Biochemical Characterization with Parathormone and Calcitonin of Isolated Bone Cells: Provisional Identification of Osteoclasts and Osteoblasts RICHARD A. LUBEN, GLENDA L. WONG, AND DAVID V. COHN Calcium Research Laboratory, Veterans Administration Hospital, Kansas City, Missouri 64128; University of Missouri-Kansas City, School of Dentistry, Kansas City, Missouri 64108; and University of Kansas School of Medicine, Kansas City, Kansas 66103 and prolyl hydroxylase were higher in the PT cells. Parathormone stimulated acid phosphatase and hyaluronate synthesis by 100-200% only in the CT cells; it inhibited alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase by 75-90% only in the PT cells. Calcitonin alone had no effect on any of these activities other than cAMP production, but it inhibited the action of parathormone in the CT cells. The sensitivities, time courses of development, and magnitudes of these hormonal effects were similar to those observed previously in intact calvaria, indicating that the isolated cell system is a reliable model for the study of bone metabolism. Based on the metabolic responses of the cells, we postulate that the CT type of populations is enriched in osteoclasts and, possibly, osteocytes, and the PT type of population is enriched in osteoblasts. (Endocrinology 99: 526, 1976)
ABSTRACT. Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately twofold increases in cAMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activities were measured in the two types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase, and general protein synthesis. Although each of these activities was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation,
S
TUDIES on the biochemistry of bone have been complicated by the extreme heterogeneity of the tissue. In order to simplify such work, several groups have studied bone metabolism, using mixed populations of cells freed from the matrix by enzymatic (1-3) or mechanical means (4,5). In such work, the effects of parathormone or calcitonin, hormones which play major roles in bone physiology, have been evaluated on parameters such as cAMP formation (6-9), nucleotide metabolism (10,11), and calcium uptake (12,13). We recently separated from mouse calvaria two types of bone cell populations that differed from each other in morphology and in the formation of cAMP in response to
parathormone and calcitonin (14,15). One type was enriched in cells that responded to both parathormone and calcitonin. The second type was enriched in cells that responded only to parathormone. In the present report, we have further characterized these cell populations on the basis of hormone-induced changes in metabolic and enzymatic activities. Our data reinforce the conclusion that the separated populations represent metabolically different cell types which respond distinctively to parathormone and calcitonin. Moreover, the responses of the separated populations are qualitatively similar to the responses of intact bone in vitro.
Received February 2, 1976. Supported in part by grants DE 1523, DE 4211, and AM 5116 from the National Institutes of Health. All correspondence about this publication should be addressed to David V. Cohn. The present address of Dr. Luben is Dept. of Biochemistry, U. of Calif., Riverside.
Materials and Methods Isolation and culture of bone cells Bone cell populations were isolated from the calvaria of 2-3-day-old mice as described previously (15). The calvaria, consisting of frontal
526
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CHARACTERIZATION OF BONE CELLS and parietal bones, were removed aseptically and gently cleansed of adherent skin and nervous tissue. Seventy-five calvaria were incubated at 22-25 C for 20 min with gentle stirring in 3 ml of a sterile enzyme solution consisting of 0.1% collagenase (Worthington crude, lot no. CLS 45M 181X), 0.05% trypsin, 0.004M EDTA, 0.137M NaCl, and 0.003M NaH,PO4, pH 7.0. The digestion medium, containing freed bone cells, was decanted and replaced with fresh enzyme solution, and digestion was resumed for 20 additional min. This procedure was repeated to yield a total of 5 such digests. The cells freed during each digestion, denoted populations 1 through 5, were collected by centrifugation at 200 x g and cultured in 10 ml of Minimal Essential Medium containing 10% fetal calf serum. The culture medium was replaced 24 h after digestion and every 48 h thereafter. Populations 1-5 were maintained in primary culture for 6 or 7 days, at which time they had grown to confluence. For each experiment, described below, the cells of the selected populations were subcultured by suspending the primary culture monolayer in Ca ++ , Mg ++ free Tyrode's solution containing 0.004M EDTA, adjusted to pH 7.0. The cells were collected by centrifugation and resuspended in culture medium, plated at 0.5-1 x 105 cells per well in plastic multiwell dishes (1.6 cm diameter wells) and allowed to attach overnight. Each experiment was initiated by adding fresh medium containing vehicle, parathonnone or calcitonin, as indicated, to the adherent cells. Measurement of cAMP The cells were exposed for 5 min at 37 C to culture medium containing 0.5 mM theophylline and 200 ng/ml of parathonnone or 110 ng/ml of calcitonin, or both. The dishes were chilled in ice; the medium was decanted; the adherent cells were rinsed 3 times with ice-cold tyrode's solution and covered with 0.5 ml ice-cold 95% ethanol containing 0.02N HC1. After standing overnight at —20 C, the cells were suspended in the acidic ethanol solution and transferred to glass tubes. Each well was rinsed with an additional 0.5 ml aliquot of ethanol-HCl, and the rinsing was combined with the cell suspension. The mixture was evaporated to dryness in a boiling water bath, and the residue was redissolved in 0.05M sodium acetate buffer, pH 4.0. The amount of cAMP in this extract was deter-
527
