- Email: [email protected]
Clonal dental pulp cells (RDP4-1, RPC-C2A) synthesize and secrete osteopontin (SPP1, 2ar)
Vol. 189, No. 2, 1992 December 15, 1992
CLONAL
BIOCHEMICAL
AND BIOPHYSICAL
DENTAL PULP CELLS (RDP4-1, AND SECRETE OSTEOPONTIN
Mika Yokota,
RESEARCH COMMUNICATIONS Pages 892-898
RPC-C2A) SYNTHESIZE (SPPl, 2ar)
Toshihiko Nagata, Hiroshi Ishida and Yoichi Wakano
Department of Periodontology and Endodontology, Tokushima University School of Dentistry, Tokushima 770, Tokushima, Japan Received
October
5,
1992
Dental pulp cells play an important role in maintaining dental mineralized tissue throughout life. Supplementary mineralization such as reparative dentin and pulp stone frequently occurs after primary dentin formation. Dental pulp cells are thought to be closely associated with such mineralization. We found that clonal rat dental pulp cells, RDP4-1 and RPC-C2A, produce and secrete osteopontin, but do not synthesize phosphophoryn which is a major noncollagenous protein found in dentin. The dental pulp osteopontin was highly phosphorylated and identified by thrombin susceptibility and immunoprecipitation with osteopontin/2ar antibody. Osteopontin synthesis markedly increased by 12-O-tetradecanoylphorbol- 13-acetate (TPA) as observed in many osteoblastic cells. This study indicates that these cells can produce osteopontin as a major phosphoprotein and suggests that the synthesis of osteopontin could be used as a characteristic marker of dental pulp cells. m 1992Academic Press,Inc.
Dental pulp, a primitive
loose connective tissue surrounded by dentin, has an
important lifelong role in maintaining dentin. Primary dentin is formed by odontoblasts, which are differentiated and aided by cells of the subodontoblastic layer in the dental pulp (1). The major noncollagenous protein in dentin is a phosphoprotein referred to as phosphophoryn (2) or highly phosphorylated phosphoprotein (PP-H) (3), which is dentin specific and synthesized by odontoblasts (4, 5). Although phosphophoryn is present in most primary dentin, it is not detected in reparative dentin which is frequently formed in the dental pulp in response to external stimuli after completion of the primary dentin (6). In general, supplementary mineralizations such as reparative dentin and pulp stone are thought to be associated not with odontoblasts but with other dental pulp cells (7, 8). However, the characteristics and functions of dental pulp cells during hard tissue formation are poorly understood mainly because of difficulties in culturing dental pulp cells. There is little information regarding the relationship between the dental pulp cells and the synthesis of phosphoprotein which is indispensable in the mineralization process (9). . . *SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; A.bbrewdua a-MEM, a-minimal essential medium; FBS, fetal bovine serum; BSA, bovine serum albumin; TPA, 12-0-tetradecanoylphorbol-13-acetate. 0006-291X/92
Copyright All righrs
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
892
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Clonal cell lines, RDP4-I and RPC-C2A, were derived from rat dental pulp from which the odontoblasts had been removed, and both lines reportedly exhibit high alkaline phosphatase activities (10, 11) which is regarded as a phenotypical marker of hard tissue forming cells (12). However, it is unclear whether these cells can produce phosphoproteins. RPC-C2A,
In this study, we found that the clonal dental pulp cells, RDP61 and
synthesize and secrete osteopontin
(SPPl, 2ar), which is a highly
phosphorylated noncollagenous protein associated with bone metabolism (13, 14, 15).
MATERIALS
AND METHODS
Materials: Culture media, a-MEM and Eagle’s MEM were purchased from Flow Laboratories (McLean, VA) and Nissui (Tokyo, Japan). Fetal bovine serum (FBS) was from Whittacker Bioproducts (Walkersville, MD). Bovine thrombin was from Sigma Chemical Co. (St.Louis, MO). L-Ascorbic acid phosphate magnesium salt n-hydrate and 12-O-tetradecanoylphorbol- 13-acetate (TPA) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Column PD-10 containing Sephadex G-25M was from Pharmacia (Uppsala, Sweden). The radioisotopes, H3[32P04], (21 mCi/ml, carrier free) and [s?j]methionine (1300 Ci/mmol) were from the Japan Energy Research Institute (Tokyo, Japan) and Amersham (Bucks, UK), respectively. Mouse monoclonal antibody to rat osteopontin was purchased from The Developmental Studies Hybridoma Bank (Iowa City, IA). Other materials used were commercial products of the highest grade available. Cell Culture: The established clonal dental pulp cell lines, RDP4-1 and RPC-C2A, were gifts from Drs. T. Kawase (10) and S. Kasugai (1 l), respectively. RDP61 cells were plated in a-MEM containing 10% FBS and antibiotics (100 U/ml penicillin G, 100 pg/ml streptomycin and 300 rug/ml amphotericin B) at 4 x lo4 cells/35mm dish. RPC-C2A cells were plated in Eagle’s MEM containing 10% FBS, 50 pig/ml ascorbic and 60 @ml kanamycin at 3 x 104 cells/35mm dish. ROS17/2.8 cells, purchased from the RGB