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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3013-3020.)
© 1997 American Heart Association, Inc.

Articles

Two Intracellular Signaling Pathways for Activation of Protein Kinase C Are Involved in Oxidized Low-Density Lipoprotein–Induced Macrophage Growth

Takeshi Matsumura; Masakazu Sakai; Shozo Kobori; Takeshi Biwa; Toru Takemura; Hirofumi Matsuda; Hideki Hakamata; Seikoh Horiuchi; ; Motoaki Shichiri

From the Departments of Metabolic Medicine (T.M., M.S., S.K., T.B., T.T., H.M., M.S.) and Biochemistry (H.H., S.H.), Kumamoto University School of Medicine, Kumamota, Japan.

Correspondence to Takeshi Matsumura, MD, Department of Metabolic Medicine, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860, Japan.

	Abstract

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Abstract Recent studies demonstrated that oxidized LDL (Ox-LDL) induces macrophage growth in vitro. The present study was undertaken to elucidate the intracellular signaling pathways for macrophage growth. Ox-LDL initiated a rapid and transient rise in intracellular free calcium ion and induced activation of membrane protein kinase C (PKC). Pertussis toxin completely inhibited the Ox-LDL–induced rise in free calcium ion and significantly inhibited macrophage growth by 50%. Moreover, PKC inhibitors calphostin C and H-7 significantly inhibited Ox-LDL–induced macrophage growth by 80%. On the other hand, phospholipase A2–treated acetylated LDL did not induce a rise in calcium but significantly activated PKC and led to significant macrophage growth that was significantly inhibited by calphostin C by 90%. These results suggest the presence of two intracellular signaling pathways for activation of PKC, a rise in calcium that was mediated by pertussis toxin–sensitive G protein and the internalization of lysophosphatidylcholine through the scavenger receptors. These two pathways may play an important role in Ox-LDL–induced macrophage growth.

Key Words: protein kinase C • G proteins • oxidized LDL • macrophage growth • atherosclerosis

	Introduction

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Macrophage-derived foam cells, which are characterized by massive deposition of cytoplasmic cholesteryl esters, are the key cellular elements in the early stage of atherosclerosis. Foam cells are known to produce various active molecules, such as cytokines and growth factors, which play an important role in the development and progression of atherosclerosis.¹ It is well known that macrophages take up chemically modified LDL, such as Ox-LDL and acetyl-LDL, through the scavenger receptor pathway and become foam cells in vitro.² Ox-LDL isolated directly from human atherosclerotic plaques³ induces foam cell formation in vitro.⁴ Thus, Ox-LDL has been considered as an atherogenic lipoprotein in vivo.

One of the characteristic events in the development of atherosclerotic lesions in arterial walls is the growth of cellular components. In particular, proliferating foam cells found in the advanced or mature stages of atherosclerosis are derived from smooth muscle cells.¹ However, recent reports emphasized that a proportion of foam cells proliferating in the atherosclerotic lesions are also derived from macrophages.^{5 6 7} Gordon et al⁵ demonstrated that 27% of cells positive for proliferating cell nuclear antigen in human coronary atherosclerotic lesions corresponded to macrophages. Furthermore, Rosenfeld and Ross⁶ demonstrated that in aortic atherosclerotic plaques of both Watanabe heritable hyperlipidemic rabbits and cholesterol-fed rabbits, 30% of cells that incorporated [³H]thymidine were derived from macrophages. Since macrophage-derived foam cells play an important role in the development of atherosclerotic lesions, it seems reasonable to expect that macrophage growth might be linked to atherosclerotic processes, probably by enhancing its progression.

Along with this line of thinking, we originally observed the capacity of Ox-LDL to stimulate the growth of starch-induced mouse peritoneal macrophages.⁸ Our subsequent studies showed that the Ox-LDL–induced macrophage growth is also observed with other macrophages, such as mouse resident peritoneal macrophages,⁹ rat resident peritoneal macrophages,¹⁰ and human monocyte-derived macrophages.¹¹ Moreover, our biochemical studies showed that endocytic internalization of lyso-PC, a major modified lipid moiety of Ox-LDL, into cells through the SR-AI/AII is essential for Ox-LDL–induced macrophage growth.¹² Considered together, these findings strongly suggest that to promote macrophage growth, Ox-LDL probably acts as a growth factor to macrophages by inducing certain intracellular signaling pathways. The present study was undertaken to characterize Ox-LDL–induced intracellular signaling pathways for the growth of mouse resident macrophages. The results indicate that a rise in intracellular calcium ion and the uptake of lyso-PC through the scavenger receptors are two potential pathways for activation of PKC that might be involved in Ox-LDL–induced macrophage growth.

