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p42 Mitogen-Activated Protein Kinase Limits Triiodothyronine-Stimulated Alkaline Phosphatase Activity in Osteoblasts
Biochemical and Biophysical Research Communications 286, 1140 –1143 (2001) doi:10.1006/bbrc.2001.5515, available online at http://www.idealibrary.com on
Activation of p44/p42 Mitogen-Activated Protein Kinase Limits Triiodothyronine-Stimulated Alkaline Phosphatase Activity in Osteoblasts Osamu Kozawa, 1 Daijiro Hatakeyama, Minoru Yoshida, Yasuka Kamiya, Chihiro Kondo, Hiroyuki Matsuno, and Toshihiko Uematsu Department of Pharmacology, Gifu University School of Medicine, Gifu 500-8705, Japan
Received August 1, 2001
It has been shown that thyroid hormone stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated whether p44/p42 mitogen-activated protein (MAP) kinase is involved in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. Triiodothyronine (T 3) markedly induced the phosphorylation of p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly enhanced the T 3-induced alkaline phosphatase activity in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by T 3 was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase takes part in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblasts and that p44/p42 MAP kinase plays an inhibitory role in the thyroid hormone-effect. © 2001 Academic Press
Key Words: triiodothyronine; alkaline phosphatase; MAP kinase; osteoblast.
Hyperthyroidism is a major cause of secondary osteoporosis (1). In hyperthyroidism, the serum levels of alkaline phosphatase and osteocalcin, markers of osteoblast phenotype, and the excretion of pyridinoline and hydroxypyridinoline cross-link, which reflects bone resorption, are elevated (2). It is recognized that both increased bone resorption and decreased bone formation contribute to the loss of bone mass by hyperthyroidism (2). Bone metabolism is regulated mainly by two functional cells, osteoblasts and osteoclasts (3). The former cells are responsible for bone formation and the latter for bone resorption. The receptor for triiodothyronine (T 3) has been shown to exist on osteoblasts 1
To whom correspondence [email protected].
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be
addressed.
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(2). It has been shown that thyroid hormone stimulates alkaline phosphatase activity and secretion of osteocalcin, insulin-like growth factors and insulin-like growth factor-binding proteins in osteoblasts and that it modulates proliferation of osteoblasts (2, 4, 5). In a previous study (6), we have reported that T 3 modulates interleukin-6 synthesis at two points in osteoblast-like MC3T3-E1 cells as follows: one is exerted at the point between adenylyl cyclase and protein kinase A, and the other is at a point downstream from protein kinase C activation. Thus, it is generally recognized that thyroid hormone physiologically acts in osteoblasts, resulting in modulating bone metabolism. However, the exact mechanism of thyroid hormone in osteoblasts has not been precisely clarified. The receptor of thyroid hormone belongs to the steroid hormone receptor superfamily (7). It is well recognized that the effects of thyroid hormone, as well as other steroid hormones, are exerted through binding to its specific intracellular receptors and subsequently activating the expression of the gene network (7). As for the signaling of thyroid hormone, it has very recently been reported that the activation of p44/p42 mitogen-activated protein (MAP) kinase, which belongs to the MAP kinase superfamily (8), is involved in the regulation of thyroid hormone-inhibited p53 transcriptional activity in human kidney cells (9). The MAP kinase superfamily plays crucial roles in intracellular signaling of a variety of agonists (8). The three MAP kinases, p38 MAP kinase, p44/p42 MAP kinase, and SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) are known as central elements used by mammalian cells to transduce the diverse messages (8). In the present study, we investigated whether p44/p42 MAP kinase is involved in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. We herein show that the T 3-stimulated alkaline phosphatase activity is negatively regulated by p44/p42 MAP kinase in these cells.
