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(Journal of Leukocyte Biology. 2002;71:329-338.)
© 2002 by Society for Leukocyte Biology

The synthetic chemoattractant peptide, Trp-Lys-Tyr-Met-Val-D-Met, enhances monocyte survival via PKC-dependent Akt activation

Yoe-Sik Bae, Youndong Kim, Jun Chul Park, Pann-Ghill Suh and Sung Ho Ryu

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previously, we showed that Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) stimulates superoxide generation and chemotactic migration in monocytes and neutrophils. In this study, we examined the effect of WKYMVm on monocyte survival. Serum starvation-induced monocyte death was attenuated in the presence of WKYMVm, which was abated when the cells were preincubated with LY294002, suggesting the involvement of phosphoinositide-3-kinase (PI 3-kinase) in the peptide-induced monocyte survival. WKYMVm stimulated ERK and Akt activity via PI 3-kinase activation in monocytes. We also investigated the signaling pathway of WKYMVm-induced ERK and Akt activation. The WKYMVm-induced ERK activation was PI 3-kinase-dependent but PKC-independent. However, Akt activation by WKYMVm was dependent not only on PI 3-kinase but also on the PKC pathway. When monocytes were incubated with WKYMVm, caspase-3 activity, which is important for cell death, was inhibited. Pretreatment of the cells with LY294002, GF109203X, and Go 6976 but not PD98059 blocked WKYMVm-induced monocyte survival and caspase-3 inhibition. In summary, the novel chemoattractant WKYMVm enhances monocyte survival via Akt-mediated pathways, and in this process, PKC and PI 3-kinase act upstream of Akt.

Key Words: PI 3-kinase • ERK • G-protein • caspase-3


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human monocytes play a critical role in human immune response. Therefore, the homeostasis of cells is important. The lifespan of monocytes is limited, and homeostasis is regulated by programmed cell death [1 ]. Circulating monocytes perform their work in the presence of growth factors, and they undergo apoptosis in the absence of growth factors [2 ]. Several extracellular stimuli regulate human monocyte survival and apoptosis. For example, the macrophage colony-stimulating factor (CSF), interleukin-1ß (IL-1ß), and the ligation of high-affinity immunoglobulin E (IgE) receptor have been suggested to regulate the cell death of monocytes [2 3 4 5 ]. Recently, CXC-chemokine platelet factor 4, which acts on monocytes via the pertussis toxin (PTX)-sensitive G-protein-coupled receptor, has been shown to promote monocyte survival [6 , 7 ]. However, the specific intracellular mechanism involved in the regulation of monocyte survival is not yet well understood.

