Biomedical subjects

Elizabeth A Eklund

Publications and source records attributed to Elizabeth A Eklund.

13 recordsLinked to original sources

PubMed2007

PU.1, interferon regulatory factor (IRF) 2, and the interferon consensus sequence-binding protein (ICSBP/IRF8) cooperate to activate NF1 transcription in differentiating myeloid cells.

Nf1 (neurofibromin 1) is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells. In previous studies, we found that the interferon consensus sequence-binding protein (ICSBP; also referred to as interferon regulatory factor 8) activates transcription of the gene encoding Nf1 (the NF1 gene) in differentiating myeloid cells. We also found that NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the interferon regulatory factor (IRF) domain of ICSBP/IRF8. In this study, we found that ICSBP/IRF8 cooperates with PU.1 and interferon regulatory factor 2 to activate a composite ets/IRF-cis element in the NF1 promoter. We found that PU.1 binds directly to the NF1-cis element, and DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the cis element. This interaction requires cytokine-induced phosphorylation of specific serine residues in the PU.1 PEST domain and of a conserved tyrosine residue in the IRF domain of IRF2. We found that ICSBP/IRF8 interaction with the NF1-cis element requires pre-binding of PU.1 and IRF2. The conserved IRF domain tyrosine in ICSBP/IRF8 is required for interaction with the DNA-bound PU.1-IRF2 heterodimer. NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation. Therefore, these studies identify a target gene for the previously observed tumor-suppressor effect of PU.1. Additionally, these studies identify a tumor-suppressor function for the "oncogenic" transcription factor, IRF2.

Cell Differentiation↗

PubMed2006

Activation of SHP2 protein-tyrosine phosphatase increases HoxA10-induced repression of the genes encoding gp91(PHOX) and p67(PHOX).

The CYBB and NCF2 genes encode the phagocyte oxidase proteins gp91(PHOX) and p67(PHOX), respectively. These genes are transcribed after the promyelocyte stage of differentiation, and transcription continues until cell death. In undifferentiated myeloid cells, homologous cis-elements in the CYBB and NCF2 genes are repressed by the homeodomain transcription factor HoxA10. During cytokine-induced myelopoiesis, tyrosine phosphorylation of HoxA10 decreases binding affinity for the CYBB and NCF2 cis-elements. This abrogates HoxA10-induced transcriptional repression as differentiation proceeds. Therefore, mechanisms involved in differentiation stage-specific HoxA10 tyrosine phosphorylation are of interest because HoxA10 phosphorylation modulates myeloid-specific gene transcription. In this study, we found that HoxA10 is a substrate for SHP2 protein-tyrosine phosphatase in undifferentiated myeloid cells. In contrast, HoxA10 is a substrate for a constitutively active mutant form of SHP2 in both undifferentiated and differentiating myeloid cells. Expression of such SHP2 mutants results in persistent HoxA10 repression of CYBB and NCF2 transcription during myelopoiesis. Both HoxA10 overexpression and activating SHP2 mutations have been described in human myeloid malignancies. Therefore, our results suggest that these mutations could cooperate, leading to decreased myeloid-specific gene transcription and functional differentiation block in myeloid cells with both defects.

Animals↗

PubMed2006

The role of HOX genes in myeloid leukemogenesis.

PURPOSE OF REVIEW: Homeodomain proteins of the Hox family play an important role in regulation of normal hematopoiesis. Substantial evidence also indicates that abnormal Hox protein expression is functionally significant in the pathogenesis of acute myeloid malignancies. The purpose of this review is to outline recent progress in understanding molecular mechanisms involved in Hox regulation of myelopoiesis and myeloid leukemogenesis. RECENT FINDINGS: Since Hox proteins function as transcription factors, recent studies have focused on identifying Hox target genes. Various approaches to this problem have been taken, including high throughput screening techniques. In these studies, expression profiles of hematopoietic cells overexpressing various Hox proteins have been analyzed to obtain initial information about potential target genes. Identification of common and unique sets of target genes for various Hox proteins will shed light on function and regulation of the Hox code in developing hematopoietic cells. SUMMARY: Recent studies have generated some intriguing information about potential Hox target genes involved in myelopoiesis and leukemogenesis. A number of issues regarding Hox protein function are unresolved, however. These issues include determining whether the effects of various Hox proteins are redundant versus antagonistic, identifying mechanisms which regulate Hox protein function and mechanisms by which Hox proteins modulate target gene transcription in a context-dependent manner.

