Advances in Bioscience and Biotechnology, 2012, 3, 592-602 ABB
http://dx.doi.org/10.4236/abb.2012.35077 Published Online September 2012 (http://www.SciRP.org/journal/abb/)
The role of Src family kinases in mediating M-CSF
receptor signaling and monocytic cell survival
Yijie Wang, Melissa G. Piper, Clay B. Marsh
Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, The Dorothy M. Davis Heart
and Lung Research Institute, Ohio State University, Columbus, USA
Received 20 June 2012; revised 28 July 2012; accepted 10 August 2012
ABSTRACT
Previously, we reported that M-CSF induced mono-
cyte survival through the activation of Akt, p38 MAPK
and Erk1/2 kinases. Here, we found that Src family
kinases were upstream of these kinases and played a
central role in regulating M-CSF-induced monocyte
survival. We observed that M-CSF promoted c-Src
activation in monocytes and MDMs in a time-de-
pendent manner. Src inhibitors reduced M-CSF-me-
diated phosphorylation of the M-CSF receptor (M-
CSFR), Akt, Erk1/2, and p38 MAPK. We also ob-
served that Src directly phosphorylated the M-CSFR.
Notably, the inhibitors blocked phosphorylation of
specific tyrosine residues within the M-CSFR. We
further demonstrated that the Src inhibitor, PP2, at-
tenuated M-CSF-induced NF-κB activation and M-
CSF-induced monocyte survival. These findings indi-
cated that Src family kinases mediate monocyte sur-
vival through the regulation of receptor phosphoryla-
tion and modulation of downstream signaling events.
Thus, we predict that targeting Src family kinases
may have therapeutic implication in inflammatory
diseases.
Keywords: Monocyte/Macrophage; M-CSF Receptor;
Src Family Kinases; Cell Survival
1. INTRODUCTION
Monocytes are derived from bone marrow progenitor
cells. Once released into circulation, a monocyte’s life-
span is 24 - 48 hours [1]. However, when a monocyte
encounters specific growth factors, inflammatory cyto-
kines or bacterial components, the monocyte enters the
tissue and differentiates into a specialized tissue macro-
phage [2,3]. Macrophage colony-stimulating factor (M-
CSF) is important in promoting maturation of monocytes
to macrophages as well as the survival of both cell types
[4,5]. Survival of monocytes and macrophages occurs
through the activation of Akt, Erk, p38 and JNK [6-8].
Previously, we reported that M-CSF activates Akt by
suppressing caspase-3 and caspase-9 activity [5].
M-CSF mediates the proliferation of monocyte pre-
cursors and their differentiation into mature phagocytes
through its interactions with the cell surface M-CSF re-
ceptor (M-CSFR) [3]. The M-CSFR is a disulfide-linked
homodimer, encoded by c-Fms proto-oncogene [9]. The
M-CSFR is a member of the receptor tyrosine kinase
(RTK) family, which includes platelet-derived growth
factor (PDGF) receptor, c-Kit and Flt3/Flt2 receptors
[10]. The M-CSFR is comprised of an Ig-like extracellu-
lar domain, juxtamembrane domain, and important in-
tracellular domains required for its catalytic activity in-
cluding ATP binding domain, kinase insert domain, and
major kinase domain [11]. Upon M-CSF stimulation, the
human M-CSFR undergoes dimerization and autophos-
phorylation of specific five tyrosine residues (561, 699,
723, 809 and 823) as well as ubiquitination leading to its
degradation and limitation of its intracellular signaling.
Phosphorylated receptor tyrosine residues are docking
sites for adaptor and signaling molecules, such as Src
family kinases, PI3-kinase/Akt and Ras/MAPK leading
to the induction of a cascade of signaling events resulting
in cell differentiation, proliferation and survival (see re-
views [12,13]).
Src protein tyrosine kinase (PTK) family members
belong to the non-receptor tyrosine kinases and are im-
plicated in multiple signaling pathways that regulate cel-
lular growth, migration, differentiation and survival (see
review [14]). There are eight Src family kinases in verte-
brates including Blk, Fyn, Fgr, Hck, Lck, Lyn, Src and
Yes. Among these kinases, Hck, Lyn and Fgr are re-
stricted to myeloid cells and B-lymphocytes, while c-Src,
Yes and Fyn are ubiquitously expressed (see review [15]).