mined by the protein-binding method of Gilman (16). This procedure allowed essentially complete recovery of cAMP, as determined by analysis of duplicate cultures containing added amounts of cAMP. Synthesis of hyaluronate The cells were treated with parathonnone and calcitonin at the indicated dosages and times. The medium was replaced with fresh medium containing, in addition to the hormones, 10/u-Ci/ml of [6-3H]glucosamine (10.1 Ci/mmol), and incubation was continued for 1 to 4 h. The medium and cells were frozen and thawed several times to lyse the cells and the resultant suspension was digested overnight at 60 C in a solution containing 0.25 mg/ml of papain, 5 mM cysteine, and 5 mM EDTA. The digests were dialyzed exhaustively against 0.05M Tris-HCl, pH 7.2, to remove free glucosamine and papain-degraded fragments, and hyaluronate was separated from other glycosaminoglycans by DEAE cellulose chromatography (17). It was shown previously that changes in the incorporation of radioactive glucosamine into hyaluronate accurately reflected similar changes in the net synthesis of hyaluronate (17,18). Decarboxylation of citrate The cells were exposed to medium containing parathonnone or calcitonin, as indicated, after which the medium was replaced with the same medium, containing, in addition, 0.1 /u,Ci/ml of [6-I4C]citric acid (4.5 mCi/mmol). After equilibration with 5% CO2 in air, each well in the dish was sealed with an adhesive plastic film (Linbro product No 64 PS), to which had been affixed on the inside surface a 0.5 cm circle of Whatman 3MM filter paper. The dishes were incubated for 2h at 37 C, then placed in ice. After 30 min, each filter paper was impregnated with 0.05 ml of Hyamine-lOX by injection through the plastic film with a 26-gauge hypodermic needle. Then the incubation medium was acidified by injecting 0.1 ml of 10% trichloroacetic acid through the film, after which the holes made in the film were sealed immediately with a small piece of adhesive film. The dishes were incubated at 37 C for 2 h to allow the HyaminelOX to absorb evolved 14CO2. Each filter paper was transferred to a toluene-based scintillation solution for assay of radioactivity. In control
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studies the quantitative recovery of 14CO2 derived from NaH14CO3 added to the culture medium was obtained using this procedure. Determination of acid and alkaline phosphatases The cells were cultured for the indicated times with parathormone or calcitonin, after which the medium was decanted and retained. The cells were rinsed twice with ice-cold Tyrode's solution. Then 0.2 ml of distilled water was added, and the cells were lysed by freezing and thawing three times. Acid and alkaline phosphatase were assayed by a modification of the method of Lowry et al. (19). Samples (0.1 ml) of cell lysate or culture medium were mixed at 0 C with 0.1 ml of acid or alkaline buffer containing 0.015M p-nitrophenylphosphate and 0.1% Triton X-100. The buffer for the assay of acid phosphatase activity was 0. 1M sodium citrate, pH 4.8; that for the assay of alkaline phosphatase activity was O.lM glycine, pH 10.3. The samples were incubated at 38 C for 30 min, placed in ice, and diluted to 2.0 ml with O.lN NaOH. Absorbance was read at 410 nm. The substrate cleavage was determined by comparing experimental samples with identically incubated blanks containing known amounts of p-nitrophenol. Collagen prolyl hydroxylase The cells tested in this assay were grown in the presence of 100 /xg/ml of ascorbic acid for at least the last 48 h of culture. After treatment for the indicated times with parathormone or calcitonin, the cell monolayers were lysed by freezing and thawing in distilled water. The suspensions were centrifuged at 20,000 x g. The activity of prolyl hydroxylase of the supernatant fluid was assayed by the procedure of Berg and Prockop (20,21), in which the stoichiometric release of 14CO2 from [l-14C]a-ketoglutarate is measured during prolyl hydroxylation by the enzyme (22). The substrate used was nonradioactive underhydroxylated collagen that was isolated from mouse calvaria after culture in the presence of a,a'dipyridyl (23). In some experiments (not shown), the activity of prolyl hydroxylase was measured by the tritium release procedure of Peterkofsky and Diblasio (23), in which [3,4- 3 H]proline-labeled underhydroxylated collagen was the substrate. Similar results were obtained using both methods of assay.
Protein synthesis The control or hormone-treated cells were incubated for an additional 2 h with [4,5-3H]leucine (43.5 Ci/mmol) at 0.2 /u.Ci/m], after which the medium was decanted. The cells were washed twice with Ca ++ , Mg++-free Tyrode's solution, resuspended in distilled water which was made 10% in trichloroacetic acid, and allowed to stand for 2 h at 0 C. The precipitate was separated by filtration on a Millipore filter. The filter was transferred to a liquid scintillation fluid and assayed for radioactivity. The total protein content of cell monolayers was measured by the method of Lowry et al. (24). Parathormone caused no changes in the total protein content of any population. Hormones and reagents Parathormone (2,000 U/mg) was prepared in our laboratory from bovine parathyroids. Synthetic salmon calcitonin (4,000 MRC units/mg) was a gift of Armour Pharmaceutical Company (Chicago, IL). Trypsin was obtained from BioQuest (Cockeysville, MD). All other reagents were analytical grade and were used without further purification. The radioactive substrates were obtained from New England Nuclear Corp. (Boston, Mass.). The purity of radioactive [3H]glucosamine and [HC]citrate was established before use, as described previously (18). The tissue-culture plastic ware was obtained variously from Falcon (Oxnard, CA) and Linbro (Hamden, CT).
Results
Figure 1 portrays the effects of parathormone and calcitonin on the cAMP content of 5 sequentially released bone cell populations in one of several such studies. The basal levels of cAMP were similar in each of the populations. Parathormone produced consistent increases of at least 2-fold in those cells released earlier in the digestive procedures (populations 1 and 2) and substantially larger increases in cAMP in those cells released later (populations 3-5). Calcitonin also elicited 2-fold increases in cAMP in populations 1 and 2 but had no effect in the other populations. These data are similar to those reported
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CHARACTERIZATION OF BONE CELLS CALCITONIN
PARATHORMONE
o FIG. 1. Effects of parathormone and calcitonin on the cAMP content of bone cell populations. The cells were tested 7 days after isolation. The dose of parathormone was 200 ng/ml and of calcitonin, 110 ng/ml. The control cAMP values (pmol per 105 cells ± SE) were: Population 1, 1.6 ± 0.2; population 2, 2.2 ± 0.3; population 3, 3.1 ± 0.1; population 4, 2.4 ± 0.2; population 5, 3.2 ± 0.4. Bars represent means ± SE. The asterisks denote significant^ < 0.02) difference from control levels; NS denotes no significant difference from control. Number of determinations = 4.
