ETA receptor mediates the signaling of endothelin-1 in osteoblast-like cells

Bone Vol. 21, No. 2 August 1997:143-146 ELSEVIER

Receptor Mediates the Signaling of Endothelin-1 in Osteoblast-Like Cells

ET A

A. S U Z U K I , 1 J. S H I N O D A , l Y. W A T A N A B E - T O M I T A , 1 N. O Z A K I , 1 Y. OISO, 1 and O. K O Z A W A 2 First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya, Japan 2 Department of Biochemistry, Institutefor Developmental Research, Aichi Human Service Center, Kasugai, Japan 4, pholipase C, Ca 2+ mobilization, and arachidonic acid release in osteoblasts including mouse osteoblast-like MC3T3-EI cells. 17"21-24 We recently showed that ET-I stimulates phosphatidylcholine-hydrolyzing phospholipase D independently of protein kinase C activated by phosphoinositide hydrolysis in MC3T3-E1 cells. 2° Three subtypes of ET receptors are recognized and denoted ETA, ETB, and ETc. 1,1L14 ET A receptors are selective for ET-1 and ET-2, whereas ETa ~ceptors bind to ET-1, ET-2, and ET-3 with equal potency. 11 ~e~l"c has also been identified, but its physiological significandd is uncertain. In bone tissue it has been shown that ET A and ET B receptors exist in osteoblasts. 13'~5,z2 ET-1 has been reported to induce Ca 2+ transients and to activate Na+-H + exchanger through ET A receptor in rat osteosarcoma UMR-106 cells,3 and it has recently been reported that ET-1 interacts with both ET A and ET B receptors to elicit Ca 2÷ transients in UMR-106 cells, 15 and that ET A and ET n receptors are coupled to phospholipase C in rat osteosarcoma ROS17/2.8 cells. 9 However, the exact roles of ET A and ETa receptorsmediated signaling in osteoblasts have not yet been fully clarified. In the present study, we examined the characteristics of the receptors mediating the ET-l-induced intracellular signaling pathway using receptor antagonists for ET A and ET B in osteoblast-like MC3T3-E1 cells. Herein, we show that the ET receptor subtype, ET A, is coupled to the three intracellular signaling pathways of ET-I: (1) phosphoinositide hydrolysis by phospholipase C; (2) phosphatidylcholine hydrolysis by phospholipase D; and (3) arachidonic acid release in MC3T3-E1 cells.

We previously reported that endothelin-1 (ET-1) stimulates phosphatidylcholine..hydrolyzing phospholipase D independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the characteristics of the receptors mediating ET-l.induced intracellular signaling pathway in MC3T3-E1 cells. Cyclo-nTrp-n-Asp-Pro-n-Val-Leu (BQ123), a selective ET A receptor antagonist, significantly inhibited the ET-l-induced formation of inositol phosphates in a dose-dependent manner in the range between 22 nmol/L (ICso) and 2.2 ixmol/L (ICso × 100). On the contrary, N-cis-2,6-dimethylpiperidinocarbonyl-I~-~/MeLeu-I)-Trp(COOMe)-o-Nle-ONa (BQ788), a selective ET n receptor antagonist, had no effect on the ET-1induced formation of inositol phosphates in the range between 1.2 nmol/L (ICso) and 120 nmol/L (ICso × 100). BQ123 significantly suppressed the ET-l-induced formation of choline dose-dependently, however, BQ788 did not affect the choline formation. BQ123 also inhibited the ET-l-indueed release of arachidonic acid, but BQ788 had little effect. The results strongly suggest that ET A receptor mediates the three intraceUular siignaling pathways of ET-I: (1) phosphoinositide hydrolysis by phospholipase C; (2) phosphatidylcholine hydrolysis by phospholipase D; and (3) arachidonic acid release in osteoblast-like cells. (Bone 21:143-146; 1997) © 1997 by Elsevier Science Inc. All rights reserved. Key Words: Endothelin-l; BQ123; BQ788; ET A receptor; ET B receptor; Osteoblast.

