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The mechanisms by which interleukin-6 (IL-6) family cytokines, which utilize the common receptor signaling subunit gp130, influence monocyte/macrophage development remain unclear. Here we have utilized macrophages devoid of either gp130-dependent STAT1/3 (gp130(Delta STAT/Delta STAT)) or extracellular signal-regulated kinases 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase (gp130(Y757F/Y757F)) activation to assess the individual contribution of each pathway to macrophage formation. While the inhibition by IL-6 of macrophage colony-stimulating factor (M-CSF)-induced colony formation observed in gp130(wt/wt) mice was abolished in gp130(Delta STAT/Delta STAT) mice, inhibition of macrophage colony formation was enhanced in gp130(Y757F/Y757F) mice. In gp130(Delta STAT/Delta STAT) bone marrow-derived macrophages (BMMs), both IL-6- and M-CSF-induced ERK1/2 tyrosine phosphorylation was enhanced. By contrast, tyrosine phosphorylation of ERK1/2 in response to M-CSF was reduced in gp130(Y757F/Y757F) BMMs, and the pattern of ERK1/2 activation in gp130 mutant BMMs correlated with their opposing responsiveness to M-CSF-induced proliferation. When compared to the level of expression in gp130(wt/wt) BMMs, c-fms expression was elevated in gp130(Delta STAT/Delta STAT) BMMs but reduced in gp130(Y757F/Y757F) BMMs. Finally, an ERK1/2 inhibitor suppressed M-CSF-induced BMM proliferation, and this result corresponded to a reduction in c-fms expression. Collectively, these results provide a functional and causal correlation between gp130-dependent ERK MAP kinase signaling and c-fms gene activation, a finding that provides a potential mechanism underlying the inhibition of M-CSF-dependent macrophage development by IL-6 family cytokines in mice.

To address the role of ras signaling in monocytic phagocytes in vivo, the expression of two dominant suppressors of in vitro ras signaling pathways, the carboxyl-terminal region of the GTPase-activating protein (GAP-C) and the DNA binding domain of the transcription factor ets-2, were targeted to this cell compartment. A 5-kb portion of the human c-fms proximal promoter was shown to direct expression of the transgenes to the monocytic lineage. As a result of the GAP-C transgene expression, ras-GTP levels were reduced in mature peritoneal macrophages by 70%. The terminal differentiation of monocytes was altered, as evidence by the accumulation of atypical monocytic cells in the blood. Mature peritoneal macrophages exhibited changes in colony-stimulating factor 1-dependent survival and structure. Further, expression of the colony-stimulating factor 1-stimulated gene urokinase plasminogen activator was inhibited in peritoneal macrophages. The results indicate that ras action is critical in monocytic cells after these cells have lost the capacity to traverse the cell cycle.

The gene encoding the receptor for macrophage colony-stimulating factor 1 (CSF-1), the c-fms protooncogene, is selectively expressed in immature and mature mononuclear phagocytes and trophoblasts. Exon 1 is expressed only in trophoblasts. Isolation and sequencing of genomic DNA flanking exon 2 of the murine c-fms gene revealed a TATA-less promoter with significant homology to human c-fms. Reverse transcriptase primer extension analysis using exon 2 primers identified multiple clustered transcription initiation sites. Their position was confirmed by RNase protection. The same primer extension products were detected in equal abundance from macrophage or nonmacrophage sources of RNA. c-fms mRNA is acutely down-regulated in primary macrophages by CSF-1, bacterial lipopolysaccharide (LPS), and phorbol myristate acetate (PMA). Each of these agents reduced the abundance of c-fms RNA detectable by primer extension using an exon 3 primer without altering the abundance of presumptive short c-fms transcripts detected with exon 2 primers. Primer extension analysis with an intron 2 primer detected products at greater abundance in nonmacrophages. Templates detected with the intronic primer were induced in macrophages by LPS, PMA, and CSF-1, suggesting that each of the agents caused a shift from full-length c-fms mRNA production to production of unspliced, truncated transcripts. The c-fms promoter functioned constitutively in the RAW264 macrophage cell line, the B-cell line MOPC.31C, and several nonhematopoietic cell lines. Macrophage-specific expression and responsiveness to selective repression by LPS and PMA was achieved by the incorporation of intron 2 into the c-fms promoter-reporter construct. The results suggest that expression of the c-fms gene in macrophages is controlled by sequences in intron 2 that act by regulating transcription elongation.

cis-acting elements found in 3′-untranslated regions (UTRs) are regulatory signals determining mRNA stability and translational efficiency. By binding a novel non-AU-rich 69-nucleotide (nt) c-fms 3′ UTR sequence, we previously identified HuR as a promoter of c-fms proto-oncogene mRNA. We now identify the 69-nt c-fms mRNA 3′ UTR sequence as a cellular vigilin target through which vigilin inhibits the expression of c-fms mRNA and protein. Altering association of either vigilin or HuR with c-fms mRNA in vivo reciprocally affected mRNA association with the other protein. Mechanistic studies show that vigilin decreased c-fms mRNA stability. Furthermore, vigilin inhibited c-fms translation. Vigilin suppresses while HuR encourages cellular motility and invasion of breast cancer cells. In summary, we identified a competition for binding the 69-nt sequence, through which vigilin and HuR exert opposing effects on c-fms expression, suggesting a role for vigilin in suppression of breast cancer progression.