Cell Bank (Tsukuba, Japan), were also used as a positive control cell line that synthesizes osteopontin (16). These cells were maintained at 37 “C in a humidified air/C02 (19: 1) atomosphere. The medium were changed routinely every second day. Metabolic Labeling: The proteins synthesized by the dental pulp cells were studied by radiolabeling the cells with 100 pCi/ml [s*PO4] or 100 pCi/ml [s5S]methionine. After 5 days in culture, confluent RDP6 1 cells were preincubated in o(-MEM containing 0.5% FBS for 6 h. The cells were then washed with phosphate buffered saline (PBS) and the radioisotope was added in fresh medium containing 0.5% FBS and antibiotics. RPC-C2A cells were incubated for 6 h on 15 day when alkaline phosphatase activity was almost peaked (1 l), followed by radioisotope labeling with [32P04] or [35S]methionine for 24 h in phosphate-free or methionine-free Eagle’s MEM respectively under the usual culture conditions. After labeling, the media were collected, desalted by gel chromatography on Column PD-10, freeze-dried then analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Some samples were followed by thrombin digestion or immunoprecipitation before applying to SDS-PAGE. Thrombin Digestion and Immunoprecipitation: Freeze dried portions of media were digested with thrombin at 37°C for 30 min in 6 pl of 10 mM Tris/HCl buffer, pH 8.0, containing 10 mM CaC12 and 1 unit of thrombin. Digestion was terminated by adding 6 pl of 4-fold-concentrated SDS-PAGE sample buffer containing 60 mg/ml dithiothreitol. Radiolabeled proteins were immunoprecipitated with a specific antibody to osteopontin/2ar by a procedure described in detail previously (17). Briefly, freeze-dried samples were dissolved in 200 pl of immunoprecipitation buffer (0.3% v/v Nonidet P-40, 0.3% v/v sodium deoxycholate, 0.1% w/v BSA in Tris/HCl buffered saline, 0.02% w/v sodium azide) in a 1.5 ml microfuge tube and incubated with 100 p1 of pre-washed Pansorbin for 2h. Simultaneously, 1.O pl of the primary antibody was coupled to 100 pl of Protein A-Sepharose. The Pansorbin was removed and the radiolabeled proteins were incubated with the primary antibody for 24 h at 4°C with gentle shaking. The radiolabeled proteins bound to the antibody were washed four times with PBSnween (PBS containing 893
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189,
No.
2,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Tween-20 and 0.1% BSA in the first three washes) then dissolved in 12 yl of SDSPAGE sample buffer containing dithiothreitol. Polyacryiamide Gel Electrophoresis: SDS-PAGE of proteins on mini-slab gels using the discontinuous Tris/glycine buffer system with 10 and 15% cross-linked linear gels was performed as described previously (18). Samples were dissolved in 10 pl sample buffer containing 1% SDS, 2 M urea and Bromophenol Blue dye. When proteins were analysed under reducing conditions, 150 pg of dithiothreitol was included. Samples were heated to 56 “C for 25 min and cooled immediately before being added to individual wells. Electrophoresis proceeded for 1h at 150 V. After separation, proteins were detected by autoradiography for [32P04], or fluorography for [35S]methionine, on FujiRX film. 0.5%
RESULTS
Phosphorylated proteins from the media of RDP4-1 and RPC-C2A cells were separated by SDS-PAGE on 10% cross-linked gels as shown in Fig.1. The [32P04]labeled analysis revealed a broad band between 50 and 70 kDa both in RDP4- 1 and RPCC2A cells. In addition to these, sharp bands were observed at 44 and 40 kDa in RDP4-1 and RPCC2A cells, respectively. There was no phosphophoryn-like band at the position of -90 kDa as reported previously (19, 20). The mobility of the major phosphorylated proteins remained the same under reducing and nonreducing conditions (not shown). The main band at 50-70 kDa on the 10% cross-linked gel shifted to 50 kDa on the 15% gel in RDP4-1 cells showing anomalous behavior (Fig. 2A). The [32P04]-labeled proteins were characterized by thrombin digestion. It has been reported that thrombin susceptibility is a characteristic of osteopontin (21). As shown in Fig. 2A, the 50 kDa band disappeared and 32 and 28 kDa fragments appeared on the 15% cross-linked gel in RDP4-1 cells. A
-Analysis of newly synthesized phosphoproteins in clonal dental DUID cells. RDP4-1 and RPC-CZA. Confluent RDP4-I and RPC-C2A cells were ia&led with 100 @i/ml [32PO4] for 24 h. Aliquots of the culture media were analysed by SDS-PAGE on 10% cross-linked minigel under reducing conditions, then the gel was processed for autoradiography. MW, molecular weight markers. The marker sizes are shown in kDa on the left. The arrows indicate the position of the major phosphorylated
protein.
894
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189, No. 2, 1992
RDP4-
A
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A
1 kDa
2
1
kDa
BIOCHEMICAL
kh
B
[3*PO4j 1
2
kDa
200 m
200 w 97 ) 69 * 46 )
[35S]-Met 1
200~ 97 *
97,
69 *
69,
46 *
30 e
46, i6-
02 Is*
30,
Fie.
Thrombin
03
digestion
and immunoprecipitation
of osteopontin.
