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Stimulatory Effect of Zinc-Chelating Dipeptide on Deoxyribonucleic Acid Synthesis in Osteoblastic MC3T3-E1 Cells
Peptides, Vol. 17, No. 7, pp. 1207-1211, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/96 $15.00 + .00
PII S0196-9781(96)00114-3
Stimulator Effect of Zinc-Chelating Dipeptide on Deoxyribonucleic Acid Synthesis in Osteoblastic MC3T3-E1 Cells MASAYOSHI
YAMAGUCHI’
AND TOMOHIRO MATSUI
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City 422, Japan Received 23 February
1996
YAMAGUCHI,M. AND T. MATSUI. Stimulator effect of zinc-chelating dipeptide on deoxyribonucleic acid synthesis in osteoblastic MC3Z’3-EI cells. PEPTIDES17(7) 1207–1211,1996.—Whetherdeoxyribonucleicacid (DNA) synthesisin osteoblastic MC3T3-E1cells is stimulatedby zinc, an activator of bone formation,was investigatedin vitro. After subculturefor 3 days, the cells were cultured for up to 3 days (72 h) with zinc sulfate or zinc-chelateddipeptide (&alanyl-L-histidinatozinc; AHZ) in the range of 10-7to 10-5M. The culture with zinc compounds( 10-5M) produceda significantincreaseof cell number, DNAcontent,and proteinconcentrationin the cells, as reportedpreviously.The culturewith zinc compounds( 10-’ and 10-5M) clearly stimulatedDNA synthesis in the homogenate,when it was estimated by the incorporationof [3H]deoxythymidine5‘triphosphateinto the DNA in the homogenateof cells. The AHZ effect was greater than that of zinc sulfate. The culture together with cycloheximide(19 M) completelyabolishedthe zinc compounds(10 M)-induced increase of DNA synthesis in the cells, suggestingthat the zinc compoundeffect is based on a newly synthesizedproteincomponent.Moreover,when zinc sulfate ( 10-’ and 10-’ M) or AHZ ( 10-8 to 10-5M) was addedinto the reactionmixturewith the homogenateof cells culturedwithout zinc compounds,the DNA synthesis was clearly increased. The effect of addition of zinc compounds( 10-6 M) on the DNA synthesiswas completelyinhibitedby the presence of staurosporine( 10-8 M), an itrhibitorof protein kinase C, or okadaicacid ( 10-’ M), an inhibitorof proteinphosphatase.The present studydemonstratesthat zinc compoundshave a stimulator effect on DNA synthesisin osteoblasticcells. Copyright 01996 Elsevier Science Inc. &Alanyl-L-histidinatozinc
DNA synthesis
Cell proliferation
IT is known that zinc is essential for growth in humans and many animals ( 1). Bone growth retardation is a common finding in various conditions associated with zinc deficiency (7,13). Recently, zinc has been demonstrated to play a physiologic role as an activator in the regulation of bone formation due to stimulating bone protein synthesis in vivo and in vitro (23,24,26). Moreover, it has been reported that ~-alanyl-L-histidinato zinc (AHZ), in which zinc is chelated to ,8-alanyl-L-histidine, has a potent effect, more than that of zinc sulfate, on bone formation and calcification in vivo and in vitro ( 19,20,22,25), suggesting a role in the therapeutics for osteoporosis. More recently, it has been demonstrated that AHZ can stimulate the proliferation of osteoblastic cells in vitro, and its effect is more intensive than zinc sulfate (6). The mechanism of AHZ action, however, has not been clarified fully. AHZ has been shown to stimulate insulin-like growth factor-I (IGF-1) production in osteoblastic cells (21), and the anabolic effect of IGF-I in osteoblastic cells is enhanced by zinc (11 ). These findings suggest that AHZ action to stimulate osteoblastic cell proliferation is partly mediated through IGF-I action. Furthermore, the
Osteoblasticcells
present study was undertaken to clarify whether zinc-chelating dipeptide can directly stimulate DNA synthesis in osteoblastic MC3T3-EI cells in vitro. It was found that zinc-chelating dipeptide has a stimulator effect on DNA synthesis in osteoblastic cells in vitro. METHODS
Chemicals
a-Modification of Eagle’s minimum essential medium (aMEM) was obtained from Flow Laboratories, Inc. (Mclean, VA). Fetal bovine serum was obtained from Bioproducts Inc. (Walhersville, MD). [Methyl- 3H]deoxythymidine 5 ‘-triphosphate ( [3H]dTTP; 2.59 TBq/rnmol) was obtained from New England Nuclear (Boston, MA). Insulin-like growth factor-I (IGF1; human recombinant) was obtained from Mrtllinckrodt Speciality Chemicals Company, Inc. (Paris, France). Bovine serum albumin (fraction V), 17/3-estradiol, cyclohexirnide, staurosporine, and okadaic acid were purchased from Sigma Chemical Co. (St. Louis, MO). ,8-Alanyl-r--histidinato zinc (AHZ) was sup-
1Requests for reprints shouldbe addressedto MasayoshiYamaguchi,Ph.D., Laboratoryof Endocrinologyand MolecularMetabolism,Graduate Schoolof NutritionalSciences,Universityof Shizuoka,52-1 Yada, ShizuokaCity 422, Japan. 1207
1208
YAMAGUCHI AND MATSUI
plied by the Zeria Pharmaceutical Co. (Tokyo, Japan). Zinc sulfate, dipicolinate (2,3 -pyridinedicarboxylic acid; neutralized with sodium hydroxide), and all other chemicals were reagent grade from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Tissue culture plastic dishes were purchased from Falcon Plastics (Los Angels, CA). Other materials used were commercial products of the highest grade available.
Statistical Analysis
Cell Culture Osteoblastic MC3T3-E1 cells were generously provided by Drs. Y. Amagai and S. Kasai (Koriyama, Japan). The cells were cultured at 37°C in a COZincubator in plastic dishes containing a-MEM supplemented with 10% fetal bovine serum (FBS) (6). They were subculture every 3 days using 0.2% trypsin plus 0.02% ethylene-diamine-tetraacetic acid (EDTA) in Ca2+/ Mgz+-free phosphate-buffered saline (PBS). For experiments, about 2.5 X 103 cell per dish were cultured for 3 days to obtain confluent monolayer in 35-mm plastic dishes containing 2 ml a-MEM with 10’ZO FBS. After the cultures, the cells were rinsed with PBS, the medium was exchanged for that containing O.1% bovine serum albumin (BSA) plus various concentrations of zinc sulfate, AHZ, IGF-1, 17&estradiol, or other chemicals, and the cells were cultured for up to 3 days (72 h). Determination
of Cell Number
After trypsinization using 0.2% trypsin plus 0.02% EDTA in Ca2+/Mg 2+-free pBS, cell numbers were determined by the use of an electronic particle counter (6). Estimation
ice-cold 6.5 mkf barbital buffer (pH 7.4) containing 0.270 polyoxyethylene ( 10) octylphenyl ether (Triton X-1OO)solution, and disrupted for 60 s with an ultrasonic device ( 11). Protein concentration in the homogenate of cells was determined by the method of Lowry et al. (8) and was expressed as the amount of protein (pg) per dish.
