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Mechanism of transcriptional activation of the immediate early gene Egr- 1
in response to PIXY321
RC Mignacca, HJ Lee, EM Kwon and KM Sakamoto
Gwynne Hazen Cherry Memorial Laboratories, Department of Pediatrics, UCLA
School of Medicine 90024-1752, USA.
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.
Volume 88,
Issue 3,
pp. 848-854,
08/01/1996
Copyright © 1996 by The American Society of Hematology
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