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
The peptide was synthesized, purified, and prepared in the Peptide Library Support Facility at Pohang University of Science and Technology (Korea), as described previously [23 24 25 26 27 28 ]. Peripheral blood mononuclear cell (PBMC) separation medium (Histopaque-1077), the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent, and fMLF [a specific agonist for formyl peptide receptor (FPR)] were purchased from Sigma Chemical Co. (St. Louis, MO), and RPMI 1640 was bought from Life Technologies (Grand Island, NY). Lipoxin A4 was purchased from Biomol Research Laboratories (Polymouth Meeting, PA). Dialyzed fetal bovine serum (FBS) and supplemented bovine calf serum were purchased from Hyclone Laboratories (Logan, UT); GF109203X, Go 6976, PD98059, and LY294002, from Calbiochem (San Diego, CA); rabbit anti-human antibodies to total and specific phospho forms of Akt and ERK, from New England Biolabs (Beverly, MA); rabbit anti-human antibodies to PKC isoforms ({alpha}, ßI, ßII, {gamma}, {delta}, {varepsilon}, and {zeta}), 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 {alpha} (TNF-{alpha}) 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
WKYMVm promotes monocyte survival in a PI 3-kinase-dependent manner.
Monocytes underwent spontaneous apoptosis within 48–96 h of serum deprivation [1 , 5 ]. To investigate the effect of WKYMVm on monocyte survival, we measured monocyte viability after WKYMVm stimulation to monocytes for several different lengths of time in a serum-free medium. Monocyte viability was enhanced by WKYMVm treatment, and this effect was most apparent after 72 h of incubation against an unstimulated control (unpublished results). When cultured for 72 h in the absence of serum, approximately 60% of the monocytes died as determined by the MTT reduction assay, and this serum starvation-induced monocyte death was attenuated in the presence of 1–1000 nM WKYMVm (Fig. 1A ). Recently some papers demonstrated that WKYMVm acts on at least two phagocyte formyl peptide receptors, FPR and FPR-like 1 (FPRL1) [29 , 30 ]. To further delineate which receptor is involved in the action of monocyte survival, we compared the effect of fMLF and lipoxin A4 (a specific agonist for FPRL1) on monocyte survival. As shown in Figure 1A , neither fMLF nor lipoxin A4 enhanced monocyte survival. To confirm that serum starvation-induced monocyte apoptosis was attenuated by WKYMVm, we performed a DNA fragmentation analysis. As shown in Figure 1B , serum deprivation caused DNA fragmentation, and addition of 100 nM WKYMVm inhibited DNA fragmentation. This result provides direct evidence that WKYMVm inhibits serum deprivation-induced monocyte apoptosis. Several studies have demonstrated that PI 3-kinase is essential for cell survival after various death-inducing treatments [36 37 38 ]. Therefore, we investigated whether PI 3-kinase plays a role in WKYMVm-induced monocyte survival using the specific PI 3-kinase inhibitor LY294002. Monocytes were pretreated with LY294002 or vehicle for 15 min before being cultured for 72 h in the medium alone or in the presence of 100 nM WKYMVm. As shown in Figure 1C , 10 µM LY294002 almost completely reversed the protective effect of WKYMVm. These results indicate that the monocyte survival by WKYMVm requires PI 3-kinase activation.



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  		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.

 
WKYMVm activates Akt in a PI 3-kinase-dependent manner
To determine whether WKYMVm stimulates Akt activation in monocytes, we analyzed the activation of Akt using an antiphospho [Ser 473]-specific Akt antibody that specifically recognizes the active form of Akt [39 ] and found that WKYMVm stimulated significant activation of Akt in monocytes, which was obvious within 1–100 nM concentrations (Fig. 2A ). The concentration dependency of WKYMVm-induced Akt activation was similar to that of peptide-induced cell survival (Fig. 1A) . When we examined the time course of WKYMVm-induced Akt activation, we found it was very rapid and transient, showing activity within 5 min of peptide treatment (Fig. 2B) . D-Trp-D-Lys-D-Tyr-D-Met-D-Val-D-Met (wkymvm) was used as a negative control for WKYMVm, because we had found previously that it could not stimulate phosphoinositide hydrolysis in leukocytes [24 ]. Prior to use, we confirmed that it could not induce the activation of Akt at 1 µM concentration in monocytes (unpublished results). It is known that Akt, upon activation, translocates from the cytosol to the membrane. We also observed that the stimulation of monocytes with 100 nM of the peptide for 2 min elicited translocation of Akt to the membrane fraction, which supported the notion that WKYMVm induces Akt activation (unpublished results). Recent data suggested that Akt is a downstream target of PI 3-kinase products, such as PI 3,4P2 and PI 3,4,5P3 [17 , 18 ], and, therefore, we checked whether PI 3-kinase has a role in WKYMVm-induced Akt activation using the PI 3-kinase-specific inhibitor LY294002. As shown in Figure 2C , pretreatment of monocytes with 10 µM LY294002 for 15 min before WKYMVm stimulation completely inhibited WKYMVm-induced Akt activation. Thus, these results indicated that WKYMVm stimulates Akt activation via PI 3-kinase activity. In our previous studies, we demonstrated that WKYMVm activates leukocytes in a PTX-sensitive manner [28 ]. In this study, we also confirmed that WKYMVm-induced Akt activation was inhibited completely by PTX but not by cholera toxin, indicating the involvement of PTX-sensitive G-protein(s) (unpublished results).