529
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I
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100
1 2 3 4 5 •" 1 2 3 4 5 CELL POPULATION
previously (15) and support our conclusion that, based on cAMP production, calvaria contain at least two types of populations: one type represented by populations 1 and 2, that is, enriched in cells that respond to both parathormone and calcitonin, and a second type, represented by populations 3, 4, and 5, that is, enriched in cells that respond to parathormone and not to calcitonin. In the present report we have termed those populations that respond in terms of cAMP formation to both calcitonin and parathormone as CT type and those that respond only to parathormone as PT type. In general, the distribution of populations into CT and PT types was similar to that shown in Fig. 1. There was some variability from one experiment to another. For example, in some experiments the maximum parathormone response was observed in populations 3 or 5 and the maximum calcitonin response in population 2. The effects of parathormone and calcitonin on several biochemical activities were assessed in each of the cell populations. We measured the decarboxylation of citrate, the synthesis of hyaluronate and pro-
tein, and the levels of alkaline phosphatase, acid phosphatase, and prolyl hydroxylase. In all of these measurements, with the exception of general protein synthesis, there were substantial differences in both basal and hormone-stimulated activities in the different cell populations (Table 1). The basal levels of these activities differed progressively from one cell population to another suggesting that a separation of metabolically different cell types had occurred. The control levels of hyaluronate synthesis and acid phosphatase were higher in the CT populations. Conversely, citrate decarboxylation, alkaline phosphatase, and prolyl hydroxylase activities were higher in the PT populations. Parathormone stimulated hyaluronate synthesis and acid phosphatase 2- to 3-fold in the CT populations but did not affect these activities in the PT populations. In contrast, the hormone reduced the levels of citrate decarboxylation, alkaline phosphatase, and prolyl hydroxylase by 75 to 90% in the PT populations but did not affect these activities in the CT populations. Calcitonin blocked the effects of parathormone
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LUBEN, WONG AND COHN
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Enilo • 1976 Vol 99 • No 2
TABLE 1. Actions of parathonnone and calcitonin on various biochemical activities in differentbone cell populations Bone cell populations PT type
CT type
Activity
Treatment
1
2
3
4
5
Hyaluronate synthesis
Control Parathonnone Calcitonin Parathonnone + Calcitonin
19.1 ± 0.9 36.8 ± 2.6* 18.3 ± 2.8
8.8 ± 0.4 20.1 ± 2.2* 11.1 ± 1.7
10.6 ± 1.2 13.4 ± 1.1 9.2 ± 1.2
5.8 ± 0.9 5.6 ± 0.8 6.9 ± 0.7
6.2 ± 0.4 5.8 ± 0.4 7.2 ± 1.6
Acid phosphatase
Control Parathonnone Calcitonin
11.1 ± 1.7 28 ± 1 42 ± 3* 29 ± 2
7.1 ± 0.5 31 ± 3 29 ± 4 27 ± 3
19 21 23
± 1 ± 3 ± 1
31
30
24
±2
21.7 ± 2.5 96 213 91
±5 ± 7* ±4
103
±5
Parathonnone + Calcitonin
Citrate decar-
Control
4.8 ± 0.4
boxylation Alkaline phosphatase
Parathonnone
4.6 ± 0.3
Prolyl hydroxylase
Control Parathonnone
Protein synthesis
Control Parathonnone
Control Parathonnone
18 ± 1 19 ± 1 0.7 ± 0.2 0.5 ± 0.1 19 ± 2 20 ± 1
9.8 ± 0.8 72 ± 2 193 ± 9* 77 ± 5 92
± 5*
3.9 ± 0.8 4.1 ± 1.6
21 ± 1 18 ±2 0.8 ± 0.1 0.9 ± 0.1 17 ± 2 18 ± 3
±2
±1
6.0 ± 1.0
10.8 ± 0.5 4.0 ± 0.2* 25 ± 2 24 ± 3
32.2 ± 8.1 5.9 ± 2.0* 78 ± 4 16 ± 1*
9.7 ± 2.4 5.6 ± 1.0* 51 ± 5 24 ± 2*
4.2 ± 1.0 1.0 ± 0.3*
18.1 ± 1.3 1.2 ± 0.2*
12.4 ± 1.1 3.1 ± 0.8*
23 ± 1 24 ± 2
24 21
21 ± 1 17 ± 2
±1 ±1
The cells used for these assays were derived from separate digestions of calvaria as described in Methods. All were tested after 6 days in primary culture except those used for hyaluronate assay which were studied after 7 days. In each case the dose of parathonnone was 200 ng/ml and that of calcitonin was 110 ng/ml. The units in which each activity is expressed are as follows: hyaluronate synthesis, dpm X 10~3 [3H]glucosamine incorporated; acid and alkaline phosphatase, nmol substrate cleaved/h; citrate decarboxylation, dpm MCO2 released x 10~3; prolyl hydroxylase, dpm I4CO2 evolved x 10~3; protein synthesis, dpm [3H]leucine incorporated.
All values are reported per 105 cells. * Significantly different from control, P < 0.01.
on the CT populations but not on the PT populations. Alone, it had no affect on any of the measured activities. A dose-response curve for the stimulation of hyaluronate synthesis in a typical CT population is shown in Fig. 2. As little as 10 ng/ml of parathormone elicited a substantial increase in synthesis. The magnitude of stimulation increased through a dose of at least 300 ng/ml, the highest tested in this experiment. The development of the effect is shown in Fig. 3. At a dose of 200 ng/ml of parathormone, there occurred substantial stimulation of hyaluronate synthesis by 1 h, which reached a maximum level within 4 h. The stimulation of acid phosphatase activity occurred more slowly (Table 2). No effect was noted at 6 h, and a maximal stimulation was observed at 24 h.
A dose-response curve for the inhibition of citrate decarboxylation by parathormone in a typical PT population is shown in Fig. 4. A dose of 10 ng/ml inhibited decarboxylation by 40% and 100 ng/ml inhibited by 80%. Inhibition became apparent by 6 h and reached maximum between 6 and 24 h (Fig. 5). The inhibition of alkaline phosphatase in the PT cells was not observed until after 6 h (Table 2). Discussion This study shows that two types of bone cell populations, which we previously characterized on the basis of their different cAMP responses to parathormone and calcitonin, exhibit other major biochemical differences. The CT cells exhibited para-
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CHARACTERIZATION OF BONE CELLS thormone-stimulated increases in hyaluronate synthesis and acid phosphatase. These effects of parathormone in CT cells were inhibited by calcitonin. The PT cells exhibited parathormone-induced inhibition of citrate decarboxylation, prolyl hydroxylase, and alkaline phosphatase activities and an insensitivity to calcitonin. These effects occurred in magnitudes, dosages, and times after treatment equivalent to those which have been observed in intact calvaria (18,25-27). Thus, with respect to the parameters studied, one can reconstmct the effects of parathormone and calcitonin in the intact calvarium by summing the effects obtained in the individual cell populations. These results make
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FIG. 3. Time course of parathormone stimulation of hyaluronate synthesis in CT type cells. Cells of population 1 were replated on the seventh day of culture. At zero h, 200 ng/ml of parathormone were added. Separate cultures were exposed to [6-3H]glucosamine for a 1 h pulse, beginning at 0, 4, or 23 h. The control value for hyaluronate synthesis was 4.4 ± 0.7 x 103 dpm per 105 cells. Values are means ± SE of 3 cultures.
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it clear that our isolated bone cells retained their biochemical uniqueness in culture and represent a useful model for the study of bone tissue. This conclusion permits us to interpret previous observations in intact tissue with greater precision. Luben and Cohn (18), for example, showed that a close correlation existed between parathormone-induced bone resorption, hyaluronate synthesis, and the inhibition of citrate decarboxylation in intact cultured calvaria. As in the present study, calcitonin blocked the action of parathormone on hyaluronate synthesis but not on citrate decarboxylation. It is now clear that the biochemical events noted by Luben and Cohn, despite their close coordination (18), had occurred in different cell types in the calvarium. Likewise, Vaes and Jacques (27) showed that parathormone enhanced the secretion of acid phosphatase and inhibited the secretion of alkaline phosphatase by cultured calvaria. Our results establish that these events were also mediated by different cell types. Moreover, several investigators showed that in vitro the effects of parathormone and calcitonin
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TABLE 2. Stimulation of acid phosphatase in CT cells and inhibition of alkaline phosphatase in PT cells by parathormone Cell population CT type Hours of treatment
Acid phosphatase 63 62 59 188
0 2 6 24
±4 ±2 ±2 ± 4*
PT type
Alkaline phosphatase 19 ± 20 ± 18 ± 20 ±
1 2 1 1
Acid phosphatase
Alkaline phosphatase
23 ± 1 18 ± 2 22 ± 1 21 ± 2
80 ± 4 79 ± 2 70 ± 3 13 ± 1*
* The CT population was prepared by mixing populations 1 and 2, the PT population by mixing populations 4 and 5. The dose of parathormone was 200 ng/ml. Units of enzyme activity are nmol substrate cleaved per h per 105 cells.
