SHORT
COMMUNICATIONS
BIOSYNTHESIS OF PHOSPHOPROTEIN RAT INCISOR ODONTOBLASTS IN IN-VITRO
BY CULTURE
E. C. MUNKSGAARD, W. S. RICHARDSON,III and W. T. BUTLER Institute of Dental Research, University of Alabama in Birmingham, University Station, Birmingham, AL 35294.U.S.A. Summary-A tissue culture system consisting of odontoblasts still attached to predentine and dentine was developed to study dentinogenesis. After incubating the odontoblasts with both C3H]-serine and [33P]-phosphate for 24 h, the protein components were extracted with 0.5 M EDTA. A major component synthesized by the odontoblasts was identified as the phosphoprotein by double labelling with C3H]-serine and [33P]-phosphate and by co-elution in two chromatographic systems with unlabelled, carrier phosphoprotein.
A soluble phosphoprotein has been isolated from bovine molars by Veis, Spector and Zamoscianyk (1972), from rabbit incisors by Richardson et al. (1977) and by Butler, Finch and DeSteno (1972) according to whom the phosphoprotein of rat incisors is a major constituent, accounting for about 10 per cent of the organic material. This polyanionic protein has unusual chemical and physical properties, being composed of more than 40 per cent phosphoserine and 35 per cent aspartic acid (Munksgaard, Butler and Richardson, 1977). Several investigators (Veis and Perry, 1967; Nawrot et a/., 1976; Termine and Conn, 1976) have suggested that the dentine phosphoprotein plays a role in the processes which lead to the conversion of predentine to dentine as mineralization occurs. This is supported by the radioautographic studies of Weinstock and Leblond (1973). Their results suggest that the phosphoprotein of rat incisors is made by odontoblasts, passes quickly through predentine and is deposited at the predentine-dentine junction. Thus, deposition would be coincident with the mineralization process. However, an unequivocal identification of the molecules labelled by the radioisotopes was not determined. To investigate the biosynthesis and deposition of dentine matrix components in a manner which will utilize definitive biochemical techniques, we have developed an organ culture system employing odontoblasts attached to predentine and dentine. Mandibular incisors were removed from l&l l-week old male Sprague-Dawley rats and immediately placed in BGJ, culture medium (Grand Island Biological Co.) supplemented with 10 per cent foetal calf serum, 0.01 per cent glutamine and 1 per cent antibiotic-antimycotic solution. Under a stereomicroscope, the apical one-third of the tooth was carefully scraped free of soft pre-enamel and periodontal tissue and cut from the rest of the incisor. This apical segment was then opened by making a longitudinal incision on the lingual side, and the pulp was carefully removed. The dentine pieces were divided into two by a longitudinal incision at the labial side. Light microscopic sections of such pieces, fixed, demineralized and
stained (Weinstock and Leblond. 1973), showed parallel layers of intact odontoblasts still attached to predentine and dentine. Furthermore, odontoblasts were the major cell type present, although a few capillaries with associated endothelial cells could be seen between the odontoblasts. The odontoblasts attached to predentine-dentine pieces were incubated for 24 h in the culture medium with SO&i/ml each of E3H]-serine and [33P]-phosphate in the presence of SOpgg/ml of ascorbic acid. Incubation was at 37°C in 5 per cent CO, humid air. The cultured pieces were then washed with fresh medium and placed in 0.5 M EDTA, pH 7.4, with 5 mg of unlabelled phosphoprotein (Munksgaard et al., 1977). After 2 h at 24”C, the dentine pieces and cellular debris were removed by centrifugation; the resulting supernatant was dialysed exhaustively against water at 4”C, lyophilized and redissolved in 5.0 ml of 0.05 M tris-HCl buffer, pH 7.4. To determine whether the phosphoprotein had been synthesized by the odontoblasts, this material was chromatographed on DEAE-cellulose. A major radioactive component recovered by this procedure eluted at a position corresponding to carrier phosphoprotein and was labelled with both [33P]-phosphate and C3H]-serine (Fig. 1). The large [33P]-phosphate-labelled peak which eluted at 110-130ml was probably inorganic phosphate. The unidentified ultraviolet-absorbing peaks which eluted prior to 350ml must have originated from the medium and the tissue, because the phosphoprotein which was added prior to extraction with EDTA eluted as a single peak at an elution volume of 380-430ml when chromatographed alone under the same conditions (not shown). To confirm further the identity of the labelled phosphoprotein, the DEAE-cellulose peak eluting at 38&430ml (Fig. 1) was dialysed against water, lyophilized and redissolved in dilute HCl, pH 1.75. It was then rechromatographed on a sulphonated polystyrene column (Munksgaard et al., 1977). This procedure yields a highly pure phosphoprotein because, at pH 1.75, the phosphoprotein from rat dentine emerges in the void volume of the column whereas 583
584
E. C. Munksgaard,
100
200
W. S. Richardson,
300 EFFLUENT
III and W. T. Butler
400 VOLUME.ml
500
600
Fig. 1. Chromatography of proteins extracted from odontoblast cultures with EDTA. The sample was applied to a 1.5 x 10 cm column of DEAE-cellulose (Whatman DE-32, microgranular) and was eluted in 0.05 M tris-HCI buffer, pH 7.4, with a linear gradient from 0 to 0.6 M NaCl over a total volume of 800 ml. The flow rate was 50 ml/h and 5-ml fractions were collected. The effluent was monitored at 223 nm with a Beckman DB-GT spectrophotometer. 0.5-ml aliquots were taken, mixed with 5 ml of scintillation fluid (Scintiverse, Fisher Scientific) and counted with a liquid scintillation counter (Beckman Instruments, model LS-233) using a narrow tritium channel.