RDP4-I and RPC-CZA cells were cultured for 24 h in the presence of 100 @i/ml [32P04]. (A)The radiolabeled proteins in the medium from RDP4-I cells (lane I) were digested with thrombin (lane 2) resulting in the loss of the 50 kDa band and the appearance of 32/28 kDa bands on 15% gel as shown by arrows. (B) Proteins labeled with [32P04] were immunoprecipitated using monoclonal antibody to rat osteopontin/2ar and visualized on a 10% gel. ROSl7/2.8 cells were used as a positive osteopontinsecreting control. Osteopontin secreted by RDP4-I cells migrated as a broad band at 58 kDa (arrow). Two forms of osteopontin, 69 and 60 kDa, were observed in RPC-C2A cells (arrows).
Fie. 3, Effect of TPA on phosphorylation of osteopontin and synthesis of osteopontin protein. RPC-C2A cells were radiolabeled with 100 pCi/ml [j2P04] (A) or 100 pCi/ml [35S]methionine (B) in the presence of IO @ml TPA (lane 2) or vehicle alone (lane I) for 24 h. Media labeled with [32PO4] were analysed by SDS-PAGE on 10% gels and the amount loaded onto each lane was normalized to equal 33,000 cells (A). Media labeled with [35S]methionine corresponding to 83,000 cells were immunoprecipitated with osteopontin antibody and the immunoprecipitated proteins were analysed on 10% SDS-PAGE gels (B). Radioactive bands were revealed by autoradiography for [32PO4] or fluorography for [3sS]methionine. Arrows indicate two forms of osteopontin labeled by [3sS]methionine. TPA increased the levels of both phosphorylated and non phosphorylated osteopontin.
similar
tendency
completely digested
was also observed
digested by thrombin
by thrombin and produced
in RPC-C2A cells, though
(not shown).
the protein
Thus a major phosphorylated
a 32/28 kDa doublet,
demonstrating
was not
protein
was
that the 50 kDa
protein on the 15% gel (SO-70 kDa on 10% gel) was osteopontin. To confirm this, the protein was immunoprecipitated using a monoclonal antibody to rat osteoponM2ar. Phosphorylated osteopontin was identified in the media of RDPb1 and RCP-C2A cells showing clear bands with almost the same migration as the osteopontin in ROS17/2.8 cells on 10% cross-linked gels (Fig. 2B). Notably, two major phosphorylated forms of osteopontin from RPC-C2A cells migrated at 69 and 60 kDa on 10% cross-linked gels. Osteopontin from RDP4-1 cells was detected as a single 58 kDa band which was slightly smaller than that from RPC-C2A cells as shown in Figs. 1 and 2B. Since osteopontin is induced by TPA (22), its effects upon osteopontin synthesis in dental pulp cells was examined. TPA at 10 rig/ml increased the intensity of the [32P04]-labeled bands from RPC-C2A cells. As shown in Fig. 3A, the intensity of two major phosophoprotein bands, one which migrated broadly at 50-70 kDa containing two forms of osteopontin and the other at 40 kDa which was not immunoprecipitated with osteopontin antibody, 895
2
Vol. 189, No. 2, 1992
BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS
proteins were were both enhanced by TPA. When [3sS]methionine-labeled immunoprecipitated, two forms of osteopontin at 69 and 60 kDa on 10% SDS-PAGE were revealed. Also, osteopontin synthesis was promoted by TPA (Fig. 3B). Though intense bands at -97 and -200 kDa, as well as several minor bands were also present in the immunoprecipitate, they were not affected by TPA.
DISCUSSION Although RDP4-1 and RPC-C2A are clonal cell lines of rat dental pulp exhibiting high alkaline phosphatase activity, whether these cells can be used to investigate the biological characteristics of dental pulp or even dentinogenesis is not well documented. One of the reasons for this is the lack of information about matrix protein components synthesized by dental pulp cells other than odontoblasts in order to explain the mechanism of hard tissue formation such as reparative dentin or pulp stone. It is understood that primary dental pulp cells synthesize collagen, proteoglycans and glycoproteins (23, 24, 25), but few studies have been reported regarding the formation of other non-collagenous proteins essential for hard tissue formation as well as its mineralization by either primary or clonal pulp cells. The results presented here indicate that both clonal dental pulp cell types synthesize and secrete phosphoprotein, which was revealed to be osteopontin and not phosphophoryn.
Our data is consistent with a histochemical
study indicating that
phosphophoryn is not detectable in reparative dentin (6). Since osteopontin is associated with mineralized tissue formation (13, 14), the feature of producing osteopontin may be regarded as an important differential marker of dental pulp cells. It is also possible that osteopontin secreted by dental pulp cells acts as a major phosphoprotein during the supplementary mineralization process. Recently Kohri et al. (26) identified osteopontin in urinary stones by means of cDNA sequencing, suggesting that osteopontin is involved in stone formation as a matrix. This fact indicates the association of osteopontin with morbid mineralization such as pulp stone. Although osteopontin is synthesized by various nonosteogenic and osteogenic cell populations (27) the protein in dental pulp must be an important factor in supplementary mineralization including reparative dentin and pulp stone. In this study, osteopontin migrated as a SO-70 kDa broad band on a 10% cross-linked gel (Fig.