Animals↗

PubMed2006

Leukemia-associated, constitutively active mutants of SHP2 protein tyrosine phosphatase inhibit NF1 transcriptional activation by the interferon consensus sequence binding protein.

Deficiency in either the interferon consensus sequence binding protein (ICSBP) or neurofibromin 1 (Nf1) increases the proliferative response of myeloid progenitor cell to hematopoietic cytokines. Consistent with this, we previously demonstrated that ICSBP activates transcription of the gene encoding Nf1 (the NF1 gene). In the studies presented here, we determine that ICSBP tyrosine phosphorylation is necessary for the activation of NF1 transcription. Since ICSBP is tyrosine phosphorylated in response to hematopoietic cytokines, these studies identify a novel pathway by which cytokine-induced posttranslational modification of ICSBP results in NF1 transcription. Nf1 subsequently inactivates cytokine-activated Ras, thereby creating a negative feedback mechanism for cytokine-induced proliferation. In these studies, we also determine that ICSBP is a substrate for SHP2 protein tyrosine phosphatase (SHP2-PTP). We find that wild-type SHP2-PTP dephosphorylates ICSBP only in undifferentiated myeloid cells. In contrast, a leukemia-associated, constitutively activated mutant form of SHP2-PTP dephosphorylates ICSBP in both myeloid progenitors and differentiating myeloid cells. Activated SHP2-PTP mutants thereby inhibit ICSBP-dependent NF1 transcription, impairing this negative feedback mechanism on cytokine-activated Ras. Therefore, these studies suggest that leukemia-associated ICSBP deficiency cooperates with leukemia-associated activating mutants of SHP2-PTP to contribute to the proliferative phenotype in myeloid malignancies.

Animals↗

PubMed2005

HoxA10 represses transcription of the gene encoding p67phox in phagocytic cells.

p67(phox) and gp91(phox) are components of the phagocyte-specific respiratory burst oxidase that are encoded by the NCF2 and CYBB genes, respectively. These genes are transcribed exclusively in myeloid cells that have differentiated beyond the promyelocyte stage. In mature phagocytes, NCF2 and CYBB transcription continues until cell death and further increases in response to IFN-gamma and other inflammatory mediators. Because p67(phox) and gp91(phox) expression profiles are similar, we hypothesize that common transcription factors interact with homologous cis elements in the CYBB and NCF2 genes to coordinate transcription. Previously, we identified a negative CYBB promoter cis element that is repressed by the homeodomain transcription factor HoxA10. We found that transcriptional repression requires HoxA10-dependent recruitment of histone deacetylase activity to the CYBB cis element. In response to IFN-gamma, phosphorylation of two tyrosine residues in the HoxA10 homeodomain decreases binding to CYBB promoter, thereby abrogating HoxA10-mediated repression. In the current studies, we investigate the possibility that HoxA10 similarly represses NCF2 transcription. We identify a sequence in the NCF2 promoter that is homologous to the HoxA10-binding CYBB cis element. We find that this NCF2 promoter sequence functions as a negative cis element that is repressed by HoxA10 in a tyrosine phosphorylation and histone deacetylase-dependent manner. Our results suggest that cytokine-stimulated pathways regulate HoxA10-mediated repression of the CYBB and NCF2 genes in differentiating myeloid cells and in mature phagocytes during the inflammatory response. Because p67(phox) and gp91(phox) are rate-limiting components for respiratory burst activity, our studies may identify rational therapeutic targets to modulate free radical generation in pathological conditions.

