INTRODUCTION

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THREE ENDOTHELINS (ETs) have been identified, namely, ET-1, ET-2, and ET-3 (22, 30). Two types of receptor for ETs have been also cloned: the A-type (ET_A) receptor (2) and the B-type (ET_B) receptor (26). Both receptors belong to the superfamily of G protein-coupled receptors with seven transmembrane helices. The ET_A receptor is activated by ET-1 and ET-2. The ET_B receptor is activated to an equal extent by all three ETs. ETs evoke diverse physiological responses, which include potent vasoconstrictive and vasopressor activities (36), regulation of neuroendocrine functions (19, 27), stimulation of the secretion of aldosterone (8) and of natriuretic peptide (7), and modulation of cell growth (4, 18). In addition, ET-1 can regulate the differentiation of adipocytes (13, 33). The effects of ETs on bone remodeling have been reported by several groups. ET-1 regulates bone resorption by osteoclasts (1, 34, 37). ETs also increase the turnover of inositol phosphate and decrease the activity of alkaline phosphatase in osteoblastic cells (10, 31, 32). However, little information is available about the effects of ETs on the differentiation and mineralization of osteoblastic cells.

It is widely believed that the vasculature plays an important role in bone remodeling under normal and pathological conditions. We have found that natriuretic peptides, such as atrial natriuretic peptide and C-type natriuretic peptide, promote the differentiation of newborn-rat calvarial osteoblast-like cells (ROB cells) (10) and of the mouse line of clonal preosteoblastic cells MC3T3-E1 (15), as well as the formation of bone by these cells via the action of cGMP in a signal-transduction pathway that is mediated by receptor guanylate cyclases. Nitric oxide, an activator of soluble guanylate cyclase, also stimulates the expression of mRNA for osteocalcin in osteoblastic cells (24). By contrast, we have demonstrated that angiotensin II decelerates the differentiation of ROB cells and the formation by ROB cells of mineralized nodules (9).

The purpose of our present study was to elucidate the contribution of ETs to the formation of bone by osteoblastic cells. We found that ET-1 and the ET_A receptor were expressed in MC3T3-E1 cells and that the addition of ET-1 to the culture medium of MC3T3-E1 cells inhibited the expression of mRNA for osteocalcin. ET-1 also attenuated the deposition of calcium when included in the medium on days 6-8. We propose, therefore, that ET-1 might act as a local factor to inhibit the maturation and mineralization of osteoblastic cells.

	MATERIALS AND METHODS

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Materials. Human ET-1, ET-2, and ET-3 were purchased from the Peptide Institute, Osaka, Japan. ETs were dissolved in 0.1% acetic acid. BQ-123 [cyclo(D-Trp-D-Asp-Pro-D-Val-Leu)], an ET_A-selective antagonist, was obtained from Research Biochemicals International, Natick, MA (14). ³²P-labeled nucleotides and ¹²⁵I-labeled ET-1 were obtained from Amersham Life Science, Buckinghamshire, UK. DMEM, -MEM, fetal bovine serum, and penicillin/streptomycin antibiotic mixture were obtained from Life Technologies, Grand Island, NY.

Cell cultures. MC3T3-E1 cells were a generous gift from Dr. M. Kumegawa (Meikai University, Sakado, Japan). Cells were maintained in 55-cm² dishes in DMEM supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin in a humidified atmosphere of 5% CO₂ in air at 37°C. After reaching 70% confluence, cells were detached by treatment with 0.05% trypsin. The cells were replated in 12-well plates (3.8 cm²/well) or 6-well plates (9.4 cm²/well) at a density of 1 × 10⁴ cells/cm² and grown in -MEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 5 mM -glycerophosphate, and 50 µg/ml ascorbic acid. During subculture, the medium, with or without ET, was replaced every 3 days.

