Prostaglandin E2 inhibition of growth in a colony-stimulating factor 1-dependent macrophage cell line

The effects of prostaglandin E2 (PGE2) were examined in a murine macrophage cell line (BAC1.2F5) that was completely dependent on colony-stimulating factor-1 (CSF-1) for both growth and survival. The addition of PGE2 to cultures of BAC1.2F5 cells resulted in the inhibition of CSF-1-induced [3H]thymidine incorporation and cell proliferation. The inhibitory effects of PGE2 were mimicked by the addition of dibutyryl-cyclic AMP, and the effectiveness of PGE2 was markedly potentiated by 1-methyl-3-isobutylxanthine, a potent inhibitor of cyclic nucleotide phosphodiesterase activity. PGE2 caused a 10-fold elevation of the intracellular cyclic AMP concentration, whereas CSF-1 neither increased cyclic AMP levels nor attenuated the rise in cyclic AMP promoted by PGE2. However, CSF-1 may indirectly regulate cyclic AMP levels since in the absence of CSF-1, BAC1.2F5 cells actively synthesized PGE2, whereas PGE2 production was abruptly terminated by the addition of CSF-1. In BAC1.2F5 cells, PGE2 increases the intracellular cyclic AMP concentration, thereby blocking cell proliferation, but does not down-regulate the CSF-1 receptor or abrogate the functions of CSF-1 necessary for cell survival.     

CSF-1-induced gene expression in macrophages: dissociation from the mitogenic response

Early gene expression associated with the mitogenic response to colony stimulating factor-1 (CSF-1) has been examined in BAC1.2F5, a CSF-1-dependent murine macrophage cell line. Stimulation of arrested cells by CSF-1 resulted in acute, transient elevation in c-fos and subsequently in c-myc mRNA levels. Dramatic, sustained elevations were observed for JE and KC mRNAs, which are induced by platelet-derived growth factor (PDGF) in 3T3 cells. The kinetics of expression of all four messages were similar to those reported in PDGF-stimulated fibroblasts, implying a program of gene expression common to these two mitogens. Granulocyte-macrophage CSF (GM-CSF) can replace CSF-1 in stimulating the growth of 2F5 cells. It induced mRNAs for c-fos, c-myc and JE but not KC. Therefore KC expression, although correlated with mitogenesis, is not required for proliferation. The effects of CSF-1 were also examined in cells cycling continuously in its absence: 2F5 cells incubated in GM-CSF and an autonomous variant subclone of 2F5. In either case, the only detected growth effect of CSF-1 was a reduction in doubling-time. Nevertheless, all four of the mRNAs induced by CSF-1 in arrested cultures of 2F5 were strongly induced with the same kinetics in these cycling cells. Thus it would appear that the functions mediated by this early-gene program are not restricted to the mitogenic stimulation of arrested cells.     

Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels.

Growth factor regulation of phosphatidylcholine (PtdCho) metabolism during the G1 stage of the cell cycle was investigated in the colony-stimulating factor 1 (CSF-1)-dependent murine macrophage cell-line BAC1.2F5. The transient removal of CSF-1 arrested the cells in G1. Incorporation of [3H]choline into PtdCho was stimulated significantly 1 h after growth factor addition to quiescent cells. Metabolic labeling experiments pointed to CTP:phosphocholine cytidylyltransferase (CT) as the rate-controlling enzyme for PtdCho biosynthesis in BAC1.2F5 cells. The amount of CT mRNA increased 4-fold within 15 min of CSF-1 addition and remained elevated for 2 h. The rise in CT mRNA levels was accompanied by a 50% increase in total CT specific activity in cell extracts within 4 h after the addition of CSF-1. CSF-1-dependent elevation of CT mRNA content was neither attenuated nor superinduced by the inhibition of protein synthesis with cycloheximide. The rate of CT mRNA turnover decreased in the presence of CSF-1 indicating that message stabilization was a key factor in determining the levels of CT mRNA. These data point to increased CT mRNA abundance as a component in growth factor-stimulated PtdCho synthesis.     

Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1

The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 stimulation.     

