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The effect of active vitamin D3 analogs and dexamethasone on the expression of osteocalcin gene in rat tibiae in vivo
Vol. 189, No. 2, 1992 December 15, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1231-l 235
THE EFFECT OF ACTIVE VITAMIN D3 ANALOGS AND DEXAMETHASONE ON THE EXPRESSION OF OSTEOCALCIN GENE IN RAT TIBIAE IN VZVO Toshihiko Ike&I, Hiroaki Kohnol, Takao Yamamuml, Ryuichi Kasail, Shuichi OhtaI, Hideo Okumura3, Junji Konishiz, Hart&i Kikuchiz, and Chohei Shigen$
1 Department of Orthopaedic Surgery and 2 Calcium Laboratory, Department of Radiology & Nuclear Medicine, Kyoto University Faculty of Medicine, Kyoto 606-01, Japan 3 Department of Orthopaedic Surgery, Ehime University Faculty of Medicine, Ehime 79 l-02, Japan Received
November
6,
1992
SUMMARY: We tested the effects of lq25dihydroxyvitamin D3 (1,25-(OH)zD$, 2P-(3-hydroxypropoxy)- la,25dihydroxyvitamin D3 (ED-7 1) and dexamethasone on osteocalcin mRNA levels in rat tibiae in vivo. Northern blot analysis showed that both 1,25-(OH)2D3 and ED-71 caused an increase in osteocalcin mRNA levels in bone: 1,25-(OH)2D3 induced a transient increase in the mRNA levels followed by a decrease in the control level by 12 h post administration. In contrast, ED-71 caused a persistent increase in osteocalcin mRNA level for seven days post administration. Serum osteocalcin levels paralleled the osteocalcin mRNA level in bone in both groups. Dexamethasone caused a marked reduction in both osteocalcin mRNA and serum osteocalcin levels. Suppressive effect of dexamethasone on osteocalcin expression was persistent for seven days at higher dose. Our results represent the fmt demonstration of the effect of active vitamin D and corticosteroid on the expression of ostcocalcin mRNA in bone in vivo. 0 1992 Academic Press, ~nc.
Osteocalcin is secreted exclusively by osteoblasts and represents the major noncollagenous protein present in bone matrix. Since the half life of osteocalcin in the circulation is reported to be only five minutes, its serum concern&on is believed to reflect secretion by osteoblasts and therefore os;eoblastic activity [l-5]. Many hormones and drugs are known to modulate osteocalcin production in bone. In particular, active forms of vitamin D increase and dexamethasone decreases the levels of serum osteocalcin in vivo and osteocalcin mRNA in osteoblasts in vitro [ 1,2,6,7]. The effect of these steroid hormones on osteocalcin gene expression in vivo, however, has not yet been studied because of the difficulty in preparing RNA from skeletal hard tissues. We as well as others have recently established the reproducible method to
1231
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
189, No. 2, 1992
quantitatively
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
extract total RNA from skeletal hard tissues [&lo].
