Abstract
Antisense oligonucleotide to the translation initiation sequence of human c-mpI reduced the proliferation of human CD34+ bone marrow cells in response to interleukin-3 (IL-3) alone or to the combination of IL-3 and thrombopoietin (TPO). To investigate the molecular basis for these cytokine interactions, we analyzed the relationship between the receptor subunits for IL-3 and TPO and determined whether both receptors activate identical signal transduction pathways. The function of the receptor subunits was characterized in transiently transfected hepatoma cells and fibroblasts by the activation of gene expression via specific regulatory elements and by the stimulation of DNA-binding activity of STAT proteins. Although c-mpl and IL-3 receptor (IL-3R) reconstituted a qualitatively comparable gene regulatory response, there was no detectable functional interaction between their respective receptor subunits. By comparing the receptor action in different cell lines, we observed that in human hepatoma cells the signaling of c-mpI was 100-fold less sensitive to TPO than in rat hepatoma cells. However, IL-3R signaling was comparable between the two cell types, suggesting that c-mpI and IL-3R do not use identical signal transducing mechanisms. The cytoplasmic domains necessary for c-mpI signaling were determined by testing deletion mutants. The membrane-proximal box 1 sequence motif was critical for gene regulation and for STAT protein activation that seemed to involve the Janus kinase 2 (JAK2). Because IL-3R was less dependent on JAK2 than c-mpI, different levels of JAK2 expression may account, in part, for the quantitative difference in IL-3 and TPO response among various cell lines.
Publication types
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Comparative Study
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Base Sequence
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Carcinoma, Hepatocellular / genetics
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Carcinoma, Hepatocellular / pathology
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Cells, Cultured
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Chlorocebus aethiops
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DNA-Binding Proteins / metabolism
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Gene Expression Regulation / drug effects*
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Genes, Reporter
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Hematopoietic Stem Cells / metabolism*
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Humans
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Interleukin-3 / physiology*
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Janus Kinase 1
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Janus Kinase 2
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Liver Neoplasms / genetics
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Liver Neoplasms / pathology
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Molecular Sequence Data
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Neoplasm Proteins*
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Oligonucleotides, Antisense / pharmacology
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Protein-Tyrosine Kinases / metabolism
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / physiology*
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Rats
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Receptors, Cytokine*
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Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / physiology
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Receptors, Immunologic / genetics
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Receptors, Immunologic / physiology*
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Receptors, Interleukin-3 / drug effects
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Receptors, Interleukin-3 / physiology
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Receptors, Thrombopoietin
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Regulatory Sequences, Nucleic Acid
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STAT1 Transcription Factor
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STAT2 Transcription Factor
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STAT3 Transcription Factor
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Sequence Deletion
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Signal Transduction*
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Species Specificity
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Thrombopoietin / pharmacology*
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Trans-Activators / metabolism
Substances
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DNA-Binding Proteins
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Interleukin-3
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Neoplasm Proteins
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Oligonucleotides, Antisense
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Proto-Oncogene Proteins
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Receptors, Cytokine
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Receptors, Granulocyte-Macrophage Colony-Stimulating Factor
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Receptors, Immunologic
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Receptors, Interleukin-3
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Receptors, Thrombopoietin
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STAT1 Transcription Factor
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STAT1 protein, human
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STAT2 Transcription Factor
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STAT3 Transcription Factor
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STAT3 protein, human
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Stat3 protein, rat
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Trans-Activators
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MPL protein, human
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Thrombopoietin
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Protein-Tyrosine Kinases
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JAK1 protein, human
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JAK2 protein, human
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Jak1 protein, rat
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Jak2 protein, rat
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Janus Kinase 1
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Janus Kinase 2