Activation of Cytosolic Phospholipase A2 by Platelet-derived Growth Factor Is Essential for Cyclooxygenase-2-dependent Prostaglandin E2 Synthesis in Mouse Osteoblasts Cultured with Interleukin-1*

(Received for publication, October 2, 1996, and in revised form, December 2, 1996)

Qing-Rong Chen Dagger §, Chisato Miyaura Dagger , Sayumi Higashi , Makoto Murakami , Ichiro Kudo , Shigeru Saito §, Takatoshi Hiraide §, Yoshinobu Shibasaki § and Tatsuo Suda Dagger par

From the Dagger  Department of Biochemistry, School of Dentistry,  Department of Health Chemistry, School of Pharmaceutical Sciences, and § Department of Orthodontics, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

The synthesis of prostaglandins (PGs) is regulated by the arachidonic acid release by phospholipase A2 (PLA2) and its conversion to PGs by cyclooxygenase (COX). In the present study, we examined the regulation of PG synthesis by interleukin (IL)-1alpha in primary mouse osteoblastic cells isolated from mouse calvaria. Although IL-1alpha greatly enhanced cox-2 mRNA expression and its protein levels, PGE2 was not produced until 24 h. When arachidonic acid was added to osteoblastic cells precultured with IL-1alpha for 24 h, PGE2 was produced within 10 min. Of several growth factors tested, platelet-derived growth factor (PDGF) specifically initiated the rapid synthesis of PGE2, which was markedly suppressed by a selective inhibitor of cox-2 (NS-398). In mouse osteoblastic cells, cytosolic PLA2 (cPLA2) mRNA and its protein were constitutively expressed and increased approximately 2-fold by IL-1alpha , but secretory PLA2 mRNA was not detected. PDGF rapidly stimulated PLA2 activity, which was blocked completely by a cPLA2 inhibitor (arachidonyltrifluoromethyl ketone). The PDGF-induced cPLA2 activation was accompanied by phosphorylation of its protein. These results indicate that cox-2 induction by IL-1alpha is not sufficient, but cPLA2 activation by PDGF is crucial for IL-1alpha -induced PGE2 synthesis in mouse osteoblasts.


INTRODUCTION

Prostaglandins (PGs)1 are potent regulators of bone metabolism that are produced mainly by osteoblasts in the bone tissue. Among several forms of PG, PGE2 is the major product of osteoblasts, and its production is regulated by several growth factors and cytokines including interleukin-1 (IL-1), parathyroid hormone, basic fibroblast growth factor (bFGF), and PGE2 itself (1-6). PGE2 is a complicated regulator of bone metabolism, potently stimulating bone resorption in vitro (7) and stimulating both bone formation and bone resorption in vivo (8). PGE2 production is involved in the mechanism of bone resorption induced by IL-1 in vitro (7, 9).

PG synthesis is regulated by two successive metabolic steps, the release of arachidonic acid from membranous phospholipids and its conversion to prostanoids. Phospholipase A2 (PLA2), the enzyme responsible for arachidonic acid release, consists of two forms, secretory PLA2 (sPLA2) and cytosolic PLA2 (cPLA2) (10-13). Secretory PLA2 requires millimolar levels of calcium for its activation and is divided into several groups, among which pancreatic (type I) and nonpancreatic (type II) subtypes are well characterized (10). Type II sPLA2 is widely distributed in several tissues and inflammatory exudates. On the other hand, cPLA2 is an intracellular enzyme that is found in various cells and tissues including macrophages, mast cells, platelets, kidney, and brain (10, 14, 15). This enzyme preferentially hydrolyzes arachidonic acid at the Sn2 position of membranous phospholipids and requires micromolar levels of calcium for its activation. Both the phosphorylation and Ca2+-dependent translocation of cPLA2 to the membranes are essential for its activation (16-20).

Cyclooxygenase (COX) is a rate-limiting enzyme for the conversion of arachidonic acid to prostanoids. Two COX genes, constitutive COX (cox-1) and inducible COX (cox-2), have been identified (21-24). These glycosylated enzymes have 60% homology in nucleic acid and amino acid sequences and a similar molecular mass (70-74 kDa). cox-1 is constitutively expressed in several mammalian tissues (21). In contrast, cox-2 is undetectable under physiological conditions but markedly induced by several cytokines and growth factors (2, 4, 6, 23, 24).

Both cox-1 and cox-2 are expressed in osteoblastic cells. cox-2 is the main enzyme regulating PG synthesis in response to several bone-resorbing factors such as IL-1, bFGF, and PGE2 in osteoblastic cells and mouse calvarial cultures (2-4, 6). We reported that IL-1alpha markedly stimulates the mRNA expression of cox-2, but not of cox-1, in primary osteoblastic cells and that IL-13 and IL-4 inhibit IL-1-induced bone resorption by suppressing cox-2-dependent PG synthesis (2). Kawaguchi et al. (4) reported that bFGF rapidly induces cox-2 expression in osteoblasts, which is responsible for bFGF-induced bone resorption.

