0003-9969392 SS.OO+ 0.00 Copyright c 1992 Pergamon Press Ltd

Archr oral Bio/. Vol. 37. No. IO, pp. 789-795, 1992 Printed in Great Britain. All rights reserved

EFFECTS OF EPIDERMAL

GROWTH

FACTOR

TRANSFORMING GROWTH FACTOR-B INSULIN-INDUCED DIFFERENTIATION RAT DENTAL PULP CELLS

AND

ON IN

R.-F. LIANG, S. NISHIMURA and S. SATO Department of Pharmacology, Meikai University School of Dentistry, Saitama 350-02. Japan (Received 12 June 1991; accepted 7 May 1992)

Summary-An established pulp cell line (RPC-CZA) was used to study the regulatory effect of insulin on dentinogenesis. Insulin increased alkaline phosphatase activity and the incorporation of [2,3-‘HI-prohne into collagenasedigestible protein, whereas [‘HI-thymidine incorporation by the cells was inhibited by insulin. The enhancing effect of insulin on alkaline phosphatase activity was inhibited by epidermal growth factor (EGF) or transforming growth factor-@ (TGF-j?). The stimulatory effect of insulin on collagen synthesis was also inhibited when insulin was combined with EGF, but was accelerated by the addition of TGF-8. Inhibitory effects of insulin on the (‘HI-thymidine incorporation were potentiated by EGF, though EGF alone strongly increased the effect; whereas the addition of TGF-fl had no significant effect on the insulin action. These findings suggest that insulin may be concerned with the differentiation of pulp cells in dentinogenesis and that EGF or TGF-j regulate the insulin effects. Key words: insulin, epidermal growth factor, transforming growth factor-j, pulp cells.

MATERIALS AND METHODS

INTRODUCTION

Dental pulp cells are able to differentiate into odontoblasts when stimulated by external irritation. However, what kinds of factors are concerned with this mechanism are unknown. Two possible pathways of pulp cell differentiation in dentine formation were suggested by experiments using pulp transplants (Yamamura, 1985). First, after several cycles of mitosis, pulp cells differentiate into odontoblasts, which are responsible for forming the dentine bridge. Second, pulp cells proliferate and de-differentiate into undifferentiated mesenchymal cells, which then differentiate into odontoblasts and form the bridge. In a previous study, we suggested that EGF and TGF-P participate in the growth and differentiation of pulp cells (Liang et al., 1990). Our findings were that several growth factors in the tissue may participate in the differentiation/dedifferentiation of the cell and that these factors must play an important part in dentinogenesis. Insulin is an important growth factor for several cells (Strauss, 1984). However, the effects of this hormone on the pulp cell are still unclear. We have now used rat clonal pulp cells (RPC-CZA), possessing high alkaline phosphatase activity and established by Kasugai, Adachi and Ogura (1988), to investigate the effect of insulin on the cells and the influence of EGF and TGF-/l on the insulin effect. Abbrevialions:

EGF, epidermal growth factor; FBS, fetal bovine serum; TCA, trichloracetic acid: TGF-p. transforming growth factor-b.

Materials

TGF-/3 (a mixture of TGF-/Is extracted from fresh, white cell-free, porcine platelets) was purchased from R&D Systems, Inc. (Minneapolis, MN, U.S.A.). EGF was obtained from Collaborative Research (Lexington, MA, U.S.A.). Insulin, ascorbic acid, N-ethylmaleimide, and collagenase were from Sigma Chemical Co. (St Louis, MO, U.S.A.). Eagle’s minimum essential medium was from Gibco Laboratories Life Technologies Inc. (Grand Island, NY, U.S.A.). TCA and tannic acid were obtained from Wako Pure Chemical Industries (Osaka, Japan); and methyl [‘HI-thymidine (2.48 x IO” Bq/mmol) and [2,3-‘HIproline (1.30 x lOI Bq/mmol), from New England Nuclear Corp. (Boston, MA, U.S.A.). Protein reagents and protein standards came from Bio-Rad (Richmond, CA, U.S.A.). The concentration of insulin to be used in this study in combination with other factors was decided from the result of its dose-response effect on alkaline phosphatase activity. The concentrations of the TGFj and EGF used were based on the dose-response experiments reported by Liang et al. (1990). Cell culture RPC-CZA, which is a clonal cell line obtained from maxillary incisor pulp cells from male Wistar rats, was generously provided by Dr S. Kasugai of Tokyo Medical and Dental University, Japan (Kasugai et al., 1988). The cells were maintained in Eagle’s minimum essential medium supplemented with 10% FBS, and routinely subcultured twice a week. These 789

R.-F. LIASG et al.

790

cells are steliate with few processes when sparse; when confluent. they become polygonal. Their population doubling time is 17.7 h. The cell density is about 1 x 10S/cm’ when they are confluent. After confluence. the number of cells increases slowly as a monolayer. The cell density does not exceed 2 x 10S/cmZ after up to 8 weeks in culture (Kasugai et al., 1988). When the cells are sparse, no alkaline phosphatase activity is detected and they proliferate rapidly: however high activity is detected when they become confluent (Kasugai et al.. 1988). We used different seeding densities so that the cells would be nearly confluent (experiments for 13H]-thymidine incorporation and alkaline phosphatase activity) or confluent (experiment for {2,3-3H]-proline incorporation) when the factors were added; that is, the cells were seeded at a density of 3 x 103cellslwell in 96-well plates for the [lH]-thymidine incorporation assay, at a density of 2 x 104cells’well in 24-well plates for the enzyme activity assay, and at 5 x IO’cells~dish in 35-mm dishes for the f2,3-‘HI-proline incorporation assay. Determination

of [-‘HI-rh>,midine incorporarion

The method for the determination of [“HIthymidine incorporation was described in detail by Liang et al, (1990). In brief. the ceils were plated in 96-well plates and grown for 2 days in Eagle’s minimal essential medium containing 10% FBS; and then the medium was replaced with fresh minimal essential medium containing 1% FBS and various concentrations of factors. and incubated For the periods described in the figure legends. [‘HjThymidine was then added to each well, the cultures incubated for another 18 h, and the relative amount of [‘HI-thymidine incorporated was determined with a liquid scintillation counter. ~~effsuremet?t of alkaline ph~sp~ase actiuit?

