Prostaglandins
43:27 I-280,
1992
INHIBITORY EFFECT OF 17 fi -BSTRADIOL ON PROSTAGLANDIN B INDUCED PHOSPHOINOSITIDB HYDROLYSIS IN OSTBOBLAST-LIKE C J&S H. Tokuda, 4. Yoneda, Y. Oiso and 0. Ko#sawa*' Firat Department of Internal Hedicine, Nagoya University School of Medicine, Nagoya 466, Japan and Department of Biochemietry, Inetitute For Developmental Research, Aichi Prefectural Colony, Kasugai 480-03, Japan
ABSTRACT the effect of estradiol on PGE2-induced We examined and CAMP production in cloned phosphoinositide hydrolysis 17 6 -Estradiol pretreatment osteoblast-like MC3T3-El cells. significantly inhibited the formation of inositol phosphates manner between 1 induced by 10 uM PGE2 in a dose-dependent pM and 10 nM. This effect of 17 a -estradiol was dependent on the time of pretreatment and submaximum inhibition was However, 17 S -estradiol had little effect observed at 4 h. on the formation of inositol phosphates induced by 20 mM The CAMP production protein activator. NaF, a GTP-binding by 176 -estradiol. These induced by PGE2 was not influenced results suggest that 17 a-estradiol modulates the signal transduction by PGE2 and that the effect seems to be exerted between PGE2 receptor and the GTP-binding protein coupled to phospholipase C in osteoblast-like MC3T3-El cells. INTRODUCTION Bone metabolism is regulated by two functional cell and osteoclasts (1). The former cells types, osteoblasts are responsible for bone formation and the latter for bone According to current understanding that the resorption. receptors of bone resorbing agents such as parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 are found in osteoblasts are also considered to play osteoblasts, important roles in the regulation of bone resorption (1). Several studies have been reported, suggesting that 17 B estradiol modulates osteoblast functions in vitro. In 17 S-estradiol reduces the osteosarcoma UMR-106 cells, growth rates, increases alkaline phosphatase activity and secretion of insulin-like growth factors (2,3). It has also been reported that prolonged incubation with 17 (3-estradiol enhances proliferation and type I collagen mRNA in _____-___-----'To whom
correspondence
should
Copyright 0 1992 Butterworth-Heinemann
be addressed
272
Prostaglandins
These actions osteoblast-like cells from rat calvaria (4). of estradiol have been confirmed by the evidence that the In addition, receptor of estrogen is in osteoblasts (5,6). it has recently been reported that 17 8-estradiol reduces PTH-stimulated CAMP production in osteosarcoma SaOS-2 cells and inhibits PTH(7) and osteoblast-like RCT cells (81, mouse calvariae (9). induced PGE2 production in cultured These results suggest that estradiol modulates the response to certain stimuli in osteoblasts. Prostaglandins are considered to be important As for PGE2, regulators of osteoblasts as autacoids (1,101. which is known as a potent bone resorbing agent (1,111, it is well documented that PGE2 modulates diverse cellular functions of osteoblasts through the activation of adenylate cyclase (11-13). Recently, it has been reported that PGE2 stimulates phosphoinositide (PI) hydrolysis in UMR-106 cells (14) and we also reported that the PGE2-induced PI hydrolysis is mediated by a pertussis toxin-sensitive GTPbinding protein (G protein) in osteoblast-like MC3T3-El cells (15) cloned from newborn mouse calvaria (16,17). In the present study, we investigated the effect of estradiol on the PI hydrolysis and the CAMP production induced by PGE2 in MC3T3-El cells. Herein, we show that 17 @-estradiol reduces PGE2-induced PI hydrolysis without affecting PGE2induced CAMP production in osteoblast-like cells. METHODS
