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

Mol Cell Biol. 1994 Sep;14(9):5975-85. doi: 10.1128/mcb.14.9.5975.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Binding Sites
  • Cyclic AMP / physiology
  • Cyclic AMP Response Element-Binding Protein / metabolism*
  • DNA-Binding Proteins / genetics*
  • Early Growth Response Protein 1
  • Gene Expression Regulation*
  • Granulocyte-Macrophage Colony-Stimulating Factor / physiology*
  • Humans
  • Immediate-Early Proteins*
  • In Vitro Techniques
  • Interleukin-3 / physiology*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oligodeoxyribonucleotides / chemistry
  • Promoter Regions, Genetic*
  • RNA, Messenger / genetics
  • Signal Transduction
  • Structure-Activity Relationship
  • Transcription Factors / genetics*
  • Transcriptional Activation

Substances

  • Cyclic AMP Response Element-Binding Protein
  • DNA-Binding Proteins
  • EGR1 protein, human
  • Early Growth Response Protein 1
  • Immediate-Early Proteins
  • Interleukin-3
  • Oligodeoxyribonucleotides
  • RNA, Messenger
  • Transcription Factors
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Cyclic AMP