All Src family kinase members possess an N-terminal
unique region, a Src homology 3 (SH3) domain which
binds proline rich motifs, a Src homology 2 (SH2) do-
main, which interacts with phosphotyrosine motifs, and a
kinase domain (see review [16]). Src family kinases act

Y. J. Wang et al. / Advances in Bioscience and Biotechnology 3 (2012) 592-602 593
in either a positive or a negative fashion. The Src family
kinases interact with the human M-CSFR at Tyr561 resi-
due (mouse Tyr559) and Tyr809 residue (mouse Tyr807) (see
review [12]). The Src family kinase member, c-Src, is
one of the most studied member. Notably, this 60 KDa
protein has two phosphorylation sites, which positively
and negatively regulates its activity to ultimately regulate
signal transduction pathways. Phosphorylation of Tyr416
activates c-Src kinase activity, while Tyr527 phosphoryla-
tion leads to inactivation of the kinase [17].
We previously reported that M-CSF induces the acti-
vation of Erk1/2, Akt, and p38 MAPK to regulate
monocyte survival [18,19]. We therefore postulated that
M-CSF induces these signaling molecules and perhaps
proximally through Src family kinase activation. We
observed that M-CSF induced c-Src activation in mono-
cytes. We also found that c-Src induced M-CSFR phos-
phorylation. Furthermore, Src family kinase inhibition
reduced M-CSF-induced receptor phosphorylation as
well as downstream activation events, including Akt,
Erk1/2, and p38 MAPK phosphorylation resulting in a
decrease in cell survival and NF-κB activation. Thus, our
data indicated that members of the Src family kinases
serve as a critical regulatory factor in M-CSF-mediated
monocyte survival.
2. MATERIALS AND METHODS
2.1. Reagents
Endotoxin-free RPMI-1640 and PBS were purchased
from Mediatec (Herndon, VA) and endotoxin-free FBS
was from Atlanta Biotec (Lawrenceville, GA). Recom-
binant human M-CSF was purchased from R&D Systems
(Minneapolis, MN). The Src kinase inhibitor, pyrrolo-
pyrimidine (PP2); the inactive analog, PP3 and the Src
family inhibitor, SU6656 were obtained from EMD
Chemicals (Gibbstown, NJ). Phospho-specific-Src Tyr416,
global phospho-tyrosine (P-Tyr-100), phospho-Akt (Ser473/
Thr308), phospho-Erk1/2 (Thr202/Tyr204) and phospho-p38
(Thr180/Tyr182) antibodies were purchased from Cell Sig-
naling Technology (Beverly, MA). Phospho-M-CSFR
Tyr699, Tyr723 and Tyr809 antibodies were obtained from
Cell Signaling Technology while phospho-M-CSFR
Tyr561 antibody was purchased from Abcam (Cambridge,
MA). M-CSFR, c-Src, Akt1 and Erk2 antibodies were
obtained from Santa Cruz Biotech (Santa Cruz, CA).
Recombinant full length active c-Src was obtained from
Millipore (Billerica, MA). All other reagents were pur-
chased from Sigma-Aldrich (St. Louis, MO) unless indi-
cated otherwise.
2.2. Cell Lines
Murine NIH-3T3 fibroblasts stably expressing the human
M-CSFR (c-Fms) and designated 3T3/fms were main-
tained in DMEM supplemented with 10% FBS and 1%
antibiotic-antimycotic (PSA: 1000 U/ml penicillin G
sodium, 1000 µg/ml streptomycin sulfate, and 250 ng/ml
amphotericin B) as described previously [20]. Prior to
M-CSF stimulation, cells were serum starved in DMEM
media on ice for 4 - 6 hours [8,20].
2.3. Purification of Peripheral Blood Monocytes
and the Generation of Monocyte-Derived
Macrophages (MDMs)
Peripheral blood monocytes were isolated from buffy
coats obtained from the American Red Cross and puri-
fied by positive selection using CD14 Monocyte Isola-
tion Kit from Miltenyi Biotech (Auburn, CA) as de-
scribed previously [19]. The purity of the monocyte
preparation was confirmed by flow cytometry (LSRII;
BD BioSciences, San Jose, CA). The CD14+ isolation
resulted in 97% ± 3% purity. After isolation, monocytes
were resuspended at 1 - 10 × 106 cells/ml in RPMI-1640
medium supplemented with 10% FBS, 10 µg/ml po-
lymyxin B and 20 ng/ml recombinant human M-CSF
then cultured overnight. The next day, cells were washed
and serum starved in RPMI-1640 medium for 2 hours.