This technique
allowed us to evaluate the effects of 1,25(OH)2D3 and ED-71, a newly synthesized active analog of 1,25(OH)zD3, as well as dexamethasone, on osteocalcin gene expression in vivo in adult rat tibiae, as reported here. MATERIALS
AND METHODS
Animals: 96 female Wistar rats (7-8 month-old, 250-280 g) were fed on a standard laboratory chow. 1,25(OH)zD3 (Duphar Co., Weesp, Netherlands) and ED-71 (a gift from Chugai Pharmaceuticals, Tokyo, Japan) were administered as a single oral dose using medium chain triglyceride (MCT, 0.25mVdose) as a vehicle. Dexamethasone-21phosphate disodium (a gift from Banyu Pharmaceuticals, Tokyo, Japan) was injected intraperitoneally at graded doses (lmg&g and lOOmg./kg) using normal saline (125 ul/dose) as a vehicle. Rats were sacrificed between 1O:OOand 13:OOat 3,6, 12, 24,72 and 168 h periods post administration. Three rats were used in each time point except for the zero hour time points for vehicle control groups that were assessed using six rats for each group. Animal protocols were conducted according to the guidelines of this institution. Prenaration of total RNA from bone: Legs were excised, tibiae were stripped off the surrounding soft tissues including the periosteum and the epiphysis and growth plate cartilage were resected. The bone marrow cavity was thoroughly washed out with 25 ml normal saline. Bone tissues, six or twelve tibiae in each group, were then minced and homogenized together, and the total RNA was extracted and analyzed by northern blot analysis as previously described [ 10,111. Northern blot analvsis: The following hybridization probes were synthesized using an auto DNA synthesizer (Pharmacia, Sweden): purified synthetic oligonucleotides of 42mer corresponding to the Cys23-Ile36 region of rat osteocalcin cDNA and of 48-mer corresponding to the Gln63-Ile78 region of rat cyclophilin cDNA as the internal control [ 121. The probes were labeled with [32P] y-ATP (New England Nuclear, Boston, MA) using T4 polynucleotide kinase (Takara Shuzo, Kyoto, Japan). Signal intensity was quantitated by a densitometer, and a ratio of signal intensity for osteocalcin (OC) versus cyclophilin (CYC) mRNAs (OC/CYC mRNA) was calculated for each time point and then normalized by the values for the time zero points of vehicle groups. Results were expressed in the mean * SD form from three northern blot analyses. Measurement of serum osteocalcin levels; The serum osteocalcin level was measured by a radioimmunoassay according to the method of &Priceet a1.[13]. Serum levels of calcium and inorganic phosphorus were determined by an auto-analyzer 1141. Statistical analvsis: Statistical significance was assessed by one-way analysis of variance and unpaired Student’s t test.
RESULTS 1,25(OH)zD3 caused a rapid increase in osteocalcin mRNA level which peaked at 6 h post administration and fell sharply thereafter [Fig.l-A and 2-A]. ED-71 also induced elevation in osteocalcin mRNA level with a peak at 6 h post administration [Fig. 1-B and 2-A], similar to 1,25(OH)zD3 treatment. However, unlike 1,25(OH)zD3, ED-71 caused persistent elevation of the osteocalcin mRNA levels for at least seven days post administration. Both 1,25(OH)zD3 and ED-7 1 resulted in significant increase in the serum osteocalcin levels [ Fig. 3-A]: 1,25(OH)zD3-induced increase in the serum osteocalcin level was rapid and occurred within 6 h after administration, while ED-7 1 caused a more gradual increase in the serum osteocalcin level which reached a plateau over 24-72 h post administration. 1232
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Figure 1. The expressionof osteocalcinmRNA in the rat tibiae after administration of A; 0. lug/kg of 1,25(OH)zD3, B; O.&/kg of ED-7 1, C, lOOmg/kgof dexamethasone, and D, lmgikg of dexamethasone.OC, osteocalcin,CYC; cyclophilin. The zero hour time points were determined using RNA samples obtained from rats treated with vehiclesonly.
Dexamethasone caused a dose-related decrease in osteocalcin mRNA levels in bone [ Fig. l-C, l-D, and 2-B]. The maximal decrease in osteocalcin mRNA levels was found around 24 h post administration. The osteocalcin mRNA level showed complete recovery to the control level by 72 h after administration of lmg/kg dexamethasone [Fig. 1-D and 2-B], while it remained at low levels throughout the observation period up to 168 h in animals administered with 100 mg/kg dexamethasone [Fig.l-C and.ZB]. The serum osteocalcin level fell rapidly to its nadir by 12 h after dexamethasone administration. The serum osteocalcin level recovered to the control level by 72 h after administration of lmg/kg dexamethasone, while it remained at low levels in animals administered with 100 mg/kg dexamethasone [Fig.3-B]. None of 1,25(OH)zD3, ED-71 and dexamethasone caused significant changes in the serum calcium and inorganic phosphorus levels (data not shown).
z 8 2 E
------+G
0
24
-
1.5
1.0
,
0 48
0
72 (h)
24
48 TIME
12 01)
Figure 2. The time course of the osteocalcin/cyclophilin mRNA ratio. A; After oral administration of O.l&kg of either 1,25(OH)zDs or ED-71. B; After intraperitoneal
administration of lmg/kg or lOOmg/kg of dexamethasone. The zero hour time points were determined using RNA samples obtained from rats treated with vehicles only.
