Rostqhdins Leukohienes and Essential Falty Acids (1992) 45, 267-274 0 Longman Group UK Ltd 1992

Proliferative Effect of PGDZ on Osteoblast-like Cells; Independent Activation of Pertussis Toxin-sensitive GTP-binding Protein from PGE2 or PGFZ, K. Tsushita, 0. Kozawa*, H. Tokuda,

Y. Oiso and H. Saito

First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466, Japan and *Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan (Reprint requests to OK) ABSTRACT.

PG& stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 PM in osteoblast-like MC3T3-El cells. PGDz had little effect on CAMP production, but caused very rapid enhancement of phosphoinositide (PI) hydrolysis dose-dependently between 10 nM and 10 PM. The formation of inositol trisphosphate (IP3) induced by PGDz reached the peak within 1 min and decreased thereafter, which is more rapid than that induced by PGE2 or PGFz, and both PG& and PGFza affected PGD&duced IPs formation additively. Pertussis toxin (PTX) inhibited both PGD&duced formation of inositol phosphates and DNA synthesis. The degree of these PTX (1 pg/ml)-induced inhibitions was similar. In addition, neomycin, a phosphdipase C inhibitor, inhibited PGDrinduced DNA synthesis as well as the formation of IP, and the patterns of both inhibitions were similar. In the cell membranes, PTXcatalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGDz. Time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGDz was apparently different from that by PG& or PGFzarThese results indicate that PGDz activates PTX-sensitive GTP-binding protein independently from PGE2 or PGF2,and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.

INTRODUCTION

be mediated through the activation of adenylate cyclase (10-11). Recently, it has been reported that PGs also induce phosphoinositide (PI) hydrolysis in osteoblasts (12, 13). In a previous report (14), we have demonstrated that the activation of protein kinase C (PKC), which is considered to be physiologically activated by diacylglycerol resulted from PI hydrolysis (15), stimulates DNA synthesis in osteoblast-like MC3T3-El cells derived from newborn mouse calvaria (16, 17). Moreover, we have shown that both PGF2, and PGEz stimulate PI hydrolysis, in which pertussis toxin (PTX)-sensitive GTP-binding protein (G protein) is involved in these cells (18, 19). As for PGD2, it has been reported to increase free cytosolic Ca*’ concentration but not CAMP production in rat osteosarcoma UMR-106 cells (6). However, the precise mechanism of PGDz-induced signal transduction in osteoblasts remains unclear. In this study, we investigated the effects of PGD2 on DNA synthesis, alkaline phosphatase (ALP) activity and intracellular signal transduction mechanism in osteoblast-like MC3T3-El cells. Our results suggest that PGD2 activates PTX-sensitive G

Prostaglandins (PGs), which are generally accepted to act through their binding to specific receptors (l), are considered to be important regulators of osteoblasts as autacoids (2, 3). As for PGD2, its various effects on proliferation and differentiation of osteoblasts have been reported (4-7). A basal release of PGDz has been reported in osteoblast-like cells from chick embryo and the release is stimulated by parathyroid hormone, thrombin, epidermal growth factor and 12-0-tetradecanoylphorbol-13acetate (8). Recently, PGD;? has been shown to be a major eicosanoid product in osteoblast-rich population from chick calvaria (9). These evidences suggest that PGDz is a potent modulator of osteoblasts and acts through paracrine or autocrine system. About the intracellular signaling systems of PGs in osteoblasts, their actions have been considered to

Date received 8 August 1991 Date accepted 30 September 1991 267

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protein independently from PGE2 or PGF2,, and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.

containing PGD2 with 0.3% FCS at 37°C for 24 h. The cells were scraped into 0.2% Nonidet P-40 and disrupted by sonication. The sonicates were centrifuged and the supernatant was assayed for ALP activity by the method of Lowry et al (21).