of DNA Synthesis
To assay DNA synthesis activity in the cells after appropriate treatment periods, the cells were washed three times with PBS, scraped into 0.5 ml of ice-cold 0.25 M sucrose solution, and disrupted for 60 s with an ultrasonic device (6). The homogenate of cells was used for the measurement of DNA synthesis. DNA synthesis in the cell homogenate was estimated by the procedure of Lynch et al. (9,10) with a minor modification. DNA synthesis was measured for 30 min at 37°C in mixtures (0.5 ml) that contained 0.18 M Tris-HCl buffer (pH 8.2), 4 mlf MgCl*, 2 mkf ATP, dGTP, dCTP, dATP (each 0.08 mkf), 0.06 mlf [3H]dTTP, dextran (Type 100 C, 2%), 2.5 mkf cadaverine, and the suspension of cell homogenate (about 20 pg DNA/ml). In separate experiments, zinc sulfate ( 10-8 to 10-4 M), AHZ ( 1O-s to 10-4 M), staurosporine ( 10-8 M), okadaic acid ( 10-7 M), or dipicolinate (10‘G to 10-4 M) were added as indicated. Reactions were stopped with 0.5 ml of 1 N NaOH, and DNA was precipitated with addition (5 ml) of ice-cold trichloroacetic acid (TCA; 10%). The DNA was then dissolved (0.5 ml of 1 N NaOH) and precipitated (5 ml of 10% TCA), and the final precipitate, dissolved in 0.5 ml of 1 N NaOH, was heated at 80”C for 5 min. Finally, the DNA was precipitated with 10% TCA and the precipitate was washed with acid, ethanol, and ether. DNA content in the cell homogenate was determined by the method of Cerriotti (2). The radioactivity in the nuclear DNA was measured in a Hyamine-toluene liquid scintillation mixture, and all the data were corrected for the incorporation with control mixture that lacked the three unlabeled deoxynucleotides. DNA synthesis was expressed as disintegrations per minute (dpm) per incubation time (minute) per milligram of nuclear DNA.
ProteinDetermination To determine protein concentration in osteoblastic cells, the cells were washed three times with PBS, scraped into 0.5 ml of
Data are expressed as the mean f SEM. Statistical differences were analyzed using Student’s t-test. Values of p < 0.05 were considered to indicate statistically significant differences. RESULTS
The alteration of DNA synthesis in osteoblastic MC3T3-E1 cells cultured with zinc compounds is shown in Fig. 1. The cells were cultured for up to 3 days (72 h) in the medium containing either vehicle, zinc sulfate ( 10-5 M), or AHZ ( 10-5 M). DNA synthesis in the homogenate of cells was measured by the incorporation of [3H]dTTP into the DNA of homogenate. The culture with zinc sulfate or AHZ caused a significant increase of DNA synthesis in the homogenate of cells obtained at 2 and 3 days of culture. The effect of AHZ was greater than that of zinc sulfate. When the cells were cultured for 3 days with either vehicle (control), zinc sulfate ( 10-5 M), or AHZ ( 10-5 M), the number of cells was significantly (p < 0.01) increased from 2.7 + 0.34 to 5. I t 0.43 or 6.9 t 0.58 ( x 104/dish; mean + SEM of six dishes), respectively. In addition, DNA content in the cells cultured with either vehicle, zinc sulfate ( 10-5 M), or AHZ ( 10-5 M) for 3 days was significantly (p < 0.01) increased from 2.9 + 0.12 to 4.2 + 0.29 or 6.1 ~ 0.38 (pg/dish; mean t SEM of six dishes), respectively. The effect of increasing concentrations of zinc compounds on DNA synthesis in osteoblastic MC3T3-E1 cells is shown in Fig. 2. The cells were cultured for 3 days in the medium containing either vehicle, zinc sulfate, or AHZ in the range of 10-7 to 10-5 M. The presence of zinc compounds ( 10-7 and 10-6 M) caused a significant increase of DNA synthesis in the cell homogenate. With the highest concentration ( 10-5 M) used, however, the ef-
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FIG. 1. Effect of zinc compoundson DNA synthesis in osteoblastic MC3T3-E1cells. The cells were culturedfor up to 3 days (72 h) in the mediumcontainingeither vehicle, zinc sulfate (10-5M), or AHZ (10-5 ~. The activity of DNA synthesisin the homogenateof cells cultured with zinc compoundswas measuredby the incorporationof [3H]dTTP into the DNA of homogenate.Each value representsthe mean t SEM of six dishes. *P < 0.05, and **p < 0.01 compared with the control (vehicle)value.Openbars: control;stripedbars: zinc sulfate;filledbars: AHZ.
Z
DIPEPTIDE AND OSTEOBLASTIC DNA SYNTHESIS
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FIG. 2. Effect of increasing concentrations of zinc compounds on DNA synthesis in osteoblastic MC3T3-E1 cells. The cells were cultured for 3 days (72 h) in the medium containing either vehicle, zinc sulfate (10-’ to 10-5 M), or AHZ (10-7 to 10-5 M). The activity of DNA synthesis in the homogenate of cells cultured with zinc compounds was measured by the incorporation of [3H]dTTP into the DNA of homogenate. Each value represents the mean 3 SEM of six dishes. *p< 0.05, and **p< 0.01 compared with the control (vehicle) value. Zinc sulfate (0), AHZ (0).
feet was weakened, although the AHZ effect showed an appreciable increase. The effect of cycloheximide, an inhibitor of protein synthesis, on DNA synthesis in osteoblastic MC3T3-E1 cells cultured with zinc compounds is shown in Fig. 3. The cells were cultured for 3 days in the medium containing either vehicle, zinc sulfate ( 10-5 M), or AHZ ( 10-5 M) in the absence and presence of cycloheximide ( 10-6 M). The presence of cycloheximide completely abolished the zinc compounds-induced increase of DNA synthesis in the cell homogenate, suggesting that the zinc effect is based on a newly synthesized protein in the cells. The culture with zinc sulfate ( 10-5 M) or AHZ (10 M) for 3 days caused a significant increase of protein concentration in the osteoblastic cells; it was significantly (p < 0.01) increased 47.7 > 1.15 (control) to 63.5 t 5.02 or 69.5 ~ 4.10 (pg/dish; mean t SEM of six dishes), respectively. The effect of zinc addition on DNA synthesis in the homogenate of osteoblastic MC3T3-E1 cells was also examined, and the result is shown in Fig. 4. The cells were cultured for 48 h in the absence of zinc compounds. Zinc compounds were added in the incubation mixture of DNA synthesis in the cell homogenate. The presence of zinc sulfate ( 10-7 and 10-6 M) for 30 min caused a significant increase of DNA synthesis in the homogenate. This increase was not seen at the concentration of 10-5 M zinc sulfate, indicating an inhibitory effect. DNA synthesis was significantly increased by the presence of AHZ in the range of 10-8 to 10-5 M. The effect was saturated at 10-7 M. Thus, the AHZ effect was greater than that of zinc sulfate. The effect of addition of staurosporine or okadaic acid on DNA synthesis in the homogenate of osteoblastic MC3T3-E1 cells containing zinc compounds is shown in Fig. 5. The cells were cultured for 48 h in the medium not containing zinc compounds. Staurosporine is an inhibitor of protein kinase C ( 18), and okadaic acid is an inhibitor of protein phosphatase (3). The both inhibitors did not have an appreciable effect on DNA synthesis in the homogenate from the cells without zinc addition. However, the presence of staurosporine ( 10 M) or okadaic acid (10 “M) with an effective concentration ( 11) completely inhib-
FIG. 3. Effect of cycloheximide, an inhibitor of protein synthesis, on DNA synthesis in osteoblastic MC3T3-E1 cells cultured with zinc compounds. The cells were cultured for 3 days (72 h) in the medium containing either vehicle, zinc sulfate (10-5 Jf), or AHZ (10-5 kf) in the absence and presence of cycloheximide (10-6 M. The activity of DNA synthesis in the homogenate of cells was measured by the incorporation of [3H]dTTP into the DNA of homogenate. Each value represents the and **p<0.01 compared with mean * SEM of six dishes. *p<0.05, the control (none) value. Open bars: control; striped bars: zinc sulfate; filled bars: AHZ.
creased DNA synthesis in the cell homogenate. Meanwhile, the presence of IGF-I ( 10-8 M) or 17~-estradiol ( 10-’ M) in the incubation mixture with or without staurosporine ( 10-8 M) or okadaic acid ( 10 M) did not have an appreciable effect on DNA synthesis. The effect of zinc compounds on DNA synthesis in osteoblastic MC3T3-E1 cells in the presence of IGF-I or 17&estradiol, which has an anabolic effect on osteoblasts (5,17), was examined, and the result is shown in Fig. 6. The cells were cultured for 3 days in the medium containing either vehicle, IGF-I ( 10-8 M), or 17@estradiol ( 10-9 M) in the presence of zinc sulfate ( 10-5 M) or AHZ ( 10-5 M). The concentration of proliferation factors used was an effective level. The culture with IGF-I or
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FIG. 4. Effect of zinc addition on DNA synthesis in the homogenate of osteoblastic MC3T3-E1 cells. The cells were cultured for 48 h in the medium without addition of zinc compounds. DNA synthesis in the homogenate of cells was measured by the incorporation of [3H]dTTP into the DNA of homogenate in the presence of zinc sulfate or AHZ in the range of 10-8to 10-5M for 30 min. Each value representsthe mean i SEM of six dishes. *p<0.01 compared with the control (none) value.
1210
YAMAGUCHI AND MATSUI
None
Staurosporine
Okadaic acid
FIG. 5. Effect of additionof staurosporineor ocadaicacid on DNAsynthesis in the homogenateof osteoblasticMC3T3-E1cells.The cells were culturedfor 48 h in the mediumnot containingzinc compounds.DNA synthesisin the cell homogenatewas measuredby the incorporationof [3H]dTTPinto the DNA of homogenate.The reactionmixturecontained zinc sulfate (10–6M) or AHZ (10”6M) in the presenceof staurosporine (10-8 M) or okadaic acid (10-7&f).Each value represents the mean z SEM of six dishes. *P <0.01 comparedwith the control (none)value. Open bars: control; stripedbars: zinc sulfate; filledbars: AHZ. 17@estradiol alone caused a significant increase of DNA synthesis in the cell homogenate. These increases were clearly enhanced by the presence of AHZ. The culture with zinc sulfate fin-ther enhanced the steroid-induced increase of DNA synthesis in the cell homogenate, but this effect was not seen in the case of IGF-I. DISCUSSION
Recently, it has been demonstrated that the culture with zinc sulfate or AHZ, in which zinc is chelated to a dipeptide (@alanyl-L-histidine), stimulates proliferation of osteoblastic cells in vitro and that this effect is dependent on protein synthesis (6). The mechanism by which zinc compounds stimulate osteoblastic cell proliferation, however, has not been fully clarified. The present study was undertaken to clarify whether zinc can directly stimulate DNA synthesis in osteoblastic MC3T3-E1 cells. The culture with zinc sulfate or AHZ produced a significant increase of DNA synthesis in osteoblastic cells in vitro, when the synthesis was estimated by the incorporation of [3H] dTTP into the nuclear DNA of cell homogenate. The culture with zinc compounds caused a significant increase of cell number and DNA content in osteoblastic cells. Thus, the present finding suggests that the effect of zinc compounds to stimulate osteoblastic cell proliferation is based on the increase of DNA synthesis in the cells. The stimulator effect of zinc compound on DNA synthesis in osteoblastic cells was completely abolished when the cells were cultured with the coexistence of cycloheximide, an inhibitor of protein synthesis. This result suggests that the action of zinc to stimulate DNA synthesis in the cells is mainly mediated through a newly synthesized protein component. The present result may further support the view that the stimulator effect of zinc compound on the proliferation of osteoblastic cells is depend on protein synthesis, as reported previously (6). Moreover, the direct effect with zinc addition into the reaction mixture of DNA synthesis in the homogenate of osteoblastic cells was examined. The addition of zinc sulfate or AHZ significantly increased DNA synthesis in the cell homogenate. This result suggests that zinc compounds can stimulate DNA synthesis depen-
dent on protein synthesis in vitro, because the metal can directly activate aminoacyl-tRNA synthetase (22). The possibility that zinc can directly stimulate, at least partly, DNA synthesis in the cells cannot be excluded. DNA polymerase is a zinc enzyme (14). However, the present of dipicolinate (10-’ to 10-4 M), which is a potent chelator of zinc ion ( 15), did not cause an appreciable alteration of DNA synthesis in the homogenate of osteoblastic cells (data not shown). In the present experimental system, the endogenous zinc in osteoblastic cells may not involve in the stimulation of DNA synthesis. The zinc compound-increased DNA synthesis in the homogenate of osteoblastic cells was clearly inhibited by the coexistence of staurosporine or okadaic acid in the reaction mixture. Staurosporine is an inhibitor of protein kinase C ( 18), and okadaic acid is an inhibitor of protein phosphatase (3 ). The presence of staurosporine or okadaic acid had no effect on DNA synthesis in the cell homogenate without zinc addition. From these results, it is assumed that zinc compounds activate protein kinase C and/ or protein phosphatase in the cell homogenate. It is possible that the stimulator effect of zinc compounds on DNA synthesis may be partly mediated through the signaling pathway of protein kinase C and protein phosphatase in osteoblastic cells. The stimulator effect of zinc-chelated dipeptide (AHZ) on DNA synthesis in osteoblastic cells was greater than that of zinc sulfate. However, the AHZ effect was dependent on zinc ion, because its effect was abolished by the coexistence of dipicolinate, a chelator of zinc ion (15). Presumably, the dipeptide makes a useful state in the revelation of zinc action on DNA synthesis in the cells. Zinc ion may bind to metallothionein in osteoblastic cells, and the activity of zinc ion may be weakened. The culture with IGF-I or 17/3-estradiol, which has an anabolic effect on osteoblastic cells (5,17), produced a significant increase of DNA synthesis in osteoblastic cells. Especially, the 17@estradiol effect was markedly enhanced by the coexistence of zinc sulfate or AHZ. The receptors of 17@-estradiol exist in the nuclei of osteoblastic cells (4). Receptors for steroid hormone have been shown to have two zinc fingers at the site of interaction with DNA ( 12). One possible mechanism of zinc
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FIG.6. Effectof IGF-Ior 17@estradiolonDNAsynthesisin osteoblastic MC3T3-E1cells culturedwith zinc compounds.The cells were cultured for 3 days (72 h) in the mediumcontainingeither vehicle, IGF-I (lO-s M), or 17&estradiol(10-’ M) in the absenceandpresenceof zinc sulfate (10-5M) or AHZ (10-5M). The activity of DNA synthesisin the homogenateof cells was measuredby the incorporationof [3H]dTTPinto the DNAof homogenate.Each value representsthe mean f SEMof six dishes. *p <0.05, and **p <0.01 comparedwith the control (none) value. #p < 0.01 compared with the value of IGF-I or 17(3-estradiol alone. Openbars: control; stripedbars: zinc sulfate; filledbars: AHZ.