PLA2 in osteoblasts, however, has not been well established. Kawaguchi et al. (25) have shown that cPLA2 mRNA is detected in neonatal mouse calvarial cultures. We reported that cPLA2 mRNA is constitutively expressed in mouse osteoblastic cells and that it is slightly but significantly increased by IL-1alpha (2).

In this study, we examined the role and regulation of PLA2 and COX in PG synthesis by primary osteoblastic cells isolated from mouse calvaria. When osteoblastic cells were incubated with IL-1alpha , both the mRNA expression and the protein level of cox-2 were greatly increased, but no PGE2 was produced. When arachidonic acid or platelet-derived growth factor (PDGF)-BB was added to osteoblastic cells precultured with IL-1alpha for 24 h, PGE2 was produced within 10 min. The rapid synthesis of PGE2 induced by PDGF was due to the activation and phosphorylation of cPLA2, indicating that the activation of cPLA2 by PDGF is essential for cox-2-dependent PGE2 synthesis in osteoblasts cultured with IL-1alpha .


MATERIALS AND METHODS

Animals and Reagents

Newborn and six-week-old male mice of the ddy, C57BL/6 and Balb/c strains, were obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan). Recombinant human IL-1alpha and epidermal growth factor (EGF) were purchased from Genzyme (Cambridge, MA). Recombinant human PDGF-BB and PDGF-AA and transforming growth factor (TGF)-alpha were obtained from Life Technologies, Inc. Recombinant human TGF-beta 1 was purchased from R&D systems (Minneapolis, MN). Recombinant bovine bFGF was obtained from Boehringer Mannheim Biochemica (Mannheim, Germany). Arachidonic acid was purchased from Serdary Research Laboratories, Inc. (London, Canada). Aspirin was obtained from Sigma. Arachidonyltrifluoromethyl ketone (AACOCF3) and NS398 were purchased from Calbiochem.

Culture of Primary Mouse Osteoblastic Cells

Primary osteoblastic cells were isolated from 1-day-old ddy strain mouse calvariae after five routine sequential digestions with 0.1% collagenase (Wako Pure Chemicals, Osaka, Japan) and 0.2% dispase (Godo Shusei, Tokyo, Japan) as described (2). Osteoblasts isolated from fractions 3-5 were combined and cultured in alpha -minimal essential medium (alpha -MEM) supplemented with 10% fetal calf serum (FCS) at 37 °C in a humidified atmosphere of 5% CO2 in air. In most experiments, osteoblastic cells were cultured for 24 h in alpha -MEM supplemented with 1% FCS and then incubated with or without IL-1alpha .

Measurement of PGE2 Content

The concentration of PGE2 in the culture medium was determined using an enzyme immunoassay (EIA) (Amersham, Aylesbury, UK). The antibody had the following cross-reactivity when calculated by the bound/free ratio: PGE2, 100%; PGE1, 7.0%; 6-keto-PGF1alpha , 5.4%; PGF2alpha , 4.3%; and PGD2, 1.0%.

Northern Blot Analysis

Total cellular RNA was extracted from osteoblastic cells using the acid guanidinium/phenol/chloroform method (2). For Northern blots, 20 µg of total RNA was resolved by electrophoresis in a 1.5% agarose-formaldehyde gel and transferred onto nylon membranes (Hybond N, Amersham Corp.) and then hybridized with a 32P-labeled cDNA probe as reported (2). The signals were densitometrically quantified using a BioImage analyzer (BAS-2000, Fuji Film, Tokyo, Japan). Mouse cox-1 and cox-2 cDNA probes were purchased from Oxford Biomedical Research, Inc. (Oxford, MI). The cDNA for cPLA2 was used as a 2.4-kilobase EcoRI fragment of mouse cPLA2 cDNA (12). The cDNA for type II sPLA2 was used as a 375-base pair EcoRI fragment of mouse type II sPLA2 cDNA (26).