l),
and two osteopontins
at 69 and 60 kDa were identified
by
immunoprecipitation in RPC-C2A cells (Fig. 2B). The reason why these two bands cannot be recognized in Fig. 1 is presumably due to the overlapping of the proteins labeled with [32PO4] on the autoradiograph. The difference in the molecular mass between dental pulp osteopontin and that previously reported may be due to species variation or the degree of post-translational modifications such as phosphorylation and sulfation (28,29). In fact, the protein of RDP4-1 cells was sulfated (data not shown). The band at 50-70 kDa on 10% gel shifted to 50 kDa on 15% gel in RDP4-1 cells (Figs. 1 and 2A). This variable mobility of osteopontin on different gels is consistent with the previously reported
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
anomalous behaviour of rat osteopontin (30, 3 1). Thrombin susceptibility is reportedly a characteristic of osteopontin (21). Phosphorylated osteopontin was degraded by thrombin digestion completely in RDPb1 cells and partially in RPC-C2A cells, indicating that these proteins were osteopontin.
Furthermore, the digestion fragments were observed as a
32/28 kDa doublet on 15% gels (Fig. 2A). A similar digestion profile was observed using rat calvarial cells, which form mineralized bone nodules (14). TPA, a potent tumor promoter, reportedly enhances the synthesis of osteopontin (22, 32). TPA increased the level of both phosphorylated and non phosphorylated osteopontin in RPC-C2A cells (Fig. 3). Although levels of a 40 kDa phosphorylated protein were also increased by TPA, this protein was not a fragment of osteopontin since it was not immunoprecipitated
with
osteopontin antibody. Several bands immunoprecipitated by the antibody were observed in [35S]methionine labeled medium (Fig. 3B).
These may include fibronectin
co-
migrating with osteopontin, a 92.5 kDa protein, and degradation products of osteopontin recognized by the antibody (32,33). Osteopontin is also synthesized by odontoblasts and secreted into predentin (34). However, osteopontin
Bronckers antibody
et al. (35) have reported that immunohistochemistry revealed faint immunostaining
with
in some but not all young
odontoblasts before formation of the primary dentin, and in predentin but not in mature odontoblasts or dentin. Thus, we speculate that osteopontin has an additional role to that of phosphophoryn during primary dentin formation and that it plays an important role as a major phosphoprotein during the supplementary mineralization that occurs as a response to external factors such as thermal, physical, or carious irritation.
ents;
We thank Drs. T. Kawase and S. Kasugai for providing the rat
clonal dental pulp cell lines, RDP4- 1 and RPC-C2A.
REFERENCES 1. 2. 3. 4. 5.
Linde, A. (1985) J. Dent. Res. 64, 523-529. Stetler-Stevenson, W.G. and Veis, A. (1983) Biochemistry 22, 4326-4335. Linde, A. (1989) Anatom. Rec. 224, 154- 166. Weinstock, M. and Leblond, C.P. (1973) J. Cell Biol. 56, 838-845. MacDougall, M., Zeichner-David, M. and Slavkin, H.C. (1989) Connect. Tiss. Res. 22, 71-77. 6. Takagi, Y. and Sasaki, S. (1986) J. Oral Pathol. 15, 463-467. 7. Trowbridge, H.O. and Kim, S. (1991) In Pathway of the Pulp (S. Cohen and R.C. Bums Ed.), ~~308-349, Mosby, St.Louis. 8. Smulson, M.H. and Sieraski, S.M. (1989) In Endodontic Therapy (F.S.Weine Ed), 74-153, Mosby, St.Louis. 9. Glimcher, M.J. (1990) In Maetabolic Bone Disease (L.V. Avioli and S.M.K. Krane, Ed.), ~~42-68, W.B. Saunders Comp., Philadelphia. 10. Kawase, T., Orikasa, M. and Suzuki, A. (1990) Bone and Mineral 11, 163-175. 11. Kasugai, S., Adachi, M. and Ogura, H. (1988) Archs. Oral. Biol. 33, 887-891. 12. Wuthier, R.E. and Register, T.C. (1985) In The Chemistry and Biology of Mineralized Tissues (W.T. Butler Ed.), pp 113- 124, EBSCO Media, Birmingham. 13. Nagata, T., Goldberg, H.A., Zhang, Q., Domenicucci, C. and Sodek, J. (1991) Matrix 11, 86-100.