Materials m-[2-3H]inositol (81.5 Ci/mmol) was purchased from Amersham. PGE2, 17 a-estradiol, 17 B-estradiol and NaF were purchased from Sigma. The CAMP radioimmunoassay kit was provided by Yamasa Shoyu Co., Chiba, Japan. Other materials and chemicals were obtained from commercial sources. Cell Culture Cloned osteoblast-like MC3T3-El cells were generously provided by Dr. M. Kumegawa (Meikai University, Sakado, Japan) and maintained in u-minimum essential medium ( a-MEM) containing 10% fetal calf serum (FCS) at 37'C in a humidified atmosphere of 5% C02/ 95% air. The cells (5 x 104) were seeded into 35-mm diameter dishes in 2 ml of a-MEM containing 10% FCS. After 5 days (just after confluency), the medium was exchanged for 2 ml of u -MEM containing 0.3% FCS. The cells were used for experiments 48 h thereafter. In the experiments for the formation of inositol phosphates, the medium was exchanged for 2 ml of inositol-free CX-MEM containing 0.3% FCS. Measurement of the Formation of Inositol Phosphates The cultured cells were labeled with w-[2-3Hlinositol
Prostaglandins
273
(3 uCi/dish) for 48 h. After the pretreatment with various doses of estradiol for indicated periods, the cells were preincubated with 10 mM LiCl in 1 ml of an assay buffer (5 acid, pH mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 7.4 containing 150 mM NaCl, 5 mM KCl, 5.5 mM glucose, 0.8 mM MgS04 and 1 mM CaC12) containing 0.01% bovine serum albumin (BSA) at 37°C for 10 min and then stimulated by PGE2 or NaF terminated by 15% for 10 min. The reaction was trichloroacetic acid. The acid supernatant was treated with diethyl ether to remove the acid and neutralized with NaOH. The supernatant was applied to a column of Dowex AGl-X8 formate form. The radioactive inositol monophosphate (IP, ), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were separated by successive elution of the column with 8 ml each of 0.1 M formic acid containing 0.2, 0.4 and 1.0 M ammonium formate, respectively (ia,i9). Measurement of the Production of CAMP After the pretreatment with various doses of estradiol for 4 h, the cells were preincubated with 0.5 mM 3isobutyl-I-methyl-xanthine in 1 ml of the assay buffer containing 0.01% BSA at 37OC for IO min and stimulated by 10 uM PGE2 for 5 min. The reaction was terminated by aspiration of the medium. Then the intracellular CAMP was extracted with 1 ml of 90% n-propanol (20). CAMP in the extracts was measured by a radioimmunoassay kit. RESULTS Ten uM PGE2 markedly stimulated the formation of IPl, cells (Fig. 1). The 4 hIP2 and IP3 in MC3T3-El pretreatment with 178 -estradiol, which by itself had no effect on PI hydrolysis (data not shown), significantly reduced the formation of each product induced by 10 uM PGE2 in a dose-dependent manner in the range between 1 pM and IO nM (Fig. I). The maximum inhibitory effects of 10 nM 17@estradiol for 4 h-pretreatment on the PGE2-stimulated formation of IP,, IP2 and IP3 were 278, 39%, and 388, respectively. As previously reported (15), PGE2 stimulated formation of each of the inositol phosphates dosedependently in the range between 10 nM and 10 PM. Ten nM 17 H -estradiol reduced the formation of IP,, IP2 and IP3 induced by various doses of PGE2 in the range between 10 nM and 10 uM to a similar extent (data not shown). This inhibitory effect of 10 nM 17 a -estradiol was dependent on the time of pretreatment with a maximal effect being reached at 4 to a h (Fig. 2). Pretreatment with 176 -estradiol for 4 h and 24 h led the PGE2-induced formation of IP3 in 38% and 43% reduction, respectively. which is structurally similar to 17 H 17 ~1 -Estradiol,
Prostaglandins
274
estradiol but has little biological activity, showed no effect on the formation of inositol phosphates induced by PGE2 (data not shown). a G protein activator (21), stimulated PI NaF, hydrolysis dose-dependently in the range between 5 mM and 40 As previously reported (15), the effect of 20 mM NaF mM. was very similar to that of 10 uM PGE2 (the formation of IP3 stimulated by 20 mM NaF was 2,280 A 151 cpm; mean & S.D. of 170 -estradiol had triplicate determinations). However, little effect on NaF-induced formation of inositol phosphates (the formation of IP3 stimulated by 20 mM NaF under 4 h-pretreatment of 10 nM 17 a -estradiol was 2,340 -+ 146 cpm).