Western Blot Analysis

Osteoblastic cells cultured in 60-mm dishes were lysed at 4 °C in 70 µl of 50 mM Tris-HCl (pH 7.4) containing 0.1% SDS, 0.5% Nonidet P-40 (BDH Laboratory, UK), 5 mM Na3VO4 (Wako), 50 µg/ml leupeptin (Sigma), 1.5 µM pepstatin A (Wako), and 1 µM phenylmethylsulfonyl fluoride (Wako). Proteins in the cell lysate were measured using a BCA protein assay kit (Pierce). Lysate protein (10 µg) was resolved on an SDS-polyacrylamide gel (10%; Daiichi Pure Chemicals Co., Tokyo, Japan) and transferred to a polyvinylidene difluoride membrane (Hybond-PVDF, Amersham Corp.). The membrane was incubated for 18 h with 5% skim milk in phosphate-buffered saline without calcium and magnesium containing 0.1% Tween (Polysciences Inc., Warrington, PA) at 4 °C to block nonspecific binding and further incubated for 2 h with polyclonal rabbit anti-cox-1 antibody (donated from Dr. W. L. Smith, Michigan State University, East Lansing, MI), anti-cox-2 antibody (Cayman Chemical Co., Ann Arbor, MI), or anti-cPLA2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:5000, 1:1000, and 1:100, respectively. After incubation with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Corp.) (1:5000 dilution) for 1 h, immunoreactive bands were stained by an enhanced chemiluminescence Western blot analysis system (Amersham Corp.).

Measurement of PLA2 Activity

Osteoblastic cells were washed once with ice-cold phosphate-buffered saline without calcium and magnesium and suspended in 1 ml of 10 mM Tris-HCl (pH 7.4) containing 1 mM EDTA, 250 mM sucrose, and 1 µM phenylmethylsulfonyl fluoride, and sonicated to make a cell lysate. Cell lysate protein (2 µg) was assayed for PLA2 activity using 2 µM 1-palmitoyl-2-[14C]arachidonyl-phosphatidylethanolamine (DuPont NEN) as a substrate in 250 µl of 50 mM Tris-HCl (pH 9.0) containing 4 mM CaCl2 (27). In some experiments, 1-palmitoyl-2-[14C]linoleoyl-phosphatidylethanolamine (Amersham Corp.) was also used as a substrate. After an incubation at 37 °C for 20 min, the reaction was stopped by adding 1.25 ml of Dole's reagent, and the amount of hydrolyzed [14C]arachidonic acid was measured as described (27).

Genomic Analysis of Type II sPLA2 Gene

DNA used in the genomic analysis of mouse type II sPLA2 gene was prepared from the brains of ddy, Balb/c and C57BL/6 mice. DNA (10 µg) was digested with BamHI, fractionated on a 0.8% agarose gel, and transferred to a Nylon membrane (Pall BioSupport, East Hill, NY) and Southern blotted as reported (28). The membrane was hybridized with 32P-labeled cDNA probe for mouse type II sPLA2 for 18 h at 42 °C in 40% formamide, 5 × SSC, 10 × Denhardt's solution, 250 µg/ml denatured salmon testicular DNA, and 1% SDS. The membrane was washed twice at 55 °C in 0.1 × SSC containing 0.1% SDS, and exposed to an x-ray film.


RESULTS

cox-2 Induction Is Not Sufficient for the IL-1alpha -induced PG Synthesis in Mouse Osteoblastic Cells

We reported that IL-1alpha markedly stimulates cox-2 mRNA expression in primary mouse osteoblastic cells (2). When mouse osteoblastic cells were cultured in alpha -MEM containing 1% FCS for 24 h and then incubated with 2 ng/ml IL-1alpha , the cytokine markedly enhanced cox-2 mRNA expression at 3 h and its protein levels at 24 h (Fig. 1, A and B). cox-1 mRNA and its protein were weakly expressed in osteoblastic cells, but they were not appreciably changed by IL-1alpha . To examine the effects of IL-1alpha on PGE2 production, we measured the concentration of PGE2 in the conditioned media collected at 24 h. Despite the marked stimulation of cox-2 mRNA expression, IL-1alpha did not stimulate PGE2 production. However, when 1 µM arachidonic acid was added to osteoblastic cells precultured with IL-1alpha for 24 h, the PGE2 levels were greatly increased within 10 min (Fig. 1C). Arachidonic acid also slightly stimulated PGE2 synthesis in the control cultures without IL-1alpha . These results suggest that cox-2 induction is not sufficient and that the simultaneous addition of arachidonic acid is essential for the IL-1alpha -induced PGE2 synthesis.