	Methods

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Chemicals
[methyl-³H]Thymidine (80 Ci/mmol) was purchased from DuPont-NEN. H-7 and HA-1004 were purchased from Seikagaku Kogyo Co. H-89 was purchased from BIOMOL Research Laboratories, Inc. PTX, PLA2 from Crotalus atrox venom, calphostin C, thapsigargin, and fura 2-AM were purchased from Sigma Chemical Co. Fura 2-AM, H-7, thapsigargin, and calphostin C were dissolved in DMSO. The final concentrations of DMSO were less than 0.1% in the culture medium, which did not affect cell viability and macrophage growth. Other chemicals were the best grade available from commercial sources.

Lipoproteins and Their Modifications
Human LDL (d=1.019 to 1.063 g/mL) was isolated by sequential ultracentrifugation from the plasma of normolipidemic subjects after overnight fasting.¹³ LDL was dialyzed against 0.15 mol/L NaCl and 1 mmol/L EDTA, pH 7.4. Acetyl-LDL was prepared by chemical modification of LDL with acetate anhydride.¹⁴ Acetyl-LDL (2 mg) was dialyzed against Tris-HCl (pH 7.4) and treated then with 5 U/mL PLA2 for 2 hours at 37°C in Tris-HCl (pH 7.4) containing 2 mmol/L CaCl2.¹⁵ Then, PLA2 was removed from acetyl-LDL by ultracentrifugation. Ox-LDL was prepared by incubation of LDL with 5 µmol/L CuSO₄ for 20 hours at 37°C followed by the addition of 1 mmol/L EDTA and cooling.^{16 17} The concentration of proteins was determined by BCA protein assay reagent (Pierce) using bovine serum albumin as a standard.¹⁸ Lipid contents of lipoproteins were determined on a Hitachi 7450 automatic analyzer using standard enzymatic methods.^{19 20 21} Determination of PC and lyso-PC was performed by the method described by Bartlett.²² Endotoxin levels associated with these lipoproteins were measured by a commercially available kit (Toxicolor system, Seikagaku Corp).

Tritiated Thymidine Incorporation Assay
Peritoneal macrophages were collected from nonstimulated male C3H/He mice (25 to 30 g) and suspended in RPMI-1640 medium (Nissui Seiyaku Co) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc), streptomycin (0.1 mg/mL), and penicillin (100 U/mL) (medium A). The peritoneal cells were adjusted to 4x10⁵ cells per milliliter for the [³H]thymidine incorporation assay.⁹ Cell suspensions (0.1 mL) were dispersed in each well of 96-well tissue-culture plates (6.4 mm in diameter, Falcon) and incubated for 90 minutes at 37°C. The nonadherent cells were removed by washing three times with 0.1 mL of prewarmed medium A. More than 98% of adherent cells were confirmed to be macrophages by both Giemsa staining and carbon particle uptake.⁹ These macrophages were cultured at 37°C in 0.1 mL of medium A in the presence of the test lipoproteins without a medium change. Eighteen hours before the termination of the experiments, 10 µL of 10 µCi/mL [³H]thymidine was added to each well and incubated for 18 hours at 37°C. The medium was discarded, and the cells were dissolved in 0.1 mL of 0.5% sodium dodecyl sulfate and subsequently precipitated with 0.1 mL of ice-cold 10% trichloroacetic acid. The resulting trichloroacetic acid–insoluble material was collected on filters with Labomash LM-101 (Labo Science). The filters were dried, and their radioactivity was counted in a liquid scintillation spectrophotometer.⁹

Cell-Counting Assay
Mouse peritoneal macrophages were collected as described above. The peritoneal cells were adjusted to 2x10⁴ cells per milliliter, and 1 mL of cell suspension was dispersed in each well of 24-well tissue-culture plates (16 mm in diameter, Falcon) and incubated for 90 minutes at 37°C. The nonadherent cells were removed by washing three times with 1 mL of prewarmed medium A. These macrophages were cultured at 37°C in 1 mL of medium A with or without the test lipoproteins. After incubation for 7 days without medium change, the adherent cells in triplicate wells were lysed in 1% (wt/vol) Triton X-100, and the number of naphthol blue-black–stained nuclei were counted in a hemocytometer as described previously.⁹

