PubMed Health⌕ Search

Biomedical subjects

K M Sakamoto

Publications and source records attributed to K M Sakamoto.

At least 37 records · Page 2Linked to original sources
PubMed1999

Analysis of functional elements in the human Egr-1 gene promoter.

The early growth response (Egr)-1 gene encoding a zinc-finger transcription factor is transiently induced in many different cell types upon various differentiation signals. However, in synovial fibroblasts of rheumatoid arthritis patients, Egr-1 is constitutively expressed at high levels, and several genes with Egr-1 binding sites in their promoter regions have been associated with disease progression of RA. We analyzed the control of Egr-1 transcription by characterizing those regulatory elements in the Egr-1 promoter that induce Egr-1 expression in fibroblasts. Using reporter gene assays and deletion mutants of the Egr-1 promoter we could demonstrate that Egr-1 transcription is mainly activated by a single serum response element, whereas other transcription factor binding sites, including binding sites for AP-1 or Egr-1, were found to play a minor role. Furthermore, we identified a novel regulatory element in the human Egr-1 promoter similar to a NF kappa-B binding site. Deletion of this element enhanced Egr-1 promoter activity in 3T3 but not in L929 fibroblasts. Stimulation by phorbolester induced only transient Egr-1 expression in 3T3 fibroblasts but a extended expression of Egr-1 in L929 cells. These data suggest that in fibroblasts the most proximal serum response element in the Egr-1 promoter represents the major activation site, whereas binding of the NFkB-like factor may serve as negative regulatory signal for Egr-1 transcription in fibroblasts.

Arthritis, Rheumatoid↗
PubMed1998

Genomic organization, 5' flanking enhancer region, and chromosomal assignment of the cell cycle gene, p55Cdc.

p55Cdc is a mammalian homologue of a family of cell cycle proteins from widely divergent species, which contains WD repeats and has been implicated in cell cycle-regulated ubiquitin-mediated proteolysis. p55Cdc is highly expressed in proliferating but not in differentiated or growth-arrested cells. The expression, phosphorylation, and degradation of this protein have been shown to be cell cycle-regulated. We analyzed a 5.3-kb genomic region that contains the entire rat p55Cdc gene. The gene contains 10 exons ranging in size from 97 to 373 bp. The promoter region has a TAT box, four E-box consensus sequences, and potential binding sites for cell cycle-specific transcription factors. In transient transfection assays, a construct containing a 1000-nucleotide p55Cdc promoter region upstream of the chloramphenicol acetyltransferase reporter gene demonstrated a 12-fold increase in transcriptional activity. Finally, using radiation hybrid mapping techniques, we localized this gene to the human chromosome, 9q13-21.

Animals↗
PubMed1998

Analysis of cell death in myeloid cells inducibly expressing the cell cycle protein p55Cdc.

p55Cdc, a cell cycle protein is expressed in cycling mammalian cells and is required for normal cell division. Expression of this protein is regulated during the cell cycle, peaking in late G1 and S. We have previously shown that constitutive expression of p55Cdc results in inhibition of granulocyte differentiation. Degradation of p55Cdc is also required for apoptosis in growth factor and serum starved cells. In the present study we prepared stably transfected cells conditionally expressing p55Cdc in response to zinc stimulation to investigate the role of inducible p55Cdc expression in apoptosis of myeloid cells. We report that inducible expression of p55Cdc in the myeloid leukemic cell line 32Dc13 resulted in increased cell death. p55Cdc overexpression led to a statistically significant decrease in the viability of 32Dc13 cells compared with that of control cells. Furthermore, cell staining and flow cytometry analysis revealed that p55Cdc-overexpressing 32Dc13 cells progressed to apoptosis much earlier than uninduced cells. These results suggest that inducible expression of p55Cdc leads to earlier increases in cell death in the absence of growth factor and serum in myeloid leukemic cells.

