Mechanism of transcriptional activation of the immediate early gene Egr-1 in response to PIXY321.
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.
Mignacca
R C
RC
Gwynne Hazen Cherry Memorial Laboratories, Department of Pediatrics, UCLA School of Medicine 90024-1752, USA.
Lee
H J
HJ
Kwon
E M
EM
Sakamoto
K M
KM
D001483:Base Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D051766:Early Growth Response Protein 1; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D017781:Genes, Immediate-Early / Q000187:drug effects*; D017930:Genes, Reporter; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis* / Q000235:genetics; D007377:Interleukin-3 / Q000494:pharmacology* / Q000627:therapeutic use; D007951:Leukemia, Myeloid / Q000473:pathology; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000187:drug effects; D016185:Receptors, Interleukin-3 / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000494:pharmacology / Q000627:therapeutic use; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D015398:Signal Transduction; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D014407:Tumor Cells, Cultured
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D007377:Interleukin-3; D009363:Neoplasm Proteins; C078524:PIXY321 fusion protein, recombinant; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D016185:Receptors, Interleukin-3; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor