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Effect of vitamin D3 on prostaglandin E2 synthesis in osteoblast-like cells
PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS Proe@mi,n\ kukorr,enesand Ecwtial '0 l.ongmanGroupLA1lYY4
Fatty AC&
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Effect of Vitamin D, on Prostaglandin
E, Synthesis in Osteoblast-like
Cells
A. Suzuki, H. Tokuda, J. Kotoyori, Y. Ito. Y. Oiso and 0. Kozawa”
of
First Depurtment Internal Medicine, Nagoya University School of Medicine. Nagoya 466, Japan. *Department Biochemistry, Institute for Developmental Research, Aichi Prefectwal Colony. Kasugai, Aichi 480-02, Jupa~ (Reprint requests to OK)
qf
ABSTRACT. We previously showed that prostaglandin (PG) Fzu stimulates PGEL synthesis in osteohlast-like MC3T3-El cells, and that the activation of protein kinase C (PKC) amplifies the effect of PGFzu through the potentiation of phospholipase AZ activity (H. Tokuda, Y. Oiso and 0. Kozawa, J Cell Biochem. 48: 262-268, 1992). In the present study, we investigated the effects of vitamin D3 on PGF,,-induced PGEz synthesis in MC3T3-El cells. The pretreatment with 1,25dihydroxyvitamin D, [1,25-(OH),D,], an active form of vitamin D,, significantly inhibited the PGF,,-induced PGE, synthesis in a dose-dependent manner in the range between 1 pM and 1 nM. This inhibitory effect of 1,25-(OH),D3 was dependent on the time of pretreatment up to 8 h. On the contrary, the pretreatment with 24,25dihydroxyvitamin D3, an inactive form of vitamin D, had little effect on the PGF,a -induced PGEz synthesis in these cells. However, the pretreatment with 1,25(OH)*DJ no longer affected the amplification of PGF&nduced PGEl synthesis by 12-Q-tetradecanoylphorbol13-acetate, a PKC activator. These results strongly suggest that 1,25-(OH),D, inhibits PGF,-induced PGEZ synthesis in osteoblast-like cells, however, the activation of PKC reverses this inhibitory effect of 1,25-(OH)*D3.
INTRODUCTION
specific intracellular receptors and subsequently activating the expression of gene network ( 13 ). The receptor for 1,25-(OH)2D, has been shown to exist in osteoblasts, including MC3T3-El cells (12, 14). It has been reported that 1.25-(OH),D, inhibits PGE,-induced Ca” mobilization in rat osteosarcoma UMR-106 cells ( 15) and adenylate cyclase activity induced by parathyroid hormone (PTH), known as a potent bone resorbing hormone, in rodent osteoblasts (16) and rat osteosarcoma ROS 17/2.8 cells (17). In addition, it has been shown that 1,25-(OH),D, decreases basal PGE production in osteoblastic cells derived from human bone (18) and induces a little increase in basal PGE,! production in MC3T3-El cells (19). In recent studies (20,21), we have also shown that the pretreatment with 1,25-(OH),D, inhibits PGE*-induced CAMP accumulation, PI hydrolysis and Ca’+ influx in MC3T3-El cells. These findings suggest that 1,25-(OH),D, affects osteoblast functions through modulating the intracellular signaling system by other bone resorbing agents. In the present study, we examined the effect of 1,25-(OH)2D3 on PGF,-induced PGE? synthesis in osteoblast-like MC3T3-El cells. Herein, we show that 1,25-(OH),D3 inhibits the PGF,,-induced PGE? synthesis in MC3T3-El cells, however, the activation of PKC reverses this inhibitory effect of 1.25(OH),D, in these cells.
Prostaglandins (PGs) are recognized to act as local modulators in osteoblasts (1). Among them, PGE2 is well-known to be a potent bone resorbing agent (1, 2), and it has been reported that PGE, is a major eicosanoid product in osteoblasts including MC3T3-El cells (2, 3), which have been derived from newborn mouse calvaria (3, 5). On the other hand, PGF2,, a less potent stimulator of bone resorption than PGE? (2, 6), has been shown to stimulate proliferation and inhibit differentiation of osteoblasts (I, 7). It has recently been reported that PGF2, stimulates PGE, production in cultured neonatal mouse calvaria (6). In previous studies (8, 9), we have demonstrated that phosphoinositide (PI) hydrolysis induced by PGF,, is a pertussis toxin-sensitive event and that the activation of protein kinase C (PKC) amplifies PGF,,-induced PGEl synthesis through the potentiation of phospholipase A1 activity in MC3T3-El cells. I ,25Dihydroxyvitamin D, [1,2~5-(OH)~D,l is wellknown as a bone resorbing agent (10-12). It is generally accepted that the effects of 1,25-(OH),D3, as well as other steroid hormones, are exerted through binding to
Date received 8 November 1993 Date accepted 20 December 1993 77
28
Prostaglandins
MATERIALS
Leukotrienes
and Essential Fatty Acids
AND METHODS
RESULTS
Materials 1,25-(OH)*D3 and 24,25dihydroxyvitamin D3 [24,25(OH),D,] dissolved in ethanol were provided by Roche Pharmaceutical Co (Tokyo, Japan). PGE2 [ i251]assay system was purchased from Amersham Japan (Tokyo, Japan). PGE2, PGF,,, 12-Q-tetradecanoylphorbol13acetate (TPA) and essentially fatty acid-free bovine serum albumin (BSA) were purchased from Sigma Chemical Co (St Louis, MO). Other materials and chemicals were obtained from commercial sources. PGE2 and PGF2, were dissolved in ethanol. TPA was dissolved in dimethyl sulfoxide. The maximum concentration of ethanol or dimethyl sulfoxide in the culture medium was 0. l%, and this did not affect the assay for PGE2.
Effects of VDJ on PGE,,-induced MC3T3-El cells
PGE2 synthesis
in
PGF?, (10 pM) increased PGEz synthesis time dependently up to 3 h in MC3T3-El cells (Fig. 1). The 8 h pretreatment. with 1,25-(OH),D,, an active form of VD, (11, 12) which by itself had little effect on PGEZ synthesis in these cells (data not shown), significantly inhibited the PGFzdinduced PGE? synthesis (Fig. 1). On the contrary, the pretreatment with 24,25-(OH),DX, an inactive form of VD? (11, 12), had little effect on the PGE, synthesis (Fig. 1). The inhibitory effect of 1,25-(OH)2Di on the PGF,,-induced PGE2 synthesis was dose-dependent in the range between 1 pM and 1 nM (Fig. 2). The inhibitory effect of 1,25-(OH),D, on PGF2,induced PGEZ synthesis was dependent on the time of pretreatment up to 8 h in MC3T3-El cells (Fig. 3).
Cell culture Cloned osteoblast-like MC3T3-El cells 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-mm diameter dishes in 2 ml of CX-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.
Effect of 1,25-(OH),D3 on the amplification by TPA of PGFza-induced PGEt synthesis in MC3T3-El cells In a previous study (9), we have shown that TPA, a potent PKC activator (22), enhances PGF,,-induced synthesis in osteoblast-like MC3T3-El cells.
I
I
I
I
I
Assay for PGE2 After the pretreatment with various doses of vitamin D, (VD,) for the indicated periods, the medium was then removed. The cultured cells were washed four times with 1 ml of an assay buffer [lo mM 4-(2-hydroxyethyl)I-piperazineethanesulfonic acid, pH 7.4, 135 mM NaCl, 5 mM KCl, 1 mM MgS04 and 1 mM CaCl,]. The cells were preincubated subsequently with 1 ml of the assay buffer containing 0.1% essentially fatty acid-free BSA at 37°C for 20 min, and then stimulated by PGF,, for the indicated periods. The medium was then collected and PGEz in the medium was measured with a radioimmunoassay kit. When indicated, the cells were pretreated with TPA for 20 min. 0
0
Determination The radioactivity of ‘Z51-samples was determined Aloka ARC-300 auto well gamma system.
Statistical
I
with an
analysis
The data were analyzed by Student’s t-test and P < 0.05 was considered significant. All data are presented as the mean + SEM of triplicate determinations.
1
2
Time
3
(h)
Effects of VD3 on PGF,,-induced PGE, synthesis in MC3T3El cells: time course of PGE: synthesis. The cultured cells were pretreated with 1 nM 1,25-(OH)2Dj (0). 1 nM 24,25-(OH),D, (0) or vehicle (A) for 8 h, and then stimulated by 10 pM PGFza for the indicated periods. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean f SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P c 0.05 compared to the value treated with 24.S(OH),D, or vehicle.
Effect of Vitamin D, on Prostaglandin
8
8
6
6
E, Synthesis m Osteoblast-like
Cells
4
OGG 0
12
I 11 -Log
10
9
(VD3), M
oT 0
’
1
’
2
I
I
I
4
6
8
29
1
Time (h)
Fig. 2 Dose-dependent effects of VD, on PGFz,-induced PGEz synthesis in MC3T3-El cells. The cultured cells were pretreated with various doses of I .25-(OHj4D, (0) or 24,25-(OHLD, (0) for 8 h. and then stimulated by IO pM PGF2,1 for 3 h. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean k SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value treated with 24.2S-(OH)2D1.
Fig. 3 Effects of VD, on PGF?,-induced PG& synthesis in MC3T3El cells: time course of pretreatment. The cultured cells were pretreated with I nM I ,2.5-(OHJ2D, (0) or I nM 24,25(OH)*D, (0) for the indicated periods, and then stimulated by 10 pM PGF,, for 3 h. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean + SEM of triplicate determinations, Similar results were obtained with two-additional and different cell preparations. *P < 0.05 compared to the value treated with 24.25.(OH):D,.
Thus, we investigated the effect of 1,25-(OH)1D, on this amplification by TPA. The 8 h pretreatment with 1.25(OH),D, ( 1 nM) had no effect on the enhancement by TPA (0.1 pM) of PGFza (10 PM)-induced PGE2 synthesis in MC3T3-El cells (Fig. 4).