RT-PCR. RNA was extracted from MC3T3-E1 cells by the acid guanidinium-phenol-chloroform method (5). Total RNA (1 µg) was reverse transcribed by Moloney murine leukemia virus reverse transcriptase, Superscript (200 units; Life Technologies), using oligo(dT) primers (5 nmol) in a 20-µl reaction mixture. Amplification of the cDNA was performed with 35 cycles of PCR in 100 µl of Pfu DNA polymerase mixture (Toyobo, Tokyo, Japan) that contained 1 µM sense primer, 5'-TGTCTTGGGAGCCGAACTCA-3', and antisense primer, 5'-GCTCGGTTGTGCGTCAACTTCTGG-3', for mouse ET-1 (537 bp) (20); 1 µM sense primer, 5'-ACAGGACTCGAAAGCTGTAG-3', and antisense primer, 5'-TTGTAAGTGAAGCACGTGC-3', for mouse ET-2 (340 bp); 1 µM sense primer, 5'-TGAGCTGCAGGACCATTGAG-3', and antisense primer, 5'-AGGACACAGGTAGCATGTTC-3', for rat ET-3 (290 bp); 1 µM sense primer, 5'-GGCGCAATCGCT-GACAATGCTGAG-3', and antisense primer, 5'-CCACGTAGATAAGGTCTCCAAGGG-3', for the rat ET_A receptor (343 bp) (25); or 1 µM sense primer, 5'-CGTGTTCGTGCTAGGC-ATCATCGG-3', and antisense primer, 5'-CGACTCCAAGAAGCAACAGCTCGA-3', for the mouse ET_B receptor (293 bp) (25). The sequence of the gene for the mouse ET_A receptor is unknown. We referred to the sequence of the rat ET_A receptor in this experiment because Perkins et al (25). had previously detected the cDNA for the mouse ET_A receptor using fragments of the rat gene for the ET_A receptor. Each reaction cycle consisted of 94°C for 1 min, 60°C for 1 min, and 72°C for 2 min. Products of PCR were subjected to electrophoresis on a 1.5% agarose gel and visualized by staining with ethidium bromide. DNA markers (molecular weight marker V; Boehringer Mannheim, Tokyo, Japan) were used as size markers.

Assay of binding of ¹²⁵I-ET-1. Cells, grown in 12-well plates (3.8 cm²/well), were washed with ice-cold PBS (pH 7.4; 20 mM sodium phosphate and 130 mM NaCl) and incubated in 0.5 ml of PBS that contained 0.2% (wt/vol) bovine serum albumin, ¹²⁵I-labeled ET-1 (920 Bq/well), and unlabeled ETs or an analog of ET at various concentrations for 1 h at 4°C. After incubation, cells were washed twice with ice-cold PBS and solubilized with 0.5 ml of 0.1 M NaOH. The radioactivity was measured with a gamma-counter (ARC-300; Aloka, Tokyo, Japan).

Northern blotting analysis. Total RNA (20 µg) was subjected to electrophoresis on a 1% agarose gel that contained 2.2 M formaldehyde and was then transferred to a MagnaGraph nylon membrane (Micron Separations, Westborough, MA). After the membrane had been baked, the RNA on the membrane was allowed to hybridize overnight with cDNAs for rat osteocalcin and glyceraldehyde-3-phosphate dehydrogenase at 42°C in 50% formamide that contained 6× SSPE (1× SSPE is 0.15 M NaCl, 15 mM NaH₂PO₄, pH 7.0, 1 mM EDTA), 2× Denhardt's solution (0.1% each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll), 1% SDS, and 100 µg/ml herring sperm DNA. Each cDNA probe was radiolabeled with a Ready-to-Go kit (Pharmacia, Uppsala, Sweden). The membrane was washed twice in 1× SSC (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) that contained 0.1% SDS at room temperature for 5 min each and twice in 1× SSC that contained 0.1% SDS at 55°C for 1 h each, and then it was exposed to an imaging plate for 4 h. The plate was analyzed with a Bioimage Analyzer (BAS 2000; Fuji Film, Tokyo, Japan).