1233
%
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.0
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8
0.5 0.25 i‘\,
120 -
so-
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$Il *
O40-
*
20 O-
*
f
,NR’DEx (lmgkg) * ,-(I l *,’
% DEX(lOOmg), 1. ** 0
TIME (h)
24
; 48
II-0 i . *,* 72
-011
Figure 3. The time courseof the serumosteocalcinlevel. A, after oral administration of O.l&kg of either 1,25(OH)zD3 or ED-71, or 0.25 ml MCI as a vehicle. B; after intraperitoneal administration of lmg/kg or lOOmg/kgof dexamethasone,or 0.125ml normal saline. *; P-zO.05,**; PcO.01(by two-tailed t test). C, The standard curve of osteocakin RIA.
DISCUSSION We have demonstrated in the present in vivo study in rats that 1,25(OHhD3 and ED-71 increase the levels of both osteocalcin mRNA in bone and serum osteocalcin while dexamethasone has an opposite effect on both parameters. Our results conform previous observations that 1,25(OH)zD3 causes the elevation of 1) serum osteocalcin levels in vivo in animals as well as human[ 1,15,16] and 2) osteocalcin production and its mRNA levels in osteoblasts in vitro [2,17]. ED-71 is a new synthetic analog of 1,25(OH)zD3 and binds to embryonic chick intestinal vitamin D receptors and rat plasma vitamin D binding protein with the affinities approximately one-eighth as low and twice as high, respectively, as those of 1,25(OH)zD3 [18]. When administered in rats, the plasma concentration of ED-71 was reported to be significantly higher than that of 1,25(OH)zD3 48 hours after equivalent oral doses [ 181. Our observation that the osteocalcin mRNA levels remained elevated above control levels over the period of one week after administration of ED71, but not 1,25(OH)zD3, appears consistent with the longer plasma half life of ED-71 compared to 1,25(OH)zD3. Dexamethasone has been shown to decrease the serum osteocalcin levels in viva in animals and human as well as its mRNA levels in vitro in cultured osteoblasts [6,7]. Our results show that dexamethasone treatment resulted in the decrease in the levels of both osteocalcin mRNA in bone and the serum osteocalcin in vivo in rats. Our data thus provide direct in vivo evidence for the notion that dexamethasone has an inhibitory effect on the transcriptional regulation of osteocalcin gene expression in bone. In the present study the decrease in serum osteocalcin levels preceded the decrease in osteocalcin mRNA levels in bone. Fortune et al. showed that in sheep 1234
B
I
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Q'@J5
C
"
168
Vol.
189,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
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glucocorticoids increase the clearance of osteocalcin from plasma [ 191. It is therefore possible that the initial rapid decline in serum osteocalcin levels following dexamethasone administration in the present study is accounted for, at least in part, by the accelerated plasma clearance of osteocalcin. Further study is necessary to test this hypothesis. ACKNOWLEDGMENTS
The authors thank Drs. Y. Nishii and K. Sato, Chugai Pharmaceuticals, Tokyo, Japan, for their help.
REFERENCES
1. Markowitz, M.E., Gundberg, C.M., and Rosen, C.F. (1987) Calcif. Tissue. Int.4& 179-183. 2. Pan, L., and Price, P. (1986) J. Bone Miner. Res. 1 (suppl. 1): A20. 3. Lian, J.B., and Gundberg, C.M. (1988) Clin. Grthoped. Rel. Res. 226: 267-291. 4. Melick, R.A., Farrugia, W., Heaton, C.L., Quelch, K.J., Scoggins, B.A., and Wark, J.D. (1988) Calcif. Tissue Int. 42: 185190. 5. Farrugia, W. and Melick, R.A. (1986) Calcif. Tissue Int. 39: 234-238. 6. Godschalk, M.F., and Downs R.W. (1988) J. Bone Miner. Res. 3: 113-115. 7. Noda, M. (1989) Endocrin. 124: 612-617. 8. Nemeth, G.G., Heydemann, A., and Bolander, M.E. (1989) Anal .Biochem. 183: 301-304. 9. Marks, SC., Jr., Mackowiak, S., Shaloub, V., Lian, J.B., and Stein, G.S. (1989) Connec. Tissue Res., 21: 107-116. 10. Ohta, S., Yamamuro, T., Lee, K., Okumura, H., Kasai, R., Hiraki, Y., Ikeda, T., Iwasaki, R., Kikuchi, H., Konishi, J., and Shigeno, C. (1991) FEBS Lett. 284: 42-45.
