- Email: [email protected]
Effects of epidermal growth factor and transforming growth factor-β on insulin-induced differentiation in rat dental pulp cells
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
I
I
Control 0.1
I
I
I
I
1
5
10
50
I
I
I
100 500 1000
Wml)
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
**
6
*** *** (I
I 5-
Insulin Control l
f
** \ MP
**
/
Control
Insulin
I;_
I 0
I
I
I
I
I
I
2
3
4
2
3
4
Incubation Fig. 2. Time incorporation confluence (in incorporation).
g B -z 5 z
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).
1312Ulo-
; i .z : 9 $ .-c ? .; 5x
32-
L I m -
Io-
9876-
x_
54-
-
Control
Ins
EGF
TGF+
Ins EtF
Ins
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
792
R.-F. LIANG
et al
50-
F 'g
40-
& E" ;; 30.= C 2
0
Control
Ins
EGF
TGF-13
IflS
Ins
EiF
T&3
EGF TG;-13
I!p E!F Y/-C ii)! -13
Fig. 4. Effect of insulin in combination with EGF and,or TGF-P on alkaline phosphatase (ALPase) activity in clonal rat pulp cells. The cells were exposed to the growth factor(s) for 48 h after they had become nearly confluent. Ins, 500 ng/ml, EGF, 0.5 ng ml; TGF-8, 0.5 ngjml. Each column and bar represent the mean + SE from five dishes. +**p < 0.001compared with control. l**p < 0.001compared with TGFq3. Op < 0.05, oop c 0.001 compared with EGF. ArAp < 0.001 compared with EGF + TGF-8. dAAp < 0.001 compared with Ins (Student’s t-test):
EGF
TGF-/3
TGF-fi
Ins t
EtjF TGF-fi
Fig. 5. ER’ectsof insulin in combination with EGF and/or TGF-8 on collagen synthesis in clonal rat pulp ceils. Once they had reached confluence the cells were exposed to the growth factor(s) for 72 h. Ins, 500 ngjml; EGF, 0.5 ng/ml; TGF-p, 0.5 ng/ml. Each column and bar represent the mean f SE from four dishes. **p < 0.01, ***p < 0.001 compared with control. 00~ < 0.01,000~ < 0.001 compared with Ins. 'p < 0.05,+ + +p < 0.001 compared with EGF. 000~ < 0.001 compared with Ins + TGF-fi. "p < 0.05 compared with TGF-fl (Student’s l-test).
Insulin-induced differentiation in pulp cells
increase in alkaline phosphatase
activity effected by of EGF. TGF-/? had a similar inhibitory effect, but weaker than that of EGF. The strongest inhibition of the insulininduced activity was found when the three factors were added simultaneously. On the contrary, the inhibition of this enzyme activity by EGF or TGF-/3 used alone was blocked by insulin (Fig. 4). Collagen synthesis assessed by the measurement of the incorporation of [2,3-‘HI-proline into collagenasedigestible protein was increased by insulin. This increase was reduced by the combination of insulin with EGF, but was potentiated by the addition of TGF-/I. EGF alone decreased the collagen synthesis, but no effect was seen when TGF-B was used alone. The combination of the three factors showed a similar effect to that of EGF alone (Fig. 5). All experiments were repeated at least three times, and the results followed the same pattern. insulin was inhibited
by the addition
DISCUSSION
Insulin has a mitogenic effect on a variety of cells in aitro, including osteoblasts, chondrocytes and fibroblasts (Strauss, 1984). In fetal rat bone, insulin at 10-9-10-8 M increases the rate of collagen synthesis in the osteoblast-rich layer (Canalis et al., 1977; Canalis. 1980). Insulin also has a stimulatory effect on the proliferation and differentiation of chondrocytes or mesenchymal cells of bone in vivo (Weiss and Reddi, 1980). More recently, insulin was reported to be concerned with the regulation of DNA synthesis, alkaline phosphatase activity, and collagen synthesis of bone cells and tissues (Schwartz et al., 1970; Stevens et al., 1981; Canalis, 1983; Bembenek, Willis and Liberti, 1982; Kream et al., 1985, 1989; Craig et al., 1989). Nagata et al. (1987) also showed that insulin enhanced glycosaminoglycan synthesis in developing bovine dental pulp cells. These reports suggest that