Arcl~s oral L&l. Vol. 35, Ko. 1. pp. 7-11, 1990 Printed in Great Britain. ,411rights reserved

Copyright

e

0003-9969/90 $3.00 + 0.00 1990Pergamon Press plc

EFFECTS OF TRANSFORMING GROWTH FACTOR-B AND EPIDERMAL GROWTH FACTOR ON CLONAL RAT PULP CELLS R.-F. LIANG,’ S. NISHIMURA,’ S. MARUYAMA,’ S. HANAZAWA,’S. KITANO’ and S. ’ Department

SATO’

of Pharmacology and ‘Department of Oral Microbiology, School of Dentistry, Meikai University, Saitama 350-02, Japan (Accepted

30 August

1989)

Summary-These factors influence proliferation and differentiation in various cell types. Their effects on a clonal cell line (RPC-C2A) having high ALPase activity were examined by assay of [3H]-thymidine incorporation and ALPase activity. Neither factor (at a dose of 0.5 ng/ml) altered the shape of the pulp ceils. DNA synthesis was not affected by transforming growth factor-p either in growing cells or in those nearly confluent, but epidermal growth factor, in doses ranging from 0.5 to long/ml, stimulated the incorporation of [‘HI-thymidine in nearly confluent cells. Both factors inhibited ALPase activity in a dose-dependent manner. Indomethacin did not affect this inhibition, suggesting that this effect of growth factors may not be mediated by prostaglandin synthesis. Inhibitory effects of ALPase antagonists (L-phenylalanine, L-homoarginine, levamisole) were not affected by transforming growth factor-b. Thus epidermal growth factor stimulates DNA synthesis and both transforming growth factor-p and epidermal growth factor inhibit ALPase activity of clonal rat pulp cells, suggesting that both factors may act as regulators of biological function, including cell differentiation, in pulp cells. Key words: ce.1 culture, dental pulp, growth factors.

INTRODUCTION Dentine formation i!i regulated by several hormones and factors, such as parathyroid hormone (Shimokawa et al., 1!)79; Kido, 1987), insulin (Nagata et al., 1987), 1,25-dihydroxyvitamin D, (Kido, 1987), calcitonin (Kido, 1987) and prostaglandin E, (Kido, 1987), which act on pulp cells. However, other factors may also play a part, and one of these is transforming growth factor-b, a multifunctional peptide that controls various functions in many cell types (Sporn et al., 1986). This factor has been isolated from several sources, such as human platelets (Assoian et al., 1983), human placenta (Frolik et al., 1983) and bovine kidney (Roberts et al., 1983). From whatever sources the factor is a homodimer with a molecular weight of approx. 25,000 Da (Assoian et al., 1983). It stimulates or inhibits cell proliferation (Robey et al., 1987; Strain et al., 1987) and differentiation (Ignotz and Massague, 198fi; Masui er al., 1986), and can either stimulate or inhibit other critical cell processes (Boerner, Resnick and Racker, 1985; Tashjian et al., 1985; Hotta and Baird, 1987). Transforming growth factor is apparently identical to the cartilage-inducing factor-A in demineralized bone matrix (Seyedin et al., 1986). Demineralized bone and tooth malrix can both induce osteoblasts from undifferentiated mesenchymal cells and induce them to produce bone (Urist, 1965; Bang and Urist,

ALPase, alkaline phosphatase; EMEM, Eagle’s minimum essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline.

Abbreviations:

1967; Reddi and Anderson, 1976). Dental pulp cells are odontoprogenitor cells (Yamamura, 1985), so transforming growth factor+ may also play a part in dentine formation. Another factor, epidermal growth factor, might also be involved in dentine formation. This factor, which has been found in male mouse submaxillary glands (Cohen, 1962) and in human urine (Cohen and Carpenter, 1975), is delivered by the bloodstream to several tissues, including mineralized tissues, and it influences tooth eruption in mice (Cohen, 1962). Epidermal growth factor also stimulates DNA synthesis and proliferation in several cell types (Carpenter and Cohen, 1975; Westermark, 1976; Gospodarowicz and Mescher, 1977), and is involved in bone metabolism (Tashjian and Levine, 1978; Canalis and Raisz, 1979; Raisz et ul., 1980; Hiramatsu et al., 1982; Kumegawa et al., 1983). The effects of transforming growth factor-p and epidermal growth factor on dental tissues have scarcely been tested. Kasugai, Adachi and Ogura (1988) have established a clonal rat pulp cell line, RPC-C2A that has a high level of ALPase activity. We have now investigated the effects of transforming growth factor-p and epidermal growth factor on these pulp cells. MATERIALS AND

METHODS

EMEM was purchased from Gibco Laboratories Life Technologies, Inc., U.S.A. FBS was from Flow Laboratories. Methyl [‘HI-thymidine was obtained from New England Nuclear Corp. (Boston, Mass., U.S.A.). Tissue culture plastic dishes were from Falcon, U.S.A. Transforming growth factor-/3, prepared

8

R.-F.