Fig. 1. cox-2 induction by IL-1alpha is not sufficient for PGE2 production in mouse osteoblastic cells. A, to detect cox-1 and cox-2 mRNAs, mouse osteoblastic cells were cultured for 24 h in alpha -MEM containing 1% FCS and then with 2 ng/ml IL-1alpha . At 3 h, total RNA was extracted and Northern blotted using 32P-labeled cox-1, cox-2, and tubulin cDNA probes. B, to detect cox-1 and cox-2 proteins under the same conditions shown in A, cell lysates were extracted from osteoblastic cells cultured with IL-1alpha for 24 h. Lysate protein (10 µg) was resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted using anti-cox-1 and anti-cox-2 antibodies as described under "Materials and Methods." C, to examine PGE2 production, osteoblastic cells were cultured under the same conditions shown in B. At the end of culture, 1 µM arachidonic acid or vehicle (ethanol) was added to the medium, and the conditioned media were collected 10 min thereafter. Concentrations of PGE2 in the conditioned media were measured by EIA. Data are expressed as the means ± S.D. of 3-6 independent experiments.
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Rapid Induction of PG Synthesis by Exogenous Arachidonic Acid or PDGF

Fig. 2A shows the time course of change in the rapid synthesis of PGE2 induced by arachidonic acid. After osteoblastic cells were cultured for 24 h with or without IL-1alpha in alpha -MEM containing 1% FCS, they were washed twice with alpha -MEM and incubated for 2-10 min in alpha -MEM containing 1 µM arachidonic acid. PGE2 synthesis was induced within 10 min. Cells cultured without IL-1alpha also synthesized PGE2 in the presence of arachidonic acid but to a lesser extent than that in IL-1alpha -treated cells. These results suggest that cox-2 induced by IL-1alpha rapidly converts exogenous arachidonic acid into PGE2 in osteoblastic cells.


Fig. 2. Effects of arachidonic acid and growth factors on the rapid induction of PGE2 synthesis in mouse osteoblastic cells. A, time course of PGE2 production induced by arachidonic acid in osteoblastic cells cultured for 24 h in the presence or absence of IL-1alpha (2 ng/ml). After culture for 24 h in alpha -MEM containing 1% FCS with (bullet ) or without (open circle ) IL-1alpha , cells were washed twice with alpha -MEM and then incubated with 1 µM arachidonic acid for 2-10 min. B, effects of various growth factors on the rapid induction of PGE2 synthesis were examined under the same conditions as shown in A. After culture for 24 h with or without IL-1alpha , cells were washed and incubated for 10 min with PDGF-BB (100 ng/ml), PDGF-AA (100 ng/ml), EGF (100 ng/ml), TGF-alpha (100 ng/ml), TGF-beta (10 ng/ml), bFGF (100 ng/ml), or vehicle (alpha -MEM). Inset, time course of PGE2 production induced by PDGF-BB (100 ng/ml) was examined under the same conditions as shown in A. PGE2 levels in the conditioned media were measured by EIA. Data are expressed as means ± S.D. of 3-6 cultures.
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To identify the factor(s) that increases the endogenous level of arachidonic acid for PG synthesis, we examined the effects of various growth factors and cytokines that may be involved in the PG synthesis by osteoblastic cells. As shown in Fig. 2B, PDGF-BB markedly and specifically stimulated the rapid synthesis of PGE2 within 10 min. PDGF-BB affected the rapid synthesis of PGE2 in a time- (2-10 min) and dose (0.1-100 ng/ml) -dependent manner (Fig. 2B; data not shown). The potency of 100 ng/ml PDGF-BB was identical to that of 1 µM arachidonic acid in PG synthesis. PGE2 synthesis was also enhanced by PDGF-AA and TGF-alpha as well, but the effects were marginal. Other growth factors such as EGF, TGF-beta , and bFGF did not affect the rapid induction of PG synthesis within 10 min. These results indicate that PDGF-BB mimics the effect of exogenous arachidonic acid upon the rapid synthesis of PGE2 by mouse osteoblastic cells.

PDGF Stimulates PLA2 Activity in Mouse Osteoblastic Cells

PG synthesis is regulated by the release of arachidonic acid from membranous phospholipids by PLA2 and its conversion into PGH2, a precursor of PGs, by COX. To examine the effect of PDGF-BB on PG synthesis, we measured PLA2 activity by the release of [14C]arachidonic acid from 1-palmitoyl-2-[14C]arachidonyl-phosphatidylethanolamine using osteoblastic cell lysates. PLA2 activity was doubled upon PDGF exposure for 10 min in the control group cultured for 24 h (Fig. 3). When osteoblastic cells were cultured for 24 h with IL-1alpha , PLA2 activity was increased to a higher level than in the control culture, and it was further stimulated by PDGF-BB (Fig. 3).