Animals↗

PubMed2005

Differential regulation of the p70 S6 kinase pathway by interferon alpha (IFNalpha) and imatinib mesylate (STI571) in chronic myelogenous leukemia cells.

The precise mechanisms by which imatinib mesylate (STI571) and interferon alpha (IFNalpha) exhibit antileukemic effects are not known. We examined the effects of IFNs or imatinib mesylate on signaling pathways regulating initiation of mRNA translation in BCR-ABL-expressing cells. Treatment of IFN-sensitive KT-1 cells with IFNalpha resulted in phosphorylation/activation of mammalian target of rapamycin (mTOR) and downstream activation of p70 S6 kinase. The IFN-activated p70 S6 kinase was found to regulate phosphorylation of S6 ribosomal protein, which regulates translation of mRNAs with oligopyrimidine tracts in the 5'-untranslated region. In addition, IFNalpha treatment resulted in an mTOR- and/or phosphatidyl-inositol 3'(PI 3') kinase-dependent phosphorylation of 4E-BP1 repressor of mRNA translation on sites that are required for its deactivation and dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. In contrast to the effects of IFNs, imatinib mesylate suppressed p70 S6 kinase activity, consistent with inhibition of BCR-ABL-mediated activation of the mTOR/p70 S6 kinase pathway. Moreover, the mTOR inhibitor rapamycin enhanced the suppressive effects of imatinib mesylate on primary leukemic granulocyte macrophage-colony-forming unit (CFU-GM) progenitors from patients with chronic myelogenous leukemia (CML). Taken altogether, our data demonstrate that IFNs and imatinib mesylate differentially regulate PI 3' kinase/mTOR-dependent signaling cascades in BCR-ABL-transformed cells, consistent with distinct effects of these agents on pathways regulating mRNA translation. They also support the concept that combined use of imatinib mesylate with mTOR inhibitors may be an appropriate future therapeutic strategy for the treatment of CML.

5' Untranslated Regions↗

PubMed2005

HOXA9 activates transcription of the gene encoding gp91Phox during myeloid differentiation.

The CYBB gene encodes gp91Phox; a component of the phagocyte respiratory burst oxidase. CYBB transcription is restricted to myeloid cells differentiated beyond the promyelocyte stage. In undifferentiated myeloid cells, the homeodomain (HD) transcription factor HoxA10 represses CYBB transcription via a cis element in the proximal promoter. During myelopoiesis, phosphorylation of conserved tyrosine residues in the HD decreases HoxA10 binding to this CYBB cis element. In the current studies, we found HoxA9 activates CYBB transcription in differentiated myeloid cells via the same cis element. We find HoxA9-mediated CYBB-transcription requires Pbx1 but is inhibited by Meis1. Additionally, phosphorylation of the conserved HD tyrosines increases HoxA9 binding to the CYBB promoter. The HOXA9 gene is involved in leukemia-associated translocations with the gene encoding Nup98, a nucleopore protein. We find expression of a Nup98-hoxA9 fusion protein blocks HoxA9-induced CYBB transcription in differentiating myeloid cells. In comparison to HoxA9, Nup98-hoxA9 has greater binding affinity for the CYBB cis element, but binding is not altered by HD tyrosine phosphorylation. Therefore, these studies identify CYBB as a common target gene repressed by HoxA10 and activated by HoxA9. These studies also suggest overexpression of Meis1 or Nup98-hoxA9 represses myeloid-specific gene transcription, thereby contributing to differentiation block in leukemogenesis.

Blotting, Western↗

PubMed2004

JAK2 is necessary and sufficient for interferon-gamma-induced transcription of the gene encoding gp91PHOX.

