JOURNAL OF BONE AND MINERAL RESEARCH Volume 7, Numbcr 10, 1992 Mary Ann Llebcrt, Inc., Publishers

0-Glycerophosphate-Induced Mineralization of Osteoid Does Not Alter Expression of Extracellular Matrix Components in Fetal Rat Calvarial Cell Cultures KUI-LA1 LEE, JANE E. AUBIN, and JOHAN N.M. HEERSCHE

ABSTRACT When fetal rat calvarial cells are cultured in medium containing vitamin C, osteoid nodules develop after approximately 15 days of culture. Upon addition of an organic phosphate (0-glycerophosphate, PGP), these nodules mineralize. We have now used this system to explore the suggestion made by others that a negative feedback may exist between matrix mineralization on the one hand and the synthesis of alkaline phosphatase and bone matrix collagen on the other by analyzing the synthesis of these proteins and the levels of their mRNAs in mineralizing and nonmineralizing cultures. Our results indicate that in the osteoid nodule-bone nodule system, matrix mineralization did not affect the mRNA levels for osteopontin, type I collagen, bone sialoprotein, or osteocalcin. Synthesis of total protein and collagen and the osteocalcin content of culture media were also not different in the mineralizing and nonmineralizing cultures. However, alkaline phosphatnse mRNA was increased in early mineralizing cultures and alkaline phosphatase activity in the cell layer was also increased in mineralizing cultures. Thus, the hypothesis that a direct negative feedback exists between mineralization and matrix protein synthesis is not supported by our experiments.

INTRODUCTION involves the synthesis of an organic matrix and its subsequent mineralization. Alkaline phosphatase activity associated with osteogenic cells plays a role in providing the phosphate necessary for initiation of mineral deposition.['-') In a series of experiments in which they compared mineralizing and nonmineralizing cultures of folded chick periosteum with respect to alkaline phosphatase activity and collagen synthesis, Tenenbaum et al.[',61found that a negative feedback might exist between matrix mineralization and the production of alkaline phosphatase and collagen. With regard to alkaline phosphatase activity, their findings agreed with the results of Genge et al.,") who found a decrease in alkaline phosphatase activity during mineralization of matrix vesicles isolated from chicken growth plate cartilage. Gerstenfeld et aI.[') reported that in bone-forming cultures of cells derived from chick calvariae, alkaline phosphatase activity was increased during the early mineralization phase of their cultures but

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ORMATION OF BONE

decreased at later culture times. In rat calvaria cells cultured for up to 24 days in a-minimum essential medium (a-MEM)with or without added 0-glycerophosphate (PGP), Aronow et al.[n) also found increased alkaline phosphatase activity in mineralizing cultures and decreased alkaline phosphatase activity in heavily mineralized cultures. In agreement with Tenenbaum et al.,19) they also found that the collagen content of mineralizing cultures was lower than in nonmineralizing cultures at all culture times, thus supporting the possibility of feedback regulation of collagen synthesis. When the same cells were cultured in a BGJb-based medium with or without PGP from day 8 of culture onward, however, alkaline phosphatase activity did not change with increasing mineralization, and collagen synthesis also did not change as a function of mineralization. Some of these reports thus suggest that negative feedback regulation may exist between mineralization of bone matrix, on the one hand, and synthesis of organic bone matrix and alkaline phosphatase on the other. Because of

MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Ontario, Canada. 1211

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the possible importance of an effect of such a feedbackregulating mechanism, we investigated whether matrix mineralization directly regulates the expression of mRNAs for matrix proteins and alkaline phosphatase and of the respective proteins in the osteoid nodule-bone nodule system in which mineralization is induced by the addition of BGP.

