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Calcium ‘homeostatic’ effect of 1α,25-dihydroxycholecal-ciferol on young adult mouse calvaria in organ culture
Mechanisms of Ageing and Development, 60 ( 1991 ) 25-34 Elsevier Scientific Publishers Ireland Ltd.
25
CALCIUM 'HOMEOSTATIC' EFFECT OF Iot,25-DIHYDROXYCHOLECALCIFEROL ON YOUNG ADULT MOUSE CALVARIA IN ORGAN CULTURE
PAUL GOLDHABER a and LUKA RABADJIJA b aProfessor of Periodontology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 and hPrincipal Research Associate in Oral Biology and Pathophysiology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 (U.S.A.) (Received August 13th, 1990) (Revision received January 18th, 1991)
SUMMARY
The effect of let,25-dihydroxycholecalciferol on calcium release from cultured young adult (75-day-old) mouse calvaria in the presence of indomethacin or dexamethasone was studied. Indomethacin (2.8 x 10-7 M) or dexamethasone (10 -8 M) abolished or considerably inhibited basal calcium release. At 2.6 x 10 -7 M, lot,25dihydroxycholecalciferol prevented the effect of either indomethacin or dexamethasone, resulting in higher calcium release levels approximating those observed in cultures containing only lot,25-dihydroxycholecalciferol.We have shown previously that in contrast to its effect on neonatal mouse calvaria in culture, la,25-dihydroxycholecalciferol either failed to enhance or even inhibited basal calcium release from cultured 75-day-old mouse calvaria. Furthermore, lt~,25-dihydroxycholecalciferol inhibited PTH- or PGE2-enhanced calcium release from such cultures. These studies demonstrated that, at the same concentration, lot,25-dihydroxycholecalciferol decreases high calcium release due to various bone resorption-stimulating factors and increases low calcium release values due to various bone resorption-inhibiting factors. This suggested that this vitamin D 3 metabolite directly regulates the degree of bone resorption at the bone tissue level resulting in a local calcium 'homeostasis'.
Key words: Bone resorption; Calcium homeostasis; Dexamethasone; h~,25-Dihydroxycholecalciferol; Indomethacin; Parathyroid hormone; Prostaglandin E2
Correspondence to: Luka Rabadjija, M.D., D.Sc., Principal Research Associate in Oral Biology and Pathophysiology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115, U.S.A. 0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
26 INTRODUCTION Recently, we reported [1,2] that at concentrations in the range 2.6 x 10 -6 M to 2.6 x 10 -s M, la,25-dihydroxycholecalciferol (1,25 D H C C ) significantly inhibited both basal bone resorption and bone resorption promoted by parathyroid hormone (PTH) or prostaglandin E2 (PGE2) in 75-day-old mouse calvaria cultures. However, at concentrations in the same dose range, 1,25 D H C C strongly stimulated bone resorption in 5-day old mouse calvaria cultures [1]. After having carried out numerous experiments with 1,25 D H C C utilizing 75-day-old mouse calvaria cultures, several interesting observations were made: (1) The basal calcium release from untreated 75-day-old mouse calvaria cultures was variable from experiment to experiment, from as little as 6.7 mg/dl to as much as 18.4 mg/dl over a 14-day culture period. (2) In those experiments where basal calcium release was high ( > 14 mg/dl), 1,25 D H C C usually showed a considerable and statistically significant inhibition of calcium release. Occasionally, in such experiments, the release of calcium promoted by P T H or PGE2 was masked. This could be demonstrated by inclusion of indomethacin in the medium, which strongly inhibited basal calcium release but had no effect on calcium release in the presence of PTH- or P G E 2 (unpublished results). Calcium release from P T H - or PGE2-containing cultures was consistently decreased by 1,25 D H C C . Moreover, the calcium in the medium was lowered to a level at or below the basal calcium release. (3) In those experiments where basal calcium release was low ( < 14 mg/dl), 1,25 D H C C did not show a statistically significant inhibition of calcium release. When basal calcium release was low, P T H or PGE2 usually caused a significant enhancement of calcium release. Again, 1,25 D H C C consistently lowered the PTH- or PGE2-enhanced calcium release to a level at or below basal calcium release. Although 1,25 D H C C did significantly lower basal calcium release in those cultures where basal calcium release was high, it was not as effective as indomethacin, which almost completely abolished calcium release from cultured 75oday-old calvaria. Therefore, the effect of 1,25 D H C C in the presence of indomethacin was tested. Surprisingly, it was found that indomethacin suppression of calcium release was blocked by 1,25 D H C C and the concentration of calcium in the medium was maintained at a level approximating basal calcium release. In view of this unexpected finding, additional experiments were performed with 1,25 D H C C and indomethacin, and, with 1,25 D H C C and dexamethasone, another inhibitor of basal calcium release [3]. MATERIALS AND METHODS Bone cultures were prepared and maintained as described previously [1,3]. Briefly, male mice, age 73--77 days, were obtained from our own breeding colony (Swiss albino, Webster strain) and were randomly assigned to the experimental groups. Cal-