Quantitation of calcium. Cells were subcultured in -MEM that contained 10% fetal bovine serum, 5 mM -glycerophosphate, 50 µg/ml ascorbic acid, and ETs at various concentrations. We measured calcium, in hydroxyapatite, in the cell layer. The layers of cells in 12-well plates (3.8 cm²/well, polystyrene) were washed with PBS and incubated with 1 ml of 2 N HCl overnight with gentle shaking. The calcium ions in each sample were quantitated by the o-cresolphthalein complexone method with a Calcium C kit (Wako Pure Chemical Industries, Osaka, Japan) (10). This kit is specific for calcium, and the limit of sensitivity is 1 µg/ml. We used the standard solution of calcium (10 mg/dl) in the kit as the standard solution in our determinations.

Measurement of the production of inositol 1,4,5-trisphosphate. Cells that had been subcultured in 6-well plates (9.4 cm²/well) for 8 days were incubated with serum-free -MEM for 3 h. This culture medium was supplemented with 10⁷ M ET-1 for various brief periods, as indicated, and then the medium was removed by aspiration and replaced with 0.5 ml of 4% perchloric acid. After incubation of the cells in perchloric acid on ice for 20 min, the supernatant was brought to pH 7.5 by titration with ice-cold 1.5 M KOH that contained 60 mM HEPES buffer. The inositol 1,4,5-trisphosphate (IP₃) generated was quantitated with a D-myo-[³H]IP₃ assay kit (Amersham Life Science).

Measurement of the accumulation of cAMP. Cells, grown in 12-well plates, were incubated with serum-free -MEM that had been supplemented with 0.5 mM 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase, at 37°C for 15 min after washing with serum-free -MEM. Osteoblastic cells were subsequently incubated at 37°C for 15 min with 10⁷ M ET. After incubation, the cells were lysed by addition of 200 µl of a 0.1 N solution of HCl that contained 5 mM EDTA. The amount of cAMP was measured with a radioimmunoassay kit from Yamasa (Chiba, Japan).

	RESULTS

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Presence of ET-1 and the ET_A receptor in osteoblastic cells. In a previous study that involved radiolabeled ligand-binding assays, we showed that osteoblast-like cells from calvariae of newborn rats (ROB cells) express the ET_A receptor (10). To identify ETs and the types of receptor present in MC3T3-E1 cells, we performed RT-PCR with primers for specific ET-1, ET-2, ET-3, and both the ET_A and ET_B receptors. Messenger RNAs for ET-1 and the ET_A receptor were detected in MC3T3-E1 cells, as shown in Fig. 1. We failed to detect the mRNAs for ET-2, ET-3, and the ET_B receptor in MC3T3-E1 cells. Binding assays on day 8 using radiolabeled ET-1 showed that ET-1, ET-2, and a specific antagonist of the ET_A receptor, BQ-123 (14), prevented the binding to receptors of radiolabeled ET-1 with similar potency (IC₅₀ values: 5.4, 5.6, and 15.5 nM, respectively), whereas ET-3 was less potent (IC₅₀: 620 nM) (Fig. 2). ET_A receptors were detected in MC3T3-E1 cells during the entire culture period (data not shown).

View larger version (69K): [in this window] [in a new window]   Fig. 1.   Detection of mRNAs for endothelin (ET)-1 and ET receptors by RT-PCR. Total RNA was isolated from MC3T3-E1 cells on days 8 and 10 and reverse transcribed. Products of amplification by PCR of cDNAs were subjected to electrophoresis on a 1.5% agarose gel and detected by staining with ethidium bromide. Arrows point to fragments of 537 bp, 343 bp, and 293 bp that correspond to cDNAs for ET-1, the ETA receptor, and the ETB receptor, respectively. Results depicted are representative of results of 2 experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Competition with 125I-labeled ET-1 for binding to MC3T3-E1 cells by unlabeled ETs or an analog. Cells, grown in 12-well plates for 8 days, were incubated at 4°C for 1 h with 125I-ET-1 (920 Bq/0.5 ml/well) in the presence of unlabeled ET-1, ET-3, or BQ-123 at various concentrations. Subsequent steps for determining the extent of binding are described in MATERIALS AND METHODS. Total binding of ET-1 was 1.66 ± 0.17 fmol/106 cells. Each value is mean ± SD of results of triplicate determinations. * P < 0.01 vs. control; # P < 0.05 vs. control.