Introduction Endothelin (ET) is a 21 amino acid peptide consisting of three isotypes, ET-1, ET-2, and ET-3, and is a potent vasoconstrictor. 8"t6'25 It is recognized that ET exerts diverse biological effects through the specific receptors present on a variety of tissues and cell types. 8'11,16 In bone tissue, ET-1 has been reported to induce bone resorption and ~to stimulate collagen and noncollagen protein synthesis in the cultured neonatal mouse calvaria and to stimulate DNA synthesis of osteoblastic cells. 21"23 It is now recognized that ET-I plays an important role in bone metabolism. 17 As for the intracellular signaling system, it has been shown that ET-1 stimulates phosphoinositide hydrolysis by phos-

Materials and Methods Materials myo-[3H]inositol (90 Ci/mmol), [methyl-3H]choline chloride (85 Ci/mmol), and [5,6,8,9,11,12,14,15-3H]arachidonic acid (208 Ci/ mmol) were purchased from Amersham Japan (Tokyo). ET-1 was purchased from Peptide Institute, Inc. (Minoh, Japan). Cyclo-o-Trp-D-Asp-Pro-D-Val-Leu (BQ123) and N-cis-2,6-dimethylpiperidinocarbonyl-L-~MeLeu-o-Trp(COOMe)-o-Nle-ONa (BQ788) were kindly provided by Banyu Pharmaceutical Co., Ltd. (Tokyo). Other materials and chemicals were obtained from commercial sources. The radioactivity of 3H-samples was determined with a Beckman LS-6000IC liquid scintillation spectrometer.

Address for correspondence and reprints: Dr. Osamu Kozawa, Department of Pharmacology, Gifu University School of Medicine, Gifu 500, Japan. © 1997by ElsevierScienceInc. All rights reserved.

143

8756-3282/97/$17.00 PII $8756-3282(97)00096-3

144

A. Suzuki et al. Endothelin-I receptor subtype in osteoblasts

Bone Vol. 21, No. 2 August 1997:143-146

Cell Culture

ql

Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvariae 6"18 were generously provided by Dr. M. Kumegawa (Meikai University, Sakado, Japan) and maintained in c~-minimum essential medium ( a - M E M ) containing 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO2/95% air. The cells (5 × 104) were seeded into 35 m m diameter dishes in 2 mL of c~-MEM containing 10% FCS.

o x

Measurement of the Formation of Choline After 5 days, the medium was exchanged for 2 mL of oL-MEM containing 0.3% FCS for 48 h. The cells were labeled with [methyl-3H]choline chloride (2 I~Ci/dish) for 24 h. The labeled cells were washed twice with 1 mL of the buffer A and subsequently incubated in 1 mL of the buffer A containing 0.01% BSA at 37°C for 20 min. The cells were then stimulated by ET-1 for 15 min. The reaction was terminated by adding 0.75 mL of ice-cold methanol. The radioactive choline was measured as previously described. 7 Briefly, the contents were transferred to tubes to which chloroform was added and placed on ice for 60 min. Chloroform and water were then added at a final ratio of 1 : 1:0.9 (chloroform:methanol:water). The tubes were centrifuged at 14,000 × g for 5 min and the upper aqueous methanolic phase was separated for analysis of the water-soluble choline-containing metabolites. Separation was conducted on a 1 mL Dowex 50-WH + column ( 2 0 0 - 4 0 0 mesh). The phase was diluted to 5 mL with water and applied to the column. Glycerophosphocholine and choline phosphates were removed with 24 mL of water, and the radioactive choline was eluted with 10 mL of 1 mol/L HC1. When indicated, the cells were pretreated with BQ123 or BQ788 for 20 rain.