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MATERIALS AND METHODS Materials. T 3 was obtained from Sigma Chemical Co. (St. Louis, MO). PD98059 and U0126 were purchased from CalbiochemNovabiochem (La Jolla, CA). Phospho-specific p44/p42 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified) and p44/p42 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified) were obtained from New England BioLabs (Beverly, MA). An enhanced chemiluminescence (ECL) Western blotting detection system was obtained from Amersham Japan (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. T 3 was dissolved in 0.1 M NaOH. PD98059 and U0126 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect assay for alkaline phosphatase activity and Western blot analysis. Cell culture. Cloned osteoblast-like MC3T3-E1 cells, which have been derived from newborn mouse calvaria (10), were maintained as previously described (11). Briefly, the cells were cultured in ␣-minimum essential medium (␣-MEM) containing 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO 2/95% air. The cells were seeded into 35-mm-diameter dishes or 90-mmdiameter dishes in ␣-MEM containing 10% FCS. After 5 days, the medium was exchanged for ␣-MEM containing 0.3% FCS. The cells were used for experiments after 48 h. Assay for alkaline phosphatase activity. The cultured cells were pretreated with PD98059 or U0126 for 60 min, and then stimulated by T 3 for 48 h in 1 ml of ␣-MEM containing 0.3% FCS. At the end of the incubation, the cells were harvested by scraping with a rubber policeman into 1 ml of 0.2% Nonidet P-40 and disrupted by sonication. After centrifugation at 1500g for 5 min of the homogenate, alkaline phosphatase activity of the supernatant was measured by the method of Lowry et al. (12). Western blot analysis of p44/p42 MAP kinase. The cultured cells were stimulated by T 3 for the indicated periods. The cells were washed twice with phosphate-buffered saline and then lysed, homogenized, and sonicated in a lysis buffer containing 62.5 mM Tris–Cl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol, and 10% glycerol. The cytosolic fraction was collected as a supernatant
FIG. 2. Effect of T 3 on the phosphorylation of p44/p42 MAP kinase in MC3T3-E1 cells. The cultured cells were incubated with 10 nM T 3 for the indicated periods. The extracts of cells were subjected to SDS–PAGE against phospho-specific p44/p42 MAP kinase antibodies or p44/p42 MAP kinase antibodies.
after centrifugation at 125,000g for 10 min at 4°C. SDS– polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli (13) in 10% polyacrylamide gels. Western blotting was performed as described previously (14) by using phosphospecific p44/p42 MAP kinase antibodies or p44/p42 MAP kinase antibodies, with peroxidase-labeled antibodies raised in goat against rabbit IgG being used as second antibodies. Peroxidase activity on the nitrocellulose sheet was visualized on x-ray film by use of an ECL Western blotting detection system. When indicated, the cells were pretreated with U0126 for 60 min. Determination. The densitometric analysis was performed using Molecular Analysis/Macintosh (Bio-Rad Laboratories, Hercules, CA). Statistical analysis. The data were analyzed by Anova followed by Bonferroni method for multiple comparisons between pairs. All data are presented as the mean ⫾ SD of triplicate determinations. Each experiment was repeated three times with similar results.
RESULTS Effects of T 3 on Alkaline Phosphatase Activity and the Phosphorylation of p44/p42 MAP Kinase in MC3T3-E1 Cells It has been shown that thyroid hormone induces alkaline phosphatase activity in osteoblasts (5). We found that T 3 stimulates the activity of alkaline phosphatase in a dose-dependent manner in the range between 0.1 nM and 0.1 M (Fig. 1). To investigate whether T 3 activates p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells, we examined the effect of T 3 on the phosphorylation of p44/p42 MAP kinase. T 3 markedly stimulated the phosphorylation of p44/p42 MAP kinase in a time-dependent manner (Fig. 2). The maximum effect of T 3 on the phosphorylation was observed 3 h after the T 3-stimulation. Effects of PD98059 or U0126 on the T 3-Induced Alkaline Phosphatase Activity in MC3T3-E1 Cells
FIG. 1. Dose-dependent effect of T 3 on alkaline phosphatase (ALP) activity in MC3T3-E1 cells. The cultured cells were stimulated by various doses of T 3 for 48 h. Each value represents the mean ⫾ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P ⬍ 0.05, compared with the control value.
