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Mechanism of thrombin-induced arachidonic acid release in osteoblast-like cells
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997)56(6),467-472
© PearsonProfessionalLtd 1997
M e c h a n i s m of t h r o m b i n - i n d u c e d a r a c h i d o n i c a c i d r e l e a s e in o s t e o b l a s t . l i k e cells A.
Suzuki,10.
K o z a w a , 2 J. S h i n o d a , 1 Y. W a t a n a b e - T o m i t a , ~ H. Saito, ~ Y. O i s o ~
1First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan 2Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Centre, Kasugai, Aichi 480-03, Japan
In a previous study, we have reported that thrombin stimulates phosphatidylcholine hydrolysis by phospholipase (PL) D, but has little effect on phosphoinositide hydrolysis by PLC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of the thrombin-induced arachidonic acid (AA) release in MC3T3E1 cells. Thrombin stimulated AA release dose dependently in the range between 0.1 and 1 U/ml. Quinacrine, a PLA2 inhibitor, suppressed the thrombin-induced AA release. In addition, quinacrine also suppressed the thrombin-induced prostaglandin E2 synthesis in these cells. On the other hand, propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, did not affect the thrombin-induced AA release. 1(6-((1713-3-Methoxyestra-1,3,5(10)-trien17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122), a PLC inhibitor, had no effect on the AA release by thrombin. In addition, 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of diacylglycerol lipase, had little effect on the thrombin-induced AA release. Neither propranolol, U-73122 nor RHC-80267 affect the thrombininduced prostaglandin E2 synthesis. These results strongly suggest that thrombin induces AA release not by phosphatidylcholine hydrolysis by PLD nor phosphoinositide hydrolysis by PLC but mainly by PLA2 in osteoblast-like cells. Summary
INTRODUCTION
Localized bone resorption is a common sequela of the most common chronic inflammatory processes. Thrombin, a multifunctional protease that plays an important role in the blood coagulation cascade through its cleavage of fibrinogen to form fibrin, is a potent agonist for a number of biological responses that may mediate inflammatory and reparative responses to vasculax injury. ~ In bone tissue, it has been reported that thrombin stimulates bone resorption in vitro, 2,3 and that thrombin induces the proliferation of osteoblastsY The existence of thrombin receptor has been reported in rat and human osteoblasts. 6 As for the intracellular signaling system in osteoblasts, it has been reported that thrombin stimulates phosphoinositide hydrolysis and increases cytosolic Ca 2+level.4,5,7,8 Received 19 September 1996 Accepted 22 October 1996 Correspondence to: O. Kozawa, Department of Pharmacology, Gifu University School of Medicine, Gifu 500, Japan. Tel. 00 81 58 265 1241; Fax. 00 81 58 265 9763.
Phosphoinositide hydrolysis results in the formation of two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol (DG)? It is well-known that DG is a physiological activator of protein kinase C (PKC).1° However, phosphoinositide hydrolysis is not the only pathway of DG formation. 11,12Phosphatidylcholine (PC) is recognized to be hydrolyzed by phospholipase D (PLD), generating the formation of choline and phosphatidic acid. 11-13 Phosphatidic acid, which by itself could be a potential intracellular mediator, can be further degraded to DG. Accumulating evidences suggest that PC-PLD plays a pivotal role in modulating cellular functions that require long-term activation of PKC, since PC is the principal phospholipid in cell membranes. 1~-~4 We have recently shown that thrombin activates PC-PLD but has only a little effect on phosphoinositide hydrolysis in osteoblastlike MC3T3-E1 cells. ~5 Prostaglandins (PGs) are generally recognized to be important local modulators in bone metabolism. *¢~7The release of arachidonic acid (AA) from bone cells or other cells adjacent to bone and the subsequent formation of PGs are considered to be involved in the mechanism of 467
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bone resorption in inflammatory processes. ~,18 PGE2 has been reported to be a potent bone resorptive agent and a major eicosanoid product in osteoblasts including osteoblast-like MC3T3-E1 cells. 16,~9It has been reported that thrombin stimulates PGE2 synthesis in osteoblastlike cells including MC3T3-E1 cells, and the thrombininduced bone resorption depends on, at least in part, PG synthesis in osteoblasts. 3,a,~a,2° It is well-known that PGs are synthesized from AA, which is released from the esterified stores of phospholipids? 1Two major pathways of AA release are generally accepted. 21 One is the activation of phospholipase A2 (PLA2) which causes liberation of AA directly from the stores of phospholipids, and the other is the liberation by DG lipase from DG that is produced from phospholipids by other phospholipases. However, the precise mechanisms of thrombin-induced AA release and PG synthesis by thrombin in osteoblasts has not yet been fully clarified. Herein, we show that thrombin induces AA release not by PC-hydrolysis by PLD nor by phosphoinositide hydrolysis by phospholipase C (PLC), but mainly by PLA2 in osteoblast-like MC3T3-E 1 cells.
