Báo cáo khoa học: cAMP inhibits CSF-1-stimulated tyrosine phosphorylation but augments CSF-1R-mediated macrophage differentiation and ERK activation potx

cAMP inhibits CSF-1-stimulated tyrosine phosphorylation
but augments CSF-1R-mediated macrophage
differentiation and ERK activation
Nicholas J. Wilson1,*, Maddalena Cross3, Thao Nguyen3 and John A. Hamilton1,2,3
1 Arthritis and Inflammation Research Centre, Department of Medicine (RMH/WH), University of Melbourne, Royal Melbourne Hospital,
Parkville, Victoria, Australia
2 Department of Medicine (RMH/WH), University of Melbourne, Royal Melbourne Hospital, Parkville, Victoria, Australia
3 CRC for Chronic Inflammatory Diseases, Department of Medicine (RMH/WH), University of Melbourne, Royal Melbourne Hospital,
Parkville, Victoria, Australia

Keywords
8BrcAMP; c-Fms; MAPK; M-CSF;
prostaglandin
Correspondence
N. J. Wilson, DNAX Research Institute,
901 California Ave., Palo Alto, CA,
94304–1104, USA
Fax: +1 650 496 1200
Tel: +1 650 496 1223
E-mail:
*Present address
DNAX Research Institute, 901 California
Ave, Palo Alto, CA, USA
(Received 25 April 2005, revised 13 June
2005, accepted 20 June 2005)
doi:10.1111/j.1742-4658.2005.04826.x

Macrophage colony stimulating factor (M-CSF) or CSF-1 controls the
development of the macrophage lineage through its receptor tyrosine kinase, c-Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation
of cellular ERK activity often occurs when cAMP levels are raised. We
have shown previously that agents that increase cellular cAMP inhibited

CSF-1-dependent proliferation in murine bone marrow-derived macrophages (BMM) which was associated with an enhanced extracellular signalregulated kinase (ERK) activity. We report here that increasing cAMP
levels, by addition of either 8-bromo cAMP (8BrcAMP) or prostaglandin
E1 (PGE1), can induce macrophage differentiation in M1 myeloid cells
engineered to express the CSF-1 receptor (M1/WT cells) and can potentiate
CSF-1-induced differentiation in the same cells. The enhanced CSF-1dependent differentiation induced by raising cAMP levels correlated with
enhanced ERK activity. Thus, elevated cAMP can promote either CSF-1induced differentiation or inhibit CSF-1-induced proliferation depending
on the cellular context. The mitogen-activated protein kinase ⁄ extracellular
signal-related protein kinase kinase (MEK) inhibitor, PD98059, inhibited
both the cAMP- and the CSF-1R-dependent macrophage differentiation of
M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1
to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1Rdependent tyrosine phosphorylation of cellular substrates, including that of
the CSF-1R itself. It appears that there are at least two CSF-1-dependent
pathway(s), one MEK/ERK dependent pathway and another controlling
the bulk of the tyrosine phosphorylation, and that cAMP can modulate
signalling through both of these pathways.

A key cytokine controlling macrophage lineage development from bone marrow precursors by proliferation
and differentiation is macrophage-colony stimulating

factor (M-CSF or CSF-1) [1]. The CSF-1 receptor
(CSF-1R) is the homodimeric receptor tyrosine kinase
(RTK), c-Fms [2]. The survival-promoting and

Abbreviations
8BrcAMP, 8-bromo cAMP; BMM, bone marrow-derived macrophages; CSF, colony stimulating factor; CSF-1R, CSF-1 receptor; EGF,
epidermal growth factor; ERK, extracellular signal-regulated kinase; FITC, fluorescein isothiocyanate; M-CSF, macrophage colony stimulating
factor; MEK, mitogen-activated protein kinase ⁄ extracellular signal-related protein kinase kinase; NGF, nerve growth factor; PGE1,
prostaglandin E1; PKA, protein kinase A; RTK, receptor tyrosine kinase; WT, wild-type.

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cAMP modulates CSF-1R signalling

proliferative actions of CSF-1 have been widely studied. Evidence supporting a possible differentiation
function for CSF-1 include the observations that a significant response to CSF-1 of human bone marrow
cells in vitro is differentiation into macrophages [3] and
that mutations in c-fms have been associated with
acute myeloid leukaemia and myelodysplastic syndromes [4]. A new model for CSF-1-induced macrophage differentiation has recently been developed by
transfecting CSF-1R into the immature M1 myeloid
cell line [5]; addition of CSF-1 to these cells led to a
rapid appearance of macrophage-like cells.
Agents which raise intracellular cAMP have often
been shown to modulate cell growth and/or differentiation most likely via protein kinase A (PKA) activation.
We, and others, have shown previously that the proliferative response to CSF-1 of murine bone marrowderived macrophages (BMM) and a subpopulation of
human monocytes is dramatically suppressed by raising
intracellular cAMP [6–8]. Our studies also indicated
that early biochemical responses of BMM to CSF-1,
namely protein synthesis, Na+/H+ exchange activity,
Na+/K+ ATPase activity and c-myc mRNA expression, were not inhibited [9]; however, the CSF-1induced mRNA expression of cyclin D1 [10], of three
genes whose products are associated with the DNA synthesis machinery (the M1 and M2 subunits of ribonucleotide reductase and proliferating cell nuclear antigen)
and of c-myc at later times following CSF-1 addition
were reduced by cAMP elevation [9]. In a number of cell
systems inhibition of extracellular signal-regulated kinase (ERK) activity by increased intracellular cAMP has
often been correlated with suppression of growth factor-induced proliferation. However, in BMM we have
reported that 8-bromo cAMP (8BrcAMP), despite being
a dramatic G1 phase proliferation inhibitor, increased
ERK activity both in the absence and presence of CSF1 in a mitogen-activated protein kinase ⁄ extracellular