In order to clarify the involvement of p44/p42 MAP kinase in the thyroid hormone-stimulated alkaline phosphatase activity, we examined the effect of PD98059, a specific inhibitor of upstream kinase that activates p44/p42 MAP kinase (15), on the activity. PD98059 significantly amplified the T 3-stimulated ac-
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FIG. 3. Effect of PD98059 on the T 3-induced alkaline phosphatase (ALP) activity in MC3T3-E1 cells. The cultured cells were pretreated with various doses of PD98059 for 60 min, and then stimulated by 10 nM T 3 for 48 h. Each value represents the mean ⫾ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P ⬍ 0.05, compared with the value of T 3 alone.
tivity of alkaline phosphatase (Fig. 3). The effect of PD98059 on the alkaline phosphatase activity was dose-dependent in the range between 1 and 30 M (Fig. 3). In addition, we examined the effect of U0126, another inhibitor of the upstream kinase that activates p44/p42 MAP kinase (16), on the T 3-stimulated alkaline phosphatase activity. The T 3-stimulated activity of alkaline phosphatase was also enhanced by U0126 (Table 1). Effect of U0126 on the Phosphorylation of p44/p42 MAP Kinase Induced by T 3 in MC3T3-E1 Cells We found that U0126 significantly inhibited the T 3stimulated phosphorylation of p44/p42 MAP kinase in
TABLE 1
Effect of U0126 on the T 3-Induced Alkaline Phosphatase Activity in MC3T3-E1 Cells U0126
T3
Alkaline phosphatase activity (nmol/min/dish)
⫺ ⫺ ⫹ ⫹
⫺ ⫹ ⫺ ⫹
6.2 ⫾ 0.8 34.1 ⫾ 2.8 7.9 ⫾ 1.2 59.0 ⫾ 4.3*
Note. The cultured cells were pretreated with 3 M U0126 for 60 min, and then stimulated by 10 nM T 3 for 48 h. Each value represents the mean ⫾ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. * P ⬍ 0.05, compared with the value of T 3 alone.
FIG. 4. Effect of U0126 on the phosphorylation of p44/p42 MAP kinase induced by T 3 in MC3T3-E1 cells. (A) The cultured cells were pretreated with 10 M U0126 for 60 min, and then stimulated by 10 nM T 3 for 3 h. The extracts of cells were subjected to SDS–PAGE against phospho-specific p44/p42 MAP kinase antibodies or p44/p42 MAP kinase antibodies. (B) The histogram shows quantitative representations of the levels of T 3-induced p44/p42 MAP kinase phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ⫾ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P ⬍ 0.05, compared with the value of T 3 alone.
MC3T3-E1 cells (Fig. 4A). According to the densitometric analysis, U0126 caused almost complete reduction in the T 3-effect (Fig. 4B). DISCUSSION In the present study, we showed that T 3 induced the phosphorylation of p44/p42 MAP kinase in osteoblastlike MC3T3-E1 cells. It is well recognized that MAP kinases are activated by phosphorylation of tyrosine and threonine residues by dual-specificity MAP kinase kinase (8, 17). It has recently been shown that thyroid hormone activates p44/p42 MAP kinase resulting in the phosphorylation of p53 in human kidney cells and inhibits the p53 transcriptional activity (9). Therefore, our results suggest that T 3 activates p44/p42 MAP kinase in MC3T3-E1 cells. To the best of our knowledge, our present study probably represents the first report showing the activation of p44/p42 MAP kinase by thyroid hormone in osteoblasts. It has been shown that thyroid hormone stimulates alkaline phosphatase activity, a marker of osteoblast phenotype, in osteoblasts (5). We confirmed the stimulation of alkaline phosphatase activity by T 3 and next examined whether p44/p42 MAP kinase is involved in the thyroid hormone-induced alkaline phosphatase activity in MC3T3-E1 cells. First, PD98059, a well-
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known inhibitor of upstream kinase (MEK I) that activates p44/p42 MAP kinase (15), significantly enhanced the T 3-stimulated activity of alkaline phosphatase. These results suggest that T 3-stimulated alkaline phosphatase activity is inhibited by activation of p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Furthermore, we showed that the T 3-stimulated activity of alkaline phosphatase was amplified by U0126, another inhibitor of upstream kinase that activates p44/p42 MAP kinase (16). We found that the T 3-induced phosphorylation of p44/p42 MAP kinase was truly inhibited by U0126. Taking these findings into account as a whole, it is most likely that T 3 activates p44/p42 MAP kinase, resulting in negatively regulating the alkaline phosphatase activity in osteoblastlike MC3T3-E1 cells. It is probable that p44/p42 MAP kinase activated by thyroid hormone limits the thyroid hormone-induced alkaline phosphatase activity in these cells. In conclusion, these results strongly suggest that the thyroid hormone-stimulated alkaline phosphatase activity is negatively regulated by thyroid hormone itselfactivated p44/p42 MAP kinase in osteoblasts. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research (09671041, 12470015) from the Ministry of Education, Science, Sports, and Culture of Japan.