27
varia were cut to form a trapezoid having approximately one 6-mm side, two 7-mm sides and one 10-mm side. Intra- and interexperimental variations in the surface of dissected area was not more than 10%. Such preparations encompassed substantial parts of the frontal bone and both parietal bones. Each calvarium was mounted individually on stainless steel grids in a small Petri dish (35 mm x 10 mm, Falcon No. 3001). Two milliliters of Dulbecco's Modified Eagle's Medium, supplemented with bovine serum albumin, antibiotics, heparin and components to be tested were added to each culture. Indomethacin and 1,25 DHCC were dissolved in ethanol. The final concentration of ethanol never exceeded 0.2% and at that concentration had no effect on bone resorption in control or PTH-treated cultures. The same amount of ethanol was added to control cultures as was present in experimental cultures. PTH was dissolved in 0.15 M NaC1. Dexamethasone was dissolved in tissue culture medium. There were 5--7 cultures per group. Cultures were placed in a CO2 incubator (Queue, Model 2210) at 37°C and maintained for 14 days. Media were changed every 2--3 days. Used media were analyzed for calcium content using a Corning Calcium Analyzer, Model 940 or Nova Biomedical 7 + 7 (Nova Biomedical, Waltham, MA). At the end of the experiment, bones were routinely fixed and processed for histological examination using hematoxylin and eosin staining. All data were subjected to analysis of variance. Standard errors were calculated from standard deviations and sample sizes. Statistical significance of the difference between groups was determined using least significant difference criterion [4]. Dulbecco's Modified Eagle's Medium and glutamine were purchased from M.A. Bioproducts, Walkerville, MD; bovine serum albumin, parathyroid hormone (synthetic, bovine, fragment 1--34), prostaglandin E2 and dexamethasone from Sigma Chemical Co., St. Louis, MO; heparin (manufactured by E. Lilly Co., Indianapolis, IN) from a local pharmaceutical distributor; and antibiotics from Grand Island Biol. Co., New York, NY. Indomethacin was kindly provided by Dr. C.A. Stone, Merck, Sharp and Dohme Research Laboratories, West Point, PA; and lc~,25-dihydroxycholecalciferol by Dr. M. Uskokovic, Hoffmann-LaRoche, Nutley, NJ. RESULTS
Table I presents net cumulative data from a series of experiments wherein the effect of indomethacin was tested in the presence of 1,25 DHCC. As shown, indomethacin alone strongly inhibited calcium release in all experiments. This inhibitory effect of indomethacin was significantly reversed by 1,25 DHCC in all 5 experiments, resulting in calcium release values approximating those observed in control cultures treated with 1,25 DHCC alone. It should be noted that 1,25 DHCC suppressed calcium release in all 5 experiments. However, the difference in calcium release between untreated control and 1,25 DHCC-treated cultures was statistically significant only in 2 experiments.
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30 Data presented in Table II are obtained from a series of experiments wherein the effect of dexamethasone was tested in the presence of 1,25 DHCC. Dexamethasone alone strongly inhibited calcium release in all experiments. Similar to the inhibitory effect of indomethacin, the inhibitory effect of dexamethasone was significantly reversed by 1,25 DHCC in all six experiments, resulting in calcium release values approximating those observed in untreated control cultures or cultures treated with 1,25 DHCC alone. In this series of the experiments, the basal calcium release values obtained from the untreated cultures were relatively low and in 5 of 6 experiments 1,25 DHCC had no inhibitory effect on calcium release as compared to the untreated controls. The finding that 1,25 DHCC elevated the depressed calcium release values due to the presence of either indomethacin or dexamethasone alone appeared to be contrary to our previous reports [1,2] demonstrating that 1,25 DHCC depressed elevated calcium release values due to the presence of PTH or PGE2. Therefore, the data from all relevant experiments were reviewed in order to determine whether any pattern would emerge from these paradoxical findings. Figure 1 summarizes the data obtained from 22 experiments wherein the effect of 1,25 DHCC (2.6 × 10 -7 M) on calcium release from 75-day-old mouse calvaria cultures was tested under various experimental conditions. The data points in each of the panels are arranged in descending order for the untreated controls (panel A) and cultures treated with PTH (panel B), PGE2 (panel C), indomethacin (panel D), and dexamethasone (panel E), respectively. Each data point represents the mean and standard error of the mean of calcium release from a treatment group containing at least 5 cultures. Also, they are arranged in such a manner that those found in the same vertical plane were part of the same experiment, thereby allowing comparison of specific groups within the same experiment. For the sake of simplicity in presenting the data, the corresponding untreated controls are not included in panels