Biomedical subjects

A Miyajima

Publications and source records attributed to A Miyajima.

At least 127 records · Page 7Linked to original sources

PubMed1995

Distribution of the mammalian Stat gene family in mouse chromosomes.

Studies of transcriptional activation by interferons and a variety of cytokines have led to the identification of a family of proteins that serve as signal transducers and activators of transcription, Stats. Here, we report that the seven mouse Stat loci map in three clusters, with each cluster located on a different mouse autosome. The data suggest that the family has arisen via a tandem duplication of the ancestral locus, followed by dispersion of the linked loci to different mouse chromosomes.

Animals↗

PubMed1995

A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors.

Cytokines manifest their function through alteration of gene expression. However, target genes for signals from cytokine receptors are largely unknown. We therefore searched for immediate-early cytokine-responsive genes and isolated a novel gene, CIS (cytokine inducible SH2-containing protein) which is induced in hematopoietic cells by a subset of cytokines including interleukin 2 (IL2), IL3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), but not by stem cell factor, granulocyte colony-stimulating factor and IL6. The CIS message encodes a polypeptide of 257 amino acids that contains an SH2 domain of 96 amino acids in the middle. To clarify the function of CIS in cytokine signal transduction, we expressed CIS in IL3-dependent hematopoietic cell lines under the control of a steroid-inducible promoter. The CIS product stably associated with the tyrosine-phosphorylated beta chain of the IL3 receptor as well as the tyrosine-phosphorylated EPO receptor. Forced expression of CIS by steroid reduced the growth rate of these transformants, suggesting a negative role of CIS in signal transduction. CIS induction requires the membrane-proximal region of the cytoplasmic domain of the EPO receptor as well as that of the common beta chain of the IL3, IL5 and GM-CSF receptor, whereas CIS binds to the receptor that is tyrosine phosphorylated by cytokine stimulation. Thus CIS appears to be a unique regulatory molecule for cytokine signal transduction.

Amino Acid Sequence↗

PubMed1995

Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways.

Signal transducers and activators of transcription (STAT) proteins play an important role in cytokine signal transduction in conjunction with Janus kinases (JAKs). MGF/STAT5 is known as prolactin regulated STAT. Here we demonstrate that interleukin 2 (IL-2) as well as erythropoietin (EPO) stimulate STAT5 and induce tyrosine phosphorylation of STAT5. These IL-2- and EPO-induced STATs have an identical DNA binding specificity and immunoreactivity. We also show that IL-4 induces a DNA binding factor which possesses similar, but distinct, DNA binding specificity from that of STAT5 and is immunologically different from STAT5. Analysis of two EPO receptor (EPOR) transfected CTLL-2 cell lines discloses that IL-2 activates JAK1 and JAK3 as well as STAT5, while EPO stimulates STAT5 and JAK2 in EPO-responsive CTLL-2 cells (ERT/E2). On the contrary, EPO activates neither JAK2 nor STAT5 in other cell lines that failed to respond to EPO (ERT cells). EPOR and JAK2 associate with each other regardless of EPO presence in ERT/E2 cells, however, such an interaction is not present in ERT cells. Thus, EPOR and JAK2 association seems to be important for EPO responsiveness in CTLL-2 cells.

Animals↗

PubMed1995

Regulation of Bcl-2 expression by oncogenic Ras protein in hematopoietic cells.

Interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) induce DNA synthesis and suppress apoptosis of hematopoietic cells. IL-3/GM-CSF exert pleiotropic functions by activating multiple signaling cascades through distinct domains of the common receptor subunit. As we previously reported, the Ras signaling pathway plays a pivotal role in suppressing apoptotic death rather than stimulating DNA synthesis in IL-3 dependent hematopoietic cells. In order to clarify the molecular basis of Ras-induced cell survival, we investigated the effect of Ras activation on the expression of Bcl-2 and its related molecules. Activation of the Ras pathway by using an inducible oncogenic Ras resulted in the rapid up-regulation of bcl-2 and bcl-xL, and the level of expression was nearly equivalent to that observed in growing cells. On the other hand, expression of bax, an antagonistic bcl-2 homologue, was not affected by oncogenic Ras or IL-3-deprivation. Thus, the Ras pathway regulates the expression of Bcl-2 and its related survival protein, and this appears to underlie the mechanism by which IL-3/GM-CSF inhibit apoptosis through activation of the Ras pathway.

