Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Korea
Correspondence: S. H. Ryu, Ph.D., Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31 Hyojadong, Pohang, 790-784, Korea. E-mail: sungho{at}postech.ac.kr
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: PI 3-kinase • ERK • G-protein • caspase-3
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mitogen-activated protein kinases (MAPKs) have been linked to the regulation of cell death in various different cell types [8 9 10 ]. Three subgroups of the MAPK family enzymes have been cloned: extracellular signal-regulated protein kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 MAP kinase. ERK activity is regarded to be important for cell growth and the inhibition of cell death [11 , 12 ]. Recently a Ser/Thr kinase, Akt, was shown to play an important role in cell survival in various cells [13 14 15 16 ]. The activation of Akt is mediated by phosphoinositide 3-kinase (PI 3-kinase) [17 , 18 ]. Activated Akt then suppresses the activity of forkhead DNA transcription factors and the activity of proapoptotic proteins, thus promoting cell survival [19 ]. Among the apoptotic proteins, caspases (especially caspase-3) have been shown to regulate cell survival in several cells [20 21 22 ]. Although ERKs and Akt seem to play crucial roles in cell survival, the functional discrimination of the two molecules has not yet been achieved, especially in human monocytes.
A peptide, Trp-Lys-Tyr-Met-Val-Met (WKYMVM), was identified among a library of peptides as a phosphoinositide (PI) hydrolysis-stimulating factor in a human B myeloma cell line (U266) [23 ]. This peptide stimulates several hematopoietic cell lines but not nonhematopoietic cells, such as fibroblasts and neuronal cells [24 , 25 ]. A more potent analogue of WKYMVM was developed by modifying the methionine at its NH2 end with a D-type amino acid [24 ]. Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) acts at subnanomolar concentrations. Several leukocytes, such as granulocytes (neutrophils, basophils, and eosinophils), monocytes, and B-lymphocytes but not T-lymphocytes [25 ], express the receptor(s) for WKYMVm. We have shown previously that stimulation of human neutrophils and monocytes with this peptide enhances superoxide generation, bactericidal activity, and chemotactic migration of the cells via the activation of distinct downstream signaling pathways of the peptide receptor [26 27 28 ]. Recently two research groups demonstrated separately that WKYMVm acts on monocytes and neutrophils by binding to the lipoxin A4 receptor, which is coupled with the PTX-sensitive G-protein(s) [29 , 30 ].
Here, we studied the effect of the peptide on monocyte survival. After
inducing monocyte cell death by culturing the cells in the absence of
serum, we found that WKYMVm enhanced monocyte survival by 
25%, and
pharmacological inhibition of PI 3-kinase significantly attenuated
WKYMVm-induced monocyte survival. WKYMVm elicited a rapid activation of
ERK and Akt as downstream of PI 3-kinase. To understand more closely
the effect of ERK and Akt on the peptide-induced monocyte survival, we
blocked mitogen-activated protein kinase kinase (MEK) and protein
kinase C (PKC) and found that their activity was absolutely necessary
for the activation of ERK and Akt, respectively. Pharmacological
inhibition of the PKC-dependent Akt pathway, but not the MEK-dependent
ERK pathway, resulted in a decrease in peptide-induced monocyte
survival.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, ßI, ßII, 
,
, 
, and 
), kindly by Dr. Y. A. Hannun (Medical University
of South Carolina, Charleston, SC); and horseradish peroxidase
(HRP)-conjugated antibodies to mouse or rabbit IgG, from Kirkegaard &
Perry (Gaithersburg, MD).
Isolation of human PBMCs
Peripheral blood was collected from healthy adult donors, and
PBMCs were separated on a Histopaque-1077 gradient. After two washings
with Hank’s buffered saline solution (HBSS) without
Ca2+ and Mg2+, the
PBMCs were suspended in RPMI 1640 medium containing 10% FBS and
incubated for 60 min at 37°C to allow the monocytes time to attach to
the culture dish. The attached monocytes were then collected as
described previously [31
]. The purity of the prepared
monocytes exceeded 85%, as confirmed by fluorescein-activated cell
sorter (FACS) analysis with anti-CD14 antibody-conjugated
phycoerythrin. The isolated cells were used immediately.
Measurement of monocyte survival
A modified MTT assay was used to quantify the effect of the
peptide on monocyte survival; the method measures mitochondrial
function as described previously [32
, 33
].