Northern blotting analysis of the level of mRNA for osteocalcin. To examine whether ETs might be involved in the maturation of osteoblastic cells, we performed Northern blotting using, as probe, the cDNA for osteocalcin, which is a marker of osteoblastic maturation. MC3T3-E1 cells were treated continuously for 10 days with 10⁷ M ET-1, ET-2, or ET-3. A final concentration of 0.0001% acetic acid was used as a control treatment. We found that ET-1 and ET-2 each markedly decreased the steady-state level of expression of the mRNA for osteocalcin in MC3T3-E1 cells (Fig. 3). ET-3 had a very weak inhibitory effect compared with that of ET-1 or ET-2.

View larger version (97K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Fig. 3.   Northern blotting analysis of mRNA for osteocalcin in cultured MC3T3-E1 cells. Total RNA was isolated from MC3T3-E1 cells after treatment with 107 M ET-1, ET-2, or ET- 3 for 10 days. Twenty micrograms of total RNA were subjected to electrophoresis on an agarose gel and were allowed to hybridize with 32P-labeled cDNA for rat osteocalcin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as described in MATERIALS AND METHODS. A: autoradiograms of mRNA probed with cDNAs for osteocalcin and GAPDH. B: quantitative analysis of the data in A. Relative levels of expression of osteocalcin mRNA were calculated after normalization by reference to the respective levels of GAPDH mRNA level. Data represent typical results of 3 separate experiments.

Effects of ETs on mineralization by osteoblastic cells. We measured the deposition of calcium by MC3T3-E1 cells that had been treated with ETs at various concentrations. As shown in Fig. 4A, ET-1 and ET-2 inhibited the deposition of calcium by MC3T3-E1 cells in a dose-dependent manner. ET-1 and ET-2 had an inhibitory effect even at 10¹¹ M. ET-1 and ET-2 were much more effective than ET-3 (Fig. 4B). Furthermore, BQ-123, a specific antagonist of the ET_A receptor, attenuated the inhibitory effect of ET-1 (Fig. 5).

View larger version (22K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effects of ETs on mineralization by MC3T3-E1 cells. Cells in 12-well plates were cultured with -MEM that contained 10% fetal bovine serum, 5 mM -glycerophosphate, 50 µg/ml L-ascorbic acid, and 107 M ET or 0.0001% acetic acid (control). Quantitative analysis of calcium ions derived from hydroxyapatite was performed as described in MATERIALS AND METHODS. A: dose-dependent decreases in the deposition of calcium in MC3T3-E1 cells. Cells were treated with ET-1 and ET-2 at various concentrations for 14 days. B: effects of ETs on calcium deposition in MC3T3-E1 cells. Cells were treated with 107 M ET for 12 days. Data are means ± SD of results from 3 or 4 wells and are typical of results of 3 separate experiments. * P < 0.01 vs. control.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Attenuation by the ETA receptor-specific antagonist BQ-123 of the ET-1-induced inhibition of mineralization by MC3T3-E1 cells. Cells in 12-well plates were cultured for 15 days with -MEM that contained 10% fetal bovine serum, 5 mM -glycerophosphate, 50 µg/ml L-ascorbic acid, and 109 M ET-1 and/or 106 M BQ-123, as indicated. Quantitative analysis of calcium ions derived from hydroxyapatite was performed as described in MATERIALS AND METHODS. Data are means ± SD of results from 3 wells. * P < 0.01 vs. control; ** P < 0.01 vs. results obtained with ET-1.

We next examined the effects of the pulsed administration of ET-1 on the deposition of calcium by MC3T3-E1 cells. The presence of 10⁷ M ET-1 on days 6-8 exclusively suppressed mineralization by MC3T3-E1 cells to the same extent as the continuous presence of 10⁷ M ET-1 from day 0 to day 16 (Fig. 6).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effects of pulsed treatment with ET-1 on mineralization by MC3T3-E1 cells. Cells in 12-well plates were cultured with -MEM that contained 10% fetal bovine serum, 5 mM -glycerophosphate, and 50 µg/ml L-ascorbic acid, and they were treated with 107 M ET-1 continuously or for the indicated periods. On day 16, calcium ions derived from hydroxyapatite were quantitated as described in MATERIALS AND METHODS. Data represent means ± SD of results from 3 wells and are typical of results of 3 separate experiments. *P < 0.01 vs. control.