Animals↗

PubMed1995

Interleukin-3 (IL-3) poor-responsive inbred mouse strains carry the identical deletion of a branch point in the IL-3 receptor alpha subunit gene.

Interleukin-3 (IL-3) stimulates colony formation of multiple lineages of hematopoietic cells. Bone marrow cells of A/J mice are nonresponsive to IL-3, and this observation has recently been correlated with aberrant mRNA splicing and impaired expression of the IL-3 receptor alpha subunit (IL-3R alpha), a binding component of the high-affinity receptors. We examined the IL-3R alpha gene in 27 inbred mouse strains and found the identical mutation, a 5-bp deletion at the branch point of intron 7, in 10 of these mouse strains. Bone marrow cells isolated from these 10 mouse strains did not express IL-3R alpha on the cell surface and did not form colonies in response to IL-3. Because the defective IL-3R alpha gene was found in several distantly related mouse strains, it appears to be a recessive allele rather than a sporadic mutation. In contrast, only 1 of 21 wild-derived mouse strains carried the 5-bp deletion in the IL-3R alpha gene. This study suggests that IL-3 function is not required for normal hematopoiesis in mice, but the retention of the IL-3 and IL-3R system may be of some selective advantage in wild populations.

Alleles↗

PubMed1995

Granulocyte macrophage-colony stimulating factor-dependent replication of polyoma virus replicon in hematopoietic cells. Analyses of receptor signals for replication and transcription.

Granulocyte macrophage-colony stimulating factor (GM-CSF) stimulates proliferation of various hematopoietic cells. Using cytoplasmic deletion mutants of the human GM-CSF receptor (hGMR) beta subunit and tyrosine kinase inhibitors, we previously showed that distinct signaling pathways of hGMR are involved in the induction of c-fos/c-jun mRNAs and of c-myc mRNA/cell proliferation. We used polyoma virus (Py) replicon to analyze the initiation of DNA replication induced by hGM-CSF in mouse BA/F3 pro-B cells expressing hGMR. hGM-CSF efficiently stimulated Py replication in the presence of Py enhancer and Py large T antigen supplied in trans. Analyses of Py enhancer mutants revealed that hGM-CSF promoted Py replication and activated transcription of the Py early promoter through the PEA3/PEBP5 region of Py enhancer. The membrane proximal region of hGMR beta subunit is required for activation of PEA3/PEBP5-dependent replication which is also required for activation of DNA synthesis in the host cells. In contrast, a more distal region which is essential for activation of c-fos and c-jun genes is required for the PEA3/PEBP5-dependent transcription of Py early promoter. These results indicate that distinct signaling pathways of hGMR are required to activate PEA3/PEBP5-dependent replication and transcription although the same enhancer is required for both activities.

Animals↗

PubMed1995

Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs.

Interleukin-3 (IL-3) is an important regulator of hemopoiesis and considerable effort has been directed towards the study of its mechanism of signal transduction. In this paper, we describe the first molecular identification of a STAT transcription factor that is activated by IL-3. STATs exist in a cytoplasmic, transcriptionally inactive form which, in response to extracellular signals, become tyrosine phosphorylated and translocate to the nucleus where they bind to specific DNA elements. Several of these DNA elements were found which bind proteins in an IL-3-responsive manner. Analysis of these bandshift complexes with available antibodies to the known STATs suggests that IL-3 activates the DNA-binding ability of STAT5, a protein which was originally characterized as a prolactin-responsive transcription factor in sheep. IL-5 and granulocyte-macrophage colony stimulating factor (GM-CSF), which share a common signaling receptor subunit with IL-3, also activate STAT5. Unexpectedly, two murine STAT5 homologs, 96% identical to each other at the amino acid level, were isolated and IL-3-dependent GAS binding could be reconstituted in COS cells transfected with IL-3 receptor and either STAT5 cDNA. In IL-3-dependent hemopoietic cells, both forms of STAT5 are expressed and activated in response to IL-3.