B, C, D, and E. As shown in panel A, where basal calcium release was high, 1,25 DHCC significantly inhibited calcium release (see asterisks). As may be seen in panel B, in all experiments except one, PTH significantly enhanced calcium release compared to the corresponding untreated control (see asterisks). Also, 1,25 D H C C significantly inhibited the calcium releaseenhancing effect of PTH in all 10 experiments (see daggers). As shown in panel C, PGE2 significantly enhanced calcium release compared to the corresponding untreated control in 2 of 4 experiments (see asterisks). As was the case with PTH, 1,25 DHCC significantly inhibited the calcium release-enhancing effect of PGE2 in all experiments (see daggers). In contrast to the calcium release-enhancing effect of PTH or PGE2, it may be seen from panel D that indomethacin significantly inhibited calcium release in all 5 experiments, as compared to the corresponding untreated controls (see asterisks). However, 1,25 DHCC essentially abolished the calcium release-inhibitory effect of indomethacin in all experiments (see daggers). In panel E, similar to the results obtained with indomethacin, it may be seen that dexamethasone also strongly inhibited basal calcium release in all 6 experiments (see
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asterisks). Again, 1,25 DHCC essentially abolished the calcium release-inhibitory effect of dexamethasone in all 6 experiments (see daggers). Histological examination of cultured bones demonstrating a modest to high calcium release showed the presence of osteoclasts and evidence of osteoclastic resorption. The most striking histological observation was that cultures containing indomethacin or dexamethasone had fewer osteoclasts and evidence of bone resorption than the untreated control cultures. The addition of 1,25 DHCC to cultures containing either indomethacin or dexamethasone appeared to reverse this effect causing a greater number of osteoclasts to be present. It is noteworthy that both these histological observations were more obvious in cultures containing higher concentrations of indomethacin (2.8 x 10 -6 M) or dexamethasone (10 -7 M) and correlated with the reversal of calcium release depression (data not shown). With regard to PTHor PGE2-stimulated bone resorption, osteoclasts were quite numerous in both these "groups. However, the addition of 1,25 D H C C did not result in any obvious diminution in number of osteoclasts present in the tissue. Similarly, in otherwise untreated cultures, 1,25 D H C C did not result in any obvious decrease in number of osteoclasts as compared to the untreated control bones. DISCUSSION
Although we have previously reported that the time course pattern of basal calcium release is different for untreated cultured young adult mouse caivaria as compared to untreated neonatal mouse calvaria, calcium release is enhanced in both age groups in response to a number of bone resorption-stimulating agents, namely, PTH or PGE2. Similarly, basal calcium release is inhibited in both age groups in response to several bone resorption-inhibiting agents, namely, indomethacin or dexamethasone [1--3]. Therefore, it was surprising to observe that the response of young adult mouse calvaria to 1,25 D H C C was entirely different compared to the response of cultured neonatal mouse calvaria. Whereas 1,25 DHCC, at all concentrations tested (2.6 x 10 -9 M to 2.6 x 10 -6 M), enhanced bone resorption in cultured neonatal mouse calvaria, this compound, at higher concentrations (2.6 x 10 -7 M and 2.6 x 10 -6 M), inhibited basal bone resorption in cultured young adult mouse calvaria [1]. Results obtained in the present study essentially confirmed and extended previously published observations to include the inhibitory effect of 1,25 DHCC on calcium release from cultured young adult mouse calvaria exposed to PGE2. In addition, what was not anticipated in the current series of experiments was the observation that, at 2.6 x 10 -7 M, 1,25 D H C C essentially abolished the bone resorptioninhibiting effect of indomethacin or dexamethasone. Presently, no explanation for this effect of 1,25 D H C C is available. It would appear that under culture conditions as described, 1,25 D H C C has the capacity to either stimulate or inhibit calcium release thereby modulating the effect of bone resorption-stimulating or bone resorption-inhibiting agents and restricting to a more intermediate range calcium
