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

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 e

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

A key cytokine controlling macrophage lineage

devel-opment 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

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: nick.wilson@dnax.org

*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 kin-ase, c-Fms cAMP has been shown to influence proliferation and differenti-ation in many cell types, including macrophages In addition, moduldifferenti-ation

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 macro-phages (BMM) which was associated with an enhanced extracellular signal-regulated 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 induced differentiation in the same cells The enhanced CSF-1-dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity Thus, elevated cAMP can promote either CSF-1-induced differentiation or inhibit CSF-1-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 differen-tiation in the M1/WT cells Surprisingly, addition of 8BrcAMP or PGE1

to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1R-dependent 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

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; PGE 1 ,

prostaglandin E 1 ; PKA, protein kinase A; RTK, receptor tyrosine kinase; WT, wild-type.

proliferative actions of CSF-1 have been widely

stud-ied Evidence supporting a possible differentiation

function for CSF-1 include the observations that a

sig-nificant 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

syn-dromes [4] A new model for CSF-1-induced

macro-phage 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

differenti-ation most likely via protein kinase A (PKA) activdifferenti-ation

We, and others, have shown previously that the

prolif-erative response to CSF-1 of murine bone

marrow-derived 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

expres-sion, were not inhibited [9]; however, the

CSF-1-induced mRNA expression of cyclin D1 [10], of three

genes whose products are associated with the DNA

syn-thesis machinery (the M1 and M2 subunits of

ribonu-cleotide 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

kin-ase (ERK) activity by increkin-ased intracellular cAMP has

often been correlated with suppression of growth

fac-tor-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

CSF-1 in a mitogen-activated protein kinase⁄ extracellular

signal-regulated protein kinase kinase

(MEK)-depend-ent 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

exam-ple, 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

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 rais-ing 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 sig-nal 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 phosphoryla-tion of the 1R or to significantly increased CSF-1R 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 Simi-lar molecuSimi-lar 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 concen-tration; 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-1-induced macrophage differentiation by transfecting the CSF-1R into a population of M1 myeloid leukemic cells lacking the CSF-1R [5]; the resultant cell popula-tion is referred to as M1/WT cells to indicate that they express the normal or wild-type receptor and to distin-guish them from populations expressing mutated

1R [5] We took the precaution of removing

CSF-1R positive cells from the starting population to avoid

any confounding influence from the endogenous

recep-tor 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

lar-ger, 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

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 demon-strates 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 coaddi-tion 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 CSF-1R was required for the action of 8BrcAMP on

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 UÆmL)1), (iii) 8BrcAMP (1 m M ), or (iv) a combination of CSF-1 (5000 UÆmL)1) and 8BrcAMP (1 m M ) 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 UÆmL)1) (dark lines), or 8BrcAMP (1 m M ) (light lines) for 72 h; cells were incubated with anti-Mac-1 IgG and FITC-conjugated anti-IgG2 b 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 UÆmL)1), 8BrcAMP (1 m M )

or a combination of CSF-1 (5000 UÆmL)1) and 8BrcAMP (1 m M ) for 72 h; cells were incubated with Mac-1 IgG and FITC-conjugated anti-IgG2bsecondary 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.

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

CSF-1R with a point mutation at tyrosine 807 thereby

redu-cing CSF-1R function The mutation at tyrosine 807

behaves like a kinase dead mutation in that upon

acti-vation 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

simi-lar manner in M1/807 cells as there is minimal tyrosine

phosphorylation of the CSF-1R after

CSF-1-stimula-tion 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

differ-entiation to similar levels to that of CSF-1 in M1/WT

cells but neither CSF-1 nor 8BrcAMP induce differen-tiation 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 auto-crine 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 differenti-ation induced by CSF-1, even at the high concentra-tion used (5000 UÆmL)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,

A

C

B

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 UÆmL)1) or 8BrcAMP (1 m M ) 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 UÆmL)1) or 8BrcAMP (1 m M ) or for 72 h; cells were incubated with anti-Mac-1 IgG and FITC-conjugated anti-IgG2 b 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 UÆmL)1) or 8BrcAMP (1 m M ) in the presence (black bars) or absence (unfilled bars) of blocking CSF-1R antibody (AFS-98, 10 lgÆmL)1) for 72 h; cells were incubated with anti-Mac-1 IgG and FITC-conjugated anti-IgG2bsecondary 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 UÆmL)1), 8BrcAMP (1 m M ) or 8BrcAMP (1 m M ) for 30 min prior to CSF-1 (5000 UÆmL)1) for the times indicated CSF-1R was im-munoprecipitated 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.

but that cAMP can somehow modulate

CSF-1R-dependent signalling even in the absence of CSF-1

cAMP inhibits CSF-1-induced tyrosine

phosphory-lation of the CSF-1R and its downstream

substrates

Following CSF-1 addition, the CSF-1R-induced

tyro-sine phosphorylation of cellular substrates, including

the CSF-1R itself, is presumed to form part of the

relevant signal transduction cascades modulating

cellu-lar 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

des-cribed above Figure 2A demonstrates that, as

expec-ted, 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

phos-phorylation of the CSF-1R is still maintained at

10 min but has returned to basal by 2 h post

stimula-tion Again we did not observe tyrosine

phosphoryla-tion 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

pro-nounced 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

fol-lowing CSF-1 treatment, due to internalization and

degradation [25–27], is only slightly more pronounced

in the presence of elevated intracellular cAMP

Import-antly 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

approxi-mately the optimal time point for this phosphorylation

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 phos-photyrosine, 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 phos-phorylation in these cells; 8BrcAMP pretreatment of M1/WT cells prior to CSF-1-stimulation also signifi-cantly suppressed CSF-1-mediated tyrosine phosphory-lation

