Cell, Vol. 63, 1073-1063,

November

30, 1990, Copyright

0 1990 by Cell Press

Expression of the M-CSF Receptor Is Controlled Fodtranscriptionally by the Dominant Actions of GM-CSF or Multi-CSF 8. C. Gliniak and L. Ft. Rohrschneider Fred Hutchinson Cancer Research Center Seattle, Washington 98104

We have isolated a murine myeloid precursor cell line (FDC-PVMAC) that simultaneously expresses receptors for multi-CSF, GM-CSF, and M-CSF (c-fms protooncogene). FDC-PI/MAC cells express high levels of c-fms mRNA and protein when grown in M-CSF, whereas growth in multi-CSF or GM-CSF caused a dramatic reduction of c-fms glycoprotein and mFlNA. Nuclear run-off assays demonstrated that c-fms transcription was not growth factor dependent and the regulation occurred posttranscriptionally. Factor switching experiments have shown that both multi-CSF and GM-CSF act dominantly and in a factor concentration dependent manner to suppress c-fms expression. In vitro agar assays of bone marrow cells grown in the presence of GM-CSF and M-CSF, individually and in combination, support the concept that GM-CSF can act dominantly to prevent monocyte/macrophage development. These results suggest that GM-CSF and multi-CSF can suppress development along the monocytelmacrophage lineage and offer a simple stochastic mechanism governing myeloid lineage restriction. Introduction Hematopoiesis is the intricately balanced process for the production of different classes of mature blood cells from a population of pleuripotent stem cells residing in the bone marrow and spleen. Because the various mature blood cells have a limited life span, the stem cells must continuously replicate and give rise to committed progenitor cells that become restricted to development along one of eight major lineages and eventually produce the mature blood cells (reviewed by Metcalf, 1989a). The growth, differentiation, and commitment to a restricted lineage are guided by a set of humoral glycoprotein growth factors collectively termed the colony-stimulating factors (CSFs) (reviewed by Metcalf, 1984; Clark and Kamen, 1987). The concentration and type of CSF encountered by individual cells during the course of development determine the ultimate lineage to which that cell will differentiate. In a steady state this provides a constant proportion of individual blood cells, yet allows rapid fluctuations in cell populations in response to changes in CSFs due to such pathologic conditions as bacterial infection or blood loss. The molecular mechanisms that direct and control lineage restriction remain poorly understood (Metcalf, 1989b). Clearly, a single CSF can guide stem cell development along a defined lineage as demonstrated by in vitro agar colony assays (Ogawa et al., 1983; Metcalf, 1984). However, little is known about how this may occur in vivo,

where developing hematopoietic cells may encounter the entire range of CSFs. There almost certainly must be interactions and communication among the various CSF receptors in signaling growth and development, as different CSF receptors are simultaneously expressed on the same hematopoietic cell (Nicola, 1987). The form this communication takes, the molecular level at which it acts, and the function it serves remain unanswered questions. We have begun to explore potential mechanisms that may influence lineage restriction by focusing on a pathway of myeloid cell development. At one point in this developmental scheme, a bipotential progenitor cell has the ability to become either a neutrophilic granulocyte or a monocyte/macrophage. Development along these two separate lineages is controlled by four molecularly distinct CSFs (Metcalf, 1984). Granulocyte CSF (G-CSF) stimulates granulocyte development almost exclusively (Burgess and Metcalf, 1980; Nicola et al., 1983) and macrophage CSF (M-CSF, or CSF-1) will cause restricted development along the monocyte/macrophage pathway (Stanley and Heard, 1977). In contrast, both multi-CSF (also called IL-3) and granulocyte-macrophage CSF (GMCSF) will produce mature cells of both lineages (Metcalf, 1984). Interestingly, the concentration of GM-CSF has a strong influence on the lineage selected. Low concentrations of GM-CSF produce few colonies, but they are predominantly monocytelmacrophage colonies (Burgess and Metcalf, 1977; Metcalf, 1980). High concentrations of GM-CSF result in the bipotential progenitor cells favoring the granulocytic pathway. These observations on bipotential progenitor cells and their individual daughter cells form the basis for a stochastic model of hematopoietic cell development regulated by the type and concentration of individual CSFs. To examine the molecular events that influence the decision of a bipotential myeloid progenitor cell to choose one lineage over the other, we have generated a cell line that expresses three of the four CSF receptors involved in myeloid development (the multi-CSF receptor, the GM-CSF receptor, and the M-CSF receptor). This cell line has some of the features of a bipotential progenitor and retains the capacity for monocyte/macrophage development in response to M-CSF. Utilizing these cells, we have begun to look at regulatory mechanisms that might determine monocyte/macrophage development. Results Isolation of c-fms Expressing FDC-PVMAC Cells The murine myeloid progenitor cell line FDC-Pl is nontumorigenic and dependent on either multi-CSF (IL-3) or GM-CSF for its survival and proliferation (Dexter et al., 1980; Hapel et al., 1984). By continuously culturing the FDC-Pl cells in only M-CSF, we have isolated a subclone of this line, designated FDC-PlIMAC, which expresses the endogenous M-CSF receptor (the c-fms proto-oncogene) and can now grow in the presence of M-CSF as the sole