MC3T3-El cells (18. 19). However, the effect of !,25(OH)?D, on PGEz synthesis induced by other bone resorbing agents has not yet been reported, and to our knowledge, this is the first report on it. In addition, we have recently shown that the pretreatment with 1,25(OH)?D, inhibits PGEz-induced CAMP accumulation, PI hydrolysis and Ca” influx in MC3T3-El cells (20, 21). Therefore, our findings as a whole strongly suggest that 1.2%(OH),D3 inhibits both the synthesis and the intracellular signaling of PGEl in osteoblast-like MC3TSEl cells. In a previous study (9), we have shown that the activation of PKC by TPA markedly amplifies the PGF,,induced arachidonic acid release and PGE, synthesis through the potentiation of PLAl activity in osteoblastlike MC3T3-El cells. We demonstrated here that 1,25of (OH)2D3 no longer affected the amplification PGF,,-induced PGE, synthesis by TPA in these ceils. These results suggest that the activation of PKC reverses the inhibitory effect of 1,25-(OH)*D3 on PGF,,-induced PGE? synthesis in osteoblast-like cells. It has been recently reported that human VDI receptor is phosphorylated by PKC (23, 24). In our present study, however, 1,25-(OH),D7 was added to the cells 8 h before the pretreatment with TPA. Namely, the pretreatment with 1,25-(OH)2D3 preceded the TPA pretreatment. It is
DISCUSSION In a previous study (9). we have shown that PGFza significantly increases PGE, synthesis in osteoblast-like MC3T3-El cells. It has been reported that PGE, is a potent bone resorbing agent and a major eicosanoid product in osteoblasts including MC3T3-El cells (l-3). On the other hand, PGFza has been reported to stimulate bone resorption but to be less potent than PGEl (2, 6). Thus, our previous finding indicates that there is a positive feedback mechanism of bone resorption induced by PGF,, in MC3T3-El cells. In the present study, we showed that the pretreatment with 1,25-(OH)2D3, an active form of VD, (11, 12), significantly reduced the PGF,,-induced PGE? synthesis, whereas 24,25-(OH)*D3, an inactive form of VD, (11, 12), had no effect on PGF?,-induced PGEz synthesis in MC3T3-El cells. It has been reported that 1,25-(OH),D3 slightly modulates the basal production of PGE in osteoblasts including
30
Prostaglandins
Leukotrienes
and Essential Fatty Acids
References
4
0
TPA 1,25-(OH2)th
m
I +
-
+
+
-
+
Fig. 4 Effect of 1,25(OH)?D, on the amplification by TPA of PGF,,-induced PGE, synthesis in MC3T3-El cells. The cultured cells were pretreated with 1 nM 1.25(OH),D, or vehicle for 8 h, then pretreated with 0.1 pM TPA for 20 min. and then stimulated by 10 pM PGF,, for 3 h. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean f SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value stimulated by PGF2, alone.
certain that TPA reversed the inhibitory effect of I,25(OH),D, on PGF,-induced PGE, synthesis in MC3T3-El cells, although it remains unclear whether the phosphorylation of VD, receptor is responsible for our finding in this study. We previously showed that the activation of PKC stimulates the DNA synthesis, and suppresses the increase in alkaline phosphatase activity a marker of mature osteoblast phenotype (25), induced by parathyroid hormone and FCS in MC3T3-El cells (26, 27). In addition, we have recently reported that PKC-activation suppresses 45Ca accumulation into extracellular matrix, namely calcification, in long-term cultured MC3T3-El cells (28). Therefore, our results as a whole strongly suggest that the activation of PKC directs osteoblasts to promoting bone resorption. In conclusion, our results strongly suggest that 1,25(OH),D, inhibits the PGFzdinduced PGE, synthesis in osteoblast-like cells, and the activation of PKC reverses this inhibitory effect of 1,25-(OH),D,. Acknowledgement This investigation was supported in part by a Grant-in-Aid for scientific research from the Ministry of Education, Science, and Culture of Japan.