As for the effects on differentiation, similar results were found for both CSF-1R and overall tyrosine phosphorylation after addition of the more physiologi-cal 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 differen-tiates 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-stimula-ted 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 acti-vation we did not observe altered kinetics of CSF-1-induced 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

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

whe-ther 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

demon-strates that PD98059 (50 lm) could effectively inhibit

CSF-1-mediated Mac-1 expression in M1/WT cells,

suggesting MEK/ERK involvement Figure 4D illus-trates 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 sup-pressed 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

F E

D

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 UÆmL)1) or 8BrcAMP (1 m M ) for the times indicated or treated with 8BrcAMP (1 m M ) 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 UÆmL)1) or PGE 1 (10 l M ) for the times indicated or treated with PGE 1 (10 l M ) 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 UÆmL)1) or PGE 1 (10 l M ) for the times indicated or treated with PGE 1 (10 l M ) 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.

cells Of more direct significance, our findings are

con-sistent with the report that human monocyte

differenti-ation 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

popula-tions in vivo, particularly at sites of inflammation, will

be exposed to prostaglandins with the potential to

ele-vate intracellular cAMP From our studies the effects

on macrophage lineage differentiation by cAMP

eleva-ting 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

CSF-1R-dependent signal transduction cascades even in the absence of CSF-1 and are consistent with the observa-tion that the CSF-1R)/) mouse shows a more severe phenotype than that of the op/op mouse with an inac-tivating mutation in the CSF-1 gene [28]; in that study the authors suggested that there may be CSF-1-inde-pendent 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

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 UÆmL)1) for 4, 10 and 30 min or with 8BrcAMP (1 m M ) 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 l M ) for 30 min prior to stimulation with either CSF-1 (5000 UÆmL)1) for 4 min or 8BrcAMP (1 m M ) 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 UÆmL)1) in the absence or presence of PD98059 (50 l M ) Cells were incubated for 72 h, washed, then incubated with anti-Mac-1 IgG and FITC-conjugated anti-IgG2 b secondary antibody, and the percent-age 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 UÆmL)1) in the absence (i) or presence (iii) of PD98059 (50 l M ), or with 8BrcAMP (1 m M ) in the absence (ii) or presence (iv) of PD98059 (50 l M ) Cells were incubated for 72 h and cellu-lar morphology was examined by light microscopy (20· magnification) This experiment was repeated six times with simicellu-lar results.

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

accom-panied 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

CSF-1 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

down-regulated during macrophage differentiation [30] In

addition, the possible relationship between the function

of PDE4 splice variants during macrophage

differenti-ation [30,31] and ERK activity [32–35] is worth

explor-ing in the light of our findexplor-ings 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

gener-ally in whole cell lysates, both in the absence and

pre-sence 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 1 They suggest that the bulk of the

CSF-1R-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

prolife-ration, the two responses may not be linked,

partic-ularly 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

[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 activa-ted CSF-1R One pathway(s) involves extensive tyro-sine 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 expres-sion, 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-trea-ted 32D myeloid cell line [37], and in CSF-1-treaCSF-1-trea-ted M1/WT cells (NJ Wilson and JA Hamilton, unpub-lished 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-depend-ent tyrosine phosphorylation reported above are con-sistent with those found in a recent paper describing inhibition by cAMP of EGF-stimulated EGFR tyro-sine phosphorylation and tyrotyro-sine phosphorylation of cellular proteins [20] but are unlike the data in other recent reports showing rapid tyrosine phosphorylation

of the EGFR in response to PGE2in 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 phos-phorylation in M1/WT cells and BMM is an addi-tional 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 phosphory-lation For example, we and others have demonstrated

a role for Src in CSF-1-mediated receptor tyrosine phosphorylation [5,39], differentiation [5] and prolifer-ation [40], while others have shown that cAMP can down-regulate Src-family members via PKA directly activating the Src inhibitory kinase, CSK [41,42] How-ever, 8BrcAMP alone was able to differentiate M1

cells only when they expressed a CSF-1R which is

cap-able 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

sub-strates cAMP may be acting upon to exert the effects

discussed Although cAMP is usually thought to

acti-vate 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

tyro-sine 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]

Interest-ingly, 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

tyro-sine 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

acti-vation by cAMP and not inhibition Using PD98059,

we provided evidence before for a partial dependence

of CSF-1-induced DNA synthesis on a

MEK/ERK-dependent 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

reports showing increased intracellular cAMP affecting RTKs and/or subsequent downstream signalling, eleva-tion 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, Austra-lia) 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 CO2in 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 fol-lowing 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 phospho-tyrosine (Upstate Biotechnology, Charlottesville, VA, USA); anti-CSF-1R IgG (Upstate Biotechnology); HRP-conjugated 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 per-formed 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 exam-ined as above

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-rab-bit 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

calcu-lated 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

previ-ously [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

contain-ing 70 IUÆmL)1 aprotinin, 10 lgÆmL)1 leupeptin, 100 mm

NaF, 0.1 mm Pefabloc and 200 lm sodium orthovanadate)

Proteins were size-separated on 10% (w/v)

SDS/polyacryl-amide 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

Hyper-film)

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

pel-lets 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/poly-acrylamide gels before western blotting

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