MATERIALS AND METHODS Materials
[5,6,8,9,11,12,14,15-3H]AA (212.1 Ci/mmol) and PGE21125I] assay system were purchased from Amersham Japan (Tokyo, Japan). Mouse a-thrombin (1300NIH unit/mg protein), quinacrine and essentially fatty acid-free bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). dl-Propranolol hydrochloride (propranolol) was purchased from Wako Pure Chemical Industries (Osaka, Japan). 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267) and 1(6((17[~-3-methoxyestra 1,3,5(10)-trien- 17-yl)amino)hexyl)- 1 H-pyrrole-2,5-dione (U-73122) were purchased from Funakoshi Pharmaceutical Co. (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. Quinacrine, propranoloI, RHC-80267 and U73122 were dissolved in dimethyl sulfoxide. The maxim u m concentration of dimethyl sulfoxide in the culture medium was 0.1%, and this did not affect the measurement of AA release or assay for PGE2. Cell culture
Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria22,23 were maintained as previously described. 24 In brief, the cells were cultured in a-minimum essential medium ((z-MEM) containing 10% fetal calf serum. (FCS) at 37°C in a humidified atmosphere of 5% C0J95% air. The cells (5 x 104) were seeded into
35-mm diameter dishes in 2 ml of cz-MEM containing 10% FCS. After 5 days, the medium was exchanged for 2 ml of a-MEM containing 0.3% FCS. The cells were used for experiments after 48 h. Measurement of AA release
The measurement of AA release was performed as previously describedY In brief, the cultured cells were labelled with [3H]AA (0.5 gCi/dish) for 24h. The medium was removed and the cells were then washed four times with 1 ml of the assay buffer [10raM 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid, pH 7.4, 135 mM NaCI, 5 mM KC1, 1 mM MgSO4 and 1 mM CaC12]. The cells were preincubated subsequently with 1 ml of the assay buffer containing 0.1% essentially fatty acid-free BSA at 3 7°C for 20 rain, and the cells were then stimulated by thrombin. The reaction was terminated by collecting the medium, and the radioactivity of the medium was determined. When indicated, the cells were pretreated with various doses of quinacrine, propranolol, U-73122 or RHC-80267 for 20 min. Assay for PGE2
Procedures were done as described under 'Measurement of AA release' except for using unlabelled cells. The cultured cells were pretreated with quinacrine, propranolol, U-73122 or RHC-80267 for 20 min, and then stimulated by 1 U/ml of thrombin. After 180 min, the medium was collected and PGE2 in the medium was measured with a radioimmunoassay kit. Determination
The radioactivity of 3H-samples were determined with a Beckman LS-6000IC liquid scintillation spectrometer, and the radioactivity of 125I-samples were determined with an Aloka ARC-600 auto well gamma system. Statistical analysis
The data were analyzed by Student's t-test and P < 0.05 was considered significant. All data are presented as the mean + SD of triplicate determinations. RESULTS Effect of thrombin on AA release in MC3T3-E1 cells
Thrombin (1 U/ml) significantly increased the AA release, compared to the control, up to 30 min, and the effect was sustained for up to 60 min in osteoblast-like MC3T3-E1 cells (Fig. 1A). The stimulative effect of thrombin was dose-dependent in the range between 0.1 and 1 U/ml
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(6), 467-472
© Pearson Professional Ltd 1997
Thrombin-induced arachidonic acid release
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Fig. 1 (A) Effect of thrombin on AA release in MC3T3-E1 cells. The labelled cells were stimulated by 1 U/ml thrombin (0) or vehicle (©) for the indicated periods. (B) Dose-dependent effect of thrombin on AA release in MC3T3-E1 cells. The labelled cells were stimulated by various doses of thrombin for 30 min. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean _+ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05, compared to the value of control.
(Fig. 1B). The maximum effect of thrombin on AA release was observed at 1 U/ml. Effect of quinacrine on AA release induced by thrombin in MC3T3-E1 cells
To elucidate whether PLA2 is involved in the thrombininduced AA release in MC3T3-E1 cells, we examined the effect of quinacrine, a PLA2 inhibitor, 26 on AA release. Quinacrine, which by itself had little effect on AA release, significantly suppressed the thrombin-induced AA release in these cells (Fig. 2). The inhibitory effect of quinacrine was dose-dependent in the range between 10 and 50 p.M.