MATERIALS AND METHODS Materials

Assay for CAMP

dye-[2~H]inositol (92 Ciimmol) and [methyl3H]thymidine (84 Ci/mmol) were obtained from Amersham. [ar-32P]NAD (800 Ci/mmol) was from Du Pont/NEN. The CAMP radioimmunoassay kit was provided by Yamasa Shoyu Co, Chiba, Japan, PGDZ, neomycin sulfate and sodium sulfate were purchased from Sigma. PTX was from Funakoshi Pharmaceutical Co, Tokyo, Japan. The protein assay reagent kit was from Pierce. Other materials and chemicals were obtained from commercial sources.

The cultured cells were pretreated with 0.5 mM 3-

isobutyl-1-methylxanthine for 10 min in 1 ml of an assay buffer (5 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM KCl, 0.8 mM MgS04, 1 mM CaC12 and 5.5 mM glucose) containing 0.01% bovine serum albumin (BSA) and stimulated by PGD2 at 37°C. After the incubation for various periods, the medium was rapidly removed and CAMP was extracted with 1 ml of 90% n-propanol (22) and determined with a radioimmunoa~ay kit. Measurement of PI hydrolysis

Cell culture Cloned osteoblast-like cells, MC3T3-El, were generously provided by Dr M. Kumegawa (Meikai University, Sakado, Japan) and maintained in 01minimum essential medium (tu-MEM) containing 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% COd95% air. The cells (5 x 10’) were seeded into 35mm diameter dishes in 2 ml of ar-MEM containing 10% FCS. After 4 days, the medium was exchanged for 2 ml of ar-MEM containing 0.3% FCS. The cells were used for experiments 48 h thereafter. For the measurement of PI hydrolysis, the medium was exchanged for 2 ml of inositol-free o-MEM containing 0.3% FCS. When indicated, the cells were pretreated with PTX for 24 h or neomycin sulfate for 30 min in 1 ml of (u-MEM prior to the incubation with 1 FM PGDz. When using neomycin sulfate, to avoid an unspecific effect of sulfate, the concentration of sulfate in the control was adjusted by sodium sulfate and the medium was buffered with 20 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) at pH 7.4.

The cultured cells were labeled with myo-[2-3H]inositol(2 &/dish) for 48 h. The labeled cells were preincubated with 1 ml of the assay buffer containing 0.01% BSA and 10 mM LiCl at 37°C for 10 min. After the preincubation, the cells were stimulated by * PGD2 for the indicated periods. When we examined the additivity of PGEz or PGF& on PGD2-induced PI hydrolysis, the cells were stimulated by a combination of 0.1 FM PGD2 and 0.1 @M PGF,,, or by a combination of 0.1 PM PGDz and 0.5 PM PGE2 simultaneously. The reaction was terminated by 15% trichloroacetic acid. The acid supernatant was treated with diethyl ether to remove the acid and then neutralized with NaOH. The supernatant was applied to a column of Dowex AGl-X8 formate form. The radioactive inositol monophosphate (IPi), 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 (23, 24). UP-ri~yIation

of membrane proteins by PTX

Crude membranes were prepared at 4°C as described previously (19). Preactivation of PTX was The cultured cells were incubated in 1 ml of (Y- performed by incubating with 50 mM Tri@-ICl, pH 7.5 containing 10 mM dithiothreitol and 1 mM ATP MEM containing PGDp and 0.3% FCS at 37°C for at a concentration of 50 pg/ml at 30°C for 15 min. 28 h. Six h before harvest, the cells were pulsed with [methyl-3H]thymidine (0.3 &i/dish). The incu- The cell membranes (100 pg protein) were previously incubated in 50 ~1 of 100 mM Hepes, pH bation was terminated by 5% trichloroacetic acid. The radioactivity in the acid-insoluble materials was 7.5 containing 2.5 mM MgC12, 100 FM GTP, 120 mM NaCl, 5 mM KCl, 15 mM sodium acetate, determined (20). 1 mM EDTA, 10 mM glucose and 0.5% BSA with 1 PM PGDz or vehicle at 37°C for various periods, Measurement of ALP activity then these samples were subsequently combined me cultured cells were incubated in 1 ml of cu-MEM with 50 ~1 of the reaction mixture (200 mM Measurement of DNA synthesis