1 Nijweide P J, Burger E H, Feyen J H M. Cells of bone: proliferation, differentiation, and hormonal regulation. Physiol Rev 1986; 66: 855-886. 2. Raisz L G. Martin T J. Prostaglandins in bone and mineral metabolism. In: Peck W A, ed. Bone and mineral research. Annual 2. Amsterdam: Elsevier, 1983: 2863 10. 3. Yokota K, Kusaka M. Oshima T et al. Stimulation of prostaglandin I$ synthesis in cloned osteoblastic cells of mouse (MC3T3-El) by epidenal growth factor. J Biol Chem 1986; 261: 15410-15415. 4 Kodama H, Amagai Y, Sudo H, Kasai S, Yamamoto S. Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Jpn J Oral Biol 1981; 23: 899-90 1. 5. Sudo H, Kodama H, Amagai Y. Yamamoto S, Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 1983; 96: 191-198. 6. Raisz L G, Alander C B, Fall P M, Simmons H A. Effects of prostaglandin Fla on bone formation and resorption in cultured neonatal mouse calvariae: role of prostaglandin E, production. Endocrinology 1990; 126: 1076-1079. 7. Hakeda Y, Hotta T, Kurihara N et al. Prostaglandin E, and Fza stimulate differentiation and proliferation. respectively, of clonal osteoblastic MC3T3-El cells by different second messengers in vitro. Endocrinology 1987: 121: 19661974. 8 Miwa M, Tokuda H, Tsushita K et al. Involvement of pertussis toxin-sensitive GTP-binding protein in prostaglandin F,,-induced phosphoinositide hydrolysis in osteoblast-like cells. Biochem Biophys Res Commun 1990; 171: 1229-1235. 9 Tokuda H, Oiso Y. Kozawa 0. Protein kinase C activation amplifies prostaglandin Fz,-induced prostaglandin E? synthesis in osteoblast-like cells. J Cell Biochem 1992: 48: 262-268. 10. Raisz L G. Trummel C L, Holick M F, DeLuca H F. 1.25. Dihydroxycholecalciferol: a potent stimulator of bone resorption in tissue culture. Science 1972: 175: 768-769. 11 Stem P H. The D vitamins and bone. Pharmacol Rev 1980; 32: 47-80. 12. DeLuca H F, Schnoes H K. Vitamin D: recent advances. Annu Rev Biochem 1983; 52: 4 11439. 13. Evans R M. The steroid and thyroid hormone receptor superfamily. Science 1988; 240: 889-895. 14. Kurihara N, Ishizuka S. Kiyoki M. Haketa Y, Ikeda K. Kumegawa M. Effects of 1,25-dihydroxyvitamin D1 on osteoblastic MC3T3-El cells. Endocrinology__ 1986; 118: 94C-947. 15. Green J, Kleeman C R, Schotland S, Ye L H. 1,25(OH)*D, blunts hormone-elevated cytosolic Ca’+ in osteoblast-like cells. Am J Physiol 1992; 263: El070-E1076. 16. Chen T L. Feldman D. Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: the effects of 1,25(OH)> vitamin D, and its interaction with glucocorticoids. Calcif Tissue Int 1984; 36: 580-585. and 11. Catherwood B D. 1,25-Dihydroxycholecalciferol glucocorticosteroid regulation of adenylate cyclase in an osteoblast-like cell line. J Biol Chem 1985; 260: 736-743. 18. MacDonald B R, Gallagher J A, Ahnfelt-Ronne I, Beresford J N, Gowen M, Russell R G G. Effects of bovine parathyroid hormone and 1,25-dihydroxyvitamin D, on the production of prostaglandins by cells derived from human bone. FEBS Lett 1984; 169: 49-52. 19. Schwarz Z, Dennis R, Bonewald L, Swain L, Gomez R, Boyan BD. Differential regulation of prostaglandin E, synthesis and phospholipase A, activity by 1,25-(OH),D, in three osteoblast-like cell lines (MC-3T3-El, ROS 17/ 2.8, and MG-63). Bone 1992; 13: 51-58. 20. Kozawa 0, Tokuda H, Kotoyori J, Suzuki A. Ito Y, Oiso Y. Modulation of prostaglandin E,-induced CaZC influx by steroid hormones in osteoblast-like cells. Prost Leuko Essent Fatty Acids 1993; 49: 71 l-714.
Effect of Vitamin D, on Prostaglandin 3 I. Tokuda H, Kotoyori .I, Suzuki A. Oiso Y, Kozawa 0. Effects of vitamin D, on signaling by prostaglandin Ez in osteoblast-like cells. J Cell Biochem 1993; 52: 220-226. 22. Nishizuka Y. The role of protein kinase C in cell surface Ggnal transduction and tumour promotion. Nature 1984: 308: 6Y3-69X. 23. Hsteh J-C, Jurutka P W. Galligan M A et al. Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 5 I. a residue crucial to its tram-activation function. Proc Natl Acad Sci USA 1991: 88: Y3 15-93 19. 24. Hsteh J-C. Jurutka P W. Nakajima S et al. Phosphorylation of the human vitamin D receptor by protein kina\c C. J Biol Chem 1993: 268: 15118-15126. 25. Stein G S. Lian J B. Owen T A. Relationship of cell
E? Synthesis in Osteoblast-like
Cells
growth to the regulation of tissue-specific gene expresston during osteoblast differentiation. FASEB J 1990: 4: 3111-3123. 26. Kozawa 0. Takatsuki K, Kotake K, Yoneda M. Oiso Y. Saito H. Possible involvement of protein kinare C in proliferation and differentiation of osteoblast-lthe cell\. FEBS Lett 1989; 243: 183-185. 27. Miwa M. Kozawa 0, Tokuda H et al. Effects 01 hypergravity on proliferation and differentiation ot osteoblast-like cells. Bone Miner 190 I: 14: 15-25. 28. Kozawa 0. Miwa M. Tokuda H, Kotoyort J. Oiao Y. Activation of protein kinase C inhibits ‘Q&accumulation in cultures of osteoblast-like cells: possible involvement of insulin-like growth factor-l. Bone Miner 1992: 19: 2355243.