Effect of U-73122 on AA release induced by thrombin in MC3T3-E1 cells
We have previously reported that thrombin has a little effect on phosphoinositide hydrolysis by PLC in MC3T3E1 cells. I5 Thus, we examined the effect of U-73122, a PLC inhibitor, 28 on the thrombin-induced AA release in MC3T3-E1 cells. U-73122 (10 gM) had little effect on both basal and thrombin (1 U/ml)-induced AA release in these cells (control, 3,121 _+ 101 cpm; 1 U/ml thrombin, 7,644 + 228cpm; 10gM U-73122, 3,066 + 137cpm; 1U/ml thrombin + 10gM U-73122, 7,531 + 395 cpm, these as measured during the stimulation for 30 min).
Effect of propranolol on AA release induced by thrombin in MC3T3-E1 cells
Effect of RHC-80267 on AA release induced by thrombin in MC3T3-E1 cells
We next examined the effect of propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, 27 on the thrombin-induced AA release in MC3T3-E 1 cells. The pretreatment with propranolol, which by itself had little effect on AA release, did not affect the thrombininduced AA release in MC3T3-E1 cells (Fig. 3A).
DG is an important cellular source of AA, which may be released by DG lipase. 29 So, we examined the effect of REIC-80267, a DG lipase inhibitor, ~° on the thrombininduced AA release in MC3T3-E1 cells. RHC-80267 did not affect either basal or thrombin-induced AA release in these cells (Fig. 3B).
© Pearson Professional Ltd 1997
Prostagtandins, Leukotrienes and Essential Fatty Acids (1997) 56(6), 467-472
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Effects of propranolol, U-73122, RH0-80267 and quinacrine on PGE2 synthesis induced by thrombin in MC3T3-E1 c e l l s
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Among eicosanoids converted from AA, PGE2 is a major product in osteoblasts including MC3T3-E 1 cells.16,19PGE2 synthesis by thrombin (1 U/ml) was not affected by either propranolol (300gM), U-73122 (10gM) or RHC-80267 (50gM) in MC3T3-E1 cells (Table). On the contrary, the pretreatment with quinacrine (50 gM) significantly suppressed the thrombin (1 U/ml)-induced PGE2 synthesis in these cells (Table).
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In the present study, we showed that thrombin stimulated the AA release time- and dose-dependently in osteoblast-like MC3T3-E1 cells. It is well-known that AA is derived from at least two possible pathways; glycerophospholipid hydrolysis by PLAa, and sequential hydrolysis of phospholipid by PLC or PLD and DG lipase) 1 We have recently reported that the sequential hydrolysis of PC by PLD and DG lipase is involved in the mechanism of endothelin-l-induced AA release in MC3T3-E1 c e l l s . 31 In the present study, we here showed that propranolol, a phosphatidic acid phosphohydrolase inhibitor, 27 did not
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Fig, 3 (A) Effect of propranotol on thrombin-induced AA release in MC3T3-E1 cells. (B) Effect of RHC-80267 on thrombin-induced AA release in MC3T3-E1 cells. The labelled cells were pretreated with various doses of (A) propranolol or (B) RHC-80267 for 20 min, and then stimulated by 1 U/ml thrombin (e) or vehicle (O) for 30 rain. Each value represents the mean +- SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(6), 467-472
© Pearson Professional Ltd 1997
Thrombin-induced arachidonic acid release
Table Effect of propranolol, U-73122, RHC-80267 and quinacrine on thrombin-induced PGE2 synthesis in MC3T3-E1 cells
PGE 2 synthesis (pg/ml) Control Propranolol U-73122 RHC-80267 Quinacrine Thrombin Thrombin + Propranolol Thrombin + U-73122 Thrombin+ RHC-80267 Thrombin+ Quinacrine
180 166 176 191 170 1,918 2,011 1,996 1,958 948
__+12 _+ 13 _+ 16 __ 19 _+ 18 _+ 146 _+ 158 _+ 92 _+ 71 _+ 88*
as well as on AA release in MC3T3-E 1 cells. Therefore, our findings suggest that the activation of PLA2 is, at least in part, involved in the m e c h a n i s m of t h r o m b i n - i n d u c e d arachidonic cascade in osteoblast-like MC3T3-E1 cells, b u t DG formation b y phospholipid hydrolysis is not. In conclusion, our findings suggest that t h r o m b i n induces AA release not b y PC-hydrolysis b y PLD nor phosphoinositide hydrolysis b y PLC but mainly b y PLA; in osteoblast-like cells.