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  		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.

 
WKYMVm stimulates ERK activation in a PI 3-kinase-dependent manner
Previously, we demonstrated that WKYMVM stimulates ERKs in human promonocytic U937 cells [40 ]. In this study, we checked the activation of ERKs by WKYMVm in human peripheral monocytes by Western blot analysis using an antibody specific to the activated form of the kinase, as described before [40 ]. Stimulation of human monocytes with various concentrations of WKYMVm caused ERKs activation in a concentration-dependent manner and showed maximal activity at 10–100 nM (Fig. 3A ). This concentration dependency correlates with WKYMVm-induced Akt activation and cell survival (Figs. 1A and 2A) . Peptide-stimulated ERK activation was found to be transient. Phosphorylation was maximal after 2–5 min and returned to the basal level after 10 min (Fig. 3B) . The involvement of PI 3-kinase in ERK activation by WKYMVm was also investigated. When the cells were preincubated with 10 µM LY294002 prior to stimulation with 100 nM WKYMVm, peptide-induced ERK activation was almost completely inhibited, which suggested a role for PI 3-kinase in this event (Fig. 3C) .



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  		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.

 
WKYMVm elicits translocation of PKC
Generally, the activation of phospholipase C (PLC) leads to the generation of PI hydrolysis, diacylglycerol, and inositol trisphosphatase. These two molecules then induce PKC activation and an intracellular calcium rise, respectively [41 , 42 ]. Because we had demonstrated previously that the stimulation of monocytes with WKYMVm elicited PI hydrolysis and intracellular calcium release [23 , 24 ], we investigated which isoform of PKC might be activated by WKYMVm in monocytes. During activation, PKC has been known to translocate from the cytosol to the membrane [43 ]. Therefore, we performed immunoblot analyses with PKC isozyme-specific antibodies after subcellular fractionation. Figure 4 shows that the stimulation of monocytes with WKYMVm elicited the translocation of PKC{alpha} 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{gamma}, {delta}, and {zeta}, we could not detect these in our system (unpublished results).



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  		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{alpha} 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.

 
Akt and ERK activations by WKYMVm are distinctly regulated: involvement of MEK and PKC
Because we found that WKYMVm stimulates PKC{alpha} 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{alpha}/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{alpha} 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.



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  		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.

 
WKYMVm-induced PKC translocation is PI 3-kinase-dependent
Our finding that WKYMVm-induced Akt activation is PI 3-kinase- and PKC-dependent led us to look for a regulatory relationship between these two enzymes in WKYMVm-stimulated intracellular signaling in monocytes. For this purpose, we investigated what effect PI 3-kinase might have on WKYMVm-induced PKC translocation. When monocytes were preincubated with the PI 3-kinase-specific inhibitor, LY294002, prior to stimulation with 100 nM WKYMVm, WKYMVm-elicited PKC translocation to the membrane fraction was blocked (Fig. 6 ). This indicated that activation of PI 3-kianse and the generation of lipid products might be a prerequisite for the translocation of classical isoforms of PKC ({alpha} and ßII) downstream of the WKYMVm receptor in monocytes.



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  		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{alpha} 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.

 
Monocyte survival by WKYMVm is Akt-dependent
Many studies have demonstrated that the activation of ERK inhibits cell apoptosis [11 , 12 ]. Recently, several studies have shown that Akt activation is also important for cell survival [13 14 15 16 ]. Figure 1 shows that WKYMVm enhanced monocyte survival. Because WKYMVm was found to stimulate ERK and Akt activation in monocytes (Figs. 2 and 3) , we attempted to elucidate the functions of ERK and Akt in WKYMVm-enhanced monocyte survival. There is no known pharmacological inhibitor that specifically inhibits Akt or ERK. Because Akt and ERK activation by WKYMVm was PKC and MEK regulation-dependent, it was possible to inhibit WKYMVm-stimulated ERK and Akt activity specifically. Thus, we examined the roles of ERK and Akt activity on WKYMVm-induced monocyte survival by blocking MEK-mediated ERK activation with PD98059 or by blocking PKC-mediated Akt activation with GF109203X and Go 6976. As shown in Figure 7 , preincubation of monocytes with Go 6976 attenuated almost completely WKYMVm-enhanced monocyte survival. When the cells were pretreated with the MEK inhibitor, PD98059, WKYMVm-enhanced monocyte survival activity was not affected. These results indicate that PKC-mediated Akt but not MEK-dependent ERK activity is important for WKYMVm-induced monocyte survival.