Cdl 1074

FDC-Pi/MAC

FDC-Pl Cl.4 GM-CSF

Multi-CSF

LIQUID CULTURE

AGAR CULTURE

GIEMSA YAYI GRUNWALD STAINING

Figure

1. Growth

Characteristics

and Morphology

of FDC-PI

and FDC-PI/MAC

Cells were grown for at least 5 days in the indicated colony-stimulating microphotometry, 14 day growth in 0.3% soft agar plus 200 U/ml factor, culture growth

hematopoietic growth factor. The FDC-Pi/MAC clone was further subcloned by picking M-CSF-dependent colonies from soft agar cultures and testing for their responsiveness to multi-CSF and GM-CSF. Each subclone from agar was able to grow in either multi-CSF, GM-CSF, or M-CSF, thereby demonstrating that these cells simultaneously express the receptors for all three hematopoietic growth factors. A single subclone, designated FDC-PlIMAC-11, was selected for further study based on its high level of M-CSF receptor protein expression (see Experimental Procedures). Switching FDC-PVMAC-11 cells from one factor to another could be accomplished with only a minimal lag in growth rate and no major loss of cell viability. Thus, the FDC-Pi/MAC-11 subclone represents a myeloid progenitor cell line in which receptors for multi-CSF, GM-CSF, and M-CSF are expressed in each cell. Growth of the parental FDC-PI or FDC-PlIMAC-11 cells in multi-CSF or GM-CSF resulted in rapidly proliferating suspension cells (doubling time = 12 hr; data not shown) that formed large, tight colonies in soft agar (Figure 1). Histological staining revealed a relatively undifferentiated blast cell morphology. In contrast, M-CSF stimulation of FDC-PVMAC-11 cells induced a more differentiated phenotype, as demonstrated by their slower rate of proliferation (doubling time = 24 hr), adherence to plastic culture dishes, and the formation of many (-30%) small and loosely packed colonies or fully dispersed colonies in agar (Figure 1). These dispersed colonies were seen only in the M-CSF-treated cultures. Cell staining was consistent with a more differentiated cell type with the appearance of larger cells with vacuolated cytoplasm, irregular plasma membranes, and more advanced nuclear differentiation.

Cells

Stimulated

by Various

CSFs

factor (200 U/ml) before analysis in liquid culture by phase contrast or May-Grunwald-Giemsa staining of cytospins of cells after 5 days liquid

These growth characteristics suggest that multi-CSF and GM-CSF generated primarily a proliferative signal, while M-CSF transmitted a signal for both growth and myeloid differentiation. These results are similar to those we obtained previously by expressing the murine c-fms proto-oncogene in FDC-Pl cells from a retroviral vector (Rohrschneider and Metcalf, 1989). Cell surface marker analysis was performed to further evaluate the extent of FDC-Pi/MAC-11 cell differentiation after M-CSF stimulation. FDC-Pl cells growing in multiCSF and FDC-PlIMAC-11 cells growing in multi-CSF or M-CSF were incubated with monoclonal antibodies to Thy-1.2,8C5, Mac-l, and Mac-3. The Thy-l.2 antigen is expressed primarily on T cells and immature myeloid cells (Basch and Berman, 1982) whereas the 8C5 antibody is believed to recognize a granulocytic-specific antigen (Holmes and Morse, 1988). Both Mac-l and Mac-3 antigens are expressed primarily by monocytelmacrophages and in lesser amounts on neutrophilic granulocytes (Springer et al., 1978; Springer, 1981). The FDC-Pl parental line grown in multi-CSF had detectable levels of Thy-l.2 antigen expression (Figure 2A), but little detectable expression of the differentiation antigens 8C5, Mac-l, or Mac-3. This is consistent with the characterization of this cell line as myeloid progenitor. FDC-PlIMAC-11 cells growing in either multi-CSF or M-CSF had lower expression levels of Thy-l.2 antigen (Figure 2A) compared with FDC-Pl cells, and comparable expression levels of the 8C5 antigen (Figure 2B). In contrast, FDC-PlIMAC-11 cells show elevated levels of both Mac-l (Figure 2C) and Mac-3 (Figure 2D) expression, with the cells growing in M-CSF expressing the highest levels. Taken together, the FDC-PVMAC-11 cells