Measurement of Cytosolic Free Calcium Concentration
The fluorescent calcium indicator fura 2 was used to monitor changes in cytosolic free calcium concentration ([Ca²⁺]_i) in mouse peritoneal macrophages.²³ The cells (1x10⁴ cells per well) were washed three times with 2 mL of Krebs-Ringer HEPES solution (in mmol/L: 128 NaCl, 5 KCl, 2.7 CaCl2, 1.2 MgSO4, 1 Na₂HPO₄, 10 glucose, and 20 HEPES, pH 7.4), followed by exposure to 4 µmol/L fura 2-AM in 2 mL of Krebs-Ringer HEPES solution for 15 minutes at 25°C. The cells were washed three times with Krebs-Ringer HEPES solution and incubated with 2 mL of Krebs-Ringer HEPES solution at 37°C. Fluorescence signals were monitored on an ARGUS-50/CA system (Hamamatsu) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. The cells were then stimulated by the test lipoproteins. In some experiments, the cells were pretreated for 5 minutes with PTX before the addition of lipoproteins.

Assay of PKC Activity
PKC activity in macrophages was assayed by MESACUP Protein Kinase Assay Kit (Medical and Biological Laboratories). Macrophages (1x10⁷ cells per well) in 2 mL of serum-free RPMI-1640 medium were treated with 50 µg/mL lipoproteins for the indicated times. The cells were washed three times with ice-cold PBS and detached from the plates with a rubber policeman. Cells were suspended in 1 mL of sample preparation buffer (in mmol/L: 5 EDTA, 10 EGTA, 50 2-mercaptoethanol, 1 PMSF, 10 benzamidine, and 50 Tris-HCl, pH 7.5) and sonicated for 30 seconds at 4°C by SONIFIER (Branson Sonic Power Co). Homogenates were centrifuged at 100 000g for 1 hour at 4°C. The supernatant was discarded and the precipitates were resuspended in 1 mL of buffer and used as the membrane fractions. The PKC activity in the membrane fractions was measured as follows. Twelve microliters of membrane fractions (5 µg) was added to 108 µL of component buffer (final concentration of reaction mixture: 3 mmol/L MgCl₂, 0.1 mmol/L ATP, 2 mmol/L CaCl₂, 50 µg/mL phosphatidylserine, 0.5 mmol/L EDTA, 1 mmol/L EGTA, 5 mmol/L 2-mercaptoethanol, and 25 mmol/L Tris-HCl, pH 7.0). One hundred microliters of this mixture was transferred to each well coated with PKC-specific peptide (RFARKGSLRQKNV) with a multichannel pipette. Each well was incubated at 25°C for 5 minutes, and the reaction was terminated by the addition of 100 µL of 20% H₃PO₄, followed by washing five times with 400 µL of PBS. To each well was added 100 µL of biotinylated antiphosphorylated tyrosine antibody (2B9), incubated at 25°C for 60 minutes, and washed five times with PBS. To each well was then added 100 µL of peroxidase-conjugated streptavidin, and samples were incubated at 25°C for 60 minutes and washed five times with PBS, followed by the addition of 100 µL of H₂O₂ and o-phenylenediamine. After incubation at 25°C for 5 minutes, 100 µL of 20% H₃PO₄ was added, and the absorbance at 492 nm was read with a microplate reader. Nonspecific serine/threonine-kinase activities were measured both in the absence of phosphatidylserine and in the presence of 2 mmol/L EGTA in component buffer. The enzyme activity specific for conventional PKC was quantitated by subtracting the nonspecific serine/threonine-kinase activities from total kinase activity in membrane fraction.

Miscellaneous
Data were expressed as mean±SD. Differences between groups were compared for statistical significance using the Student's t test. A probability value less than 5% was considered significant. The experimental protocol was approved by the Human Ethics Review Committee and the Ethics Review Committee for Animal Experimentation of our institution.