Involvement of CSF-1 in generating a stroma-independent hematopoietic stem cell line

The hematopoietic stem cell line, Myl-D7, is maintained by a self-renewing stem cell population that spontaneously generates myeloid, lymphoid, and erythroid progeny. MS-5 stromal cells are necessary for the growth of Myl-D7 cells. One component of the Myl-D7 cells proliferation activity released by MS-5 stromal cells was enriched by Q sepharose fractionation and shown to be colony stimulating factor-1 (CSF-1) by Western blotting, BAC1.2F5 cell bioassay and inhibition of Myl-D7 proliferation by CSF-1 antibody. The requirement of Myl-D7 cells for CSF-1 was also demonstrated independently by selecting for rare, stroma-independent Myl-D7 mutant clones able to grow without stroma and additional factors. Eighty-nine stroma-independent mutant clones were obtained and belonged to two classes. The majority of mutants did not secrete any growth promoting activity. The second, rarer class of mutants releases a factor that stimulates proliferation/survival for up to several months and approximately half of the secretors express high levels of CSF-1 mRNA. Wild type Myl-D7 grown with supernatants from the secretor cells retained the stem cell phenotype. These data suggest that CSF-1 may act as a key factor in stroma-regulated hematopoiesis and cell-cell interaction.     

An altered repertoire of fos/jun (AP-1) at the onset of replicative senescence.

With multiple divisions in culture, normal diploid cells suffer a loss of growth potential that leads to replicative senescence and a finite replicative capacity. Using quantitative RT-PCR, we have monitored mRNA expression levels of c-fos, c-jun, JunB, c-myc, p53, H-ras, and histone H4 during the replicative senescence of human fibroblasts. The earliest and the largest changes in gene expression occurred in c-fos and junB at mid-senescence prior to the first slowing in cell growth rates. The basal level of c-fos mRNA decreased to one-ninth that of the early-passage levels, while junB declined to one-third and c-jun expression remained constant. The decline in the basal c-fos mRNA level in mid-senescence should lead to an increase in Jun/Jun AP-1 homodimers at the expense of Fos/Jun heterodimers and may trigger a cascade of further changes in c-myc, p53, and H-ras expression in late-passage senescent fibroblasts.     

Induction of clonal monocyte-macrophage tumors in vivo by a mouse c-myc retrovirus: rearrangement of the CSF-1 gene as a secondary transforming event.

A mouse retrovirus containing the c-myc oncogene was found to induce tumors of mononuclear phagocytic cells in vivo. All tumors expressed the c-myc retroviral gene but not the endogenous c-myc gene (with one exception), and virtually all tumors were clonal with a unique proviral integration. This observation, coupled with a lag time in tumor formation, suggests that a second event, in addition to c-myc proviral integration, is necessary for the generation of neoplastic cells in vivo. All of the tumor cells expressed high levels of mRNA for both the putative colony-stimulating factor 1 (CSF-1) receptor (c-fms proto-oncogene product), as well as the c-fos proto-oncogene. Although all of the tumor cells proliferated in culture without the addition of exogenous CSF-1, which is required for the proliferation of primary macrophages partially transformed by the same c-myc retrovirus, several phenotypes were observed with respect to the expression of CSF-1 and granulocyte-macrophage CSF and to their growth factor responsiveness. The proliferation of one tumor, which secreted high levels of CSF-1, was blocked by specific anti-CSF-1 serum. This tumor was found to express altered CSF-1 mRNA and to have a DNA rearrangement at the CSF-1 locus. In this particular case, the data indicate that a CSF-1 gene rearrangement was the secondary event in development of the tumor. The pleiotropy of phenotypes among the other tumors indicated that there are a variety of other mechanisms for such secondary events which can be investigated with this system.     

Identification of tyrosine 706 in the kinase insert as the major colony-stimulating factor 1 (CSF-1)-stimulated autophosphorylation site in the CSF-1 receptor in a murine macrophage cell line.