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MATERIALS AND METHODS

Cell cultures Rat calvaria cells were isolated by sequential digestion of 21 day fetal rat calvariae with an enzyme mixture containing collagenase. ( l o ) Populations 11-V were pooled and plated into T-75tissue culture flasks in a-MEM containing 10% fetal calf serum (FCS) and antibiotics and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO1. After 24 h, cells were trypsinized, replated at 5 x 10' cells per 100 mm tissue culture dish, and cultured in aMEM containing 10% FCS and antibiotics as before but supplemented with 50 pg/ml of ascorbic acid and lo-' M dexamethasone (supplemented media; Sigma). Media were t=72H changed every 2 or 3 days. At approximately 15 days, when osteoid nodules were visible, the medium was changed to supplemented medium with 10 mM BGP and cultures were continued for another 8, 24, 48, or 72 h. During these last 72 h, the medium was changed every 12 h in these cultures and in the control cultures (without BGP) to prevent calcium and phosphate depletion caused by the FIG. 1. Rat calvarial cells were cultured in medium conrapid mineralization. ['I Mineralization was evaluated using taining 50 pg/ml ascorbic acid until osteoid nodules were formed (day 15). BGP at 10 mM was added at day 15 ( I = "Ca uptake and von Kossa's staining.('.'' 0), and cultures were fixed and stained with von Kossa's technique after 8, 24, and 72 h. Osteoid nodules present in "Ca uptake cultures mineralize progressively over the 3 day period. After culture with or without OGP, the cultures were labeled for 2 h with .'Ca (0.1 pCi/ml culture medium; ICN Biochemicals Canada, Ltd., Mississauga, Ontario, Canada). After labeling, the medium was removed and the cells were washed three times 5 minutes each in cold phosphate-buffered saline (PBS) and harvested with a rubber scraper. The cells and matrix were dissolved in 10% formic acid for 24 h and counted using a scintillation counter.

RNA extraction Total RNA was extracted by the method of Auffray and Rougeon.(IL1RNA (20 pg) from each sample was run on an 1% agarose formaldehyde gel. Samples were stained with ethidium bromide to visualize the 18s and 28s rRNA subunits before being transferred onto a 0.2 mm nitrocellulose filter and immobilized by baking at 80°C for 2 h. Prehybridization and hybridization were performed in fivefold Denhardt's solution, fivefold SSC (SSC = 0.15 M NaCl, 15 mM trisodium citrate, pH 7.0), 50 mM sodium monophosphate, 50% formamide, and 250 pg/ml of salmon testes DNA for 16-22 h. cDNA probes for porcine osteopontin (full-length cDNA plus 97 bases of the 5'-untranslated region).'"' rat type I collagen (cDNA for the entire 3'-noncoding region and one-half of the C terminus of the propeptide of the a-I chain of collagen type I),(131 human alkaline phosphatase (full-length cDNA, EcoRI digest

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FIG. 2. "Ca uptake in nodule cultures with and without addition of 10 mM BGP to the culture media. Cultures as described in Fig. 1 were labeled with "Ca for 2 h at the times indicated as described in Materials and Methods. "Ca uptake increased progressively during the 72 h period in the presence of 10 mM BGP. +Significantly different from control cultures (p < 0.05).

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MINERALIZATION IN CALVARlAL CELL CULTURES

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FIG. 3. Quantitative densitometry of mRNA levels of collagen type I (COLL), bone sialoprotein (BSP), osteopontin (OP), osteocalcin (OC), and alkaline phosphatase (AP). Autoradiographs were scanned using an LKB-Gelscan XL (Pharmacia, Canada). The integrated peak area of each mRNA was first expressed as the ratio to tubulin mRNA for each individual measurement. The bar groups represent the ratios of these mRNA levels in mineralizing and nonmineralizing for the two experiments combined. All values were normalized, with the ratio to tubulin mRNA at 8 h given the value 1. Means, SD, and significance are calculated. Results represent mean f SD of four measurements. +Significantly different from control (-/3GP) cultures (p < 0.05).

of pAT153, 2.5 kb insert),'l'' rat bone sialoprotein and osteocalcin (cDNA generated with specific primers by polymerase chain reaction (PCR); rat bone sialoprotein (BSP) cDNA: 1808 bp, 15 bp 5' from transcription initiation site to 1823 bp 3'; rat osteocalcin cDNA: 327 bp, 153 bp 5' from transcription initiation site to 480 bp 3'; Dr.