ZINC-CHELATING DIPEPTIDE AND OSTEOBLASTIC DNA SYNTHESIS compound may be to potentate the interaction of the steroid– receptor complex with DNA at that site. The present finding suggests that zinc plays a cell physiologic role in the enhancement of anabolic effect of estrogen on osteoblastic cells, although the cloned cells do not fulfill all of the physiological criteria for normal osteoblasts in vitro. It is well known that ovarian hormone
1211
deficiency at menopause stimulates bone loss ( 16). Zinc may have a pathophysiologic role in the development of osteoporosis. In conclusion, it has been demonstrated that zinc compounds have a stimulato~ effect on DNA synthesis in osteoblastic cells in vitro. The present finding may provide a possible mechanism of zinc action to stimulate the proliferation of osteoblastic cells.
REFERENCES 1. Burch, R. E.; Hahn, H. K. J.; Sullivan,J. F. Newer aspects of the
roles of zinc, manganese, and copper in human nutrition. Clin. Chem.21:501–520; 1975. 2. Ceriotti,G. Determinationof nucleicacids in animaltissues.J. Biol. Chem. 214:39–77; 1955. 3. Cohen,P.; Klumpp,S.; Shelling,D. L. An improvedprocedurefor 4.
5. 6.
7.
8. 9. 10. 11, 12.
13.
identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett. 250:596–600; 1989. Eriksen, E. F.; Colvard, D. S.; Berg, N. J.; Graham, M. L.; Mann, K. G.; Spelsberg, T. C.; Riggs, B, L. Evidence of estrogen receptors in normal human osteoblast-like cells. Science 241:84–86; 1988. Gray, T. K.; Flyynn, T. C.; Gray, K. M.; Nabel, L. M. 17/3-Estradiol acts directly on the clonal osteoblastic cell line. Proc. Nat]. Acad. Sci. USA 84:6267–6271; 1987. Hashizume, M.; Yamaguchi, M. Stimulator effect of ,f?-alanyl-Lhistidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells. Mol. Cell. Biochem. 122:59– 64; 1993. Hurley, L. S.; Gowan, J.; Milhaoud, G. Calcium metabolism in manganese-deficient and zinc-deficient rats. Proc. Soc. Exp. Biol. Med. 130:856–860; 1969. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. Lynch, W. E.; Brown, R. F.; Umeda, T.; Langreth, S. G.; Lieberman, I. Synthesis of deoxyribonucleic acid by isolated liver nuclei. J. Biol, Chem. 245:391 1–3916; 1970. Lynch, W. E.; Umeda, T.; Uyeda, M,; Lieberman, I. Nature of the deoxyribonucleic acid made by isolated liver nuclei. Biochem. Biophys. Acta 287:28–37; 1972. Matsui, T.; Yamaguchi, M. Zinc modulation of insulin-like growth factor’s effect in osteoblastic MC3T3-E1 cells. Peptides 16:1063– 1068; 1995. McDonnell, D. P.; Mongelsdorf, D. J.; Pike, J, W.; Haussler, M. R.; O’Malley, B. W. Molecular cloning of complementary DNA encoding the avian receptor for vitamin D. Science 235:1214– 1217; 1987. Oner, G.; Bhaumick, B.; Bala, R. M. Effect of zinc deficiency on serum somatomedin levels and skeletal growth in young rats. Endocrinology 114:1860–1863; 1984.
14. Parisi, A. F.; Vallee, B. L. Zinc metalloenzyme: Characteristics and significance in biology and medicine. Am. J. Clin. Nutr. 22: 1222– 1230; 1969. 15. Pocker, Y.; Fong, C. T. 0. Kinetics of inactivation of erythrocyte carbonic anhydrase by sodium 2,6-pyridinedicarboxy late. Biochemistry 19:2045–2050; 1980. 16. Riggs, B. L.; Jowsey, J.; Kelly, P. J.; Jones, J. D.; Maher, F. T. Effect of sex hormones on bone in primary osteoporosis. J, Clin. Invest. 48:1065-1072; 1969. 17. Schmid, C.; Guler, H. P.; Rowe, D.; Froesch, E. R. Insulin-like growth factor I regulates type I procollagen mRNA steady state levels in bone of rats. Endocrinology 125:1575– 1580; 1989. 18. Tamaoki, T.; Nomoto, H.; Takahashi, I.; Kate, Y,; Morimoto, M.; Tomita, F. Staurosporin, a potent inhibitor of phospholipid/Ca2+ dependent protein kinase. Biochem. Biophys. Res. Commun. 135:397-402; 1986. 19. Yamaguchi, M. @Alanyl-L-histidinato zinc: A potent activator in bone formation. Curr. Med. Chem. 1:356–365; 1995. 20. Yamaguchi, M. ~-Alanyl-L-histidinato zinc and bone resorption. Gen. Pharrnacol. 26:1 179–1 183; 1995. 21. Yamaguchi, M.; Hashizume, M. Effect of@-alanyl-L-histidinato zinc on protein compounds in osteoblastic MC3T3-E1 cells: Increase in osteocalcin, insulin-like growth factor-I and transforming growth factor-~, Mol. Cell. Biochem. 136:163-169; 1994. 22. Yamaguchi, M.; Kishi, S.; Hashizume, M. Effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase. Peptides 15:1367– 1371; 1994. 23. Yamaguchi, M.; Oishi, H.; Suketa, Y. Stimulator effect of zinc on bone formation in tissue culture. Biochem. Pharmacol. 36:4007– 4012; 1987. 24. Yamaguchi, M.; Oishi, H.; Suketa, Y. Zinc stimulation of bone protein synthesis in tissue culture. Activation of aminoacyl-tRNA synthetase. Biochem. Pharrnacol. 37:4075–4080; 1988. 25. Yamaguchi, M.; Ozaki, K. A new zinc compound, ,8-akmyl-L.-histidinato zinc, stimulates bone growth in weanling rats. Res. Exp, Med. (Berl.) 190:105-110; 1990. 26. Yamaguchi, M.; Yamaguchi, R, Action of zinc on bone metabolism in rats. Increase in alkaline phosphatase activity and DNA content. Biochem. Pharmacol. 35:773–777; 1986.