signal-regulated protein kinase kinase (MEK)-dependent manner [11]. It was also found that an acute but not
a sustained elevation of c-fos mRNA expression due to
8BrcAMP was also MEK dependent [11].
As human monocytes differentiate in vitro, increases
in intracellular cAMP levels occur [12]; in addition,
increasing the cAMP levels in the human myeloid cell
lines, U937 and HL60, promoted their differentiation
into macrophage-like cells [13–15]. Raising intracellular
cAMP in other cellular systems can also regulate their
differentiation in response to various stimuli] for example, cAMP can enhance osteoclast differentiation by
receptor activator of NF-jB ligand (RANKL) [16] and
neuronal differentiation by nerve growth factor (NGF)
or epidermal growth factor (EGF) [17]. Although the
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signaling mechanism(s) underlying the differentiating
promoting effect of cAMP remains unclear there are
reports that for cAMP-induced neurite outgrowth from
PC12 cells, ERK activation is greatly enhanced [18],
while other reports suggest that activation of the EGF
receptor (EGFR) can occur [17]. Others have shown
that the EGF-induced tyrosine phosphorylation and
kinase activity of the EGFR can be inhibited by raising intracellular cAMP which was mediated by direct
phosphorylation by PKA of a particular serine residue
of the EGFR [19]. These studies suggest that cAMP
can modulate RTK activity and affect downstream signal transduction.
Given the above background on cAMP-dependent
biology, we explored in this study the effects of

enhanced cAMP levels on CSF-1-induced macrophage
differentiation in M1/WT cells, i.e. M1 cells expressing
the normal or ‘wild-type’ CSF-1R [5]. We report that
agents that increase intracellular cAMP, namely
8BrcAMP and prostaglandin E1 (PGE1), potentiate
the CSF-1-induced M1/WT differentiation. We also
show that increasing intracellular cAMP in the
absence of CSF-1 can induce differentiation of M1/
WT cells but not in M1 cells lacking a functional
CSF-1R. While not leading to tyrosine phosphorylation of the CSF-1R or to significantly increased CSF1R degradation, 8BrcAMP and PGE1 dramatically
reduced both basal and CSF-1R-dependent tyrosine
phosphorylation, including that of the CSF-1R itself.
In spite of this suppressed CSF-1-dependent tyrosine
phosphorylation an increase in CSF-1-dependent ERK
activity was noted in the presence of these agents. Similar molecular changes were made in CSF-1-treated
BMM where elevated cAMP suppresses proliferation.
Thus CSF-1-stimulated ERK activity is another early
CSF-1-dependent biochemical response which is not
suppressed by increasing intracellular cAMP concentration; it would appear that these particular responses
are independent of a pathway(s) involving most of the
CSF-1R-dependent tyrosine phosphorylation.

Results
cAMP augments CSF-1-induced differentiation
of M1/WT cells
We have previously established a model of CSF-1induced macrophage differentiation by transfecting the
CSF-1R into a population of M1 myeloid leukemic
cells lacking the CSF-1R [5]; the resultant cell population is referred to as M1/WT cells to indicate that they
express the normal or wild-type receptor and to distinguish them from populations expressing mutated
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N. J. Wilson et al.

A

cAMP modulates CSF-1R signalling

B

C

Fig. 1. 8BrcAMP augments CSF-1-induced differentiation in M1/WT cells. (A) M1/WT cells were either (i) untreated, (ii) treated with CSF-1
(5000 mL)1), (iii) 8BrcAMP (1 mM), or (iv) a combination of CSF-1 (5000 mL)1) and 8BrcAMP (1 mM) for 24 h. Cell morphology was
examined by light microscopy (20· magnification). This experiment was repeated six times with similar results; a representative experiment
is shown. (B) M1/WT and M1/parental cells were either untreated (dashed lines) or treated with CSF-1 (5000 mL)1) (dark lines), or
8BrcAMP (1 mM) (light lines) for 72 h; cells were incubated with anti-Mac-1 IgG and FITC-conjugated anti-IgG2b secondary antibody and the
median fluorescence intensity of Mac-1 staining was determined by flow cytometry. This experiment was repeated four times with similar
results; representative FACS plots are shown. (C) M1/WT cells were either untreated or treated with CSF-1 (5000 mL)1), 8BrcAMP (1 mM)
or a combination of CSF-1 (5000 mL)1) and 8BrcAMP (1 mM) for 72 h; cells were incubated with anti-Mac-1 IgG and FITC-conjugated antiIgG2b secondary antibody and the percentage of Mac-1 positive staining cells was determined by flow cytometry. This experiment was
repeated four times with similar results; means and standard deviation are shown.