Trams-Modulation 1075

of c-fms

Expression

Figure 2. Expression of Differentiated Cell Surface Markers on FDC-PI and FDC-PI/MAC Cells

Thy-l .2

Cells were grown for 5 days in liquid culture with the indicated colony-stimulating factor (200 U/ml) before analysis. Antigen expression on FDC-Pl cl.4 cells in multi-CSF (thin line) was compared with FDC-PlIMAC-11 cells stimulated with multi-CSF (thick line) or M-CSF (dashed line). Cells were incubated with Thy1.2 monoclonal antibody (A), BC5 monoclonal antibody (B), Mac-l monoclonal antibody (C), Mac-3 monoclonal antibody(D), or with an antic-fms polyclonal serum (E), and then stained with a secondary phycoarythrin-conjugated antibody. The fluorescence intensity was analyzed by flow cytometry.

Mac-3 --en--

-

FDC-Pl

-

FDC-PI/MAC

Multi-CSF

FDC-PI/MAC

M-CSF

-

Multi-CSF

Fluorescence Intensity -

growing in either multi-CSF or M-CSF appear to be more differentiated than FDC-Pl cells, and stimulation of these cells with M-CSF promoted the most differentiated phenotype, which appeared to be primarily along the monocytelmacrophage pathway. The M-CSF receptor is identical to the c-fms protooncogene (Sherr et al., 1985) and the level of c-fms surface expression was determined in FDC-Pl cells and FDC-Pi/MAC-11 cells grown in different factors (Figure 2E). As expected from our previous results (Rohrschneider and Metcalf, 1989) there was no cell surface expression of c-fms proteins on the parental FDC-Pl line, as confirmed by immunoprecipitation analysis (see results below). In contrast, FDC-PVMAC-11 cells grown in the presence of M-CSF expressed elevated levels of c-fms protein. The same cells in multi-CSF did not express detectable amounts of c-fms protein above background. The suppression of c-fms surface protein expression by multiCSF was also observed in our previous studies of c-fms proteins expressed in FDC-Pl cells from a retroviral vector (Rohrschneider and Metcalf, 1989). The c-fms expressed in the FDC-PVMAC-11 cells appears to be a functional receptor, as judged by rapid down-regulation in response to M-CSF and strong kinase activity in vitro (data not shown). These results indicate that the expression of c-fms was suppressed by multi-CSF

c-fms Expression Is Suppressed Posttranscriptionally Previous studies have demonstrated that both multi-CSF and GM-CSF could down-regulate the level of cell surface receptors for both M-CSF and G-CSF in freshly isolated bone marrow cells (Walker et al., 1985). The down-regulation of unoccupied receptors by a heterologous growth factor has been termed trans-modulation (Walker et al., 1985; Nicola, 1987). However, owing primarily to a lack of antibodies and cDNA probes to these receptors, very little is known about frans-modulation at the molecular level. Using the FDC-PlIMAC-11 line, which simultaneously expresses the receptors for multi-CSF, GM-CSF, and M-CSF, we determined whether trans-modulation of the M-CSF receptor occurred in response to multi-CSF or GM-CSF stimulation. The expression level of c-fms glycoprotein and mRNA in FDC-Pl and FDC-PlIMAC-11 cells grown in various factors is shown in Figure 3. Expression of c-fms glycoprotein was undetectable in the parental FDC-Pl line grown in multi-CSF (Figure 3A, lane 2). In contrast, the expression level of both the intracellular precursor (gp140) and cell surface form of c-fms (gp185) was very strong in FDCPVMAC-11 cells grown continuously in the presence of MCSF (lane 4) or switched from M-CSF to no growth factor for 12 hr (lane 3). As expected, in the continuous presence

Cell 1076

A.