Ashwani Gupta, unpublished), rat a-tubulin [excised with EcoRI from the Xgtll vector and with Psi1 from pILTI subcloned in pSP65 RNA synthesis vector'16'], and mouse glyceraldehyde-3-phosphate dehydrogenase (MGAP, cDNA insert of 1050 bp excised with PsrI from pBR322)'l'.ln1 were labeled with d ' P - d C T P (NEN, 3000

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Ci/mmol) using an oligolabeling kit (Pharmacia, LKB Biotechnology, Inc.). The filter was washed in twofold SSC and 0.1 Vo sodium dodecyl sulfate (SDS) for 45-60 minutes and SSC and 0.1 To SDS for 30-40 minutes at 55°C and exposed to Kodak X-Omat AR film at -70°C. The same blot was used for probing with different cDNA probes. The amount of mRNA in each lane was standardized to that of tubulin and MGAP. Densitometric scanning of the gels was performed on an LKB-Pharmacia Ultrascan XL (Pharmacia, LKB Biotechnology, Inc.) linked with an IBM computer. Tubulin and MGAP mRNA were probed on each blot to correct for unequal loading of RNA. The ratio of tubulin to MGAP was the same in mineralizing versus nonmineralizing cultures and did not change with time in culture after initiation of mineralization. Care was taken t o expose the films such that the density of the autoradiographs did not exceed the linearity of the densitometric quantitation.

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Osteocalcin assay Media were removed from the cultures at various times after the addition of 10 mM PGP, and osteocalcin was measured directly by radioimmunoassay (Biomedical Technologies, Inc., Stoughton, MA).

Assay of alkaline phosphatase activity Alkaline phosphatase activity in cell layer extracts and media was determined by measuring the release of p-nitrophenol from p-nitrophenylphosphate (Sigma, St. Louis, MO) at p H 10.3.[1sl

TB

Protein assay Total protein in the cell layer was determined as described by Bradford.[’O’ FIG. 4. Representative blot, illustrating the expression of mRNAs of bone matrix proteins and alkaline phosphatase in mineralizing and nonmineralizing cultures. Total RNA At the indicated times after addition of BGP, the cells was extracted from control (without BGP, -), and BGPwere washed three times with 2 ml methionine-free Dulbec- treated cultures (+) at the indicated times and equal co’s modified Eagle’s medium (DMEM) and 0.2% (VOV amounts analyzed by Northern blotting. Collagen type I vol) FCS and then pulse-labeled for 30 minutes at 37°C in (COLL), alkaline phosphatase (AP), osteopontin (OP), bone sialoprotein (BSP), osteocalcin (OC), tubulin (TB), 1 ml of the same medium containing 50 pCi [”Slmethionine (specific activity 800 Ci/mmol; NEN) and 50 pg/ml of and mouse glyceraldehyde-3-phosphate dehydrogenase ascorbic acid. Following the pulse period, cells were (MGAP) mRNA are shown. washed twice with a-MEM containing 0.2% FCS and then cultured in this medium for an additional 4 h. The media from the 4 h chase period were collected, proteolytic en- lyzed by SDS-PAGE (polyacrylamide gel electrophoresis) zyme inhibitors were added (10 pM phenylmethylsulfonyl under reducing conditions using 5-20Vo gradient gels in the fluoride, 0.2 mM EDTA, and 50 pM benzamidine; Sigma), discontinuous buffer system of Laemmli. [ I 1 ) One-quarter and the media were dialyzed against three changes of volume of fourfold electrophoresis sample buffer was water. Cell layers were washed three times with 2 ml ice- added to equal aliquots of media and cell layer samples cold PBS, scraped with a rubber scraper, collected in 1 ml and heated t o 60°C for 20 minutes before loading. Followice-cold PBS containing proteolytic enzyme inhibitors as ing electrophoresis, gels were prepared for fluorography as before, and sonicated on ice for 20 s at 17 J/s with a Bran- described by Bonner and Laskey.Ia2’ Dried gels were exson 185 sonifier. Aliquots of the radiolabeled media and posed to Kodak SB-5 x-ray film for 2-5 days at -70°C. the cell layer were then analyzed for radioactivity by scin- Molecular mass markers radiolabeled by reductive methtillation counting. The radiolabeled proteins were also ana- ylation were myosin (200 kD), phosphorylase B (97.4 kD),