100
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Endo • 1976 Vol 99 • No 2
LUBEN, WONG AND COHN
532
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FIG. 4. Dose-response relationship of parathormone and citrate decarboxylation in PT cells. Cells of population 4 were replated on the seventh day after isolation and cultured for 48 h in the presence of the indicated concentrations of parathormone. The control value for citrate decarboxylation was 17.0 ± 1.8 x 103 dpm per 105 cells. Values are means ± SE of 4 cultures.
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FIG. 5. Time course of development of parathormone effects on citrate decarboxylation in PT type cells. Cells of population 4 were replated on the sixth day after isolation and were cultured for the indicated times with 200 ng/ml of parathormone. The control value for citrate decarboxylation was 14.6 ± 1.9 x 103 dpm per 105 cells. Values are mean ± SE for 4 cultures.
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CHARACTERIZATION OF BONE CELLS CT populations are enriched in cells which express the metabolic characteristics of osteoclasts. It is possible that this population also contains osteocytes, since these cells are believed to have several metabolic characteristics in common with osteoclasts (37). It is less likely, however, that osteocytes could be the predominant cell type in the CT population, because of the relative scarcity of osteocytes in the calvaria used as a source of the isolated cells (38). Alkaline phosphatase and collagen synthesis activities are considered characteristic of osteoblasts (39), and both have been shown to be decreased by parathormone in bone in vitro (27,28,31,40). Both activities were highest in the PT cells, using prolyl hydroxylase as a marker of collagen synthesis. Moreover, both activities were decreased by parathormone. Hence, we conclude that PT populations are enriched in cells which express characteristics of osteoblasts. We must yet determine whether the cells in the PT and CT populations were derived either from mature osteoblasts and osteoclasts, from precursor cells which differentiated in culture, or from as yet unrecognized hormone-responsive bone cells. Of equal importance is the need to evaluate in these cells other metabolic reactions characteristic of bone cells, such as calcium transport and matrix dissolution and formation, and to determine the effects of other bone-active agents such as vitamin D metabolites and prostaglandins. Acknowledgment We wish to acknowledge the excellent technical assistance of Ms. M. Mohler.
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4. Flanagan, B., and G. Nichols, Jr., Fed Proc 22: 553, 1963. 5. Smith, D. M., C. C. Johnston, Jr., and A. R. Severson, CalcifTiss Res 11: 56, 1973. 6. Peck, W. A., J. Carpenter, K. Messinger, and D. DeBra, Endocrinology 92: 692, 1973. 7. Rodan, S. B., and G. A. RodanJ Biol Chem 249: 3068, 1974. 8. Smith, D. M., and C. C. Johnston, Jr., Endocrinology 96: 1261, 1975. 9. Mahgoub, A., and H. Sheppard, Biochem Biophys Res Commun 62: 901, 1975. 10. Peck, W. A., J. Carpenter, and K. Messinger, Endocrinology 94: 148, 1974. 11. Peck, W. A., K. Messinger, G. Kimmich, and J. Carpenter, Endocrinology 95: 289, 1974. 12. Dziak, R., and J. BrandJ Cell Physiol 84: 85, 1974. 13. Dziak, R., and P. H. Stern, Endocrinology 97: 1281, 1975. 14. Wong, G., and D. Cohn, Nature 252: 713, 1974. 15. Wong, G. L., and D. V. Cohn, Proc Natl Acad Sci USA 72: 3167, 1975. 16. Gilman, A., Proc Natl Acad Sci USA 67: 305, 1970. 17. Luben, R. A., J. F. Goggins, and L. G. Raisz, Endocrinology 94: 737, 1975. 18. Luben, R. A., and D. V. Cohn, Endocrinology 98: 411, 1976. 19. Lowry, O. H., N. R. Roberts, M. L. Wu, W. S. Hixon, and E. J. Crawford, / Biol Chem 207: 19, 1954. 20. Berg, R. A., and D. J. Prockop, / Biol Chem 248: 1175, 1973. 21. Rao, W. W. Y., R. A. Berg, and D. J. Prockop, Biochim Biophys Ada 411: 202, 1975. 22. Rhoads, R. E., and S. Udenfriend, Proc Natl Acad Sci USA 60: 1473, 1968. 23. Peterkofsky, B., and R. DiBlasio, Anal Biochem 66: 279, 1975. 24. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall,/ Biol Chem 193: 265, 1951. 25. Wolinsky, I., and D. V. Cohn, Endocrinology 84: 28, 1969. 26. Chu, L. L. H., R. R. MacGregor, J. W. Hamilton, and D. V. Cohn, Endocrinology 89: 1425, 1971. 27. Vaes, G., and P. Jacques, Biochem J 97: 380, 1965. 28. Goldhaber, P., B. D. Stern, M. J. Glimcher, and J. Chao, In Talmage, R. V., and L. Belanger (eds.), Parathyroid Hormone and Thyrocalcitonin (Calcitonin), Excerpta Medica Foundation, Amsterdam, 1968, p. 182. 29. Brand, J. S., and L. G. Raisz, Endocrinology 90: 479, 1972. 30. Atkins, D., and M. Peacock, J Endocrinol 64: 573, 1975. 31. Dietrich, J. W., E. M. Canalis, D. M. Maina, and L. G. Raisz, Endocrinology 98: 943, 1976. 32. Hancox, N. M., Biology of Bone, Cambridge University Press, 1972, p. 63.
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33. Owen, M., and M. R. Shetlar, Nature 220: 1335, 1968. 34. Johnston, C. C , Jr., D. M. Smith, and A. R. Severson, In Talmage, R. V., and P. L. Munson (eds), Calcium, Parathyroid Hormone and the Calcitonins, Excerpta Medica Foundation, Amsterdam, 1972, p. 327. 35. Walker, D. G., Calcif Tiss Res 9: 296, 1972. 36. Doty, S. B., B. H. Schofield, and R. A. Robinson, In Talmage, R. V., and L. F. Belanger (eds.), Parathyroid Hormone and Thyrocalcitonin
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Endo • 1976 Vo! 99 • No 2
(Calcitonin), Excerpta Medica Foundation, Amsterdam, 1968, p. 169. Baylink, D., and J. Wergedal, In Nichols, G., and R. H. Wasserman (eds.), Cellular Mechanisms for Calcium Transfer and Homeostasis, Academic Press, New York, 1971, p. 257. Aaron, J. E., Calcif Tiss Res 12: 259, 1973. Rasmussen, H., and P. Bordier, The Physiological and Cellular Basis of Metabolic Bone Disease, Williams and Wilkins, Baltimore, 1974, p. 185. Vaes, G. M., and G. Nichols, Jr., Endocrinology 70: 546, 1962.