Fig. 3. Effect of PDGF on PLA2 activity in mouse osteoblastic cells. After culture for 24 h in alpha -MEM containing 1% FCS with or without IL-1alpha (2 ng/ml), cells were washed twice with alpha -MEM and then incubated with PDGF-BB (100 ng/ml) or vehicle (alpha -MEM) for 10 min. PLA2 activity was assayed in 2 µg of cell lysate protein. Using 2 µM 1-palmitoyl-2-[14C]arachidonyl-phosphatidylethanolamine as the substrate, the release of [14C]arachidonic acid was detected as described under "Materials and Methods." Data are expressed as the means ± S.D. of four independent experiments.
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In Mouse Osteoblastic Cells cPLA2 Is Involved in PG Synthesis

Type II sPLA2 and cPLA2 are involved in the production of eicosanoids in various cells and tissues (29-32). We determined which PLA2 is the enzyme responsible for PGE2 synthesis in mouse osteoblastic cells by means of Northern and Western blot analysis. Fig. 4A shows that cPLA2 mRNA was constitutively expressed in mouse osteoblastic cells, and it increased at 6 h in the presence of IL-1alpha . Concomitantly, cPLA2 protein was detected in osteoblastic cells, and the level was almost doubled at 24 h in the presence of IL-1alpha (Fig. 4B). On the other hand, no sPLA2 mRNA was detected in mouse osteoblastic cells by Northern blot analysis (Fig. 5B) and reverse transcription polymerase chain reaction (data not shown).


Fig. 4. Effects of IL-1alpha on the expression of cPLA2 mRNA and its protein levels in mouse osteoblastic cells. Osteoblastic cells were cultured for 24 h in alpha -MEM containing 1% FCS with or without IL-1alpha (2 ng/ml). A, to detect cPLA2 mRNA, total RNA was extracted at 6 and 24 h and Northern blotted using 32P-labeled cDNA probe of cPLA2 as described under "Materials and Methods." B, to detect cPLA2 protein, cell lysates were extracted from osteoblastic cells at 6 and 24 h, and then 10 µg of cell lysate protein was resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted using anti-cPLA2 antibody as described under "Materials and Methods."
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Fig. 5. Genomic analysis of mouse sPLA2 and the lack of sPLA2 mRNA expression in mouse osteoblastic cells. A, genomic analysis of sPLA2 gene was performed using Balb/c, C57BL/6, and ddy strains of mice. DNA was prepared from the brains of each strain. Genomic DNA (10 µg) was digested with BamHI and Southern blotted using the 32P-labeled cDNA probe of mouse sPLA2 described under "Materials and Methods." +/+, normal sPLA2 gene; -/-, disrupted sPLA2 gene. B, expression of sPLA2 and cPLA2 mRNAs in mouse osteoblastic cells derived from three strains of mice. After culture for 24 h with IL-1alpha (2 ng/ml) in alpha -MEM containing 1% FCS, total RNA was extracted from osteoblastic cells of various mouse strains and Northern blotted using 32P-labeled cDNA probes of sPLA2 and cPLA2. C, PDGF-dependent rapid synthesis of PGE2 was measured in mouse osteoblastic cells cultured with IL-1alpha . After culture for 24 h with IL-1alpha (2 ng/ml) in alpha -MEM containing 1% FCS, cells were washed twice with alpha -MEM and then treated with PDGF-BB (100 ng/ml) or vehicle (alpha -MEM) for 10 min. PGE2 levels in the conditioned media were measured by EIA. Data are expressed as the means ± S.D. of 3-6 cultures.
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The murine sPLA2 gene is naturally disrupted in some inbred mouse strains such as C57BL/6 (28, 33). Therefore, we performed genomic analysis of sPLA2 in ddy mice which was used in this study. C57BL/6 mice had a disrupted sPLA2 genotype but that of Balb/c mice was normal, as reported (28, 33). Ddy mice had a normal sPLA2 genotype (Fig. 5A). In accordance with the sPLA2 genotype, sPLA2 mRNAs were expressed at high levels in the small intestine of Balb/c and ddy mice but not in that of C57BL/6 (data not shown). We did not detect sPLA2 mRNA in osteoblastic cells from C57BL/6, Balb/c, and ddy mice. In contrast, cPLA2 mRNA was expressed in osteoblastic cells from all of the mouse strains tested (Fig. 5B). Despite differences in the sPLA2 genotype, the osteoblastic cells from all of the strains of mice similarly responded to IL-1alpha and PDGF in PG synthesis (Fig. 5C), suggesting that cPLA2 is the enzyme responsible for arachidonic acid release in mouse osteoblastic cells. To confirm this notion, we used 1-palmitoyl-2-[14C]arachidonyl-phosphatidylethanolamine and 1-palmitoyl-2-[14C]linoleoyl-phosphatidylethanolamine as a substrate and compared PLA2 activities in osteoblastic cells (Table I). It is reported that cPLA2, but not sPLA2, preferentially hydrolyzes arachidonic acid at the Sn2 position of phospholipids (12-15). As shown in Table I, arachidonyl-phosphatidylethanolamine was a selective substrate for the PLA2 activity in mouse osteoblastic cells. Furthermore, the PLA2 activity was completely inhibited by 1 µM AACOCF3, a competitive inhibitor of cPLA2 (Table I) and by EDTA (data not shown). These results indicate that cPLA2 is selectively expressed and is the enzyme responsible for arachidonic acid release in mouse osteoblastic cells.