Figure 3. Expression of c-fms Glycoprotein and mRNA in FDC-Pl and FDC-PI/MAC Cells Stimulated with Various Colony-Stimulating Factors

(A) Ceils were immunoprecipitated with preimmune or anti-c-fms serum, transferred to nitrocellulose. and then immunoblotted with anti-c-fms serum. FDC-Pl cl.4 cells grown in multi-CSF for 5 days were precipitated with preimmune serum (lane 1) or anti-c-fms serum (lane 2). FDC-PlIMAC-11 cells grown in M-CSF c-fms -28s and then switched to no factor for 12 hr (lane 3) grown continuously in M-CSF (lane 4) or -18s grown in GM-CSF or multi-CSF for 5 days (lanes 5 and 6, respectively) were precipitated with anti-c-fms serum. -Actin (B) Northern blot analysis of c-fms mRNA lev123456 els. RNA was isolated for FDC-Pl cl.4 cells grown in GM-CSF or multi-CSF (lanes 1 and 2, respectively) or FDC-PI/MAC-11 cells grown in no factor for 12 hr (lane 3) M-CSF (lane 4) GM-CSF (lane 5). or multi-CSF (lane 6). Approximately 5 wg of poly(A)+ RNA was analyzed for each sample and hybridized to 3zP-labeled c-fms or actin probes.

of M-CSF the ligand-bound receptor is rapidly internalized and degraded, and therefore the cell surface form of the receptor is present in lesser amounts. The level of the intracellular precursor, gp140, was not affected by the presence or absence of M-CSF. FDC-PVMAC-11 cells grown in either GM-CSF (lane 5) or multi-CSF (lane 6) had a greatly reduced level of both intracellular and cell surface c-fms expression. These results suggest that the observed trans-modulation was not merely due to internalization and degradation of the mature cell surface receptor; rather, it resulted from suppression of the overall expression. Densitometer scanning of the autoradiogram indicated that the gp165 was reduced lOO-fold in the presence of multi-CSF or GM-CSF, compared with the gp16.5 in no factor. The expression of the processed 4.2 kb c-fms mRNA (Rothwell and Rohrschneider, 1987) paralleled the pattern of glycoprotein expression (Figure 3B). Only a faint transcript was seen in FDC-Pl and FDC-PlIMAC-11 cells grown in GM-CSF (lanes 1 and 5, respectively) or multiCSF (lanes 2 and 6, respectively), whereas more than a loo-fold increase in the amount of the 4.2 kb c-fms mRNA transcript was seen in FDC-PVMAC-11 cells switched to medium containing no growth factor for 12 hr or grown continuously in M-CSF (lanes 3 and 4, respectively). An additional transcript of 6.5 kb was detectable in all lanes and was expressed independently of the growth factor conditions. A similar transcript has been observed in the murine myeloid WEHI-3B cell line (Gonda and Metcalf, 1984) and this may represent a primary unprocessed c-fms transcript; however, the exact nature of this species remains unknown. Rehybridization of the same blot with an actin probe demonstrated that actin expression is not controlled in the same factor-dependent manner and that relatively equivalent amounts are expressed under all growth conditions. The suppression of c-fms expression at both the glycoprotein and mRNA levels suggested that the fransmodulation of c-fms did not occur at a translational or posttranslational level. Rather, the control point must occur at

the level of transcription, transport out of the nucleus, and/or mRNA accumulation in the cytoplasm. Nuclear run-off assays (Figure 4) revealed that c-fms transcription was not growth factor dependent and relatively equal transcription levels were detectable in both FDC-Pl and FDCPi/MAC-11 cells. Therefore, the suppression of c-fms expression by multi-CSF and GM-CSF occurs posttranscriptionally and presumably through the stabilization and accumulation of cytoplasmic mRNA. Switching FDCPI/MAC-11 cells from M-CSF to no growth factor for 12 hr did result in an apparent reduction of transcription (2x) and suggested that M-CSF stimulation of c-fms may provide some enhancement of its own transcription. However, as will be described below, the posttranscriptional stabilization of c-fms mRNA occurs independently of M-CSF stimulation of c-fms transcription, and the expression of c-fms mRNA can occur exclusively through a posttranscriptional process. In contrast to c-fms, the equivalent level of actin transcription and the ubiquitous pattern of mRNA expression would suggest that actin represents

Figure 4. Analysis of the Relative Levels of c-fms Gene in FDC-Pl and FDC-PlIMAC Cells

Transcription

Nuclear run-off assays were performed on nuclei isolated from FDCPl cl.4 cells grown in multi-CSF and on FDC-PlIMAC-11 cells grown in multi-CSF, GM-CSF, M-CSF, or no factor (12 hr). 3zP-labeled nuclear RNA was hybridized to the indicated plasmid DNAs.

Trans-Modulation 1077

of c-fms

Expression

GM-CSF

GM-CSF-MA-CSF 0

6

12

2.4

4.8

7.2

Q

6

-

GM-CSFwM-CSF

GM-CSF

?