Prior to M-CSF (100 ng/ml) stimulation, the cells were
treated with the Src inhibitors, SU6656 or PP2, or the
control inactive analog, PP3 as indicated. Since mono-
cytes cultured for 24 hours in M-CSF are not fully dif-
ferentiated macrophages, we refer to these cells as
monocytes throughout this paper.
To obtain monocyte-derived macrophages (MDMs),
freshly isolated monocytes were incubated in RPMI-1640
medium containing 10% FBS, 10 µg/ml polymyxin B and
20 ng/ml M-CSF in a 37˚C CO2 incubator for 5 - 6 days.
M-CSF was replenished and maintained in the culture by
the addition of 20 ng/ml of M-CSF every other day. Dif-
ferentiated MDMs were serum-starved overnight at 37˚C
before being re-stimulated with M-CSF [19,20].
Prior to stimulation, monocytes were serum starved in
RPMI-1640 medium at 37˚C incubator for two hours.
MDMs were serum starved at 37˚C in a CO2 incubator
overnight. The next day, the media was replaced and the
cells were serum starved for another two hours prior to
stimulation.
2.4. Immunoprecipitation and Immunoblotting
Monocytes or MDMs were lysed on ice for 15 minutes in
1× lysis buffer (Cell Signaling Technology) containing
protease inhibitor cocktail III (EMD Chemicals). Lysates
were cleared of insoluble material then the protein con-
centration was determined by BioRad DC protein kit
(BioRad, Hercules, CA). The samples were separated on
10% Bis-Tris SDS-PAGE gel with MOPS running buffer,
transferred to a nitrocellulose membrane, probed with the

Y. J. Wang et al. / Advances in Bioscience and Biotechnology 3 (2012) 592-602
594
indicated antibodies, and detected by ECL (Amersham
Biosciences, Piscataway, NJ). The ECL signal was quan-
tified using the Quantity One densitometry program
(BioRad). Phosphorylation data was normalized to total
protein controls or β-actin and expressed as fold-change
from non-stimulated samples.
2.5. In Vitro Kinase Assay
After serum starvation, 3T3/fms cells were lysed in cell
lysis buffer and equal amount of protein (0.8 - 1 mg) was
immunoprecipitated with irrelevant antibody control or
M-CSFR antibody overnight at 4˚C. The immune com-
plexes were then incubated with protein G beads (Life
Technologies, Grand Island, NY) at 4˚C for 1 hour. The
beads were washed three times in ice-cold lysis buffer,
then twice in ice-cold kinase buffer containing 25 mM
Tris-HCl (pH 7.5), 5 mM beta-glycerophosphate, 2 mM
dithiothreitol (DTT), 0.1 mM Na3VO4 and 10 mM MgCl2.
The beads were pre-incubated in 20 μl kinase buffer with
10 μm of PP2, PP3 or SU6656 at 37˚C for 30 minutes
prior to the addition of 100 μM ATP, 20 ng of recombi-
nant active c-Src or both then incubated at 30˚C for an
additional 30 minutes. The reactions were subjected to
SDS-PAGE analysis on a 10% SDS polyacrylamide gel.
After electrophoresis, proteins were transferred onto a
nitrocellulose membrane, probed with phosphotyrosine
antibodies, and reblotted with M-CSFR antibodies. M-
CSFR phosphorylation was quantified by comparing
band densitometry of phospho-M-CSFR and normalizing
these values to the total M-CSFR signals in the respec-
tive lanes.
2.6. Caspase-3 Analysis
Caspase-3 activity assay was described previously [21].
Briefly, monocytes (5 × 106 cell/condition) were treated
with inhibitors for 30 minutes prior to the addition of
M-CSF (100 ng/ml) then incubated overnight at 37˚C.
The next day, both the suspension and adherent cells
were collected and caspase-3 activity was analyzed using
the fluorogenic substrate Ac-DEVD-AMC (N-acetyl-
(Asp-Glu-Val-Asp)-(7-amino-4-methylcoumarin)) (EMD
Chemicals) as measured with Cytofluor 4000 fluorome-
ter (Perseptive, Framingham, MA). The linear change of
the fluorescence of hydrolyzed free AMC was used to
calculate caspase-3 activity. Protein concentration was
measured using BCA protein assay kit (Therma Scien-
tific, Rockford, IL). Caspase-3 activity is presented as
the relative fold-increase in the individual sample read-
out over M-CSF-stimulated sample readout per total
protein and are expressed as the mean ± SEM.