Amino Acid Sequence↗

PubMed1995

Structure of the gene encoding the alpha subunit of the human interleukin 3 receptor.

Interleukin 3 is a cytokine that stimulates proliferation and differentiation of hematopoietic progenitor cells. Its receptor consists of two subunits, an interleukin 3-specific alpha subunit and a beta subunit shared by garanulocyte-macrophage colony stimulating factor and interleukin 5 receptors. In this paper, we determined the genomic structure of the alpha subunit of the human interleukin 3 receptor, which spans approximately 40 kb and has 12 exons. We found that the genomic structures of the alpha subunits of the human interleukin 3 and granulocyte-macrophage colony stimulating factor receptors are very similar. They possess a unique additional intron in the 'C domain', which is absent in the alpha subunit of the interleukin 5 receptor. These results suggest a shared evolutionary pathway of these two genes.

Base Sequence↗

PubMed1995

High affinity chimeric human granulocyte-macrophage colony-stimulating factor receptor carrying the cytoplasmic domain of the beta subunit but not the alpha subunit transduces growth promoting signals in Ba/F3 cells.

Granulocyte-macrophage colony-stimulating factor receptor (GMR) is composed of two distinct subunits alpha and beta, and the cytoplasmic domains of both subunits are essential to transduce signals. We further analyzed the role of the cytoplasmic domain of each subunit by constructing chimeric subunits, designated alpha/beta and beta/alpha, by exchanging cytoplasmic domains of the alpha and beta subunits of hGMR. Reconstituted high-affinity chimeric hGMRs, hGMR(alpha/beta,beta/alpha) and hGMR(alpha/beta,beta), as well as the wild type hGMR(alpha,beta), transduced signals in Ba/F3 cells. These observations indicate that the original configuration between the extracellular and the cytoplasmic domains of the hGMR(alpha,beta) subunits is not obligatory, and that hGMR(alpha/beta,beta) transduced signals through the cytoplasmic domain of the beta subunit in an oligomeric form, without involvement of the cytoplasmic domain of the alpha subunit.

Animals↗

PubMed1995

Impaired interleukin-3 (IL-3) response of the A/J mouse is caused by a branch point deletion in the IL-3 receptor alpha subunit gene.

Interleukin-3 (IL-3) alone does not support hematopoietic colony formation of bone marrow cells from the A/J mouse. To elucidate the molecular lesion in A/J mice, we examined expression of the alpha and beta subunits of the IL-3 receptor (IL-3R). While IL-3R beta was normally expressed, IL-3R alpha was not detectable on the surface of A/J-derived cells by antibody staining. Genetic linkage analysis using recombinant inbred mouse strains between A/J and IL-3-responsive C57BL/6 indicated that the IL-3R alpha gene locus was responsible for the impaired IL-3 response in A/J mice. Molecular cloning and characterization of A/J-derived IL-3R alpha cDNA revealed that it lacked the sequence corresponding to exon 8, which encodes 10 amino acid residues in the extracellular domain. The aberrant splicing was due to a 5 base pair deletion at the branch point in intron 7 and was reproduced in heterologous cells by transfecting with an IL-3R alpha minigene carrying the deleterious intron. The A/J-specific abnormal form of IL-3R alpha was localized inside the cells, but not on the cell surface, providing the molecular basis for the impaired IL-3 response in the A/J mouse.

Amino Acid Sequence↗

PubMed1995

Multi-colony stimulating activity of interleukin 5 (IL-5) on hematopoietic progenitors from transgenic mice that express IL-5 receptor alpha subunit constitutively.