CSF-1R [5]. We took the precaution of removing CSF1R positive cells from the starting population to avoid
any confounding influence from the endogenous receptor. As shown before [5] and again in Fig. 1A, panel ii,
the M1/WT cells rapidly (within 24-72 h) differentiate
into macrophage-like cells upon treatment with CSF-1,
with the cells becoming more irregular in appearance
and adhering to the tissue culture surface; cells lacking
the transfected receptor (M1/parental) do not respond
to CSF-1 [5]. We show in Fig. 1A, panel iv, that

8BrcAMP, a stable analogue of cAMP, can enhance
the macrophage-like morphological changes induced in
M1/WT cells by CSF-1; more cells become much larger, acquire long projections, have a more granular
appearance and adhere strongly to the tissue culture
surface. Similar results (data not shown) were obtained
by treating CSF-1-stimulated M1/WT cells with PGE1
(1 lm) which raises intracellular cAMP by activating
adenylate cyclase. Treatment of M1/WT cells with
8BrcAMP (Fig. 1A, panel iii) or PGE1 (data not
shown) alone had a slight morphologic effect at 24 h.
We next examined the influence of elevated cAMP
on expression of the integrin, Mac-1, another marker
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of CSF-1-induced macrophage differentiation in M1/
WT cells [5,20]. It can be seen in Fig. 1B that
8BrcAMP can up-regulate Mac-1 expression in M1/
WT cells to a similar level to that seen for CSF-1,
while neither agent could up-regulate its expression in
M1/parental cells. Figure 1C shows quantitative data
on the increase in Mac-1 expression due to both
8BrcAMP and CSF-1 in M1/WT cells and demonstrates that 8BrcAMP can augment the enhanced
expression induced by CSF-1. The effect of 8BrcAMP
alone on M1/WT cell differentiation but not on that in
M1/parental cells suggests that cAMP may interact in
some way with CSF-1R-associated signaling to induce
M1 cell differentiation (see below). It should be noted
that the actual percentage of Mac-1 positive cells
induced by CSF-1 alone was not increased by coaddition of 8BrcAMP (data not shown).
cAMP requires a functional CSF-1R to induce

differentiation of M1 cells
In order to determine whether a fully functional CSF1R was required for the action of 8BrcAMP on
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N. J. Wilson et al.

B

A

C

D

Fig. 2. 8BrcAMP-mediated M1 cell differentiation is dependent on a functional CSF-1R. (A) M1/WT cells and M1/807 cells were either
untreated or treated with CSF-1 (5000 mL)1) or 8BrcAMP (1 mM) for the times indicated. CSF-1R was immunoprecipitated from protein
lysates with an anti-CSF-1R IgG, proteins separated by 1D SDS/PAGE, western blotted and probed with an anti-phosphotyrosine IgG. (B)
M1/parental, M1/WT and M1/807 cells were either untreated or treated with CSF-1 (5000 mL)1) or 8BrcAMP (1 mM) or for 72 h; cells
were incubated with anti-Mac-1 IgG and FITC-conjugated anti-IgG2b secondary antibody and the percentage of Mac-1 positive staining cells
was determined by flow cytometry. This experiment was repeated four times with similar results; means and standard deviation are shown.
(C) M1/WT cells were either untreated or treated with CSF-1 (5000 mL)1) or 8BrcAMP (1 mM) in the presence (black bars) or absence
(unfilled bars) of blocking CSF-1R antibody (AFS-98, 10 lgỈmL)1) for 72 h; cells were incubated with anti-Mac-1 IgG and FITC-conjugated
anti-IgG2b secondary antibody and the percentage of Mac-1 positive staining cells was determined by flow cytometry. This experiment was
repeated four times with similar results; means and standard deviations are shown. (D) M1/WT cells were either untreated or treated with
CSF-1 (5000 mL)1), 8BrcAMP (1 mM) or 8BrcAMP (1 mM) for 30 min prior to CSF-1 (5000 mL)1) for the times indicated. CSF-1R was immunoprecipitated from protein lysates with an anti-CSF-1R IgG, proteins separated by 1D SDS/PAGE, western blotted and probed with an
anti-phosphotyrosine IgG (pCSF-1R) or with an anti-CSF-1R IgG (CSF-1R). A representative blot of three independent experiments is shown.