Isolated human monocytes were plated on 96-well plates
(5x104-8x104 cells/well) and maintained
overnight in complete medium. Cells were then changed to serum-free
medium in the absence or presence of various concentrations of WKYMVm.
Cells were then pretreated with several inhibitors (LY294002, PD98059,
GF109203X, and Go 6976) for indicated lengths of time prior to the
addition of the peptide to investigate the intracellular signaling
associated with WKYMVm-induced monocyte survival. After 72 h, the
medium was aspirated from the wells, and 10 µl MTT reagent (1 mg/ml)
was added to each well. The cells were then incubated for 2 h at
37°C and lysed by adding 50 µl dimethylsulfoxide and shaking for 20
min. The optical density at 570 nm was read with an enzyme-linked
immunosorbent assay (ELISA) reader (EL312e, Bio-Tek Instruments,
Winooski, VT). To rule out the possible contaminating effect of
endotoxin on monocyte survival, we confirmed that the vehicle and
peptide solution contain endotoxin at a level below the detection
limits of the assay using an endotoxin detection kit (Sigma Chemical
Co.). We also confirmed that WKYMVm did not affect monocyte
differentiation to macrophages or affect the secretion of inflammatory
cytokine tumor necrosis factor (TNF-) by monitoring
morphological changes or by using an ELISA assay, respectively
(unpublished results).
DNA fragmentation analysis
The DNA fragmentation study was performed as described before
[5
]. In brief, cells were gently lysed for 30 min at
4°C in a buffer containing 5 mM Tris buffer (pH 7.4), 20 mM
ethylenediaminetetraacetate (EDTA), and 0.5% Triton X-100. After
centrifugation at 12,000 g for 5 min, supernatants
containing the soluble fragmented DNA were collected and extracted with
phenol/chloroform/isoamyl alchol (25:24:1, v/v/v), and the DNA was
precipitated with ethanol and pelleted by centrifugation at 12,000
g for 15 min at 4°C. The pellet was washed with 70%
ethanol, dried, and dissolved in distilled water containing DNase-free
RNase (0.4 mg/ml). The samples were incubated for 30 min at 37°C and
then electrophoresed through a 1.8% (wt/vol) agarose gel containing
ethidium bromide and visualized under the UV light.
Stimulation of human monocytes with WKYMVm
The prepared human monocytes were aliquoted into a 2 x
106 cells and stimulated at the indicated concentrations of
WKYMVm for the indicated lengths of time. In some experiments, the
cells were pretreated with certain inhibitors to specific enzymes for
15 min or 60 min prior to the addition of stimulators. After
stimulation, the cells were washed with serum-free RPMI and lysed in
lysis buffer [20 mM Hepes, pH 7.2, 10% glycerol, 150 mM NaCl, 1%
Triton X-100, 50 mM NaF, 1 mM Na3VO4, 10
µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl
fluoride (PMSF)]. The detergent-insoluble materials were pelleted by
centrifugation (12,000 g, 15 min, at 4°C), and the soluble
supernatant fraction was removed and stored at -80°C or used
immediately. Protein concentrations in the lysates were determined
using Bradford protein assay reagent.
PKC translocation analysis
Prepared monocytes were stimulated with 100 nM WKYMVm for
various lengths of time in serum-free RPMI. After discarding the
reaction buffer, the cells were extracted in homogenizing buffer [20
mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM
ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic
acid (EGTA), 1 mM EDTA, 1 µM PMSF, 10 µg/ml leupeptin, and 10
µg/ml aprotinin] using a sonicator. The cell lysates obtained were
centrifuged at 100,000 g for 45 min at 4°C in a Beckman
TL-100s ultracentrifuge. The supernatants were collected and saved as
the cytosol fraction, and the pellets were washed with 0.1 ml
homogenizing buffer and resuspended in this buffer containing 1%
Triton X-100 to solubilize the particulates (particulate fractions).
Electrophoresis and immunoblot analysis
Protein samples were prepared for electrophoresis. The proteins
in the samples were then separated in an 8% sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) using the buffer system
described by King and Laemmli [34
]. Following
electrophoresis, the proteins were blotted onto a nitrocellulose
membraneand were then blocked by incubating with TTBS (Tris-buffered
saline, 0.05% Tween-20) containing 5% nonfat, dry milk. Subsequently,
the membranes were incubated with antiphospho-ERK antibody,
antiphospho-Akt antibody, or anti-Akt antibody and washed with TBS. PKC
isozyme-specific antibody was incubated for the PKC translocation
assay. Antigen-antibody complexes were visualized after incubating the
membrane with 1:5000 diluted goat anti-rabbit IgG or goat anti-mouse
IgG antibody, coupled to HRP, and detected by enhanced
chemiluminescence.