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14. Nagata, T., Bellows, C.G., Kasugai, S., Butler, W.T. and Sodek, J. (1991) Biochem. J. 274, 513-520. 15. Reinholt, F.P., Hultenby, K. and Heinegard, D. (1990) Proc. Natl. Acad. Sci. USA. 87, 4473-4475. 16. Noda, M., Yoon, K., Prince, C.W., Butler, W.T. and Rodan, G.A. (1988) J. Biol. Chem. 263, 13916-13921. 17. Wrana, J.L., Maeno, M., Hawrylyshyn, B., Yao, K.-L., Domenicucci, C. and Sodek, J. (1988) J. Cell Biol. 106, 915-924. 18. Overall, C.M., Wrana, J.L. and Sodek, J. (1989) J. Biol. Chem. 264, 1860-1869. 19. DiMuzio, M.T., Bhown, M., Walton, R.K. and Butler, W.T. (1985) In The Chemistry and Biology of Mineralized Tissues (W.T. Butler, Ed.), ~~296-302, EBSCO Media, Birmingham. 20. Rahima, M.M., Sabsay, B., Wu, C.B. and Veis, A. (1989) Connect. Tiss. Res. 22, 79-90. 21. Senger, D.R., Peruzzi, CA., Gracey, C.F., Papadopoulos, A. and Tenen, D.G. (1988) Cancer Res. 48, 5770-5774. 22. Craig, A.,M., Smith, J.H. and Denhardt, D.T. (1989) J. Biol. Chem. 264, 96829689. 23. Shuttleworth, C.A. (1990) In Dynamic Aspects of Dental Pulp, (R. Inoki, T. Kudo and M. Olgart, Ed.), ~~239-257, Chapman and Hall, London. 24. Shuttleworth, C.A., Berry, L., Bloxsome, C. and Wilson, N.H.F. (1982) Archs. Oral Biol. 27, 729-733. 25. Nagata, T., Kido, J., Hamasaki, A., Ishida, H. and Wakano, Y. (1991) J. Endodon. 17, 594-597. 26. Kohri, K., Suzuki, Y., Yoshida, K., Yamamoto, K., Amasaki, N., Yamate,.T., Umekawa, T., Iguchi, M., Shinohara, H. and Kurita, T. (1992) Biochem. Biophys. Res. Commun. 184, 859-864. 27. Butler, W.T. (1991) J. Biol. Buccale. 19, 83-89. 28. Nagata, T., Todescan, R., Goldberg, H.A., Zhang, Q. and Sodek, J. (1989) Biochem. Biophys. Res. Commun. 165, 234-240. 29. Singh, K., DeVouge, M. and Mukherjee, B.B. (1990) J. Biol. Chem. 265, 1869618701. 30. Prince, C.W., Oosawa, T., Butler, W.T., Tomana, M., Bhown, A.S., Bhown, M. and Schrohenloher, R.E. (1986) J. Biol. Chem. 262, 2900-2907. 31. Kubota, T., Zhang, Q,, Wrana, J.L., Ber, R., Aubin, J.E., Butler, W.T. and Sodek, J. (1989) Biochem. Biophys. Res. Commun. 162, 1453-1459. 32. Kasugai, S., Zhang, Q., Overall, C.M., Wrana, J.L., Butler, W.T. and Sodek, J. (1991) Bone and Mineral. 13,235-250. 33. Nemir, M., DeVouge, M.W. and Mukherjee, B.B. (1989) J. Biol. Chem. 264, 18202- 18208. 34. Mark, M.P., Butler, W.T., Prince, C.W., Finkelman, R.D. and Ruth, J.-V. (1988) Differentiation 37, 123- 136. 35. Bronckers, A.L.J.J., Lyaruu, D.M. and Woltgens, J.H.M. (1989) Connect. Tiss. Res. 22, 65-70.
898
CLONAL
BIOCHEMICAL
AND BIOPHYSICAL
DENTAL PULP CELLS (RDP4-1, AND SECRETE OSTEOPONTIN
Mika Yokota,
RESEARCH COMMUNICATIONS Pages 892-898
RPC-C2A) SYNTHESIZE (SPPl, 2ar)
Toshihiko Nagata, Hiroshi Ishida and Yoichi Wakano
Department of Periodontology and Endodontology, Tokushima University School of Dentistry, Tokushima 770, Tokushima, Japan Received
October
5,
1992
Dental pulp cells play an important role in maintaining dental mineralized tissue throughout life. Supplementary mineralization such as reparative dentin and pulp stone frequently occurs after primary dentin formation. Dental pulp cells are thought to be closely associated with such mineralization. We found that clonal rat dental pulp cells, RDP4-1 and RPC-C2A, produce and secrete osteopontin, but do not synthesize phosphophoryn which is a major noncollagenous protein found in dentin. The dental pulp osteopontin was highly phosphorylated and identified by thrombin susceptibility and immunoprecipitation with osteopontin/2ar antibody. Osteopontin synthesis markedly increased by 12-O-tetradecanoylphorbol- 13-acetate (TPA) as observed in many osteoblastic cells. This study indicates that these cells can produce osteopontin as a major phosphoprotein and suggests that the synthesis of osteopontin could be used as a characteristic marker of dental pulp cells. m 1992Academic Press,Inc.
Dental pulp, a primitive
loose connective tissue surrounded by dentin, has an
important lifelong role in maintaining dentin. Primary dentin is formed by odontoblasts, which are differentiated and aided by cells of the subodontoblastic layer in the dental pulp (1). The major noncollagenous protein in dentin is a phosphoprotein referred to as phosphophoryn (2) or highly phosphorylated phosphoprotein (PP-H) (3), which is dentin specific and synthesized by odontoblasts (4, 5). Although phosphophoryn is present in most primary dentin, it is not detected in reparative dentin which is frequently formed in the dental pulp in response to external stimuli after completion of the primary dentin (6). In general, supplementary mineralizations such as reparative dentin and pulp stone are thought to be associated not with odontoblasts but with other dental pulp cells (7, 8). However, the characteristics and functions of dental pulp cells during hard tissue formation are poorly understood mainly because of difficulties in culturing dental pulp cells. There is little information regarding the relationship between the dental pulp cells and the synthesis of phosphoprotein which is indispensable in the mineralization process (9). . . *SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; A.bbrewdua a-MEM, a-minimal essential medium; FBS, fetal bovine serum; BSA, bovine serum albumin; TPA, 12-0-tetradecanoylphorbol-13-acetate. 0006-291X/92
Copyright All righrs
$4.00
0 1992 by Academic Press, of reproduction in any form
Inc. reserved.