2.7. Annexin V/PI Apoptosis Assay
Annexin V/PI cell apoptosis assay was performed as
described previously [19] using the Annexin V-FITC
apoptosis detection kit (BD PharMingen, San Diego,
CA). Briefly, human monocytes (5 × 106) were incubated
with inhibitors for 30 minutes in serum free X-vivo
(Lonza Walkersville Inc., Walkersville, MD) before the
addition of M-CSF (100 ng/ml) then incubated overnight
at 37˚C. The cells were removed from the culture dish
using Accutase (eBioscience, San Diego, CA), then stained
with Annexin V-FITC and PI. The cells were analyzed by
flow cytometry. Annexin V-FITC and PI double positive
cells were considered apoptotic cells for the statistical
analysis.
2.8. Transfection of Monocytes and NF-κB
Activity Assay
Transient transfection of primary human monocytes was
performed as described previously [19] using the Amaxa
Nucleofector system (Lonza, Walkersville, MD). The
NF-κB-luc secreted alkaline phosphatase (SEAP) re-
porter assay was performed as described previously [20].
Briefly, monocytes (15 × 106) isolated from buffy coats
were resuspended in Monocyte Transfection Solution
(Lonza) containing 2 µg of NF-κB-Luc construct and
transfected using the Amaxa program Y-01. The cells
were immediately washed in warm X-vivo medium and
plated in triplicate in 24-well plates. After an hour, the
monocytes were incubated with inhibitors for 30 minutes
in X-vivo medium before addition of M-CSF (100 ng/ml)
then cultured overnight at 37˚C. Luciferase production
was measured with the Luciferase Assay System using a
Luminometer (Promega, Madison, WI). Data are pre-
sented as the relative fold increase in luciferase produc-
tion over nonstimulated mock transfected samples and
are expressed as the mean ± SEM.
2.9. Statistical Analysis
All data are expressed as the mean ± SEM derived from
at least three independent studies. Statistical analysis was
performed with SPSS17 software (SPSS Inc. Chicago, IL)
by using independent sample t-test. Statistical signifi-
cance was defined as p < 0.05.
3. RESULTS
3.1. M-CSF Induced Src Activation in
Human Monocytes and MDM
In this study, we hypothesized that Src family kinases are
involved in M-CSF-regulated survival pathways in both
monocytes and macrophages. Since Src family kinases
are tyrosine phosphorylated following M-CSF stimula-
tion in fibroblast cell lines overexpressing c-Fms [22],
we first examined whether M-CSF directly activated
c-Src as proximal kinase, in primary human monocytes

Y. J. Wang et al. / Advances in Bioscience and Biotechnology 3 (2012) 592-602 595
or MDMs. Therefore, cells were treated in the presence
or absence of M-CSF at the indicated time and western
blot analysis was performed to detect active phospho-
c-Src (Tyr416). In both primary monocytes and MDMs,
phosphorylated c-Src (Tyr416) was apparent after 2 min-
utes of M-CSF stimulation (Figures 1(a)-(d)). Significant
increase of c-Src phosphorylation was observed after 5
and 10 minutes of M-CSF stimulation in monocytes,
while c-Src phosphorylation was significantly increased
within 2 minutes of M-CSF stimulation in MDMs. The
phosphorylation then returned to basal levels after 20
minutes of stimulation in both monocytes and MDMs.
family [24], we were interested in determining whether
the M-CSFR is also a substrate for Src family kinases. To
test this hypothesis, we treated the cells with Src kinase
inhibitors, PP2 or SU6656, or an inactive analogue, PP3,
prior to M-CSF stimulation and then examined M-CSFR
phosphorylation. Since M-CSFR is expressed at very low
levels in monocytes and is up-regulated during macro-
phage differentiation [11], we utilized MDMs to examine
M-CSFR phosphorylation. As shown in Figures 2(a) and
(b), tyrosine phosphorylation of the M-CSFR was en-
hanced in response to M-CSF, but was significantly re-
duced by the PP2 inhibitor, but not inactive analogue
PP3. Similar reduction in receptor phosphorylation was
observed in cells treated with M-CSF and the SU6656
inhibitor. Importantly, using trypan blue exclusion, we
did not detect non-specific cytotoxicity from the inhibi-
tors (data not shown). These data suggested that the
-CSFR is a substrate for Src family kinases.