REFERENCES 1. Khosla, S., and Melton, III, L. J. (1995) Secondary osteoporosis. In Osteoporosis: Etiology, Diagnosis and Management, 2nd ed. (Riggs, L. G., and Melton, III, L. J., Eds.), pp. 183–204, Lippincott-Raven Publishers, Philadelphia. 2. Stern, P. H. (1996) Thyroid hormone and bone. In Principles of Bone Biology (Bilezikian, J. P., Raisz, L. G., Rodan, G. A., Eds.), pp. 521–531, Academic Press, San Diego. 3. Nijweide, P. J., Burger, E. H., and Feyen, J. H. M. (1986) Cells of bone: Proliferation, differentiation, and hormonal regulation. Physiol. Rev. 66, 855– 886. 4. Rizzoli, R., Poser, J., and Burgi, U. (1986) Nuclear thyroid hormone receptor in cultured bone cells. Metabolism 35, 71–74.
5. Kasono, K., Sato, K., Han, D. C., Fujii, Y., Tsushima, T., and Shizume, K. (1988) Stimulatory effect of alkaline phosphatase activity by thyroid hormone in mouse osteoblast-like (MC3T3E1): A possible mechanism of hyperalkaline phosphatasia in hyperthyroidism. Bone Miner. 4, 355–363. 6. Tokuda, H., Kozawa, O., Harada, A., Isobe, K., and Uematsu, T. (1998) Triiodothyronine modulates interleukin-6 synthesis in osteoblasts: Inhibition in protein kinase A and C pathways. Endocrinology 139, 1300 –1305. 7. Evans, R. M. (1988) The steroid and thyroid hormone receptor superfamily. Science 240, 889 – 895. 8. Widmann, C., Gibson, S., Jarpe, M. B., and Johnson, G. L. (1999) Mitogen-activated protein kinase: Conservation of a threekinase module from yeast to human. Physiol. Rev. 79, 143–180. 9. Shih, A., Lin, H. Y., Davis, F. B., and Davis, P. J. (2001) Thyroid hormone promotes serine phosphorylation of p53 by mitogenactivated protein kinase. Biochemistry 40, 2870 –2878. 10. Sudo, H., Kodama, H., Amagai, Y., Yamamoto, S., and Kasai, S. (1983) In vivo differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J. Cell. Biol. 96, 191–198. 11. Kozawa, O., Tokuda, H., Miwa, M., Kotoyori, J., and Oiso, Y. (1992) Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells. Exp. Cell. Res. 198, 130 –134. 12. Lowry, O. H., Roberts, N. R., Wu, M. L., Hixon, W. S., and Crawford, E. J. (1954) The quantitative histochemistry of brain. II. Enzyme measurements. J. Biol. Chem. 207, 19 –37. 13. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. 14. Kato, K., Ito, H., Hasegawa, K., Inaguma, Y., Kozawa, O., and Asano, T. (1996) Modulation of the stress-induced synthesis of hsp27 and ␣B-crystallin by cyclic AMP in C6 rat glioma cells. J. Neurochem. 66, 946 –950. 15. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) PD98059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo. J. Biol. Chem. 270, 27489 –27494. 16. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. (1998) Identification of a novel inhibitor of mitogen-activated protein kinase. J. Biol. Chem. 273, 18623– 18632. 17. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation ob tyrosine and threonine. J. Biol. Chem. 270, 7420 –7426.
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Activation of p44/p42 Mitogen-Activated Protein Kinase Limits Triiodothyronine-Stimulated Alkaline Phosphatase Activity in Osteoblasts Osamu Kozawa, 1 Daijiro Hatakeyama, Minoru Yoshida, Yasuka Kamiya, Chihiro Kondo, Hiroyuki Matsuno, and Toshihiko Uematsu Department of Pharmacology, Gifu University School of Medicine, Gifu 500-8705, Japan
Received August 1, 2001
It has been shown that thyroid hormone stimulates the activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblasts. In the present study, we investigated whether p44/p42 mitogen-activated protein (MAP) kinase is involved in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. Triiodothyronine (T 3) markedly induced the phosphorylation of p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly enhanced the T 3-induced alkaline phosphatase activity in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by T 3 was reduced by U0126. These results strongly suggest that p44/p42 MAP kinase takes part in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblasts and that p44/p42 MAP kinase plays an inhibitory role in the thyroid hormone-effect. © 2001 Academic Press
Key Words: triiodothyronine; alkaline phosphatase; MAP kinase; osteoblast.