c
26 24 22 * 20 **
** 18 R 4 2 0E! 0
12
11 10
9
8
17P-EstradiolC-logMI
Fig. 1. 17 B -Estradiol effect on PGE2-induced formation of inositol phosphates in MC3T3-El cells. The [3H]inositollabeled cells were pretreated with various doses of 17 B estradiol for 4 h, then stimulated by 10 uM PGE2 (0) for 10 min. Open circles (0) indicate the value of inositol phosphates without PGE2 stimulation or estradiol pretreatment. Each value (A), IP]; (H), IP2; (C), IP3. represents the mean -+ S.D. of triplicate determinations. These representative of three results are independent experiments. **ptO.Ol compared to control. *Q
43:27 I-280,
1992
INHIBITORY EFFECT OF 17 fi -BSTRADIOL ON PROSTAGLANDIN B INDUCED PHOSPHOINOSITIDB HYDROLYSIS IN OSTBOBLAST-LIKE C J&S H. Tokuda, 4. Yoneda, Y. Oiso and 0. Ko#sawa*' Firat Department of Internal Hedicine, Nagoya University School of Medicine, Nagoya 466, Japan and Department of Biochemietry, Inetitute For Developmental Research, Aichi Prefectural Colony, Kasugai 480-03, Japan
ABSTRACT the effect of estradiol on PGE2-induced We examined and CAMP production in cloned phosphoinositide hydrolysis 17 6 -Estradiol pretreatment osteoblast-like MC3T3-El cells. significantly inhibited the formation of inositol phosphates manner between 1 induced by 10 uM PGE2 in a dose-dependent pM and 10 nM. This effect of 17 a -estradiol was dependent on the time of pretreatment and submaximum inhibition was However, 17 S -estradiol had little effect observed at 4 h. on the formation of inositol phosphates induced by 20 mM The CAMP production protein activator. NaF, a GTP-binding by 176 -estradiol. These induced by PGE2 was not influenced results suggest that 17 a-estradiol modulates the signal transduction by PGE2 and that the effect seems to be exerted between PGE2 receptor and the GTP-binding protein coupled to phospholipase C in osteoblast-like MC3T3-El cells. INTRODUCTION Bone metabolism is regulated by two functional cell and osteoclasts (1). The former cells types, osteoblasts are responsible for bone formation and the latter for bone According to current understanding that the resorption. receptors of bone resorbing agents such as parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 are found in osteoblasts are also considered to play osteoblasts, important roles in the regulation of bone resorption (1). Several studies have been reported, suggesting that 17 B estradiol modulates osteoblast functions in vitro. In 17 S-estradiol reduces the osteosarcoma UMR-106 cells, growth rates, increases alkaline phosphatase activity and secretion of insulin-like growth factors (2,3). It has also been reported that prolonged incubation with 17 (3-estradiol enhances proliferation and type I collagen mRNA in _____-___-----'To whom
correspondence
should
Copyright 0 1992 Butterworth-Heinemann
be addressed
272
Prostaglandins
These actions osteoblast-like cells from rat calvaria (4). of estradiol have been confirmed by the evidence that the In addition, receptor of estrogen is in osteoblasts (5,6). it has recently been reported that 17 8-estradiol reduces PTH-stimulated CAMP production in osteosarcoma SaOS-2 cells and inhibits PTH(7) and osteoblast-like RCT cells (81, mouse calvariae (9). induced PGE2 production in cultured These results suggest that estradiol modulates the response to certain stimuli in osteoblasts. Prostaglandins are considered to be important As for PGE2, regulators of osteoblasts as autacoids (1,101. which is known as a potent bone resorbing agent (1,111, it is well documented that PGE2 modulates diverse cellular functions of osteoblasts through the activation of adenylate cyclase (11-13). Recently, it has been reported that PGE2 stimulates phosphoinositide (PI) hydrolysis in UMR-106 cells (14) and we also reported that the PGE2-induced PI hydrolysis is mediated by a pertussis toxin-sensitive GTPbinding protein (G protein) in osteoblast-like MC3T3-El cells (15) cloned from newborn mouse calvaria (16,17). In the present study, we investigated the effect of estradiol on the PI hydrolysis and the CAMP production induced by PGE2 in MC3T3-El cells. Herein, we show that 17 @-estradiol reduces PGE2-induced PI hydrolysis without affecting PGE2induced CAMP production in osteoblast-like cells. METHODS