33 release values that would otherwise be extremely high or extremely low. This direct regulating effect of 1,25 DHCC on bone tissue suggests that bone might contain a local calcium 'homeostatic' mechanism that could be of physiological importance. Apparently, this local mechanism is 1,25 DHCC-dependent and is not as well developed in neonatal bones suggesting that this capacity develops as a function of age. It would be of interest to determine whether this regulating mechanism continues to function with the same efficiency as the animal ages or whether advanced age leads to dysfunction thereby resulting in pathological alterations of bone. Since this mechanism appears to respond to either a 'systemic', bone resorptionstimulating agent, such as PTH, or a local bone resorption-stimulating agent, such as PGE2, it appears to have the capacity to modulate locally either systemic or locally produced factors that influence bone resorption. It is conceivable that some of the results presented here might be explained in part by a modulating effect of 1,25 DHCC on the lipid composition and permeability of cell membranes [5,6], which might in turn influence calcium movement into and out of bone. Gunasekaran et al. [71 have reported that the addition of 1,25 DHCC to calvaria cultures from 6-week-old quail resulted in a fall in medium total and ionic calcium concentrations. Of interest was the fact that they could not demonstrate any effect of 1,25 DHCC on net calcium movement in cultures of calvaria removed from quail up to the age of 2 weeks. They suggested that the results with older quail were due to a direct stimulatory effect in vitro of 1,25 DHCC on calcium movement into bone. However, since these authors reported that PTH had no effect on the medium calcium in their 6-week-old quail calvaria cultures, it is evident that this culture system does not respond to bone resorption-stimulating factors comparable to the response obtained with neonatal or young adult mouse calvaria. Histological observations made in the present study are consistent with the concept that the calcium homeostatic activity of bone cells in the presence of 1,25 DHCC is related to the differentiation and function of osteoclasts. Whereas indomethacin and dexamethasone apparently inhibited the formation of osteoclasts, the presence of 1,25 DHCC stimulated their development and function, as determined histologically and by calcium release. On the other hand, where high calcium release is reduced in the presence of 1,25 DHCC, as is the case in PTH- or PGE2treated cultures or in some control cultures, there was no obvious diminution in the number of osteoclasts, suggesting that the inhibition of calcium release by 1,25 DHCC in these cases was due primarily to an effect on the function of osteoclasts. It should be noted that 1,25 DHCC has been shown to inhibit PTH-enhanced cyclic AMP production in cultured bone cells or bone tissues [8--10]. In other tissue culture systems, 1,25 DHCC inhibited the proliferation of murine granulocytemacrophage progenitor cells, and, also, induced murine leukemia cells to differentiate into macrophages [ 11,12]. Such diverse actions of 1,25 DHCC suggest a pivotal role for this vitamin D 3 metabolite in the local regulation of bone remodeling above and beyond its well recognized role in systemic calcium homeostasis.
34 ACKNOWLEDGEMENTS T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e t h e skilled t e c h n i c a l a s s i s t a n c e o f J o a n J e n n ings, L o r r a i n e S t e v e n s , a n d B e v e r l y B e r e s f o r d . T h i s s t u d y was s u p p o r t e d in p a r t by t h e G e l f a n d F u n d at the H a r v a r d S c h o o l o f D e n t a l M e d i c i n e a n d t h e N a t i o n a l Institutes o f H e a l t h G r a n t A G - 0 2 8 9 9 . REFERENCES 1 P. Goldhaber and L. Rabadjija, Age-dependent stimulation or inhibition of calcium release from bone cultures by hx,25-(OH)2D 3. Mech. Ageing Dev., 49 (1989) 199--209. 2 P. Goldhaber and L. Rabadjija, Calcium 'homeostatic' effect of l a,25-(OH)2D 3 on adult mouse calvaria cultures. J. Dent. Res. (spec. issue), 68 0989) 967 (abstr. 803). 3 P. Goldhaber, L. Rabadjija and S.L. Swartz, Effect of age on calcium release from mouse calvaria in tissue culture. Mech. Ageing Dev., 34 (1986) 273--288. 4 G.W. Snedecor and W.G. Cochran, Statistical Methods, 6th edn., The Iowa State University Press, Ames, 1976. 5 A.W. Norman and F.P. Ross, Vitamin D secosteroids: Unique molecules with both hormone and possible membranophilic properties. Life Sci., 24 (1979) 759--770. 6 B.R.C. Kurnik, M. Huskey and K. Hruska, 1,25-dihydroxycholecalciferol stimulates renal phosphate transport by directly altering membrane phosphatidylcholine composition. Biochim. Biophys. Acta, 917 0987) 81--85. 7 S. Gunasekaran, G.E. Hall and A.D. Kenny, Vitamin D and avian bone in vitro: Stimulation of calcium movement into Japanese quail calvaria. Calcif. Tissue Int., 39 (1986) 396--403. 8 G.L. Wong, R.A. Luben and D.V. Cohn, 1,25-dihydroxycholecalciferol and parathormone: Effects on isolated osteoclast-like and osteoblast-like cells. Science, 197 (1977) 663--665. 9 M.P.M. Hermann-Erlec and P.J. Gaillard, The effects of 1,25-dihydroxycholecalciferol on embryonic bone in vitro: A biochemical and histological study. Calcif. Tissue Res., 25 (1978) I l l--118. l0 B.D. Catherwood, 1,25-dihydroxycholecalciferol and glucocorticoids regulation ofadenylate cyclase in an osteoblast-like cell line. J. Biol. Chem., 260 (1984) 736--743. I I C. Miyaura, E. Abe, H. Nomura, Y. Nishii and T. Suda, h x,25-dihydroxyvitamin D3 suppresses proliferation of routine granulocyte-macrophage progenitor cell (CFU-C). Biochem. Biophys. Res. Commun., 108 0982) 1728--1733. 12 E. Abe, C. Miyaura, H. Sakagami, M. Takeda, K. Konno, T. Yamazaki, S. Yoshiki and T. Suda, Differentiation of mouse myeloid leukemia cells induced by h x,25-dihydroxyvitamin D 3. Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 4990----4994.