3
Fatty AC&
tlYY4)Sl,27-31
Effect of Vitamin D, on Prostaglandin
E, Synthesis in Osteoblast-like
Cells
A. Suzuki, H. Tokuda, J. Kotoyori, Y. Ito. Y. Oiso and 0. Kozawa”
of
First Depurtment Internal Medicine, Nagoya University School of Medicine. Nagoya 466, Japan. *Department Biochemistry, Institute for Developmental Research, Aichi Prefectwal Colony. Kasugai, Aichi 480-02, Jupa~ (Reprint requests to OK)
qf
ABSTRACT. We previously showed that prostaglandin (PG) Fzu stimulates PGEL synthesis in osteohlast-like MC3T3-El cells, and that the activation of protein kinase C (PKC) amplifies the effect of PGFzu through the potentiation of phospholipase AZ activity (H. Tokuda, Y. Oiso and 0. Kozawa, J Cell Biochem. 48: 262-268, 1992). In the present study, we investigated the effects of vitamin D3 on PGF,,-induced PGEz synthesis in MC3T3-El cells. The pretreatment with 1,25dihydroxyvitamin D, [1,25-(OH),D,], an active form of vitamin D,, significantly inhibited the PGF,,-induced PGE, synthesis in a dose-dependent manner in the range between 1 pM and 1 nM. This inhibitory effect of 1,25-(OH),D3 was dependent on the time of pretreatment up to 8 h. On the contrary, the pretreatment with 24,25dihydroxyvitamin D3, an inactive form of vitamin D, had little effect on the PGF,a -induced PGEz synthesis in these cells. However, the pretreatment with 1,25(OH)*DJ no longer affected the amplification of PGF&nduced PGEl synthesis by 12-Q-tetradecanoylphorbol13-acetate, a PKC activator. These results strongly suggest that 1,25-(OH),D, inhibits PGF,-induced PGEZ synthesis in osteoblast-like cells, however, the activation of PKC reverses this inhibitory effect of 1,25-(OH)*D3.
INTRODUCTION
specific intracellular receptors and subsequently activating the expression of gene network ( 13 ). The receptor for 1,25-(OH)2D, has been shown to exist in osteoblasts, including MC3T3-El cells (12, 14). It has been reported that 1.25-(OH),D, inhibits PGE,-induced Ca” mobilization in rat osteosarcoma UMR-106 cells ( 15) and adenylate cyclase activity induced by parathyroid hormone (PTH), known as a potent bone resorbing hormone, in rodent osteoblasts (16) and rat osteosarcoma ROS 17/2.8 cells (17). In addition, it has been shown that 1,25-(OH),D, decreases basal PGE production in osteoblastic cells derived from human bone (18) and induces a little increase in basal PGE,! production in MC3T3-El cells (19). In recent studies (20,21), we have also shown that the pretreatment with 1,25-(OH),D, inhibits PGE*-induced CAMP accumulation, PI hydrolysis and Ca’+ influx in MC3T3-El cells. These findings suggest that 1,25-(OH),D, affects osteoblast functions through modulating the intracellular signaling system by other bone resorbing agents. In the present study, we examined the effect of 1,25-(OH)2D3 on PGF,-induced PGE? synthesis in osteoblast-like MC3T3-El cells. Herein, we show that 1,25-(OH),D3 inhibits the PGF,,-induced PGE? synthesis in MC3T3-El cells, however, the activation of PKC reverses this inhibitory effect of 1.25(OH),D, in these cells.
Prostaglandins (PGs) are recognized to act as local modulators in osteoblasts (1). Among them, PGE2 is well-known to be a potent bone resorbing agent (1, 2), and it has been reported that PGE, is a major eicosanoid product in osteoblasts including MC3T3-El cells (2, 3), which have been derived from newborn mouse calvaria (3, 5). On the other hand, PGF2,, a less potent stimulator of bone resorption than PGE? (2, 6), has been shown to stimulate proliferation and inhibit differentiation of osteoblasts (I, 7). It has recently been reported that PGF2, stimulates PGE, production in cultured neonatal mouse calvaria (6). In previous studies (8, 9), we have demonstrated that phosphoinositide (PI) hydrolysis induced by PGF,, is a pertussis toxin-sensitive event and that the activation of protein kinase C (PKC) amplifies PGF,,-induced PGEl synthesis through the potentiation of phospholipase A1 activity in MC3T3-El cells. I ,25Dihydroxyvitamin D, [1,2~5-(OH)~D,l is wellknown as a bone resorbing agent (10-12). It is generally accepted that the effects of 1,25-(OH),D3, as well as other steroid hormones, are exerted through binding to
Date received 8 November 1993 Date accepted 20 December 1993 77
28
Prostaglandins
MATERIALS
Leukotrienes
and Essential Fatty Acids
AND METHODS
RESULTS
Materials 1,25-(OH)*D3 and 24,25dihydroxyvitamin D3 [24,25(OH),D,] dissolved in ethanol were provided by Roche Pharmaceutical Co (Tokyo, Japan). PGE2 [ i251]assay system was purchased from Amersham Japan (Tokyo, Japan). PGE2, PGF,,, 12-Q-tetradecanoylphorbol13acetate (TPA) and essentially fatty acid-free bovine serum albumin (BSA) were purchased from Sigma Chemical Co (St Louis, MO). Other materials and chemicals were obtained from commercial sources. PGE2 and PGF2, were dissolved in ethanol. TPA was dissolved in dimethyl sulfoxide. The maximum concentration of ethanol or dimethyl sulfoxide in the culture medium was 0. l%, and this did not affect the assay for PGE2.