Transduction of the ET-1 signal in MC3T3-E1 cells. To elucidate the signaling pathway triggered by ET-1 in MC3T3-E1 cells, we measured the rates of production of both IP₃ on day 8 and cAMP on day 9 when ET-1 most effectively influenced the mineralization by MC3T3-E1 cells, as shown in Fig. 6. The production of IP₃ in MC3T3-E1 cells was stimulated by ET-1, with a peak between 15 and 30 s after exposure to ET-1 (Fig. 7A). By contrast, ET-1 and ET-3 had no significant effect on the level of cAMP compared with the control (Fig. 7B). Moreover, ET-1 had no effect on the production of cAMP during the entire culture period (data not shown).

View larger version (17K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Fig. 7.   Signal-transduction pathways activated by ET-1 in MC3T3-E1 cells. A: ET-1-induced accumulation of inositol 1,4,5-trisphosphate (IP3) in MC3T3-E1 cells. On day 8, cells in 6-well plates were exposed to 107 M ET-1 for 0-120 s at 37°C. Subsequent steps for determination of levels of IP3 are described in MATERIALS AND METHODS. B: effects of ET-1 and ET-3 on intracellular production of cAMP. On day 9, cells in 12-well plates were exposed to 107 M ET-1 or ET-3 for 15 min in the presence of 0.5 M 3-isobutyl-1-methylxanthine, and then amounts of intracellular cAMP were determined by a radioimmunoassay. * P < 0.01 vs. 0 s. NS, not significantly different from control.

	DISCUSSION

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In this study, we demonstrated that ET-1 might affect osteoblastic metabolism via the ET_A receptor, acting as an autocrine/paracrine regulator. We next demonstrated that ETs decreased the level of mRNA for osteocalcin, which is known as a marker of the maturation of osteoblastic cells. ETs also acted, via the ET_A receptor, to inhibit mineralization by MC3T3-E1 cells in a dose-dependent manner. It appeared that phosphatidylinositol turnover might be involved in the action of ET-1 in MC3T3-E1 cells, and all our results together suggest that ETs might be local modulators of osteoblastic function in bone.

There are some reports of the regulation by ETs of the metabolism of cartilage and bone. ET-1 and the ET_A receptor have been shown to be involved in the formation of bone and cartilage that is derived from the branchial arch by use of mice deficient in ET-1 (20) and the ET_A receptor. Such mice have poorly developed mandibular and thyroid cartilage and mandibular and temporal bone. We also reported the presence of ET_A receptors on the perichondrium in rat tracheal and xiphoid cartilage and in the fetal rat epiphysis and showed that ET-1 increased the rate of incorporation of thymidine into cartilage tissue (21). ET-1 also regulates osteoclastic bone resorption (1, 34, 37). Localization of ET-1 in the osteoclasts indicates that osteoclasts are target cells for ET-1 (28). Perkins et al. (25) showed that ET-1 stimulates osteoblastic production of interleukin-6 (IL-6), an active mediator of osteoclasts. Their result indicates that ET-1 might regulate the generation of osteoclasts through the production of IL-6 by osteoblasts. Thus ETs appear to be involved in both the resorption and the formation of bone. However, little information on the effects of ETs on the maturation and mineralization of osteoblastic cells has been reported. In the present study, we showed that ETs inhibit the maturation and the mineralization of MC3T3-E1 cells. Other investigators and we ourselves (25, 31, 32) have also demonstrated that ET-1 and/or the ET_A receptor are expressed in osteoblastic cells. These results support the hypothesis that ETs are among the local regulators of osteoblastic functions in bone, as well as in cartilage and osteoclasts.