macrophage-like differentiation of M1 cells, we tested
8BrcAMP and CSF-1 addition on M1/parental cells
and on M1/807 cells, i.e. M1 cells which express CSF1R with a point mutation at tyrosine 807 thereby reducing CSF-1R function. The mutation at tyrosine 807
behaves like a kinase dead mutation in that upon activation there is minimal tyrosine phosphorylation of
either the CSF-1R or downstream substrates, including
overall tyrosine phosphorylation and that of specific
substrates such as Shc and p42/44 MAPK [20].
Figure 2A shows that this mutation behaves in a similar manner in M1/807 cells as there is minimal tyrosine
phosphorylation of the CSF-1R after CSF-1-stimulation compared with M1/WT cells. We and others have
previously reported that myeloid cells expressing the
tyrosine 807 mutation do not differentiate in response
to CSF-1 [20,21]. Using these cells and the percentage
of Mac-1 positive cells as a readout for differentiation
it can be seen in Fig. 2B that 8BrcAMP induces differentiation to similar levels to that of CSF-1 in M1/WT
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cells but neither CSF-1 nor 8BrcAMP induce differentiation in M1/parental or M1/807 cells (Fig. 2B); the
coaddition of CSF-1 and 8BrcAMP does not result in
differentiation of either M1/parental or M1/807 cells
(data not shown). Similar results were obtained when
PGE1 (1 lm) was used to raise intracellular cAMP and
when Mac-1 expression was monitored (data not
shown).
These data indicate that differentiation of M1 cells
by increased cAMP concentrations is dependent upon
a functional CSF-1R. To assess the possibility of autocrine production of CSF-1 leading to M1/WT cell
differentiation in response to 8BrcAMP we used a
blocking antibody against the CSF-1R. The blocking
antibody effectively inhibited M1/WT cell differentiation induced by CSF-1, even at the high concentration used (5000 mL)1), but had no effect on
8BrcAMP induced differentiation (Fig. 2C). These

results suggest that cAMP does not induce M1/WT
cell differentiation via autocrine production of CSF-1,
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N. J. Wilson et al.

but that cAMP can somehow modulate CSF1R-dependent signalling even in the absence of CSF-1.
cAMP inhibits CSF-1-induced tyrosine phosphorylation of the CSF-1R and its downstream
substrates
Following CSF-1 addition, the CSF-1R-induced tyrosine phosphorylation of cellular substrates, including
the CSF-1R itself, is presumed to form part of the
relevant signal transduction cascades modulating cellular responses [20–24]. It might therefore be expected
that cAMP elevation could enhance CSF-1R-induced
tyrosine phosphorylation in M1/WT cells and maybe
induce tyrosine phosphorylation of the CSF-1R itself
as part of the differentiation induction program described above. Figure 2A demonstrates that, as expected, CSF-1 induces a rapid and transient tyrosine
phosphorylation of the CSF-1R; however, addition of
8BrcAMP does not lead to tyrosine phosphorylation
of the CSF-1R. To ensure that 8BrcAMP treatment
did not simply induce tyrosine phosphorylation of the
CSF-1R with different kinetics to CSF-1 we extended
the time course of CSF-1 and 8BrcAMP treatment.
Figure 2D shows that CSF-1-induced tyrosine phosphorylation of the CSF-1R is still maintained at
10 min but has returned to basal by 2 h post stimulation. Again we did not observe tyrosine phosphorylation of the CSF-1R induced by 8BrcAMP treatment at
any time point. Surprisingly, given that the addition of
8BrcAMP and CSF-1 together results in more pronounced differentiation of M1/WT cells, we found that
8BrcAMP treatment could suppress the rapid tyrosine
phosphorylation of the CSF-1R induced by CSF-1
(Fig. 2D). The effect of 8BrcAMP was still maintained

even up to 2 h after CSF-1 addition. The effects on
CSF-1R tyrosine phosphorylation are unlikely due to
reduced CSF-1R levels because it can also be seen in
Fig. 2D that the reduction in total CSF-1R levels following CSF-1 treatment, due to internalization and
degradation [25–27], is only slightly more pronounced
in the presence of elevated intracellular cAMP. Importantly treatment with 8BrcAMP alone for up to 30
mins, i.e. the pretreatment time before CSF-1 addition,
does not reduce the levels of the CSF-1R in the M1/
WT cells.
We extended these findings by examining the effects
of cAMP on CSF-1-induced tyrosine phosphorylation
of cellular substrates. Figure 3A confirms the effect of
8BrcAMP addition on the transient CSF-1-induced
CSF-1R tyrosine phosphorylation at the 4 min time
point of following CSF-1 addition, which is approximately the optimal time point for this phosphorylation
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cAMP modulates CSF-1R signalling

and for downstream substrates [20]. It can be seen in
Fig. 3D, as indicated by probing western blots of
whole cell lysates with antibodies raised against phosphotyrosine, that CSF-1 treatment of M1/WT cells
leads to a rapid and transient tyrosine phosphorylation
of many cellular proteins. In contrast, 8BrcAMP again
does not induce tyrosine phosphorylation in M1/WT
cells and appears to suppress the basal tyrosine phosphorylation in these cells; 8BrcAMP pretreatment of
M1/WT cells prior to CSF-1-stimulation also significantly suppressed CSF-1-mediated tyrosine phosphorylation.
As for the effects on differentiation, similar results
were found for both CSF-1R and overall tyrosine
phosphorylation after addition of the more physiological stimulus, PGE1 (Fig. 3B,E, respectively).

