PROSTAGLANTlllWJXJKOTRlENES ANDESS-FATTPACIDS Prostaglandms Leukotrienes and Eswxi.4 0 Longman Group UK Ltd ,992
Fatty Acids (1992) 46. S-295
Mechanism of Prostaglandin EJnduced Arachidonic Acid Release in Osteoblast-like Cells: Independence from Phosphoinositide Hydrolysis 0. Kozawa, H. Tokuda*, M. Miwa*, Y. Takahashi”,
N. Ozaki* and Y. Oiso*
Department of Biochemistry. Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan and *First Department of Internal Medicine, Nagoya Universio School of Medicine, Nagoya 466, Japan (Reprint requests to OK) ABSTRACT.
We previously reported that pertussis toxin (PTX)-sensitive GTP-binding protein is involved in the coupling of prostaglandin E, (PGE,) receptor to phospholipase C in osteohlast-like MC3T3-El cells (1). In the present study, we analyzed the mechanism of PGE,-induced arachidonic acid (AA) release in MC3T3-El cells. PGEz stimulated the release of AA and the formation of inositol trisphosphate (IP,) dose dependently in the range between 1 nM and 10 ,uM. The effect of PGE, on AA release (ED,, was 80 nM) was more potent than that on IP, formation (ED,, was 0.8 PM). Quinacrine, a phospholipase A2 inhibitor, suppressed the PGE2induced AA release but had little effect on the IP, formation. NaF, a GTP-binding protein activator, mimicked PGE, by stimulating the AA release. The AA release stimulated by a combination of PGE, and NaF was not additive. PTX had little effect on the PGE,-induced AA release. These results strongly suggest that the AA release and the phosphoinositide hydrolysis are separately stimulated by PGE, in osteoblast-like cells, and the PGE,-induced AA release is mediated by PTX-insensitive GTP-binding protein.
INTRODUCTION
It is well known that prostaglandins are synthesized from arachidonic acid (AA), which is released from the esterified stores of phospholipids (4). Two major pathways of AA release are generally accepted (4). One is the activation of phospholipase A2 which causes liberation of AA directly from the stores of phospholipids, and the other is the sequential PI hydrolysis by phospholipase C and glycerol lipases. In addition, accumulating evidence suggests that GTP-binding protein is coupled to phospholipase A2 as well as phospholipase C (15). While, it has been reported that PGE? is a major eicosanoid product in osteoblasts including MC3T3-El cells (5,16), and that PGE, synthesis is induced by several exogenous stimuli such as epidermal growth factor (16), tumor necrosis factor (17), transforming growth factors ( 18) and platelet-derived growth factor (19). Thus, it is considerd that PGE? plays an important role as an autocrine and/or paracrine factor in osteoblasts. However, the precise mechanism of the AA release and the autocrine mechanism of prostaglandins in osteoblasts remains unclear. In this study, we investigated the mechanism of PGE,-induced AA release in osteoblast-like MC3T3-El cells. Our results suggest that PGE, stimulates AA release and PI hydrolysis via independent pathways and PTX-insensitive GTP-binding protein is involved in the PGEz-induced AA release in osteoblast-like cells.
Prostaglandins (PGs), which are generally accepted to act through their binding to specific receptors (2), are considered to be important modulators of osteoblasts as autacoids (3, 4). Among them, prostaglandin E2 (PGE2) is known as a potent bone resorbing agent (5). In osteoblasts, evidence is accumulating that the effects of PGE2 are mediated through both CAMP production and phosphoinositide (PI) hydrolysis (6-9). It is generally accepted that, in response to a variety of agonists, phosphoinsitides are hydrolyzed by phospholipase C, resulting in the formation of diacylglycerol and inositol phosphates. Among these products, diacylglycerol and inositol trisphosphate (IP,) serve as messengers for the activation of protein kinase C and the mobilization of intracellular Ca2+, respectively (10, 1 l), In addition, it is well known that GTP-binding protein(s) is involved in the coupling of the receptor to phospholipase C as well as the adenylate cyclase-CAMP system (12). In a previous report (1). we have shown that pertussis toxin (PTX)-sensitive GTP-binding protein is involved in PGE,-induced PI hydrolysis in osteoblast-like MC3T3El cells derived from newborn mouse calvaria (13, 14). Date received 8 January 1992 Date accepted 10 February 1992 291
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and Essential Fatty Acids
MATERIALS AND METHODS Materials [5, 6, 8, 9, 11, 12, 14, 15-3H]AA (76 Ci/mmol) was obtained from Du Pont/NEN. myo-[2-‘H]Inositol (81.5 Ci/mmol) was purchased from Amersham. PGE?, NaF and quinacrine were purchased from Sigma. PTX was purchased from Funakoshi Pharmaceutical Co, Tokyo, Japan. Other materials and chemicals were obtained from commercial sources. Cell culture Cloned osteoblast-like cells, MC3T3-El, were generously provided by Dr M. Kumegawa (Meikai University, Sakado, Japan) and maintained in a-minimum essential medium (a-MEM) containing 10% fetal calf serum (FCS) at 37 “C in a humidified atmosphere of 5% CO,! 95% air. The cells (5 x 104) were seeded into 35 mmdiameter dishes in 2 ml of a-MEM containing 10% FCS. After 4 days, the medium was exchanged for 2 ml of a-MEM containing 0.3% FCS. The cells were used for experiments 48 h thereafter. For the measurement of the formation of IP,, the medium was exchanged for 2 ml of inositol-free a-MEM containing 0.3% FCS. When indicated, the cells were pretreated with quinacrine for 20 min or PTX for 24 h in 1 ml of a-MEM prior to the stimulation by PGE2.