0

p

1,22,44,87,2

ME

1,22,44j7?HrS

t

-

DME

12244; I I

7;HrS

28s

18s M-CSFp

t

GM-CSF I,2

2,4

4,8

7,2

M-CSF C!

2

1 f

DME 2,4

4,8

77

Hrs

FDC-PI/MAC

Figure 6. Coincubation of FDC-PI/MAC Cells with Multi-CSF CSF and M-CSF and Its Effect on c-fms mRNA Levels

Figure

5. Factor

Switching

Effects

on the Expression

of c-fms

FDC-Pl cl.4 ceils (top) or FDC-PVMAC-11 cells (middle) growing in GM-CSF (200 U/ml) were spun down, washed thoroughly, and then switched to M-CSF (ZOO U/ml) or no factor (DMEM plus 10% FBS) for the indicated time. FDC-PI/MAC-l1 cells growing in M-CSF (bottom) were switched in the same way to GM-CSF (200 U/ml) or no factor. Approximately IO6 viable cells at each time point were immunoprecipitated with anti-c-fms. transferred to nitrocellulose, and then immunoblotted with anti-c-fms serum.

a gene controlled primarily at the transcriptional both FDC-Pl and FDC-PVMAC-11 cells.

level in

Multi-CSF and GM-CSF Act Dominantly to Suppress c-fms Expression Both multi-CSF and GM-CSF can reduce the expression of c-fms in FDC-PVMAC-11 cells. However, it was not clear whether M-CSF generated a positive signal to promote stabilization of the c-fms mRNA, or whether the other two growth factors each acted dominantly to destabilize the c-fms mRNA. Factor switching experiments were performed to determine if M-CSF was required for c-fms expression (Figure 5). Switching both FDC-Pl (Figure 5, top) and FDC-PlIMAC-11 (Figure 5, middle) cells from GM-CSF to either M-CSF or no factor resulted in the appearance of c-fms glycoproteins with similar kinetics. In both cases, c-fms glycoprotein could be detected within 6 hr after factor switching and appeared to reach a steady-state level within 12-24 hr. This demonstrated that the expression of c-fms was not M-CSF dependent and the removal of GMCSF was sufficient for the reexpression of c-fms. Similar results were obtained in factor switching experiments with multi-CSF (data not shown). Thus, even though the transcription of c-fms appears to be enhanced by M-CSF (see

or GM-

FDC-PI/MAC-II cells were incubated for 46 hr with 200 U/ml M-CSF (lane I), multi-CSF (lane 2). GM-CSF (lane 3) multi-CSF plus M-CSF (lane 4) or GM-CSF plus M-CSF (lane 5). Approximately 5 ug of poly(A)+ RNA was analyzed for each sample and hybridized to 3zP-labeled probes for c-fms and actin.

Figure 4) the expression of c-ims occurs independently of M-CSF stimulation of transcription and is modulated primarily by the actions of multi-CSF and GM-CSF. Further evidence for the dominant action of these factors can be seen in switching FDC-Pi/MAC-11 cells from M-CSF to GM-CSF or to medium containing no growth factor (Figure 5, bottom). The expression of c-fms is extinguished between 6 and 12 hr following switching into GM-CSF, whereas removal of the growth factor results in the upregulation of the cell surface form and the continued expression of c-fms glycoproteins throughout the 72 hr period that these cells remain viable. The analysis of c-fms expression has clearly demonstrated that multi-CSF and GM-CSF can each suppress c-fms at both the glycoprotein and mRNA levels. To determine if these factors could suppress c-fms expression in the presence of M-CSF, FDC-PlIMAC-11 ceils were cultured with both factors simultaneously and then analyzed for c-fms mRNA expression (Figure 6). FDC-Pi/MAC-11 cells cultured with M-CSF (lane l), multi-CSF (lane 2) or GM-CSF (lane 3) alone resulted in the expected expression pattern for c-fms mRNA. Culturing FDC-PVMAC-11 cells with multi-CSF plus M-CSF (lane 4) or GM-CSF plus M-CSF (lane 5) resulted in c-fms expression levels similar to multi-CSF or GM-CSF stimulation alone. Thus, both multi-CSF and GM-CSF can suppress c-fms expression even in the presence of M-CSF. The above results demonstrate that multi-CSF and GM-

Cdl 1076

UNITS 0

IO

of GM-CSF 50

100

250

500

Table 1. Morphology Purified CSFs

Factor

Added

GM-CSF IO 50 500 2500

Figure

M-CSF (U/ml) 10 100 250

18S-

GM-CSF M-CSF

7. Titration

Effect

of GM-CSF

on c-fms

mFiNA

Levels

FDC-PVMAC-11 cells were grown in GM-CSF, spun down and washed thoroughly with DMEM plus 10% FBS. and then switched for 10 hr to no factor (DMEM plus 10% F&S), 10, 50, 100, 250, or 500 U/ml GMCSF. Approximately 5 pg of poly(A)+ RNA was analyzed for each sample and hybridized to 32P-labeled c-fms or actin probes.