Table I.

Characterization of PLA2 activity in mouse osteoblastic cells

Osteoblastic cells were cultured for 24 h in alpha -MEM containing 1% FCS with or without IL-1alpha (2 ng/ml), washed twice with alpha -MEM, and then incubated for 10 min with vehicle (alpha -MEM) or PDGF-BB (100 ng/ml). Using 2 µM 1-palmitoyl-2-[14C]arachidonyl-phosphatidylethanolamine (Ara-PE) and 1-palmitoyl-2-[14C]linoleoyl-phosphatidylethanolamine (Lino-PE) as a substrate, PLA2 activity was measured as described under "Materials and Methods." To examine the effects of AACOCF3 on PLA2 activity, [14C]Ara-PE was used as the substrate and AACOCF3 (1 µM) was added to the cell lysate 30 min before the substrate. Data are expressed as the means ± S.D. of four independent experiments. UD, undetectable.
Treatment
PLA2 activity
24 h 10 min Ara-PE Lino-PE Ara PE + AACOCF3 (1 µM)

pmol/min/mg protein
Vehicle Vehicle 103  ± 6 11  ± 9 UD
Vehicle PDGF 203  ± 4 19  ± 9 UD
IL-1alpha Vehicle 171  ± 19 18  ± 5 UD
IL-1alpha PDGF 415  ± 30 36  ± 11 UD

PDGF Stimulates Phosphorylation of cPLA2 in Mouse Osteoblastic Cells

The experimental results described above suggest that PDGF rapidly activates cPLA2 in mouse osteoblastic cells. It appears that the cPLA2 activation is associated with increased phosphorylation of the protein, which causes a decreased mobility in SDS-polyacrylamide gel electrophoresis (16-18). To determine if the PDGF-induced rapid stimulation of PLA2 activity is accompanied by the mobility change in the cPLA2 protein, we performed Western blot analyses. Exposing osteoblastic cells to PDGF for 10 min decreased the mobility of cPLA2 (Fig. 6), suggesting that PDGF stimulates phosphorylation of this protein. IL-1alpha did not affect the phosphorylation of cPLA2 by PDGF (Fig. 6). These results suggest that PDGF phosphorylates and activates cPLA2, which in turn triggers arachidonic acid release.


Fig. 6. PDGF induces cPLA2 phosphorylation in mouse osteoblastic cells. After culture for 24 h with or without IL-1alpha (2 ng/ml) in alpha -MEM containing 1% FCS, osteoblastic cells were washed twice with alpha -MEM and then incubated with PDGF-BB (100 ng/ml) or vehicle (alpha -MEM) for 10 min. At the end of culture, lysates were extracted from osteoblastic cells, and 10 µg of protein was resolved by SDS-polyacrylamide gel electrophoresis for 5 h at 20 mA and then immunoblotted using anti-cPLA2 antibody as described under "Materials and Methods."
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Effects of COX Inhibitors on the PG Synthesis Regulated by PDGF and IL-1alpha in Mouse Osteoblastic Cells

COX is essential for the conversion of arachidonic acid into prostanoids. As described in Fig. 1, mouse osteoblastic cells expressed both cox-1 and cox-2 mRNA and proteins, but IL-1alpha preferentially stimulated cox-2 expression. To study the contribution of cox-1 and cox-2 to IL-1alpha -induced PGE2 synthesis in osteoblasts, we examined the effects of COX inhibitors on the rapid induction of PGE2 synthesis by PDGF in IL-1alpha -treated osteoblastic cells. When cells were preincubated for 24 h with aspirin to block preexistent cox-1 and cox-2 activities, washed, and further cultured for 24 h with or without IL-1alpha , the PDGF-dependent rapid synthesis of PGE2 was partially suppressed (Fig. 7). In contrast, NS-398, a selective inhibitor of cox-2, added 3 h before IL-1alpha , completely blocked the PGE2 production (Fig. 7). These results indicate that the induction of cox-2 by IL-1alpha is also critical for the rapid synthesis of PGE2 triggered by PDGF.