The interleukin 3 (IL-3), IL-5, and granulocyte/macrophage colony-stimulating factor receptors consist of a cytokine-specific alpha subunit and the common beta subunit. Whereas IL-3 stimulates various lineages of hematopoietic cells, including multipotential progenitors, IL-5 acts mainly as an eosinophil lineage-specific factor. To investigate whether the lineage specificity of IL-5 is due to restricted expression of the IL-5 receptor alpha subunit (IL-5R alpha), we generated transgenic mice that express the mouse IL-5R alpha constitutively by phosphoglycerate kinase promoter. The transgenic mouse expressed IL-5R alpha ubiquitously, and the bone marrow cells formed various types of colonies, including multi-lineage colonies, in response to IL-5. IL-5 also supported formation of both multi-lineage and blast cell colonies from dormant progenitors of the 5-fluorouracil-treated transgenic mice. The cells composing the blast cell colony gave rise to many colonies including multi-lineage colonies when they were replated in secondary culture containing either Il-5 or IL-3. There was no significant difference in replating efficiency or in types of secondary colonies between IL-5- and IL-3-stimulated cultures. Conversely, the cells from the IL-3-induced blast cell colonies of the transgenic mice proliferated in response to either IL-3 or IL-5. Thus, the development of the progenitors can be equally supported by either IL-5 or IL-3, suggesting that intracellular signals from the IL-3R can be replaced by those from IL-5. These results strongly suggest that the lineage specificity of IL-5 is mainly due to the restricted expression of IL-5R alpha.

Animals↗

PubMed1995

The murine interleukin-3 receptor alpha subunit gene: chromosomal localization, genomic structure, and promoter function.

The interleukin-3 receptor (IL-3R) is composed of alpha and beta subunits, members of the class I cytokine receptor family. Here we describe isolation and characterization of the chromosomal gene for the mouse IL-3R alpha subunit (mIL-3R alpha). Whereas the human IL-3R alpha gene is tightly linked with the granulocyte-macrophage colony-stimulating factor receptor alpha subunit (GM-CSFR alpha) gene in the pseudoautosomal region of the X and Y chromosomes, the mIL-3R alpha gene (II3ra) is located in the proximal region of mouse chromosome 14, separated from the mouse GM-CSFR alpha gene, which is on chromosome 19. The mIL-3R alpha gene spans about 10 kb and is divided into 12 exons. All the exon-intron boundaries possess the splicing junction consensus sequences (5'GT-AG3'), and the whole genomic structure is similar to those of the previously reported class I cytokine receptor genes. There are two major transcription initiation sites that are located at 215 and 188 nucleotides upstream of the initiator codon. The promoter region is GC-rich and contains potential binding sites for GATA, Ets, c-myb,, Sp1, Ap-2, and G-C boxes, but not a typical TATA or CAAT sequence. A fusion gene containing 0.8 kb of the 5' noncoding sequence linked to the firefly luciferase gene directed the transcription in mouse mast cells but not in fibroblasts or T cells, suggesting that this promoter functions in a cell type-specific manner. Further sequential deletion of the 5' region suggests two potential regulatory regions for transcription of the mIL-3R alpha gene.

3T3 Cells↗

PubMed1995

Human IL-3 receptor signaling: rapid induction of phosphatidylcholine hydrolysis is independent of protein kinase C but dependent on tyrosine phosphorylation in transfected NIH 3T3 cells.