PII S0196-9781(96)00114-3
Stimulator Effect of Zinc-Chelating Dipeptide on Deoxyribonucleic Acid Synthesis in Osteoblastic MC3T3-E1 Cells MASAYOSHI
YAMAGUCHI’
AND TOMOHIRO MATSUI
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City 422, Japan Received 23 February
1996
YAMAGUCHI,M. AND T. MATSUI. Stimulator effect of zinc-chelating dipeptide on deoxyribonucleic acid synthesis in osteoblastic MC3Z’3-EI cells. PEPTIDES17(7) 1207–1211,1996.—Whetherdeoxyribonucleicacid (DNA) synthesisin osteoblastic MC3T3-E1cells is stimulatedby zinc, an activator of bone formation,was investigatedin vitro. After subculturefor 3 days, the cells were cultured for up to 3 days (72 h) with zinc sulfate or zinc-chelateddipeptide (&alanyl-L-histidinatozinc; AHZ) in the range of 10-7to 10-5M. The culture with zinc compounds( 10-5M) produceda significantincreaseof cell number, DNAcontent,and proteinconcentrationin the cells, as reportedpreviously.The culturewith zinc compounds( 10-’ and 10-5M) clearly stimulatedDNA synthesis in the homogenate,when it was estimated by the incorporationof [3H]deoxythymidine5‘triphosphateinto the DNA in the homogenateof cells. The AHZ effect was greater than that of zinc sulfate. The culture together with cycloheximide(19 M) completelyabolishedthe zinc compounds(10 M)-induced increase of DNA synthesis in the cells, suggestingthat the zinc compoundeffect is based on a newly synthesizedproteincomponent.Moreover,when zinc sulfate ( 10-’ and 10-’ M) or AHZ ( 10-8 to 10-5M) was addedinto the reactionmixturewith the homogenateof cells culturedwithout zinc compounds,the DNA synthesis was clearly increased. The effect of addition of zinc compounds( 10-6 M) on the DNA synthesiswas completelyinhibitedby the presence of staurosporine( 10-8 M), an itrhibitorof protein kinase C, or okadaicacid ( 10-’ M), an inhibitorof proteinphosphatase.The present studydemonstratesthat zinc compoundshave a stimulator effect on DNA synthesisin osteoblasticcells. Copyright 01996 Elsevier Science Inc. &Alanyl-L-histidinatozinc
DNA synthesis
Cell proliferation
IT is known that zinc is essential for growth in humans and many animals ( 1). Bone growth retardation is a common finding in various conditions associated with zinc deficiency (7,13). Recently, zinc has been demonstrated to play a physiologic role as an activator in the regulation of bone formation due to stimulating bone protein synthesis in vivo and in vitro (23,24,26). Moreover, it has been reported that ~-alanyl-L-histidinato zinc (AHZ), in which zinc is chelated to ,8-alanyl-L-histidine, has a potent effect, more than that of zinc sulfate, on bone formation and calcification in vivo and in vitro ( 19,20,22,25), suggesting a role in the therapeutics for osteoporosis. More recently, it has been demonstrated that AHZ can stimulate the proliferation of osteoblastic cells in vitro, and its effect is more intensive than zinc sulfate (6). The mechanism of AHZ action, however, has not been clarified fully. AHZ has been shown to stimulate insulin-like growth factor-I (IGF-1) production in osteoblastic cells (21), and the anabolic effect of IGF-I in osteoblastic cells is enhanced by zinc (11 ). These findings suggest that AHZ action to stimulate osteoblastic cell proliferation is partly mediated through IGF-I action. Furthermore, the
Osteoblasticcells
present study was undertaken to clarify whether zinc-chelating dipeptide can directly stimulate DNA synthesis in osteoblastic MC3T3-EI cells in vitro. It was found that zinc-chelating dipeptide has a stimulator effect on DNA synthesis in osteoblastic cells in vitro. METHODS
Chemicals
a-Modification of Eagle’s minimum essential medium (aMEM) was obtained from Flow Laboratories, Inc. (Mclean, VA). Fetal bovine serum was obtained from Bioproducts Inc. (Walhersville, MD). [Methyl- 3H]deoxythymidine 5 ‘-triphosphate ( [3H]dTTP; 2.59 TBq/rnmol) was obtained from New England Nuclear (Boston, MA). Insulin-like growth factor-I (IGF1; human recombinant) was obtained from Mrtllinckrodt Speciality Chemicals Company, Inc. (Paris, France). Bovine serum albumin (fraction V), 17/3-estradiol, cyclohexirnide, staurosporine, and okadaic acid were purchased from Sigma Chemical Co. (St. Louis, MO). ,8-Alanyl-r--histidinato zinc (AHZ) was sup-
1Requests for reprints shouldbe addressedto MasayoshiYamaguchi,Ph.D., Laboratoryof Endocrinologyand MolecularMetabolism,Graduate Schoolof NutritionalSciences,Universityof Shizuoka,52-1 Yada, ShizuokaCity 422, Japan. 1207
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YAMAGUCHI AND MATSUI
plied by the Zeria Pharmaceutical Co. (Tokyo, Japan). Zinc sulfate, dipicolinate (2,3 -pyridinedicarboxylic acid; neutralized with sodium hydroxide), and all other chemicals were reagent grade from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Tissue culture plastic dishes were purchased from Falcon Plastics (Los Angels, CA). Other materials used were commercial products of the highest grade available.
Statistical Analysis
Cell Culture Osteoblastic MC3T3-E1 cells were generously provided by Drs. Y. Amagai and S. Kasai (Koriyama, Japan). The cells were cultured at 37°C in a COZincubator in plastic dishes containing a-MEM supplemented with 10% fetal bovine serum (FBS) (6). They were subculture every 3 days using 0.2% trypsin plus 0.02% ethylene-diamine-tetraacetic acid (EDTA) in Ca2+/ Mgz+-free phosphate-buffered saline (PBS). For experiments, about 2.5 X 103 cell per dish were cultured for 3 days to obtain confluent monolayer in 35-mm plastic dishes containing 2 ml a-MEM with 10’ZO FBS. After the cultures, the cells were rinsed with PBS, the medium was exchanged for that containing O.1% bovine serum albumin (BSA) plus various concentrations of zinc sulfate, AHZ, IGF-1, 17&estradiol, or other chemicals, and the cells were cultured for up to 3 days (72 h). Determination
of Cell Number
After trypsinization using 0.2% trypsin plus 0.02% EDTA in Ca2+/Mg 2+-free pBS, cell numbers were determined by the use of an electronic particle counter (6). Estimation
ice-cold 6.5 mkf barbital buffer (pH 7.4) containing 0.270 polyoxyethylene ( 10) octylphenyl ether (Triton X-1OO)solution, and disrupted for 60 s with an ultrasonic device ( 11). Protein concentration in the homogenate of cells was determined by the method of Lowry et al. (8) and was expressed as the amount of protein (pg) per dish.