The receptor for colony-stimulating factor 1 (CSF-1) is a ligand-activated protein-tyrosine kinase. It has been shown previously that the CSF-1 receptor is phosphorylated on serine in vivo and that phosphorylation on tyrosine can be induced by stimulation with CSF-1. We studied the phosphorylation of the CSF-1 receptor by using the BAC1.2F5 murine macrophage cell line, which naturally expresses CSF-1 receptors. Two-dimensional tryptic phosphopeptide mapping showed that the CSF-1 receptor is phosphorylated on several different serine residues in vivo. Stimulation with CSF-1 at 37 degrees C resulted in rapid phosphorylation on tyrosine at one major site and one or two minor sites. We identified the major site as Tyr-706. The identity of Tyr-706 was confirmed by mutagenesis. This residue is located within the kinase insert domain. There was no evidence that Tyr-973 (equivalent to Tyr-969 in the human CSF-1 receptor) was phosphorylated following CSF-1 stimulation. When cells were stimulated with CSF-1 at 4 degrees C, additional phosphotyrosine-containing phosphopeptides were detected and the level of phosphorylation of the individual phosphotyrosine-containing phosphopeptides was substantially increased. In addition, we show that CSF-1 receptors are capable of autophosphorylation at six to eight major sites in vitro.     

Elevated expression of proto-oncogenes during interleukin-5-induced growth and differentiation of murine B lineage cells

Interleukin 5 (IL-5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. To elucidate IL-5-mediated intracellular mechanisms, we have established IL-5-dependent and -independent murine early B cell lines, J6 and MJ88-1, respectively, and examined the effect of IL-5 on the expression of proto-oncogenes during proliferation. Two- to 3.5-fold increases in the levels of c-myb, c-myc, c-fos, and c-fms mRNA were observed in J6 cells, compared with those in MJ88-1 cells. Further, a role of IL-5 in the proto-oncogene expression during differentiation was examined by using thymidine-treated murine B-cell chronic leukemia BCL1-B20 cells with growth arrest. After 4-day culture, the amount of IgM secreted from BCL1-B20 cells was augmented 4-6 fold in the presence of IL-5. Although expression of c-myb, c-fos, and c-fms mRNA did not change, only c-myc mRNA expression was elevated within 30 min of stimulation with IL-5 and reached a maximal level by 1 hr. Addition of phorbol 12-myristate 13-acetate (PMA) or IL-4 to the culture of BCL1-B20 cells inhibited both the IL-5-mediated augmentation of IgM secretion and the elevated expression of c-myc mRNA. These findings suggest that the IL-5 signal may be associated with the up-regulation of c-myc expression.     

A novel pathway from phosphorylation of tyrosine residues 239/240 of Shc, contributing to suppress apoptosis by IL-3.

Interleukin 3 (IL-3) not only induces DNA synthesis of haematopoietic cells but also maintains their viability by suppressing apoptosis. IL-3 stimulates tyrosine phosphorylation of the Shc adaptor protein and thereby formation of a complex of Shc with Grb2 at phosphorylated tyrosine (Y) residue 317-Shc. This pathway is implicated in Ras/mitogen-activated protein kinase (MAPK) activation towards c-fos gene expression. We examined the possible involvement of Shc in the antiapoptotic activity of IL-3. Conditional overexpression of the Shc SH2 domain, a dominant-negative mutant of Shc, was found to induce apoptosis of IL-3-dependent Ba/F3 cells along with a reduction of c-myc gene expression. Apoptosis was rescued by the exogenously introduced c-myc gene. Since we identify novel tyrosine phosphorylation sites of Shc: Y239 and Y240, their role on cell survival was tested by mutational analysis. Ba/F3 cells expressing mutant Shc Y317F, which is unable to stimulate efficiently the Ras pathway, still showed resistance to apoptosis. However, cells expressing Shc Y239/240F, which is able to stimulate the Ras pathway, were sensitive to apoptosis. In these cells, induction of the c-myc gene was reduced. These findings suggest that a new signalling pathway for cell survival is generated from Y239/240 of Shc to the nuclei involving c-myc gene expression.     