	Results

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Lipid Contents of Lipoproteins
Before the experiments, we determined lipid contents of lipoproteins used in the present study. As shown in Table 1, total cholesterol, cholesteryl esters, and phospholipids in Ox-LDL were slightly lower than those in LDL, acetyl-LDL, and PLA2-treated acetyl-LDL. Oxidation of LDL significantly increased lyso-PC contents from 30 nmol/mg protein to 480 nmol/mg protein. Moreover, treatment of acetyl-LDL with PLA2 increased a significant conversion of PC to lyso-PC from 40 nmol/mg protein to 620 nmol/mg protein. A lyso-PC content in PLA2-treated acetyl-LDL was higher than that in Ox-LDL. Levels of thiobarbituric acid-reactive substances in LDL, Ox-LDL, acetyl-LDL, or PLA2-treated acetyl-LDL were 2.5, 88, 3.2, and 3.6 nmol malondialdehyde per milligram protein, respectively, indicating that oxidation did not occur to acetyl-LDL by treatment with PLA2. Endotoxin levels associated with these lipoproteins were <1 pg/µg protein. Moreover, the viability of macrophages was not affected by endotoxin at a concentration <1 ng/mL under our experimental conditions (data not shown).

View this table: [in this window] [in a new window]   Table 1. Lipid Contents of Lipoproteins

Effect of Ox-LDL on Intracellular Calcium Concentration in Macrophages
To elucidate the intracellular signaling pathways in Ox-LDL–induced macrophage proliferation, we first examined [Ca²⁺]_i in macrophages using digital fluorescent microscopy. Addition of Ox-LDL to these macrophages resulted in an instantaneous rise in [Ca²⁺]_i followed by a return to the baseline level within 5 minutes (Fig 1A). The effect of Ox-LDL on [Ca²⁺]_i was dose dependent (Fig 2). In contrast, both LDL and acetyl-LDL had no effect on [Ca²⁺]_i in these cells (Figs 1B, 1C, and 2). Moreover, PLA2-treated acetyl-LDL, which possessed growth-stimulating activity for macrophages (Table 2), did not induce a rise in [Ca²⁺]_i (Fig 1D). Even when the cells were pretreated with acetyl-LDL, Ox-LDL retained its capacity to induce a rise in [Ca²⁺]_i in these macrophages (Fig 1E). Pretreatment of macrophages with thapsigargin significantly inhibited Ox-LDL–induced rise in [Ca²⁺]_i (Fig 1F), suggesting that the rise in [Ca²⁺]_i was derived from intracellular calcium store. This notion was supported by the observation that Ox-LDL–induced rise in [Ca²⁺]_i was inhibited neither by calcium channel blockers, such as nifedipine and nicardipine nor by calcium chelator EGTA (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 1. Effect of lipoproteins on cytosolic calcium concentration in macrophages. Mouse resident peritoneal macrophages (1x104 cells per well) were incubated in 2 mL of Krebs-Ringer HEPES solution at 37°C with 50 µg/mL Ox-LDL (A), 50 µg/mL LDL (B), 50 µg/mL acetyl-LDL (C), 50 µg/mL PLA2-treated acetyl-LDL (D), 50 µg/mL Ox-LDL preincubated with 50 µg/mL acetyl-LDL (E), or 50 µg/mL Ox-LDL preincubated with 2 µmol/L thapsigargin (F). Arrows indicate the time of addition of these samples. Cytosolic free calcium concentrations were determined as described under "Methods." Data represent the mean of five separate experiments. SD is within 5%.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of Ox-LDL on cytosolic calcium concentration in macrophages. Experiments were identical to those of Fig 1. Mouse resident peritoneal macrophages (1x104 cells per well) were incubated in 2 mL of Krebs-Ringer HEPES solution with the indicated concentrations of Ox-LDL (), LDL (), acetyl-LDL(), or PLA2-treated acetyl-LDL (). Cytosolic free calcium concentrations were determined as described under "Methods." Data represent the mean±SD of peak cytosolic calcium concentration of five separate experiments. SD is within 5%.