25
CALCIUM 'HOMEOSTATIC' EFFECT OF Iot,25-DIHYDROXYCHOLECALCIFEROL ON YOUNG ADULT MOUSE CALVARIA IN ORGAN CULTURE
PAUL GOLDHABER a and LUKA RABADJIJA b aProfessor of Periodontology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 and hPrincipal Research Associate in Oral Biology and Pathophysiology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115 (U.S.A.) (Received August 13th, 1990) (Revision received January 18th, 1991)
SUMMARY
The effect of let,25-dihydroxycholecalciferol on calcium release from cultured young adult (75-day-old) mouse calvaria in the presence of indomethacin or dexamethasone was studied. Indomethacin (2.8 x 10-7 M) or dexamethasone (10 -8 M) abolished or considerably inhibited basal calcium release. At 2.6 x 10 -7 M, lot,25dihydroxycholecalciferol prevented the effect of either indomethacin or dexamethasone, resulting in higher calcium release levels approximating those observed in cultures containing only lot,25-dihydroxycholecalciferol.We have shown previously that in contrast to its effect on neonatal mouse calvaria in culture, la,25-dihydroxycholecalciferol either failed to enhance or even inhibited basal calcium release from cultured 75-day-old mouse calvaria. Furthermore, lt~,25-dihydroxycholecalciferol inhibited PTH- or PGE2-enhanced calcium release from such cultures. These studies demonstrated that, at the same concentration, lot,25-dihydroxycholecalciferol decreases high calcium release due to various bone resorption-stimulating factors and increases low calcium release values due to various bone resorption-inhibiting factors. This suggested that this vitamin D 3 metabolite directly regulates the degree of bone resorption at the bone tissue level resulting in a local calcium 'homeostasis'.
Key words: Bone resorption; Calcium homeostasis; Dexamethasone; h~,25-Dihydroxycholecalciferol; Indomethacin; Parathyroid hormone; Prostaglandin E2
Correspondence to: Luka Rabadjija, M.D., D.Sc., Principal Research Associate in Oral Biology and Pathophysiology, Harvard School of Dental Medicine, 188 Longwood Avenue, Boston, MA 02115, U.S.A. 0047-6374/91/$03.50 Printed and Published in Ireland
© 1991 Elsevier Scientific Publishers Ireland Ltd.
26 INTRODUCTION Recently, we reported [1,2] that at concentrations in the range 2.6 x 10 -6 M to 2.6 x 10 -s M, la,25-dihydroxycholecalciferol (1,25 D H C C ) significantly inhibited both basal bone resorption and bone resorption promoted by parathyroid hormone (PTH) or prostaglandin E2 (PGE2) in 75-day-old mouse calvaria cultures. However, at concentrations in the same dose range, 1,25 D H C C strongly stimulated bone resorption in 5-day old mouse calvaria cultures [1]. After having carried out numerous experiments with 1,25 D H C C utilizing 75-day-old mouse calvaria cultures, several interesting observations were made: (1) The basal calcium release from untreated 75-day-old mouse calvaria cultures was variable from experiment to experiment, from as little as 6.7 mg/dl to as much as 18.4 mg/dl over a 14-day culture period. (2) In those experiments where basal calcium release was high ( > 14 mg/dl), 1,25 D H C C usually showed a considerable and statistically significant inhibition of calcium release. Occasionally, in such experiments, the release of calcium promoted by P T H or PGE2 was masked. This could be demonstrated by inclusion of indomethacin in the medium, which strongly inhibited basal calcium release but had no effect on calcium release in the presence of PTH- or P G E 2 (unpublished results). Calcium release from P T H - or PGE2-containing cultures was consistently decreased by 1,25 D H C C . Moreover, the calcium in the medium was lowered to a level at or below the basal calcium release. (3) In those experiments where basal calcium release was low ( < 14 mg/dl), 1,25 D H C C did not show a statistically significant inhibition of calcium release. When basal calcium release was low, P T H or PGE2 usually caused a significant enhancement of calcium release. Again, 1,25 D H C C consistently lowered the PTH- or PGE2-enhanced calcium release to a level at or below basal calcium release. Although 1,25 D H C C did significantly lower basal calcium release in those cultures where basal calcium release was high, it was not as effective as indomethacin, which almost completely abolished calcium release from cultured 75oday-old calvaria. Therefore, the effect of 1,25 D H C C in the presence of indomethacin was tested. Surprisingly, it was found that indomethacin suppression of calcium release was blocked by 1,25 D H C C and the concentration of calcium in the medium was maintained at a level approximating basal calcium release. In view of this unexpected finding, additional experiments were performed with 1,25 D H C C and indomethacin, and, with 1,25 D H C C and dexamethasone, another inhibitor of basal calcium release [3]. MATERIALS AND METHODS Bone cultures were prepared and maintained as described previously [1,3]. Briefly, male mice, age 73--77 days, were obtained from our own breeding colony (Swiss albino, Webster strain) and were randomly assigned to the experimental groups. Cal-
27