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Tri$-ICl, pH 8.0 containing 5 PM [~x-~~P]NAD (1000-2000 cpm/pmol), 20 mM thymidine, 1 mM EDTA, 2 mM dithiothreitol, 2 mM L-dimyristoyl and 20 &ml preactivated phosphatidylcholine PTX) and incubated at 30°C for 60 min. The reaction was terminated by adding 400 ~1 of 25 mM Tri@-ICI, pH 8.8 containing 192 mM glycine and 0.1% sodium dodecyl sulfate (SDS). The sample was subjected to SDS-polyacrylamide gel electrophoresis (11% polyacrylamide) as specified by Laemmli (25) and processed by autoradiography. Determinations The radioactivity of 3H-samples was determined with a Beckman LS 5OOOTD liquid scintillation spectrometer. Protein concentrations were determined by using a protein assay reagent kit with BSA as a reference protein. Statistical analysis

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PGD2 t-log MI Fig. 1 Dose-dependent stimulation of [%L]thymidineincorporation by PGD, in MC3T3-El cells. The cells were incubated with various doses of PGD, for 28 h. Six h before harvest, the cells were pulsed with [%]thymidine (0.3 @/dish). Each value represents the mean + SD of triplicate determinations.

All data are presented as the mean f SD of triplicate determinations. Data were analyzed by analysis of variance followed by Student’s t-test and a p < 0.05 was considered significant.

RESULTS Effects of PGDz on DNA synthesis and ALP activity To know how PGD2 influences cellular functions of osteoblast-like MC3T3-El cells, we studied the effects of PGD, on DNA synthesis and ALP activity, regarded as a marker of cell proliferation and of mature osteoblast phenotype (26)) respectively. PGD2 stimulated [3H]thymidine-incorporation into the acid-insoluble materials of these cells potently in a dose-dependent manner in the range between 10 nM and 10 PM (Fig. 1). PGD2 inhibited ALP activity in MC3T3-El cells in a dose-dependent manner between 10 nM and 10 PM (Fig. 2). PGDz (10 PM) resulted in the reduction of 63% compared with the value without PGD,. Effect of PGDz on CAMP production PGD2 had little effect on CAMP production in these cells. The maximum effect was observed at 1 min; control: 13.8 + 3.6 pmol/dish, 10 PM PGD;!: 19.2 + 4.8 pmol/dish. There was no significant difference from control. Effect of PGD2 on PI hydrolysis PGD2 markedly stimulated the formation of IP, , IP2

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Fig. 2 Dose-dependent inhibition of ALP activity by PGD, in MC3T3-El cells. The cells were incubated with various doses of PGD, for 24 h. Each value represents the mean + SD of triplicate determinations.

and IP3 in MC3T3-El cells (Fig. 3). The formation of IP3 reached the peak within 1 min and decreased thereafter. The PGDZ-induced formation of IP,, IP2 and IP3 was dose-dependent between 10 nM and 10 PM (Fig. 4). When the cells were stimulated by a combination of PGD2 and PGF2, or by a combination of PGD, and PGEz for 1 min, both PGF2, and PGE;? affected the PGDTinduced 1P3 formation additively, increasing it about 40% more than that stimulated by PGD2 alone; control: 322 + 20 cpm,

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and Essential Fatty Acids 3,

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TIME hnin) Fig. 3 The formation of IP,, IP, and IP, induced by PGD, in MC3T3-El cells: PTX-effect. The PHjnositol-labeled cells were pretreated with 1 p g/ml PTX (o----o) or vehicle (e-0) for 24 h and then stimulated by 10 FM PGD, for indicated periods. (A), IP,; (B), IP,; (C), IP,. Each value represents the mean + SD of triplicate determinations.

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Fig. 4 Dose-dependent stimulation of the formation of IP,, IP, and IP, induced by PGD, in MC3T3-El cells: PTX-effect. The [~~inositoI-ladled cells were pretreated with 1 &ml PTX (o-----o) or vehicle (e-e) for 24 h and then stimulated by various doses of PGD, for 1 min. (A), IP,; (B), IP,; (C), IP,. Each value represents the mean + SD of triplicate determinations.