Measurement of Arachidonic Acid Release After 5 days, the medium was exchanged for 2 mL of a - M E M containing 0.3% FCS for 48 h. The cells were labeled with [3H]arachidonic acid (0.5 ~zCi/dish) for 24 h. The labeled cells were washed four times with 1 mL of an assay buffer B [10 mmol/L HEPES, pH 7.4, 135 mmol/L NaC1, 5 mmol/L KC1, 1 mmolfL MgSO4, and 1 mmol/L CaC12] and subsequently preincubated with 1 mL of the buffer containing 0.1% essentially fatty

z

T

I

o o~ G) r" O. ~) O r" O. m 0 ,~_ uJ 0 c

Measurement of the Formation of Inositol Phosphates After 5 days, the medium was exchanged for 2 mL of inositolfree a - M E M containing 0.3% FCS and the cells were used for the experiment after 48 h. The cells were labeled with myo[3H]inositol (3 p~Ci/dish) for 48 h. The labeled cells were preincubated with 10 mmol/L LiC1 for 10 min in 1 mL of an assay buffer A [5 mmol/L 4-(2-hydroxyethyl)-l-piperazineethane-sulfonic acid (HEPES), pH 7.4, 150 mmol/L NaC1, 5 mmol/L KC1, 5.5 mmol/L glucose, 0.8 mmol/L MgSO4, and 1 mmol/L CaClz] containing 0.01% bovine serum albumin (BSA) at 37°C. The cells were then stimulated by ET-1 for 15 rain. The reaction was terminated by adding 1 mL of 30% trichloroacetic acid. The radioactive inositol phosphates were measured as previously described. ]9 Briefly, the acid supernatant was treated with diethyl ether to remove the acid and neutralized with 0.1 mol/L NaOH. The supematant was applied to a 1 mL Dowex AG l-X8 column (100-200 mesh, formate form). The radioactive inositol phosphates were eluted from the column with 8 mL of 0.1 mol/L formic acid containing 1 mol/L ammonium formate. When indicated, the cells were pretreated with BQ123 or BQ788 for 20 min.

15,

,

co

E U.

10

iI

I

I

I

1

10

100

Dose (Ics0 x)

Figure 1. Effects of BQ123 or BQ788 on the ET-l-induced formation of inositol phosphates in MC3T3-E 1 cells. The labeled cells were pretreated with various doses of BQ123 (IC5o for ET A receptors = 22 nmol/L) (filled and open circles) or BQ788 (IC5o for ET B receptors = 1.2 nmol/L) (filled and open diamonds) for 20 min, and then stimulated by 0.1 txmol/L ET-1 (filled circles and diamonds) or vehicle (open circles and diamonds) for 15 min. Each value represents the mean --_ SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05 compared with the value of ET-I alone.

acid-free BSA at 37°C for 20 rain. The cells were then stimulated by ET- 1 for 30 min. After the indicated periods, the medium was collected and the radioactivity of the medium was determined. When indicated, the cells were pretreated with BQ123 or BQ788 for 20 min.

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 --SEM of triplicate determinations.

Results Effect of BQ123 or BQ788 on ET-l-lnduced Formation of Inositol Phosphates in MC3T3-E1 Cells To characterize the functioning receptor for ET-1-induced intracellular signaling in osteoblast-like MC3T3-E1 cells, we used a more selective ET A receptor antagonist, BQ123, 4 and a more selective ETB receptor antagonist, BQ788. 5 We first examined the effect of BQ123 or BQ788 on the ET-l-induced formation of inositol phosphates in these cells. BQ123 (IC5o value for ET A receptor = 22 nmol/L), which alone did not affect basal levels, significantly inhibited the ET-l-induced formation of inositol phosphates in a dose-dependent manner in the range between 22 nmol/L (IC5o) and 2.2 ixmol/L (IC5o × 100) in MC3T3-E1 cells (Figure 1). The maximum inhibitory effect of BQ123 on the ET-l-induced formation of inositol phosphates was about 79%. However, the BQ788 (IC5o value for ET B receptor = 1.2 nmol/L) had little effect on the ET-l-induced formation of inositol phosphates in the range between 1.2 nmol/L (IC5o) and 120 nmol/L (IC5o × 100) in these cells (Figure 1).

Bone Vol. 21, No. 2 August 1997:143-146

A. Suzuki et al. Endothelin-1 receptor subtype in osteoblasts

145

20 x

5

E ¢9 o

15" t~

x

E I,.