Fig. 7. Effects of COX inhibitors on the rapid synthesis of PGE2 regulated by IL-1alpha and PDGF in mouse osteoblastic cells. After culture for 24 h with or without IL-1alpha (2 ng/ml) in alpha -MEM containing 1% FCS, osteoblastic cells were washed twice with alpha -MEM and then incubated with PDGF-BB (100 ng/ml) for 10 min. Cells were incubated with aspirin (1 µg/ml) for 24 h before IL-1alpha , washed twice with alpha -MEM, and cultured with or without IL-1alpha . NS-398 (1 µM) was added 3 h before IL-1alpha and then cells were cultured with or without IL-1alpha in the presence of NS-398. All cells were incubated with PDGF for 10 min at the end of culture. The PGE2 level in the conditioned media was measured by EIA. Data are expressed by the means ± S.D. of 3-6 independent experiments.
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DISCUSSION

The present study indicates that cox-2 induction is not sufficient but that cPLA2-dependent arachidonic acid release is essential for the PGE2 synthesis induced by IL-1alpha . Arachidonic acid release was mediated by cPLA2 but not sPLA2, and the activation of cPLA2 accompanying its phosphorylation was crucial for its function in mouse osteoblastic cells. We also found that PDGF-BB markedly stimulated the phosphorylation and activation of cPLA2, which triggered the rapid induction of PGE2 synthesis in mouse osteoblastic cells. Raisz and his co-authors (4) have reported that osteoblasts cultured in serum-free medium do not produce PGs although cox-2 is induced by cytokines such as bFGF. We reproduced their results. Furthermore, the activation of cPLA2 was essential for the PGE2 synthesis induced by IL-1alpha in mouse osteoblastic cells.

PLA2 is the enzyme responsible for the release of arachidonic acid, and it consists of low molecular mass (14 kDa) sPLA2 and high molecular mass (60-110 kDa) cPLA2 (10-13). The former is linked to prolonged PG synthesis stimulated by cytokines in various cells such as mesangial cells and macrophage-like P388 D1 cells (29, 30). In this study, we did not detect sPLA2 mRNA expression in mouse osteoblastic cells collected from the ddy strain of mice. The sPLA2 gene is naturally disrupted in some inbred strains of mice such as C57BL/6, 129/Sv, and A/J (28, 33). Therefore, it is possible that the absence of sPLA2 mRNA in osteoblastic cells was due to natural disruption of the gene in ddy mice. However, the sPLA2 gene was normal and sPLA2 mRNA was expressed at high levels in the small intestine of ddy mice (Fig. 5). MacPhee et al. (33) have reported that the sPLA2 gene is a major candidate for the mom1 locus, which is closely associated with the development of intestinal tumors and characterized in multiple intestinal neoplasia strain of mice. They also showed that there is 100% concordance between the sPLA2 genotype and tumor susceptibility. This suggests that the disruption of sPLA2 could develop intestinal polyps and tumors. In this study, sPLA2 was not detected in mouse osteoblastic cells, but PGE2 synthesis was similarly induced by IL-1alpha and PDGF in all of the strains of mice tested (Balb/c, C57BL/6, and ddy). These observations suggest that there is a difference in the gene regulation and biological significance of sPLA2 between intestine and bone.

Cytosolic PLA2 is an arachidonyl-selective PLA2 (14, 15). It is suggested that calcium (Ca2+)-dependent translocation to the membrane is essential for the functional activation of cPLA2. Clark et al. (12) reported that cPLA2 possesses a Ca2+-dependent phospholipid-binding domain at the amino terminus of cPLA2. In addition, Lin et al. (17) reported that the activation of PLA2 is associated with the increased phosphorylation of its protein. The phosphorylation of cPLA2 is thought to be mediated by mitogen-activated protein kinase and/or protein kinase C (17, 34). The phosphorylation and translocation of cPLA2 to the membranes are important in the arachidonic acid release and PG synthesis in several cells, for example, macrophages regulated by colony-stimulating factor 1 (35), platelets by thrombin (18), smooth muscle cells by PDGF (32), and endothelial cells by bFGF (36). The present study showed that the activity and phosphorylation of cPLA2 are stimulated by PDGF in mouse osteoblastic cells. It has been reported that PDGF stimulates calcium influx and elevates the level of intracellular calcium in mouse osteoblastic cells (37). When mouse osteoblastic cells were treated with thapsigargin for 1 h which has the capacity to deplete intracellular calcium (32), or EDTA for 10 min, PDGF-induced PGE2 synthesis was significantly suppressed (data not shown). The activity of cPLA2 induced by PDGF was also completely abolished by EDTA (data not shown). These results suggest that PDGF induces functional activation of cPLA2 in mouse osteoblastic cells and that both the elevation of intracellular calcium and the phosphorylation of the cPLA2 protein are indeed involved in the process of the activation. IL-1 activates cPLA2 for the arachidonic acid release and also increases its protein level in some cells, such as rat mesangial cells and human fibroblasts (31, 38, 39). We also found that IL-1alpha increases cPLA2 protein levels in mouse osteoblastic cells (Fig. 4). However, IL-1alpha induced neither rapid stimulation of cPLA2 activity nor its phosphorylation in mouse osteoblastic cells (data not included). Furthermore, the increase in cPLA2 protein by IL-1alpha was not accompanied by concomitant PGE2 synthesis until 24 h, despite the marked induction of cox-2. Therefore, we concluded that IL-1alpha does not induce PGE2 synthesis without PDGF.