Materials m-[2-3H]inositol (81.5 Ci/mmol) was purchased from Amersham. PGE2, 17 a-estradiol, 17 B-estradiol and NaF were purchased from Sigma. The CAMP radioimmunoassay kit was provided by Yamasa Shoyu Co., Chiba, Japan. Other materials and chemicals were obtained from commercial sources. Cell Culture Cloned osteoblast-like MC3T3-El cells were generously provided by Dr. M. Kumegawa (Meikai University, Sakado, Japan) and maintained in u-minimum essential medium ( a-MEM) containing 10% fetal calf serum (FCS) at 37'C in a humidified atmosphere of 5% C02/ 95% air. The cells (5 x 104) were seeded into 35-mm diameter dishes in 2 ml of a-MEM containing 10% FCS. After 5 days (just after confluency), the medium was exchanged for 2 ml of u -MEM containing 0.3% FCS. The cells were used for experiments 48 h thereafter. In the experiments for the formation of inositol phosphates, the medium was exchanged for 2 ml of inositol-free CX-MEM containing 0.3% FCS. Measurement of the Formation of Inositol Phosphates The cultured cells were labeled with w-[2-3Hlinositol
Prostaglandins
273
(3 uCi/dish) for 48 h. After the pretreatment with various doses of estradiol for indicated periods, the cells were preincubated with 10 mM LiCl in 1 ml of an assay buffer (5 acid, pH mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 7.4 containing 150 mM NaCl, 5 mM KCl, 5.5 mM glucose, 0.8 mM MgS04 and 1 mM CaC12) containing 0.01% bovine serum albumin (BSA) at 37°C for 10 min and then stimulated by PGE2 or NaF terminated by 15% for 10 min. The reaction was trichloroacetic acid. The acid supernatant was treated with diethyl ether to remove the acid and neutralized with NaOH. The supernatant was applied to a column of Dowex AGl-X8 formate form. The radioactive inositol monophosphate (IP, ), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were separated by successive elution of the column with 8 ml each of 0.1 M formic acid containing 0.2, 0.4 and 1.0 M ammonium formate, respectively (ia,i9). Measurement of the Production of CAMP After the pretreatment with various doses of estradiol for 4 h, the cells were preincubated with 0.5 mM 3isobutyl-I-methyl-xanthine in 1 ml of the assay buffer containing 0.01% BSA at 37OC for IO min and stimulated by 10 uM PGE2 for 5 min. The reaction was terminated by aspiration of the medium. Then the intracellular CAMP was extracted with 1 ml of 90% n-propanol (20). CAMP in the extracts was measured by a radioimmunoassay kit. RESULTS Ten uM PGE2 markedly stimulated the formation of IPl, cells (Fig. 1). The 4 hIP2 and IP3 in MC3T3-El pretreatment with 178 -estradiol, which by itself had no effect on PI hydrolysis (data not shown), significantly reduced the formation of each product induced by 10 uM PGE2 in a dose-dependent manner in the range between 1 pM and IO nM (Fig. I). The maximum inhibitory effects of 10 nM 17@estradiol for 4 h-pretreatment on the PGE2-stimulated formation of IP,, IP2 and IP3 were 278, 39%, and 388, respectively. As previously reported (15), PGE2 stimulated formation of each of the inositol phosphates dosedependently in the range between 10 nM and 10 PM. Ten nM 17 H -estradiol reduced the formation of IP,, IP2 and IP3 induced by various doses of PGE2 in the range between 10 nM and 10 uM to a similar extent (data not shown). This inhibitory effect of 10 nM 17 a -estradiol was dependent on the time of pretreatment with a maximal effect being reached at 4 to a h (Fig. 2). Pretreatment with 176 -estradiol for 4 h and 24 h led the PGE2-induced formation of IP3 in 38% and 43% reduction, respectively. which is structurally similar to 17 H 17 ~1 -Estradiol,
Prostaglandins
274
estradiol but has little biological activity, showed no effect on the formation of inositol phosphates induced by PGE2 (data not shown). a G protein activator (21), stimulated PI NaF, hydrolysis dose-dependently in the range between 5 mM and 40 As previously reported (15), the effect of 20 mM NaF mM. was very similar to that of 10 uM PGE2 (the formation of IP3 stimulated by 20 mM NaF was 2,280 A 151 cpm; mean & S.D. of 170 -estradiol had triplicate determinations). However, little effect on NaF-induced formation of inositol phosphates (the formation of IP3 stimulated by 20 mM NaF under 4 h-pretreatment of 10 nM 17 a -estradiol was 2,340 -+ 146 cpm).
c
26 24 22 * 20 **
** 18 R 4 2 0E! 0
12
11 10
9
8
17P-EstradiolC-logMI
Fig. 1. 17 B -Estradiol effect on PGE2-induced formation of inositol phosphates in MC3T3-El cells. The [3H]inositollabeled cells were pretreated with various doses of 17 B estradiol for 4 h, then stimulated by 10 uM PGE2 (0) for 10 min. Open circles (0) indicate the value of inositol phosphates without PGE2 stimulation or estradiol pretreatment. Each value (A), IP]; (H), IP2; (C), IP3. represents the mean -+ S.D. of triplicate determinations. These representative of three results are independent experiments. **ptO.Ol compared to control. *Q
