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 enhanc ed biological activity of this molecule in comparison with GM-CSF or I L-3 alone or in combination. Experiments were performed to study the m echanisms resulting in PIXY321-induced egr-1 expression in human myelo id leukemic cells (TF-1), Transfections of egr-1 promoter constructs r evealed that PIXY321 stimulation resulted in fourfold induction of the -116 and -600 nucleotide (nt) constructs. We transfected a -116 nt co nstruct 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, PI XY321 stimulation resulted in 2.5-fold induction of a SRE-CRE-containi ng construct (P < .05), suggesting that the SRE and CRE are sufficient for PIXY321 responsiveness. Electrophoretic mobility shift assays (EM SA) 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 const itutively phosphorylated in TF-1 cells grown on PIXY321 before growth factor and serum starvation. However, in TF-1 cells grown on GM-CSF be fore starvation, CREB phosphorylation was observed 10 minutes after PI XY321 stimulation. Furthermore, EMSAs with PIXY321-stimulated and -uns timulated extracts demonstrated the presence of specific proteins that recognize the SRE. Our data demonstrate that transcriptional regulati on of egr-1 by PIXY321 is mediated by the CRE and SRE. (C) 1996 by The American Society of Hematology.