Measurement of caspase activity
Caspase activity was measured as described previously
[35
]. Briefly, cells were sonicated in a buffer of 20 mM
Hepes, pH 7.25, 1 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 5 mM
dithiothreitol (DTT), 10 µg/ml leupeptin, and 1 mM PMSF. The cleared
lysates (containing 15 µg protein) were incubated at 37°C for
1 h in a buffer of 20 mM Hepes, pH 7.25, 10% sucrose, 0.1%
CHAPS, and 10 mM DTT with 50 µM fluorogenic substrates
DEVD-7-amino-4-methylcoumarin (AMC). AMC release was measured
using a 7620 Microplate Fluorometer at 460 nm using an excitation
wavelength of 360 nm.
Statistical analysis
Results are expressed as means ± SE from the
number of determinations indicated. The Student’s t-test
was used to compare individual treatments with their respective control
values. In the figure legends, * and ** indicate significant
differences at the P < 0.05 and P <
0.01 probability levels, respectively, as compared with the values
obtained from untreated human monocytes. # Indicates P < 0.05 in comparison with values from human monocytes treated with
WKYMVm.
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 View larger version (32K): [in a new window] |
Figure 1. WKYMVm enhances monocyte survival via the PI 3-kinase pathway.
Monocytes were incubated in serum-free RPMI medium in the absence or
presence of various concentrations of WKYMVm, fMLF, or lipoxin
A4 for 72 h (A). Monocytes were incubated in serum-free
RPMI in the absence or presence of 100 nM WKYMVm for 72 h.
Cytoplasmic DNA was extracted and analyzed as described in Materials
and Methods (B). Monocytes were preincubated with vehicle or 10 µM
LY294002 for 15 min prior to treatment with 100 nM WKYMVm or vehicle
alone (C). Monocyte survival was determined using an MTT reduction
assay, as described in Materials and Methods. Results are represented
as means ± SE (n=9; A and C). * and **,
P < 0.05 and P < 0.01, respectively,
when compared with vehicle-treated cells. #, P < 0.05
when compared with WKYMVm-treated cells.
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 View larger version (19K): [in a new window] |
Figure 2. WKYMVm stimulates Akt activation in a PI 3-kinase-dependent manner in
monocytes. Monocytes were stimulated with various concentrations of
WKYMVm for 2 min (A) or with 100 nM WKYMVm for various periods of time
(B). The cells were preincubated with vehicle or 10 µ LY294002 for 15
min prior to treatment with 100 nM WKYMVm or vehicle alone for 2 min
(C). Each sample (30 µg protein) was subjected to 8% SDS-PAGE.
Phosphorylated Akt and total Akt were determined by immunoblot analysis
with antiphospho-Akt or Akt antibodies. Akt phosphorylation was
quantified by densitometry. Results are expressed as means ±
SE of five independent experiments. * and **,
P < 0.05 and P < 0.01, respectively,
compared with vehicle-treated cells. #, P < 0.05 when
compared with WKYMVm-treated cells.
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 View larger version (19K): [in a new window] |
Figure 3. WKYMVm stimulates ERK activation in a PI 3-kinase-dependent manner in
monocytes. Monocytes were stimulated with various concentrations of
WKYMVm for 2 min (A) or with 100 nM WKYMVm for various periods of time
(B). Cells were preincubated with vehicle or 10 µM LY294002 for 15
min prior to treatment with 100 nM WKYMVm or vehicle alone for 2 min
(C). Each sample (30 µg protein) was subjected to 8% SDS-PAGE, and
phosphorylated ERK was determined by immunoblot analysis with
antiphospho-ERK antibody. ERK phosphorylation was quantified by
densitometry. Results are presented as the means ± SE
of five independent experiments. * and **, P < 0.05
and P < 0.01, respectively, when compared with
vehicle-treated cells. #, P < 0.05 when compared with
WKYMVm-treated cells.
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and ßII to the membrane. This PKC
translocation was also a transient event, exhibiting its maximal effect
0.5–5 min after stimulation (Fig. 4)
. PKCßI, too, although only
slightly, translocated to the membrane (unpublished results). Although
it has been shown that human monocytes express other PKC isoforms such
as PKC, , and , we could not detect these in our system
(unpublished results).