Measurement of AA release The cultured cells were labeled with [3H]AA (0.3 @I dish) for 24 h. The medium was then removed and the cells were washed with 1 ml of buffer A (10 mM 4-(2hydroxyethyl)-1 -piperazineethanesulfonic acid, pH 7.4, containing 135 mM NaCl, 5 mM KCl, 1 mM MgSO, and 1 mM CaCl,) four times. The cells were preincubated subsequently in 1 ml of buffer A containing 0.1% essentially fatty acid-free bovine serum albumin (BSA) at 37 “C for 20 min, then the cells were stimulated by various doses of PGE, or NaF. After the indicated periods, the medium was collected and the radioactivity of the medium was determined.
eluted from the column with 8 ml of 0.1 M formic acid containing 1 M ammonium formate (20, 21).
Determination The radioactivity of 3H-samples was determined with an Aloka LSC 3 100 liquid scintillation spectrometer.
Statistical analysis All data are presented as the mean + SD of triplicate determinations. The data were analyzed by Student’s t-test.
RESULTS Effect of PGE, on AA release and IP, formation PGE, stimulated both the release of AA and IP, formation time dependently in osteoblast-like MC3T3-El cells (Figs 1 & 4). The AA release increased gradually up to 30 min, whereas the formation of IP, reached a plateau within 10 min and sustained it up to 30 min. Both the AA release and the IP, formation were stimulated by PGE, in a dose-dependent manner in the range between 1 nM and 10 FM (Fig. 2). The effect of PGE, on AA release (ED,, was 80 nM) was more potent than that on IP, formation (ED,, was 0.8 PM).
Effect of quinacrine on PGE,-induced AA release and IP, formation To elucidate whether the activation of phospholipase A2 is involved in the PGE,-induced AA release, we examined the effect of quinacrine, a phospholipase A2 inhibitor (22), on this reaction. Pretreatment with 10 PM quinacrine, which by itself had little effect on AA release, significantly inhibited the PGE,-induced AA release (Fig. 3). Quinacrine (10 PM) led to the reduction of 66% in the PGE,-induced AA release. On the other hand, quinacrine (10 PM) had little effect on the PGE,induced IP, formation (Fig. 4).
Effect of NaF on PGE,-induced AA release Measurement of IP, formation The cultured cells were labeled with myo-[3H]inositol (3 pCi/dish) for 48 h. The labeled cells were pretreated with 10 mM LiCl at 37 “C for 10 min in 1 ml of buffer A containing 0.01% BSA. The cells were then stimulated by PGE*. The reaction was terminated by 15% trichloroacetic acid. The acid supematant was treated with diethyl ether to remove the acid and neutralized with NaOH. The supematant was applied to a column of Dowex AGI-X8 formate form. To remove inositol monophosphate and inositol bisphosphate, 8 ml of 0.1 M formic acid containing 0.4 M ammonium formate was applied to the column. The radioactive IP, was then
We next examined the effect of NaF, known as a GTPbinding protein activator (12), on the PGE,-induced AA release. NaF mimicked PGE, by stimulating the AA release (Fig. 5). The effect of 40 mM NaF was very similar to that of 10 PM PGE,. The release of AA stimulated by a combination of PGE2 (10 PM) and NaF (40 mM) was not additive.
Effect of PTX on PGE,-induced AA release The pretreatment with PTX in the range between 1 nM and 1 pM had little effect on the PGE,-induced release of AA (Fig. 6).
Arachidonic
Acid Release by PGE?
PGE2
Cont.
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6
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Fig. 1 Effect of PC& on the AA release in MC3T3-El cells. The [3H]AA-labeled cells were stimulated by 10pM PGEz (0) or vehicle (0) for the indicated periods, then the release of AA was determined. Each value represents the mean + SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
0
Cont.
5
Log (PGEz), M
Fig. 2 Dose-dependent effect of PGEz on the AA release and the formation of IP, in MC3T3-El cells. The cultured cells were stimulated by various doses of PGEz for 30 min, then the release of AA (0) and the formation of IP, (fi) were determined. Values are expressed as a net increase compared with control. Each value represents the mean f SDof triplicate determinations, Similar results were obtained with two additional and different cell preparations.
Quinacrine Fig. 3 Effect of quinacrine on the PCiE,-induced AA release in MC3T3-El cells. The (‘H]AA-labeled cells were pretreated with IO PM quinacrine or vehicle for 20 min. then stimulated by 10 PM PGEz for 30 min. Each value represents the mean f SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *Significantly different from the value stimulated by PGEL without quinacrine pretreatment (p4.05).