As referred to above, CSF-1-induced proliferation of
BMM is inhibited by cAMP elevation [6,9]. It can be
observed that CSF-1-induced tyrosine phosphorylation
of cellular proteins and, more specifically, the CSF-1R
is also reduced by 8BrcAMP in BMM (Fig. 3F,C,
respectively) and is therefore not restricted to M1/WT
cells. The effects on CSF-1R tyrosine phosphorylation
are again not due to reduced CSF-1R levels.
cAMP enhances CSF-1-induced ERK activation
The above data suggest that CSF-1-induced tyrosine
phosphorylation is not critical for the CSF-1-induced
macrophage differentiation in M1/WT cells as there is
an inverse correlation between the effects of raised
cAMP on these two parameters. We recently showed
that 8BrcAMP enhances CSF-1-induced ERK activity
in proliferating BMM via a MEK-dependent pathway
[11]. We therefore decided to explore whether this
might also be the case for a cell system which differentiates in response to CSF-1. In order to do this we
probed western blots of lysates treated similarly to
those shown in Fig. 3D-F with anti-phospho-ERK
IgG. Figure 3D,E (lower panels) show that CSF-1
addition results in rapid ERK activation in M1/WT
cells; the analogous BMM data can be seen in Fig. 3F.
Pretreatment of both cell types with 8BrcAMP and
M1/WT cells with PGE1 enhanced the CSF-1-stimulated ERK activity with some slight stimulation even in
the absence of CSF-1. Although we found elevated
cAMP led to an increase in CSF-1-induced ERK activation we did not observe altered kinetics of CSF-1induced ERK activation by pretreatment of BMM
with 8BrcAMP as an explanation for the increased
activation (Fig. 4A). The increase in CSF-1-induced
ERK activation by cAMP is perhaps surprising given

the suppression of tyrosine phosphorylation noted.
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N. J. Wilson et al.

A

B

C

D

E

F

Fig. 3. cAMP inhibits CSF-1-induced tyrosine phosphorylation but augments CSF-1-induced ERK activation. (A) M1/WT cells were either
untreated or treated with CSF-1 (5000 mL)1) or 8BrcAMP (1 mM) for the times indicated or treated with 8BrcAMP (1 mM) for 30 min prior
to CSF-1 stimulation for 4 min. CSF-1R was immunoprecipitated from lysates with an anti-CSF-1R IgG, proteins were separated by 1D SDS/
PAGE, western blotted and probed with an anti-phosphotyrosine IgG (pCSF-1R) (upper panel) or anti-CSF-1R IgGs (middle panel). The IgG
heavy chain band is shown as a loading control (lower panel). (B) M1/WT cells were either untreated or treated with CSF-1 (5000 mL)1) or
PGE1 (10 lM) for the times indicated or treated with PGE1 (10 lM) for 30 min prior to CSF-1 stimulation for 4 min. Protein lysates were
made and treated as in (A). (C) BMM cells and lysates were treated exactly as described in (A). (D) M1/WT cells were treated as in (A),
whole cell lysates were separated by 1D SDS/PAGE, western blotted and probed with either anti-phosphotyrosine IgGs (a-PY) (upper panel)
or anti-phospho-ERK IgGs (pERK) (middle panel). Total ERK is shown as a loading control (lower panel). (E) M1/WT cells were either
untreated or treated with CSF-1 (5000 mL)1) or PGE1 (10 lM) for the times indicated or treated with PGE1 (10 lM) for 30 min prior to

CSF-1 stimulation for 4 min. Protein lysates were made and treated as in (D). (F) BMM cells and lysates were treated exactly as described
in (D). All experiments were repeated four times, with representative blots shown.

ERK activation is required for M1/WT cell
differentiation in response to CSF-1 and 8BrcAMP
Seeing that ERK activation correlated with the degree
of differentiation in M1/WT cells, we determined whether the MEK inhibitor, PD98059, might suppress the
macrophage-like differentiation. We have previously
shown that PD98059 (50 lm) inhibited the increased
ERK activity in CSF-1-treated BMM suggesting MEK
dependence [11]; we confirm that this result is also true
for M1/WT cells (Fig. 4B) and show that PD98059
also inhibits the weak ERK activation induced by
8BrcAMP alone in M1/WT cells. Figure 4C demonstrates that PD98059 (50 lm) could effectively inhibit
CSF-1-mediated Mac-1 expression in M1/WT cells,
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suggesting MEK/ERK involvement. Figure 4D illustrates that PD98059 addition inhibited both CSF-1
and 8BrcAMP-mediated M1/WT cell differentiation
as determined by morphology after 72 h incubation. It
was also found that the differentiation induced by
8BrcAMP or PGE1 in the presence of CSF-1 was suppressed by the inclusion of PD98059 or UO126 (data
not shown). Thus a MEK/ERK pathway would
appear to be necessary for the differentiating actions
of both CSF-1 and cAMP in M1/WT cells.