The level of expression of mRNA for osteocalcin in MC3T3-E1 cells that had been treated continuously with ET-1 was only ~20-30% of the steady-state level, as shown in Fig. 3. Osteocalcin is a specific product of osteoblastic cells, and the expression of mRNA for osteocalcin is observed in osteoblastic cells at the maturation stage. These results strongly suggest that ET-1 inhibits the differentiation of osteoblastic cells. Recently, ET was reported to increase the level of mRNA for osteocalcin in rat osteosarcoma cells (29). Differences in the effects of ET-1 on the expression of osteocalcin might reflect differences in cell culture conditions (methods of administration of ET-1), differences between species (rat versus mouse), and the type of osteoblast model used (osteosarcoma cells versus normal osteoblastic cells).

Continuous culture in the presence of ET-1 or ET-2 resulted in inhibition of the deposition of calcium by MC3T3-E1 cells. Moreover, ET-1 inhibited the mineralization of MC3T3-E1 cells when present only from day 6 to day 8, as shown in Fig. 5. We also showed that the level of the ET_A receptor did not change in MC3T3-E1 cells during the culture period. These results suggest that ET-1 might regulate the expression of mineralization-related genes from day 6 to day 8. We are now using the technique of differential-display PCR to identify the genes whose expression is regulated by endothelins. Transforming growth factor (TGF)- has also been reported to inhibit the formation of nodules after a short pulse in the initial culture (12). It will be of interest to pursue the relationship, in terms of signaling, between ET-1 and TGF- in osteoblastic cells.

In a previous study (16), we showed that 8-bromo-cAMP inhibits both the synthesis of alkaline phosphatase (ALPase) and the formation by ROB cells of mineralized nodules, in a model of bone formation in vitro. Parathyroid hormone (PTH), which activates adenylate cyclase in osteoblastic cells, has been reported to inhibit the activity of ALPase and the mineralization of osteoblastic cells via production of cAMP, depending on the exposure time in vitro (17). Therefore, we postulated that cAMP might be a candidate for a second messenger of ET-1 in MC3T3-E1 cells. However, ET-1 did not affect the production of intracellular cAMP in MC3T3-E1 cells during culture from day 3 to day 14. Takuwa et al. (32) reported similar results with confluent MC3T3-E1 cells. These results indicate that ET-1 does not stimulate adenylate cyclase in MC3T3-E1 cells, unlike PTH and prostaglandin (PG) E₂ (6). However, ET-1 can modulate calcium signaling via phospholipase C in various types of osteoblastic cell (31, 32). In our cell culture system, ET-1 stimulated the production of IP₃ (Fig. 7A). PGF₂ has been shown to decrease the activity of ALPase, via the accumulation of inositol phosphate, in MC3T3-E1 cells (11). Signaling by inositol phosphate in response to ET-1 might be involved in the inhibition of differentiation of osteoblastic cells and of mineralization by osteoblastic cells. Recently, the presence of phospholipase D was demonstrated in MC3T3-E1 cells, and the enzyme was shown to hydrolyze phosphatidylcholine (31).

In the present study, we found that ETs retarded the maturation and mineralization of osteoblastic cells. ET-1 from vascular endothelial cells might act on osteoblasts since bone is rich in blood vessels. However, we showed that osteoblastic cells expressed mRNAs for ET-1 and the ET_A receptor. These results suggest that ET-1 derived from osteoblasts might regulate some aspect of osteoblastic differentiation. Our present findings suggest that a relationship might exist between bone cells and ETs in the local environment.

Perspectives

We have proposed that vasoconstrictors, such as angiotensin II (9) and ET-1, might retard the differentiation of and formation of bone by osteoblastic cells. Spontaneously hypertensive rats are known as models of osteoporosis (3, 35). The vasocontractile effects of ET-1 are greater in spontaneously hypertensive rats than in normotensive Wistar-Kyoto rats (23). These observations suggest that certain relationships are likely to exist between bone cells and vasoactive peptides in the local environment. It might even be useful to examine whether antagonists of the ET_A receptor, such as BQ-123, could be effective in the treatment of osteoporosis.