During the inflammatory response, interferon-gamma (IFN-gamma) increases transcription of the gene encoding gp91PHOX, a respiratory burst oxidase component. This gene (referred to as the CYBB gene) is transcribed in phagocytic cells differentiated beyond the promyelocyte stage, and transcription continues until cell death. Previous investigations identified a positive regulatory element in the proximal CYBB promoter referred to as the hematopoiesis-associated factor 1 (HAF1)-cis element. This element is activated by a multiprotein complex, which includes the IFN consensus sequence-binding protein (ICSBP). Interaction of this complex with the HAF1-cis element requires ICSBP tyrosine phosphorylation, which is induced by IFN-gamma stimulation of phagocytic cells. Previous studies also identified a negative cis element in the CYBB promoter. This element is repressed by the homeodomain protein HoxA10. HoxA10 tyrosine phosphorylation, which occurs in response to IFN-gamma, decreases HoxA10 DNA binding and therefore repression of CYBB transcription. In these studies, we determine Janus tyrosine kinase 2 (JAK2) activation is necessary and sufficient for IFN-gamma-induced CYBB transcription in phagocytic cells and also for ICSBP and HoxA10 tyrosine phosphorylation. Consistent with these results, we find JAK2 activation is sufficient to induce ICSBP interaction with the HAF1 element and abolish HoxA10 binding to the CYBBrepressor element. Therefore, these findings provide direct demonstration of JAK2 dependence of IFN-gamma-induced CYBB transcription. In addition, these results identify a mechanism mediating this effect.

Chloramphenicol↗

PubMed2004

The interferon consensus sequence-binding protein activates transcription of the gene encoding neurofibromin 1.

Deficiency of the interferon consensus sequence-binding protein (ICSBP) is associated with increased myeloid cell proliferation in response to hematopoietic cytokines. However, previously identified ICSBP target genes do not indicate a mechanism for this "cytokine hypersensitivity." In these studies, we identify the gene encoding neurofibromin 1 (Nf1) as an ICSBP target gene, by chromatin immunoprecipitation. Additionally, we find decreased Nf1 expression in bone marrow-derived myeloid cells from ICSBP-/- mice. Since Nf1 deficiency is also associated with cytokine hypersensitivity, our results suggested that NF1 is a functionally significant ICSBP target gene. Consistent with this, we find that the hypersensitivity of ICSBP-/- myeloid cells to granulocyte monocyte colony-stimulating factor (GM-CSF) is reversed by expression of the Nf1 GAP-related domain. We also find that treatment of ICSBP-deficient myeloid cells with monocyte colony-stimulating factor (M-CSF) results in sustained Ras activation, ERK phosphorylation, and proliferation associated with impaired Nf1 expression. These M-CSF effects are reversed by ICSBP expression in ICSBP-/- cells. Consistent with this, we find that ICSBP activates the NF1 promoter in myeloid cell line transfectants and identify an ICSBP-binding NF1 cis element. Therefore, the absence of ICSBP leads to Nf1 deficiency, impairing down-regulation of Ras activation by GM-CSF or M-CSF. These results suggest that one mechanism of increased myeloid proliferation, in ICSBP-deficient cells, is decreased NF1 gene transcription. This novel ICSBP function provides insight into regulation of myelopoiesis under normal conditions and in myeloproliferative disorders.

Animals↗

PubMed2004

Hematology grants workshop.

Obtaining research funding is of utmost importance for academic hematologists, especially those engaged in basic science research. Several funding mechanisms are available to researchers working in the fields of Hematology and/or Oncology. This chapter will discuss various issues relating to applications for National Institutes of Health (NIH) R01 grants, the classic funding mechanism for investigator-initiated research. It will also discuss other opportunities for funding from other government agencies, including the Department of Veterans Affairs and the Department of Defense. We will also provide a summary of the process by which NIH R01 applications are reviewed and some guidelines and tips for the successful writing of R01 grants or their equivalents from other agencies.