View this table: [in this window] [in a new window]   Table 2. Inhibitory Effect of Pertussis Toxin on OX-LDL–Induced Macrophage Growth Determined by Counting of Solubilized Nuclei

Effect of PTX on Intracellular Calcium Concentration and Macrophage Proliferation
In the next step, we compared the effects of PTX on Ox-LDL–induced [Ca²⁺]_i rise and subsequent macrophage growth. Pretreatment of macrophages with 10 ng/mL PTX for 5 minutes inhibited Ox-LDL–induced [Ca²⁺]_i rise (Fig 3). As shown in Fig 4, PTX significantly inhibited Ox-LDL–induced increase in [³H]thymidine incorporation in a dose-dependent manner up to 10 ng/mL, with a maximal inhibition of macrophage growth of 50%. Under these conditions, PTX alone up to 100 ng/mL did not affect cell viability determined by both the cell-counting assay using trypan blue staining and the release of lactic dehydrogenase from cells (data not shown). Parallel experiments with the cell-counting assay also showed that the cell number increased by 20 µg/mL Ox-LDL was inhibited by 52% with 10 ng/mL PTX (Table 2). In contrast, PLA2-treated acetyl-LDL–induced increase in cell number was not affected by PTX (Table 2). These results suggested that the initial but transient increase in [Ca²⁺]_i induced by Ox-LDL was probably related to Ox-LDL–induced macrophage growth and also suggested the presence of other signaling pathway(s) for PLA2-treated acetyl-LDL–induced macrophage growth.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of PTX on cytosolic calcium concentration in macrophages. Mouse resident peritoneal macrophages (1x104 cells per well) in 2 mL of Krebs-Ringer HEPES solution were preincubated at 37°C with 100 µL of PBS (A) or PTX at a final concentration of 10 ng/mL (B), followed by the addition of 100 µL of Ox-LDL to a final concentration of 50 µg/mL. Cytosolic free calcium concentrations were determined as described under "Methods." Data represent the mean of five separate experiments. SD is within 5%.

View larger version (23K): [in this window] [in a new window]   Figure 4. Inhibitory effect of PTX on Ox-LDL–induced macrophage growth determined by [3H]thymidine incorporation assay. Mouse resident peritoneal macrophages (4x104 cells per well) were incubated at 37°C for 6 days in 1 mL of medium A with the indicated concentrations of PTX in the absence () or presence () of 20 µg/mL Ox-LDL. During the last 18 hours of incubation, cells in each well were chased with [3H]thymidine, harvested, and the radioactivity was determined as described under "Methods." Data represent the mean±SD of four separate experiments.

Involvement of PKC in Ox-LDL–Induced Macrophage Growth
Fig 5 shows the time-course study of the membrane PKC activity. After the addition of 20 µg/mL Ox-LDL to these cells, the enzyme activity increased 2.2-fold above the basal level at 5 minutes and further increased to 4.4-fold above the basal level at 10 minutes, followed by a rapid decline close to the baseline level at 20 minutes. However, PKC seemed to be still activated to some extent even 30 minutes after the addition of Ox-LDL. PLA2-treated acetyl-LDL also activated the membrane PKC, whose level was less than that activated by Ox-LDL. Both LDL and acetyl-LDL failed to stimulate the membrane PKC activity in these cells (Fig 5).

View larger version (21K): [in this window] [in a new window]   Figure 5. Effect of Ox-LDL on PKC activity in macrophages. Mouse resident peritoneal macrophages (1x107 cells per well) in 2 mL of serum free RPMI-1640 medium were incubated at 37°C for the indicated time with 20 µg/mL Ox-LDL (), 20 µg/mL LDL (), 20 µg/mL acetyl-LDL (), or 20 µg/mL PLA2-treated acetyl-LDL (). The membrane PKC activity was determined as described under "Methods." Data represent the mean±SD of four separate experiments.