ERK5/BMK1 is indispensable for optimal colony-stimulating factor 1 (CSF-1)-induced proliferation in macrophages in a Src-dependent fashion

CSF-1, by binding to its high-affinity receptor CSF-1R, sustains the survival and proliferation of monocyte/macrophages, which are central cells of innate immunity and inflammation. The MAPK ERK5 (also known as big MAPK-1, BMK1, or MAPK7) is a 98-kDa molecule sharing high homology with ERK1/2. ERK5 is activated by oxidative stress or growth factor stimulation. This study was undertaken to characterize ERK5 involvement in macrophage signaling that is elicited by CSF-1. Exposure to the CSF-1 of primary human macrophages or murine macrophage cell lines, as well as murine fibroblasts expressing ectopic CSF-1R, resulted in a rapid and sustained increase of ERK5 phosphorylation on activation-specific residues. In the BAC1.2F5 macrophage cell line, ERK5 was also activated by another mitogen, GM-CSF, while macrophage activators such as LPS or IFN-gamma and a number of nonproliferative cytokines failed. Src family kinases were found to link the activation of CSF-1R to that of ERK5, whereas protein kinase C or the serine phosphatases PP1 and PP2A seem not to be involved in the process. Treatment of macrophages with ERK5-specific small interfering RNA markedly reduced CSF-1-induced DNA synthesis and total c-Jun phosphorylation and expression, while increasing the expression of the cyclin-dependent kinase inhibitor p27. Following CSF-1 treatment, the active form of ERK5 rapidly translocated from cytosol to nucleus. Taken together, the results reported in this study show that ERK5 is indispensable for optimal CSF-1-induced proliferation and indicate a novel target for its control.     

Colony-stimulating factor-1 receptor utilizes multiple signaling pathways to induce cyclin D2 expression

Colony-stimulating factor-1 (CSF-1) induces expression of immediate early gene, such as c-myc and c-fos and delayed early genes such as D-type cyclins (D1 and D2), whose products play essential roles in the G1 to S phase transition of the cell cycle. Little is known, however, about the cytoplasmic signal transduction pathways that connect the surface CSF-1 receptor to these genes in the nucleus. We have investigated the signaling mechanism of CSF-1-induced D2 expression. Analyses of CSF-1 receptor autophosphorylation mutants show that, although certain individual mutation has a partial inhibitory effect, only multiple combined mutations completely block induction of D2 in response to CSF-1. We report that at least three parallel pathways, the Src pathway, the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, and the c-myc pathway, are involved. Induction of D2 is partially inhibited in Src(-/-) bone marrow-derived macrophages and by Src inhibitor PP1 and is enhanced in v-Src-overexpressing cells. Activation of myc's transactivating activity selectively induces D2 but not D1. Blockade of c-myc expression partially blocks CSF-1-induced D2 expression. Complete inhibition of the MEK/ERK pathway causes 50% decrease of D2 expression. Finally, simultaneous inhibition of Src, MEK activation, and c-myc expression additively blocks CSF-1-induced D2 expression. This study indicates that multiple signaling pathways are involved in full induction of a single gene, and this finding may also apply broadly to other growth factor-inducible genes.     

Isolation and characterization of a cloned growth factor dependent macrophage cell line, BAC1.2F5

The SV40 transformed murine macrophage cell line, BAC1, proliferates in response to the colony stimulating factor, CSF-1 (Schwarzbaum et al., J. Immunol., 132:1158, 1984). In order to obtain a cell line suitable for biochemical and genetic studies of CSF-1 signal transduction, clones of BAC1 were established. Clones ranged from being completely autonomous to being completely dependent on CSF-1 for growth. Cells of one clone (2F5), which proliferated in response to either CSF-1 or granulocyte-macrophage CSF (GM-CSF) were characterized in detail. The kinetics of receptor-mediated internalization and intracellular destruction of CSF-1 were comparable to the kinetics observed with peritoneal exudate macrophages. CSF-1 was shown to regulate cell spreading, cell survival, protein degradation, and the duration of the G1 and S phases of the cell cycle. The 2F5 clone therefore exhibits a number of CSF-1 stimulated responses and is being used for genetic and biochemical studies of CSF-1 action.