Discussion
We have provided evidence that cAMP can enhance
CSF-1-induced macrophage differentiation in M1/WT
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cAMP modulates CSF-1R signalling

A

C

B

D

Fig. 4. ERK activation is required for M1/WT cell differentiation in response to CSF-1 or 8BrcAMP. (A) M1/WT cells were treated with CSF-1
(5000 mL)1) for 4, 10 and 30 min or with 8BrcAMP (1 mM) for 30 min prior to the same CSF-1 time course. Protein lysates were collected
and proteins separated by 1D SDS/PAGE, western blotted and probed with anti-phospho-ERK IgGs (pERK). Blots were stripped and reprobed
with an anti-ERK IgG (ERK) as a loading control. (B) M1/WT cells were treated with (+) and without ()) PD98059 (50 lM) for 30 min prior to
stimulation with either CSF-1 (5000 mL)1) for 4 min or 8BrcAMP (1 mM) for 30 min. Protein lysates were collected and proteins separated
by 1D SDS/PAGE, western blotted and probed with anti-phospho-ERK IgGs (pERK). Blots were stripped and reprobed with an anti-ERK IgG
(ERK) as a loading control. (C) M1/WT cells were treated with CSF-1 (5000 mL)1) in the absence or presence of PD98059 (50 lM). Cells
were incubated for 72 h, washed, then incubated with anti-Mac-1 IgG and FITC-conjugated anti-IgG2b secondary antibody, and the percentage of Mac-1 positive staining cells was determined by flow cytometry. This experiment was repeated four times with similar results;
means and standard deviation are shown. (D) M1/WT cells were treated with CSF-1 (5000 mL)1) in the absence (i) or presence (iii) of
PD98059 (50 lM), or with 8BrcAMP (1 mM) in the absence (ii) or presence (iv) of PD98059 (50 lM). Cells were incubated for 72 h and cellular morphology was examined by light microscopy (20· magnification). This experiment was repeated six times with similar results.

cells. Of more direct significance, our findings are consistent with the report that human monocyte differentiation is accompanied by an increase in intracellular
cAMP [12]. Also, as discussed previously in the context
of suppression of CSF-1-driven macrophage lineage
proliferation [6,9], it is likely that macrophage populations in vivo, particularly at sites of inflammation, will
be exposed to prostaglandins with the potential to elevate intracellular cAMP. From our studies the effects

on macrophage lineage differentiation by cAMP elevating agents should now be considered as a possibility.
Even though further experiments are required to
exclude formally the involvement of endogenous
CSF-1, our data with neutralizing antibody (Fig. 2C)
suggest that cAMP can somehow interact with CSFFEBS Journal 272 (2005) 4141–4152 ª 2005 FEBS

1R-dependent signal transduction cascades even in the
absence of CSF-1 and are consistent with the observation that the CSF-1R)/) mouse shows a more severe
phenotype than that of the op/op mouse with an inactivating mutation in the CSF-1 gene [28]; in that study
the authors suggested that there may be CSF-1-independent activation of the CSF-1R.
In the experiments reported above we extended our
prior studies on the inhibition by elevated cAMP on
the CSF-1-dependent proliferation in BMM [9] by
comparing some of the responses in CSF-1-treated
M1/WT cells. The overall biological response to CSF-1
in each of these two cell populations is determined by
the nature of the target cell, i.e. either differentiation
or proliferation. Increases in cAMP in M1/WT cells
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cAMP modulates CSF-1R signalling

potentiated the differentiation induced by CSF-1 while
inhibiting the mitogenic action of CSF-1 on BMM
[10]. However, these particular biological responses to
elevated cAMP [10] may not be so different as they
both involve cell cycle exit; for BMM it is also
possible, by analogy with human monocytes [12] and
other cell types [18,29], that the cell cycle exit in

response to elevated cAMP may ultimately be accompanied by differentiation. In support of this proposed
analogy in the responses of the two cell types and the
possible usefulness of the M1/WT cells as a model for
understanding the early biochemical responses to CSF1 in normal cell types, cAMP promoted an increase in
ERK activity in both CSF-1-treated M1/WT cells and
BMM [11]. In the context of our findings and the
reports describing increases in cAMP associated with
macrophage differentiation [12–15], it is worth noting
that the cAMP phosphodiesterase, PDE4, is downregulated during macrophage differentiation [30]. In
addition, the possible relationship between the function
of PDE4 splice variants during macrophage differentiation [30,31] and ERK activity [32–35] is worth exploring in the light of our findings above.
In spite of the potentiation of the CSF-1-induced
differentiation and ERK activity in M1/WT cells by
cAMP elevation, we found, perhaps surprisingly, that
instead of there being more tyrosine phosphorylation
there was in fact a reduction in the degree of tyrosine
phosphorylation of CSF-1R and that was found generally in whole cell lysates, both in the absence and presence of CSF-1. Again supporting the analogy drawn
above between the early responses to CSF-1 in both
cell types, these changes in tyrosine phosphorylation
were similar in CSF-1-treated BMM. These findings
with tyrosine phosphorylation raise questions about
the significance of acute CSF-1R-dependent tyrosine
phosphorylation for the subsequent cellular responses
to CSF-1. They suggest that the bulk of the CSF1R-dependent tyrosine phosphorylation in M1/WT
cells, including that of CSF-1R itself, is not critical for
CSF-1-dependent differentiation and ERK activity.
For BMM, even though it could be interpreted that
the reduced CSF-1-dependent tyrosine phosphorylation
due to enhanced cAMP correlates with reduced proliferation, the two responses may not be linked, particularly as CSF-1-dependent ERK activity is also
enhanced by elevated cAMP in this cell type. In our

previous studies with CSF-1-stimulated BMM we
could find no evidence for a suppression by raised
intracellular cAMP on early responses such as Na+/
H+ antiport activity, Na+/K+ ATPase activity,
protein synthesis, etc. [9]; however, we also reported a
MEK-dependent increase in c-fos mRNA expression
4148