892
Vol.
189, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Clonal cell lines, RDP4-I and RPC-C2A, were derived from rat dental pulp from which the odontoblasts had been removed, and both lines reportedly exhibit high alkaline phosphatase activities (10, 11) which is regarded as a phenotypical marker of hard tissue forming cells (12). However, it is unclear whether these cells can produce phosphoproteins. RPC-C2A,
In this study, we found that the clonal dental pulp cells, RDP61 and
synthesize and secrete osteopontin
(SPPl, 2ar), which is a highly
phosphorylated noncollagenous protein associated with bone metabolism (13, 14, 15).
MATERIALS
AND METHODS
Materials: Culture media, a-MEM and Eagle’s MEM were purchased from Flow Laboratories (McLean, VA) and Nissui (Tokyo, Japan). Fetal bovine serum (FBS) was from Whittacker Bioproducts (Walkersville, MD). Bovine thrombin was from Sigma Chemical Co. (St.Louis, MO). L-Ascorbic acid phosphate magnesium salt n-hydrate and 12-O-tetradecanoylphorbol- 13-acetate (TPA) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Column PD-10 containing Sephadex G-25M was from Pharmacia (Uppsala, Sweden). The radioisotopes, H3[32P04], (21 mCi/ml, carrier free) and [s?j]methionine (1300 Ci/mmol) were from the Japan Energy Research Institute (Tokyo, Japan) and Amersham (Bucks, UK), respectively. Mouse monoclonal antibody to rat osteopontin was purchased from The Developmental Studies Hybridoma Bank (Iowa City, IA). Other materials used were commercial products of the highest grade available. Cell Culture: The established clonal dental pulp cell lines, RDP4-1 and RPC-C2A, were gifts from Drs. T. Kawase (10) and S. Kasugai (1 l), respectively. RDP61 cells were plated in a-MEM containing 10% FBS and antibiotics (100 U/ml penicillin G, 100 pg/ml streptomycin and 300 rug/ml amphotericin B) at 4 x lo4 cells/35mm dish. RPC-C2A cells were plated in Eagle’s MEM containing 10% FBS, 50 pig/ml ascorbic and 60 @ml kanamycin at 3 x 104 cells/35mm dish. ROS17/2.8 cells, purchased from the RGB Cell Bank (Tsukuba, Japan), were also used as a positive control cell line that synthesizes osteopontin (16). These cells were maintained at 37 “C in a humidified air/C02 (19: 1) atomosphere. The medium were changed routinely every second day. Metabolic Labeling: The proteins synthesized by the dental pulp cells were studied by radiolabeling the cells with 100 pCi/ml [s*PO4] or 100 pCi/ml [s5S]methionine. After 5 days in culture, confluent RDP6 1 cells were preincubated in o(-MEM containing 0.5% FBS for 6 h. The cells were then washed with phosphate buffered saline (PBS) and the radioisotope was added in fresh medium containing 0.5% FBS and antibiotics. RPC-C2A cells were incubated for 6 h on 15 day when alkaline phosphatase activity was almost peaked (1 l), followed by radioisotope labeling with [32P04] or [35S]methionine for 24 h in phosphate-free or methionine-free Eagle’s MEM respectively under the usual culture conditions. After labeling, the media were collected, desalted by gel chromatography on Column PD-10, freeze-dried then analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Some samples were followed by thrombin digestion or immunoprecipitation before applying to SDS-PAGE. Thrombin Digestion and Immunoprecipitation: Freeze dried portions of media were digested with thrombin at 37°C for 30 min in 6 pl of 10 mM Tris/HCl buffer, pH 8.0, containing 10 mM CaC12 and 1 unit of thrombin. Digestion was terminated by adding 6 pl of 4-fold-concentrated SDS-PAGE sample buffer containing 60 mg/ml dithiothreitol. Radiolabeled proteins were immunoprecipitated with a specific antibody to osteopontin/2ar by a procedure described in detail previously (17). Briefly, freeze-dried samples were dissolved in 200 pl of immunoprecipitation buffer (0.3% v/v Nonidet P-40, 0.3% v/v sodium deoxycholate, 0.1% w/v BSA in Tris/HCl buffered saline, 0.02% w/v sodium azide) in a 1.5 ml microfuge tube and incubated with 100 p1 of pre-washed Pansorbin for 2h. Simultaneously, 1.O pl of the primary antibody was coupled to 100 pl of Protein A-Sepharose. The Pansorbin was removed and the radiolabeled proteins were incubated with the primary antibody for 24 h at 4°C with gentle shaking. The radiolabeled proteins bound to the antibody were washed four times with PBSnween (PBS containing 893