Although tyrosine kinases are clearly activated after ligand engagement of the human IL-3R in both hematopoietic and nonhematopoietic cytoplasmic environments, a role for phospholipid hydrolysis and protein kinase C in IL-3R signal transduction is emerging. We have used NIH 3T3 cells transiently transfected with human IL-3R alpha- and beta-subunits to study phosphatidylcholine hydrolysis in response to human IL-3. We have found that NIH 3T3 cells expressing the complete human IL-3R respond to human IL-3 with a rapid and sustained increase in sn-1'2'-diacylglycerol. Accompanying this was a rapid increase in intracellular levels of phosphorylcholine. The protein kinase C inhibitor H-7, however, was not effective in inhibiting phosphatidylcholine hydrolysis in response to human IL-3 in NIH 3T3 cells expressing the human receptor. Thus the human IL-3R induces a rapid protein-kinase-C-independent hydrolysis induced by the murine receptor. Simultaneous with the increase in phosphatidylcholine hydrolysis induced by the murine receptor. Simultaneous with the increase in diacylglycerol levels was an increase in membrane-bound protein kinase C enzyme activity. Immunoblotting with isoform-specific Abs against protein kinase C showed that, whereas the zeta-isoform is constitutively membrane bound, the alpha-isoform of protein kinase C is translocated to the membrane in response to IL-3. Activation of phosphatidylcholine hydrolysis and protein kinase C activation required both alpha- and beta-receptor subunits. To determine the relationship of tyrosine phosphorylation to the activation of phosphatidylcholine hydrolysis and protein kinase C translocation, we used the specific and structurally unrelated tyrosine kinase inhibitors genistein and herbimycin. Both inhibitors effectively blocked human IL-3-induced tyrosine phosphorylation. In addition, both inhibitors blocked phosphatidylcholine hydrolysis and protein kinase C translocation. These data, combined with our previous report showing that c-jun induction by IL-3 is dependent on protein kinase C, suggest that, in hematopoietic and nonhematopoietic cells expressing the human IL-3R, phosphatidylcholine hydrolysis and protein kinase C are downstream effectors of tyrosine phosphorylation in the IL-3 signal transduction cascade resulting in immediate early response gene induction.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Suppression of apoptotic death in hematopoietic cells by signalling through the IL-3/GM-CSF receptors.

Interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) exert their biological functions through acting on a specific receptor which consists of a ligand-specific alpha subunit and the shared common beta subunit. Inhibition by genistein of a subset of IL-3/GM-CSF-mediated signals, including c-myc induction, resulted in the abrogation of DNA synthesis, however, IL-3 still protected cells from apoptotic cell death. Conversely, a C-terminal truncated form of the GM-CSF receptor, which is missing a critical cytoplasmic region required for activation of the Ras/Raf-1/MAP kinase pathway, induced DNA synthesis, but failed to prevent cell death in response to GM-CSF. Consequently, cells died by apoptosis in the presence of GM-CSF, despite displaying a transient mitogenic response. However, expression of activated Ras protein complemented defective signalling through the mutant receptor and supported long-term proliferation in concert with GM-CSF. These results indicate that IL-3 and GM-CSF prevent apoptosis of hematopoietic cells by activating a signalling pathway distinct from the induction of DNA synthesis and that long-term cell proliferation requires the activation of both pathways.

Animals↗

PubMed1995

Cis-acting DNA elements of mouse granulocyte-macrophage colony-stimulating factor gene responsive to Fc epsilon receptor cross-linking stimulation in the mouse mast cell line MC/9.

Mouse mast cells produce many kinds of cytokines in response to cross-linking of high affinity Fc epsilon receptor (Fc epsilon RI). Among these cytokines, granulocyte-macrophage CSF (GM-CSF) gene induction in mouse mast cells has been reported to be regulated at both the transcriptional level and the post-transcriptional level. We analyzed the mechanism of the transcriptional regulation of GM-CSF gene induction through Fc epsilon RI cross-linking stimulation in the mouse mast cell line MC/9. In MC/9, the GM-CSF gene was activated transcriptionally by Fc epsilon RI cross-linking stimulation. The 5' deletion analysis of GM-CSF gene promoter indicated that the 5' boundary of the responsive promoter region lay between positions -113 and -95. When the deletion was extended to positions -72 or -60, the stimulatory effect was significantly diminished. We then examined 3' deletion of pmGMCAT -113 from position -60. This analysis indicated that the 3' boundary lay between positions -84 and -72. No subfragments of the region spanning positions -113 to -72 could cover the full induction level. A site-directed mutagenesis experiment revealed that the sequence spanning positions -108 to -72 was needed for full activation. These data indicate that GM-CSF gene in mast cells is activated mainly through the sequence spanning positions -108 to -72.