REFERENCES
The cells were pretreated with 300 gM propranolol, 10 pM U-73122, 50 taM RHC-80267, 50 taM quinacrine or vehicle for 20 min, and then stimulated with 1 U/ml thrombin for 180 min. Each value represents the mean -+ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05, corn,pared to the value of thrombin alone.
affect the t h r o m b i n - i n d u c e d AA release in osteoblast-like MC3T3-E1 cells. We previously demonstrated that thrombin stimulates PC hydrolysis b y PLD in MC3T3-E1 cells, and that t h r o m b i n induces DG formation mainly t h r o u g h PC hydrolysis b y PLD in these cellsJ 5 DG is recognized to be an important cellular source of AA which m a y be generated subsequently b y DG lipase. 29 In addition, we s h o w e d that RHC-80267, an inhibitor of DG lipase, 3° did not affect the t h r o m b i n - i n d u c e d AA release in MC3T3-E 1 cells. Thus, it is unlikely that DG formation i n d u c e d by PC hydrolysis b y PLD is involved in the thrombini n d u c e d AA release in MC3T3-E1 cells. We previously f o u n d that t h r o m b i n has a little effect on phosphoinositide hydrolysis b y PLC in MC3T3-E1 cells, is In this study, we s h o w e d that U-73122, w h i c h is k n o w n to inhibit PLC, 2s had little effect on the AA release by t h r o m b i n in MC3T3-E1 cells. Therefore, our findings suggest that DG formation b y t h r o m b i n is not involved in the p a t h w a y of AA release in osteoblast-like MC3T3-E1 cells. On the other hand, we here s h o w e d that quinacrine, a PLA2 inhibitor, 26 significantly suppressed the thrombini n d u c e d AA release in MC3T3-E1 cells. Therefore, our findings suggest that t h r o m b i n induces the AA release mainly t h r o u g h PLA2 activation in MC3T3-E 1 cells. The release of AA is metabolized to a variety of bioactive substances such as PGs. 21 PGE2 has b e e n reported to be one of the major eicosanoid p r o d u c t in osteoblasts including MC3T3-E1 cells, 16,19 and it has been reported that t h r o m b i n stimulates PGE2 synthesis in osteoblastlike cells. 3,s,~s,2° In the present study, we confirmed that t h r o m b i n induces PGE2 synthesis in MC3T3-E1 cells, and s h o w e d that quinacrine suppressed the thrombininduced PGE2 synthesis in these cells. On the other hand, we s h o w e d that propranolol, RHC80267 and U-73122 had little effect on the t h r o m b i n - i n d u c e d PGE2 synthesis © Pearson Professional Ltd 1997
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1. Fenton J. W. II. Regulation of thrombin generation and functions. Semin Thromb Hem 1988; 14: 229-235. 2. Gustafson G. T., Lerner U. Thrombin, a stimulator of bone resorption. Biosci Rep 1983; 3: 255-261. 3. Hoffman O., Klaushofer K., Koller K., Peterlik M., Mavreas T., Stern P. Indomethacin inhibits thrombin-, but not thyroxinestimulated resorption of fetal rat limb bones. Prostaglandins 1986; 31: 601-608. 4. Tatakis D. N., Dolce C., Dziak R. Thrombin's effects on osteoblastic cells I. Cytosolic calcium and phosphoinositides. Biochem Biophys Res Commun 1989; 164:119-127. 5. Babich M., King L. K., Nissenson R. A. Thrombin stimulates inositol phosphate production and intracellular free calcium by a pertussis toxin-insensitive mechanism in osteosarcoma cells. Endocrinology 1990; 126: 948-954. 6. Jenkins A. L., Bootman M. D., Taylor C. W., Mackie E.J., Stone S. K Characterization of the receptor responsible for thrombininduced intracellular calcium responses in osteoblast-like cells. JBiol Chem 1993; 268: 21433-21437. 7. Partridge N. C., Hillyard C. L., Nolan R. D., Martin T.J. Regulation of prostaglandin production by osteoblast-rich calvarial cells. Prostaglandins 1985; 30: 527-539. 8. Ljunggren O., Johansson H., Ljunghall S., Lerner U. H. Thrombin increases cytoplasmic Ca2+and stimulates formation of prostaglandin E=in the osteoblastic cell line MC3T3-E1. Bone Miner 1991; 12: 81-90. 9. Berridge M.J. Inositol trisphosphate and calcium signalling. Nature 1993; 361: 315-325. 10. Nishizuka Y. Studies and perspectives of protein kinase C. Science 1986; 233:305-312.
l 1. Exton J. H. Signaling through phosphatidylchotine breakdown. JBiol Chem 1990; 265: 1-4. 12. Zeisel S. H. Choline phospholipids: signal transduction and carcinogenesis. FASEBJ 1993; 7: 551-557. 13. Billah M. M., Anthes J. C. The regulation and cellular functions of phosphatidylcholine hydrolysis. BiochemJ 1990; 269:281-291.