Protein synthesis

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MINERALIZATION IN CALVARIAL CELL CULTURES

staining material (Fig. 1). Mineralization assessed by the presence of von Kossa’s staining material correlated with 45Caaccumulation: after a lag period of approximately 8 h, 45Ca uptake increased with time in cultures containing 30 BGP but not in cultures without PGP (Fig. 2). These C .-0 ) results confirm our previous To determine whether BGP-induced mineralization L o caused changes in steady-state levels of mRNA for bone matrix proteins and alkaline phosphatase, total RNA from 10 mineralizing and nonmineralizing cultures was extracted at m C various times during a 72 h period of incubation with PGP and analyzed by Northern blot analysis. The results of two 0 independent experiments with two samples at each time (10mMRGP) + - + - + - + point were combined and are shown in Fig. 3. Absorbance 8 24 48 72 H values were normalized against the 8 h values. First, the reFIG. 5. Osteocalcin content of the culture media in min- sults show an increase in the level of mRNA for all RNAs eralizing and nonmineralizing cultures. Osteocalcin was studied over the 72 h culture period in both mineralizing measured by radioimmunoassay in aliquots of media from and nonmineralizing cultures, reflecting continuing growth cells grown with and without PGP for the times indicated. Each bar represents the mean f SD of a group of three and development of osteoid-bone nodules during this period. No systematic differences between mineralizing and samples expressed per milligram protein in the cell layer. nonmineralizing cultures were seen of the mRNA levels for osteopontin, osteocalcin, collagen type I, or bone sialoprobovine serum albumin (68 kD), ovalbumin (43 kD), car- tein. Alkaline phosphatase mRNA levels were significantly bonic anhydrase (30 kD), /3-lactoglobulin (18.4 kD), lyso- increased in mineralizing cultures at the 8 h time point but zyme (14.3 kD), bovine trypsin inhibitor (6.2 kD), and in- not at the other time points (Fig. 3). A representative comsulin A and B chain (2.3 and 3.4 kD). posite of one of the blots from these experiments is shown in Fig. 4. We subsequently measured osteocalcin content, collagen Collagen synthesis synthesis, and alkaline phosphatase activity in the cell layer To quantify collagen synthesis, aliquots of [3SS]methio- and/or the culture media of mineralizing and nonmineralnine-labeled media and cell layer samples were digested izing cultures at 24 and 72 h. Osteocalcin levels in the cul~ ~ M Tris ture media of mineralizing and nonmineralizing cultures with 25 pg purified bacterial c o I l a g e n a ~ e ~in*0.05 HCI buffer, pH 7.5, containing 5 mM CaCI, and 0.5 mM were the same (Fig. 5). Only media osteocalcin was meaN-ethylrnaleimide for 2 h at 37°C. Nondigested proteins sured since Gerstenfeld et al. l’) showed that osteocalcin were precipitated with ice-cold 7% wt/vol trichloroacetic content was 10-fold higher in the media than in the cell acid (TCA) and removed by centrifugation at 10,OOO x g. layer in both mineralizing and nonmineralizing cultures. The pellet was washed twice and extracted at 90°C for 20 The percentage of collagen synthesized in mineralizing and minutes with 7% wt/vol TCA to check the efficiency of nonmineralizing cultures in both cell layers and media was the collagenase digestion. Samples of the pellet, solubilized also similar (Fig. 6). Total protein in the cell layer of minin 200 pl 70% formic acid (noncollagenous protein), the eralizing and nonmineralizing cultures was also not differhot TCA extract (nondigested collagen), and the TCA ent (72 h values: nonmineralizing cultures, 1.05 0.05 mg supernatant (collagen) were analyzed for radioactivity. per culture; mineralizing cultures, 1.05 f 0.02 mg per culCounts soluble in “hot TCA,” that is, residual collagen, ture). The overall protein profiles in media and cell layers were added to the collagenase-digestible counts. Collagen were also similar for mineralizing and nonmineralizing culsynthesis was expressed as the amount of [35S]methionine tures (Fig. 7). in collagen (collagen and nondigested collagen) as a perAlkaline phosphatase activity in the cell layer of minercentage of total protein (collagen, nondigested collagen, alizing cultures was significatnly higher than that in nonand noncollagenous protein). mineralizing cultures at 8, 24, 48, and 72 h after addition of BGP (Fig. 8). Medium alkaline phosphatase activity was very low compared to the activity in the cell layer, was Statistics lower in mineralizing cultures at 8 and 24 h, not different Differences between groups were evaluated using the in the two types of cultures at 48 h, and slightly higher in two-tailed Student’s [-test. mineralizing cultures at 72 h.