Hyperthyroidism is a major cause of secondary osteoporosis (1). In hyperthyroidism, the serum levels of alkaline phosphatase and osteocalcin, markers of osteoblast phenotype, and the excretion of pyridinoline and hydroxypyridinoline cross-link, which reflects bone resorption, are elevated (2). It is recognized that both increased bone resorption and decreased bone formation contribute to the loss of bone mass by hyperthyroidism (2). Bone metabolism is regulated mainly by two functional cells, osteoblasts and osteoclasts (3). The former cells are responsible for bone formation and the latter for bone resorption. The receptor for triiodothyronine (T 3) has been shown to exist on osteoblasts 1
To whom correspondence [email protected].
should
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
be
addressed.
E-mail:
(2). It has been shown that thyroid hormone stimulates alkaline phosphatase activity and secretion of osteocalcin, insulin-like growth factors and insulin-like growth factor-binding proteins in osteoblasts and that it modulates proliferation of osteoblasts (2, 4, 5). In a previous study (6), we have reported that T 3 modulates interleukin-6 synthesis at two points in osteoblast-like MC3T3-E1 cells as follows: one is exerted at the point between adenylyl cyclase and protein kinase A, and the other is at a point downstream from protein kinase C activation. Thus, it is generally recognized that thyroid hormone physiologically acts in osteoblasts, resulting in modulating bone metabolism. However, the exact mechanism of thyroid hormone in osteoblasts has not been precisely clarified. The receptor of thyroid hormone belongs to the steroid hormone receptor superfamily (7). It is well recognized that the effects of thyroid hormone, as well as other steroid hormones, are exerted through binding to its specific intracellular receptors and subsequently activating the expression of the gene network (7). As for the signaling of thyroid hormone, it has very recently been reported that the activation of p44/p42 mitogen-activated protein (MAP) kinase, which belongs to the MAP kinase superfamily (8), is involved in the regulation of thyroid hormone-inhibited p53 transcriptional activity in human kidney cells (9). The MAP kinase superfamily plays crucial roles in intracellular signaling of a variety of agonists (8). The three MAP kinases, p38 MAP kinase, p44/p42 MAP kinase, and SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) are known as central elements used by mammalian cells to transduce the diverse messages (8). In the present study, we investigated whether p44/p42 MAP kinase is involved in the thyroid hormone-stimulated alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. We herein show that the T 3-stimulated alkaline phosphatase activity is negatively regulated by p44/p42 MAP kinase in these cells.
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MATERIALS AND METHODS Materials. T 3 was obtained from Sigma Chemical Co. (St. Louis, MO). PD98059 and U0126 were purchased from CalbiochemNovabiochem (La Jolla, CA). Phospho-specific p44/p42 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified) and p44/p42 MAP kinase antibodies (rabbit polyclonal IgG, affinity purified) were obtained from New England BioLabs (Beverly, MA). An enhanced chemiluminescence (ECL) Western blotting detection system was obtained from Amersham Japan (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. T 3 was dissolved in 0.1 M NaOH. PD98059 and U0126 were dissolved in dimethyl sulfoxide. The maximum concentration of dimethyl sulfoxide was 0.1%, which did not affect assay for alkaline phosphatase activity and Western blot analysis. Cell culture. Cloned osteoblast-like MC3T3-E1 cells, which have been derived from newborn mouse calvaria (10), were maintained as previously described (11). Briefly, the cells were cultured in ␣-minimum essential medium (␣-MEM) containing 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere of 5% CO 2/95% air. The cells were seeded into 35-mm-diameter dishes or 90-mmdiameter dishes in ␣-MEM containing 10% FCS. After 5 days, the medium was exchanged for ␣-MEM containing 0.3% FCS. The cells were used for experiments after 48 h. Assay for alkaline phosphatase activity. The cultured cells were pretreated with PD98059 or U0126 for 60 min, and then stimulated by T 3 for 48 h in 1 ml of ␣-MEM containing 0.3% FCS. At the end of the incubation, the cells were harvested by scraping with a rubber policeman into 1 ml of 0.2% Nonidet P-40 and disrupted by sonication. After centrifugation at 1500g for 5 min of the homogenate, alkaline phosphatase activity of the supernatant was measured by the method of Lowry et al. (12). Western blot analysis of p44/p42 MAP kinase. The cultured cells were stimulated by T 3 for the indicated periods. The cells were washed twice with phosphate-buffered saline and then lysed, homogenized, and sonicated in a lysis buffer containing 62.5 mM Tris–Cl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol, and 10% glycerol. The cytosolic fraction was collected as a supernatant
FIG. 2. Effect of T 3 on the phosphorylation of p44/p42 MAP kinase in MC3T3-E1 cells. The cultured cells were incubated with 10 nM T 3 for the indicated periods. The extracts of cells were subjected to SDS–PAGE against phospho-specific p44/p42 MAP kinase antibodies or p44/p42 MAP kinase antibodies.