¢D

"O 0p. 0

4

10 t13

-I-

O t-

"6 tO

O °"O N tO ~3 t-

cO

E U.

---•l

I

I

I

1

10

100

.

3

Dose Ocs0

Control

Figure 2. Effects of BQI23 or BQ788 on the ET-l-induced formation of choline in MC3T3-E1 cells. The labeled cells were pretreated with various doses of BQ 123 (ICso for E T A receptors = 22 nmol/L) (filled and open circles) or BQ788 (IC5o for ET B receptors = 1.2 nmol/L) (filled and open diamonds) for 20 min, and then stimulated by 0.1 ixmol/L ET-1 (filled circles and diamonds) or vehicle (open circles and diamonds) for 15 min. Each value represents the mean _+ SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05 compared with the value of ET-1 alone.

~_

ET-1

BQ123

BQ788

ET-1

ET-1

+

+

Figure

3. Effects of BQ123 or BQ788 on the ET-l-induced release of arachidonic acid in MC3T3-E1 cells. The labeled cells were pretreated with 2.2 wmol/L (IC5o for ET A receptors × 100) BQ123 or 120 nmol/L (IC5o for ET B receptors × 100) BQ788 for 20 min, and then stimulated by 0.1 i~mol/L ET-1 or vehicle for 30 min. Each value represents the mean _+ SEM of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *p < 0.05 compared with the value of ET-1 alone.

Discussion Effect of BQ123 or BQ788 on ET-l-lnduced Formation of Choline in MC3T3-E,! Cells We previously reported that ET-1 stimulates phosphatidylcholine-hydrolyzing phospholipase D independently of protein kinase C activated by phosphoinositide hydrolysis in MC3T3-E1 cells. 19 W e next examined the effect of BQ123 or BQ788 on the ET-1-induced formation of choline in these cells. BQ123, which alone did not affect basal levels, significantly suppressed the ET-l-induced formation of choline in MC3T3-E1 cells 2). The inhibitory effect of BQ123 was dose dependent in the range between 22 nmol/L (ICso) and 2.2 I~mol/L (ICso × 100). The maximum effect of BQ123 on the ET-l-induced formation of choline was 77%. On the contrary, BQ788 did not affect the ET-l-induced formation of choline in the range between 1.2 nmol/L (ICso) and 120 nmol/L (ICso × 100) in these cells (Figure 2).

(Figure

Effect of BQ123 or BQ788 on ET-l-lnduced Release of Arachidonic Acid in MC3T3-E1 Cells We examined the effects of these two ET receptor antagonists on the ET-1-induced release of arachidonic acid. We confirmed that ET-1 stimulated arachidonic acid release in osteoblast-like MC3T3-E1 cells. BQ123 [2.2 ixmol/L (IC5o × 100)], which by itself had no effect on arachidonic acid release (data not shown), significantly inhibited the ET-l-induced arachidonic acid in these cells (Figure 3). The inhibitory effect of BQ123 on the ET-l-induced release of arachidonic acid was about 77%. However, BQ788 [120 nmol/L (IC5o × 100)] had little effect on the ET-l-induced release of arachidonic acid in these cells (Figure

3).

