The hematopoietic growth factors regulate the growth and differentiation of myeloid and extramedullary cells of diverse embryologic origin. Granulocyte-macrophage colony stimulating factor (GM-CSF) is a notably pleiotropic hematopoietic growth factor which is being evaluated in clinical trials designed to restore hematologic cell number and function. The effects of GM-CSF on monocyte-macrophages are particularly noteworthy. In contrast to its more restricted effects on other cell types, GM-CSF induces both growth and differentiation of macrophages. The subcellular mechanism of action of GM-CSF in target cells in general, and macrophages in particular, is unknown. We have recently examined the effect of GM-CSF on the expression of the immediate early response gene, Egr-1. The regulation of Egr-1 was selected for study because it is expressed within minutes following mitogenic stimuli, is induced by diverse stimuli in proliferating and non-proliferating (post-mitotic) cell types, and encodes a protein with three zinc finger domains which confer DNA binding and transcriptional regulatory properties. We have demonstrated that GM-CSF produces a dose-dependent increase in Egr-1 mRNA in tissue macrophages within 30 minutes through a protein kinase C-independent pathway which does not require protein synthesis. Moreover, the increase in Egr-1 mRNA induced by GM-CSF is mediated, in part, by an increase in transcription of Egr-1. In recent experiments, we have successfully transfected peritoneal macrophages with an Egr-1-chloramphenicol acetyltransferase reporter construct using DEAE-dextran. We propose to pursue these initial observations by further characterizing the mechanism of GM-CSF's effect on Egr-1 expression and to define the role of Egr-1 as a nuclear intermediary in transmitting the effect of GM-CSF on responsive cell types such as macrophages. This research proposal is comprised of three major objectives. First, we will define the cis-acting elements in the 5' region of the Egr-1 gene which enhance Egr-1 transcription in response to GM-CSF. Second, the effect of GM-CSF on the level, intracellular localization, and phosphorylation state of Egr-1 protein will be defined. Third, we will pursue two experimental approaches to determine the role of Egr-1 in transmitting the effect of GM-CSF on macrophage proliferation and function. First, we will attempt to inhibit the function of endogenous Egr-1 protein by over-expressing only the zinc finger domains of Egr-1. Second, we will attempt to decrease Egr-1 protein synthesis using anti-sense oligonucleotides. These studies are ultimately intended to provide insight into the mechanisms whereby the growth and differentiation of tissue macrophages are regulated by GM-CSF.
| Kamine, J; Elangovan, B; Subramanian, T et al. (1996) Identification of a cellular protein that specifically interacts with the essential cysteine region of the HIV-1 Tat transactivator. Virology 216:357-66 |
| Li, C; Mitchell, D H; Coleman, D L (1994) Analysis of Egr-1 protein induction in murine peritoneal macrophages treated with granulocyte-macrophage colony-stimulating factor. Yale J Biol Med 67:269-76 |