14. Nishizuka Y. lntracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 1992; 258: 607-614.
15. Suzuki A., Kozawa O., Shinoda J., Watanabe Y., Saito H., Oiso Y. Thrombin induces proliferation of osteoblast-like cells through phosphatidylcholine hydrolysis. J Cell Physio11996; 168: 209-216. 16. Raisz L. G., Martin T.J. Prostaglandins in bone and mineral metabolism. J Bone Miner Res 1984; 2:286-310. 17. Nijweide P.J., Burger E. H., Feyen J. H. M. Cells of bone: proliferation, differentiation, and hormonal regulation. Phys Rev 1986; 66: 855-886. 18. Lerner U. H. Regulation of bone metabolism by the kallikrein-
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kinin system, the coagulation cascade, and the acute phase reactants. Oral Surg Oral Med Oral Patho11994; 78: 481-493. Yokota tC, Kusaka M., Ohshima T., et al. Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3 -E 1) by epidermal growth factor. J Biol Chem 1986; 261: 15410-15415. Leis H.J., Zach D., Huber E., Zierman L., Gleisppach H., Windischofer W. Extracellular Ca2+ sensing by the osteoblastlike cell line, MC3T3-E1. Cell Calcium 1994; 15: 47-456. Smith W. L. The eicosanoids and their biochemical mechanisms of action. BiochemJ 1989; 259: 315-324. Kodama H., Amagai Y., Sudo H., Kasai S., Yamamoto S. Establishment of a clonal osteoblastic cell line from newborn mouse calvaria. Jpn J Oral Biol 1981; 23:899-901. Sudo H., Kodama H., Amagai Y., Yamamoto S., Kasai S. In vitro differentiation and calcification in a new clonal osteogenic ceil line derived from newborn mouse calvaria. J Cell Bio11983; 96: 191-198. Kozawa O., Suzuki A., Kotoyori J., et al. Prostaglandin Fa~ activates phospholipase D independently from activation of protein kinase C. J Cell Biochem 1994; 55: 373-379. Suzuki A., KotoyoriJ., Kozawa O., Oiso Y. Prostaglandin E2 is a
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potential mediator of extracellular action in osteoblast-like cells. CelI Adh Commun 1993; 1:113-118. Lapetina E. G., Billah M. M., Cuatrecasas P. The phosphatidylinositol cycle and the regulation of arachidonic acid production. Nature 1981; 292: 367-369. Pappu A. S., Hauser G. Propranolol-induced inhibition of rat brain cytoplasmic phosphatidate phosphohydrolase. Neurochem Res 1983; 8: 1565-1575. Bleasdale J. E., Thakur N. R., Gremban R. S., et al. Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophfls. Jpharmacol Exp Ther 1990; 255: 756-768. Bell R. L., Kennerly D. A., Stanford N., Majerns P. W. Diglyceride lipase: A pathway for arachidonic acid release from human platelets. Proc Natl Acad Sci USA 1979; 76: 3238-3241. Sutherland C. A., Amin D. Relative activities of rat and dog platelet phospholipase A2 and diglyceride lipase. J Biol Chem 1982; 257: 14006-14010. Kozawa O., Suzuki A., Shinoda J., Ozaki N., Oiso Y., Uematsu T. Involvement of phospholipase D activation in endothelin-1induced release of arachidonic acid in osteoblast-like cells. J Cell Biochem in press.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(6), 467-472
© Pearson Professional Ltd 1997
© PearsonProfessionalLtd 1997
M e c h a n i s m of t h r o m b i n - i n d u c e d a r a c h i d o n i c a c i d r e l e a s e in o s t e o b l a s t . l i k e cells A.
Suzuki,10.
K o z a w a , 2 J. S h i n o d a , 1 Y. W a t a n a b e - T o m i t a , ~ H. Saito, ~ Y. O i s o ~
1First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan 2Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Centre, Kasugai, Aichi 480-03, Japan
In a previous study, we have reported that thrombin stimulates phosphatidylcholine hydrolysis by phospholipase (PL) D, but has little effect on phosphoinositide hydrolysis by PLC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of the thrombin-induced arachidonic acid (AA) release in MC3T3E1 cells. Thrombin stimulated AA release dose dependently in the range between 0.1 and 1 U/ml. Quinacrine, a PLA2 inhibitor, suppressed the thrombin-induced AA release. In addition, quinacrine also suppressed the thrombin-induced prostaglandin E2 synthesis in these cells. On the other hand, propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, did not affect the thrombin-induced AA release. 1(6-((1713-3-Methoxyestra-1,3,5(10)-trien17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U-73122), a PLC inhibitor, had no effect on the AA release by thrombin. In addition, 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of diacylglycerol lipase, had little effect on the thrombin-induced AA release. Neither propranolol, U-73122 nor RHC-80267 affect the thrombininduced prostaglandin E2 synthesis. These results strongly suggest that thrombin induces AA release not by phosphatidylcholine hydrolysis by PLD nor phosphoinositide hydrolysis by PLC but mainly by PLA2 in osteoblast-like cells. Summary
INTRODUCTION
Localized bone resorption is a common sequela of the most common chronic inflammatory processes. Thrombin, a multifunctional protease that plays an important role in the blood coagulation cascade through its cleavage of fibrinogen to form fibrin, is a potent agonist for a number of biological responses that may mediate inflammatory and reparative responses to vasculax injury. ~ In bone tissue, it has been reported that thrombin stimulates bone resorption in vitro, 2,3 and that thrombin induces the proliferation of osteoblastsY The existence of thrombin receptor has been reported in rat and human osteoblasts. 6 As for the intracellular signaling system in osteoblasts, it has been reported that thrombin stimulates phosphoinositide hydrolysis and increases cytosolic Ca 2+level.4,5,7,8 Received 19 September 1996 Accepted 22 October 1996 Correspondence to: O. Kozawa, Department of Pharmacology, Gifu University School of Medicine, Gifu 500, Japan. Tel. 00 81 58 265 1241; Fax. 00 81 58 265 9763.