varia were cut to form a trapezoid having approximately one 6-mm side, two 7-mm sides and one 10-mm side. Intra- and interexperimental variations in the surface of dissected area was not more than 10%. Such preparations encompassed substantial parts of the frontal bone and both parietal bones. Each calvarium was mounted individually on stainless steel grids in a small Petri dish (35 mm x 10 mm, Falcon No. 3001). Two milliliters of Dulbecco's Modified Eagle's Medium, supplemented with bovine serum albumin, antibiotics, heparin and components to be tested were added to each culture. Indomethacin and 1,25 DHCC were dissolved in ethanol. The final concentration of ethanol never exceeded 0.2% and at that concentration had no effect on bone resorption in control or PTH-treated cultures. The same amount of ethanol was added to control cultures as was present in experimental cultures. PTH was dissolved in 0.15 M NaC1. Dexamethasone was dissolved in tissue culture medium. There were 5--7 cultures per group. Cultures were placed in a CO2 incubator (Queue, Model 2210) at 37°C and maintained for 14 days. Media were changed every 2--3 days. Used media were analyzed for calcium content using a Corning Calcium Analyzer, Model 940 or Nova Biomedical 7 + 7 (Nova Biomedical, Waltham, MA). At the end of the experiment, bones were routinely fixed and processed for histological examination using hematoxylin and eosin staining. All data were subjected to analysis of variance. Standard errors were calculated from standard deviations and sample sizes. Statistical significance of the difference between groups was determined using least significant difference criterion [4]. Dulbecco's Modified Eagle's Medium and glutamine were purchased from M.A. Bioproducts, Walkerville, MD; bovine serum albumin, parathyroid hormone (synthetic, bovine, fragment 1--34), prostaglandin E2 and dexamethasone from Sigma Chemical Co., St. Louis, MO; heparin (manufactured by E. Lilly Co., Indianapolis, IN) from a local pharmaceutical distributor; and antibiotics from Grand Island Biol. Co., New York, NY. Indomethacin was kindly provided by Dr. C.A. Stone, Merck, Sharp and Dohme Research Laboratories, West Point, PA; and lc~,25-dihydroxycholecalciferol by Dr. M. Uskokovic, Hoffmann-LaRoche, Nutley, NJ. RESULTS
Table I presents net cumulative data from a series of experiments wherein the effect of indomethacin was tested in the presence of 1,25 DHCC. As shown, indomethacin alone strongly inhibited calcium release in all experiments. This inhibitory effect of indomethacin was significantly reversed by 1,25 DHCC in all 5 experiments, resulting in calcium release values approximating those observed in control cultures treated with 1,25 DHCC alone. It should be noted that 1,25 DHCC suppressed calcium release in all 5 experiments. However, the difference in calcium release between untreated control and 1,25 DHCC-treated cultures was statistically significant only in 2 experiments.
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30 Data presented in Table II are obtained from a series of experiments wherein the effect of dexamethasone was tested in the presence of 1,25 DHCC. Dexamethasone alone strongly inhibited calcium release in all experiments. Similar to the inhibitory effect of indomethacin, the inhibitory effect of dexamethasone was significantly reversed by 1,25 DHCC in all six experiments, resulting in calcium release values approximating those observed in untreated control cultures or cultures treated with 1,25 DHCC alone. In this series of the experiments, the basal calcium release values obtained from the untreated cultures were relatively low and in 5 of 6 experiments 1,25 DHCC had no inhibitory effect on calcium release as compared to the untreated controls. The finding that 1,25 DHCC elevated the depressed calcium release values due to the presence of either indomethacin or dexamethasone alone appeared to be contrary to our previous reports [1,2] demonstrating that 1,25 DHCC depressed elevated calcium release values due to the presence of PTH or PGE2. Therefore, the data from all relevant experiments were reviewed in order to determine whether any pattern would emerge from these paradoxical findings. Figure 1 summarizes the data obtained from 22 experiments wherein the effect of 1,25 DHCC (2.6 × 10 -7 M) on calcium release from 75-day-old mouse calvaria cultures was tested under various experimental conditions. The data points in each of the panels are arranged in descending order for the untreated controls (panel A) and cultures treated with PTH (panel B), PGE2 (panel C), indomethacin (panel D), and dexamethasone (panel E), respectively. Each data point represents the mean and standard error of the mean of calcium release from a treatment group containing at least 5 cultures. Also, they are arranged in such a manner that those found in the same vertical plane were part of the same experiment, thereby allowing comparison of specific groups within the same experiment. For the sake of simplicity in presenting the data, the corresponding untreated controls are not included in panels B, C, D, and E. As shown in panel A, where basal calcium release was high, 1,25 DHCC significantly