PGD*: 1240 k 42 cpm, PGF&O6 + 48 cpm, PGE2: 1020 k 36 cpm, PGDz and PGF2& 1760 + 66 cpm*, PGD2 and PGE2: 1625 f: 54 cpm*. (*p C 0.05, compared to the value of PGD2, PGF2, or PGE2 alone.)

Effects of PTX on PGD+duced and DNA synthesis

PI hydrolysis

The formation of IP1, IP2 and IP3 induced by PGD2 was partially inhibited by the pretreatment of PTX (1 &ml) (Figs 3 & 4). The inhibition by PTX was

Effect of PGD, on Osteoblasts

Effects of neomycin on PGD&duced hydrolysis and DNA synthesis

z

271

PI

To further elucidate the role of PI hydrolysis in the PGD;?-induced DNA synthesis, we examined the effect of neomycin, which is known to be a potent phospholipase C inhibitor (27, 28). Neomycin significantly inhibited the PGDz-induced DNA synthesis dose-dependently as well as the formation of IPs, and the patterns of both inhibitions seem to be similar (Fig. 6).

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Effect of PGDl on PTX-cataIyzed ADP-ribosylation of 40-kDa protein -

PGO2

PGO2

PiX (1 rghl) Fig. 5 Effect of PTX on PGD,-induced DNA synthesis. After the pretreatment with 1 pg/ml PTX or vehicle for 24 h, the cells were stimulated by 1 PM PGD,. Values are expressed as a net increase compared with control. Each value represents the mean + SD of triplicate determinations. * Significantly different from control (p < 0.05). (Comparison of means was made using Student’s t-test.)

dose-dependent between 1 ng/ml and 1 kg/ml (data not shown). PTX (1 &ml) caused about 30% reduction of the formation of inositol phosphates induced by 10 PM PGD2. PGDzinduced DNA synthesis was also suppressed by PTX (Fig. 5). The degree of inhibition by PTX (1 &ml) of the PGDz induced DNA synthesis appears to be similar to that of the formation of inositol phosphates.

We have previously shown that PTX catalyzed ADP-ribosylation of a protein with an M, of about 40 000 in the cell membranes (18, 19). To clarify the coupling between PGD2 receptor and the PTX substrate, we monitored alterations in PTXcatalyzed ADP-ribosylation of the 40-kDa protein after treatment with PGD;!. Preincubation of the membranes with 1 PM PGDz in the presence of 2.5 mM MgClz and 100 JLM GTP attenuated subsequent ADP-ribosylation of the 40-kDa protein time-dependently. The attenuation reached the peak at 10 min and decreased thereafter up to 30 min (Fig. 7).

DISCUSSION In the present study, we showed that PGDz caused very rapid enhancement of PI hydrolysis, which was inhibited by PTX. PTX is well known to catalyze ADP-ribosylation of the o-subunit of certain G

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Fii. 6 Effect of neomycin on the PGD,-induced [3H]thymidine-incorporation (A) and the formation of [H]IP, (B). The cells were first pretreated with neomycin for 30 min and then stimulated by 1 PM PGD, for 28 h (A) and 1 min (B), respectively. Values are expressed as a net increase compared with control. Each value represents the mean Ifr SD of triplicate determinations.

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40-kDa -

Fig. 7 Effect of PGD, on PTX-catalyzed ADP-ribosylation of a 40-kDa protein in MC3T3-El cell membranes. Membranes were pretreated with 1 PM PGD, or vehicle for indicated periods, then PTX-catalyzed ADP-ribosylation was performed.