Phosphoinositide hydrolysis results in the formation of two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol (DG)? It is well-known that DG is a physiological activator of protein kinase C (PKC).1° However, phosphoinositide hydrolysis is not the only pathway of DG formation. 11,12Phosphatidylcholine (PC) is recognized to be hydrolyzed by phospholipase D (PLD), generating the formation of choline and phosphatidic acid. 11-13 Phosphatidic acid, which by itself could be a potential intracellular mediator, can be further degraded to DG. Accumulating evidences suggest that PC-PLD plays a pivotal role in modulating cellular functions that require long-term activation of PKC, since PC is the principal phospholipid in cell membranes. 1~-~4 We have recently shown that thrombin activates PC-PLD but has only a little effect on phosphoinositide hydrolysis in osteoblastlike MC3T3-E1 cells. ~5 Prostaglandins (PGs) are generally recognized to be important local modulators in bone metabolism. *¢~7The release of arachidonic acid (AA) from bone cells or other cells adjacent to bone and the subsequent formation of PGs are considered to be involved in the mechanism of 467
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bone resorption in inflammatory processes. ~,18 PGE2 has been reported to be a potent bone resorptive agent and a major eicosanoid product in osteoblasts including osteoblast-like MC3T3-E1 cells. 16,~9It has been reported that thrombin stimulates PGE2 synthesis in osteoblastlike cells including MC3T3-E1 cells, and the thrombininduced bone resorption depends on, at least in part, PG synthesis in osteoblasts. 3,a,~a,2° It is well-known that PGs are synthesized from AA, which is released from the esterified stores of phospholipids? 1Two major pathways of AA release are generally accepted. 21 One is the activation of phospholipase A2 (PLA2) which causes liberation of AA directly from the stores of phospholipids, and the other is the liberation by DG lipase from DG that is produced from phospholipids by other phospholipases. However, the precise mechanisms of thrombin-induced AA release and PG synthesis by thrombin in osteoblasts has not yet been fully clarified. Herein, we show that thrombin induces AA release not by PC-hydrolysis by PLD nor by phosphoinositide hydrolysis by phospholipase C (PLC), but mainly by PLA2 in osteoblast-like MC3T3-E 1 cells.
MATERIALS AND METHODS Materials
[5,6,8,9,11,12,14,15-3H]AA (212.1 Ci/mmol) and PGE21125I] assay system were purchased from Amersham Japan (Tokyo, Japan). Mouse a-thrombin (1300NIH unit/mg protein), quinacrine and essentially fatty acid-free bovine serum albumin (BSA) were purchased from Sigma Chemical Co. (St. Louis, MO). dl-Propranolol hydrochloride (propranolol) was purchased from Wako Pure Chemical Industries (Osaka, Japan). 1,6-Bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267) and 1(6((17[~-3-methoxyestra 1,3,5(10)-trien- 17-yl)amino)hexyl)- 1 H-pyrrole-2,5-dione (U-73122) were purchased from Funakoshi Pharmaceutical Co. (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. Quinacrine, propranoloI, RHC-80267 and U73122 were dissolved in dimethyl sulfoxide. The maxim u m concentration of dimethyl sulfoxide in the culture medium was 0.1%, and this did not affect the measurement of AA release or assay for PGE2. Cell culture
Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria22,23 were maintained as previously described. 24 In brief, the cells were cultured in a-minimum essential medium ((z-MEM) containing 10% fetal calf serum. (FCS) at 37°C in a humidified atmosphere of 5% C0J95% air. The cells (5 x 104) were seeded into
35-mm diameter dishes in 2 ml of cz-MEM containing 10% FCS. After 5 days, the medium was exchanged for 2 ml of a-MEM containing 0.3% FCS. The cells were used for experiments after 48 h. Measurement of AA release
The measurement of AA release was performed as previously describedY In brief, the cultured cells were labelled with [3H]AA (0.5 gCi/dish) for 24h. The medium was removed and the cells were then washed four times with 1 ml of the assay buffer [10raM 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid, pH 7.4, 135 mM NaCI, 5 mM KC1, 1 mM MgSO4 and 1 mM CaC12]. The cells were preincubated subsequently with 1 ml of the assay buffer containing 0.1% essentially fatty acid-free BSA at 3 7°C for 20 rain, and the cells were then stimulated by thrombin. The reaction was terminated by collecting the medium, and the radioactivity of the medium was determined. When indicated, the cells were pretreated with various doses of quinacrine, propranolol, U-73122 or RHC-80267 for 20 min. Assay for PGE2