of DNA Synthesis
To assay DNA synthesis activity in the cells after appropriate treatment periods, the cells were washed three times with PBS, scraped into 0.5 ml of ice-cold 0.25 M sucrose solution, and disrupted for 60 s with an ultrasonic device (6). The homogenate of cells was used for the measurement of DNA synthesis. DNA synthesis in the cell homogenate was estimated by the procedure of Lynch et al. (9,10) with a minor modification. DNA synthesis was measured for 30 min at 37°C in mixtures (0.5 ml) that contained 0.18 M Tris-HCl buffer (pH 8.2), 4 mlf MgCl*, 2 mkf ATP, dGTP, dCTP, dATP (each 0.08 mkf), 0.06 mlf [3H]dTTP, dextran (Type 100 C, 2%), 2.5 mkf cadaverine, and the suspension of cell homogenate (about 20 pg DNA/ml). In separate experiments, zinc sulfate ( 10-8 to 10-4 M), AHZ ( 1O-s to 10-4 M), staurosporine ( 10-8 M), okadaic acid ( 10-7 M), or dipicolinate (10‘G to 10-4 M) were added as indicated. Reactions were stopped with 0.5 ml of 1 N NaOH, and DNA was precipitated with addition (5 ml) of ice-cold trichloroacetic acid (TCA; 10%). The DNA was then dissolved (0.5 ml of 1 N NaOH) and precipitated (5 ml of 10% TCA), and the final precipitate, dissolved in 0.5 ml of 1 N NaOH, was heated at 80”C for 5 min. Finally, the DNA was precipitated with 10% TCA and the precipitate was washed with acid, ethanol, and ether. DNA content in the cell homogenate was determined by the method of Cerriotti (2). The radioactivity in the nuclear DNA was measured in a Hyamine-toluene liquid scintillation mixture, and all the data were corrected for the incorporation with control mixture that lacked the three unlabeled deoxynucleotides. DNA synthesis was expressed as disintegrations per minute (dpm) per incubation time (minute) per milligram of nuclear DNA.
ProteinDetermination To determine protein concentration in osteoblastic cells, the cells were washed three times with PBS, scraped into 0.5 ml of
Data are expressed as the mean f SEM. Statistical differences were analyzed using Student’s t-test. Values of p < 0.05 were considered to indicate statistically significant differences. RESULTS
The alteration of DNA synthesis in osteoblastic MC3T3-E1 cells cultured with zinc compounds is shown in Fig. 1. The cells were cultured for up to 3 days (72 h) in the medium containing either vehicle, zinc sulfate ( 10-5 M), or AHZ ( 10-5 M). DNA synthesis in the homogenate of cells was measured by the incorporation of [3H]dTTP into the DNA of homogenate. The culture with zinc sulfate or AHZ caused a significant increase of DNA synthesis in the homogenate of cells obtained at 2 and 3 days of culture. The effect of AHZ was greater than that of zinc sulfate. When the cells were cultured for 3 days with either vehicle (control), zinc sulfate ( 10-5 M), or AHZ ( 10-5 M), the number of cells was significantly (p < 0.01) increased from 2.7 + 0.34 to 5. I t 0.43 or 6.9 t 0.58 ( x 104/dish; mean + SEM of six dishes), respectively. In addition, DNA content in the cells cultured with either vehicle, zinc sulfate ( 10-5 M), or AHZ ( 10-5 M) for 3 days was significantly (p < 0.01) increased from 2.9 + 0.12 to 4.2 + 0.29 or 6.1 ~ 0.38 (pg/dish; mean t SEM of six dishes), respectively. The effect of increasing concentrations of zinc compounds on DNA synthesis in osteoblastic MC3T3-E1 cells is shown in Fig. 2. The cells were cultured for 3 days in the medium containing either vehicle, zinc sulfate, or AHZ in the range of 10-7 to 10-5 M. The presence of zinc compounds ( 10-7 and 10-6 M) caused a significant increase of DNA synthesis in the cell homogenate. With the highest concentration ( 10-5 M) used, however, the ef-
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FIG. 1. Effect of zinc compoundson DNA synthesis in osteoblastic MC3T3-E1cells. The cells were culturedfor up to 3 days (72 h) in the mediumcontainingeither vehicle, zinc sulfate (10-5M), or AHZ (10-5 ~. The activity of DNA synthesisin the homogenateof cells cultured with zinc compoundswas measuredby the incorporationof [3H]dTTP into the DNA of homogenate.Each value representsthe mean t SEM of six dishes. *P < 0.05, and **p < 0.01 compared with the control (vehicle)value.Openbars: control;stripedbars: zinc sulfate;filledbars: AHZ.
Z
DIPEPTIDE AND OSTEOBLASTIC DNA SYNTHESIS
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FIG. 2. Effect of increasing concentrations of zinc compounds on DNA synthesis in osteoblastic MC3T3-E1 cells. The cells were cultured for 3 days (72 h) in the medium containing either vehicle, zinc sulfate (10-’ to 10-5 M), or AHZ (10-7 to 10-5 M). The activity of DNA synthesis in the homogenate of cells cultured with zinc compounds was measured by the incorporation of [3H]dTTP into the DNA of homogenate. Each value represents the mean 3 SEM of six dishes. *p< 0.05, and **p< 0.01 compared with the control (vehicle) value. Zinc sulfate (0), AHZ (0).
feet was weakened, although the AHZ effect showed an appreciable increase. The effect of cycloheximide, an inhibitor of protein synthesis, on DNA synthesis in osteoblastic MC3T3-E1 cells cultured with zinc compounds is shown in Fig. 3. The cells were cultured for 3 days in the medium containing either vehicle, zinc sulfate ( 10-5 M), or AHZ ( 10-5 M) in the absence and presence of cycloheximide ( 10-6 M). The presence of cycloheximide completely abolished the zinc compounds-induced increase of DNA synthesis in the cell homogenate, suggesting that the zinc effect is based on a newly synthesized protein in the cells. The culture with zinc sulfate ( 10-5 M) or AHZ (10 M) for 3 days caused a significant increase of protein concentration in the osteoblastic cells; it was significantly (p < 0.01) increased 47.7 > 1.15 (control) to 63.5 t 5.02 or 69.5 ~ 4.10 (pg/dish; mean t SEM of six dishes), respectively. The effect of zinc addition on DNA synthesis in the homogenate of osteoblastic MC3T3-E1 cells was also examined, and the result is shown in Fig. 4. The cells were cultured for 48 h in the absence of zinc compounds. Zinc compounds were added in the incubation mixture of DNA synthesis in the cell homogenate. The presence of zinc sulfate ( 10-7 and 10-6 M) for 30 min caused a significant increase of DNA synthesis in the homogenate. This increase was not seen at the concentration of 10-5 M zinc sulfate, indicating an inhibitory effect. DNA synthesis was significantly increased by the presence of AHZ in the range of 10-8 to 10-5 M. The effect was saturated at 10-7 M. Thus, the AHZ effect was greater than that of zinc sulfate. The effect of addition of staurosporine or okadaic acid on DNA synthesis in the homogenate of osteoblastic MC3T3-E1 cells containing zinc compounds is shown in Fig. 5. The cells were cultured for 48 h in the medium not containing zinc compounds. Staurosporine is an inhibitor of protein kinase C ( 18), and okadaic acid is an inhibitor of protein phosphatase (3). The both inhibitors did not have an appreciable effect on DNA synthesis in the homogenate from the cells without zinc addition. However, the presence of staurosporine ( 10 M) or okadaic acid (10 “M) with an effective concentration ( 11) completely inhib-
FIG. 3. Effect of cycloheximide, an inhibitor of protein synthesis, on DNA synthesis in osteoblastic MC3T3-E1 cells cultured with zinc compounds. The cells were cultured for 3 days (72 h) in the medium containing either vehicle, zinc sulfate (10-5 Jf), or AHZ (10-5 kf) in the absence and presence of cycloheximide (10-6 M. The activity of DNA synthesis in the homogenate of cells was measured by the incorporation of [3H]dTTP into the DNA of homogenate. Each value represents the and **p<0.01 compared with mean * SEM of six dishes. *p<0.05, the control (none) value. Open bars: control; striped bars: zinc sulfate; filled bars: AHZ.
