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

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.Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the proliferation and maturation of hematopoietic progenitor cells and enhances the differentiated functions of mature granulocytes and monocytes/macrophages (12, 18). The pleiotropic biological activities of GM-CSF are mediated by a high-affinity receptor which consists of an alpha subunit that binds ligand with low affinity and a beta subunit that confers high-affinity binding to the receptor-ligand complex and is required for signal transduction. Both subunits are members of the hematopoietin receptor superfamily, which includes the receptors for interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, and IL-7, granulocyte colony-stimulating factor, erythropoietin, growth hormone, and prolactin (1, 7). The 120-kDa beta subunit of the human GM-CSF receptor (13-common) is also shared with the high-affinity human IL-3 and IL-5 receptors (23). Phosphorylation of similar sets of proteins has been demonstrated in factor-responsive cells stimulated by either GM-CSF or 20). In addition, pathways involving p2lras, c-Raf, and p42,4...

Serine/threonine phosphorylation in cytokine signal transduction

“…[86][87][88][89][90][91][92] The regulation of these transcription factors and kinases in response to cytokines was discussed by Dr Kathleen Sakamoto (UCLA, Los Angeles, CA, USA). [86][87][88][89][90][91][92] Dr Sakamoto described studies to determine the differential roles of the ERK1 and ERK2 proteins in regulation of CREB phosporylation and Egr-1 transcription in response to GM-CSF. Stimulation of TF-1 cells with GM-CSF results in the phosphorylation of p90 Rsk on threonine 359.…”

Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism

“…[26][27][28][29][30][31][32] The CREB and Egr-1 transcription factors could stimulate GM-CSF transcription and result in autocrine transformation. Aberrant activation of the Raf/MEK/ERK pathway has been shown in other cell systems to induce autocrine growth factor expression.…”

“…p90 Rsk can sequentially activate the CREB and Egr transcription factors that bind specific promoter regions to induce gene transcription. [21][22][23][24][25][26][27] Recently, it has also been shown that ERK2 can phosphorylate Bcl-2 and this results in an anti-apoptotic response. 14,18,20 Evidence suggests that this pathway is intimately associated with the control of the apoptotic machinery in myelo-monocytic cells.…”

Combined effects of aberrant MEK1 activity and BCL2 overexpression on relieving the cytokine dependency of human and murine hematopoietic cells

“…Activation of ERK1 and ERK2 ultimately results in the activation of transcription factors, such as Elk-1 and CREB, that regulate gene expression. [31][32][33][34] N-terminal deletion of the MEK1 negative regulatory domain results in a constitutively activated oncoprotein that can transform NIH-3T3 and Chinese hamster ovary cells and abrogate the cytokine dependency of certain hematopoietic cells. [35][36][37][38][39] In addition, evidence suggests that the Raf pathway is intimately associated with the control of the apoptotic machinery in myelomonocytic cells.…”