To elucidate the role of PKC activation in Ox-LDL–induced macrophage growth, the effect of a PKC inhibitor, calphostin C, on Ox-LDL–induced macrophage growth was examined. On incubation of macrophages with different concentrations of calphostin C, the Ox-LDL–induced increase in [³H]thymidine incorporation was suppressed in a dose-dependent manner, with 50% inhibition of 50 nmol/L calphostin C and the maximal inhibition of 80% at 200 nmol/L (Fig 6). Incubation of macrophages with 2 or 10 µmol/L H-7, another PKC inhibitor, also suppressed Ox-LDL–induced increase in [³H]thymidine incorporation by 80% (Fig 6). On the other hand, HA-1004, an inhibitor of cyclic GMP–dependent and cyclic AMP–dependent protein kinase, and H-89, an inhibitor of cyclic AMP–dependent protein kinase, had almost no effect on this system. To further confirm the results obtained by [³H]thymidine incorporation assay, we also assessed the effect of calphostin C on Ox-LDL–induced macrophage growth by cell counting. As shown in Table 3, the increased number of cells induced by Ox-LDL was 1.9-fold above the basal level. When macrophages were cultured under the identical conditions but in the presence of calphostin C, the increase in cell numbers was only 1.2-fold above the basal level, indicating the effective suppression of cell growth by this drug. Moreover, calphostin C significantly inhibited the increased number of cells induced by PLA2-treated acetyl-LDL (Table 3). Under these conditions, calphostin C had no cytotoxic effect on these cells; more than 95% of the cells were viable, as confirmed by microscopic examination after trypan blue staining and incorporation of [³H]leucine into protein (data not shown). These results support a possible role of PKC but not cyclic GMP–dependent and cyclic AMP–dependent protein kinase in Ox-LDL–induced macrophage growth. In support for this notion, TPA alone could induce macrophage growth, which was suppressed by calphostin C (Table 3), and when the PKC activity of these macrophages was downregulated by 24 hours' pretreatment with 1 µmol/L TPA, Ox-LDL–induced macrophage growth was significantly inhibited (data not shown).

View larger version (53K): [in this window] [in a new window]   Figure 6. Inhibitory effect of protein kinase inhibitors on Ox-LDL–induced macrophage growth. Mouse resident peritoneal macrophages (4x104 cells per well) were incubated at 37°C for 6 days with 20 µg/mL Ox-LDL in the absence or presence of the indicated concentrations of calphostin C, H-7, HA-1004, or H-89. During the last 18 hours of incubation, cells in each well were chased with [3H]thymidine, harvested, and the radioactivity was determined as described under "Methods." Data represent the mean±SD of four separate experiments.

View this table: [in this window] [in a new window]   Table 3. Inhibitory Effect of Calphostin C on Ox-LDL–Induced Macrophage Growth Determined by Counting of Solubilized Nuclei

	Discussion

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PKC is involved in agonist-induced cellular responses in T lymphocytes such as those induced by hormones, neurotransmitters, and certain growth factors.²⁴ The enzyme is activated by high concentrations of diacylglycerol and calcium in membranes, which are derived from agonist-induced hydrolysis of inositol phospholipid.²⁴ However, lyso-PC generated from PC, through the action of cytosolic PLA2, is also involved in prolonged activation of PKC in T lymphocytes and HL-60 cells.²⁵ In fact, sustained activation of PKC is essential for long-term cellular responses such as cell proliferation and differentiation.²⁵ In addition, we recently demonstrated that the endocytic internalization of lyso-PC through the SR-AI/AII may play an important role in Ox-LDL–induced macrophage growth.¹² Thus, it is reasonable to assume that PKC may be activated by Ox-LDL. The present study has clearly shown that two Ox-LDL–induced phenomena, the activation of PKC (Fig 5) and macrophage growth, were significantly inhibited in common by PKC inhibitors such as calphostin C and H-7 (Fig 6 and Table 3). Moreover, TPA alone significantly induced macrophage growth, which was inhibited by calphostin C (Table 3). Furthermore, downregulation of PKC by pretreatment of macrophages with TPA led to inhibition of macrophage growth (data not shown). However, HA-1004 or HA-89 had no effect on Ox-LDL–induced macrophage growth. These results suggest an important role of PKC but not cyclic GMP–dependent nor cyclic AMP–dependent protein kinases in Ox-LDL–induced macrophage growth. This conclusion is supported by the results of Hamilton and Dientsman,^{26 27} who demonstrated the growth-stimulating activity of phorbol esters for mouse peritoneal–elicited macrophages.

It is likely that the increased [Ca²⁺]_i is derived from intracellular calcium store, because pretreatment of macrophages with thapsigargin significantly inhibited Ox-LDL–induced rise in [Ca²⁺]_i (Fig 1F) and because neither calcium channel blockers nor EGTA affected Ox-LDL–induced calcium rise (data not shown). This notion was supported by Shackelfold et al,²⁸ who demonstrated that Ox-LDL initiated a rapid increase in phosphatidylinositol bisphosphate hydrolysis into inositol triphosphate, which in turn enhanced calcium release from the endoplasmic reticulum in mouse macrophages. Our results also showed that Ox-LDL–induced [Ca²⁺]_i rise was completely inhibited by PTX (Fig 3). Moreover, PTX inhibited Ox-LDL–induced macrophage growth by 50% (Fig 4 and Table 2). These results suggest that the rapid release of Ca²⁺ from the endoplasmic reticulum is likely to be involved in Ox-LDL–induced macrophage proliferation.