LIANG

et al.

from porcine platelets and reportedly 96% pure, was purchased from R&D Systems Inc. (Minneapolis, Minn., U.S.A.). Epidermal growth factor was obtained from Collaborative Research, Lexington, Mass., U.S.A. Indomethacin and ALPase inhibitors (L-phenylalanine, L-homoarginine, levamisole) were from Sigma Chemical Co., St Louis, MO., U.S.A. Protein reagents and protein standards were obtained from Bio-Rad (Richmond, Calif., U.S.A.). Cell culture

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The RPC-C2A cell line isolated from the dental pulp of male Wistar strain rats was generously provided by Dr S. Kasugai of the Tokyo Medical and Dental University, Japan (Kasugai et al., 1988). The cells were cultured in EMEM containing 10% FBS in plastic dishes (60 cm2) in a CO, incubator at 37°C and subcultured twice a week. For each experiment the cells were plated in 24- or 96-well plates with seeding densities of 1 x lo’, 3 x 103, or 2 x 104cells/well.

01 Control

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Fig. 1. Lack of effect of transforming growth factor-p and epidermal growth factor on DNA synthesis in clonal rat pulp cells at the early stage (seeding density = 1 x 10’ cells/well) of cell growth. Each point and bar represent the mean + SE from 5 wells. 0, Epidermal growth factor; 0, transforming growth factor-b.

DNA synthesis

The cells were plated in EMEM containing 10% FBS in Falcon 96-well, flat-bottomed tissue culture plates at a density of 1 x lo3 or 3 x 10’ cells per well. After being cultured for 48 h, when the cells (1 x I03/well) reached 30% confluence (called the early stage of cell growth hereafter) or later when they (3 x lO’/well) were nearly confluent (called the late stage of cell growth hereafter), they were washed with PBS and then given fresh medium containing 1% FBS with various concentrations of either transforming or epidetmal growth factor. After a further 2 days of culture, 10 ~1 of medium containing 0.5 pCi/ml [3H]-thymidine was added to each well. After incubation for 18 h the medium was removed and the cells were washed with PBS, treated with PBSEDTA containing 0.25% trypsin for 20min, and then harvested on glass filters using an automatic cell harvester (Lab. Scien. Co., Tokyo, Japan). Finally, each sample was added to a toluene-base scintillation fluid and radioactivity was measured in an Aloka LSC-903 liquid scintillation counter. The results were expressed as the arithmetic mean of counts/min [3H]thymidine incorporation with SD of 4 cultures. ALPase

ous ALPase types (Bell, 1972; Moss, 1975; Goldstein, Rogers and Harris, 1980). L-Phenylalanine, Lhomoarginine and levamisole with final concentrations of 10-4-10-’ M were used as the inhibitors of ALPase. Enzyme samples were prepared from control and transforming growth factor-/I-treated cells and incubated at 37°C for 30 min with the inhibitors, after which ALPase activity was determined as described above. Other details

Details of experimental addition of indomethacin, legends.

procedure, including the can be found in figure

RESULTS

The pulp cells had a fibroblast-like morphology at low cell density; at near confluence, they became cuboidal and formed a pavement-like arrangement. No conspicuous morphological changes occurred after the addition of transforming growth factor-j? or epidermal growth factor at 0.5 @ml. In the early

activity

When the cells had nearly reached confluence, they were rinsed twice with PBS, scraped off their dishes with a rubber stirrer in 0.2% Nodinet P-40, and sonicated. The homogenate was centrifuged for 10min at 3OOOg, and the supernatant was used as the enzyme sample. ALPase activity was assayed by the method of Lowry et al. (1954), using sodium p-nitrophenyl phosphate as substrate. The amount of p-nitrophenol liberated was measured with a spectrophotometer set at a wavelength of 410 nm. One unit of enzyme was defined as the activity causing the release of 1 nmol of product/min under standard assay conditions. Protein content was determined using the Bradford (1976) method, with bovine serum albumin as a standard. Characterization

of ALPase

The ALPase in the cells was characterized using three inhibitors which readilv distinguished the vari-

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Fig. 2. Effect of transforming growth factor-8 and epidermal growth factor on DNA synthesis in clonal rat pulp cells at the late stage (seeding density = 3 x lO’cells/weIl) of cell growth. Each point and bar are the mean f SE from 5 wells. 0, Epidermal growth factor; 0, transforming growth factor-b. *p < 0.05, yp < 0.001 compared with control.

Effects of transforming and epidermal growth factor

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Fig. 3. Effect of transient exposure to transforming growth factor-b on the expression of ALPase activity in clonal rat pulp cells. The pulp cells were cultured with transforming growth factor-b (O.Sng/ml) for 24, 48 or 72 h and then rinsed two times with EMEM. The culture was continued

until confluent in the absence of transforming growth factor-/3and then assayed for enzyme activity. Each column and bar is the mean f SE from 4 wells. *p < 0.001 compared with control. tp < 0.01, $p