after centrifugation at 125,000g for 10 min at 4°C. SDS– polyacrylamide gel electrophoresis (PAGE) was performed by the method of Laemmli (13) in 10% polyacrylamide gels. Western blotting was performed as described previously (14) by using phosphospecific p44/p42 MAP kinase antibodies or p44/p42 MAP kinase antibodies, with peroxidase-labeled antibodies raised in goat against rabbit IgG being used as second antibodies. Peroxidase activity on the nitrocellulose sheet was visualized on x-ray film by use of an ECL Western blotting detection system. When indicated, the cells were pretreated with U0126 for 60 min. Determination. The densitometric analysis was performed using Molecular Analysis/Macintosh (Bio-Rad Laboratories, Hercules, CA). Statistical analysis. The data were analyzed by Anova followed by Bonferroni method for multiple comparisons between pairs. All data are presented as the mean ⫾ SD of triplicate determinations. Each experiment was repeated three times with similar results.
RESULTS Effects of T 3 on Alkaline Phosphatase Activity and the Phosphorylation of p44/p42 MAP Kinase in MC3T3-E1 Cells It has been shown that thyroid hormone induces alkaline phosphatase activity in osteoblasts (5). We found that T 3 stimulates the activity of alkaline phosphatase in a dose-dependent manner in the range between 0.1 nM and 0.1 M (Fig. 1). To investigate whether T 3 activates p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells, we examined the effect of T 3 on the phosphorylation of p44/p42 MAP kinase. T 3 markedly stimulated the phosphorylation of p44/p42 MAP kinase in a time-dependent manner (Fig. 2). The maximum effect of T 3 on the phosphorylation was observed 3 h after the T 3-stimulation. Effects of PD98059 or U0126 on the T 3-Induced Alkaline Phosphatase Activity in MC3T3-E1 Cells
FIG. 1. Dose-dependent effect of T 3 on alkaline phosphatase (ALP) activity in MC3T3-E1 cells. The cultured cells were stimulated by various doses of T 3 for 48 h. Each value represents the mean ⫾ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P ⬍ 0.05, compared with the control value.
In order to clarify the involvement of p44/p42 MAP kinase in the thyroid hormone-stimulated alkaline phosphatase activity, we examined the effect of PD98059, a specific inhibitor of upstream kinase that activates p44/p42 MAP kinase (15), on the activity. PD98059 significantly amplified the T 3-stimulated ac-
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FIG. 3. Effect of PD98059 on the T 3-induced alkaline phosphatase (ALP) activity in MC3T3-E1 cells. The cultured cells were pretreated with various doses of PD98059 for 60 min, and then stimulated by 10 nM T 3 for 48 h. Each value represents the mean ⫾ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P ⬍ 0.05, compared with the value of T 3 alone.