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  		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.

 
WKYMVm inhibits caspase-3 activity in monocytes
Caspase is a member of a family of proteases that are involved in proteolytic cleavage of cellular proteins during apoptosis [46 ]. Many studies have shown that apoptosis is accompanied by the activation of caspases, especially caspase-3 [20 21 22 ]. We studied the effect of WKYMVm on caspase-3 activity during monocyte apoptosis using its substrate DEVD-AMC. When the cells were incubated with various concentrations of WKYMVm, caspase-3 activity was inhibited (Fig. 8A ). Because caspase-3 is known to be cleaved during its activation process [47 ], we confirmed the effect of WKYMVm on caspase-3 activity by Western blot analysis using an antibody that recognizes caspase-3 (pro form). Treatment with various concentrations of WKYMVm inhibited the cleavage of caspase-3, a mark for its activation induced by culturing the cells in the absence of growth factors (Fig. 8A , inset). The concentration dependency of WKYMVm-induced caspase-3 inhibition correlates with that of WKYMVm-induced cell survival, as shown in Figure 1A . The regulatory mechanism of the WKYMVm-induced cell survival and caspase-3 activation was examined further using several pharmacological inhibitors. The peptide-induced caspase-3 inhibitory effect was blocked almost completely by PI 3-kinase inhibitor (LY294002) and two kinds of PKC inhibitors (GF109203X and Go 6976) but not by MEK inhibitor (PD98059; Fig. 8B ). These results coincide with PI 3-kinase and the PKC-dependent Akt activation as well as the enhancement of cell survival via the Akt pathway (Fig. 7) .



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  		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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we examined the effects of WKYMVm on monocyte survival. Monocyte survival was found to be enhanced in the presence of WKYMVm, and this effect was PI 3-kinase-dependent. Treatment of monocytes with WKYMVm caused ERK and Akt activation as a downstream signaling event of the WKYMVm-induced PI 3-kinase pathway. The regulation of ERK and Akt activation is distinct by being dependent on the MEK or PKC activity, respectively. Although WKYMVm-induced ERK activation is MEK-dependent and not affected by PKC, Akt activation by the peptide is PKC-dependent and not affected by MEK. WKYMVm-induced monocyte survival and caspase-3 inhibition were attenuated by PI 3-kinase inhibitor and PKC inhibitors but not by MEK inhibitor. We suggest that PI 3-kinase activation and PKC-dependent Akt, but not ERK activation, play significant roles in the enhancement of monocyte cell survival.

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{alpha} and ßII and that WKYMVm-induced Akt activation could be inhibited by Go 6976, a PKC{alpha}/ß-specific inhibitor, we concluded that classical PKC isozymes (especially PKC{alpha} 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{delta}-specific inhibitor, attenuated the PMA-induced effect, suggesting a negative role for PKC{delta} in Akt activation. However, Li et al. [52 ] showed that the overexpression of PKC{alpha} 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{alpha} 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.


   ACKNOWLEDGEMENTS
 
This work was supported by the Highly Advanced National Project and National Research Laboratory of Ministry of Science and Technology and by the Korean Center for Cellular Signaling Research. We thank D. S. Cho and his colleagues for kind preparation of peripheral blood leukocytes.

Received November 27, 2000; revised October 1, 2001; accepted October 1, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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