11. Chomczynski, P. and Sacchi, N. (1987) Anal. B&hem. 162: 156-159. 12. Nanes, MS., Rubin, J., Titus, L., Hendy, G.N., and Catherwood, B. (1991) Endocrinology. 128: 2577-2582. 13. Price, P.A., Parthemore, J.B., and Deftos, L.J. (1980) J. Clin. Invest. 66: 878883.
14. Kitamura, N., Shigeno, C., Shiomi, K., Lee, K., Ohta, S.. Sone, T., Katsushima, S., Tadamura, E., Kousaka, T., Yamamoto, I., Dokoh. S., and Konishi, J. (1990) J. Clin. Endocrinol. Metab. 70: 252-263. 15. Lian, J.B., Cames, D.L., and Glimcher, M.J.(1987) Endocrinology 120: 21232130. 16. Price, P.A., and Baukol, S.A. (1981) B&hem. Biophys. Res. Commun. 99: 928-935. 17. Price, P.A., andBauko1, S.A. (1980) J. Biol. Chem. 255: 11660-11663. 18. Okano, T., Tsugawa, N., Masuda, S., Takeuchi, A., Kobayashi, T., Takita, Y., and Nishii, Y. (1989) B&hem. Biophys. Res. Commun. 163: 1444-1449. 19. Fortune, C.L., Farrugia, W., Tresham, J., Scoggins, B.A., and Wark, J.D. (1989) Endocrinology 124: 2785-2790.
1235
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 1231-l 235
THE EFFECT OF ACTIVE VITAMIN D3 ANALOGS AND DEXAMETHASONE ON THE EXPRESSION OF OSTEOCALCIN GENE IN RAT TIBIAE IN VZVO Toshihiko Ike&I, Hiroaki Kohnol, Takao Yamamuml, Ryuichi Kasail, Shuichi OhtaI, Hideo Okumura3, Junji Konishiz, Hart&i Kikuchiz, and Chohei Shigen$
1 Department of Orthopaedic Surgery and 2 Calcium Laboratory, Department of Radiology & Nuclear Medicine, Kyoto University Faculty of Medicine, Kyoto 606-01, Japan 3 Department of Orthopaedic Surgery, Ehime University Faculty of Medicine, Ehime 79 l-02, Japan Received
November
6,
1992
SUMMARY: We tested the effects of lq25dihydroxyvitamin D3 (1,25-(OH)zD$, 2P-(3-hydroxypropoxy)- la,25dihydroxyvitamin D3 (ED-7 1) and dexamethasone on osteocalcin mRNA levels in rat tibiae in vivo. Northern blot analysis showed that both 1,25-(OH)2D3 and ED-71 caused an increase in osteocalcin mRNA levels in bone: 1,25-(OH)2D3 induced a transient increase in the mRNA levels followed by a decrease in the control level by 12 h post administration. In contrast, ED-71 caused a persistent increase in osteocalcin mRNA level for seven days post administration. Serum osteocalcin levels paralleled the osteocalcin mRNA level in bone in both groups. Dexamethasone caused a marked reduction in both osteocalcin mRNA and serum osteocalcin levels. Suppressive effect of dexamethasone on osteocalcin expression was persistent for seven days at higher dose. Our results represent the fmt demonstration of the effect of active vitamin D and corticosteroid on the expression of ostcocalcin mRNA in bone in vivo. 0 1992 Academic Press, ~nc.