Procedures were done as described under 'Measurement of AA release' except for using unlabelled cells. The cultured cells were pretreated with quinacrine, propranolol, U-73122 or RHC-80267 for 20 min, and then stimulated by 1 U/ml of thrombin. After 180 min, the medium was collected and PGE2 in the medium was measured with a radioimmunoassay kit. Determination
The radioactivity of 3H-samples were determined with a Beckman LS-6000IC liquid scintillation spectrometer, and the radioactivity of 125I-samples were determined with an Aloka ARC-600 auto well gamma system. Statistical analysis
The data were analyzed by Student's t-test and P < 0.05 was considered significant. All data are presented as the mean + SD of triplicate determinations. RESULTS Effect of thrombin on AA release in MC3T3-E1 cells
Thrombin (1 U/ml) significantly increased the AA release, compared to the control, up to 30 min, and the effect was sustained for up to 60 min in osteoblast-like MC3T3-E1 cells (Fig. 1A). The stimulative effect of thrombin was dose-dependent in the range between 0.1 and 1 U/ml
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(6), 467-472
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Thrombin-induced arachidonic acid release
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Fig. 1 (A) Effect of thrombin on AA release in MC3T3-E1 cells. The labelled cells were stimulated by 1 U/ml thrombin (0) or vehicle (©) for the indicated periods. (B) Dose-dependent effect of thrombin on AA release in MC3T3-E1 cells. The labelled cells were stimulated by various doses of thrombin for 30 min. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean _+ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05, compared to the value of control.
(Fig. 1B). The maximum effect of thrombin on AA release was observed at 1 U/ml. Effect of quinacrine on AA release induced by thrombin in MC3T3-E1 cells
To elucidate whether PLA2 is involved in the thrombininduced AA release in MC3T3-E1 cells, we examined the effect of quinacrine, a PLA2 inhibitor, 26 on AA release. Quinacrine, which by itself had little effect on AA release, significantly suppressed the thrombin-induced AA release in these cells (Fig. 2). The inhibitory effect of quinacrine was dose-dependent in the range between 10 and 50 p.M.
Effect of U-73122 on AA release induced by thrombin in MC3T3-E1 cells
We have previously reported that thrombin has a little effect on phosphoinositide hydrolysis by PLC in MC3T3E1 cells. I5 Thus, we examined the effect of U-73122, a PLC inhibitor, 28 on the thrombin-induced AA release in MC3T3-E1 cells. U-73122 (10 gM) had little effect on both basal and thrombin (1 U/ml)-induced AA release in these cells (control, 3,121 _+ 101 cpm; 1 U/ml thrombin, 7,644 + 228cpm; 10gM U-73122, 3,066 + 137cpm; 1U/ml thrombin + 10gM U-73122, 7,531 + 395 cpm, these as measured during the stimulation for 30 min).
Effect of propranolol on AA release induced by thrombin in MC3T3-E1 cells
Effect of RHC-80267 on AA release induced by thrombin in MC3T3-E1 cells
We next examined the effect of propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, 27 on the thrombin-induced AA release in MC3T3-E 1 cells. The pretreatment with propranolol, which by itself had little effect on AA release, did not affect the thrombininduced AA release in MC3T3-E1 cells (Fig. 3A).
DG is an important cellular source of AA, which may be released by DG lipase. 29 So, we examined the effect of REIC-80267, a DG lipase inhibitor, ~° on the thrombininduced AA release in MC3T3-E1 cells. RHC-80267 did not affect either basal or thrombin-induced AA release in these cells (Fig. 3B).
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Effects of propranolol, U-73122, RH0-80267 and quinacrine on PGE2 synthesis induced by thrombin in MC3T3-E1 c e l l s
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Among eicosanoids converted from AA, PGE2 is a major product in osteoblasts including MC3T3-E 1 cells.16,19PGE2 synthesis by thrombin (1 U/ml) was not affected by either propranolol (300gM), U-73122 (10gM) or RHC-80267 (50gM) in MC3T3-E1 cells (Table). On the contrary, the pretreatment with quinacrine (50 gM) significantly suppressed the thrombin (1 U/ml)-induced PGE2 synthesis in these cells (Table).