It is now well established that the chromatin of genes expressed in specific hematopoietic lineages is already partly reorganized towards an active state in hematopoietic stem cells (HSCs) and multipotent progenitors, and a number of such genes are expressed at a low level prior to lineage commitment (10, 12, 15, 18, 33). During progressive lineage restriction and cell fate specification this promiscuous gene expression program is then restricted by upregulation of lineage-appropriate genes and silencing of lineage-inappropriate genes (8, 24). These observations indicate that lineage-specific gene priming must occur at an early stage of HSC development. However, due to the low abundance of HSCs and multipotent progenitors little is known about the mechanistic details of how such priming events are achieved and how an active chromatin structure is established that supports high-level transcription later in development. PU.1, a member of the Ets family of DNA-binding proteins, is a transcription factor that is critical for the development of myeloid lineages such as monocytes and granulocytes. Deletion of the PU.1 gene leads to defects in myelopoiesis, including loss of monocytes and macrophages (22, 28). Early myeloid progenitors are generated in PU.1-deficient mice, albeit at reduced numbers, but their differentiation is blocked (6). PU.1−/− myeloid progenitors fail to undergo macrophage differentiation and do not express the colony-stimulating factor 1 (CSF-1) receptor gene (c-fms), one of the most important genes regulating macrophage survival and proliferation. This gene is absolutely required for macrophage development (4). However, rescue of PU.1−/− myeloid progenitor cells with a c-fms expression vector restores macrophage progenitor growth and proliferation but not macrophage differentiation, indicating that PU.1 regulates a larger program of macrophage gene expression (6). c-fms belongs to a class of myeloid genes which are already expressed at a low level in HSCs (24, 34). Tissue-specific expression of c-fms mRNA is regulated by well-defined promoter and intronic enhancer elements (Fig. ​(Fig.1).1). The promoter used in macrophages is a TATA-less promoter, with multiple purine-rich elements bound by Ets family transcription factors (26). Tissue-restricted high-level expression of the c-fms gene is dependent upon the c-fms intron regulatory element termed FIRE, within the first intron (11, 27). Both the promoter and FIRE are bound by PU.1 in macrophages (5, 6, 13, 36). We previously showed by in vivo footprinting that the c-fms locus is already partly occupied by transcription factors in HSCs and becomes fully occupied in committed myeloid progenitor cells (34). In contrast, cell surface expression of CSF-1 receptor protein and high levels of mRNA are readily detected only in committed macrophage precursors (31) and their progeny. An initial mechanistic explanation of why this was the case was provided by in vivo footprinting studies demonstrating that the increase in c-fms mRNA expression during macrophage differentiation correlates with a dynamic assembly and disassembly of transcription factor complexes on the FIRE enhancer (31). However, the molecular details of this dynamic behavior are unknown because the identities of the specific factors and cofactors recruited were not determined in the previous study. It is also not known whether other transcription factors can bind to c-fms in the absence of PU.1 and to what extent chromatin of c-fms is reorganized in PU.1−/− cells. FIG. 1. Map of the mouse c-fms locus and induction of c-fms expression on addition of OHT. (A) Chromatin structure of mouse c-fms locus regulatory regions around the proximal promoter with indications of the transcription factor binding sites, the localization ... To address the above questions, we examined the chromatin fine structure of c-fms by performing in vivo footprinting experiments and chromatin immunoprecipitation (ChIP) assays. For a model system we employed a myeloid progenitor cell line derived from PU.1-deficient mice, which cannot differentiate into macrophages but can proliferate in the presence of interleukin-3. In contrast to wild-type myeloid progenitor cells, the c-fms locus was not occupied by any transcription factors in the PU.1−/− cells. To further study the role of PU.1 in the regulation of the c-fms locus, we employed a well-established derivative of the PU.1−/− cell line (PUER) that expresses an inducible form of PU.1 (36). Significantly, induction of PU.1 in PUER cells that resulted in restoration of macrophage differentiation led to in vivo transcription factor occupancy at the c-fms locus. The promoter was very rapidly occupied by transcription factors, whereas it took significantly longer for the same transcription factors to assemble at FIRE and for elevated levels of c-fms mRNA to be expressed. This delayed kinetics could be explained by our finding that formation of an active enhancer complex at FIRE required the induction of at least one secondary transcription factor, Egr-2, by PU.1. These observations suggest a two-step mechanism of c-fms activation which involves the promoter being active in early progenitor cells, thereby enabling low-level c-fms mRNA expression, whereas activation of FIRE occurs at a later developmental time during the course of macrophage differentiation. We suggest that this mechanism ensures that high levels of c-fms mRNA and CSF-1 receptor protein are expressed only in cells destined to be CSF-1 responsive.

The macrophage colony-stimulating factor receptor encoded by the c-fms gene is expressed in vascular intimal smooth muscle cells isolated from atherosclerotic lesions. A combination of platelet-derived growth factor-BB and epidermal growth factor induces stable expression of c-fms in normal vascular medial smooth muscle cells. The mechanism by which these growth factors induce c-fms expression has now been investigated in an attempt to gain insight into the events that underlie the phenotypic conversion of vascular smooth muscle cells in atherosclerosis. Deletion analysis of the c-fms promoter revealed that the region including a binding site for transcription factor PU.1 was required for transcriptional activity in human aortic medial smooth muscle cells. Mutation in the PU.1 binding site markedly reduced promoter activity. Northern (RNA) blot analysis demonstrated that growth factors induced the expression of PU.1 mRNA in vascular medial smooth muscle cells and that PU.1 mRNA was expressed in vascular intimal smooth muscle cells. PU.1 antisense oligonucleotides inhibited growth factor-induced c-fms expression and foam cell formation. These results suggest that transcription factor PU.1 plays an essential role in the phenotypic conversion of vascular smooth muscle cells to macrophagelike cells by mediating the induction of c-fms.