N. J. Wilson et al.

[11]. Therefore, like M1/WT differentiation, there are a
number of CSF-1-dependent responses that are intact
in BMM treated with cAMP elevating agents. These
responses also inversely correlate with the majority of
CSF-1-induced tyrosine phosphorylation.
Based on the analogous acute biochemical responses
of CSF-1-treated M1/WT cells and BMM to elevated
intracellular cAMP we propose that there are at least
two independent pathways emanating from the activated CSF-1R. One pathway(s) involves extensive tyrosine phosphorylation of numerous substrates, including
the CSF-1R, and can be blocked by increases in
cAMP, while the other(s) is MEK/ERK-dependent.
We previously reported observations in CSF-1-treated
BMM consistent with this model where we found that
the antiproliferative effect of 8BrcAMP correlated with
reduced cyclin D1 and delayed c-myc mRNA expression, while PD98059 addition did not lower such
mRNA expression [36]. However, when 8BrcAMP was
combined with PD98059, dramatic apoptosis was
noted in CSF-1-treated BMM [36], in the CSF-1-treated 32D myeloid cell line [37], and in CSF-1-treated
M1/WT cells (NJ Wilson and JA Hamilton, unpublished observation). These data indicate that the two
proposed CSF-1-dependent pathways converge at some

point to promote cell survival in some cell types with
this convergence being critical for this parameter.
The suppressive effects of cAMP on CSF-1-dependent tyrosine phosphorylation reported above are consistent with those found in a recent paper describing
inhibition by cAMP of EGF-stimulated EGFR tyrosine phosphorylation and tyrosine phosphorylation of
cellular proteins [20] but are unlike the data in other
recent reports showing rapid tyrosine phosphorylation
of the EGFR in response to PGE2 in colon cancer cells
[38] and of the EGFR and NGFR in PC12 cells by
raised intracellular cAMP [17]. Therefore, as for the
work of Barbier et al. for EGF-treated cells [20], it
would seem that CSF-1R-dependent tyrosine phosphorylation in M1/WT cells and BMM is an additional example where RTK-dependent tyrosine
phosphorylation is inhibited by cAMP, possibly via
PKA activity. Whether this inhibition is actually at the
level of CSF-1R itself is unknown as it is possible that
a downstream tyrosine kinase(s) may be responsible
for much of the CSF-1-dependent tyrosine phosphorylation. For example, we and others have demonstrated
a role for Src in CSF-1-mediated receptor tyrosine
phosphorylation [5,39], differentiation [5] and proliferation [40], while others have shown that cAMP can
down-regulate Src-family members via PKA directly
activating the Src inhibitory kinase, CSK [41,42]. However, 8BrcAMP alone was able to differentiate M1
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N. J. Wilson et al.

cells only when they expressed a CSF-1R which is capable of leading to cellular differentiation, suggesting
that cAMP may in fact interact with an active CSF-1R
itself in some way.
We are presently unsure as to which direct substrates cAMP may be acting upon to exert the effects
discussed. Although cAMP is usually thought to activate PKA it has recently been shown that increasing

cellular cAMP can activate the guanine nucleotide
exchange factors, exchange protein activated cAMP
(Epac)1 and Epac2, independently of PKA activation
[43,44]. It has been demonstrated that Epac does not
alter cAMP-mediated ERK activation but can mediate
other cAMP-stimulated events, such as exocytosis and
cell adhesion. Epac1 has also recently been shown to
be expressed in macrophages where it is responsible
for cAMP-mediated suppression of phagocytosis [45].
Whether Epac activation may also alter CSF-1R tyrosine phosphorylation is unknown. Others have shown
that increases in cAMP can trans-modulate the EGFR
[17,19,38] and that PKA can directly phosphorylate
the EGFR on a particular serine residue [19]. Interestingly, the CSF-1R also contains a serine residue in a
PKA consensus phosphorylation motif, which is
located within the kinase-insert region (NJ Wilson and
JA Hamilton, unpublished observation). Although we
are currently determining the significance of this serine
on CSF-1R tyrosine phosphorylarion we cannot rule
out the possibility that cAMP, through PKA or Epac,
may instead activate a tyrosine phosphatase which
results in the observed decrease in both basal and
CSF-1-induced tyrosine phosphorylation.
In contrast to the data for EGFR-dependent tyrosine phosphorylation reported by Pai et al. [38], the
surprising result for both M1/WT cells and BMM is
that there is still elevation of CSF-1-induced ERK activation by cAMP and not inhibition. Using PD98059,
we provided evidence before for a partial dependence
of CSF-1-induced DNA synthesis on a MEK/ERKdependent pathway [36] while others, using a similar
strategy, reported that ERK activity is essential for
CSF-1-induced proliferation in Bac1.2F5 and BMM
cells [46,47]; from our findings above it would appear

for CSF-1-induced differentiation in M1/WT cells that
such a pathway(s) is critical. However, whether other
nonspecific effects of PD98059 are occurring over time
need to be excluded before definitive conclusions can
be drawn about the significance of the MEK/ERK
pathway in CSF-1-treated cells.
The significance of the pathway(s) involving the bulk
of the CSF-1R-dependent tyrosine phosphorylation
and which lies downstream of cAMP action awaits
clarification. However, given the increasing number of
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cAMP modulates CSF-1R signalling

reports showing increased intracellular cAMP affecting
RTKs and/or subsequent downstream signalling, elevation of cAMP may be a common mechanism by which
cells alter the cellular outcome of RTK activation.