3.2. Src Kinase Inhibitors Reduced
M-CSF-Induced M-CSFR Phosphorylation
Previous research reported that the PDGF receptor is a
substrate for Src kinases [23]. Since both the M-CSFR
and PDGFR are in the same tyrosine kinase receptor M
(a) (b)
(c) (d)
Figure 1. M-CSF increased c-Src phosphorylation. (a) and (b) Freshly isolated human monocytes (10 × 106/condition) or (c) and (d)
MDM (10 × 106/condition) were stimulated with 100 ng/ml M-CSF for the indicated times. Cells were lysed and c-Src phosphoryla-
tion was measured by western blot analysis using an antibody recognizing the phospho-Src Tyr416. Densitometry quantification of the
blots comparing phosphorylated c-Src Tyr416 to total c-Src (lower panel). Data are expressed as the mean ± SEM from three inde-
pendent donors (*p < 0.05).

Y. J. Wang et al. / Advances in Bioscience and Biotechnology 3 (2012) 592-602
596
(a) (b)
Figure 2. Src kinase inhibition reduces M-CSFR phosphorylation in primary MDMs. (a) MDMs (10 × 106/condition) were serum
starved overnight and pretreated with DMSO solvent, 10 μM of PP2, PP3 or SU6656 for 30 minutes prior to stimulation with 100
ng/ml M-CSF for 5 minutes. Proteins were extracted from the cells and equal amount of protein was resolved on a 10% SDS-PAGE
gel, and immunoblotted with phosphotyrosine antibody. The blots were then immunoblotted with total M-CSFR antibody; (b)
M-CSFR phosphorylation was quantified by densitometry and the phospho-M-CSFR band was compared to total M-CSFR in the
respective lanes. Data are expressed as the mean ± SEM from three independent donors (*p < 0.05 compare with DMSO + M-CSF
treated samples).
Since global tyrosine phosphorylation of the M-CSFR
was reduced in the presence of M-CSF and Src kinase
inhibitors, we examined whether this inhibition affected
a specific tyrosine residue. To analysis the phosphoryla-
tion status of specific tyrosine residues, we utilized the
3T3/fms cell line to ensure sufficient M-CSFR levels to
be visualized by western blot analysis. The 3T3/fms cells
were pretreated with either PP2 or SU6656 prior to
stimulation with M-CSF then divided among separate
gels for western blot analysis with each phospho-tyrosine
antibody as indicated. Similar to MDMs, Src inhibition
reduced M-CSF mediated phosphorylation in 3T3/fms
cells (Figure 3(a)). In the presence of M-CSF and either
Src kinase inhibitors, PP2 or SU6656, as indicated, re-
duced M-CSFR tyrosine phosphorylation at residues 699,
723 and 809 was apparent with (Figure 3(a)) while
Tyr561 was relatively unchanged. Notably, Tyr561 of the
M-CSFR is the Src docking site (see review [13]).
3.3. The M-CSFR Is a Substrate for c-Src
To further confirm that the M-CSFR can be directly
phosphorylated by Src kinases, we performed an in vitro
kinase activity assay. To perform the kinase assay, the
immunoprecipitated M-CSFR from 3T3/fms cells was
incubated with or without recombinant active c-Src pro-
tein (Figures 3(b) and (c)). Due to intrinsic kinase activ-
ity of the receptor, we detected M-CSFR phosphorylation
in the presence of ATP alone (Figure 3(c), ATP) or ATP
and the inactive analog PP3 (Figure 3(b), PP3 + ATP).