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Tween-20 and 0.1% BSA in the first three washes) then dissolved in 12 yl of SDSPAGE sample buffer containing dithiothreitol. Polyacryiamide Gel Electrophoresis: SDS-PAGE of proteins on mini-slab gels using the discontinuous Tris/glycine buffer system with 10 and 15% cross-linked linear gels was performed as described previously (18). Samples were dissolved in 10 pl sample buffer containing 1% SDS, 2 M urea and Bromophenol Blue dye. When proteins were analysed under reducing conditions, 150 pg of dithiothreitol was included. Samples were heated to 56 “C for 25 min and cooled immediately before being added to individual wells. Electrophoresis proceeded for 1h at 150 V. After separation, proteins were detected by autoradiography for [32P04], or fluorography for [35S]methionine, on FujiRX film. 0.5%
RESULTS
Phosphorylated proteins from the media of RDP4-1 and RPC-C2A cells were separated by SDS-PAGE on 10% cross-linked gels as shown in Fig.1. The [32P04]labeled analysis revealed a broad band between 50 and 70 kDa both in RDP4- 1 and RPCC2A cells. In addition to these, sharp bands were observed at 44 and 40 kDa in RDP4-1 and RPCC2A cells, respectively. There was no phosphophoryn-like band at the position of -90 kDa as reported previously (19, 20). The mobility of the major phosphorylated proteins remained the same under reducing and nonreducing conditions (not shown). The main band at 50-70 kDa on the 10% cross-linked gel shifted to 50 kDa on the 15% gel in RDP4-1 cells showing anomalous behavior (Fig. 2A). The [32P04]-labeled proteins were characterized by thrombin digestion. It has been reported that thrombin susceptibility is a characteristic of osteopontin (21). As shown in Fig. 2A, the 50 kDa band disappeared and 32 and 28 kDa fragments appeared on the 15% cross-linked gel in RDP4-1 cells. A
-Analysis of newly synthesized phosphoproteins in clonal dental DUID cells. RDP4-1 and RPC-CZA. Confluent RDP4-I and RPC-C2A cells were ia&led with 100 @i/ml [32PO4] for 24 h. Aliquots of the culture media were analysed by SDS-PAGE on 10% cross-linked minigel under reducing conditions, then the gel was processed for autoradiography. MW, molecular weight markers. The marker sizes are shown in kDa on the left. The arrows indicate the position of the major phosphorylated
protein.
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A
1 kDa
2
1
kDa
BIOCHEMICAL
kh
B
[3*PO4j 1
2
kDa
200 m
200 w 97 ) 69 * 46 )
[35S]-Met 1
200~ 97 *
97,
69 *
69,
46 *
30 e
46, i6-
02 Is*
30,
Fie.
Thrombin
03
digestion
and immunoprecipitation
of osteopontin.
RDP4-I and RPC-CZA cells were cultured for 24 h in the presence of 100 @i/ml [32P04]. (A)The radiolabeled proteins in the medium from RDP4-I cells (lane I) were digested with thrombin (lane 2) resulting in the loss of the 50 kDa band and the appearance of 32/28 kDa bands on 15% gel as shown by arrows. (B) Proteins labeled with [32P04] were immunoprecipitated using monoclonal antibody to rat osteopontin/2ar and visualized on a 10% gel. ROSl7/2.8 cells were used as a positive osteopontinsecreting control. Osteopontin secreted by RDP4-I cells migrated as a broad band at 58 kDa (arrow). Two forms of osteopontin, 69 and 60 kDa, were observed in RPC-C2A cells (arrows).
Fie. 3, Effect of TPA on phosphorylation of osteopontin and synthesis of osteopontin protein. RPC-C2A cells were radiolabeled with 100 pCi/ml [j2P04] (A) or 100 pCi/ml [35S]methionine (B) in the presence of IO @ml TPA (lane 2) or vehicle alone (lane I) for 24 h. Media labeled with [32PO4] were analysed by SDS-PAGE on 10% gels and the amount loaded onto each lane was normalized to equal 33,000 cells (A). Media labeled with [35S]methionine corresponding to 83,000 cells were immunoprecipitated with osteopontin antibody and the immunoprecipitated proteins were analysed on 10% SDS-PAGE gels (B). Radioactive bands were revealed by autoradiography for [32PO4] or fluorography for [3sS]methionine. Arrows indicate two forms of osteopontin labeled by [3sS]methionine. TPA increased the levels of both phosphorylated and non phosphorylated osteopontin.
similar
tendency
completely digested
was also observed
digested by thrombin
by thrombin and produced
in RPC-C2A cells, though
(not shown).