Academic Medical Centers↗

PubMed2003

HoxA10 represses gene transcription in undifferentiated myeloid cells by interaction with histone deacetylase 2.

The homeodomain proteins, HoxA10 and Pbx1a, interact with negative cis elements to repress gene transcription in undifferentiated myeloid cells. The CYBB and NCF2 genes, which encode the gp91PHOX and p67PHOX proteins, are two such HoxA10-Pbx1a target genes. In previous studies, we found that HoxA10-Pbx1a represses transcription of these genes by two mechanisms: competition for DNA binding with transcriptional activators and endogenous repression activity. In these studies, we identify a novel molecular mechanism of endogenous transcriptional repression by HoxA10-Pbx1a. Endogenous repression activity of other Hox-Pbx1a complexes requires recruitment of transcriptional co-repressor proteins by Pbx1a. In contrast, our investigations have determined that HoxA10 has Pbx1a-independent endogenous repression activity. We find that this transcriptional repression activity is abrogated by histone deacetylase inhibitors, suggesting involvement of co-repressor proteins. Consistent with this, we identify HoxA10 amino acids 224-249 as a Pbx1-independent repression domain, which interacts with histone deacetylase 2. We have determined that this HoxA10 domain is not conserved with other Abd Hox proteins, although homology exists with other transcription factors and co-repressors. Understanding the roles different Hox proteins play in myeloid differentiation is a challenging problem. Our results suggest that insight into this problem can be obtained from biochemical characterization of the various molecular mechanisms of Hox protein function.

Blotting, Northern↗

PubMed2002

SHP1 protein-tyrosine phosphatase regulates HoxA10 DNA binding and transcriptional repression activity in undifferentiated myeloid cells.

The homeodomain protein HoxA10 interacts with negative cis elements to repress gene transcription in undifferentiated myeloid cells. The CYBB and NCF2 genes, which encode the gp91(PHOX) and p67(PHOX) proteins, are two such HoxA10 target genes. During interferon gamma-induced myeloid differentiation, tyrosine phosphorylation decreases HoxA10 DNA binding affinity and transcriptional repression. Therefore, decreased HoxA10 repression contributes to increased CYBB and NCF2 transcription in differentiating myeloid cells. The current studies investigate modulation of HoxA10 repression activity during myelopoiesis. We determine that phosphorylation of tyrosine residues in the conserved homeodomain decreases HoxA10-DNA binding. We also determine that interaction of the homeodomain phosphotyrosine residues with an adjacent domain in the HoxA10 protein is necessary for decreased DNA binding affinity. Since SHP1 protein-tyrosine phosphatase antagonizes myeloid differentiation and decreases CYBB and NCF2 transcription, we investigated the influence of SHP1-protein-tyrosine phosphatase (PTP) on HoxA10 tyrosine phosphorylation. We find that SHP1-PTP activity increases HoxA10 target gene repression in undifferentiated myeloid cells. Consistent with this, SHP1-PTP interacts with HoxA10 and decreases homeodomain-tyrosine phosphorylation. These investigations suggest that SHP1-PTP activity, in undifferentiated myeloid cells, influences HoxA10 repression of myeloid-specific genes. Therefore, increased HoxA10 repression of myeloid gene transcription is a molecular mechanism for SHP1 inhibition of myeloid differentiation.

Animals↗

PubMed2002

Hyposialylation of integrins stimulates the activity of myeloid fibronectin receptors.

Despite numerous reports suggesting that beta(1) integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that beta(1) integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the beta(1) integrin subunit becomes hyposialylated, suggesting that the beta(1) integrin is a substrate for this enzyme. The expression of hyposialylated beta(1) integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified alpha(5)beta(1) integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated beta(1) integrins are more adhesive to fibronectin, although desialylation of alpha(5) subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the beta(1) integrin family of cell adhesion receptors.

Amidohydrolases↗