The inhibition by PTX of Ox-LDL–induced [Ca²⁺]_i rise and macrophage growth (Figs 3 and 4, Table 2) suggests the presence of a G-protein–coupled Ox-LDL receptor that initiates the intracellular signal(s) and finally leads to macrophage growth. Most G-protein–linked receptors identified so far have a common structure: seven membrane-spanning domains connected by extracellular and intracellular loops.²⁹ According to a general model of chemokine-mediated signal transduction events in phagocytes, an appropriate ligand-receptor interaction causes the activation of a PTX-sensitive G-protein with subsequent induction of phospholipase C activity.³⁰ This leads to the accumulation of both diacylglycerol and cytosolic inositol trisphosphate³¹ ; the former directly stimulates PKC activity, whereas the latter in turn initiates mobilization of intracellular Ca²⁺ from the endoplasmic reticulum, leading to PKC activation.^{32 33} Several scavenger receptors have been reported, which include the SR-AI/AII,³⁴ the Fc receptor,³⁵ CD-36,³⁶ SR-BI,³⁷ SR-CI,³⁸ macrosialin,³⁹ and MARCO.⁴⁰ However, none of them posses a seven-transmembrane domain, a characteristic feature of the G-protein–coupled receptor. Further studies are needed, therefore, to identify the receptor involved in the Ox-LDL–induced rise in [Ca²⁺]_i.

Considering the findings of this and our previous studies,^{9 12} we could assume the intracellular events that lead to macrophage growth. Two intracellular signaling pathways, activation of G-protein and the endocytic internalization of lyso-PC, are responsible for Ox-LDL–mediated macrophage growth. In the first pathway, binding of Ox-LDL to its receptor on macrophage plasma membranes initiates activation of a PTX-sensitive G-protein and then induces phospholipase C activity, to produce diacylglycerol and inositol triphosphate. PKC is known to be activated directly by diacylglycerol or indirectly by inositol triphosphate via an increase in [Ca²⁺]_i. Well established as major endocytic receptors, the SR-AI/AII are also known to modulate a variety of macrophage functions, such as endocytosis of modified proteins,⁴¹ cell adhesion,⁴² and the release of lipoprotein lipase.⁴³ However, intracellular signaling in response to the ligand binding to the SR-AI/AII has not been well established. Recently, Claus et al⁴⁴ demonstrated that acetyl-LDL could activate PKC in P388D1 cells, which was inhibited by polyinosinic acid, suggesting that the SR-AI/AII could induce intracellular signals. However, since acetyl-LDL neither initiated [Ca²⁺]_i rise nor activated PKC in mouse peritoneal macrophages (Figs 1, 2, and 5), it seemed unlikely that the receptors for acetyl-LDL, such as the SR-AI/AII, MARCO, or SR-CI, are involved in increased [Ca²⁺]_i in Ox-LDL–induced macrophage growth. An exact reason is unclear why the PKC was activated by acetyl-LDL in P388D1 cells⁴⁴ but not in mouse macrophage in the present study. It could be due to the difference in cell types and/or experimental conditions. However, PLA2-treated acetyl-LDL could stimulate both PKC (Fig 5) and macrophage growth (Table 2). Moreover, our recent study showed that PLA2-treated acetyl-LDL did not show a significant growth-stimulating capacity for the SR-AI/AII knockout mice, while Ox-LDL–induced growth of these macrophages was 30% of that of the wild–type littermates.¹² These results suggest that the internalization of lyso-PC in Ox-LDL into the cells through the SR-AI/AII may also cause the activation of PKC, leading to macrophage growth, whereas involvement of other receptors for Ox-LDL could not be completely ruled out. This notion was supported by the present results that PTX completely inhibited the rise in [Ca²⁺]_i but produced a 50% decrease in macrophage growth (Figs 3 and 4 and Table 2). Thus, two independent pathways for PKC activation might act synergically to induce Ox-LDL–induced macrophage growth. The PKC family is reported to comprise at least 11 different subspecies of serine/threonine protein kinase,⁴⁵ such as conventional PKC (, ß1, ß2, and ) and novel PKC (, , , and ). Moreover, atypical subspecies of PKC were also reported recently.⁴⁵ The present study suggested the presence of two pathways for activation of PKC. Thus, it is possible to speculate that different PKC isozyme(s) were activated by these pathways. Further studies using anti-PKC isoform-specific antibodies are needed to identify the PKC isoform(s) involved in Ox-LDL–induced macrophage growth.