0
10
Time
20
30
(min)
Fig. 4 Effect of quinacrine on the PGE&duced formation of IPI in MC3T3-El cells. The [‘Hlinositol-labeled cells were pretreated with IO /fM quinacrine ((1) or vehicle (0) for 20 min. then stimulated by 10 PM PGE, for the indicated periods. Each value represents the mean f SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
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and Essential Fatty Acids
DISCUSSION
Cont.
PGE2
NaF
PGE2 N+aF
Fig. 5 Effect of NaF on the PGE,-induced AA release in MC3T3-El cells. The [3H]AA-labeled cells were stimulated by 10 PM PGE2, 40 mM NaF or their combination for 30 min. Each value represents the mean f SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
-6
r-7
r----l t
"Oh+1
- Log (PTX),
g/ml
Fig. 6 Effect of PTX on the PGE,-induced AA release in MC3T3El cells. The [3H]AA-labe1ed cells were pretreated with various doses of PTX for 24 h, then stimulated by 10 PM PGE, for 30 min. Values are expressed as a net increase compared with control. Each value represents the mean _+SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
In the present study, we showed that NaF, a GTPbinding protein activator (12), mimicked PGE2 by stimulating the release of AA, and the AA release stimulated by a combination of PGE2 and NaF was not additive in osteoblast-like MC3T3-El cells. These results suggest that PGE,-induced AA release is mediated by GTP-binding protein in these cells. Moreover, we demonstrated that the PGE,-induced AA release was not affected by PTX pretreatment. It is well known that PTX catalyzes ADP-ribosylation of the a-subunit of certain GTP-binding proteins including Gi and G,, and causes uncoupling of receptor to these GTP-binding proteins (11, 23, 24). So, these evidences suggest that PTX-insensitive GTP-binding protein is involved in PGE,-induced AA release in osteblast-like MC3T3-El cells. We have previously reported that PTX-sensitive GTP-binding protein is involved in the coupling of PGE2 receptor and phospholipase C in MC3T3-El cells (1). These results might allow the speculation that PGE2 induces AA release separately from PI hydrolysis by phospholipase C in osteoblast-like MC3T3-El cells. We first compared the time-course of PGE,-induced AA release with that of PGE,-induced IP, formation in MC3T3-El cells and demonstrated that the AA release increased gradually up to 30 min, whereas the formation of IP, reached a plateau almost within 10 min and sustained it up to 30 min. Thus, it seems that the difference between both of the time-dependent curves are consistent with our notion that PGE? separately stimulates AA release and PI hydrolysis in these cells. We next compared the PGE,-induced AA release with the PGE,induced IP, formation in dose-dependency and showed that EDSo levels of PGE, affecting IP, formation were lo-fold higher than those affecting AA release. From these findings, it seems certain that PGE, stimulates AA release and PI hydrolysis via independent pathways in osteoblast-like MC3T3-El cells. In addition, we showed that quinacrine, a phospholipase A, inhibitor (22), significantly inhibited the PGE,-induced AA release in MC3T3-El cells, while quinacrine had little effect on the PGE,-induced IP, formation in these cells. Namely, quinacrine inhibited the AA release induced by PGE, without affecting the PGE,-induced PI hydrolysis. These results suggest that the major pathway of PGE,-induced AA release is the activation of phospholipase A, in these cells. Therefore, in osteoblast-like MC3T3-El cells, it is very likely that PGE, activates both phospholipase C and phospholipase AZ via distinct GTP-binding proteins, PTX-sensitive and insensitive ones, respectively. In a previous report (25), we have shown that exogenous PGE? increases DNA synthesis and decreases alkaline phosphatase activity, in turn, PGE2 promotes the proliferation and suppresses the differentiation of MC3T3-El cells, since alkaline phosphatase activity is
Arachidonic
considered to be a mature osteoblast phenotype (26). Taking into account the results in this study, it is possible that effects of PGE* are modulated by selfinduced AA release resulting in the synthesis of PGE,, which has been reported to be a major eicosanoid product in osteoblasts including MC3T3-El cells (5. 16). So, it seems that PGE, modulates osteoblast functions through the so-called positive feedback mechanism demonstrated here. In conclusion, our results strongly suggest that the AA release and the PI hydrolysis are separately stimulated by PGE, in osteoblast-like cells, and PTX-insensitive GTP-binding protein is involved in the AA release.
12.
13.
14.
15.
16.