proteins including Gi and G,, and causes uncoupling of receptor to these G proteins (29-31). So, these evidences suggest that PTX-sensitive G protein is involved in PGD2-induced PI hydrolysis in osteoblast-like MC3T3-El cells. We have previously reported that both PGF2, and PGE2 stimulate PI hydrolysis in PTX-sensitive manner in these cells (18, 19). It must be considered that these PGs, which resemble one another structurally (l), might act through the same pathway. However, it seems unlikely from following reasons. First, the time course of the formation of IPs induced by these PGs was quite different. PGD2 caused very rapid enhancement of PI hydrolysis; IPs reached the peak within 1 min and decreased thereafter (Fig. 3). In contrast, as described previously (18, 19), the formation of IPs induced by PGF,, increases gradually up to 5 min and sustained up to 60 min (18), and the formation of IPs induced by PGEz is intermediate of them (19). Second, the additive effects of both PGF2, and PGE2 on the PGDzinduced IPs formation were observed in combination experiments. Third, the time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGDz was apparently different from each other. The attenuation by PGDz was maximum at 10 min and recovered gradually up to 30 min, but the attenuation by PGF*, is in a time-dependent manner up to 60 min (18) and that by PGEz is timedependent up to 30 min (19). When G proteincoupled receptor is stimulated by an agonist, the heterotrimeric G protein (@y-complex) is dissociated into its o-and fly-subunits (29). It is known that the a-subunit of PTX-sensitive G protein is ADP-ribosylated by this toxin only when it is in the

inactive heterotrimeric form, not in the active dissociated form (32). Therefore, the reduction in the PTX substrate activity of G protein is thought to reflect the dissociation of their subunits (33, 34). In addition, the recovery of the activity seems to reflect the re-association of their subunits into inactive copy-complex. So, our findings that PGD2 attenuated PTX-catalyzed ADP-ribosylation, which was maximum at 10 min and recovered thereafter, seem likely that PGDzinduced dissociation of the G protein into its active state was followed by re-association of the G protein. On the other hand, as we previously reported (18, 19), the attenuation induced by PGF2, or PGE2 is continued for more than 10 min, though the mechanism is not clear. These evidences suggest that PGD2 activates PTXsensitive G protein in a different fashion from PGE2 or PGFz~ We also examined the effect of PGD2 on adenylate cyclase-CAMP system, another important intracellular signal transduction system. As shown in this study, PGD2 had little effect on CAMP production in MC3T3-El cells, as well as reported in UMR-106 cells (6). In the signal transduction systems of PGD2 in MC3T3-El cells, our results suggest that adenylate cyclase-CAMP system is not a major pathway. About the effects of PGDz on the cellular function of these cells, we showed here that PGD2 increased DNA synthesis and decreased ALP activity, in turn, PGD2 stimulated the proliferation and suppressed the differentiation of mouse osteoblast-like MC3T3-El cells, since ALP activity is considered to be a mature osteoblast phenotype (26). As described above, PGD2 caused significant

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enhancement of PI hydrolysis, which has been known that PI is hydrolyzed by FhosFhoii~ase C, resuIting in the fo~at~o~ of inositoX phosphates and dia~y~~~y~ero~, the Iatter activates PKC (15,329, We have previously reported that the activation of PKC stimulates DNA synthesis and suppresses ALP activity in osteoblast-like MC3T3-El cells (14). So, it is possible that PGDz induces PI hydrolysis and subsequently activates PIK, resulting in proliferation of these cells. Therefore, we examined the effect of neomy~i~~ a phus~bu~iF~~ C inhibitor (27, 28), on PG~~indu~ed DNA synthesis We showed that neomycin inhibited PGD~-induced DNA synthesis as well as the formation of IP3, and the patterns of both inhibitions seem to be similar. Thus, these evidences indicate that proliferative effect of PGDz on osteobl~t”like MC3T3-El celfs is mediated through PI hydrofysis. In addition, we showed that PGDTinduced DNA synthesis was also inhibited by PTX and that the degree of PTX (I ~~ml)-induced inhibition appeared to be sirni~~r to that of the formation of inositol phosphates, These data support our notions, in conclusion, our results suggest that PGD, activates PTX-sensitive G protein inde~nde~t~y from PGE2 or PGF2, and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.

This ~~~~~~~g~~~n was ~uppor&xl in partby a Grant-hi-Aid for Scientific Research from the Ministry of Edueatian, Science and Culture of Japan and by a grant from the Suzuken ~ern~ri~~ Foundation.

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Proliferative effect of PGD2 on osteoblast-like cells; independent activation of pertussis toxin-sensitive GTP-binding protein from PGE2 or PGF2 alpha.

PGD2 stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 microM in osteoblast-like MC3T3-E1 cel...
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