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RESULTS Cells isolated from fetal rat calvaria and cultured in DISCUSSION a-MEM with 10% serum and 50 p g h l of ascorbic acid We investigated whether OGP-induced mineralization of form numerous osteoid nodules by day 15 of culture. These nodules mineralize upon the addition of 10 mM osteoid nodules directly influences the synthesis and/or the BGP as evidenced by the accumulation of von Kossa’s activity of alkaline phosphatase and of several bone matrix

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FIG. 6. Effect of PGP addition on collagen synthesis in media and cell layers. Cultures were pulse labeled with [3SS]methionineat the indicated times. Equal aliquots of samples were digested with purified bacterial collagenase. The amount of radioactivity in the collagenase-digestible fraction (collagen), the hot TCA-soluble fraction (nondigested collagen), and the formic acid-solubilized pellet (noncollagenous protein) was analyzed. Results represent the percentage of [35S]methioninein the collagenase-digestible fraction plus the hot TCA fraction of triplicate determinations from one experiment. Values for mineralizing and nonmineralizing cultures were not significantly different.

proteins. We found that the steady-state levels of mRNA for osteopontin, osteocalcin, collagen. and bone sialoprotein, the synthesis of collagen and total protein, and osteocalcin content were similar in mineralizing and nonmineralizing cultures. This indicates that negative feedback regulation does not exist between mineralization and organic matrix deposition. However, alkaline phosphatase mRNA levels were increased significantly early during the SGP-induced mineralization, and alkaline phosphatase activity associated with the cell layer was increased in mineralizing compared to nonmineralizing cultures at all time points during the 72 h of PGP-induced mineralization. Using cultures continuously exposed to PGP during the bone formation stage, Aronow et a1.ln1and Gerstenfeld et al.['] also reported an increase in alkaline phosphatase activity coincident with or just before the onset of mineralization and when the cultures were actively mineralizing but reported a decrease in alkaline phosphatase activity during the later phases of culture, when the cultures were heavily mineralized and mineralization progressed at a slower rate. Thus, increased alkaline phosphatase activity appears to be associated with early mineralization in these and our studies. The decrease in alkaline phosphatase in older cultures may reflect terminal differentiation of osteoblasts into lining cells and osteocytes and a concomitant decrease in the number of alkaline phosphatase-positive osteoblasts and osteoprogenitor cells. In matrix vesicles isolated from chicken growth plate cartilage".21 and in mineralizing cultures of folded chick periosteum, mineralization is also associated with decreased alkaline phosphatase activity. Genge et al.[21hypothesize that the observed loss of alkaline phosphatase activity may be caused by a loss of zinc and magnesium from the active site of the enzyme secondary to mineral deposition. However, the results in mineralizing cell cultures we report here and those of Aronow et al.[71and Gerstenfeld et al.[nlindicate that mineralization per se does not decrease alkaline phosphatase activity. The steady-state levels of osteocalcin mRNA and the amount of osteocalcin in the culture medium were not different in mineralizing and nonmineralizing cultures, suggesting that tissue mineralization does not regulate osteocalcin synthesis. This observation agrees with the data of Poliard et al.,lz4]who found no differences in the osteocalcin mRNA levels of mineralizing and nonmineralizing mouse osteogenic cells, and is also consistent with the observations of Aronow et al.,[nl who showed similar osteocalcin levels in mineralizing and nonmineralizing cultures in a-MEM-based media. Because Tenenbaum et al.l9] reported that in folded chick periosteum cultured for 6 days in the continuous presence of OGP collagen synthesis was less than in similar cultures grown without BGP, we measured collagen [a,(I)] mRNA levels, total collagen synthesis, and percentage collagen synthesis in mineralizing and nonmineralizing cultures. We found no differences between these two types of cultures and concluded that in our system levels of collagen mRNA and percentage of collagen synthesis are not directly regulated by BGP-induced mineralization. We also found no differences in total protein profile or total pro-

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MINERALIZATION IN CALVARIAL CELL CULTURES

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FIG. 7. Total protein synthesis in control cultures (without PGP) and cultures with added PGP. Cultures were treated with 10 mM PGP for the indicated times and pulse labeled subsequently with 75 pCi/ml of ["S]methionine for 30 minutes. Equal aliquots of radioactivity from the cell layer (A) and chase medium (B) were analyzed under reducing conditions in 7.5% linear (medium proteins) and 5520% gradient (cell layer) SDS-PAGE gels. Banding patterns in PGP-treated cultures and control cultures were similar after 24 and 72 h in both culture medium and cell layer.