Acknowledgement This investigation Scientific Research of Japan.
was supported in part by a Grant-in-Aid for from Ministry of Education, Science and Culture
References I. Tokuda, H., Kozawa, 0.. Yoneda, M.. Oiso, Y., Takatsuki, K., Asano,T. and Kato, K. Possible coupling of prostagiandin Ez receptor with pertussis toxin-sensitive guanine nucleotide-binding protein in osteoblast-like cells. J. Biochem. 109: 229-233, 1991. 2. Samuelsson, B., Goldyne, M., Granstrom. E.. Hamberg, M., Hammarstrom. S. and Malmsten, C. Prostaglandins and thromboxanes. Annu. Rev. Biochem. 47: 997-1029. 1978. 3. Nijweide, P. J., Burger, E. H. and Feyen J. H. M. Cells of bone: proliferation, differentiation, and hormonal regulation. Physiol. Rev. 66: 855-886, 1986. 4. Smith, W. L. The eicosanoids and their biochemical mechanism of action. Biochem. J. 259: 3 15-324. 1989. 5. Raisz, L. G. and Martin, T. J. Prostaglandins in bone and mineral metabolism. Excerpta Med. 286310, 1984. 6. Atkins, D. and Martin, T. J. Rat osteogenic sarcoma cells: effects of some prostaglandins. their metabolites and analogues on cyclic AMP production. Prostaglandins 13: 861-871, 1977. 7. Partridge, N. C.. Alcom, D., Michelangeli, V. P.. Kemp. B. E., Ryan, G. B. and Martin T. J. Functional properties of hormonally responsive cultured normal and malignant rat ostoblastic cells. Endocrinoloav 108: 213-219, 1981. 8. Hakeda, Y., Nakatani, Y., Hiram&t, M.. Kurihara, N., Tsunoi, M., Ikeda, E. and Kumegawa. M. Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells. clone MC3T3-El. J. Biochem. 97: 97-104, 1985. 9. Yamaguchi, D. T., Hahn T. J.. Beeker, T. G.. Kleeman. C. R. and Muallem, S. Relationship of CAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells. J. Biol. Chem. 263: 10745-10753, 1988. 10. Nishizuka, Y. Studies and perspectives of protein kinase C. Science 233: 305-312, 1986. II. Berridge. M. J. and Irvine, R. F. Inositol trisphosphate.
17.
18.
19.
20
21
22
23
24
25
26
Acid Release by PGEz
novel second messenger in cellular signal transduction. Nature 312: 315-321. 1984. Gilman, A. G. G proteins: transducers of receptorgenerated signals. Annu. Rev. Biochem. 56: 615-649, 1987. Kodama, H.. Amagai, Y., Sudo, H.. Kasai. S. and Yamamoto, S. Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Jpn. J. Oral Biol. 23: 899-901. 1981. Sudo, H.. Kodama. H., Amagai, Y.. Yamamoto, S. and Kasai, S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell Biol. 96: 191-198. 1983. Bimbaumer, L., Abramowitz, J. and Brown. A. M. Receptor-effector coupling by G proteins. Biochim. Biophys. Acta 103 1: 163-224, 1990. Yokota, K., Kusaka. M., Oshima. T.. Yamamoto. S.. Kurihara. N., Yoshino, T. and Kumegawa, M. Stimulation of prostaglandin Ez synthesis in cloned osteoblastic cells of mouse (MC3T3-El) by epidermal growth factor. J. Biol. Chem. 261: 4481150. 1986. Sate, K.. Kasano. F., Fujii. Y., Kawakami. M.. Tsushima. T. and Sizume. K. Tumor necrosis factor type a (cachectin) stimulates mouse osteoblast-like cells (MC3T3-El f to produce macrophage-colony stimulating activity and prostaglandin Ez. Biochem. Biophys. Res. Commun. 145: 323-329. 1987. Tashijian, A. H., Jr., Voelkel. E. F.. Lazzaro, M.. Singer, F. R.. Roberts. A. B. Derynck, R., Winkler. M. E. and Levine, L. a and B Human transforming growth factors stimulate prostaglandin production and bone resorption in cultured mouse calvaria. Proc. Nat]. Acad. Sci. USA 82: 45354538, 1985. Shupnik, M. A.. Antoniades. N. H. and Tashijian. A. H.. Jr. Platelet-derived growth factor increases prostaglandin production and decreases epidennal growth factor receptors in human osteosarcoma cells. Life Sci. 30: 347-353, 1982. Betridge. M. J., Dawson. R. M. C.. Downes. C. P., Heslop. J.P. and Irvine, R.F. Changes in the level of inositol phosphates after agonist dependent hydrolysis of membrane phosphoinositides. Biochem. J. 2 I?: 473-482. 1983. Betridge, M. J.. Heslop. J. P., Irvine, R. F. and Brown. K. D. lnositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to plateletderived growth factor. Biochem. J. 222: 195-201. 1984. Lapetina. E. G.. Billah. M. M. and Cuatrecasas, P. The phosphatidylinositol cycle and the regulation of arachidonic acid production. Nature 292: 367-369, 198 I. Ui. M. Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. Trends Pharmacol. Sci. 5: 777-279. 1984. Stemweis. P. C. and Robishaw, J. D. Isolation of two proteins with high affinity for guanine nucleotide from membranes of bovine brain. J. Biol. Chem. 259: 13806 13813. 1984. Miwa. M., Kozawa. O., Tokuda, H., Kawakubo. A.. Yoneda, M.. Oiso, Y. and Takatsuki. K. Effects of hypergravity on proliferation and differentiation of osteoblast-like cells. Bone Miner. 14: 15-25, 1991. Peck, W. A.. Birge. S. J. and Fedak, S. A. Bone cells: biochemical and biological studies after enzymatic isolation. Science 146: 1476-1477. 1964.