In samples treated with both recombinant active c-Src
and ATP (PP3 + Src + ATP or Src + ATP), we observed
increased M-CSFR phosphorylation compared to sam-
ples treated with ATP only (ATP or PP3 + ATP) (Figures
3(b) and (c)). Interestingly, there was a 54% ± 6% and
42% ± 18% decrease in M-CSFR phosphorylation in the
presence ATP alone with either PP2 or SU6656, respec-
tively. This observation reflects the ability of the inhibi-
tors to non-selectively interfere with intrinsic kinase ac-
tivity of the M-CSFR. Using Src kinase inhibitors in the
presence of ATP and recombinant active c-Src, we ob-
served further reduction in M-CSFR phosphorylation
compared to the control samples. Notably, there was a
75% ± 3% reduction in receptor phosphorylation com-
paring PP2 + c-Src + ATP samples to PP3 + c-Src + ATP
samples (Figure 3(b)). Likewise, there was an 82% ±
8% reduction in M-CSFR phosphorylation in SU6656 +
c-Src + ATP samples compared to c-Src + ATP samples
(Figure 3(c)). As expected, treatment with the inhibitors
with or without recombinant active c-Src in the absence
of ATP did not induce M-CSFR phosphorylation. Thus,
our data indicated that the M-CSFR is a substrate for Src
family kinases.
3.4. Inhibition of Src Kinases Reduce
M-CSF-Induced Intracellular Signaling
and NF-κB Activity in Human
Monocyte and MDM
Our previous work identified that M-CSF and reactive
oxygen species (ROS) induces Akt, Erk1/2 and p38
MAPK phosphorylation in human monocytes [18,19].

Y. J. Wang et al. / Advances in Bioscience and Biotechnology 3 (2012) 592-602 597
(a) (b)
(c)
Figure 3. The M-CSFR is a substrate for Src kinases. (a) 3T3/fms cells (5 × 106/condition) were serum starved overnight
and then pretreated with DMSO solvent, 10 μM of PP2, PP3 or SU6656 for 30 minutes prior to stimulation with 100 ng/ml
M-CSF for 5 minutes. Equal amount of protein was separated on multiple 10% SDS-PAGE gels and immunoblotted with
phospho-tyrosine antibody or specific M-CSFR phospho-tyrosine antibodies as indicated. To confirm equal loading, the
blots were immunoblotted with total M-CSFR antibody and β-actin antibody; (b) and (c) In vitro kinase assay was per-
formed using protein lysates from 3T3/fms cells treated with or without Src kinase inhibitors PP2 (b) and SU6656 (c) that
were immunoprecipitated with the M-CSFR antibody. The M-CSFR immune complexes were incubated with ATP or c-Src
as indicated then subjected to western blot analysis with phospho-tyrosine antibody to measure to M-CSFR phosphoryla-
tion. M-CSFR phosphorylation was quantified by densitometry and normalized to the total M-CSFR signals in the respec-
tive lanes. The fold decrease of PP2-treated samples compared to the corresponding PP3-treated samples (Figure 3(b),
lower panel) or the SU6656-treated samples over the corresponding control samples (Figure 3(c), lower panel) is shown.
Data are expressed as the mean ± SEM from three independent experiments.

Y. J. Wang et al. / Advances in Bioscience and Biotechnology 3 (2012) 592-602
598
Therefore, we next examined the role of Src family
kinases in M-CSFR-mediated activation of these signal-
ing molecules in monocytes and MDMs. Since these
cells contain multiple Src kinase members, we used the
general Src tyrosine kinase inhibitor, PP2 instead of try-
ing to use multiple siRNAs. Monocytes and MDMs were
treated with either PP2 or the control, PP3, for 30 min-
utes prior to M-CSF stimulation and then phosphoryla-
tion of the signaling molecules was examined. As pre-
dicted, PP2, but not the inactive analogue PP3, signifi-
cantly reduced M-CSF-induced phosphorylation of Akt,
Erk1/2 and p38 in human monocytes (Figures 4(a) and
(b)) and MDMs (Figures 4(c) and (d)).
3.5. Inhibition of Src Kinases Reduce
M-CSF-Induced NF-κB Activity in
Human Monocyte
Since NF-κB activity is important in monocyte survival
and function [19,20], we next examined the role of Src
family kinases on M-CSF-induced NF-κB activation in
monocytes. To examine NF-κB activity, we utilized a
luciferase reporter system. Monocytes transiently trans-
fected with a NF-κB-Luc construct were cultured in the
absence or presence of Src kinase inhibitors with M-CSF.
Following, M-CSF stimulation, NF-κB-transcriptional
activity was increased in monocytes (Figure 5). Notably,
the kinase inhibitor PP2 reduced M-CSF-induced NF-κB
activity (p < 0.05).
