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 m yeloid progenitor cell proliferation and enhances the function of term inally differentiated effector cells. Interleukin-3 (IL-3) stimulation results in the proliferation and maturation of early bone marrow prog enitor cells. These activities are mediated by non-tyrosine kinase-con taining 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 earl y growth response gene 1 (egr-1) in the human factor-dependent cell li ne TF-1. To define the mechanism of early response gene induction by G M CSF and IL-3, growth factor- and serum-starved TF-1 cells transfecte d,vith recombinant constructs containing sequences of the human egr-1 promoter were stimulated with GM-CSF or IL-3. A 116-nucleotide (nt) re gion of the egr-1 promoter which contains sequences inducible by GM-CS F 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 regio n 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. Nucl ear extracts from GM-CSF- or IL-3-stimulated but not unstimulated TF-1 cells contain factors which specifically bind to the Egr-1-binding si te in the nt -600 to -480 region of the promoter. Electrophoretic mobi lity shift assays were performed with antibodies against the Egr-1 pro tein to demonstrate the presence of the protein product in the shifted complex. Our studies suggest that the Egr-1 protein may further stimu late transcription of the egr-1 gene in response to GM-CSF as a second ary event.