Effects of VDJ on PGE,,-induced MC3T3-El cells
PGE2 synthesis
in
PGF?, (10 pM) increased PGEz synthesis time dependently up to 3 h in MC3T3-El cells (Fig. 1). The 8 h pretreatment. with 1,25-(OH),D,, an active form of VD, (11, 12) which by itself had little effect on PGEZ synthesis in these cells (data not shown), significantly inhibited the PGFzdinduced PGE? synthesis (Fig. 1). On the contrary, the pretreatment with 24,25-(OH),DX, an inactive form of VD? (11, 12), had little effect on the PGE, synthesis (Fig. 1). The inhibitory effect of 1,25-(OH)2Di on the PGF,,-induced PGE2 synthesis was dose-dependent in the range between 1 pM and 1 nM (Fig. 2). The inhibitory effect of 1,25-(OH),D, on PGF2,induced PGEZ synthesis was dependent on the time of pretreatment up to 8 h in MC3T3-El cells (Fig. 3).
Cell culture Cloned osteoblast-like MC3T3-El cells 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-mm diameter dishes in 2 ml of CX-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.
Effect of 1,25-(OH),D3 on the amplification by TPA of PGFza-induced PGEt synthesis in MC3T3-El cells In a previous study (9), we have shown that TPA, a potent PKC activator (22), enhances PGF,,-induced synthesis in osteoblast-like MC3T3-El cells.
I
I
I
I
I
Assay for PGE2 After the pretreatment with various doses of vitamin D, (VD,) for the indicated periods, the medium was then removed. The cultured cells were washed four times with 1 ml of an assay buffer [lo mM 4-(2-hydroxyethyl)I-piperazineethanesulfonic acid, pH 7.4, 135 mM NaCl, 5 mM KCl, 1 mM MgS04 and 1 mM CaCl,]. The cells were preincubated subsequently with 1 ml of the assay buffer containing 0.1% essentially fatty acid-free BSA at 37°C for 20 min, and then stimulated by PGF,, for the indicated periods. The medium was then collected and PGEz in the medium was measured with a radioimmunoassay kit. When indicated, the cells were pretreated with TPA for 20 min. 0
0
Determination The radioactivity of ‘Z51-samples was determined Aloka ARC-300 auto well gamma system.
Statistical
I
with an
analysis
The data were analyzed by Student’s t-test and P < 0.05 was considered significant. All data are presented as the mean + SEM of triplicate determinations.
1
2
Time
3
(h)
Effects of VD3 on PGF,,-induced PGE, synthesis in MC3T3El cells: time course of PGE: synthesis. The cultured cells were pretreated with 1 nM 1,25-(OH)2Dj (0). 1 nM 24,25-(OH),D, (0) or vehicle (A) for 8 h, and then stimulated by 10 pM PGFza for the indicated periods. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean f SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P c 0.05 compared to the value treated with 24.S(OH),D, or vehicle.
Effect of Vitamin D, on Prostaglandin
8
8
6
6
E, Synthesis m Osteoblast-like
Cells
4
OGG 0
12
I 11 -Log
10
9
(VD3), M
oT 0
’
1
’
2
I
I
I
4
6
8
29
1
Time (h)
Fig. 2 Dose-dependent effects of VD, on PGFz,-induced PGEz synthesis in MC3T3-El cells. The cultured cells were pretreated with various doses of I .25-(OHj4D, (0) or 24,25-(OHLD, (0) for 8 h. and then stimulated by IO pM PGF2,1 for 3 h. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean k SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value treated with 24.2S-(OH)2D1.
Fig. 3 Effects of VD, on PGF?,-induced PG& synthesis in MC3T3El cells: time course of pretreatment. The cultured cells were pretreated with I nM I ,2.5-(OHJ2D, (0) or I nM 24,25(OH)*D, (0) for the indicated periods, and then stimulated by 10 pM PGF,, for 3 h. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean + SEM of triplicate determinations, Similar results were obtained with two-additional and different cell preparations. *P < 0.05 compared to the value treated with 24.25.(OH):D,.
Thus, we investigated the effect of 1,25-(OH)1D, on this amplification by TPA. The 8 h pretreatment with 1.25(OH),D, ( 1 nM) had no effect on the enhancement by TPA (0.1 pM) of PGFza (10 PM)-induced PGE2 synthesis in MC3T3-El cells (Fig. 4).