3.6. Activation of Src Kinases Is Important in
M-CSF Mediated Survival in Monocytes
Previously, our laboratory reported that the activation of
Akt, Erk1/2 and NF-κB is important in monocyte sur-
vival [5,7]. Since inhibition of Src family kinases pre-
vented M-CSF-induced receptor phosphorylation as well
as downstream activation of signaling path ways in
(a) (b)
(c) (d)
Figure 4. Inhibition of Src kinases suppresses M-CSF-induced Akt, Erk1/2 and p38 phosphorylation in primary hu-
man monocytes and MDMs. Freshly isolated monocytes (a) or MDMs (c) were incubated with DMSO solvent, PP2
or PP3 (10 μM) for 30 minutes before stimulation with M-CSF at 37˚C for 5 minutes. Cells were lysed and equal
amount of protein was resolved using a 10% SDS-PAGE gel then immunoblotted with the indicated antibodies.
Equal loading was confirmed by reblotting the membranes with β-actin antibody. Phosphorylation levels were quan-
tified by comparing the densitometry of the phosphorylated protein bands to β-actin. The data are presented as the
mean ± SEM from three independent donors (*p < 0.05).

Y. J. Wang et al. / Advances in Bioscience and Biotechnology 3 (2012) 592-602 599
Figure 5. Src kinase inhibition decreases M-CSF-induced NF-
κB activity. Freshly isolated monocytes (15 × 106) were trans-
fected with 2 µg of NF-κB-Luc construct or eGFP control con-
struct. After an hour, the monocytes were incubated with in-
hibitors for 30 minutes in X-vivo medium before addition of
M-CSF (100 ng/ml) then cultured overnight at 37˚C. Cells were
lysed and luciferase activity was measured using a luminometer
to detect luciferase activity. Data are presented as the relative
fold-increase in non-stimulated mock transfected samples and
are expressed as the mean ± SEM from three independent do-
nors done in triplicate (*p < 0.05 compared with DMSO alone
treated samples).
monocytes, we next examined if cell survival was altered
by Src family kinase inhibition. As shown in Figure 6(a),
non-stimulated cells (DMSO) had high levels of cas-
pase-3 activity compared to M-CSF-treated cells (DMSO
+ M-CSF) (p < 0.05). As expected, the Src family kinase
inhibitor PP2, but not the inactive analogue PP3, induced
caspase-3 activity in M-CSF-stimulated monocytes.
As an alternative method of measuring cell apoptosis,
we measured Annexin V/PI staining in the treated cells
by flow cytometry. Apoptotic cells were designated as
Annexin V+/PI− (early apoptosis) and Annexin V+/PI+
populations (late apoptosis) while viable cells were An-
nexin V−/PI−. Compared to DMSO treated cells, M-CSF
protected monocytes from apoptosis, but inhibition of
Src family kinases with PP2 in the presence of M-CSF
significantly reduced the survival of the monocytes (Fig-
ure 6(b)). Collectively, these data showed that Src family
kinases regulate M-CSF-induced survival of human
monocyte survival through NF-κB activation as well as
other pathways in monocytes.
4. DISCUSSION
Previously, we found M-CSF modulates monocyte sur-
vival by activating Akt, p38 MAPK, and Erk1/2 kinases
[5,18,19]. In this study, we focused on understanding the
role of Src family kinases as proximal signaling interme-
diaries in downstream networks activated by M-CSF in
monocytes and MDMs. We found cross-talk between Src
family kinases and the M-CSFR. Specifically, inhibition
(a)
(b)
Figure 6. Src kinase inhibition decreases M-CSF-induced cell
survival. Freshly isolated human monocytes (5 × 106) were
pretreated with DMSO solvent, PP2 or PP3 (10 μM) in RPMI
1640 medium for 30 minutes then stimulated with 100 ng/ml
M-CSF overnight. (a) Cells were lysed and caspase-3 activity
was measured with DEVD-AMC as substrate. Caspase-3 activ-
ity is presented as the relative fold increase compared to M-
CSF-stimulated sample (DMSO + M-CSF). Data are expressed
as the mean ± SEM from three independent donors done in
triplicate (*p < 0.05 compared with DMSO + M-CSF treated
sample); (b) Cells were removed from the plate using Accutase
and stained with Annexin V/PI then analyzed by flow cytome-
try. Data are presented as percent of viable cells (Annexin
V−/PI−) and expressed as mean ± SEM from three independent
donors (*p < 0.05 compared to DMSO + M-CSF treated sam-
ples).
of Src family kinase activity in monocytes and MDMs
reduced the activation of Akt, Erk1/2 and p38 MAPK
and induced monocyte apoptosis. Moreover, inhibition of
Src family kinases decreased NF-κB transcriptional ac-
tivity after M-CSF stimulation. Our data indicate that Src
family kinases play central roles in the M-CSF-mediated
primary myeloid cell survival.