 View larger version (34K): [in a new window] |
Figure 4. WKYMVm elicits PKC translocation to the membrane fraction. Monocytes
were stimulated with 100 nM WKYMVm for the indicated periods. At
specific times, the cells were disrupted by sonication in hypotonic
conditions. The samples were then fractionated by centrifugation at
100,000 g for 60 min at 4°C. Aliquots containing 30 µg
particulates (membrane fractions) were separated by 8% SDS-PAGE and
immunoblot-analyzed with anti-PKC or ßII antibodies. PKC in the
membrane fraction was quantified by densitometry. Results represent the
means ± SE of three independent experiments. * and
**, P < 0.05 and P < 0.01,
respectively, when compared with vehicle-treated cells.
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and ßII
activation in monocytes, we examined the role played by PKC in
WKYMVm-induced Akt and ERK activation using two kinds of PKC inhibitor:
GF109203X, which is a pan PKC inhibitor, and Go 6976, a
PKC/ß-specific inhibitor. GF109203X and Go 6976 inhibited almost
completely WKYMVm-stimulated Akt activation, but these two inhibitors
had no effect on WKYMVm-stimulated ERK activation (Fig. 5A
). These results suggest that PKC (possibly PKC and ßII)
activation is required for WKYMVm-induced Akt activation but not for
the ERK activation in monocytes. Many studies have demonstrated that
ERK activation by various extracellular stimuli is dependent on MEK
activity [44
, 45
]. Pretreatment of
monocytes with the MEK inhibitor, PD98059, completely blocked
WKYMVm-induced ERK activation (Fig. 5B)
. The effect of MEK on
WKYMVm-induced Akt activation was also tested using the same inhibitor.
PD98059 did not affect WKYMVm-induced Akt activation, suggesting that
WKYMVm stimulates Akt activation via a MEK-independent pathway.
 View larger version (35K): [in a new window] |
Figure 5. Distinctive regulation of WKYMVm-stimulated Akt and ERK activation.
Monocytes were preincubated with 5 µM GF109203X, 5 µM Go 6976 for
15 min (A), or 50 µM PD98059 for 60 min (B) prior to stimulation with
100 nM WKYMVm. After 2 min of stimulation with WKYMVm, the cells were
lysed in lysis buffer, as described in Materials and Methods. Each
sample containing 30 µg proteins was subjected to 8% SDS-PAGE and
immunoblot analysis with antiphospho-Akt or antiphospho-ERK antibodies.
Kinase phosphorylations were quantified by densitometry. Results
represent the means ± SE of three independent
experiments. **, P < 0.01 compared with the
vehicle-treated cells. #, P < 0.05 when compared with
WKYMVm-treated cells.
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and ßII) downstream of the WKYMVm
receptor in monocytes.
 View larger version (35K): [in a new window] |
Figure 6. WKYMVm-induced PKC translocation is PI 3-kinase-dependent. Isolated
monocytes were preincubated with 50 µM LY294002 or vehicle only for
15 min prior to stimulation with 100 nM WKYMVm for 5 min. The cells
were then disrupted by sonication under hypotonic conditions and
fractionated, as described in Materials and Methods. Aliquots of 30
µg particulates (membrane fractions) were separated by 8% SDS-PAGE
and analyzed by immunoblot using anti-PKC or ßII antibodies. PKC
in the membrane fraction was quantified by densitometry. Results are
represented as means ± SE of three independent
experiments. **, P < 0.01 when compared with
the vehicle-treated cells. #, P < 0.05 when compared
with WKYMVm-treated cells.
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 View larger version (23K): [in a new window] |
Figure 7. Role of Akt activation in WKYMVm-induced monocyte survival. To
determine the role of the Akt and ERK pathways in WKYMVm-induced
monocyte survival, monocytes were incubated with 100 nM WKYMVm with and
without the ERK pathway inhibitor PD98059 (50 µM) or with the
Akt pathway inhibitors (5 µM GF109203X or 5 µM Go 6976).
Monocyte survival was determined using the MTT reduction assay as
described in Materials and Methods. Results are represented as the
mean ± SE (n=6). **, P <
0.01 compared with the vehicle-treated cells. #, P <
0.05 when compared with WKYMVm-treated cells.