MC3T3-El cells (18. 19). However, the effect of !,25(OH)?D, on PGEz synthesis induced by other bone resorbing agents has not yet been reported, and to our knowledge, this is the first report on it. In addition, we have recently shown that the pretreatment with 1,25(OH)?D, inhibits PGEz-induced CAMP accumulation, PI hydrolysis and Ca” influx in MC3T3-El cells (20, 21). Therefore, our findings as a whole strongly suggest that 1.2%(OH),D3 inhibits both the synthesis and the intracellular signaling of PGEl in osteoblast-like MC3TSEl cells. In a previous study (9), we have shown that the activation of PKC by TPA markedly amplifies the PGF,,induced arachidonic acid release and PGE, synthesis through the potentiation of PLAl activity in osteoblastlike MC3T3-El cells. We demonstrated here that 1,25of (OH)2D3 no longer affected the amplification PGF,,-induced PGE, synthesis by TPA in these ceils. These results suggest that the activation of PKC reverses the inhibitory effect of 1,25-(OH)*D3 on PGF,,-induced PGE? synthesis in osteoblast-like cells. It has been recently reported that human VDI receptor is phosphorylated by PKC (23, 24). In our present study, however, 1,25-(OH),D7 was added to the cells 8 h before the pretreatment with TPA. Namely, the pretreatment with 1,25-(OH)2D3 preceded the TPA pretreatment. It is
DISCUSSION In a previous study (9). we have shown that PGFza significantly increases PGE, synthesis in osteoblast-like MC3T3-El cells. It has been reported that PGE, is a potent bone resorbing agent and a major eicosanoid product in osteoblasts including MC3T3-El cells (l-3). On the other hand, PGFza has been reported to stimulate bone resorption but to be less potent than PGEl (2, 6). Thus, our previous finding indicates that there is a positive feedback mechanism of bone resorption induced by PGF,, in MC3T3-El cells. In the present study, we showed that the pretreatment with 1,25-(OH)2D3, an active form of VD, (11, 12), significantly reduced the PGF,,-induced PGE? synthesis, whereas 24,25-(OH)*D3, an inactive form of VD, (11, 12), had no effect on PGF?,-induced PGEz synthesis in MC3T3-El cells. It has been reported that 1,25-(OH),D3 slightly modulates the basal production of PGE in osteoblasts including
30
Prostaglandins
Leukotrienes
and Essential Fatty Acids
References
4
0
TPA 1,25-(OH2)th
m
I +
-
+
+
-
+
Fig. 4 Effect of 1,25(OH)?D, on the amplification by TPA of PGF,,-induced PGE, synthesis in MC3T3-El cells. The cultured cells were pretreated with 1 nM 1.25(OH),D, or vehicle for 8 h, then pretreated with 0.1 pM TPA for 20 min. and then stimulated by 10 pM PGF,, for 3 h. Values for unstimulated cells have been subtracted from each data point. Each value represents the mean f SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P < 0.05 compared to the value stimulated by PGF2, alone.
certain that TPA reversed the inhibitory effect of I,25(OH),D, on PGF,-induced PGE, synthesis in MC3T3-El cells, although it remains unclear whether the phosphorylation of VD, receptor is responsible for our finding in this study. We previously showed that the activation of PKC stimulates the DNA synthesis, and suppresses the increase in alkaline phosphatase activity a marker of mature osteoblast phenotype (25), induced by parathyroid hormone and FCS in MC3T3-El cells (26, 27). In addition, we have recently reported that PKC-activation suppresses 45Ca accumulation into extracellular matrix, namely calcification, in long-term cultured MC3T3-El cells (28). Therefore, our results as a whole strongly suggest that the activation of PKC directs osteoblasts to promoting bone resorption. In conclusion, our results strongly suggest that 1,25(OH),D, inhibits the PGFzdinduced PGE, synthesis in osteoblast-like cells, and the activation of PKC reverses this inhibitory effect of 1,25-(OH),D,. Acknowledgement This investigation was supported in part by a Grant-in-Aid for scientific research from the Ministry of Education, Science, and Culture of Japan.