the protein
Thus a major phosphorylated
a 32/28 kDa doublet,
demonstrating
was not
protein
was
that the 50 kDa
protein on the 15% gel (SO-70 kDa on 10% gel) was osteopontin. To confirm this, the protein was immunoprecipitated using a monoclonal antibody to rat osteoponM2ar. Phosphorylated osteopontin was identified in the media of RDPb1 and RCP-C2A cells showing clear bands with almost the same migration as the osteopontin in ROS17/2.8 cells on 10% cross-linked gels (Fig. 2B). Notably, two major phosphorylated forms of osteopontin from RPC-C2A cells migrated at 69 and 60 kDa on 10% cross-linked gels. Osteopontin from RDP4-1 cells was detected as a single 58 kDa band which was slightly smaller than that from RPC-C2A cells as shown in Figs. 1 and 2B. Since osteopontin is induced by TPA (22), its effects upon osteopontin synthesis in dental pulp cells was examined. TPA at 10 rig/ml increased the intensity of the [32P04]-labeled bands from RPC-C2A cells. As shown in Fig. 3A, the intensity of two major phosophoprotein bands, one which migrated broadly at 50-70 kDa containing two forms of osteopontin and the other at 40 kDa which was not immunoprecipitated with osteopontin antibody, 895
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proteins were were both enhanced by TPA. When [3sS]methionine-labeled immunoprecipitated, two forms of osteopontin at 69 and 60 kDa on 10% SDS-PAGE were revealed. Also, osteopontin synthesis was promoted by TPA (Fig. 3B). Though intense bands at -97 and -200 kDa, as well as several minor bands were also present in the immunoprecipitate, they were not affected by TPA.
DISCUSSION Although RDP4-1 and RPC-C2A are clonal cell lines of rat dental pulp exhibiting high alkaline phosphatase activity, whether these cells can be used to investigate the biological characteristics of dental pulp or even dentinogenesis is not well documented. One of the reasons for this is the lack of information about matrix protein components synthesized by dental pulp cells other than odontoblasts in order to explain the mechanism of hard tissue formation such as reparative dentin or pulp stone. It is understood that primary dental pulp cells synthesize collagen, proteoglycans and glycoproteins (23, 24, 25), but few studies have been reported regarding the formation of other non-collagenous proteins essential for hard tissue formation as well as its mineralization by either primary or clonal pulp cells. The results presented here indicate that both clonal dental pulp cell types synthesize and secrete phosphoprotein, which was revealed to be osteopontin and not phosphophoryn.
Our data is consistent with a histochemical
study indicating that
phosphophoryn is not detectable in reparative dentin (6). Since osteopontin is associated with mineralized tissue formation (13, 14), the feature of producing osteopontin may be regarded as an important differential marker of dental pulp cells. It is also possible that osteopontin secreted by dental pulp cells acts as a major phosphoprotein during the supplementary mineralization process. Recently Kohri et al. (26) identified osteopontin in urinary stones by means of cDNA sequencing, suggesting that osteopontin is involved in stone formation as a matrix. This fact indicates the association of osteopontin with morbid mineralization such as pulp stone. Although osteopontin is synthesized by various nonosteogenic and osteogenic cell populations (27) the protein in dental pulp must be an important factor in supplementary mineralization including reparative dentin and pulp stone. In this study, osteopontin migrated as a SO-70 kDa broad band on a 10% cross-linked gel (Fig.
l),
and two osteopontins
at 69 and 60 kDa were identified
by
immunoprecipitation in RPC-C2A cells (Fig. 2B). The reason why these two bands cannot be recognized in Fig. 1 is presumably due to the overlapping of the proteins labeled with [32PO4] on the autoradiograph. The difference in the molecular mass between dental pulp osteopontin and that previously reported may be due to species variation or the degree of post-translational modifications such as phosphorylation and sulfation (28,29). In fact, the protein of RDP4-1 cells was sulfated (data not shown). The band at 50-70 kDa on 10% gel shifted to 50 kDa on 15% gel in RDP4-1 cells (Figs. 1 and 2A). This variable mobility of osteopontin on different gels is consistent with the previously reported
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anomalous behaviour of rat osteopontin (30, 3 1). Thrombin susceptibility is reportedly a characteristic of osteopontin (21). Phosphorylated osteopontin was degraded by thrombin digestion completely in RDPb1 cells and partially in RPC-C2A cells, indicating that these proteins were osteopontin.
Furthermore, the digestion fragments were observed as a
32/28 kDa doublet on 15% gels (Fig. 2A). A similar digestion profile was observed using rat calvarial cells, which form mineralized bone nodules (14). TPA, a potent tumor promoter, reportedly enhances the synthesis of osteopontin (22, 32). TPA increased the level of both phosphorylated and non phosphorylated osteopontin in RPC-C2A cells (Fig. 3). Although levels of a 40 kDa phosphorylated protein were also increased by TPA, this protein was not a fragment of osteopontin since it was not immunoprecipitated
with
osteopontin antibody. Several bands immunoprecipitated by the antibody were observed in [35S]methionine labeled medium (Fig. 3B).
These may include fibronectin
co-
migrating with osteopontin, a 92.5 kDa protein, and degradation products of osteopontin recognized by the antibody (32,33). Osteopontin is also synthesized by odontoblasts and secreted into predentin (34). However, osteopontin
Bronckers antibody
et al. (35) have reported that immunohistochemistry revealed faint immunostaining
with
in some but not all young
odontoblasts before formation of the primary dentin, and in predentin but not in mature odontoblasts or dentin. Thus, we speculate that osteopontin has an additional role to that of phosphophoryn during primary dentin formation and that it plays an important role as a major phosphoprotein during the supplementary mineralization that occurs as a response to external factors such as thermal, physical, or carious irritation.
ents;
We thank Drs. T. Kawase and S. Kasugai for providing the rat
clonal dental pulp cell lines, RDP4- 1 and RPC-C2A.
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