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Fatty Acids (1992) 46. S-295
Mechanism of Prostaglandin EJnduced Arachidonic Acid Release in Osteoblast-like Cells: Independence from Phosphoinositide Hydrolysis 0. Kozawa, H. Tokuda*, M. Miwa*, Y. Takahashi”,
N. Ozaki* and Y. Oiso*
Department of Biochemistry. Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan and *First Department of Internal Medicine, Nagoya Universio School of Medicine, Nagoya 466, Japan (Reprint requests to OK) ABSTRACT.
We previously reported that pertussis toxin (PTX)-sensitive GTP-binding protein is involved in the coupling of prostaglandin E, (PGE,) receptor to phospholipase C in osteohlast-like MC3T3-El cells (1). In the present study, we analyzed the mechanism of PGE,-induced arachidonic acid (AA) release in MC3T3-El cells. PGEz stimulated the release of AA and the formation of inositol trisphosphate (IP,) dose dependently in the range between 1 nM and 10 ,uM. The effect of PGE, on AA release (ED,, was 80 nM) was more potent than that on IP, formation (ED,, was 0.8 PM). Quinacrine, a phospholipase A2 inhibitor, suppressed the PGE2induced AA release but had little effect on the IP, formation. NaF, a GTP-binding protein activator, mimicked PGE, by stimulating the AA release. The AA release stimulated by a combination of PGE, and NaF was not additive. PTX had little effect on the PGE,-induced AA release. These results strongly suggest that the AA release and the phosphoinositide hydrolysis are separately stimulated by PGE, in osteoblast-like cells, and the PGE,-induced AA release is mediated by PTX-insensitive GTP-binding protein.
INTRODUCTION
It is well known that prostaglandins are synthesized from arachidonic acid (AA), which is released from the esterified stores of phospholipids (4). Two major pathways of AA release are generally accepted (4). One is the activation of phospholipase A2 which causes liberation of AA directly from the stores of phospholipids, and the other is the sequential PI hydrolysis by phospholipase C and glycerol lipases. In addition, accumulating evidence suggests that GTP-binding protein is coupled to phospholipase A2 as well as phospholipase C (15). While, it has been reported that PGE? is a major eicosanoid product in osteoblasts including MC3T3-El cells (5,16), and that PGE, synthesis is induced by several exogenous stimuli such as epidermal growth factor (16), tumor necrosis factor (17), transforming growth factors ( 18) and platelet-derived growth factor (19). Thus, it is considerd that PGE? plays an important role as an autocrine and/or paracrine factor in osteoblasts. However, the precise mechanism of the AA release and the autocrine mechanism of prostaglandins in osteoblasts remains unclear. In this study, we investigated the mechanism of PGE,-induced AA release in osteoblast-like MC3T3-El cells. Our results suggest that PGE, stimulates AA release and PI hydrolysis via independent pathways and PTX-insensitive GTP-binding protein is involved in the PGEz-induced AA release in osteoblast-like cells.
Prostaglandins (PGs), which are generally accepted to act through their binding to specific receptors (2), are considered to be important modulators of osteoblasts as autacoids (3, 4). Among them, prostaglandin E2 (PGE2) is known as a potent bone resorbing agent (5). In osteoblasts, evidence is accumulating that the effects of PGE2 are mediated through both CAMP production and phosphoinositide (PI) hydrolysis (6-9). It is generally accepted that, in response to a variety of agonists, phosphoinsitides are hydrolyzed by phospholipase C, resulting in the formation of diacylglycerol and inositol phosphates. Among these products, diacylglycerol and inositol trisphosphate (IP,) serve as messengers for the activation of protein kinase C and the mobilization of intracellular Ca2+, respectively (10, 1 l), In addition, it is well known that GTP-binding protein(s) is involved in the coupling of the receptor to phospholipase C as well as the adenylate cyclase-CAMP system (12). In a previous report (1). we have shown that pertussis toxin (PTX)-sensitive GTP-binding protein is involved in PGE,-induced PI hydrolysis in osteoblast-like MC3T3El cells derived from newborn mouse calvaria (13, 14). Date received 8 January 1992 Date accepted 10 February 1992 291
292
Prostaglandins
Leukotrienes
and Essential Fatty Acids
MATERIALS AND METHODS Materials [5, 6, 8, 9, 11, 12, 14, 15-3H]AA (76 Ci/mmol) was obtained from Du Pont/NEN. myo-[2-‘H]Inositol (81.5 Ci/mmol) was purchased from Amersham. PGE?, NaF and quinacrine were purchased from Sigma. PTX was purchased from Funakoshi Pharmaceutical Co, Tokyo, Japan. Other materials and chemicals were obtained from commercial sources. Cell culture Cloned osteoblast-like cells, MC3T3-El, were generously provided by Dr M. Kumegawa (Meikai University, Sakado, Japan) and maintained in a-minimum essential medium (a-MEM) containing 10% fetal calf serum (FCS) at 37 “C in a humidified atmosphere of 5% CO,! 95% air. The cells (5 x 104) were seeded into 35 mmdiameter dishes in 2 ml of a-MEM containing 10% FCS. After 4 days, the medium was exchanged for 2 ml of a-MEM containing 0.3% FCS. The cells were used for experiments 48 h thereafter. For the measurement of the formation of IP,, the medium was exchanged for 2 ml of inositol-free a-MEM containing 0.3% FCS. When indicated, the cells were pretreated with quinacrine for 20 min or PTX for 24 h in 1 ml of a-MEM prior to the stimulation by PGE2.