Experimental procedures
Cells and media
M1 murine myeloid cells were a gift from N Nicola (Walter
and Eliza Hall Institute, Melbourne, Australia) and were
maintained in DMEM (Trace Biosciences, Sydney, Australia) with 10% newborn calf serum (Trace Biosciences) at
37 °C in 5% humidified CO2. M1 cells expressing wild-type
(WT)-CSF-1R (M1/WT) or Y807F-CSF-1R (M1/807) were
constructed as described previously [5,20]. BMM were
obtained as described previously [48] and maintained at
37 °C in 5% humidified CO2 in RPMI with 10% fetal calf
serum and 30% L-cell conditioned medium, as a source of
CSF-1. The BMM were deprived of CSF-1 for 18 h to

growth-arrest them before the start of the experiments.

Antibodies and reagents
Mac-1-expressing hybridoma cells were from the American
Tissue Culture Collection (Manassus, VA, USA). The following reagents were purchased as follows: phospho-ERK
antibody (New England Biolabs, Inc., Beverly, MA, USA);
ERK antibodies (Santa Cruz Biotechnology Inc., Santa
Cruz, CA, USA); 4G-10 antibody raised against phosphotyrosine (Upstate Biotechnology, Charlottesville, VA,
USA); anti-CSF-1R IgG (Upstate Biotechnology); HRPconjugated rabbit anti-goat and swine anti-rabbit IgGs
(DAKO, Glostrup, Denmark); PD98059 (New England
Biolabs, Inc., Ipswich, MA, USA); PGE1 and PGE2
(ICN); and the sodium salt of 8BrcAMP (Sigma Chemical
Co., St Louis, MO, USA). Recombinant human CSF-1
[49] was donated by Chiron Corp., Emeryville, USA.
Assays to determine the levels of lipopolysaccharide (LPS)
in PGE1, PGE2, 8BrcAMP and CSF-1 were routinely performed. Reagents found negative for LPS were used in all
experiments.

Light microscopy
Cell morphology was examined with a Leica inverted
microscope prior to image acquisition with a Sony digital
Hyper HAD colour video camera (Sony Corporation,
Tokyo, Japan) and Leica q500mc Windows software
(Leica, Cambridge, UK). In experiments assessing the role
of ERK inhibition on M1/WT cell differentiation cells were
treated for 72 h with the MEK inhibitor, PD98059 (50 lm),
with either CSF-1 or 8BrcAMP, and the morphology examined as above.

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cAMP modulates CSF-1R signalling

Mac-1 staining
Mac-1 staining was performed as described previously [20].
Briefly, 2 · 105 cells were incubated with hybridoma cell
supernatant containing antibody raised against Mac-1 or
50 lL isotype-matched control (rat anti-mouse IgG2b), left
on ice for 1.5 h, washed with ice cold NaCl/Pi, incubated
with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG, left on ice for a further 30 min and finally washed
with ice cold NaCl/Pi again. Fluorescence was measured
using a FACS Calibur flow cytometer (Becton Dickinson,
San Jose, CA, USA). Acquisition was restricted to 10 000
events for each sample and Mac-1 positive cells were calculated by subtracting the isotype-matched control value from
the Mac-1 positive value. Median fluorescence intensity was
determined by calculating the median of Mac-1 fluorescence
for 10 000 events.

Western blot analysis
Western blot analysis was performed as described previously [20]. Briefly, cells were harvested at 1 · 107 cells in
300 lL lysis buffer (5 mm EDTA, 25 mm Hepes pH 7.4,
100 mm NaCl, 1% Triton X-100 and 10% glycerol containing 70 ImL)1 aprotinin, 10 lgỈmL)1 leupeptin, 100 mm
NaF, 0.1 mm Pefabloc and 200 lm sodium orthovanadate).
Proteins were size-separated on 10% (w/v) SDS/polyacrylamide gels and then transferred to Hybond C (Amersham,
Baulkham Hills, NSW, Australia). Membranes were then
immunoblotted with appropriate antibody and subjected to
chemiluminescence (Amersham ECL reagents and Hyperfilm).

Immunoprecipitation
Cytosolic lysates were prepared as follows: 1 · 107 cells

were scraped in lysis buffer (as above) and left on ice for
10 min before centrifugation at 5000 g for 5 min. The pellets were discarded and immunoprecipitations performed
by incubating lysates (100 lg) with 1 lg of antibodies raised
against CSF-1R overnight at 4 °C. Twenty microlitres of a
50% (v/v) slurry of Protein A-Sepharose 4B (Pharmacia,
Uppsala, Sweden) were added to the lysates and incubated
for 30 min at 4 °C. An equal volume of 2· SDS/PAGE
sample buffer was added and the immunoprecipitates were
boiled for 5 min and separated on 10% (w/v) SDS/polyacrylamide gels before western blotting.

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