Animals↗

PubMed1995

Interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5 transduce signals through two forms of STAT5.

Recently, JAK2 kinase was found to be one of the tyrosine kinases activated by interleukin-3 (IL-3) in target cells. JAK2 belongs to a family of kinases that act upstream of transcription factors called STATs. STATs exist in the cytoplasm as latent, transcriptionally inactive forms until, in response to extracellular signals, they become phosphorylated on tyrosine residues, translocate to the nucleus, and bind to specific DNA elements. Because IL-3 activates JAK2, we searched for the STAT(s) that might transduce IL-3 signals. Several lines of evidence suggest that IL-3 uses the murine homologue of STAT5, a factor originally purified from sheep. Unexpectedly, during isolation of the murine homologue, we found two highly related molecules that we have designated STAT5A and STAT5B.

Animals↗

PubMed1995

Synergistic action of interleukin-10 (IL-10) with IL-3, IL-4 and stem cell factor on colony formation from murine mast cells in culture.

We examined the effects of interleukin-10 (IL-10) on colony formation from two different types of murine mast cells, bone marrow-derived mast cells (BMMC) and serosal mast cells (SMC), using a methylcellulose culture method. IL-10 alone did not induce colony formation from either phenotype. However, BMMC and SMC produced colonies in the presence of IL-10 in combinations with IL-3, IL-4 or stem cell factor (SCF), but they responded in different manners. IL-10 enhanced the IL-3-dependent colony formation from BMMC and induced the colony formation from BMMC synergistically with IL-4 or SCF dose-dependently, although IL-4 and SCF were not active alone. The most significant synergism was observed between IL-10 and IL-4. The addition of IL-10 to the cultures of BMMC in the presence of two or three factors enhanced the colony formation induced by IL-3 plus IL-4, and inhibited the colony formation induced by IL-3 plus SCF or IL-3, IL-4 plus SCF. In the colony formation from SMC, IL-10 synergized with IL-3 but not with IL-4 or SCF. IL-10 in combination with two or three factors enhanced the colony formation from SMC induced by IL-3 plus IL-4, but did not affect the colony formation induced by other combinations among IL-3, IL-4 and SCF. These findings indicate that IL-10 plays an important role in the proliferation of murine mast cells.

Animals↗

PubMed1995

Roles of the cytoplasmic domains of the alpha and beta subunits of human granulocyte-macrophage colony-stimulating factor receptor.

The high-affinity and functional granulocyte-macrophage colony-stimulating factor receptor (GMR) is composed of two distinct subunits, alpha and beta; and the cytoplasmic domain of the beta subunit is essential to transduce growth-promoting signals. In contrast to the beta subunit, the role of the alpha subunit is not well characterized. We examined the requirement of the cytoplasmic domain of the alpha subunit and its functional region by deletion analyses. We demonstrated that the cytoplasmic domain of the alpha subunit, especially 29 amino acids residues near the transmembrane domain, was absolutely required for various signaling events including activation of immediate early genes, induction of tyrosine phosphorylation of cellular proteins, and cell growth. We further analyzed the role of the cytoplasmic domain of each subunit by constructing chimeric subunits, designated alpha/beta and beta/alpha, by exchanging cytoplasmic domains of the alpha and beta subunits of human (h) GMR. Reconstituted high-affinity chimeric hGMRs, hGMR(alpha/beta,beta/alpha) and hGMR(alpha/beta,beta), transduced signals at levels similar to the wild type hGMR(alpha,beta) in Ba/F3 cells and in NIH3T3 cells. These observations indicate that the original configuration between the extracellular and the cytoplasmic domains of the hGMR(alpha,beta) subunits is not required and that hGMR(alpha/beta,beta) transduced signals through the cytoplasmic domain of the beta subunit in an oligomeric form, without involvement of the cytoplasmic domain of the alpha subunit. Therefore human granulocyte-macrophage colony-stimulating factor signals are mainly transduced through the beta subunit, and the cytoplasmic domain of the alpha subunit is likely to activate the beta subunit in the normal hGMR.

3T3 Cells↗