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 View larger version (22K): [in a new window] |
Figure 8. WKYMVm inhibits caspase-3 activity in a PI 3-kinase- and PKC-dependent
manner. Monocytes were incubated with various concentrations of WKYMVm
(A) or 100 nM WKYMVm in the presence or absence of LY294002 (10 µM),
PD98059 (50 µM), GF109203X (5 µM), or Go 6976 (5 µM) (B). After
72 h, the caspase activity was determined by the ability of
cellular extracts (15 µg protein) to cleave DEVD-AMC as described in
Materials and Methods. For Western blot analysis, the cells were lysed,
and the samples (each 30 µg total protein) were subjected to
SDS-PAGE, blotted, and probed with anticaspase-3 (pro form) antibody.
The arrowheads indicate caspase-3 (pro form), and the figure is
representative of at least three independent experiments. Results
represent means ± SE (n=3). **,
P < 0.01 when compared with vehicle-treated cells. #,
P < 0.05 when compared with WKYMVm-treated cells.
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According to previous studies, WKYMVm is a potent ligand for FPR and FPRL1 [29 , 30 ]. To identify the receptor involved in WKYMVm-induced monocyte survival, we tested the effect of fMLF (as an FPR agonist) and lipoxin A4 (as an FPRL1 agonist) on monocyte survival. Neither fMLF nor lipoxin A4 enhanced monocyte survival (Fig. 1A) . Costimulation of monocytes with fMLF and lipoxin A4 also could not enhance monocyte survival (unpublished results). Recently, Christophe et al. [48 ] demonstrated that WKYMVm is an agonist for the monocyte-expressed chemoattractant receptor FPRL2. We also observed that WKYMVm could induce an additional calcium increase in fMLF- and lipoxin A4-treated monocytes, suggesting that WKYMVm might bind other receptor(s) in the cells (unpublished results). Taken together, it will be possible that WKYMVm enhances monocyte survival via activating on FPRL2 or an unidentified receptor but not on FPR or FPRL1.
Many studies have shown that cell survival is controlled by various extracellular stimuli, including growth factors, such as epidermal growth factor and macrophage-CSF [3 , 4 , 49 ]. Recently, leukocytic cell survival based on chemoattractants that act via PTX-sensitive G-protein-coupled receptor(s) has been described [7 , 50 ]. Platelet factor 4, a CXC-chemokine, was shown to promote monocyte survival, although the intracellular mechanism has not been revealed yet [7 ]. Recently, IL-8, one of the CXC-chemokines, was shown to attenuate human neutrophils apoptosis, and it has been suggested that the process is mediated by PI 3-kinase/ERK activation [50 ]. Although Akt has been viewed as an important molecule in the regulation of cell survival by several extracellular stimuli in various cell types, the role of Akt downstream of the chemoattractant receptors that are coupled to PTX-sensitive G-protein in monocyte survival has not been shown. In this study, we demonstrate that the activation of the PI 3-kinase pathway is critical for WKYMVm-induced monocyte survival. In the case of WKYMVm-induced signaling, ERK and Akt were activated downstream of PI 3-kinase. Because PI 3-kinase is important for WKYMVm-induced cell survival, we looked at the roles of ERK and Akt, two downstream molecules of PI 3-kinase in the WKYMVm-induced signaling in monocytes. It is interesting that we found that the inhibition of Akt but not ERK completely blocked not only WKYMVm-induced cell survival but also caspase-3 inhibition. Thus, we suggest that Akt activation is a critical determinant of monocyte survival stimulated by a chemoattractant.
The regulation of Akt by various extracellular stimuli has been shown.
Many groups have demonstrated that PI 3-kinase activation is a
prerequisite for Akt activation in various cell types
[17
, 18
]. In our study, we also found that
Akt activation by WKYMVm is PI 3-kinase-dependent (Fig. 2C)
. Through
experiments targeted at the signaling pathway of WKYMVm-induced Akt
activation, we found that PKC activation is required for Akt activation
(Fig. 5A) . Bearing in mind that WKYMVm induces the translocation of
PKC and ßII and that WKYMVm-induced Akt activation could be
inhibited by Go 6976, a PKC/ß-specific inhibitor, we concluded
that classical PKC isozymes (especially PKC and ßII) may be
involved in Akt activation by WKYMVm. The role of PKC in Akt activation
has not been studied extensively yet. Recently, Zheng et al.