5
v--~~
1.2
--
%
ABSORBANCE j3,Pl SERINE PHOSPHATE [SHj
140 120 B
N. 0.6 t?
100 5 g
f
60
3
60
q
g
0.4
;! z 8 0
4o
si
20
$
0 5
IO
15 EFFLUENT
20 VOLUME.
25
30
ml
Fig. 2. Re-chromatography of the phosphoprotein peak from DEAE-cellulose (Fig. 1) on a 0.5 x 100 cm column of sulphonated polystyrene (AG 5OW-X2, Bio-Rad Laboratories). The column was equilibrated and eluted with dilute HCl, pH 1.75, by gravity flow. The effluent was monitored at 226nm and the l&ml fractions were assayed for radioactivity as described in Fig. 1.
adhering impurities are strongly bound to the resin (Munksgaard et al., 1977). The material synthesized by odontoblasts which was doubly labelled with C3H]-serine and [33P]-phosphate, co-chromatographed with carrier phosphoprotein (Fig. 2). Identical results were obtained using crude EDTA extract not chromatographed on DEAE-cellulose. When the extract was treated with 1 mg/ml of pepsin at pH 1.75 for 3 h at 24”C, the radioactive phosphoprotein emerged in the same position of the sulphonated polystyrene column as in Fig. 2 with no loss of radioactivity. Our data establish that the odontoblast-dentine cultures are viable and capable of synthesizing a phosphate- and serine-containing material which cochromatographs with unlabelled phosphoproteins isolated from rat dentine. This radioactive product is apparently resistant to pepsin treatment, as is the dentine phosphoprotein and phosvitin, another highlyphosphorylated protein (Clarke, 1973). In other experiments, we have shown that the odontoblast cultures synthesize and secrete collagen, and deposit it in predentine (E. C. Munksgaard, M. Rhodes, R. Mayne and W. T. Butler, unpublished). It thus appears that our culture system is capable of reproducing the biosynthetic in-vitro events which lead to the formation of predentine and its transformation to dentine. The system should prove valuable in
answering certain critical questions concerning the biosynthesis of the matrix components and how they are involved in dentinogenesis.
REFERENCES
Butler W. T., Finch J. E., Jr. and DeSteno C. V. 1972. Chemical character of proteins in rat incisors. Biochim. biophys. Acta 257, 167-171. Clark R. S. 1973. Amino acid sequence of a cyanogen bromide cleavage peptide from hens egg phosvitin. Eiochim. biophys. Acta 310, 174187. Munksgaard E. C., Butler W. T. and Richardson W. S., III. 1977. Phosphoprotein from dentin. New approaches to achieve and assess purity. Prep. Biochem. 7, 321-331. Nawrot C. F., Campbell D. J., Schroeder J. K. and Van Valkenburg M. 1976. Dental phosphoprotein-induced formation of hydroxylapatite during in-vitro synthesis of amorphous calcium phosphate. Biochemistry 15,
34453449. Richardson W. S., Beegle W. F., Butler W. T. and Munksgaard E. C. 1977. The phosphoprotein of rabbit incisors. J. dent. Rex 56, 233-237. Termine J. D. and Conn K. M. 1976. Inhibition of apatite formation by phosphorylated metabolites and macromolecules. Calc. Tiss. Res. 22, 149-157. Veis A. and Perry A. 1967. The phosphoprotein of dentin matrix. Biochemistry 6, 2409-2416.
Biosynthesis of phosphoprotein Veis A., Spector A. R. and Zamoscianyk H. 1972. The isolation of an EDTA-soluble phosphoprotein from mineralizing bovine dentin. Biochim. biophys. Acta 257, 404413.
0.“.
23 1
t
by odontoblasts
585
Weinstock M. and Leblond C. P. 1973. Radioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentin of the rat incisor. J. Ceil Biol. 56, 838-845.