creased DNA synthesis in the cell homogenate. Meanwhile, the presence of IGF-I ( 10-8 M) or 17~-estradiol ( 10-’ M) in the incubation mixture with or without staurosporine ( 10-8 M) or okadaic acid ( 10 M) did not have an appreciable effect on DNA synthesis. The effect of zinc compounds on DNA synthesis in osteoblastic MC3T3-E1 cells in the presence of IGF-I or 17&estradiol, which has an anabolic effect on osteoblasts (5,17), was examined, and the result is shown in Fig. 6. The cells were cultured for 3 days in the medium containing either vehicle, IGF-I ( 10-8 M), or 17@estradiol ( 10-9 M) in the presence of zinc sulfate ( 10-5 M) or AHZ ( 10-5 M). The concentration of proliferation factors used was an effective level. The culture with IGF-I or
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FIG. 4. Effect of zinc addition on DNA synthesis in the homogenate of osteoblastic MC3T3-E1 cells. The cells were cultured for 48 h in the medium without addition of zinc compounds. DNA synthesis in the homogenate of cells was measured by the incorporation of [3H]dTTP into the DNA of homogenate in the presence of zinc sulfate or AHZ in the range of 10-8to 10-5M for 30 min. Each value representsthe mean i SEM of six dishes. *p<0.01 compared with the control (none) value.
1210
YAMAGUCHI AND MATSUI
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Staurosporine
Okadaic acid
FIG. 5. Effect of additionof staurosporineor ocadaicacid on DNAsynthesis in the homogenateof osteoblasticMC3T3-E1cells.The cells were culturedfor 48 h in the mediumnot containingzinc compounds.DNA synthesisin the cell homogenatewas measuredby the incorporationof [3H]dTTPinto the DNA of homogenate.The reactionmixturecontained zinc sulfate (10–6M) or AHZ (10”6M) in the presenceof staurosporine (10-8 M) or okadaic acid (10-7&f).Each value represents the mean z SEM of six dishes. *P <0.01 comparedwith the control (none)value. Open bars: control; stripedbars: zinc sulfate; filledbars: AHZ. 17@estradiol alone caused a significant increase of DNA synthesis in the cell homogenate. These increases were clearly enhanced by the presence of AHZ. The culture with zinc sulfate fin-ther enhanced the steroid-induced increase of DNA synthesis in the cell homogenate, but this effect was not seen in the case of IGF-I. DISCUSSION
Recently, it has been demonstrated that the culture with zinc sulfate or AHZ, in which zinc is chelated to a dipeptide (@alanyl-L-histidine), stimulates proliferation of osteoblastic cells in vitro and that this effect is dependent on protein synthesis (6). The mechanism by which zinc compounds stimulate osteoblastic cell proliferation, however, has not been fully clarified. The present study was undertaken to clarify whether zinc can directly stimulate DNA synthesis in osteoblastic MC3T3-E1 cells. The culture with zinc sulfate or AHZ produced a significant increase of DNA synthesis in osteoblastic cells in vitro, when the synthesis was estimated by the incorporation of [3H] dTTP into the nuclear DNA of cell homogenate. The culture with zinc compounds caused a significant increase of cell number and DNA content in osteoblastic cells. Thus, the present finding suggests that the effect of zinc compounds to stimulate osteoblastic cell proliferation is based on the increase of DNA synthesis in the cells. The stimulator effect of zinc compound on DNA synthesis in osteoblastic cells was completely abolished when the cells were cultured with the coexistence of cycloheximide, an inhibitor of protein synthesis. This result suggests that the action of zinc to stimulate DNA synthesis in the cells is mainly mediated through a newly synthesized protein component. The present result may further support the view that the stimulator effect of zinc compound on the proliferation of osteoblastic cells is depend on protein synthesis, as reported previously (6). Moreover, the direct effect with zinc addition into the reaction mixture of DNA synthesis in the homogenate of osteoblastic cells was examined. The addition of zinc sulfate or AHZ significantly increased DNA synthesis in the cell homogenate. This result suggests that zinc compounds can stimulate DNA synthesis depen-
dent on protein synthesis in vitro, because the metal can directly activate aminoacyl-tRNA synthetase (22). The possibility that zinc can directly stimulate, at least partly, DNA synthesis in the cells cannot be excluded. DNA polymerase is a zinc enzyme (14). However, the present of dipicolinate (10-’ to 10-4 M), which is a potent chelator of zinc ion ( 15), did not cause an appreciable alteration of DNA synthesis in the homogenate of osteoblastic cells (data not shown). In the present experimental system, the endogenous zinc in osteoblastic cells may not involve in the stimulation of DNA synthesis. The zinc compound-increased DNA synthesis in the homogenate of osteoblastic cells was clearly inhibited by the coexistence of staurosporine or okadaic acid in the reaction mixture. Staurosporine is an inhibitor of protein kinase C ( 18), and okadaic acid is an inhibitor of protein phosphatase (3 ). The presence of staurosporine or okadaic acid had no effect on DNA synthesis in the cell homogenate without zinc addition. From these results, it is assumed that zinc compounds activate protein kinase C and/ or protein phosphatase in the cell homogenate. It is possible that the stimulator effect of zinc compounds on DNA synthesis may be partly mediated through the signaling pathway of protein kinase C and protein phosphatase in osteoblastic cells. The stimulator effect of zinc-chelated dipeptide (AHZ) on DNA synthesis in osteoblastic cells was greater than that of zinc sulfate. However, the AHZ effect was dependent on zinc ion, because its effect was abolished by the coexistence of dipicolinate, a chelator of zinc ion (15). Presumably, the dipeptide makes a useful state in the revelation of zinc action on DNA synthesis in the cells. Zinc ion may bind to metallothionein in osteoblastic cells, and the activity of zinc ion may be weakened. The culture with IGF-I or 17/3-estradiol, which has an anabolic effect on osteoblastic cells (5,17), produced a significant increase of DNA synthesis in osteoblastic cells. Especially, the 17@estradiol effect was markedly enhanced by the coexistence of zinc sulfate or AHZ. The receptors of 17@-estradiol exist in the nuclei of osteoblastic cells (4). Receptors for steroid hormone have been shown to have two zinc fingers at the site of interaction with DNA ( 12). One possible mechanism of zinc
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FIG.6. Effectof IGF-Ior 17@estradiolonDNAsynthesisin osteoblastic MC3T3-E1cells culturedwith zinc compounds.The cells were cultured for 3 days (72 h) in the mediumcontainingeither vehicle, IGF-I (lO-s M), or 17&estradiol(10-’ M) in the absenceandpresenceof zinc sulfate (10-5M) or AHZ (10-5M). The activity of DNA synthesisin the homogenateof cells was measuredby the incorporationof [3H]dTTPinto the DNAof homogenate.Each value representsthe mean f SEMof six dishes. *p <0.05, and **p <0.01 comparedwith the control (none) value. #p < 0.01 compared with the value of IGF-I or 17(3-estradiol alone. Openbars: control; stripedbars: zinc sulfate; filledbars: AHZ.