Osteocalcin is secreted exclusively by osteoblasts and represents the major noncollagenous protein present in bone matrix. Since the half life of osteocalcin in the circulation is reported to be only five minutes, its serum concern&on is believed to reflect secretion by osteoblasts and therefore os;eoblastic activity [l-5]. Many hormones and drugs are known to modulate osteocalcin production in bone. In particular, active forms of vitamin D increase and dexamethasone decreases the levels of serum osteocalcin in vivo and osteocalcin mRNA in osteoblasts in vitro [ 1,2,6,7]. The effect of these steroid hormones on osteocalcin gene expression in vivo, however, has not yet been studied because of the difficulty in preparing RNA from skeletal hard tissues. We as well as others have recently established the reproducible method to
1231
0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
189, No. 2, 1992
quantitatively
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
extract total RNA from skeletal hard tissues [&lo].
This technique
allowed us to evaluate the effects of 1,25(OH)2D3 and ED-71, a newly synthesized active analog of 1,25(OH)zD3, as well as dexamethasone, on osteocalcin gene expression in vivo in adult rat tibiae, as reported here. MATERIALS
AND METHODS
Animals: 96 female Wistar rats (7-8 month-old, 250-280 g) were fed on a standard laboratory chow. 1,25(OH)zD3 (Duphar Co., Weesp, Netherlands) and ED-71 (a gift from Chugai Pharmaceuticals, Tokyo, Japan) were administered as a single oral dose using medium chain triglyceride (MCT, 0.25mVdose) as a vehicle. Dexamethasone-21phosphate disodium (a gift from Banyu Pharmaceuticals, Tokyo, Japan) was injected intraperitoneally at graded doses (lmg&g and lOOmg./kg) using normal saline (125 ul/dose) as a vehicle. Rats were sacrificed between 1O:OOand 13:OOat 3,6, 12, 24,72 and 168 h periods post administration. Three rats were used in each time point except for the zero hour time points for vehicle control groups that were assessed using six rats for each group. Animal protocols were conducted according to the guidelines of this institution. Prenaration of total RNA from bone: Legs were excised, tibiae were stripped off the surrounding soft tissues including the periosteum and the epiphysis and growth plate cartilage were resected. The bone marrow cavity was thoroughly washed out with 25 ml normal saline. Bone tissues, six or twelve tibiae in each group, were then minced and homogenized together, and the total RNA was extracted and analyzed by northern blot analysis as previously described [ 10,111. Northern blot analvsis: The following hybridization probes were synthesized using an auto DNA synthesizer (Pharmacia, Sweden): purified synthetic oligonucleotides of 42mer corresponding to the Cys23-Ile36 region of rat osteocalcin cDNA and of 48-mer corresponding to the Gln63-Ile78 region of rat cyclophilin cDNA as the internal control [ 121. The probes were labeled with [32P] y-ATP (New England Nuclear, Boston, MA) using T4 polynucleotide kinase (Takara Shuzo, Kyoto, Japan). Signal intensity was quantitated by a densitometer, and a ratio of signal intensity for osteocalcin (OC) versus cyclophilin (CYC) mRNAs (OC/CYC mRNA) was calculated for each time point and then normalized by the values for the time zero points of vehicle groups. Results were expressed in the mean * SD form from three northern blot analyses. Measurement of serum osteocalcin levels; The serum osteocalcin level was measured by a radioimmunoassay according to the method of &Priceet a1.[13]. Serum levels of calcium and inorganic phosphorus were determined by an auto-analyzer 1141. Statistical analvsis: Statistical significance was assessed by one-way analysis of variance and unpaired Student’s t test.
RESULTS 1,25(OH)zD3 caused a rapid increase in osteocalcin mRNA level which peaked at 6 h post administration and fell sharply thereafter [Fig.l-A and 2-A]. ED-71 also induced elevation in osteocalcin mRNA level with a peak at 6 h post administration [Fig. 1-B and 2-A], similar to 1,25(OH)zD3 treatment. However, unlike 1,25(OH)zD3, ED-71 caused persistent elevation of the osteocalcin mRNA levels for at least seven days post administration. Both 1,25(OH)zD3 and ED-7 1 resulted in significant increase in the serum osteocalcin levels [ Fig. 3-A]: 1,25(OH)zD3-induced increase in the serum osteocalcin level was rapid and occurred within 6 h after administration, while ED-7 1 caused a more gradual increase in the serum osteocalcin level which reached a plateau over 24-72 h post administration. 1232
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1992
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Figure 1. The expressionof osteocalcinmRNA in the rat tibiae after administration of A; 0. lug/kg of 1,25(OH)zD3, B; O.&/kg of ED-7 1, C, lOOmg/kgof dexamethasone, and D, lmgikg of dexamethasone.OC, osteocalcin,CYC; cyclophilin. The zero hour time points were determined using RNA samples obtained from rats treated with vehiclesonly.