inhibited calcium release (see asterisks). As may be seen in panel B, in all experiments except one, PTH significantly enhanced calcium release compared to the corresponding untreated control (see asterisks). Also, 1,25 D H C C significantly inhibited the calcium releaseenhancing effect of PTH in all 10 experiments (see daggers). As shown in panel C, PGE2 significantly enhanced calcium release compared to the corresponding untreated control in 2 of 4 experiments (see asterisks). As was the case with PTH, 1,25 DHCC significantly inhibited the calcium release-enhancing effect of PGE2 in all experiments (see daggers). In contrast to the calcium release-enhancing effect of PTH or PGE2, it may be seen from panel D that indomethacin significantly inhibited calcium release in all 5 experiments, as compared to the corresponding untreated controls (see asterisks). However, 1,25 DHCC essentially abolished the calcium release-inhibitory effect of indomethacin in all experiments (see daggers). In panel E, similar to the results obtained with indomethacin, it may be seen that dexamethasone also strongly inhibited basal calcium release in all 6 experiments (see
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asterisks). Again, 1,25 DHCC essentially abolished the calcium release-inhibitory effect of dexamethasone in all 6 experiments (see daggers). Histological examination of cultured bones demonstrating a modest to high calcium release showed the presence of osteoclasts and evidence of osteoclastic resorption. The most striking histological observation was that cultures containing indomethacin or dexamethasone had fewer osteoclasts and evidence of bone resorption than the untreated control cultures. The addition of 1,25 DHCC to cultures containing either indomethacin or dexamethasone appeared to reverse this effect causing a greater number of osteoclasts to be present. It is noteworthy that both these histological observations were more obvious in cultures containing higher concentrations of indomethacin (2.8 x 10 -6 M) or dexamethasone (10 -7 M) and correlated with the reversal of calcium release depression (data not shown). With regard to PTHor PGE2-stimulated bone resorption, osteoclasts were quite numerous in both these "groups. However, the addition of 1,25 D H C C did not result in any obvious diminution in number of osteoclasts present in the tissue. Similarly, in otherwise untreated cultures, 1,25 D H C C did not result in any obvious decrease in number of osteoclasts as compared to the untreated control bones. DISCUSSION
Although we have previously reported that the time course pattern of basal calcium release is different for untreated cultured young adult mouse caivaria as compared to untreated neonatal mouse calvaria, calcium release is enhanced in both age groups in response to a number of bone resorption-stimulating agents, namely, PTH or PGE2. Similarly, basal calcium release is inhibited in both age groups in response to several bone resorption-inhibiting agents, namely, indomethacin or dexamethasone [1--3]. Therefore, it was surprising to observe that the response of young adult mouse calvaria to 1,25 D H C C was entirely different compared to the response of cultured neonatal mouse calvaria. Whereas 1,25 DHCC, at all concentrations tested (2.6 x 10 -9 M to 2.6 x 10 -6 M), enhanced bone resorption in cultured neonatal mouse calvaria, this compound, at higher concentrations (2.6 x 10 -7 M and 2.6 x 10 -6 M), inhibited basal bone resorption in cultured young adult mouse calvaria [1]. Results obtained in the present study essentially confirmed and extended previously published observations to include the inhibitory effect of 1,25 DHCC on calcium release from cultured young adult mouse calvaria exposed to PGE2. In addition, what was not anticipated in the current series of experiments was the observation that, at 2.6 x 10 -7 M, 1,25 D H C C essentially abolished the bone resorptioninhibiting effect of indomethacin or dexamethasone. Presently, no explanation for this effect of 1,25 D H C C is available. It would appear that under culture conditions as described, 1,25 D H C C has the capacity to either stimulate or inhibit calcium release thereby modulating the effect of bone resorption-stimulating or bone resorption-inhibiting agents and restricting to a more intermediate range calcium
33 release values that would otherwise be extremely high or extremely low. This direct regulating effect of 1,25 DHCC on bone tissue suggests that bone might contain a local calcium 'homeostatic' mechanism that could be of physiological importance. Apparently, this local mechanism is 1,25 DHCC-dependent and is not as well developed in neonatal bones suggesting that this capacity develops as a function of age. It would be of interest to determine whether this regulating mechanism continues to function with the same efficiency as the animal ages or whether advanced age leads to dysfunction thereby resulting in pathological alterations of bone. Since this mechanism appears to respond to either a 'systemic', bone resorptionstimulating agent, such as PTH, or a local bone resorption-stimulating agent, such as PGE2, it