Vol. 2, 273-278, June 1991 Cell Growth & Differentiation 273 Posttranscriptional Regulation of the Zinc Finger- encoding EGR-1 Gene by Granulocyte-Macrophage Colony-stimulating Factor in Human U-937 Monocytic Leukemia Cells: Involvement of a Pertussis Toxin-sensitive G Protein’ Steven H. Bernstein,2 Surender M. Kharbanda, Matthew L Sherman, Vikas P. Sukhatme, and Donald W. Kufe Laboratory of Clinical Pharmacology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 021 15 [S. H. B., S. M. K., M. L. S., D. W. K.], and Departments of Medicine and Molecular Genetics and Cell Biology, Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637 [V. P. 5.] Abstract The EGR-1 gene is an immediate early response gene encoding a zinc finger DNA-binding protein. The present studies have examined the regulation of EGR-1 gene expression in human U-937 monocytic leukemia cells treated with granulocyte-macrophage colony- stimulating factor (GM-CSF). The results demonstrate that GM-CSF rapidly and transiently increases EGR-1 gene expression in U-937 cells. Similar findings were obtained in GM-CSF-treated human monocytes. We also show that the regulation of EGR-1 expression by GM-CSF is a pertussis toxin-sensitive event. The results of nuclear run-on assays further demonstrate that the EGR-1 gene is constitutively transcribed in untreated U-937 cells and that GM-CSF has little effect on this rate of transcription. Inhibition of protein synthesis with cycloheximide also had no detectable effect on EGR-1 gene transcription but was associated with superinduction of EGR-1 mRNA levels in GM-CSF- treated cells. Moreover, the half-life of GM-CSF- induced EGR-1 transcripts was prolonged from 33 to 70 mm following inhibition of protein synthesis. Taken together, the results indicate that GM-CSF activates signaling pathways which regulate EGR-1 gene expression through a pertussis toxin-sensitive G protein and that these events increase EGR-1 mRNA levels by a posttranscriptional mechanism. This GM-CSF- dependent regulation of EGR-1 expression may provide a mechanism for transducing signals to the nucleus that are involved in the control of gene transcription. Introduction GM-CSF3 is a 22-kiiodaiton glycoprotemn regulating the Received 1/29/91. 1 This investigation was supported in part by American Cancer Society Physician’s Research Training Grant PRTF-i20 (S. H. B.), USPHS Grants CA42802 and CA01092 (M. L. S.), and a Burroughs Wellcome Clinical Pharmacology Scholar Award (D. W. K.). 2 To whom requests for reprints should be addressed, at Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. 3 The abbreviations used are: GM-CSF, granulocyte-macrophage colony- survival, proliferation, and differentiation of hemopoietic progenitor cells (1). There are certain insights available regarding the regulation of GM-CSF secretion from ac- cessory cells (1). However, little is known about the signals through which GM-CSF mediates its cellular response. Recent work has characterized a variety of genes whose expression is rapidly and transiently induced when quiescent fibrobiasts are stimulated with serum or growth factors. Many of these genes code for DNA- binding transcription factors. Two major groups of these Immediate early response genes” include the leucine zipper family of proteins, including c-los (2, 3), Ira-i (4), lra-2 (5), los-B (6), c-jun (7, 8), jun-B (9), and jun-D (10), and the zinc finger-binding proteins, EGR-i (11) [zil/268 (12), NGF1-A (13), Krox 24 (14), and TIS-8 (15)], EGR-2 (16) [Krox 20 (17)], EGR-3, and EGR-4.4 The ECR-i gene encodes a 533-amino acid polypep- tide which includes three tandem repeat units of 28-30 amino acids conforming to the consensus sequence for zinc binding (11). The EGR-i protein thus belongs to a family of zinc finger transcription factors, including SW 15 (18), Spi (19), and TFI11A (20). The EGR-1 gene is induced in a variety of cell types following both mitogenic and differentiating signals. For example, this gene is ex- pressed after mitogenic stimulation of quiescent BALB/ c-3T3 cells by serum, platelet-derived growth factor, or fibroblast growth factor (21, 22). Furthermore, the EGR- 1 gene is transiently induced during cardiac and neuronal differentiation of the pleuripotent EC line P1951801A1 (11). Nerve growth factor induction of neuronal differ- entiation in PC12 rat pheochromocytoma cells is also associated with a rapid stimulation of ECR-1 expression (11). Finally, the EGR-1 gene is expressed following mem- brane depolarization both in vitro and in vivo (11). Pre- vious studies have demonstrated that the induction of EGR-i expression is similar to that of c-los (ii) in many situations, and that this coordinate regulation may be mediated by the presence of serum response elements present in both the ECR-1 and c-los promoters (23). However, there is little information available regarding mechanisms responsible for regulation of the ECR-1 gene. The present work has examined the effects of GM- CSF on EGR-i expression. Previous studies have dem- onstrated that GM-CSF induces monocytic differentiation stimulating factor; kb, kilobase(s); CHX, cycloheximide; G protein, gua- nine nucleotide-binding protein; cDNA, complementary DNA; SSC, standard saline citrate; SDS, sodium dodecyl sulfate. 4 V. P. Sukhatme, unpublished data. 5 C. Xinmin and V. P. Sukhatme, unpublished observations.