Measurement of AA release The cultured cells were labeled with [3H]AA (0.3 @I dish) for 24 h. The medium was then removed and the cells were washed with 1 ml of buffer A (10 mM 4-(2hydroxyethyl)-1 -piperazineethanesulfonic acid, pH 7.4, containing 135 mM NaCl, 5 mM KCl, 1 mM MgSO, and 1 mM CaCl,) four times. The cells were preincubated subsequently in 1 ml of buffer A containing 0.1% essentially fatty acid-free bovine serum albumin (BSA) at 37 “C for 20 min, then the cells were stimulated by various doses of PGE, or NaF. After the indicated periods, the medium was collected and the radioactivity of the medium was determined.
eluted from the column with 8 ml of 0.1 M formic acid containing 1 M ammonium formate (20, 21).
Determination The radioactivity of 3H-samples was determined with an Aloka LSC 3 100 liquid scintillation spectrometer.
Statistical analysis All data are presented as the mean + SD of triplicate determinations. The data were analyzed by Student’s t-test.
RESULTS Effect of PGE, on AA release and IP, formation PGE, stimulated both the release of AA and IP, formation time dependently in osteoblast-like MC3T3-El cells (Figs 1 & 4). The AA release increased gradually up to 30 min, whereas the formation of IP, reached a plateau within 10 min and sustained it up to 30 min. Both the AA release and the IP, formation were stimulated by PGE, in a dose-dependent manner in the range between 1 nM and 10 FM (Fig. 2). The effect of PGE, on AA release (ED,, was 80 nM) was more potent than that on IP, formation (ED,, was 0.8 PM).
Effect of quinacrine on PGE,-induced AA release and IP, formation To elucidate whether the activation of phospholipase A2 is involved in the PGE,-induced AA release, we examined the effect of quinacrine, a phospholipase A2 inhibitor (22), on this reaction. Pretreatment with 10 PM quinacrine, which by itself had little effect on AA release, significantly inhibited the PGE,-induced AA release (Fig. 3). Quinacrine (10 PM) led to the reduction of 66% in the PGE,-induced AA release. On the other hand, quinacrine (10 PM) had little effect on the PGE,induced IP, formation (Fig. 4).
Effect of NaF on PGE,-induced AA release Measurement of IP, formation The cultured cells were labeled with myo-[3H]inositol (3 pCi/dish) for 48 h. The labeled cells were pretreated with 10 mM LiCl at 37 “C for 10 min in 1 ml of buffer A containing 0.01% BSA. The cells were then stimulated by PGE*. The reaction was terminated by 15% trichloroacetic acid. The acid supematant was treated with diethyl ether to remove the acid and neutralized with NaOH. The supematant was applied to a column of Dowex AGI-X8 formate form. To remove inositol monophosphate and inositol bisphosphate, 8 ml of 0.1 M formic acid containing 0.4 M ammonium formate was applied to the column. The radioactive IP, was then
We next examined the effect of NaF, known as a GTPbinding protein activator (12), on the PGE,-induced AA release. NaF mimicked PGE, by stimulating the AA release (Fig. 5). The effect of 40 mM NaF was very similar to that of 10 PM PGE,. The release of AA stimulated by a combination of PGE2 (10 PM) and NaF (40 mM) was not additive.
Effect of PTX on PGE,-induced AA release The pretreatment with PTX in the range between 1 nM and 1 pM had little effect on the PGE,-induced release of AA (Fig. 6).
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PGE2
Cont.
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Fig. 1 Effect of PC& on the AA release in MC3T3-El cells. The [3H]AA-labeled cells were stimulated by 10pM PGEz (0) or vehicle (0) for the indicated periods, then the release of AA was determined. Each value represents the mean + SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
0
Cont.
5
Log (PGEz), M
Fig. 2 Dose-dependent effect of PGEz on the AA release and the formation of IP, in MC3T3-El cells. The cultured cells were stimulated by various doses of PGEz for 30 min, then the release of AA (0) and the formation of IP, (fi) were determined. Values are expressed as a net increase compared with control. Each value represents the mean f SDof triplicate determinations, Similar results were obtained with two additional and different cell preparations.
Quinacrine Fig. 3 Effect of quinacrine on the PCiE,-induced AA release in MC3T3-El cells. The (‘H]AA-labeled cells were pretreated with IO PM quinacrine or vehicle for 20 min. then stimulated by 10 PM PGEz for 30 min. Each value represents the mean f SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *Significantly different from the value stimulated by PGEL without quinacrine pretreatment (p4.05).
