Research in our laboratory is aimed at understanding the cellular and molecular mechanisms that govern colony stimulating factor‐1 (CSF‐1) gene expression. Our hypothesis is that a basal set of trans‐acting factors is bound to the CSF‐1 gene during fibroblast proliferation, resulting in constitutive CSF‐1 gene expression. Modulation of CSF‐1 gene transcription by growth‐arrest (decrease) or stimulation of growth‐arrested fibroblasts (re‐initiate) is mediated by changes in the basal set of factors bound and/or by the addition of stimulus‐specific factors. We have extended our hypothesis to include other cell types (monocytes) to determine if mechanisms used to control CSF‐1 gene expression in fibroblasts are unique or represent common nontissue‐specific regulatory mechanisms. Analysis of CSF‐1‐CAT reporter constructs in transiently transfected fibroblasts and monocytes was used to identify CSF‐1 genomic sequences that affect transcriptional activity. DNase 1 protection, electrophoretic mobility shift, and methylation interference assays were used to identify the putative cis‐acting elements. Results of our study suggest multiple trans‐acting factors may regulate CSF‐1 gene expression; some may be tissue specific, while others, such as AP1, CTF/NF1, Spl, and Sp3, are shared in common. Mol Reprod Dev 46:39–45, 1997. © 1997 Wiley‐Liss, Inc.
Circulating and tissue-specific monocytes/macrophages, through production of hydrolytic enzymes and growth factors, can dramatically affect the local tissue environment. Colony-stimulating factor-1 (CSF-1) is a key regulator of monocyte/macrophage cell activity. CSF-1 is produced by stromal elements, including fibroblasts, which are found in all tissues. To understand at the molecular level how changes in CSF-1 gene transcription are initiated in fibroblasts, we set out to identify the cis-acting elements and cognate trans-acting factor(s) that bind regulatory regions of the mouse CSF-1 gene. Analysis of heterologous reporter constructs containing the mouse CSF-1 promoter linked to the bacterial chloramphenicol acetyltransferase (CAT) gene in transiently transfected fibroblasts identified a cis-acting element located between base pairs -88 and -43 of the CSF-1 gene. Electrophoretic mobility-shift assays (EMSAs) and DNase I protection assays with nuclear extracts isolated from proliferating fibroblasts revealed distinct protein binding to the region spanning base pairs -90 to -68. Results from methylation interference assays suggest CTF/NF1 or a CTF/NF1-like factor is the cognate trans-acting factor. Mutation of the putative CTF/NF1 binding site in the CSF-1 promoter lead to a modest decrease in promoter activity in transiently transfected fibroblasts and monocytes. Therefore, we have demonstrated that CTF/NF1 or a CTF/NF1-like protein binds to the CSF-1 gene promoter; however, binding of the CTF/NF1-like protein alone does not significantly effect changes in CSF-1 gene promoter activity.
At the site of a wound or an infection, localized production of colony-stimulating factor-1 (CSF-1) by resident macrophages is chemotactic for circulating monocytes. Several intracellular signaling pathways, including those initiated in response to activation of phospholipase A2 (PLA2) have been proposed to play a role in the regulation of CSF-1 gene expression. The goal of these studies was to determine whether PLA2 is required for the initial increase in CSF-1 gene expression in serum- or IL-1 alpha-stimulated growth-arrested fibroblasts. IL-1 alpha- or serum-stimulation of growth-arrested fibroblasts had no effect on PLA2 enzyme activity and inhibitors of cytosolic or Ca(2+)-independent PLA2 activity had no effect on IL-1 alpha- or serum-mediated increases in CSF-1 mRNA levels. High concentrations of the PLA2 inhibitors, 4-bromophenacyl lactone and quinacrine, resulted in a generalized decrease in cellular mRNA levels. Our results, obtained in fibroblasts, suggest treatment with 4-bromophenacyl lactone or quinacrine, instead of inhibiting PLA2 activity specifically, results in a generalized depression of cellular mRNA levels. These data demonstrate that the initial increase in CSF-1 gene expression in growth-arrested fibroblasts treated with serum or IL-1 alpha occurs through a PLA2-independent mechanism.
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