CSF act dominantly to control c-fms expression at the mRNA level and suggest that the extent of suppression may be determined by the concentration of factor present. To test the possibility that the dominant suppressing effect of GM-CSF may be titratable, FDC-PlIMAC-11 cells were grown in various concentrations of GM-CSF and then analyzed for the level of c-fms transcript (Figure 7). Switching FDC-Pi/MAC-11 cells to medium containing no growth factor for 10 hr resulted in the reexpression of the c-fms transcript to a level similar to cells growing continuously in M-CSF (unpublished data). Thus, the level of c-fmS expression in the absence of any hematopoietic growth factor serves as a control transcript level to compare the concentration effect of GM-CSF on c-fms expression. As little as 10 U/ml GM-CSF caused a dramatic reduction (75%) in the level of cytoplasmic c-fms mRNA. Concentrations of 50-500 U/ml GM-CSF reduced the level of c-fms transcript to barely detectable levels. Therefore, the extent of c-fms trans-modulation is dependent on the concentration of GM-CSF present, and concentrations of less than 10 U/ml could reduce c-fms expression by more than 50% in vitro. Interestingly, the 6.5 kb transcript is not suppressed by increasing GM-CSF concentrations, and only the processed cytoplasmic transcript is trans-modulated by GMCSF. No change in actin levels were observed as would be expected for a stable, transcriptionally regulated mRNA. Modulation of Bone Marrow Colony Development by GM-CSF and M-CSF Purified myeloid CSFs can promote the proliferation and differentiation of both granulocytes and macrophages from mouse bone marrow cells when grown in semisolid

Colonies

Stimulated

by

Percentage of Colonies (Actual Number)

Total Colonies per Culture

M

92 147 165 165

36 16 12 10

GM

G

(U/ml)

28S-

Actin -

of Bone Marrow

(35) (27) (19) (16)

30 40 44 44

(26) (59) (73) (73)

32 42 44 46

(29) (61) (73) (76)

40 65 65

95 (36) 95 (62) 96 (63)

5 (2) 5 (3) 2 (2)

0 (0) 0 (0) 0 (0)

(IO U/ml) plus (250 U/ml)

171

50 (65)

35 (60)

15 (26)

GM-CSF M-CSF

(50 U/ml) plus (250 U/ml)

175

21 (36)

47 (62)

32 (57)

GM-CSF M-CSF

(500 U/ml) plus (250 U/ml)

164

10 (19)

50 (92)

40 (73)

GM-CSF M-CSF

(2500 U/ml) (250 U/ml)

165

11 (20)

51 (94)

36 (71)

plus

Approximately 75,000 BALWc bone marrow cells were plated in 1 ml of 0.3% soft agar cultures and grown for 7 days at 37°C and 10% COP with the indicated factors. Abbreviations: M, macrophage; GM, granulocyte-macrophage; G, neutrophilic granulocyte.

agar cultures (Ogawa et al., 1963; Metcalf, 1964). The colonies formed in this assay are clonal and represent the differentiation of a single progenitor cell. Single lineage colony development results from the stimulation of unipotential cells committed to one differentiation lineage, whereas development of mixed lineage colonies results from the stimulation of earlier bipotential or multipotential precursor cells not yet restricted to a specific lineage. The number and distribution of individual colonies are directly influenced by the concentration and specific type of CSF. Multi-CSF and GM-CSF can stimulate both bipotential and unipotential progenitor cells, whereas M-CSF and G-CSF stimulate primarily the differentiation of cells committed to the monocyte/macrophage and neutrophilic granulocyte lineages, respectively. Therefore, although it is not possible to isolate large quantities of bipotential myeloid progenitor cells (GM-CFC) for biochemical analysis, it is possible to detect these cells based on the type of colonies they produce in an agar assay, and this reflects the quantity and type of progenitor cells present in the bone marrow. Soft agar assays on mouse bone marrow cells were performed using GM-CSF and M-CSF, alone and in combination, to determine if GM-CSF can act dominantly to suppress M-CSF-mediated monocytelmacrophage differentiation. As seen in Table 1, GM-CSF alone promoted the differentiation of macrophage, granulocyte, and mixed granulocyte-macrophage colonies. At low concentrations (10 U/ml) the percentage of colonies of each morphology was evenly distributed among the three types of potential