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ABSTRACT. Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately twofold increases in cAMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activities were measured in the two types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase, and general protein synthesis. Although each of these activities was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation,
S
TUDIES on the biochemistry of bone have been complicated by the extreme heterogeneity of the tissue. In order to simplify such work, several groups have studied bone metabolism, using mixed populations of cells freed from the matrix by enzymatic (1-3) or mechanical means (4,5). In such work, the effects of parathormone or calcitonin, hormones which play major roles in bone physiology, have been evaluated on parameters such as cAMP formation (6-9), nucleotide metabolism (10,11), and calcium uptake (12,13). We recently separated from mouse calvaria two types of bone cell populations that differed from each other in morphology and in the formation of cAMP in response to
parathormone and calcitonin (14,15). One type was enriched in cells that responded to both parathormone and calcitonin. The second type was enriched in cells that responded only to parathormone. In the present report, we have further characterized these cell populations on the basis of hormone-induced changes in metabolic and enzymatic activities. Our data reinforce the conclusion that the separated populations represent metabolically different cell types which respond distinctively to parathormone and calcitonin. Moreover, the responses of the separated populations are qualitatively similar to the responses of intact bone in vitro.
Received February 2, 1976. Supported in part by grants DE 1523, DE 4211, and AM 5116 from the National Institutes of Health. All correspondence about this publication should be addressed to David V. Cohn. The present address of Dr. Luben is Dept. of Biochemistry, U. of Calif., Riverside.
Materials and Methods Isolation and culture of bone cells Bone cell populations were isolated from the calvaria of 2-3-day-old mice as described previously (15). The calvaria, consisting of frontal
526
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CHARACTERIZATION OF BONE CELLS and parietal bones, were removed aseptically and gently cleansed of adherent skin and nervous tissue. Seventy-five calvaria were incubated at 22-25 C for 20 min with gentle stirring in 3 ml of a sterile enzyme solution consisting of 0.1% collagenase (Worthington crude, lot no. CLS 45M 181X), 0.05% trypsin, 0.004M EDTA, 0.137M NaCl, and 0.003M NaH,PO4, pH 7.0. The digestion medium, containing freed bone cells, was decanted and replaced with fresh enzyme solution, and digestion was resumed for 20 additional min. This procedure was repeated to yield a total of 5 such digests. The cells freed during each digestion, denoted populations 1 through 5, were collected by centrifugation at 200 x g and cultured in 10 ml of Minimal Essential Medium containing 10% fetal calf serum. The culture medium was replaced 24 h after digestion and every 48 h thereafter. Populations 1-5 were maintained in primary culture for 6 or 7 days, at which time they had grown to confluence. For each experiment, described below, the cells of the selected populations were subcultured by suspending the primary culture monolayer in Ca ++ , Mg ++ free Tyrode's solution containing 0.004M EDTA, adjusted to pH 7.0. The cells were collected by centrifugation and resuspended in culture medium, plated at 0.5-1 x 105 cells per well in plastic multiwell dishes (1.6 cm diameter wells) and allowed to attach overnight. Each experiment was initiated by adding fresh medium containing vehicle, parathonnone or calcitonin, as indicated, to the adherent cells. Measurement of cAMP The cells were exposed for 5 min at 37 C to culture medium containing 0.5 mM theophylline and 200 ng/ml of parathonnone or 110 ng/ml of calcitonin, or both. The dishes were chilled in ice; the medium was decanted; the adherent cells were rinsed 3 times with ice-cold tyrode's solution and covered with 0.5 ml ice-cold 95% ethanol containing 0.02N HC1. After standing overnight at —20 C, the cells were suspended in the acidic ethanol solution and transferred to glass tubes. Each well was rinsed with an additional 0.5 ml aliquot of ethanol-HCl, and the rinsing was combined with the cell suspension. The mixture was evaporated to dryness in a boiling water bath, and the residue was redissolved in 0.05M sodium acetate buffer, pH 4.0. The amount of cAMP in this extract was deter-
527
mined by the protein-binding method of Gilman (16). This procedure allowed essentially complete recovery of cAMP, as determined by analysis of duplicate cultures containing added amounts of cAMP. Synthesis of hyaluronate The cells were treated with parathonnone and calcitonin at the indicated dosages and times. The medium was replaced with fresh medium containing, in addition to the hormones, 10/u-Ci/ml of [6-3H]glucosamine (10.1 Ci/mmol), and incubation was continued for 1 to 4 h. The medium and cells were frozen and thawed several times to lyse the cells and the resultant suspension was digested overnight at 60 C in a solution containing 0.25 mg/ml of papain, 5 mM cysteine, and 5 mM EDTA. The digests were dialyzed exhaustively against 0.05M Tris-HCl, pH 7.2, to remove free glucosamine and papain-degraded fragments, and hyaluronate was separated from other glycosaminoglycans by DEAE cellulose chromatography (17). It was shown previously that changes in the incorporation of radioactive glucosamine into hyaluronate accurately reflected similar changes in the net synthesis of hyaluronate (17,18). Decarboxylation of citrate The cells were exposed to medium containing parathonnone or calcitonin, as indicated, after which the medium was replaced with the same medium, containing, in addition, 0.1 /u,Ci/ml of [6-I4C]citric acid (4.5 mCi/mmol). After equilibration with 5% CO2 in air, each well in the dish was sealed with an adhesive plastic film (Linbro product No 64 PS), to which had been affixed on the inside surface a 0.5 cm circle of Whatman 3MM filter paper. The dishes were incubated for 2h at 37 C, then placed in ice. After 30 min, each filter paper was impregnated with 0.05 ml of Hyamine-lOX by injection through the plastic film with a 26-gauge hypodermic needle. Then the incubation medium was acidified by injecting 0.1 ml of 10% trichloroacetic acid through the film, after which the holes made in the film were sealed immediately with a small piece of adhesive film. The dishes were incubated at 37 C for 2 h to allow the HyaminelOX to absorb evolved 14CO2. Each filter paper was transferred to a toluene-based scintillation solution for assay of radioactivity. In control
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Endo • 1976 \'ol 99 • No 2
LUBEN, WONG AND COHN
studies the quantitative recovery of 14CO2 derived from NaH14CO3 added to the culture medium was obtained using this procedure. Determination of acid and alkaline phosphatases The cells were cultured for the indicated times with parathormone or calcitonin, after which the medium was decanted and retained. The cells were rinsed twice with ice-cold Tyrode's solution. Then 0.2 ml of distilled water was added, and the cells were lysed by freezing and thawing three times. Acid and alkaline phosphatase were assayed by a modification of the method of Lowry et al. (19). Samples (0.1 ml) of cell lysate or culture medium were mixed at 0 C with 0.1 ml of acid or alkaline buffer containing 0.015M p-nitrophenylphosphate and 0.1% Triton X-100. The buffer for the assay of acid phosphatase activity was 0. 1M sodium citrate, pH 4.8; that for the assay of alkaline phosphatase activity was O.lM glycine, pH 10.3. The samples were incubated at 38 C for 30 min, placed in ice, and diluted to 2.0 ml with O.lN NaOH. Absorbance was read at 410 nm. The substrate cleavage was determined by comparing experimental samples with identically incubated blanks containing known amounts of p-nitrophenol. Collagen prolyl hydroxylase The cells tested in this assay were grown in the presence of 100 /xg/ml of ascorbic acid for at least the last 48 h of culture. After treatment for the indicated times with parathormone or calcitonin, the cell monolayers were lysed by freezing and thawing in distilled water. The suspensions were centrifuged at 20,000 x g. The activity of prolyl hydroxylase of the supernatant fluid was assayed by the procedure of Berg and Prockop (20,21), in which the stoichiometric release of 14CO2 from [l-14C]a-ketoglutarate is measured during prolyl hydroxylation by the enzyme (22). The substrate used was nonradioactive underhydroxylated collagen that was isolated from mouse calvaria after culture in the presence of a,a'dipyridyl (23). In some experiments (not shown), the activity of prolyl hydroxylase was measured by the tritium release procedure of Peterkofsky and Diblasio (23), in which [3,4- 3 H]proline-labeled underhydroxylated collagen was the substrate. Similar results were obtained using both methods of assay.