1 Nijweide P J, Burger E H, Feyen J H M. Cells of bone: proliferation, differentiation, and hormonal regulation. Physiol Rev 1986; 66: 855-886. 2. Raisz L G. Martin T J. Prostaglandins in bone and mineral metabolism. In: Peck W A, ed. Bone and mineral research. Annual 2. Amsterdam: Elsevier, 1983: 2863 10. 3. Yokota K, Kusaka M. Oshima T et al. Stimulation of prostaglandin I$ synthesis in cloned osteoblastic cells of mouse (MC3T3-El) by epidenal growth factor. J Biol Chem 1986; 261: 15410-15415. 4 Kodama H, Amagai Y, Sudo H, Kasai S, Yamamoto S. Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Jpn J Oral Biol 1981; 23: 899-90 1. 5. Sudo H, Kodama H, Amagai Y. Yamamoto S, Kasai S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 1983; 96: 191-198. 6. Raisz L G, Alander C B, Fall P M, Simmons H A. Effects of prostaglandin Fla on bone formation and resorption in cultured neonatal mouse calvariae: role of prostaglandin E, production. Endocrinology 1990; 126: 1076-1079. 7. Hakeda Y, Hotta T, Kurihara N et al. Prostaglandin E, and Fza stimulate differentiation and proliferation. respectively, of clonal osteoblastic MC3T3-El cells by different second messengers in vitro. Endocrinology 1987: 121: 19661974. 8 Miwa M, Tokuda H, Tsushita K et al. Involvement of pertussis toxin-sensitive GTP-binding protein in prostaglandin F,,-induced phosphoinositide hydrolysis in osteoblast-like cells. Biochem Biophys Res Commun 1990; 171: 1229-1235. 9 Tokuda H, Oiso Y. Kozawa 0. Protein kinase C activation amplifies prostaglandin Fz,-induced prostaglandin E? synthesis in osteoblast-like cells. J Cell Biochem 1992: 48: 262-268. 10. Raisz L G. Trummel C L, Holick M F, DeLuca H F. 1.25. Dihydroxycholecalciferol: a potent stimulator of bone resorption in tissue culture. Science 1972: 175: 768-769. 11 Stem P H. The D vitamins and bone. Pharmacol Rev 1980; 32: 47-80. 12. DeLuca H F, Schnoes H K. Vitamin D: recent advances. Annu Rev Biochem 1983; 52: 4 11439. 13. Evans R M. The steroid and thyroid hormone receptor superfamily. Science 1988; 240: 889-895. 14. Kurihara N, Ishizuka S. Kiyoki M. Haketa Y, Ikeda K. Kumegawa M. Effects of 1,25-dihydroxyvitamin D1 on osteoblastic MC3T3-El cells. Endocrinology__ 1986; 118: 94C-947. 15. Green J, Kleeman C R, Schotland S, Ye L H. 1,25(OH)*D, blunts hormone-elevated cytosolic Ca’+ in osteoblast-like cells. Am J Physiol 1992; 263: El070-E1076. 16. Chen T L. Feldman D. Modulation of PTH-stimulated cyclic AMP in cultured rodent bone cells: the effects of 1,25(OH)> vitamin D, and its interaction with glucocorticoids. Calcif Tissue Int 1984; 36: 580-585. and 11. Catherwood B D. 1,25-Dihydroxycholecalciferol glucocorticosteroid regulation of adenylate cyclase in an osteoblast-like cell line. J Biol Chem 1985; 260: 736-743. 18. MacDonald B R, Gallagher J A, Ahnfelt-Ronne I, Beresford J N, Gowen M, Russell R G G. Effects of bovine parathyroid hormone and 1,25-dihydroxyvitamin D, on the production of prostaglandins by cells derived from human bone. FEBS Lett 1984; 169: 49-52. 19. Schwarz Z, Dennis R, Bonewald L, Swain L, Gomez R, Boyan BD. Differential regulation of prostaglandin E, synthesis and phospholipase A, activity by 1,25-(OH),D, in three osteoblast-like cell lines (MC-3T3-El, ROS 17/ 2.8, and MG-63). Bone 1992; 13: 51-58. 20. Kozawa 0, Tokuda H, Kotoyori J, Suzuki A. Ito Y, Oiso Y. Modulation of prostaglandin E,-induced CaZC influx by steroid hormones in osteoblast-like cells. Prost Leuko Essent Fatty Acids 1993; 49: 71 l-714.
Effect of Vitamin D, on Prostaglandin 3 I. Tokuda H, Kotoyori .I, Suzuki A. Oiso Y, Kozawa 0. Effects of vitamin D, on signaling by prostaglandin Ez in osteoblast-like cells. J Cell Biochem 1993; 52: 220-226. 22. Nishizuka Y. The role of protein kinase C in cell surface Ggnal transduction and tumour promotion. Nature 1984: 308: 6Y3-69X. 23. Hsteh J-C, Jurutka P W. Galligan M A et al. Human vitamin D receptor is selectively phosphorylated by protein kinase C on serine 5 I. a residue crucial to its tram-activation function. Proc Natl Acad Sci USA 1991: 88: Y3 15-93 19. 24. Hsteh J-C. Jurutka P W. Nakajima S et al. Phosphorylation of the human vitamin D receptor by protein kina\c C. J Biol Chem 1993: 268: 15118-15126. 25. Stein G S. Lian J B. Owen T A. Relationship of cell
E? Synthesis in Osteoblast-like
Cells
growth to the regulation of tissue-specific gene expresston during osteoblast differentiation. FASEB J 1990: 4: 3111-3123. 26. Kozawa 0. Takatsuki K, Kotake K, Yoneda M. Oiso Y. Saito H. Possible involvement of protein kinare C in proliferation and differentiation of osteoblast-lthe cell\. FEBS Lett 1989; 243: 183-185. 27. Miwa M. Kozawa 0, Tokuda H et al. Effects 01 hypergravity on proliferation and differentiation ot osteoblast-like cells. Bone Miner 190 I: 14: 15-25. 28. Kozawa 0. Miwa M. Tokuda H, Kotoyort J. Oiao Y. Activation of protein kinase C inhibits ‘Q&accumulation in cultures of osteoblast-like cells: possible involvement of insulin-like growth factor-l. Bone Miner 1992: 19: 2355243.
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