-CSFR, thereby allowing Mo differentiation and maturation. We hence propose that these miRs function as a master gene complex underlying the Monocytopoiesis gene program, at least in part through the regulation of two pivotal proteins, AML1 and M-CSFR. This study also sheds light on other relevant aspects:the regulatory action of miR-17/20/106 action on AML1 mRNA is restricted to the Mo lineage, implying a role for lineage-specific factor(s) to modulate the interaction of these miRs with their mRNA target;the subtle complexity of mechanisms controlling the transcription/processing of the miR-17-5p-92 cluster, implied by the differential expression patterns of the miRs comprised in the 17-5p-92 polycistron in Mo culture;the leukemogenesis mediated by AML1 fusion proteins.
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micrornas 17 5p 20a 106a function as a master gene complex controlling Monocytopoiesis through aml1 targeting
Blood, 2006Co-Authors: Laura Fontana, Paolo Greco, Francesca Liuzzi, Elvira Pelosi, Ugo Testa, Ercole Brunetti, Carlo M. Croce, Cesare PeschleAbstract:MicroRNAs (miRs) are a novel regulatory class of non-coding, single-stranded RNAs of ~ 22 nucleotides, identified in plants and animals. MiRs repress protein expression at post-transcriptional level, mostly through base pairing to the 3′ untranslated region (UTR) of the target mRNA, thus leading to its degradation and/or reduced translation. MiRs have been shown to control basic biological functions, such as cell proliferation and differentiation. However, little is known on their role in hematopoiesis. Furthermore, in diverse types of cancer miRs may function as “oncomirs”. We investigated the role of miR-17-5p, -20a and -106a, located in the miR-17-5p-92 and miR-106a-92 clusters respectively, in monocytic-macrophage (Mo) differentiation-maturation. In unilineage Mo culture generated by cord blood (CB) hematopoietic progenitor cells (HPC), these miRs are gradually and markedly downmodulated, whereas AML1 is upmodulated at protein but not at mRNA level. The acute myeloid leukaemia-1 protein (AML1, also known as Runt-related transcription factor 1, Runx1) is the DNA-binding subunit of the hematopoietic transcription factor CBF, which plays a pivotal role in Mo differentiation-maturation. Luciferase assay shows that miR-17/20/106 bind the AML1 mRNA 3′UTR, implying that during Monocytopoiesis their decline unblocks AML1 translation. Mo culture transfection with miR-17/20/106 stimulates blast cell proliferation and inhibits differentiation-maturation: this is coupled with AML1 translation blockade, leading in turn to inhibition of M-CSF receptor (M-CSFR) expression. Equivalent results were observed in Mo clonogenic culture of HPCs. Knockdown of endogenous miR-17/20/106 by anti-miR oligonucleotide transfection causes opposite effects. Altoghether, our studies indicate that miR-17/20/106 downmodulation during Monocytopoiesis “unblocks” the translation of AML1, which in turn transactivates the M-CSFR, thereby allowing Mo differentiation and maturation. We hence propose that these miRs function as a master gene complex underlying the Monocytopoiesis gene program, at least in part through the regulation of two pivotal proteins, AML1 and M-CSFR. This study also sheds light on other relevant aspects: the regulatory action of miR-17/20/106 action on AML1 mRNA is restricted to the Mo lineage, implying a role for lineage-specific factor(s) to modulate the interaction of these miRs with their mRNA target; the subtle complexity of mechanisms controlling the transcription/processing of the miR-17-5p-92 cluster, implied by the differential expression patterns of the miRs comprised in the 17-5p-92 polycistron in Mo culture; the leukemogenesis mediated by AML1 fusion proteins.
David J. Curtis - One of the best experts on this subject based on the ideXlab platform.
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The TAL1/SCL transcription factor regulates cell cycle progression and proliferation in differentiating murine bone marrow monocyte precursors.