appears to have the capacity to modulate locally either systemic or locally produced factors that influence bone resorption. It is conceivable that some of the results presented here might be explained in part by a modulating effect of 1,25 DHCC on the lipid composition and permeability of cell membranes [5,6], which might in turn influence calcium movement into and out of bone. Gunasekaran et al. [71 have reported that the addition of 1,25 DHCC to calvaria cultures from 6-week-old quail resulted in a fall in medium total and ionic calcium concentrations. Of interest was the fact that they could not demonstrate any effect of 1,25 DHCC on net calcium movement in cultures of calvaria removed from quail up to the age of 2 weeks. They suggested that the results with older quail were due to a direct stimulatory effect in vitro of 1,25 DHCC on calcium movement into bone. However, since these authors reported that PTH had no effect on the medium calcium in their 6-week-old quail calvaria cultures, it is evident that this culture system does not respond to bone resorption-stimulating factors comparable to the response obtained with neonatal or young adult mouse calvaria. Histological observations made in the present study are consistent with the concept that the calcium homeostatic activity of bone cells in the presence of 1,25 DHCC is related to the differentiation and function of osteoclasts. Whereas indomethacin and dexamethasone apparently inhibited the formation of osteoclasts, the presence of 1,25 DHCC stimulated their development and function, as determined histologically and by calcium release. On the other hand, where high calcium release is reduced in the presence of 1,25 DHCC, as is the case in PTH- or PGE2treated cultures or in some control cultures, there was no obvious diminution in the number of osteoclasts, suggesting that the inhibition of calcium release by 1,25 DHCC in these cases was due primarily to an effect on the function of osteoclasts. It should be noted that 1,25 DHCC has been shown to inhibit PTH-enhanced cyclic AMP production in cultured bone cells or bone tissues [8--10]. In other tissue culture systems, 1,25 DHCC inhibited the proliferation of murine granulocytemacrophage progenitor cells, and, also, induced murine leukemia cells to differentiate into macrophages [ 11,12]. Such diverse actions of 1,25 DHCC suggest a pivotal role for this vitamin D 3 metabolite in the local regulation of bone remodeling above and beyond its well recognized role in systemic calcium homeostasis.
34 ACKNOWLEDGEMENTS T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e t h e skilled t e c h n i c a l a s s i s t a n c e o f J o a n J e n n ings, L o r r a i n e S t e v e n s , a n d B e v e r l y B e r e s f o r d . T h i s s t u d y was s u p p o r t e d in p a r t by t h e G e l f a n d F u n d at the H a r v a r d S c h o o l o f D e n t a l M e d i c i n e a n d t h e N a t i o n a l Institutes o f H e a l t h G r a n t A G - 0 2 8 9 9 . REFERENCES 1 P. Goldhaber and L. Rabadjija, Age-dependent stimulation or inhibition of calcium release from bone cultures by hx,25-(OH)2D 3. Mech. Ageing Dev., 49 (1989) 199--209. 2 P. Goldhaber and L. Rabadjija, Calcium 'homeostatic' effect of l a,25-(OH)2D 3 on adult mouse calvaria cultures. J. Dent. Res. (spec. issue), 68 0989) 967 (abstr. 803). 3 P. Goldhaber, L. Rabadjija and S.L. Swartz, Effect of age on calcium release from mouse calvaria in tissue culture. Mech. Ageing Dev., 34 (1986) 273--288. 4 G.W. Snedecor and W.G. Cochran, Statistical Methods, 6th edn., The Iowa State University Press, Ames, 1976. 5 A.W. Norman and F.P. Ross, Vitamin D secosteroids: Unique molecules with both hormone and possible membranophilic properties. Life Sci., 24 (1979) 759--770. 6 B.R.C. Kurnik, M. Huskey and K. Hruska, 1,25-dihydroxycholecalciferol stimulates renal phosphate transport by directly altering membrane phosphatidylcholine composition. Biochim. Biophys. Acta, 917 0987) 81--85. 7 S. Gunasekaran, G.E. Hall and A.D. Kenny, Vitamin D and avian bone in vitro: Stimulation of calcium movement into Japanese quail calvaria. Calcif. Tissue Int., 39 (1986) 396--403. 8 G.L. Wong, R.A. Luben and D.V. Cohn, 1,25-dihydroxycholecalciferol and parathormone: Effects on isolated osteoclast-like and osteoblast-like cells. Science, 197 (1977) 663--665. 9 M.P.M. Hermann-Erlec and P.J. Gaillard, The effects of 1,25-dihydroxycholecalciferol on embryonic bone in vitro: A biochemical and histological study. Calcif. Tissue Res., 25 (1978) I l l--118. l0 B.D. Catherwood, 1,25-dihydroxycholecalciferol and glucocorticoids regulation ofadenylate cyclase in an osteoblast-like cell line. J. Biol. Chem., 260 (1984) 736--743. I I C. Miyaura, E. Abe, H. Nomura, Y. Nishii and T. Suda, h x,25-dihydroxyvitamin D3 suppresses proliferation of routine granulocyte-macrophage progenitor cell (CFU-C). Biochem. Biophys. Res. Commun., 108 0982) 1728--1733. 12 E. Abe, C. Miyaura, H. Sakagami, M. Takeda, K. Konno, T. Yamazaki, S. Yoshiki and T. Suda, Differentiation of mouse myeloid leukemia cells induced by h x,25-dihydroxyvitamin D 3. Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 4990----4994.