Tram-Modulation 1079

of c-fms

Expression

colonies. Increasing the concentration of GM-CSF resulted in a plateau of about 165 total colonies per CUltUR?, suggesting that a saturation of precursor cells capable of being stimulated by GM-CSF had been reached. The colony morphology at higher GM-CSF concentrations shifted to primarily granulocyte and mixed granulocyte-macrophage. In contrast, the lineage-specific factor M-CSF promoted macrophage development almost exclusively throughout all concentrations used, and no pure granulocytic colonies were observed. The combination of 10 U/ml GM-CSF plus 250 U/ml M-CSF resulted in more total colonies per culture than either factor alone, suggesting that both GM-CSF and M-CSF maintained their ability to stimulate progenitor cell development. However, the distribution of colony morphologies suggested that the two factors did not act in a simple additive manner. The number of macrophage colonies formed was nearly the same as when cells were grown in M-CSF alone, but GM-CSF plus M-CSF stimulated a large increase in mixed granulocyte-macrophage colony development. This suggested that M-CSF is still capable of stimulating the monocytelmacrophage lineage in the presence of a low concentration of GM-CSF and that GM-CSF plus M-CSF is able to stimulate an additional subpopulation of bipotential cells not stimulated by GMCSF alone. Increasing the concentration of GM-CSF with a constant level of M-CSF (250 U/ml) resulted in the gradual suppression of macrophage colony development. The percentage and actual number of macrophage colonies observed with a combination of factors was very similar to the number of macrophage colonies observed with a high GM-CSF concentration alone. In contrast, the stimulation of neutrophilic granulocyte colony formation (in actual numbers) was almost identical to GM-CSF stimulation alone. Interestingly, the effect of GM-CSF appeared titratable with the maximum macrophage suppression and granulocytic stimulation occurring between 50-100 U/ml. Therefore, GM-CSF appears to act dominantly to M-CSF with the ability to suppress development along the monocyte/macrophage lineage. Once again, the major difference between a high GM-CSF concentration alone and in combination with M-CSF was observed in the percentage and actual number of mixed granulocyte-macrophage colonies stimulated, and suggested that this combination of factors retained the ability to stimulate a subpopulation of bipotential progenitor cells. Multi-CSF also appeared to have a dominant effect compared with that of M-CSF, and it suppressed the number of colonies stimulated by M-CSF in agar assays (data not shown). However, presumably because multi-CSF acts on very early progenitor cells, high concentrations of multi-CSF alone produced substantial macrophage development, and therefore, the results obtained with M-CSF plus multi-CSF were not as clear as those obtained with M-CSF plus GM-CSF. Discussion The FCC-P1 cell line was originally isolated from normal mouse bone marrow cultures for its ability to continuously

proliferate in the presence of multi-CSF (Dexter et al., 1980). These nontumorigenic myeloid progenitor cells require either multi-CSF or GM-CSF for proliferation and survival (Hapel et al., 1984). In previous experiments we demonstrated that introduction of a murine M-CSF receptor into these cells by retroviral expression resulted in an M-CSF-dependent differentiation (Rohrschneider and Metcalf, 1989). This fact, along with the observation that the M-CSF receptor is coordinately expressed with differentiation induction in the monocytelmacrophage lineage (Guilbert and Stanley, 1980; Byrne et al., 1981), suggests that an important control point in regulating development along this lineage involves the expression of the M-CSF receptor itself. Therefore, to examine possible control mechanisms in M-CSF receptor expression, we isolated a subclone of the FDC-Pl line that expressed the endogenous M-CSF receptor (the c-fms proto-oncogene). Clones of these cells simultaneously expressed the receptors for M-CSF, GM-CSF, and multi-CSF and were used to explore communication and regulation among these receptors. Tram-Modulation of c-fms Occurs Posttranscriptionally The expression of c-fms in FDC-Pi/MAC cells is efficiently trans-modulated by both multi-CSF and GM-C% The Vans-modulation of c-fms could occur at a number of posttranscriptional stages such as mRNA processing, transport out of the nucleus, or mRNA stability in the cytoplasm. However, defects in mRNA processing or transport would be expected to result in the accumulation of mRNA at the particle step immediately before the block. Since we only observe the c-fms transcript during growth in M-CSF or after switching to no factor, we believe that c-fms is regulated posttranscriptionally by the enhanced stabilization and accumulation of the mRNA in the cytoplasm. In agreement with this conclusion, recent work on 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation of the leukemic promyelocytic HL-60 cell line has suggested that c-fms is posttranscriptionally stabilized by a labile protein factor (Weber et al., 1989). Preliminary data analyzing multi-CSWGM-CSF-stimulated trans-modulation in FDC-PlIMAC cells suggests that posttranscriptional regulation of c-fms is more complex than the simple activation/inactivation of a labile stabilizing factor and appears to involve both stabilizing and destabilizing factors (unpublished data). The posttranscriptional stabilization and degradation of many cellular transcripts appears to be mediated primarily through signals within the 3’untranslated region of the transcript. Specifically, one or more AUUUA motifs and AU-rich sequences in the 3’ untranslated region are common features of many posttranscriptionally regulated mRNAs(Shaw and Kamen, 1986), although signals within the protein-coding sequences of c-fos appear to be important for its degradation (Kabnick and Housman, 1988). The 3’ untranslated region of c-fms contains two AUUUA motifs but no stretch of AU-rich sequences. The role, if any, that the AUUUA motifs play in the Vans-modulation of c-fms is not known. How the 3’ untranslated region of a genes transcript regulates its ability remains very poorly understood. Re-