[51
] demonstrated that PMA inhibited insulin-like growth
factor-induced Akt activation in PC-12 cells and that rottlerin, a
PKC-specific inhibitor, attenuated the PMA-induced effect,
suggesting a negative role for PKC in Akt activation. However, Li et
al. [52
] showed that the overexpression of PKC in 32D
myeloid progenitor cells greatly enhanced endogenous Akt activity and
that this was correlated with a suppression of the onset of apoptosis
by cytokine withdrawal. Based on our findings that WKYMVm binds to a
PTX-sensitive G-protein-coupled receptor, that it activates PKC and
ßII, and that WKYMVm-stimulated PKC activation is required for Akt
activation and monocyte survival, we suggest that classical isoforms of
PKC may have a positive regulatory effect on Akt activation in
monocytes.
Our data show that PI 3-kinase and PKC operate upstream of Akt during WKYMVm-induced signaling (Figs. 2C and 5A) . The relationship between PI 3-kinase and PKC on WKYMVm-induced signaling was also examined. To elucidate the effect of PI 3-kinase and PKC activation upon WKYMVm stimulation, the cells were preincubated with LY294002, and then PKC translocation was assayed. LY294002 inhibited WKYMVm-induced PKC translocation, which suggested that PI 3-kinase activity was required for PKC activation (Fig. 6) . To test the possibility that PI 3-kinase acted upstream of PLC in WKYMVm-induced signaling, we examined the effect of LY294002 on WKYMVm-stimulated PI hydrolysis and intracellular calcium rise. Pretreatment of the cells with LY294002 at concentrations up to 50 µM prior to stimulation with WKYMVm had no effect on WKYMVm-induced total inositol-phosphate formation and intracellular calcium rise (unpublished results). These results indicate that PI 3-kinase does not act upstream of PLC in WKYMVm-induced intracellular signaling. Several previous studies have indicated that the products of PI 3-kinase, PI 3,4P2 and PI 3,4,5P3, are necessary for the activation of not only novel PKC but also classical PKC isozymes [53 , 54 ]. It has been proposed that to enable PI 3-kinase to activate PKC, phosphoinositide-dependent kinase-1 has to be activated by PI 3-kinase products, which, in turn, controls the phosphorylation of conventional PKC isozymes [54 ]. From these studies, we may deduce that PKC probably acts downstream of PI 3-kinase and upstream of Akt in the WKYMVm-induced signaling pathway leading to monocyte survival. The details of their involvement of PI 3-kinase in the PKC activation should be further investigated.
In our study of the mechanisms involved in WKYMVm-induced monocyte survival, we found that WKYMVm inhibited caspase-3 activation in the cells (Fig. 8A) . One of the proposed mechanisms by which Akt promotes cell survival is via its ability to phosphorylate caspase-9. Phosphorylated caspase-9 is resistant to cleavage by apoptosis-promoting activating factor-1 complex [55 ]. Caspase-9 is an important factor in the activation of caspase-3, which is a protease of pivotal importance in the apoptosis program [55 ]. In our study, we demonstrated that WKYMVm not only stimulated Akt activation but also inhibited caspase-3 activation in monocytes (Figs. 2A and 8A) . Our results suggest that WKYMVm-induced monocyte survival could be mediated by Akt-mediated caspase-9 phosphorylation (which equals suppression of its activation) and the suppression of caspase-3 activity.
Human monocytes undergo spontaneous apoptosis upon culture in vitro [56 ]. Because serum deprivation increases spontaneous monocyte apoptosis dramatically, and this spontaneous apoptosis can be inhibited by treatment of growth factors or several stimuli, monocyte apoptosis by serum deprivation is one of the in vitro model systems for the study of the regulation of apoptosis [56 ]. The spontaneous monocyte apoptosis has been shown to mediate by the interaction of Fas (CD95) and Fas ligand (FasL) on the surface of the cells [56 ]. Serum deprivation increased the expression of FasL on the monocyte surface [56 ]. Because WKYMVm attenuated serum deprivation-induced monocyte apoptosis, it should be possible for WKYMVm to modulate Fas- and FasL-induced apoptosis. In future work, it will be necessary to determine whether WKYMVm affects the expression of FasL or affects the signaling of Fas/FasL-induced apoptosis in human monocytes.
In summary, we have demonstrated that the chemoattractant peptide WKYMVm enhances monocyte survival and that this effect is PI 3-kinase-dependent. Upon studying the downstream signaling pathway leading to monocyte survival, we found that Akt but not ERK activation is critical for monocyte survival. With regard to the regulatory mechanism of Akt activity, we suggest here for the first time that the stimulation of classical PKCs modulates the G-protein-coupled receptor-induced activation of Akt and cell survival by inhibiting caspase-3 activity.
Received November 27, 2000; revised October 1, 2001; accepted October 1, 2001.
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