The cells were plated in 24-well piates and grown for 2 days in Eagle’s minimal essential medium containing 10% FBS so that they would be nearly confluent. The cells were then incubated with fresh medium containing 1% FBS and factors for another 2 days. Alkaline phosphatase activity in the ceils was measured by the method of Lowry et al. (1954) using y,$-n;,ophenyl phosphate. as substrate (Liang et al.,

volume of 0.1 M tris-HCI buffer, pH 7.5, containing 100 mM CaClz and 2 mM ~-ethylmalemide was added. The amount of [2,3-‘HI-proline incorporated into collagenase-digestible protein was measured as described by Peterkofsky and Diegelmann (1971). Radioactivity was measured with an Aloka LSC-903 liquid scintillation counter. RESULTS

Changes in the alkaline phosphatase activity of RPC-C2A cells following the addition of several concentrations of insulin to the medium are shown in Fig. 1. A dose-de~ndent increase in the activity was found from 100 up to lOOOngjm1. From this result, we selected 500 ng/ml for use in subsequent experiments. Changes in the [‘HI-thymidine and [2,3-‘HIproline incorporation of the cells over several days after the addition of insulin to the medium are shown in Fig. 2. A peak of [‘HI-thymidine inco~oration was found at the third day in both control and insulintreated cells, but the [3HI-thymidine incorporation by insulin-treated cells was significantly lower than that by the control cells [Fig. 2(a)]. A significant increase in [2,3-‘HI-proline incorporation in response to insulin was seen by 2 days of exposure, and the incorporation reached its maximum by the fourth day [Fig. 2(b)]. In the study on the interaction between insulin and EGF and/or TGF-P, a marked increase in incorporation of [‘HI-thymidine was observed on treatment with EGF alone, though TGF-fi had no significant effect and the increase was diminished by the addition of insulin. The inhibitory effect of insulin on incorporation of [3H]-thymidine was potentiated by EGF, though it was unaffected by the addition of TGF-fi. No significant effect was observed when these three factors were added simultaneousIy and the results compared with the control value (Fig. 3). The

Colltlgen qxthesis

Cells were fed with 10% FBS Eagle’s minimal essential medium supplemented with 50 pg/ml ascorbic acid in 35.mm dishes until they reached confluence. Then the cultures were given fresh medium containing 1% FBS with insulin, TGF-fl and/or EGF and incubated for another 3 days. The cells were iabelled for the last 18 h with 3.7 x IO4Bq/ml [2,33H]-proline. The cell layers were scraped into I ml of 0.2 M NaOH and homogenized with a glass homogenizer. Protein in the homogenate was precipitated by addition of 0.2 ml of 50% TCA containing 5% tannic acid and washed three times with 10% TCA containing 1% tannic acid. The acid-precipitable material was collected by centrifugation. The pellets were extracted with acetone, and then dried and resolubilized in 0.5 ml of 0.05 M NaOH. The solution was neutralized with 1 M HCI and then an equal

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Control 0.1

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5

10

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100 500 1000

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Fig. I. Dose-dependent effect of insulin on alkaline phosphatase (ALPase) activity in clonal rat pulp cells. The cells were incubated with each concg~tration of insulin for 48 h after they had become nearly confluent. Each point represents the mean from four dishes. **p < 0.01. ***p < O.M)l compared with control (Student’s r-test).

Insulin-induced differentiation in pulp cells

791

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Control

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Incubation Fig. 2. Time incorporation confluence (in incorporation).

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period (days)

Incubation

period (days)

course of the effect of insulin (500 ng/ml) on [JH]-thymidine and [2.3-‘HI-proline into clonal rat pulp cells. The cells were exposed to insulin when they reached near the case of [‘HI-thymidine incorporation) or confluence (in the case of [2,3-‘HI-proline Each point and bar represent the mean k SE from four dishes. *p < 0.005. **p < 0.01, ***p < 0.001 compared with control (Student’s r-test).

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TGF+

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EGF

TGi-/3 TGt-/3

Ins EG' TGj-fi

Fig. 3. EtTects of insulin in combination with EGF and/or TGF-8 on [‘HI-thymidine incorporation into clonal rat pulp cells. The cells were exposed to the factor(s) for 48 h after they had become nearly confluent. Ins, 500 ng/ml, EGF, 0.5 ng/ml; TGF-B, 0.5 ng/ml. Each column and bar represent the mean k SE from five dishes. l p ~0.05. l**p

Effects of epidermal growth factor and transforming growth factor-beta on insulin-induced differentiation in rat dental pulp cells.

An established pulp cell line (RPC-C2A) was used to study the regulatory effect of insulin on dentinogenesis. Insulin increased alkaline phosphatase a...
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