Cell 1080

cent evidence has suggested that the 3’ AU-rich sequences can be recognized differentially by transacting factors and can mediate both the stability and instability of the same transcript depending on the cell type in which it is expressed (S&ruler and Cole, 1988). Direct evidence has been provided for a transacting stabilizing factor that stabilizes the transferrin receptor in response to ferritin levels (MulIner et al., 1989). In this system, a 90 kd stabilizing protein has’been shown to bind to a series of stemloop mRNA structures within the 3’ untranslated region, and it has been proposed that this regulates the use of instability elements also within this region (reviewed by Klausner and Harford, 1989). Experiments are currently underway to determine which domains of the c-fms transcript mediate its trans-modulation in FDC-PlIMAC cells. The identification of a control element for expression of a hematopoietic growth factor receptor suggests another step that may be relevant to the development of some leukemias. It is possible that the constitutive destabilization of mRNA for receptors that promote hematopoietic cell differentiation could contribute to some forms of leukemia. Implications for Hematopoiesis and Lineage Restriction The critical role of CSFs in the development of specialized blood cells has long been established (Ogawa et al., 1983; Metcalf, 1984). Myeloid cell development is currently believed to involve a hierarchy of CSF action where multiCSF and GM-CSF can act on both early progenitor cells and committed lineage cells, whereas the actions of M-CSF and G-CSF are restricted primarily to the monocytelmacrophage and neutrophil granulocyte lineages, respectively. Although M-CSF and G-CSF may also be able to act on early progenitor cells and enhance their proliferation and survival in combination with multi-CSF (Ikebuchi et al., 1988; Koike et al., 1988) their primary effect individually is to promote lineage-specific differentiation. Unique cell surface receptors exist for each of these factors and they are differentially expressed on developing cells in a manner that reflects the cellular sensitivity described above (Nicola, 1987). Therefore, the regulation of receptor expression appears to be a critical regulatory point in this hierarchy and ultimately must influence the lineage restriction of a bipotential progenitor cell. Factor switching experiments with FDC-PlIMAC cells suggest that this hierarchy may be maintained almost exclusively by the dominant actions of multi-CSF and GM-CSF. Moreover, the extent of c-fms suppression is titratable over a relatively narrow range of factor concentration ( 3,000 CilmM (ICN). Run-off transcription products were purified through G-50 spin columns and hybridized to 5 pg of the denatured plasmids pGEM2, ~755 (c-fms), and pAC269 (actin). Plasmids were denatured in 0.3 M NaOH for 1 hr at 65’%, neutralized with 2 M ammonium acetate, and then slot blotted onto nitrocellulose. Filters were hybridized for 48 hr and washed twice with 0.1 x SSC, 0.1% SDS at room temperature for 5 min and then once in 2 x SSC with 2 pglml RNAase A for 30 min at room temperature.

Acknowledgments We thank Don Metcalf and Kristen Carlberg for critical review of the manuscript and lmmunex Corp. for supplying the murine GM-CSF and multi-CSF. This work was supported by grants from the American Cancer Society (RD-287) and the National Cancer Institute (CA 40987 and CA 20551). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “‘advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

July 18, 1990; revised

September

14, 1990.

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