Conversion of arachidonic acid into PGH2 is mediated by cox-1 and cox-2. Mouse osteoblastic cells expressed both cox-1 and cox-2, but IL-1alpha specifically stimulated cox-2 expression. The rapid induction of PGE2 synthesis induced by PDGF in osteoblastic cells cultured with IL-1alpha was markedly suppressed by NS-398, a selective inhibitor of cox-2. These results indicate that the endogenous arachidonic acid released by PDGF-dependent activation of cPLA2 is essential for IL-1alpha -induced PGE2 synthesis. Morita et al. (40) examined the subcellular location of cox-1 and cox-2 in mouse 3T3 fibroblasts using quantitative confocal immunofluorescence microscopy studies. They found that both cox-1 and cox-2 were distributed in the endoplasmic reticulum (ER) and nuclear envelope (NE) but not in the plasma membrane. Glover et al. (19) and Schievella et al. (20) have shown that cPLA2 is translocated into ER and NE, but not into the plasma membrane, through a Ca2+-dependent mechanism. These results suggest that arachidonic acid is produced and metabolized at the NE and ER. Murakami et al. (41) reported that there is a linkage between cPLA2 and cox-1 in c-kit ligand-primed, IgE-dependent PGD2 synthesis in mouse mast cells. A linkage between cPLA2 and cox-2 has also been suggested in monocytes and fibroblasts (42, 43). Therefore, it is likely that PG synthesis is mediated by cPLA2 and COX cooperatively, both of which may be co-localized in NE and ER.

PG synthesis in osteoblasts is regulated by several factors including IL-1, parathyroid hormone, bFGF, TGFs, and PGE2 itself (1-6). Studies have indicated that cox-2 is the major enzyme regulating PG synthesis in osteoblasts. Cytokines such as IL-1 and bFGF greatly stimulated cox-2-dependent PGE2 production in mouse long bone and calvarial cultures (2, 4). However, osteoblastic cells cultured in serum-free medium or medium containing only 1% FCS did not produce PGE2 despite the marked induction of cox-2 by these cytokines (Fig. 1, Refs. 2 and 4). Preliminary experiments showed that IL-1alpha markedly stimulated PGE2 production, when osteoblastic cells were cultured in medium containing 10% FCS (data not shown, Refs. 6 and 44). Adding 10% FCS significantly stimulated PLA2 activity in cultures of mouse osteoblastic cells (data not shown). The discrepancy in PG synthesis between organ and cell culture may be due to the presence or absence of a bone marrow-derived growth factor(s) that activates cPLA2 to release arachidonic acid. To identify the factor(s) in bone marrow that activates cPLA2 needs further investigation.

Several lines of evidence have suggested that PDGF is an important regulator of bone metabolism. PDGF is stored in bone matrix (45) and stimulates both bone formation and bone resorption (46-48). PDGF is a major growth factor in serum. However, the fact that PDGF is produced by human osteoblasts and mouse bone marrow-derived stromal cells (49, 50) indicates that PDGF is also a local factor produced by bone cells. The present study showed that PDGF is a potent stimulator of PGE2 synthesis in osteoblasts. Bone resorption induced by PDGF is suppressed by indomethacin (48). Thus, we speculate that PDGF is an important local regulator of bone metabolism by activating cPLA2 in bone.

In conclusion, IL-1alpha -induced cox-2 induction is not sufficient for PGE2 production by osteoblasts. The rapid activation of cPLA2 by PDGF is essential for cox-2-dependent PGE2 synthesis. The two-step regulation of PGE2 synthesis, cPLA2 activation by PDGF, and cox-2 induction by IL-1alpha will help understand the role of PGE2 in several metabolic bone diseases.


FOOTNOTES

*   This work was supported by Grants-in-Aid 06404067 and 05671552 from the Ministry of Science, Education and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence and reprint requests should be addressed: Dept. of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan. Tel.: 813-3784-8162; Fax: 813-3784-5555.
1    The abbreviations used are: PG, prostaglandin; IL-1, interleukin-1; bFGF, basic fibroblast growth factor; PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; COX, cyclooxygenase; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; TGF, transforming growth factor; FCS, fetal calf serum; AACOCF3, arachidonyltrifluoromethyl ketone; ER, endoplasmic reticulum; NE, nuclear envelope; EIA, enzyme immunoassay; alpha -MEM, alpha -minimal essential medium.

Acknowledgment

We thank Dr. W. L. Smith (Michigan State University, East Lansing, MI) for his generous gift of antibody for cox-1.


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