Protein synthesis The control or hormone-treated cells were incubated for an additional 2 h with [4,5-3H]leucine (43.5 Ci/mmol) at 0.2 /u.Ci/m], after which the medium was decanted. The cells were washed twice with Ca ++ , Mg++-free Tyrode's solution, resuspended in distilled water which was made 10% in trichloroacetic acid, and allowed to stand for 2 h at 0 C. The precipitate was separated by filtration on a Millipore filter. The filter was transferred to a liquid scintillation fluid and assayed for radioactivity. The total protein content of cell monolayers was measured by the method of Lowry et al. (24). Parathormone caused no changes in the total protein content of any population. Hormones and reagents Parathormone (2,000 U/mg) was prepared in our laboratory from bovine parathyroids. Synthetic salmon calcitonin (4,000 MRC units/mg) was a gift of Armour Pharmaceutical Company (Chicago, IL). Trypsin was obtained from BioQuest (Cockeysville, MD). All other reagents were analytical grade and were used without further purification. The radioactive substrates were obtained from New England Nuclear Corp. (Boston, Mass.). The purity of radioactive [3H]glucosamine and [HC]citrate was established before use, as described previously (18). The tissue-culture plastic ware was obtained variously from Falcon (Oxnard, CA) and Linbro (Hamden, CT).
Results
Figure 1 portrays the effects of parathormone and calcitonin on the cAMP content of 5 sequentially released bone cell populations in one of several such studies. The basal levels of cAMP were similar in each of the populations. Parathormone produced consistent increases of at least 2-fold in those cells released earlier in the digestive procedures (populations 1 and 2) and substantially larger increases in cAMP in those cells released later (populations 3-5). Calcitonin also elicited 2-fold increases in cAMP in populations 1 and 2 but had no effect in the other populations. These data are similar to those reported
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CHARACTERIZATION OF BONE CELLS CALCITONIN
PARATHORMONE
o FIG. 1. Effects of parathormone and calcitonin on the cAMP content of bone cell populations. The cells were tested 7 days after isolation. The dose of parathormone was 200 ng/ml and of calcitonin, 110 ng/ml. The control cAMP values (pmol per 105 cells ± SE) were: Population 1, 1.6 ± 0.2; population 2, 2.2 ± 0.3; population 3, 3.1 ± 0.1; population 4, 2.4 ± 0.2; population 5, 3.2 ± 0.4. Bars represent means ± SE. The asterisks denote significant^ < 0.02) difference from control levels; NS denotes no significant difference from control. Number of determinations = 4.
529
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•T-
200
I
O 400 u NS NS NS
100
1 2 3 4 5 •" 1 2 3 4 5 CELL POPULATION
previously (15) and support our conclusion that, based on cAMP production, calvaria contain at least two types of populations: one type represented by populations 1 and 2, that is, enriched in cells that respond to both parathormone and calcitonin, and a second type, represented by populations 3, 4, and 5, that is, enriched in cells that respond to parathormone and not to calcitonin. In the present report we have termed those populations that respond in terms of cAMP formation to both calcitonin and parathormone as CT type and those that respond only to parathormone as PT type. In general, the distribution of populations into CT and PT types was similar to that shown in Fig. 1. There was some variability from one experiment to another. For example, in some experiments the maximum parathormone response was observed in populations 3 or 5 and the maximum calcitonin response in population 2. The effects of parathormone and calcitonin on several biochemical activities were assessed in each of the cell populations. We measured the decarboxylation of citrate, the synthesis of hyaluronate and pro-
tein, and the levels of alkaline phosphatase, acid phosphatase, and prolyl hydroxylase. In all of these measurements, with the exception of general protein synthesis, there were substantial differences in both basal and hormone-stimulated activities in the different cell populations (Table 1). The basal levels of these activities differed progressively from one cell population to another suggesting that a separation of metabolically different cell types had occurred. The control levels of hyaluronate synthesis and acid phosphatase were higher in the CT populations. Conversely, citrate decarboxylation, alkaline phosphatase, and prolyl hydroxylase activities were higher in the PT populations. Parathormone stimulated hyaluronate synthesis and acid phosphatase 2- to 3-fold in the CT populations but did not affect these activities in the PT populations. In contrast, the hormone reduced the levels of citrate decarboxylation, alkaline phosphatase, and prolyl hydroxylase by 75 to 90% in the PT populations but did not affect these activities in the CT populations. Calcitonin blocked the effects of parathormone
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LUBEN, WONG AND COHN
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Enilo • 1976 Vol 99 • No 2
TABLE 1. Actions of parathonnone and calcitonin on various biochemical activities in differentbone cell populations Bone cell populations PT type
CT type
Activity
Treatment
1
2
3
4
5
Hyaluronate synthesis
Control Parathonnone Calcitonin Parathonnone + Calcitonin
19.1 ± 0.9 36.8 ± 2.6* 18.3 ± 2.8
8.8 ± 0.4 20.1 ± 2.2* 11.1 ± 1.7
10.6 ± 1.2 13.4 ± 1.1 9.2 ± 1.2
5.8 ± 0.9 5.6 ± 0.8 6.9 ± 0.7
6.2 ± 0.4 5.8 ± 0.4 7.2 ± 1.6
Acid phosphatase
Control Parathonnone Calcitonin
11.1 ± 1.7 28 ± 1 42 ± 3* 29 ± 2
7.1 ± 0.5 31 ± 3 29 ± 4 27 ± 3
19 21 23
± 1 ± 3 ± 1
31
30
24
±2
21.7 ± 2.5 96 213 91
±5 ± 7* ±4
103
±5
Parathonnone + Calcitonin
Citrate decar-
Control
4.8 ± 0.4
boxylation Alkaline phosphatase
Parathonnone
4.6 ± 0.3
Prolyl hydroxylase
Control Parathonnone
Protein synthesis
Control Parathonnone
Control Parathonnone
18 ± 1 19 ± 1 0.7 ± 0.2 0.5 ± 0.1 19 ± 2 20 ± 1
9.8 ± 0.8 72 ± 2 193 ± 9* 77 ± 5 92
± 5*
3.9 ± 0.8 4.1 ± 1.6
21 ± 1 18 ±2 0.8 ± 0.1 0.9 ± 0.1 17 ± 2 18 ± 3
±2
±1
6.0 ± 1.0
10.8 ± 0.5 4.0 ± 0.2* 25 ± 2 24 ± 3
32.2 ± 8.1 5.9 ± 2.0* 78 ± 4 16 ± 1*
9.7 ± 2.4 5.6 ± 1.0* 51 ± 5 24 ± 2*
4.2 ± 1.0 1.0 ± 0.3*
18.1 ± 1.3 1.2 ± 0.2*
12.4 ± 1.1 3.1 ± 0.8*
23 ± 1 24 ± 2
24 21
21 ± 1 17 ± 2
±1 ±1
The cells used for these assays were derived from separate digestions of calvaria as described in Methods. All were tested after 6 days in primary culture except those used for hyaluronate assay which were studied after 7 days. In each case the dose of parathonnone was 200 ng/ml and that of calcitonin was 110 ng/ml. The units in which each activity is expressed are as follows: hyaluronate synthesis, dpm X 10~3 [3H]glucosamine incorporated; acid and alkaline phosphatase, nmol substrate cleaved/h; citrate decarboxylation, dpm MCO2 released x 10~3; prolyl hydroxylase, dpm I4CO2 evolved x 10~3; protein synthesis, dpm [3H]leucine incorporated.