In the present study, we showed that BQ123 significantly inhibited the ET- 1-induced formation of inositol phosphates in osteoblast-like MC3T3-E1 cells, but BQ788 had no effect. BQ123 is known to be a more selective antagonist for ET A receptor. 4 Its ICso value for ET A receptors is 22 nmol/L, whereas that for ET B receptors is 9.7 t.tmol/L. On the other hand, BQ788 is known to be a more selective antagonist for ET B receptor. 5 Its ICso value for ET B receptors is 1.2 nmol/L, whereas that for ET A receptors is 280 nmol/L. It is well recognized that phosphoinositide is hydrotyzed by phospholipase C, resulting in the formation of diacylglycerol and inositol phosphates. 2,t° Thus, these results suggest that ET-l-induced phosphoinositide hydrolysis by phospholipase C is mediated not through ET B receptor but ET A receptor in osteoblast-like MC3T3-E1 cells. Green et al.3 reported that ET-1 induces Ca 2+ transients and activates Na+-H + exchanger through ET A receptor in rat osteosarcoma UMR-106 cells, but they did not show the data about ET B receptormediated signaling in their work. It has recently been reported that both ET A and ET B receptors mediate the ET- 1-elicited C a 2 + transients in UMR-106 cells, 15 and that both ET A and ET B receptors are coupled to phospholipase C in rat osteosarcoma ROS 17/2.8 cells. 9 We here showed that ET-1 activates phospholipase C not through ET B receptor but ET A receptor in MC3T3-E1 cells. It has been reported that osteoblast-like MC3T3-E1 cells in culture exhibit a temporal sequence of development, and show several phenotypes such as preosteoblasts and mature osteoblasts. 12"26 This discordance may be due to the difference of cell species or phenotypes. W e previously reported that ET-1 activates phosphatidylcholine-hydrolyzing phospholipase D independently of phosphoinositide hydrolysis in these cells. 2° Here, BQ123 inhibited the ET-l-induced formation of choline as well as that of inositol phosphates, and BQ788 had no effect on the ET-l-induced formation of choline in

146

A. Suzuki et al. Endothelin-1 receptor subtype in osteoblasts

MC3T3-E1 cells. Therefore, from this finding, it is probable that ET-1-induced phosphatidylcholine hydrolysis by phospholipase D is mediated also through ETA receptor in MC3T3-E1 cells. In addition, we demonstrated that ET- 1 stimulated arachidonic acid release and that BQ123 inhibited the arachidonic acid release induced by ET-1 in MC3T3-E1 cells. Thus, this result suggests that ET-l-induced release of arachidonic acid is mediated through ETA receptor as well as the ET-l-induced hydrolysis of phosphoinositide and phosphatidylcholine in osteoblast-like MC3T3-E1 cells. In conclusion, our results strongly suggest that ETA receptor mediates the three intracellular signaling pathways of ET-I: (1) phosphoinositide hydrolysis by phospholipase C; (2) phosphatidylcholine hydrolysis by phospholipase D; and (3) arachidonic acid release in osteoblast-like cells. References 1. Arai, H., Hori, S., Aramori, I., Ohkubo, H., and Nakanishi, S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348: 730-732; 1990. 2. Berridge, M. J. and lrvine, R. F. Inositol trisphosphate, a novel second messenger in cellular signal transducfion. Nature 312:315-321; 1984. 3. Green, J., Foellmer, O., Kleeman, C. R., and Basic, M. M. Endothelin receptor in osteoblastic cells is coupled to multiple messenger signals. Am J Pbysiol 267:C1329-C1337; 1994. 4. Ihara, M., Noguchi, K., Saeki, T. et al. Biological profiles of highly potent novel endothelin antagonists selective for the ET A receptor. Life Sci 50:247255; 1992. 5. Ishikawa, K., Ihara, M., Noguchi, K. et al. Biological and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ788. Proc Natl Acad Sci USA 91:4892-4896; 1994. 6. Kodama, H., Amagai, Y., Sudo, H., Kasai, S., and Yamamoto, S. Establishment of a clonal osteoblastic cell line from newborn mouse calvaria. Jpn J Oral Biol 23:899-901; 1981. 7. Kozawa, O., Suzuki, A., Kotoyori, J., Tokuda, H., Watanabe, Y., Ito, Y., and Oiso, Y. Prostaglandin F2= activates phospholipase D independently from activation of protein kinase C in osteoblast-like cells. J Cell Biochem 55:375379; 1994. 8. Masaki, T. Endothelins: Homeostatic and compensatory actions in the circulatory and endocrine systems. Endocrinol Rev 14:256-268; 1993. 9. Nambi, P., Wu, H.-L., Lipshutz, D., and Prabhakar, U. Identification and characterization of endothelin receptors on rat osteoblastic osteosarcoma cells: Down-regulation by 1,25-dihydroxy-vitamin D 3. Mol Pharmacol 47:266-271; 1995. 10. Nishizuka, Y. Studies and perspectives of protein kinase C. Science 233:305312; 1986. l 1. Pollock, D. M., Keith, T. L., and Highsmith, R. F. Endothelin receptors and calcium signaling. FASEB J 9:1196-1204; 1995. 12. Quarles, L. D., Yohay, D. A., Lever, L. W., Caton, R., and Wenstrup, R. J.