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FIG. 8. Alkaline phosphatase activity in cell layers (A) and media (B) of mineralizing and nonmineralizing cultures. Alkaline phosphatase activity was assayed using the method of Lowry and expressed per milligram protein as assayed by the Bradford method. Results represent the mean f SD of triplicate measurements of a representative experiment. *Significantly different from control (-PGP) cultures (p < 0.05).

tein synthesis in the media and cell layers of mineralizing and nonmineralizing cultures. This agrees with the observations of Aronow et al. in mineralizing versus nonmineralizing rat calvaria cell bone-forming cultures but not with those of Gerstenfeld et al., who found that total protein synthesis was decreased in mineralizing chick osteoblast bone-forming cultures. Thus, in contrast to the observations in rat calvaria cultures, collagen synthesis in chicken osteoblast bone-forming cultures appears to decrease in mineralizing versus nonmineralizing cultures even at early

stages of mineralization. Although the reasons for the discrepancies are not yet known, one possibility is a different population makeup, that is, the balance of less mature and more mature cells, in the different populations (see also Ref. 8). In our experiments, the levels of mRNA for osteopontin and bone sialoprotein were the same in nonmineralizing and mineralizing cultures. Poliard et a1.'"' also found no changes in the expression of mRNA for osteopontin in the mineralizing cultures of mouse osteogenic cells. The results

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of Nagata et al.,‘’’’ who evaluated the osteopontin and bone sialoprotein content of culture media, cell layers, and matrices of mineralizing and nonmineralizing rat calvaria cell cultures, also suggest that the synthesis of these proteins is similar in mineralizing and nonmineralizing cultures and that both osteopontin and bone sialoprotein bind to the mineral phase after deposition of the mineral. All these results suggest that mineralization per se does not regulate the synthesis of either bone sialoprotein or osteopontin. In summary, this study has shown that the steady-state levels of mRNAs for osteocalcin, bone sialoprotein, osteopontin, and collagen are not affected by PGP-induced mineralization of osteoid matrix. Similarly, medium osteocalcin content and collagen synthesis were the same in mineralizing and nonmineralizing cultures. Interestingly, alkaline phosphatase activity was higher in mineralizing cultures than in nonmineralizing cultures, and mRNA levels for alkaline phosphatase also were higher during early mineralization. This suggests that in mineralizing systems the progression of mineralization is not necessarily associated with reduced alkaline phosphatase activity and may indicate that in the initial stages of mineralization alkaline phosphatase activity and alkaline phosphatase mRNA levels are increased as a consequence of the mineralization process. Our results also indicate that mineralization of osteoid matrix has no negative feedback effect on alkaline phosphatase activity and matrix protein production by the associated osteoblast population.