All values are reported per 105 cells. * Significantly different from control, P < 0.01.
on the CT populations but not on the PT populations. Alone, it had no affect on any of the measured activities. A dose-response curve for the stimulation of hyaluronate synthesis in a typical CT population is shown in Fig. 2. As little as 10 ng/ml of parathormone elicited a substantial increase in synthesis. The magnitude of stimulation increased through a dose of at least 300 ng/ml, the highest tested in this experiment. The development of the effect is shown in Fig. 3. At a dose of 200 ng/ml of parathormone, there occurred substantial stimulation of hyaluronate synthesis by 1 h, which reached a maximum level within 4 h. The stimulation of acid phosphatase activity occurred more slowly (Table 2). No effect was noted at 6 h, and a maximal stimulation was observed at 24 h.
A dose-response curve for the inhibition of citrate decarboxylation by parathormone in a typical PT population is shown in Fig. 4. A dose of 10 ng/ml inhibited decarboxylation by 40% and 100 ng/ml inhibited by 80%. Inhibition became apparent by 6 h and reached maximum between 6 and 24 h (Fig. 5). The inhibition of alkaline phosphatase in the PT cells was not observed until after 6 h (Table 2). Discussion This study shows that two types of bone cell populations, which we previously characterized on the basis of their different cAMP responses to parathormone and calcitonin, exhibit other major biochemical differences. The CT cells exhibited para-
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CHARACTERIZATION OF BONE CELLS thormone-stimulated increases in hyaluronate synthesis and acid phosphatase. These effects of parathormone in CT cells were inhibited by calcitonin. The PT cells exhibited parathormone-induced inhibition of citrate decarboxylation, prolyl hydroxylase, and alkaline phosphatase activities and an insensitivity to calcitonin. These effects occurred in magnitudes, dosages, and times after treatment equivalent to those which have been observed in intact calvaria (18,25-27). Thus, with respect to the parameters studied, one can reconstmct the effects of parathormone and calcitonin in the intact calvarium by summing the effects obtained in the individual cell populations. These results make
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FIG. 3. Time course of parathormone stimulation of hyaluronate synthesis in CT type cells. Cells of population 1 were replated on the seventh day of culture. At zero h, 200 ng/ml of parathormone were added. Separate cultures were exposed to [6-3H]glucosamine for a 1 h pulse, beginning at 0, 4, or 23 h. The control value for hyaluronate synthesis was 4.4 ± 0.7 x 103 dpm per 105 cells. Values are means ± SE of 3 cultures.
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3 10 30 100 300 PARATHORMONE, ng/ml FlG. 2. Dose-response relationship of parathormone and hyaluronate synthesis in CT type cells. Cells of population 2 were replated on the seventh day after isolation and cultured for 24 h in the presence of the indicated concentrations of parathormone. [6-3H]Glucosamine was present for the last 4 h of culture. The control value for hyaluronate synthesis was 1.7 ± 0.4 x 103 dpm per 105 cells. Values are means ± SE of 4 cultures.
it clear that our isolated bone cells retained their biochemical uniqueness in culture and represent a useful model for the study of bone tissue. This conclusion permits us to interpret previous observations in intact tissue with greater precision. Luben and Cohn (18), for example, showed that a close correlation existed between parathormone-induced bone resorption, hyaluronate synthesis, and the inhibition of citrate decarboxylation in intact cultured calvaria. As in the present study, calcitonin blocked the action of parathormone on hyaluronate synthesis but not on citrate decarboxylation. It is now clear that the biochemical events noted by Luben and Cohn, despite their close coordination (18), had occurred in different cell types in the calvarium. Likewise, Vaes and Jacques (27) showed that parathormone enhanced the secretion of acid phosphatase and inhibited the secretion of alkaline phosphatase by cultured calvaria. Our results establish that these events were also mediated by different cell types. Moreover, several investigators showed that in vitro the effects of parathormone and calcitonin
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TABLE 2. Stimulation of acid phosphatase in CT cells and inhibition of alkaline phosphatase in PT cells by parathormone Cell population CT type Hours of treatment
Acid phosphatase 63 62 59 188
0 2 6 24
±4 ±2 ±2 ± 4*
PT type
Alkaline phosphatase 19 ± 20 ± 18 ± 20 ±
1 2 1 1
Acid phosphatase
Alkaline phosphatase
23 ± 1 18 ± 2 22 ± 1 21 ± 2
80 ± 4 79 ± 2 70 ± 3 13 ± 1*
* The CT population was prepared by mixing populations 1 and 2, the PT population by mixing populations 4 and 5. The dose of parathormone was 200 ng/ml. Units of enzyme activity are nmol substrate cleaved per h per 105 cells.