Dexamethasone caused a dose-related decrease in osteocalcin mRNA levels in bone [ Fig. l-C, l-D, and 2-B]. The maximal decrease in osteocalcin mRNA levels was found around 24 h post administration. The osteocalcin mRNA level showed complete recovery to the control level by 72 h after administration of lmg/kg dexamethasone [Fig. 1-D and 2-B], while it remained at low levels throughout the observation period up to 168 h in animals administered with 100 mg/kg dexamethasone [Fig.l-C and.ZB]. The serum osteocalcin level fell rapidly to its nadir by 12 h after dexamethasone administration. The serum osteocalcin level recovered to the control level by 72 h after administration of lmg/kg dexamethasone, while it remained at low levels in animals administered with 100 mg/kg dexamethasone [Fig.3-B]. None of 1,25(OH)zD3, ED-71 and dexamethasone caused significant changes in the serum calcium and inorganic phosphorus levels (data not shown).
z 8 2 E
------+G
0
24
-
1.5
1.0
,
0 48
0
72 (h)
24
48 TIME
12 01)
Figure 2. The time course of the osteocalcin/cyclophilin mRNA ratio. A; After oral administration of O.l&kg of either 1,25(OH)zDs or ED-71. B; After intraperitoneal
administration of lmg/kg or lOOmg/kg of dexamethasone. The zero hour time points were determined using RNA samples obtained from rats treated with vehicles only.
1233
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189, No. 2, 1992
BIOCHEMICAL
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1.0
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0.5 0.25 i‘\,
120 -
so-
I /’
$Il *
O40-
*
20 O-
*
f
,NR’DEx (lmgkg) * ,-(I l *,’
% DEX(lOOmg), 1. ** 0
TIME (h)
24
; 48
II-0 i . *,* 72
-011
Figure 3. The time courseof the serumosteocalcinlevel. A, after oral administration of O.l&kg of either 1,25(OH)zD3 or ED-71, or 0.25 ml MCI as a vehicle. B; after intraperitoneal administration of lmg/kg or lOOmg/kgof dexamethasone,or 0.125ml normal saline. *; P-zO.05,**; PcO.01(by two-tailed t test). C, The standard curve of osteocakin RIA.
DISCUSSION We have demonstrated in the present in vivo study in rats that 1,25(OHhD3 and ED-71 increase the levels of both osteocalcin mRNA in bone and serum osteocalcin while dexamethasone has an opposite effect on both parameters. Our results conform previous observations that 1,25(OH)zD3 causes the elevation of 1) serum osteocalcin levels in vivo in animals as well as human[ 1,15,16] and 2) osteocalcin production and its mRNA levels in osteoblasts in vitro [2,17]. ED-71 is a new synthetic analog of 1,25(OH)zD3 and binds to embryonic chick intestinal vitamin D receptors and rat plasma vitamin D binding protein with the affinities approximately one-eighth as low and twice as high, respectively, as those of 1,25(OH)zD3 [18]. When administered in rats, the plasma concentration of ED-71 was reported to be significantly higher than that of 1,25(OH)zD3 48 hours after equivalent oral doses [ 181. Our observation that the osteocalcin mRNA levels remained elevated above control levels over the period of one week after administration of ED71, but not 1,25(OH)zD3, appears consistent with the longer plasma half life of ED-71 compared to 1,25(OH)zD3. Dexamethasone has been shown to decrease the serum osteocalcin levels in viva in animals and human as well as its mRNA levels in vitro in cultured osteoblasts [6,7]. Our results show that dexamethasone treatment resulted in the decrease in the levels of both osteocalcin mRNA in bone and the serum osteocalcin in vivo in rats. Our data thus provide direct in vivo evidence for the notion that dexamethasone has an inhibitory effect on the transcriptional regulation of osteocalcin gene expression in bone. In the present study the decrease in serum osteocalcin levels preceded the decrease in osteocalcin mRNA levels in bone. Fortune et al. showed that in sheep 1234
B
I
023~kiln0Pns/~l, 1 ._.-.-.-.-. A.-.-.-.-.-.-.-.-. ,il: _._. L: 10 SALINE 0’
Q'@J5
C
"
168
Vol.