Animals↗
PubMed1997

Characterization of cis-acting sequences and trans-acting signals regulating early growth response 1 and c-fos promoters through the granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) activates a set of genes such as c-fos, jun, myc, and early growth response gene 1 (egr-1). Studies on BA/F3 cells that express hGM-CSF receptor (hGMR) showed that two different signaling pathways controlled by distinct regions within the beta subunit are involved in activation of c-fos/c-jun genes and in c-myc, respectively. However, the region(s) of the beta subunit responsible for activation of the egr-1 gene and other regulatory genes has not been identified. We describe here how egr-1 promoter is activated by hGMR through two regions of the beta subunit, with these regions being required for activation of the c-fos promoter. Coexpression of dominant negative (dn) Ras (N17ras) or dn JAK2 almost completely suppressed the activation of egr-1 and c-fos promoters. Deletion analysis of egr-1 promoter showed two cis-acting regions responsible for activation by hGM-CSF or mouse interleukin-3 (mIL-3), one between nucleotide positions (nt) -56 and -116, and the other between nt -235 and -480, which contains tandem repeats of the serum response element (SRE) sites. Similar experiments with the c-fos promoter showed that cis-acting regions containing the SRE/AP-1 sites is sufficient for activation by hGM-CSF. Based on these observations, we propose that signaling pathways activating egr-1 and c-fos promoters are controlled by SRE elements, either through the same or overlapping pathways that involve JAK2 and Ras.

Animals↗
PubMed1996

Over-expression of p55Cdc inhibits granulocyte differentiation and accelerates apoptosis in myeloid cells.

p55Cdc is a protein identified in cycling mammalian cells. It is highly expressed in proliferating but not in differentiated or growth-arrested cells. Structurally, p55Cdc is similar to the Cdc4 and Cdc20 proteins, which have been proposed to regulate DNA synthesis and mitosis in Saccharomyces cerevisiae. To define the role of p55Cdc during myelopoiesis, we studied the expression and regulation of this protein in response to the hematopoietic growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte-colony stimulating factor (G-CSF). We analysed the time course of expression of p55Cdc in response to GM-CSF and G-CSF stimulation in the murine factor-dependent myeloid leukemic cell line, 32Dc13, and demonstrated differential regulation of p55Cdc in response to these two growth factors. Over-expression of p55Cdc resulted in acceleration of apoptosis in growth factor- and serum-free conditions, although no difference was observed in the rate of cell proliferation. Decreases in p55Cdc protein levels correlated with cells undergoing apoptosis. p55Cdc over-expression also inhibited granulocyte differentiation of 32Dc13 cells treated with G-CSF. Our studies suggest that p55Cdc regulation is critical for normal cell cycle control during myeloid cell proliferation and differentiation.

Animals↗
PubMed1996

Mechanism of transcriptional activation of the immediate early gene Egr-1 in response to PIXY321.

Studies with the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-3 (IL-3) fusion protein, PIXY321, demonstrated enhanced biological activity of this molecule in comparison with GM-CSF or IL-3 alone or in combination. Experiments were performed to study the mechanisms resulting in PIXY321-induced egr-1 expression in human myeloid leukemic cells (TF-1). Transfections of egr-1 promoter constructs revealed that PIXY321 stimulation resulted in fourfold induction of the -116 and -600 nucleotide (nt) constructs. We transfected a -116 nt construct containing a deletion of the cyclic AMP response element (CRE) or mutation in the serum response element (SRE) and demonstrated that both the SRE and CRE are necessary for maximal induction. However, PIXY321 stimulation resulted in 2.5-fold induction of a SRE-CRE-containing construct (P < .05), suggesting that the SRE and CRE are sufficient for PIXY321 responsiveness. Electrophoretic mobility shift assays (EMSA) revealed that the CRE binding protein (CREB) was phosphorylated on serine 133 in PIXY321-stimulated but not -unstimulated extracts from cells cultured in GM-CSF. By Western analysis and EMSA, CREB was constitutively phosphorylated in TF-1 cells grown on PIXY321 before growth factor and serum starvation. However, in TF-1 cells grown on GM-CSF before starvation, CREB phosphorylation was observed 10 minutes after PIXY321 stimulation. Further-more, ENSAs with PIXY321-stimulated and -unstimulated extracts demonstrated the presence of specific proteins that recognize the SRE. Our data demonstrate that transcriptional regulation of egr-1 by PIXY321 is mediated by the CRE and SRE.

Base Sequence↗
PubMed1996

Regulation of egr-1 gene expression by retinoic acid in a human growth factor-dependent cell line.