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6. Tenenbaum HC, McCulloch CAG, Fair C, Birek C 1989 The regulatory effect of phosphates on bone metabolism in vitro. Cell Tissue Res 257:555-563. 7. Gerstenfeld LC, Chipman SD, Glowacki J , Lian JB 1987 Expression of differentiated function by mineralizing cultures of chicken osteoblasts. Dcv Biol 11249-60. 8. Aronow MA, Gerstenfeld LC, Owen TA, Tassinari MS, Stein GS, Lian JB 1990 Factors that promote progressive development of the osteoblast phenotype in cultured fetal rat calvarial cells. J Cell Physiol 143:213-221. 9. Tenenbaum HC, Limeback H. McCulloch CAG, Mamujee H. Sukhu B, Torontali M 1992 Osteogenic phase-specific COregulation of collagen synthesis and mineralization by B-glycerophosphate in chick periostcal cultures. Bone 13:129- 138. 10. Rao LG, Ny B, Brunette DM, Hccrsche JNM 1977 Parathyroid hormone and prostaglandin E, - response in a selected population of bone cells after repeated storage and subculture at -80°C. Endocrinology 100:1233-1241. 11. Auffray C, Rougeon F 1980 Purification of mouse immunoglobulin heavy-chain messenger RnAs from total myeloma tumor RNA. Eur J Biochem 107303-314. 12. Wrana JL. Zhang Q. Sodek J 1989 Full length cDNA sequence of porcine secreted phosphoprotein-1 (SPP-1. osteopontin). Nucleic Acids Res 17:10119. 3. Genovese C, Rowe D, Kream B 1984 Construction of DNA sequences complementary to rat a, and az collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1.25-dihydroxyvitamin D. Biochemistry 23: 6210-6216. 4. Weiss MJ, Henthorn PS, Lafferty MA, Slaughter C, Raducha M, Harris H 1986 lsolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc Natl Acad Sci USA 83:7182-7186. IS. Lemischka IR, Farmer S. Racaniello VR. Sharp PA 1981 ACKNOWLEDGMENTS Nucleotide sequence and evolution of a mammalian a-tubulin messenger RNA. J Mol Biol 151:lOl-120. All the probes were gifts: MGAP from Dr. Ann Cham- 16. Melton DA, Krieg PA, Rebagliati MR, Maniatis T. Zinn K. bers, London, Ontario, Canada; collagen type I from Dr. Green MR 1984 Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids conDavid Rowe; osteopontin from Dr. Jeff Wrana; alkaline taining a bacteriophage SP6 promoter. Nucleic Acids Res 12: phosphatase from Dr. Mitchell Weiss; and osteocalcin and 7035-7056. bone sialoprotein from Dr. Ashwani Gupta. We thank Dr. Jaro Sodek for advice on the protein synthesis measure- 17. Edwards DR, Parfett CLJ, Denhardt DT 1985 Transcriptional regulation of two serum-induced RNAs in mouse fiments. Data for parts of this paper were presented at the broblasts: Equivalence of one species to B2 repetitive eleASBMR meeting, Atlanta, GA, August 1990 (J Bone ments. Mol Cell Biol 5:3280-3288. Miner Res. 6, Suppl. 2, Abstract 36). 18. Edwards DR, Denhardt DT 1985 A study of mitochondria1 and nuclear transcription with cloned cDNA probes. Exp Cell Res 57:127-143. REFERENCES 19. Lowry OH 1955 Micromethods for the assay of enzyme. 11. Specific procedures. Alkaline phosphatase. Methods Enzymol 4:371-372. 1. Wuthier RE 1987 Mechanism of de novo mineral formation 20. Bradford MM 1976 A rapid and sensitive method for the by matrix vesicles. Connect Tissue Res 2227-33. quantitation of microgram quantities of protein utilizing the 2. Genge BR, Sauer GR, Wu LNY, McLean FM. Wuthier RE principle of protein-dye binding. Anal Biochem 72248-254. 1988 Correlation between loss of alkaline phosphatase activity and accumulation of calcium during matrix vesicle-medi- 21. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680ated mineralization. J Biol Chem %3:18513-18519. 685. 3. Bellows CG, Hccrsche JNM, Aubin JE 1992 Inorganic phosphate added exogenously or released from B-glycerophos- 22. Bonner WM, Laskey RA 1974 A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide phate initiates mineralization of osteoid nodules in vitro. gels. Eur J Biochem 46:83-88. Bone Miner 17:15-29. 4. BeIlows CG, Aubin JE, Heersche JNM 1991 Initiation and 23. Peterkofsky B, Diegelmann R 1971 Use of a mixture of proteinase-free collagenases for the specific assay of radioactive progression of mineralization of bone nodules formed in collagen in the presence of other proteins. Biochemistry 10: vitro: The role of alkaline phosphatase and organic phos988-994. phate. Bone Miner 14:27-40. 5. Tenenbaum HC 1987 Levamisole and inorganic pyrophos- 24. Poliard A, Lamblin D, Marie P, Buc MH. Kellermann 0 1991 Gene expression during in vitro mineralization in an phate inhibit 8-glycerophosphate-inducedmineralization of bone formed in vitro. Bone Miner 3:13-16. osteogenic cell line derived from mouse teratocarcinoma.

MINERALIZATION IN CALVARIAL CELL CULTURES ASBMR Abstract 246. J Bone Miner Res qsuppl. 1):336. 25. Nagata T, Bellow CG, Kasugai S, Butler WT, Sodek J 1991 Biosynthesis of bone proteins, SPP-I (secreted phosphoprofein-1, osteopontin) BSP (bone sialoprotein) and SPARC (osteonectin) in association with mineralized tissue formation by fetal rat calvarial cells in culture. Biochem J 274513-520.

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K.L. Lee MRC Group in Periodontal Physiology 4384 Medical Sciences Bldg. University of Toronto Toronto, Ontario, Canada MSS IAB Received for publication December 21. 1991; in revised form March 19, 1992; accepted May 6, 1992.

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beta-Glycerophosphate-induced mineralization of osteoid does not alter expression of extracellular matrix components in fetal rat calvarial cell cultures.

When fetal rat calvarial cells are cultured in medium containing vitamin C, osteoid nodules develop after approximately 15 days of culture. Upon addit...
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