189,
No.
2,
1992
BIOCHEMICAL
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BIOPHYSICAL
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glucocorticoids increase the clearance of osteocalcin from plasma [ 191. It is therefore possible that the initial rapid decline in serum osteocalcin levels following dexamethasone administration in the present study is accounted for, at least in part, by the accelerated plasma clearance of osteocalcin. Further study is necessary to test this hypothesis. ACKNOWLEDGMENTS
The authors thank Drs. Y. Nishii and K. Sato, Chugai Pharmaceuticals, Tokyo, Japan, for their help.
REFERENCES
1. Markowitz, M.E., Gundberg, C.M., and Rosen, C.F. (1987) Calcif. Tissue. Int.4& 179-183. 2. Pan, L., and Price, P. (1986) J. Bone Miner. Res. 1 (suppl. 1): A20. 3. Lian, J.B., and Gundberg, C.M. (1988) Clin. Grthoped. Rel. Res. 226: 267-291. 4. Melick, R.A., Farrugia, W., Heaton, C.L., Quelch, K.J., Scoggins, B.A., and Wark, J.D. (1988) Calcif. Tissue Int. 42: 185190. 5. Farrugia, W. and Melick, R.A. (1986) Calcif. Tissue Int. 39: 234-238. 6. Godschalk, M.F., and Downs R.W. (1988) J. Bone Miner. Res. 3: 113-115. 7. Noda, M. (1989) Endocrin. 124: 612-617. 8. Nemeth, G.G., Heydemann, A., and Bolander, M.E. (1989) Anal .Biochem. 183: 301-304. 9. Marks, SC., Jr., Mackowiak, S., Shaloub, V., Lian, J.B., and Stein, G.S. (1989) Connec. Tissue Res., 21: 107-116. 10. Ohta, S., Yamamuro, T., Lee, K., Okumura, H., Kasai, R., Hiraki, Y., Ikeda, T., Iwasaki, R., Kikuchi, H., Konishi, J., and Shigeno, C. (1991) FEBS Lett. 284: 42-45.
11. Chomczynski, P. and Sacchi, N. (1987) Anal. B&hem. 162: 156-159. 12. Nanes, MS., Rubin, J., Titus, L., Hendy, G.N., and Catherwood, B. (1991) Endocrinology. 128: 2577-2582. 13. Price, P.A., Parthemore, J.B., and Deftos, L.J. (1980) J. Clin. Invest. 66: 878883.
14. Kitamura, N., Shigeno, C., Shiomi, K., Lee, K., Ohta, S.. Sone, T., Katsushima, S., Tadamura, E., Kousaka, T., Yamamoto, I., Dokoh. S., and Konishi, J. (1990) J. Clin. Endocrinol. Metab. 70: 252-263. 15. Lian, J.B., Cames, D.L., and Glimcher, M.J.(1987) Endocrinology 120: 21232130. 16. Price, P.A., and Baukol, S.A. (1981) B&hem. Biophys. Res. Commun. 99: 928-935. 17. Price, P.A., andBauko1, S.A. (1980) J. Biol. Chem. 255: 11660-11663. 18. Okano, T., Tsugawa, N., Masuda, S., Takeuchi, A., Kobayashi, T., Takita, Y., and Nishii, Y. (1989) B&hem. Biophys. Res. Commun. 163: 1444-1449. 19. Fortune, C.L., Farrugia, W., Tresham, J., Scoggins, B.A., and Wark, J.D. (1989) Endocrinology 124: 2785-2790.
1235