tivity of alkaline phosphatase (Fig. 3). The effect of PD98059 on the alkaline phosphatase activity was dose-dependent in the range between 1 and 30 M (Fig. 3). In addition, we examined the effect of U0126, another inhibitor of the upstream kinase that activates p44/p42 MAP kinase (16), on the T 3-stimulated alkaline phosphatase activity. The T 3-stimulated activity of alkaline phosphatase was also enhanced by U0126 (Table 1). Effect of U0126 on the Phosphorylation of p44/p42 MAP Kinase Induced by T 3 in MC3T3-E1 Cells We found that U0126 significantly inhibited the T 3stimulated phosphorylation of p44/p42 MAP kinase in
TABLE 1
Effect of U0126 on the T 3-Induced Alkaline Phosphatase Activity in MC3T3-E1 Cells U0126
T3
Alkaline phosphatase activity (nmol/min/dish)
⫺ ⫺ ⫹ ⫹
⫺ ⫹ ⫺ ⫹
6.2 ⫾ 0.8 34.1 ⫾ 2.8 7.9 ⫾ 1.2 59.0 ⫾ 4.3*
Note. The cultured cells were pretreated with 3 M U0126 for 60 min, and then stimulated by 10 nM T 3 for 48 h. Each value represents the mean ⫾ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. * P ⬍ 0.05, compared with the value of T 3 alone.
FIG. 4. Effect of U0126 on the phosphorylation of p44/p42 MAP kinase induced by T 3 in MC3T3-E1 cells. (A) The cultured cells were pretreated with 10 M U0126 for 60 min, and then stimulated by 10 nM T 3 for 3 h. The extracts of cells were subjected to SDS–PAGE against phospho-specific p44/p42 MAP kinase antibodies or p44/p42 MAP kinase antibodies. (B) The histogram shows quantitative representations of the levels of T 3-induced p44/p42 MAP kinase phosphorylation obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ⫾ SD of triplicate determinations. Similar results were obtained with two additional and different cell preparations. *P ⬍ 0.05, compared with the value of T 3 alone.
MC3T3-E1 cells (Fig. 4A). According to the densitometric analysis, U0126 caused almost complete reduction in the T 3-effect (Fig. 4B). DISCUSSION In the present study, we showed that T 3 induced the phosphorylation of p44/p42 MAP kinase in osteoblastlike MC3T3-E1 cells. It is well recognized that MAP kinases are activated by phosphorylation of tyrosine and threonine residues by dual-specificity MAP kinase kinase (8, 17). It has recently been shown that thyroid hormone activates p44/p42 MAP kinase resulting in the phosphorylation of p53 in human kidney cells and inhibits the p53 transcriptional activity (9). Therefore, our results suggest that T 3 activates p44/p42 MAP kinase in MC3T3-E1 cells. To the best of our knowledge, our present study probably represents the first report showing the activation of p44/p42 MAP kinase by thyroid hormone in osteoblasts. It has been shown that thyroid hormone stimulates alkaline phosphatase activity, a marker of osteoblast phenotype, in osteoblasts (5). We confirmed the stimulation of alkaline phosphatase activity by T 3 and next examined whether p44/p42 MAP kinase is involved in the thyroid hormone-induced alkaline phosphatase activity in MC3T3-E1 cells. First, PD98059, a well-
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Vol. 286, No. 5, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
known inhibitor of upstream kinase (MEK I) that activates p44/p42 MAP kinase (15), significantly enhanced the T 3-stimulated activity of alkaline phosphatase. These results suggest that T 3-stimulated alkaline phosphatase activity is inhibited by activation of p44/p42 MAP kinase in osteoblast-like MC3T3-E1 cells. Furthermore, we showed that the T 3-stimulated activity of alkaline phosphatase was amplified by U0126, another inhibitor of upstream kinase that activates p44/p42 MAP kinase (16). We found that the T 3-induced phosphorylation of p44/p42 MAP kinase was truly inhibited by U0126. Taking these findings into account as a whole, it is most likely that T 3 activates p44/p42 MAP kinase, resulting in negatively regulating the alkaline phosphatase activity in osteoblastlike MC3T3-E1 cells. It is probable that p44/p42 MAP kinase activated by thyroid hormone limits the thyroid hormone-induced alkaline phosphatase activity in these cells. In conclusion, these results strongly suggest that the thyroid hormone-stimulated alkaline phosphatase activity is negatively regulated by thyroid hormone itselfactivated p44/p42 MAP kinase in osteoblasts. ACKNOWLEDGMENT This work was supported in part by a Grant-in-Aid for Scientific Research (09671041, 12470015) from the Ministry of Education, Science, Sports, and Culture of Japan.
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