Bone Vol. 21, No. 2 August 1997:143-146

13.

14.

15.

16. 17.

18.

19.

20. 21.

22.

23.

24.

25.

26.

Distinct proliferative and differentiated stages of murine MC3T3-EI cells in culture: An in vitro model of osteoblast development. J Bone Miner Res 7:683-692; 1992. Sakurai, T., Morimoto, H., Kasuya, Y., Takuwa, Y., Nakauchi, H., Masaki, T., and Goto, K. Level of ET B receptor mRNA is down-regulated by endothelins through decreasing the intracellular stability of mRNA molecules. Biochem Biophys Res Commun 186:342-347; 1992. Sakurai, T., Yanagisawa, M., Takuwa, Y., Miyazaki, H., Kimura, S., Goto, K., and Masaki, T. Cloning of a cDNA encoding a nonisopeptide selective subtype of the endothelin receptor. Nature 348:732-735; 1990. Semler, D., Ohlstein, E. H., Nambi, P., Slater, C., and Stern, P. H. Endothelin- 1 evoked calcium transients in UMR-106 osteoblastic osteosarcoma cells are mediated through endothelinA and endothelinB receptors. J Pharmacol Exp 272:1052-1058; 1995. Simonson, M. S. and Dunn, M. J. Cellular signaling by peptides of the endothelin gene family. FASEB J 4:2989-3000; 1990. Stern, P. H., Tatrai, A., Semler, D. E., Lee, S. K., Lakatos, P., Strieleman, P. J., Tarjan, G., and Sanders, J. L. Endothelin receptors, second messengers, and actions in bone. J Nutr 125:2028S-2032S; 1995. Sudo, H., Kodama, H., Amagai, Y., Yamamoto, S., and Kasai, S. In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96:191-198; 1983. Suzuki, A., Kozawa, O., Saito, H., and Oiso, Y. Effect of prostaglandin F2~ on Ca2÷ influx in osteoblast-like cells: Function of tyrosine kinase. J Cell Biochem 54:487-493; 1994. Suzuki, A., Oiso, Y., and Kozawa, O. Effect of endothelin- 1 on phospholipase D activity in osteoblast-like cells. Mol Cell Endocrinol 105:193-196; 1994. Takuwa, Y., Ohue, Y., Takuwa, N., and Yamashita, K. Endothelin-1 activates phospholipase C and mobilizes Ca z+ from extra- and intracellular pools in osteoblastic cells. Am J Physiol 257:E797-E803; 1989. Takuwa, Y., Masaki, T., and Yamashita, K. The effect of the endothelin family peptides on cultured osteoblastic cells from rat calvariae. Biochem Biophys Res Commun 170:998-1005; 1990. Tatrai, A., Foster, S., Lakatos, P., Shankar, G., and Stern, P. H. Endothelin-I actions on resorption, collagen and noncollagen protein synthesis, and phosphatidylinositol turnover in bone organ cultures. Endocrinology 131:603-607; 1992. Tatrai, A., Lakatos, P., Thompson, S., and Stern, P. H. Effects of endothelin-I on signal transduction in UMR-106 osteoblastic cells. J Bone Miner Res 7:1201-1209; 1992. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411-415; 1988. Yohay, D. A., Zhang, J., Thrailhill, K. M., Arthur, J. M., and Quarles, L. D. Role of serum in the developmental expression of alkaline phosphatase in MC3T3-EI osteoblasts. J Cell Physiol 158:467-475; 1994.

Date Received: December 2, 1996 Date Revised: April 3, 1997 Date Accepted." April 24, 1997