Retinoic acid (RA) has profound suppressive effects on growth and survival of human growth factor-dependent cell line, M07e. Treatment of M07e cells by RA reduced expression of egr-1 gene, while the levels of c-myc gene expression remained similar. Suppression of egr-1 gene expression by RA was dosage-dependent and reached maximum at 4 h after RA addition. The decay of egr-1 mRNA was similar in M07e cells treated with or without RA. The transcriptional activity of the promoter region up to -600 or -480 bp upstream of the egr-1 gene was greatly reduced by RA treatment. These data suggest that biological effects of RA on hematopoietic cells may, in part, be mediated by transcriptional suppression of egr-1 gene through its promoter region within -480 bp.

DNA-Binding Proteins↗
PubMed1995

Granulocyte-macrophage colony-stimulating factor induces the transcriptional activation of egr-1 through a protein kinase A-independent signaling pathway.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) rapidly and transiently induces the transcriptional activation of the early growth response gene-1 (egr-1) in the human factor-dependent myeloid leukemic cell line, TF-1. We previously demonstrated that the cAMP response element (CRE) is required for GM-CSF-induced egr-1 expression and that phosphorylation of CREB on serine 133 plays a critical role during GM-CSF signal transduction. To determine whether GM-CSF activates signaling pathways through a protein kinase A-dependent or -independent pathway, we measured cAMP levels following GM-CSF or forskolin treatment of TF-1 cells. Forskolin but not GM-CSF stimulation resulted in an increase in cAMP levels. Transient transfection assays with TF-1 cells were also performed with a -116-nucleotide egr-1 promoter construct and the protein kinase inhibitor, PKI. Although PKI inhibited forskolin induction of the -116-nucleotide construct, it did not affect GM-CSF stimulation of this construct. In the present study, we demonstrated that GM-CSF induces egr-1 expression through a protein kinase A-independent pathway.

Cell Line↗
PubMed1995

Transcriptional activation of egr-1 by granulocyte-macrophage colony-stimulating factor but not interleukin 3 requires phosphorylation of cAMP response element-binding protein (CREB) on serine 133.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) stimulate the proliferation and maturation of myeloid progenitor cells following interaction with heterodimeric receptors that share a common beta subunit required for signal transduction. Our previous studies have demonstrated that GM-CSF and IL-3 activate signaling pathways which converge upon a cAMP response element-binding protein (CREB)-binding site of the human immediate early response gene (early growth response gene-1, egr-1) promoter. Using electromobility supershift assays and antibodies directed against CREB phosphorylated on serine 133, we show that CREB is phosphorylated on serine 133 in response to GM-CSF or IL-3 stimulation. We demonstrate that phosphorylation of CREB on serine 133 substantially contributes to transcriptional activation of egr-1 in response to GM-CSF but not IL-3. These studies suggest that phosphorylation of CREB may play different roles during signal transduction, resulting in unique and overlapping biological functions in myeloid cells.

Cells, Cultured↗
PubMed1994

Granulocyte-macrophage colony-stimulating factor and interleukin-3 signaling pathways converge on the CREB-binding site in the human egr-1 promoter.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates myeloid progenitor cell proliferation and enhances the function of terminally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow progenitor cells. These activities are mediated by non-tyrosine kinase-containing receptors which consist of ligand-specific alpha subunits that complex with a common beta subunit required for signal transduction. Both GM-CSF and IL-3 rapidly and transiently induce expression of early growth response gene 1 (egr-1) in the human factor-dependent cell line TF-1. To define the mechanism of early response gene induction by GM-CSF and IL-3, growth factor- and serum-starved TF-1 cells transfected with recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) region of the egr-1 promoter which contains sequences inducible by GM-CSF and IL-3 was defined. DNase I footprint analysis identified a 20-nt region, including nt -57 to -76, which contains a potential cyclic AMP (cAMP) response element (CRE). Electrophoretic mobility shift assays performed with CREB antibody confirmed the presence of CREB in the DNA-binding complex. Mutational analysis of the cytokine-responsive region of the egr-1 promoter revealed that both the cAMP response and serum response elements are required for induction by GM-CSF and IL-3. Nuclear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding site in the nt -600 to -480 region of the promoter. Electrophoretic mobility shift assays were performed with antibodies against the Egr-1 protein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimulate transcription of the egr-1 gene in response to GM-CSF as a secondary event.