COMMUNICATIONS
BIOSYNTHESIS OF PHOSPHOPROTEIN RAT INCISOR ODONTOBLASTS IN IN-VITRO
BY CULTURE
E. C. MUNKSGAARD, W. S. RICHARDSON,III and W. T. BUTLER Institute of Dental Research, University of Alabama in Birmingham, University Station, Birmingham, AL 35294.U.S.A. Summary-A tissue culture system consisting of odontoblasts still attached to predentine and dentine was developed to study dentinogenesis. After incubating the odontoblasts with both C3H]-serine and [33P]-phosphate for 24 h, the protein components were extracted with 0.5 M EDTA. A major component synthesized by the odontoblasts was identified as the phosphoprotein by double labelling with C3H]-serine and [33P]-phosphate and by co-elution in two chromatographic systems with unlabelled, carrier phosphoprotein.
A soluble phosphoprotein has been isolated from bovine molars by Veis, Spector and Zamoscianyk (1972), from rabbit incisors by Richardson et al. (1977) and by Butler, Finch and DeSteno (1972) according to whom the phosphoprotein of rat incisors is a major constituent, accounting for about 10 per cent of the organic material. This polyanionic protein has unusual chemical and physical properties, being composed of more than 40 per cent phosphoserine and 35 per cent aspartic acid (Munksgaard, Butler and Richardson, 1977). Several investigators (Veis and Perry, 1967; Nawrot et a/., 1976; Termine and Conn, 1976) have suggested that the dentine phosphoprotein plays a role in the processes which lead to the conversion of predentine to dentine as mineralization occurs. This is supported by the radioautographic studies of Weinstock and Leblond (1973). Their results suggest that the phosphoprotein of rat incisors is made by odontoblasts, passes quickly through predentine and is deposited at the predentine-dentine junction. Thus, deposition would be coincident with the mineralization process. However, an unequivocal identification of the molecules labelled by the radioisotopes was not determined. To investigate the biosynthesis and deposition of dentine matrix components in a manner which will utilize definitive biochemical techniques, we have developed an organ culture system employing odontoblasts attached to predentine and dentine. Mandibular incisors were removed from l&l l-week old male Sprague-Dawley rats and immediately placed in BGJ, culture medium (Grand Island Biological Co.) supplemented with 10 per cent foetal calf serum, 0.01 per cent glutamine and 1 per cent antibiotic-antimycotic solution. Under a stereomicroscope, the apical one-third of the tooth was carefully scraped free of soft pre-enamel and periodontal tissue and cut from the rest of the incisor. This apical segment was then opened by making a longitudinal incision on the lingual side, and the pulp was carefully removed. The dentine pieces were divided into two by a longitudinal incision at the labial side. Light microscopic sections of such pieces, fixed, demineralized and
stained (Weinstock and Leblond. 1973), showed parallel layers of intact odontoblasts still attached to predentine and dentine. Furthermore, odontoblasts were the major cell type present, although a few capillaries with associated endothelial cells could be seen between the odontoblasts. The odontoblasts attached to predentine-dentine pieces were incubated for 24 h in the culture medium with SO&i/ml each of E3H]-serine and [33P]-phosphate in the presence of SOpgg/ml of ascorbic acid. Incubation was at 37°C in 5 per cent CO, humid air. The cultured pieces were then washed with fresh medium and placed in 0.5 M EDTA, pH 7.4, with 5 mg of unlabelled phosphoprotein (Munksgaard et al., 1977). After 2 h at 24”C, the dentine pieces and cellular debris were removed by centrifugation; the resulting supernatant was dialysed exhaustively against water at 4”C, lyophilized and redissolved in 5.0 ml of 0.05 M tris-HCl buffer, pH 7.4. To determine whether the phosphoprotein had been synthesized by the odontoblasts, this material was chromatographed on DEAE-cellulose. A major radioactive component recovered by this procedure eluted at a position corresponding to carrier phosphoprotein and was labelled with both [33P]-phosphate and C3H]-serine (Fig. 1). The large [33P]-phosphate-labelled peak which eluted at 110-130ml was probably inorganic phosphate. The unidentified ultraviolet-absorbing peaks which eluted prior to 350ml must have originated from the medium and the tissue, because the phosphoprotein which was added prior to extraction with EDTA eluted as a single peak at an elution volume of 380-430ml when chromatographed alone under the same conditions (not shown). To confirm further the identity of the labelled phosphoprotein, the DEAE-cellulose peak eluting at 38&430ml (Fig. 1) was dialysed against water, lyophilized and redissolved in dilute HCl, pH 1.75. It was then rechromatographed on a sulphonated polystyrene column (Munksgaard et al., 1977). This procedure yields a highly pure phosphoprotein because, at pH 1.75, the phosphoprotein from rat dentine emerges in the void volume of the column whereas 583
584
E. C. Munksgaard,
100
200
W. S. Richardson,
300 EFFLUENT
III and W. T. Butler
400 VOLUME.ml
500
600
Fig. 1. Chromatography of proteins extracted from odontoblast cultures with EDTA. The sample was applied to a 1.5 x 10 cm column of DEAE-cellulose (Whatman DE-32, microgranular) and was eluted in 0.05 M tris-HCI buffer, pH 7.4, with a linear gradient from 0 to 0.6 M NaCl over a total volume of 800 ml. The flow rate was 50 ml/h and 5-ml fractions were collected. The effluent was monitored at 223 nm with a Beckman DB-GT spectrophotometer. 0.5-ml aliquots were taken, mixed with 5 ml of scintillation fluid (Scintiverse, Fisher Scientific) and counted with a liquid scintillation counter (Beckman Instruments, model LS-233) using a narrow tritium channel.