0
10
Time
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(min)
Fig. 4 Effect of quinacrine on the PGE&duced formation of IPI in MC3T3-El cells. The [‘Hlinositol-labeled cells were pretreated with IO /fM quinacrine ((1) or vehicle (0) for 20 min. then stimulated by 10 PM PGE, for the indicated periods. Each value represents the mean f SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
294
Prostaglandins
Leukotrienes
and Essential Fatty Acids
DISCUSSION
Cont.
PGE2
NaF
PGE2 N+aF
Fig. 5 Effect of NaF on the PGE,-induced AA release in MC3T3-El cells. The [3H]AA-labeled cells were stimulated by 10 PM PGE2, 40 mM NaF or their combination for 30 min. Each value represents the mean f SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
-6
r-7
r----l t
"Oh+1
- Log (PTX),
g/ml
Fig. 6 Effect of PTX on the PGE,-induced AA release in MC3T3El cells. The [3H]AA-labe1ed cells were pretreated with various doses of PTX for 24 h, then stimulated by 10 PM PGE, for 30 min. Values are expressed as a net increase compared with control. Each value represents the mean _+SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
In the present study, we showed that NaF, a GTPbinding protein activator (12), mimicked PGE2 by stimulating the release of AA, and the AA release stimulated by a combination of PGE2 and NaF was not additive in osteoblast-like MC3T3-El cells. These results suggest that PGE,-induced AA release is mediated by GTP-binding protein in these cells. Moreover, we demonstrated that the PGE,-induced AA release was not affected by PTX pretreatment. It is well known that PTX catalyzes ADP-ribosylation of the a-subunit of certain GTP-binding proteins including Gi and G,, and causes uncoupling of receptor to these GTP-binding proteins (11, 23, 24). So, these evidences suggest that PTX-insensitive GTP-binding protein is involved in PGE,-induced AA release in osteblast-like MC3T3-El cells. We have previously reported that PTX-sensitive GTP-binding protein is involved in the coupling of PGE2 receptor and phospholipase C in MC3T3-El cells (1). These results might allow the speculation that PGE2 induces AA release separately from PI hydrolysis by phospholipase C in osteoblast-like MC3T3-El cells. We first compared the time-course of PGE,-induced AA release with that of PGE,-induced IP, formation in MC3T3-El cells and demonstrated that the AA release increased gradually up to 30 min, whereas the formation of IP, reached a plateau almost within 10 min and sustained it up to 30 min. Thus, it seems that the difference between both of the time-dependent curves are consistent with our notion that PGE? separately stimulates AA release and PI hydrolysis in these cells. We next compared the PGE,-induced AA release with the PGE,induced IP, formation in dose-dependency and showed that EDSo levels of PGE, affecting IP, formation were lo-fold higher than those affecting AA release. From these findings, it seems certain that PGE, stimulates AA release and PI hydrolysis via independent pathways in osteoblast-like MC3T3-El cells. In addition, we showed that quinacrine, a phospholipase A, inhibitor (22), significantly inhibited the PGE,-induced AA release in MC3T3-El cells, while quinacrine had little effect on the PGE,-induced IP, formation in these cells. Namely, quinacrine inhibited the AA release induced by PGE, without affecting the PGE,-induced PI hydrolysis. These results suggest that the major pathway of PGE,-induced AA release is the activation of phospholipase A, in these cells. Therefore, in osteoblast-like MC3T3-El cells, it is very likely that PGE, activates both phospholipase C and phospholipase AZ via distinct GTP-binding proteins, PTX-sensitive and insensitive ones, respectively. In a previous report (25), we have shown that exogenous PGE? increases DNA synthesis and decreases alkaline phosphatase activity, in turn, PGE2 promotes the proliferation and suppresses the differentiation of MC3T3-El cells, since alkaline phosphatase activity is
Arachidonic
considered to be a mature osteoblast phenotype (26). Taking into account the results in this study, it is possible that effects of PGE* are modulated by selfinduced AA release resulting in the synthesis of PGE,, which has been reported to be a major eicosanoid product in osteoblasts including MC3T3-El cells (5. 16). So, it seems that PGE, modulates osteoblast functions through the so-called positive feedback mechanism demonstrated here. In conclusion, our results strongly suggest that the AA release and the PI hydrolysis are separately stimulated by PGE, in osteoblast-like cells, and PTX-insensitive GTP-binding protein is involved in the AA release.
12.
13.
14.
15.
16.
Acknowledgement This investigation Scientific Research of Japan.