Base Sequence↗
PubMed1994

Signal transduction by granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors.

Granulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin-3 (IL-3) are cytokines which stimulate myeloid bone marrow progenitor cell proliferation and maturation. GM-CSF also enhances the function of terminally differentiated effector cells including neutrophils, monocytes and eosinophils. Both growth factors exhibit similar biological activities on overlapping cell populations which are mediated by high affinity receptors. These receptors share a common beta subunit necessary for signal transduction. The receptors for GM-CSF and IL-3 are members of the hematopoietin receptor superfamily and consequently lack intrinsic tyrosine kinase activity. Several kinases, including JAK2 and raf-1, and other downstream molecules are likely to be responsible for the functional redundancy demonstrated by GM-CSF and IL-3 in factor-responsive cells. This review discusses recent findings which elucidate the signaling pathways activated by these two cytokines.

Amino Acid Sequence↗
PubMed1992

HTLV-I and HTLV-II tax trans-activate the human EGR-1 promoter through different cis-acting sequences.

The immediate-early response gene, EGR-1, encodes a zinc finger-containing transcription factor that is involved in growth and differentiation of a variety of cell types. EGR-1 is induced in normal T cells following mitogenic stimulation and has recently been shown to be constitutively expressed in human T-cell leukemia virus type I (HTLV-I)- and type II (HTLV-II)-transformed T-cell lines. The trans-activating protein of HTLV-I, Tax, has been demonstrated to trans-activate promoters of a number of cellular genes, some of which may be critical in regulating T-cell proliferation. In this study, we examine the effect of Tax on expression of EGR-1 in three T-cell lines and demonstrate that both HTLV-I and -II Tax are capable of trans-activating human EGR-1 recombinant promoter constructs. Interestingly, HTLV-I and -II Tax trans-activate the human EGR-1 promoter through different promoter regions in the Jurkat cell line, suggesting that HTLV-I and -II Tax may lead to constitutive expression of EGR-1 through different signaling pathways. Deregulated expression of EGR-1 may contribute to uncontrolled cell growth and transformation during early stages of T-cell activation in HTLV-I and -II-infected cells.

Base Sequence↗
PubMed1991

Clinical applications of human granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates myeloid cell proliferation and maturation and enhances the function of terminally differentiated effector cells. Phase I and II clinical trials have demonstrated mild to moderate toxicities at doses of less than 30 micrograms/kg/day. These studies suggest a potential role for this growth factor to stimulate myelopoiesis in patients with aplastic anemia, myelodysplastic syndromes, AIDS, chemotherapy-induced myelosuppression, chronic neutropenia, and following bone marrow transplantation. The potential clinical uses of GM-CSF will depend on results of studies designed to optimize its therapeutic efficacy.

Acquired Immunodeficiency Syndrome↗
PubMed1991

The biology and clinical applications of granulocyte-macrophage colony-stimulating factor.

GM-CSF is a hematopoietic growth factor that regulates myeloid cell proliferation and maturation and enhances the function of terminally differentiated effector cells. Results of clinical trials with GM-CSF in a number of disease states suggest a potential role of this growth factor to stimulate hematopoiesis. Future use of GM-CSF will depend on further studies to optimize its therapeutic efficacy.

Biology↗
PubMed1991

5' upstream sequence and genomic structure of the human primary response gene, EGR-1/TIS8.

EGR-1/TIS8 is a primary response gene that encodes a zinc finger containing protein and is induced in a number of cell types by a variety of ligands. We have isolated and mapped the human EGR-1/TIS8 gene and sequenced the 5' upstream flanking region. A 'TATA' homology and several putative regulatory elements, including two Sp1 sites, five serum response-like elements, two cAMP response-like elements, an EGR-1 binding site (EBS), and a tetra-decanoyl phorbol acetate (TPA)-responsive element have been identified within 700 nucleotides of the upstream region. We demonstrated that a 500-base pair fragment, which includes several of these possible regulatory sequences, is functional and responsive to TPA in transient transfection assays. A further understanding of the regulation and function of human EGR-1/TIS8 gene expression may provide insight into the mechanisms that control normal and neoplastic proliferation of human cells.

Animals↗