5
v--~~
1.2
--
%
ABSORBANCE j3,Pl SERINE PHOSPHATE [SHj
140 120 B
N. 0.6 t?
100 5 g
f
60
3
60
q
g
0.4
;! z 8 0
4o
si
20
$
0 5
IO
15 EFFLUENT
20 VOLUME.
25
30
ml
Fig. 2. Re-chromatography of the phosphoprotein peak from DEAE-cellulose (Fig. 1) on a 0.5 x 100 cm column of sulphonated polystyrene (AG 5OW-X2, Bio-Rad Laboratories). The column was equilibrated and eluted with dilute HCl, pH 1.75, by gravity flow. The effluent was monitored at 226nm and the l&ml fractions were assayed for radioactivity as described in Fig. 1.
adhering impurities are strongly bound to the resin (Munksgaard et al., 1977). The material synthesized by odontoblasts which was doubly labelled with C3H]-serine and [33P]-phosphate, co-chromatographed with carrier phosphoprotein (Fig. 2). Identical results were obtained using crude EDTA extract not chromatographed on DEAE-cellulose. When the extract was treated with 1 mg/ml of pepsin at pH 1.75 for 3 h at 24”C, the radioactive phosphoprotein emerged in the same position of the sulphonated polystyrene column as in Fig. 2 with no loss of radioactivity. Our data establish that the odontoblast-dentine cultures are viable and capable of synthesizing a phosphate- and serine-containing material which cochromatographs with unlabelled phosphoproteins isolated from rat dentine. This radioactive product is apparently resistant to pepsin treatment, as is the dentine phosphoprotein and phosvitin, another highlyphosphorylated protein (Clarke, 1973). In other experiments, we have shown that the odontoblast cultures synthesize and secrete collagen, and deposit it in predentine (E. C. Munksgaard, M. Rhodes, R. Mayne and W. T. Butler, unpublished). It thus appears that our culture system is capable of reproducing the biosynthetic in-vitro events which lead to the formation of predentine and its transformation to dentine. The system should prove valuable in
answering certain critical questions concerning the biosynthesis of the matrix components and how they are involved in dentinogenesis.
REFERENCES
Butler W. T., Finch J. E., Jr. and DeSteno C. V. 1972. Chemical character of proteins in rat incisors. Biochim. biophys. Acta 257, 167-171. Clark R. S. 1973. Amino acid sequence of a cyanogen bromide cleavage peptide from hens egg phosvitin. Eiochim. biophys. Acta 310, 174187. Munksgaard E. C., Butler W. T. and Richardson W. S., III. 1977. Phosphoprotein from dentin. New approaches to achieve and assess purity. Prep. Biochem. 7, 321-331. Nawrot C. F., Campbell D. J., Schroeder J. K. and Van Valkenburg M. 1976. Dental phosphoprotein-induced formation of hydroxylapatite during in-vitro synthesis of amorphous calcium phosphate. Biochemistry 15,
34453449. Richardson W. S., Beegle W. F., Butler W. T. and Munksgaard E. C. 1977. The phosphoprotein of rabbit incisors. J. dent. Rex 56, 233-237. Termine J. D. and Conn K. M. 1976. Inhibition of apatite formation by phosphorylated metabolites and macromolecules. Calc. Tiss. Res. 22, 149-157. Veis A. and Perry A. 1967. The phosphoprotein of dentin matrix. Biochemistry 6, 2409-2416.
Biosynthesis of phosphoprotein Veis A., Spector A. R. and Zamoscianyk H. 1972. The isolation of an EDTA-soluble phosphoprotein from mineralizing bovine dentin. Biochim. biophys. Acta 257, 404413.
0.“.
23 1
t
by odontoblasts
585
Weinstock M. and Leblond C. P. 1973. Radioautographic visualization of the deposition of a phosphoprotein at the mineralization front in the dentin of the rat incisor. J. Ceil Biol. 56, 838-845.