was supported in part by a Grant-in-Aid for from Ministry of Education, Science and Culture
References I. Tokuda, H., Kozawa, 0.. Yoneda, M.. Oiso, Y., Takatsuki, K., Asano,T. and Kato, K. Possible coupling of prostagiandin Ez receptor with pertussis toxin-sensitive guanine nucleotide-binding protein in osteoblast-like cells. J. Biochem. 109: 229-233, 1991. 2. Samuelsson, B., Goldyne, M., Granstrom. E.. Hamberg, M., Hammarstrom. S. and Malmsten, C. Prostaglandins and thromboxanes. Annu. Rev. Biochem. 47: 997-1029. 1978. 3. Nijweide, P. J., Burger, E. H. and Feyen J. H. M. Cells of bone: proliferation, differentiation, and hormonal regulation. Physiol. Rev. 66: 855-886, 1986. 4. Smith, W. L. The eicosanoids and their biochemical mechanism of action. Biochem. J. 259: 3 15-324. 1989. 5. Raisz, L. G. and Martin, T. J. Prostaglandins in bone and mineral metabolism. Excerpta Med. 286310, 1984. 6. Atkins, D. and Martin, T. J. Rat osteogenic sarcoma cells: effects of some prostaglandins. their metabolites and analogues on cyclic AMP production. Prostaglandins 13: 861-871, 1977. 7. Partridge, N. C.. Alcom, D., Michelangeli, V. P.. Kemp. B. E., Ryan, G. B. and Martin T. J. Functional properties of hormonally responsive cultured normal and malignant rat ostoblastic cells. Endocrinoloav 108: 213-219, 1981. 8. Hakeda, Y., Nakatani, Y., Hiram&t, M.. Kurihara, N., Tsunoi, M., Ikeda, E. and Kumegawa. M. Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells. clone MC3T3-El. J. Biochem. 97: 97-104, 1985. 9. Yamaguchi, D. T., Hahn T. J.. Beeker, T. G.. Kleeman. C. R. and Muallem, S. Relationship of CAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells. J. Biol. Chem. 263: 10745-10753, 1988. 10. Nishizuka, Y. Studies and perspectives of protein kinase C. Science 233: 305-312, 1986. II. Berridge. M. J. and Irvine, R. F. Inositol trisphosphate.
17.
18.
19.
20
21
22
23
24
25
26
Acid Release by PGEz
novel second messenger in cellular signal transduction. Nature 312: 315-321. 1984. Gilman, A. G. G proteins: transducers of receptorgenerated signals. Annu. Rev. Biochem. 56: 615-649, 1987. Kodama, H.. Amagai, Y., Sudo, H.. Kasai. S. and Yamamoto, S. Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Jpn. J. Oral Biol. 23: 899-901. 1981. Sudo, H.. Kodama. H., Amagai, Y.. Yamamoto, S. and Kasai, S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell Biol. 96: 191-198. 1983. Bimbaumer, L., Abramowitz, J. and Brown. A. M. Receptor-effector coupling by G proteins. Biochim. Biophys. Acta 103 1: 163-224, 1990. Yokota, K., Kusaka. M., Oshima. T.. Yamamoto. S.. Kurihara. N., Yoshino, T. and Kumegawa, M. Stimulation of prostaglandin Ez synthesis in cloned osteoblastic cells of mouse (MC3T3-El) by epidermal growth factor. J. Biol. Chem. 261: 4481150. 1986. Sate, K.. Kasano. F., Fujii. Y., Kawakami. M.. Tsushima. T. and Sizume. K. Tumor necrosis factor type a (cachectin) stimulates mouse osteoblast-like cells (MC3T3-El f to produce macrophage-colony stimulating activity and prostaglandin Ez. Biochem. Biophys. Res. Commun. 145: 323-329. 1987. Tashijian, A. H., Jr., Voelkel. E. F.. Lazzaro, M.. Singer, F. R.. Roberts. A. B. Derynck, R., Winkler. M. E. and Levine, L. a and B Human transforming growth factors stimulate prostaglandin production and bone resorption in cultured mouse calvaria. Proc. Nat]. Acad. Sci. USA 82: 45354538, 1985. Shupnik, M. A.. Antoniades. N. H. and Tashijian. A. H.. Jr. Platelet-derived growth factor increases prostaglandin production and decreases epidennal growth factor receptors in human osteosarcoma cells. Life Sci. 30: 347-353, 1982. Betridge. M. J., Dawson. R. M. C.. Downes. C. P., Heslop. J.P. and Irvine, R.F. Changes in the level of inositol phosphates after agonist dependent hydrolysis of membrane phosphoinositides. Biochem. J. 2 I?: 473-482. 1983. Betridge, M. J.. Heslop. J. P., Irvine, R. F. and Brown. K. D. lnositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to plateletderived growth factor. Biochem. J. 222: 195-201. 1984. Lapetina. E. G.. Billah. M. M. and Cuatrecasas, P. The phosphatidylinositol cycle and the regulation of arachidonic acid production. Nature 292: 367-369, 198 I. Ui. M. Islet-activating protein, pertussis toxin: a probe for functions of the inhibitory guanine nucleotide regulatory component of adenylate cyclase. Trends Pharmacol. Sci. 5: 777-279. 1984. Stemweis. P. C. and Robishaw, J. D. Isolation of two proteins with high affinity for guanine nucleotide from membranes of bovine brain. J. Biol. Chem. 259: 13806 13813. 1984. Miwa. M., Kozawa. O., Tokuda, H., Kawakubo. A.. Yoneda, M.. Oiso, Y. and Takatsuki. K. Effects of hypergravity on proliferation and differentiation of osteoblast-like cells. Bone Miner. 14: 15-25, 1991. Peck, W. A.. Birge. S. J. and Fedak, S. A. Bone cells: biochemical and biological studies after enzymatic isolation. Science 146: 1476-1477. 1964.
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