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In the present study, we showed that thrombin stimulated the AA release time- and dose-dependently in osteoblast-like MC3T3-E1 cells. It is well-known that AA is derived from at least two possible pathways; glycerophospholipid hydrolysis by PLAa, and sequential hydrolysis of phospholipid by PLC or PLD and DG lipase) 1 We have recently reported that the sequential hydrolysis of PC by PLD and DG lipase is involved in the mechanism of endothelin-l-induced AA release in MC3T3-E1 c e l l s . 31 In the present study, we here showed that propranolol, a phosphatidic acid phosphohydrolase inhibitor, 27 did not
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Fig, 3 (A) Effect of propranotol on thrombin-induced AA release in MC3T3-E1 cells. (B) Effect of RHC-80267 on thrombin-induced AA release in MC3T3-E1 cells. The labelled cells were pretreated with various doses of (A) propranolol or (B) RHC-80267 for 20 min, and then stimulated by 1 U/ml thrombin (e) or vehicle (O) for 30 rain. Each value represents the mean +- SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations.
Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(6), 467-472
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Thrombin-induced arachidonic acid release
Table Effect of propranolol, U-73122, RHC-80267 and quinacrine on thrombin-induced PGE2 synthesis in MC3T3-E1 cells
PGE 2 synthesis (pg/ml) Control Propranolol U-73122 RHC-80267 Quinacrine Thrombin Thrombin + Propranolol Thrombin + U-73122 Thrombin+ RHC-80267 Thrombin+ Quinacrine
180 166 176 191 170 1,918 2,011 1,996 1,958 948
__+12 _+ 13 _+ 16 __ 19 _+ 18 _+ 146 _+ 158 _+ 92 _+ 71 _+ 88*
as well as on AA release in MC3T3-E 1 cells. Therefore, our findings suggest that the activation of PLA2 is, at least in part, involved in the m e c h a n i s m of t h r o m b i n - i n d u c e d arachidonic cascade in osteoblast-like MC3T3-E1 cells, b u t DG formation b y phospholipid hydrolysis is not. In conclusion, our findings suggest that t h r o m b i n induces AA release not b y PC-hydrolysis b y PLD nor phosphoinositide hydrolysis b y PLC but mainly b y PLA; in osteoblast-like cells.
REFERENCES
The cells were pretreated with 300 gM propranolol, 10 pM U-73122, 50 taM RHC-80267, 50 taM quinacrine or vehicle for 20 min, and then stimulated with 1 U/ml thrombin for 180 min. Each value represents the mean -+ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05, corn,pared to the value of thrombin alone.
affect the t h r o m b i n - i n d u c e d AA release in osteoblast-like MC3T3-E1 cells. We previously demonstrated that thrombin stimulates PC hydrolysis b y PLD in MC3T3-E1 cells, and that t h r o m b i n induces DG formation mainly t h r o u g h PC hydrolysis b y PLD in these cellsJ 5 DG is recognized to be an important cellular source of AA which m a y be generated subsequently b y DG lipase. 29 In addition, we s h o w e d that RHC-80267, an inhibitor of DG lipase, 3° did not affect the t h r o m b i n - i n d u c e d AA release in MC3T3-E 1 cells. Thus, it is unlikely that DG formation i n d u c e d by PC hydrolysis b y PLD is involved in the thrombini n d u c e d AA release in MC3T3-E1 cells. We previously f o u n d that t h r o m b i n has a little effect on phosphoinositide hydrolysis b y PLC in MC3T3-E1 cells, is In this study, we s h o w e d that U-73122, w h i c h is k n o w n to inhibit PLC, 2s had little effect on the AA release by t h r o m b i n in MC3T3-E1 cells. Therefore, our findings suggest that DG formation b y t h r o m b i n is not involved in the p a t h w a y of AA release in osteoblast-like MC3T3-E1 cells. On the other hand, we here s h o w e d that quinacrine, a PLA2 inhibitor, 26 significantly suppressed the thrombini n d u c e d AA release in MC3T3-E1 cells. Therefore, our findings suggest that t h r o m b i n induces the AA release mainly t h r o u g h PLA2 activation in MC3T3-E 1 cells. The release of AA is metabolized to a variety of bioactive substances such as PGs. 21 PGE2 has b e e n reported to be one of the major eicosanoid p r o d u c t in osteoblasts including MC3T3-E1 cells, 16,19 and it has been reported that t h r o m b i n stimulates PGE2 synthesis in osteoblastlike cells. 3,s,~s,2° In the present study, we confirmed that t h r o m b i n induces PGE2 synthesis in MC3T3-E1 cells, and s h o w e d that quinacrine suppressed the thrombininduced PGE2 synthesis in these cells. On the other hand, we s h o w e d that propranolol, RHC80267 and U-73122 had little effect on the t h r o m b i n - i n d u c e d PGE2 synthesis © Pearson Professional Ltd 1997
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Prostaglandins, Leukotrienes and Essential Fatty Acids (1997) 56(6), 467-472
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