The well-established paradigm is M-CSF binds the
M-CSFR, leading to receptor dimerization and auto-
phosphorylation, providing docking sites for signaling
molecules. Src family kinases bind the phosphorylated
Tyr561 residue in human M-CSFR (or Tyr559 in the murine
M-CSFR) resulting in their activation and downstream
signaling events [12]. Since there are nine Src family
kinase members in myeloid cells and their function are
redundant, it is not feasible to use molecular and genetic

Y. J. Wang et al. / Advances in Bioscience and Biotechnology 3 (2012) 592-602
600
approaches to reduce these kinases. Targeting c-Src alone
with siRNA or a dominant negative construct was not
effective in our hands. Thus, requiring the use of the
pharmacological Src family kinase inhibitors PP2 or
SU6656 to effective inhibits the activity of all Src family
kinase members.
Since, we observed that M-CSFR phosphorylation was
reduced by inhibition of Src kinases this suggested that
Src family kinases also mediate M-CSFR phosphoryla-
tion, similar to the finding that the PDGF receptor is a
substrate for Src kinases [25]. Interestingly, the mutant
Y559F murine M-CSFR reduces M-CSFR phosphoryla-
tion [26], while mutations at tyrosine residues 706, 921
or 947 do not affect receptor tyrosine phosphorylation [3].
In the presence of Src inhibitors, phosphorylation of the
Tyr561 of the human M-CSFR was relatively unchanged
but reduced at other residues. The in vitro kinase activity
assay using human M-CSFR further demonstrated that
the receptor could serve as a substrate for c-Src. Similar
to our findings, Galvagni and colleagues recently re-
ported the Src-dependent phosphorylation of VEGFR-3
in endothelial cells [27]. Furthermore, PP2 reduced EGF-
induced VEGFR-3 receptor phosphorylation [27]. Inter-
estingly, ROS can activate Src kinases independent of
receptor activation [28]. In turn, ROS-activated Src
kinases mediate PDGF receptor phosphorylation [28].
We also observed that ROS mediate M-CSFR phos-
phorylation through a Src-dependent pathway (data not
shown). Since, the M-CSFR belongs to the same RTK
family as VEGF and PDGF receptors, it is likely this
phosphorylation feedback loop is conserved among RTKs.
The role of Src family kinase activation in regulating
monocyte survival and proliferation has been reported. In
microglia, GM-CSF activates the Src family kinase, Hck,
which is involved in the activation of PI-3K/Akt cell
proliferation and survival [29]. Our laboratory reported
that Lyn, another Src family kinase, negatively regulates
M-CSF-induced monocyte survival by recruiting SHIP1
to the cell membrane and down regulating PI-3K/Akt
activation [30]. Moreover, Lyn−/− macrophages have in-
creased Akt activity in response to M-CSF [30]. Consis-
tent with these observations, we found that Src family
kinases are important in M-CSF-induced activation of
PI-3K/Akt and Erk pathways [10]. Most recently, Rovida
and colleagues reported Src kinase inhibitors reduced
M-CSF-induced Akt and Erk5 phosphorylation in human
and mouse macrophage cell lines [31]. In our study, we
found that pharmacological inhibition of Src family
kinases reduced tyrosine phosphorylation of the M-
CSFR at tyrosine residues 699, 723 and 809 that provide
docking sites for intracellular signaling proteins such as
Grb2-SOS, PI3-Kinase and Ras that regulate cellular
survival pathways in monocytes (see review [13]). Taken
together, these observations indicate that Src kinases are
pivotal in regulating M-CSF signaling as well as survival
pathways.
In summary, this study demonstrates that Src family
kinases play a central role in primary mononuclear
phagocytes survival by contributing to M-CSFR activa-
tion and downstream signaling events. In conditions such
as chronic inflammation where there is prolonged sur-
vival of macrophages, it is possible that targeting Src
kinases to limit cell survival and tissue damage may be a
beneficial therapeutic option. Current studies are under-
way to examine the activation of Src kinases in chronic
inflammatory diseases.
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