Molecular and cellular biology, 2010Co-Authors: Soumyadeep Dey, David J. Curtis, Stephen M. Jane, Stephen J. BrandtAbstract:Monocytopoiesis involves the stepwise differentiation in the bone marrow (BM) of common myeloid precursors (CMPs) to monocytes. The basic helix-loop-helix transcription factor TAL1/SCL plays a critical role in other hematopoietic lineages, and while it had been reported to be expressed by BM-derived macrophages, its role in Monocytopoiesis had not been elucidated. Using cell explant models of monocyte/macrophage (MM) differentiation, one originating with CMPs and the other from more committed precursors, we characterized the phenotypic and molecular consequences of inactivation of Tal1 expression ex vivo. While Tal1 knockout had minimal effects on cell survival and slightly accelerated terminal differentiation, it profoundly inhibited cell proliferation and decreased entry into and traversal of the G(1) and S phases. In conjunction, steady-state levels of p16(Ink4a) mRNA were increased and those of Gata2 mRNA decreased. Chromatin immunoprecipitation analysis demonstrated the association of Tal1 and E47, one of its E protein DNA-binding partners, with an E box-GATA sequence element in intron 4 of the Gata2 gene and with three E boxes upstream of p16(Ink4a). Finally, wild-type Tal1, but not a DNA binding-defective mutant, rescued the proliferative defect in Tal1-null MM precursors. These results document the importance of this transcription factor in cell cycle progression and proliferation during Monocytopoiesis and the requirement for direct DNA binding in these processes.
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Novel Role of the TAL1/SCL Transcription Factor in Murine Monocytopoiesis.
Blood, 2008Co-Authors: Soumyadeep Dey, David J. Curtis, Stephen M. Jane, Stephen J. BrandtAbstract:Abstract The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev., 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-ã-activated macrophages. To elucidate Tal1’sfunctions in murine Monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1fl/fl/lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1-null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1,PU.1, Csfr1, Msr1 (mouse scavenger receptor), Cd68, or Il6ra. Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining ofTal1-null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.
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novel role of the tal1 scl transcription factor in murine Monocytopoiesis
Blood, 2008Co-Authors: Soumyadeep Dey, David J. Curtis, Stephen M. Jane, Stephen J. BrandtAbstract:The basic helix-loop-helix (bHLH) transcription factor TAL1/SCL plays a critical role in hematopoiesis and vascular remodeling. A mouse Tal1 cDNA was first cloned from a bone marrow (BM) macrophage cDNA library, and we and others observed expression ofTal1 protein by BM mononuclear cells. To characterize Tal1 expression during monocyte/macrophage differentiation, we isolated common myeloid precursors (CMPs) from BM of 3-5 week old C57BL/6J mice and induced them to terminally differentiate according to a published method (Genes & Dev., 16:1721, 2002). Using real-time PCR analysis,Tal1 mRNA was expressed in a biphasic pattern from CMP to post-mitotic macrophage, including lipopolysaccharide- and interferon-a-activated macrophages. To elucidate Tal1’sfunctions in murine Monocytopoiesis we deleted the Tal1 gene in murine BM monocytes and monocytic precursors in culture. To that end, C57BL/6 mice with loxP sequences flanking the third coding exon of Tal1 were bred with C57BL/6 mice with a lacZ gene replacing Tal1 coding exons 1, 2, and 3. Tal1 fl/fl /lacZ progeny were identified by PCR genotyping, and BM mononuclear cells were cultured with mouse interleukin-3 and macrophage colony-stimulating factor (M-CSF). To render the cells Tal1 -null, Cre coding sequences were introduced with the MSCV-GFP retroviral vector and GFP-positive cells were then sorted and cultured with M-CSF alone. Real-time PCR analysis showed near-total abolition of Tal1 mRNA expression in Cre-transduced relative to vector-transduced cells. Gene expression analysis for other transcripts showed an approximately 4-foldreduction in Gata2 expression over the same culture period but no difference in Aml1 , PU.1 , Csfr1 , Msr1 (mouse scavenger receptor), Cd68 , or Il6ra . Biologically, the most significant effect of Tal1 knockout was on cell number, which increased by 80% in control cells but not at all in Tal1-null cells. Transduction of wild-type BM monocytes with MSCV-GFP-Cre (or the parental MSCV-GFP) vector had no effect on cell proliferation, precluding any nonspecific or toxic effect of Cre (or retroviral infection) in this cell type. Dye dilution analysis of virus-transduced cells with the fluorescent membrane-intercalating dye PKH26 revealed a delay and absolute reduction in proliferation of Tal1-null compared to control cells. In contrast, little or no difference was noted in annexin V staining of Tal1 -null compared to heterozygous knockout (knock-in) cells, indicating a lack of effect on apoptosis. Finally, serial analysis of CD31 and Ly6c expression in differentiating Tal1 hemizygous and nullizygous BM monocytes showed that loss of Tal1 caused a slight acceleration in terminal monocyte-macrophage differentiation. In summary, these studies confirm our earlier finding that the Tal1 gene is expressed in differentiating mouse BMmonocytes. In addition, they reveal a novel function of this bHLH transcription factor in proliferation of murine monocyte/macrophage precursors. Finally, they place Tal1upstream of Gata2 in cells of this lineage.