ABSTRACT
Studies with the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-3 (IL-3) fusion protein, PIXY321, demonstrated enhanced biological activity of this molecule in comparison with GM-CSF or IL-3 alone or in combination. Experiments were performed to study the mechanisms resulting in PIXY321-induced egr-1 expression in human myeloid leukemic cells (TF-1). Transfections of egr-1 promoter constructs revealed that PIXY321 stimulation resulted in fourfold induction of the -116 and -600 nucleotide (nt) constructs. We transfected a -116 nt construct containing a deletion of the cyclic AMP response element (CRE) or mutation in the serum response element (SRE) and demonstrated that both the SRE and CRE are necessary for maximal induction. However, PIXY321 stimulation resulted in 2.5-fold induction of a SRE-CRE-containing construct (P < .05), suggesting that the SRE and CRE are sufficient for PIXY321 responsiveness. Electrophoretic mobility shift assays (EMSA) revealed that the CRE binding protein (CREB) was phosphorylated on serine 133 in PIXY321-stimulated but not -unstimulated extracts from cells cultured in GM-CSF. By Western analysis and EMSA, CREB was constitutively phosphorylated in TF-1 cells grown on PIXY321 before growth factor and serum starvation. However, in TF-1 cells grown on GM-CSF before starvation, CREB phosphorylation was observed 10 minutes after PIXY321 stimulation. Further-more, ENSAs with PIXY321-stimulated and -unstimulated extracts demonstrated the presence of specific proteins that recognize the SRE. Our data demonstrate that transcriptional regulation of egr-1 by PIXY321 is mediated by the CRE and SRE.
Subject(s)
DNA-Binding Proteins/biosynthesis , Genes, Immediate-Early/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Immediate-Early Proteins/biosynthesis , Interleukin-3/pharmacology , Transcription Factors/biosynthesis , Transcription, Genetic/drug effects , Base Sequence , Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins/genetics , Early Growth Response Protein 1 , Gene Expression Regulation, Leukemic/drug effects , Genes, Reporter , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , Humans , Immediate-Early Proteins/genetics , Interleukin-3/therapeutic use , Leukemia, Myeloid/pathology , Molecular Sequence Data , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Phosphorylation , Promoter Regions, Genetic , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/drug effects , Receptors, Interleukin-3/drug effects , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , Regulatory Sequences, Nucleic Acid , Sequence Deletion , Signal Transduction , Transcription Factors/genetics , Transfection , Tumor Cells, CulturedABSTRACT
Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) stimulate the proliferation and maturation of myeloid progenitor cells following interaction with heterodimeric receptors that share a common beta subunit required for signal transduction. Our previous studies have demonstrated that GM-CSF and IL-3 activate signaling pathways which converge upon a cAMP response element-binding protein (CREB)-binding site of the human immediate early response gene (early growth response gene-1, egr-1) promoter. Using electromobility supershift assays and antibodies directed against CREB phosphorylated on serine 133, we show that CREB is phosphorylated on serine 133 in response to GM-CSF or IL-3 stimulation. We demonstrate that phosphorylation of CREB on serine 133 substantially contributes to transcriptional activation of egr-1 in response to GM-CSF but not IL-3. These studies suggest that phosphorylation of CREB may play different roles during signal transduction, resulting in unique and overlapping biological functions in myeloid cells.
Subject(s)
Cyclic AMP Response Element-Binding Protein/metabolism , DNA-Binding Proteins/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Immediate-Early Proteins , Interleukin-3/pharmacology , Retroviridae Proteins, Oncogenic/biosynthesis , Signal Transduction , Transcription Factors , Transcription, Genetic , Cells, Cultured , DNA-Binding Proteins/genetics , Early Growth Response Protein 1 , Hematopoietic Stem Cells , Humans , Immunoblotting , Phosphorylation , Protein Binding , Protein Processing, Post-Translational , Recombinant Proteins/biosynthesis , Retroviridae Proteins, Oncogenic/genetics , Serine/metabolismABSTRACT
Granulocyte-macrophage colony stimulating factor (GM-CSF) and Interleukin-3 (IL-3) are cytokines which stimulate myeloid bone marrow progenitor cell proliferation and maturation. GM-CSF also enhances the function of terminally differentiated effector cells including neutrophils, monocytes and eosinophils. Both growth factors exhibit similar biological activities on overlapping cell populations which are mediated by high affinity receptors. These receptors share a common beta subunit necessary for signal transduction. The receptors for GM-CSF and IL-3 are members of the hematopoietin receptor superfamily and consequently lack intrinsic tyrosine kinase activity. Several kinases, including JAK2 and raf-1, and other downstream molecules are likely to be responsible for the functional redundancy demonstrated by GM-CSF and IL-3 in factor-responsive cells. This review discusses recent findings which elucidate the signaling pathways activated by these two cytokines.