ZINC-CHELATING DIPEPTIDE AND OSTEOBLASTIC DNA SYNTHESIS compound may be to potentate the interaction of the steroid– receptor complex with DNA at that site. The present finding suggests that zinc plays a cell physiologic role in the enhancement of anabolic effect of estrogen on osteoblastic cells, although the cloned cells do not fulfill all of the physiological criteria for normal osteoblasts in vitro. It is well known that ovarian hormone
1211
deficiency at menopause stimulates bone loss ( 16). Zinc may have a pathophysiologic role in the development of osteoporosis. In conclusion, it has been demonstrated that zinc compounds have a stimulato~ effect on DNA synthesis in osteoblastic cells in vitro. The present finding may provide a possible mechanism of zinc action to stimulate the proliferation of osteoblastic cells.
REFERENCES 1. Burch, R. E.; Hahn, H. K. J.; Sullivan,J. F. Newer aspects of the
roles of zinc, manganese, and copper in human nutrition. Clin. Chem.21:501–520; 1975. 2. Ceriotti,G. Determinationof nucleicacids in animaltissues.J. Biol. Chem. 214:39–77; 1955. 3. Cohen,P.; Klumpp,S.; Shelling,D. L. An improvedprocedurefor 4.
5. 6.
7.
8. 9. 10. 11, 12.
13.
identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett. 250:596–600; 1989. Eriksen, E. F.; Colvard, D. S.; Berg, N. J.; Graham, M. L.; Mann, K. G.; Spelsberg, T. C.; Riggs, B, L. Evidence of estrogen receptors in normal human osteoblast-like cells. Science 241:84–86; 1988. Gray, T. K.; Flyynn, T. C.; Gray, K. M.; Nabel, L. M. 17/3-Estradiol acts directly on the clonal osteoblastic cell line. Proc. Nat]. Acad. Sci. USA 84:6267–6271; 1987. Hashizume, M.; Yamaguchi, M. Stimulator effect of ,f?-alanyl-Lhistidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells. Mol. Cell. Biochem. 122:59– 64; 1993. Hurley, L. S.; Gowan, J.; Milhaoud, G. Calcium metabolism in manganese-deficient and zinc-deficient rats. Proc. Soc. Exp. Biol. Med. 130:856–860; 1969. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275; 1951. Lynch, W. E.; Brown, R. F.; Umeda, T.; Langreth, S. G.; Lieberman, I. Synthesis of deoxyribonucleic acid by isolated liver nuclei. J. Biol, Chem. 245:391 1–3916; 1970. Lynch, W. E.; Umeda, T.; Uyeda, M,; Lieberman, I. Nature of the deoxyribonucleic acid made by isolated liver nuclei. Biochem. Biophys. Acta 287:28–37; 1972. Matsui, T.; Yamaguchi, M. Zinc modulation of insulin-like growth factor’s effect in osteoblastic MC3T3-E1 cells. Peptides 16:1063– 1068; 1995. McDonnell, D. P.; Mongelsdorf, D. J.; Pike, J, W.; Haussler, M. R.; O’Malley, B. W. Molecular cloning of complementary DNA encoding the avian receptor for vitamin D. Science 235:1214– 1217; 1987. Oner, G.; Bhaumick, B.; Bala, R. M. Effect of zinc deficiency on serum somatomedin levels and skeletal growth in young rats. Endocrinology 114:1860–1863; 1984.
14. Parisi, A. F.; Vallee, B. L. Zinc metalloenzyme: Characteristics and significance in biology and medicine. Am. J. Clin. Nutr. 22: 1222– 1230; 1969. 15. Pocker, Y.; Fong, C. T. 0. Kinetics of inactivation of erythrocyte carbonic anhydrase by sodium 2,6-pyridinedicarboxy late. Biochemistry 19:2045–2050; 1980. 16. Riggs, B. L.; Jowsey, J.; Kelly, P. J.; Jones, J. D.; Maher, F. T. Effect of sex hormones on bone in primary osteoporosis. J, Clin. Invest. 48:1065-1072; 1969. 17. Schmid, C.; Guler, H. P.; Rowe, D.; Froesch, E. R. Insulin-like growth factor I regulates type I procollagen mRNA steady state levels in bone of rats. Endocrinology 125:1575– 1580; 1989. 18. Tamaoki, T.; Nomoto, H.; Takahashi, I.; Kate, Y,; Morimoto, M.; Tomita, F. Staurosporin, a potent inhibitor of phospholipid/Ca2+ dependent protein kinase. Biochem. Biophys. Res. Commun. 135:397-402; 1986. 19. Yamaguchi, M. @Alanyl-L-histidinato zinc: A potent activator in bone formation. Curr. Med. Chem. 1:356–365; 1995. 20. Yamaguchi, M. ~-Alanyl-L-histidinato zinc and bone resorption. Gen. Pharrnacol. 26:1 179–1 183; 1995. 21. Yamaguchi, M.; Hashizume, M. Effect of@-alanyl-L-histidinato zinc on protein compounds in osteoblastic MC3T3-E1 cells: Increase in osteocalcin, insulin-like growth factor-I and transforming growth factor-~, Mol. Cell. Biochem. 136:163-169; 1994. 22. Yamaguchi, M.; Kishi, S.; Hashizume, M. Effect of zinc-chelating dipeptides on osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase. Peptides 15:1367– 1371; 1994. 23. Yamaguchi, M.; Oishi, H.; Suketa, Y. Stimulator effect of zinc on bone formation in tissue culture. Biochem. Pharmacol. 36:4007– 4012; 1987. 24. Yamaguchi, M.; Oishi, H.; Suketa, Y. Zinc stimulation of bone protein synthesis in tissue culture. Activation of aminoacyl-tRNA synthetase. Biochem. Pharrnacol. 37:4075–4080; 1988. 25. Yamaguchi, M.; Ozaki, K. A new zinc compound, ,8-akmyl-L.-histidinato zinc, stimulates bone growth in weanling rats. Res. Exp, Med. (Berl.) 190:105-110; 1990. 26. Yamaguchi, M.; Yamaguchi, R, Action of zinc on bone metabolism in rats. Increase in alkaline phosphatase activity and DNA content. Biochem. Pharmacol. 35:773–777; 1986.