The downstream signaling pathway from PKC activation to macrophage proliferation remains unknown. However, two possible pathways may be operating. First, PKC induces macrophage proliferation directly via activation of MAP kinase. Recently, Kusuhara et al⁴⁶ demonstrated that Ox-LDL activated MAP kinase of rat smooth muscle cells via PKC activation, suggesting that Ox-LDL–induced macrophage growth might be mediated through activation of MAP kinase after activation of PKC. Ox-LDL was also shown to activate MAP kinase of human macrophages, but it was not known whether activation of MAP kinase might be mediated by PKC activation.⁴⁶ Further studies will be needed to elucidate the involvement of MAP kinase activation via PKC in Ox-LDL–induced macrophage growth. Alternatively, PKC stimulates the induction of growth factors such as macrophage-colony stimulating factor or GM-CSF, which is able to induce macrophage growth in an autocrine or paracrine fashion. In a series of preliminary experiments, we noted that anti–GM-CSF antibody caused 80% inhibition of Ox-LDL–induced macrophage growth. Incubation with Ox-LDL resulted in a significant release of GM-CSF from mouse peritoneal macrophages in the culture medium, a process that was inhibited by calphostin C (T. Biwa and S. Horiuchi, unpublished observations, 1997). Thus, it is likely that GM-CSF may act as a growth factor in autocrine or paracrine fashion in Ox-LDL–induced macrophage growth. The presence of a signaling pathway linking PKC activation to GM-CSF induction has been described recently. Tsuboi et al⁴⁷ demonstrated that cooperation among AP-1–, NF-B–, and NF-AT–binding sequences was required for the induction of GM-CSF, which was located downstream of PKC- and Ca²⁺-signaling pathways in T lymphocytes. Moreover, Ares et al⁴⁸ reported that Ox-LDL could induce the activation of AP-1 in smooth muscle cells. Therefore, it is reasonable to speculate that induction of GM-CSF via activation of certain nuclear transcription factors after PKC activation may be involved in Ox-LDL–induced macrophage growth.

In conclusion, we have shown here that Ox-LDL initiates a rapid and transient increase in [Ca²⁺]_i from intracellular calcium store and induces PKC activation in macrophages. PTX inhibits Ox-LDL–induced [Ca²⁺]_i rise completely and Ox-LDL–induced macrophage growth significantly, whereas PKC inhibitors, such as calphostin C and H-7, show a significant inhibition for Ox-LDL–induced macrophage growth. On the other hand, PLA2-treated acetyl-LDL activates PKC and stimulates macrophage growth but failed to induce a rise in [Ca²⁺]_i. Macrophage growth induced by PLA2-treated acetyl-LDL was inhibited by calphostin C but not by PTX. These results suggest that PKC activation by both the G-protein–mediated rise in [Ca²⁺]_i and the endocytic internalization of lyso-PC plays an essential role in Ox-LDL–induced macrophage growth.

	Selected Abbreviations and Acronyms

 

acetyl-LDL = acetylated LDL GM-CSF = granulocyte/macrophage-colony stimulating factor lyso-PC = lysophosphatidylcholine MAP = mitogen-activated protein Ox-LDL = oxidized LDL PC = phosphatidylcholine PKC = protein kinase C PLA2 = phospholipase A2 PTX = pertussis toxin SR-AI/AII = scavenger receptor AI/AII TPA = phorbol 12-myristate 13-acetate

	Acknowledgments

 
This work was supported in part by a research grant from the Scientific Research Fund of the Ministry of Education, Science, and Culture, Japan (No. 05670871 and 06671041). We are grateful to Dr Naofumi Tokutomi at the Department of Pharmacology, Kumamoto University School of Medicine, for determination of intracellular calcium concentration.

Received January 28, 1997; accepted May 26, 1997.

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