100
-
Endo • 1976 Vol 99 • No 2
LUBEN, WONG AND COHN
532
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FIG. 4. Dose-response relationship of parathormone and citrate decarboxylation in PT cells. Cells of population 4 were replated on the seventh day after isolation and cultured for 48 h in the presence of the indicated concentrations of parathormone. The control value for citrate decarboxylation was 17.0 ± 1.8 x 103 dpm per 105 cells. Values are means ± SE of 4 cultures.
25-
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FIG. 5. Time course of development of parathormone effects on citrate decarboxylation in PT type cells. Cells of population 4 were replated on the sixth day after isolation and were cultured for the indicated times with 200 ng/ml of parathormone. The control value for citrate decarboxylation was 14.6 ± 1.9 x 103 dpm per 105 cells. Values are mean ± SE for 4 cultures.
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CHARACTERIZATION OF BONE CELLS CT populations are enriched in cells which express the metabolic characteristics of osteoclasts. It is possible that this population also contains osteocytes, since these cells are believed to have several metabolic characteristics in common with osteoclasts (37). It is less likely, however, that osteocytes could be the predominant cell type in the CT population, because of the relative scarcity of osteocytes in the calvaria used as a source of the isolated cells (38). Alkaline phosphatase and collagen synthesis activities are considered characteristic of osteoblasts (39), and both have been shown to be decreased by parathormone in bone in vitro (27,28,31,40). Both activities were highest in the PT cells, using prolyl hydroxylase as a marker of collagen synthesis. Moreover, both activities were decreased by parathormone. Hence, we conclude that PT populations are enriched in cells which express characteristics of osteoblasts. We must yet determine whether the cells in the PT and CT populations were derived either from mature osteoblasts and osteoclasts, from precursor cells which differentiated in culture, or from as yet unrecognized hormone-responsive bone cells. Of equal importance is the need to evaluate in these cells other metabolic reactions characteristic of bone cells, such as calcium transport and matrix dissolution and formation, and to determine the effects of other bone-active agents such as vitamin D metabolites and prostaglandins. Acknowledgment We wish to acknowledge the excellent technical assistance of Ms. M. Mohler.
References 1. Peck, W. A., S. J. Birge, and S. A. Fedak, Science 146: 1476, 1964. 2. Dziak, R., and J. S. Brand,/ Cellular Physiol 84: 75, 1974. 3. Binderman, I., D. Duskin, A. Harell, E. Katzir (Katchalski), and L. SachsJ Cell Biol 61:427,1974.
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4. Flanagan, B., and G. Nichols, Jr., Fed Proc 22: 553, 1963. 5. Smith, D. M., C. C. Johnston, Jr., and A. R. Severson, CalcifTiss Res 11: 56, 1973. 6. Peck, W. A., J. Carpenter, K. Messinger, and D. DeBra, Endocrinology 92: 692, 1973. 7. Rodan, S. B., and G. A. RodanJ Biol Chem 249: 3068, 1974. 8. Smith, D. M., and C. C. Johnston, Jr., Endocrinology 96: 1261, 1975. 9. Mahgoub, A., and H. Sheppard, Biochem Biophys Res Commun 62: 901, 1975. 10. Peck, W. A., J. Carpenter, and K. Messinger, Endocrinology 94: 148, 1974. 11. Peck, W. A., K. Messinger, G. Kimmich, and J. Carpenter, Endocrinology 95: 289, 1974. 12. Dziak, R., and J. BrandJ Cell Physiol 84: 85, 1974. 13. Dziak, R., and P. H. Stern, Endocrinology 97: 1281, 1975. 14. Wong, G., and D. Cohn, Nature 252: 713, 1974. 15. Wong, G. L., and D. V. Cohn, Proc Natl Acad Sci USA 72: 3167, 1975. 16. Gilman, A., Proc Natl Acad Sci USA 67: 305, 1970. 17. Luben, R. A., J. F. Goggins, and L. G. Raisz, Endocrinology 94: 737, 1975. 18. Luben, R. A., and D. V. Cohn, Endocrinology 98: 411, 1976. 19. Lowry, O. H., N. R. Roberts, M. L. Wu, W. S. Hixon, and E. J. Crawford, / Biol Chem 207: 19, 1954. 20. Berg, R. A., and D. J. Prockop, / Biol Chem 248: 1175, 1973. 21. Rao, W. W. Y., R. A. Berg, and D. J. Prockop, Biochim Biophys Ada 411: 202, 1975. 22. Rhoads, R. E., and S. Udenfriend, Proc Natl Acad Sci USA 60: 1473, 1968. 23. Peterkofsky, B., and R. DiBlasio, Anal Biochem 66: 279, 1975. 24. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall,/ Biol Chem 193: 265, 1951. 25. Wolinsky, I., and D. V. Cohn, Endocrinology 84: 28, 1969. 26. Chu, L. L. H., R. R. MacGregor, J. W. Hamilton, and D. V. Cohn, Endocrinology 89: 1425, 1971. 27. Vaes, G., and P. Jacques, Biochem J 97: 380, 1965. 28. Goldhaber, P., B. D. Stern, M. J. Glimcher, and J. Chao, In Talmage, R. V., and L. Belanger (eds.), Parathyroid Hormone and Thyrocalcitonin (Calcitonin), Excerpta Medica Foundation, Amsterdam, 1968, p. 182. 29. Brand, J. S., and L. G. Raisz, Endocrinology 90: 479, 1972. 30. Atkins, D., and M. Peacock, J Endocrinol 64: 573, 1975. 31. Dietrich, J. W., E. M. Canalis, D. M. Maina, and L. G. Raisz, Endocrinology 98: 943, 1976. 32. Hancox, N. M., Biology of Bone, Cambridge University Press, 1972, p. 63.
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33. Owen, M., and M. R. Shetlar, Nature 220: 1335, 1968. 34. Johnston, C. C , Jr., D. M. Smith, and A. R. Severson, In Talmage, R. V., and P. L. Munson (eds), Calcium, Parathyroid Hormone and the Calcitonins, Excerpta Medica Foundation, Amsterdam, 1972, p. 327. 35. Walker, D. G., Calcif Tiss Res 9: 296, 1972. 36. Doty, S. B., B. H. Schofield, and R. A. Robinson, In Talmage, R. V., and L. F. Belanger (eds.), Parathyroid Hormone and Thyrocalcitonin
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38. 39.
40.
Endo • 1976 Vo! 99 • No 2
(Calcitonin), Excerpta Medica Foundation, Amsterdam, 1968, p. 169. Baylink, D., and J. Wergedal, In Nichols, G., and R. H. Wasserman (eds.), Cellular Mechanisms for Calcium Transfer and Homeostasis, Academic Press, New York, 1971, p. 257. Aaron, J. E., Calcif Tiss Res 12: 259, 1973. Rasmussen, H., and P. Bordier, The Physiological and Cellular Basis of Metabolic Bone Disease, Williams and Wilkins, Baltimore, 1974, p. 185. Vaes, G. M., and G. Nichols, Jr., Endocrinology 70: 546, 1962.
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