insulin may play a part in hard tissue formation and dentinogenesis. Collagen is the major organic component of dental pulp. In citro incorporation of [“HI-proline into rat (Orlowski and Doyle, 1976) and rabbit (Shuttleworth, Ward and Hirschmann, 1979) pulp suggested a high rate of metabolic activity in this collagen. Collagen is also indispensable in the initial stage of dentine formation. Alkaline phosphatase plays an important part in hard tissue development, and is used as a marker enzyme of osteoblastic differentiation (Yee, 1985; Fritsch ef al., 1985; Wlodarski and Reddi, 1986). In dental pulp, Oida, Suzuki and Sasaki (1979) found that alkaline phosphatase activity is present in the odontoblastic, subodontoblastic, and pulp cell layer, with the activity being highest in the subodontoblastic layer. This indicates that alkaline phosphatase activity may be important for the differentiation of undifferentiated mesenchymal pulp cells to differentiated odontoblast cells. We found that insulin decreased [)H]-thymidine incorporation, an indicator of proliferation, in clonal rat pulp cells whereas it increased the alkaline phosphatase activity and collagen synthesis in these cells, which have a relatively high alkaline phosphatase activity even in the absence of the hormone. There is
793
a possibility that these results might have been due to an increase in cell number, but the fact that [‘HIthymidine incorporation by insulin-treated cells was decreased compared with that by control cells at all times suggests that this was not the case. The reason why [3HI-thymidine incorporation was decreased by insulin in the case of RPC-C2A cells but increased in other types of cells is not understood, but it does show that insulin inhibits the proliferation and accelerates the differentiation of these cells. On the other hand, TGF-/I and EGF are also involved in bone metabolism (Tashjian and Levine, 1978; Noda and Rodan, 1986; Pfeilschifter, D’Souza and Mundy, 1987), and they may play a part as modulators of regulatory mechanisms of differentiation and/or proliferation of pulp cells (Liang et al., 1988). We found that EGF increased the [‘HIthymidine incorporation and inhibited the alkaline phosphatase activity and the (2,3-‘HI-proline incorporation, which suggests that EGF stimulates cell proliferation and inhibits differentiation. Hata ef al. (1990) showed that EGF inhibits synthesis of type I collagen in mouse molar organ culture, which agrees with our results on [2,3-‘HI-proline incorporation. The effects of TGF-/I on the alkaline phosphatase activity of these cells were the same as its effects on bone cells (Kasperk et al., 1990; Ber ef al., 1991). It is reported that TGF-/I stimulates the synthesis of type I collagen in bone cells (Bortell ef al., 1990; Ber et al., 1991). However, we did not find such an effect of TGF-P on (2,3-‘HI-proline incorporation. Our finding is similar to that of Chen, Mallory and Chang (1989) who, using osteoblastic cells from fetal rat calvariae, showed that TGF-/l inhibited alkaline phosphatase activity in a dose-dependent manner but failed to increase collagen synthesis, even at a dose as high as 20mg/ml. This finding, as well as ours, is also in sharp contrast with the response of primary cultures of rat skin fibroblasts (Chen er al., 1989). The effects of various growth factors used singly or in combination are quite variable, depending on the target cells examined. For example, the combination of TGF-/I and’or EGF with insulin inhibits the stimulatory effect of insulin on [-‘HI-thymidine incorporation in epithelium of the rat intestinal crypt (IEC-6) (Kurokowa, Lynch and Podolsky, 1987). It is also reported that the stimulatory effect of insulin and EGF on DNA synthesis in adult rat hepatocytes was inhibited by TGF-/? (Nakamura et al., 1985) but insulin plus EGF stimulated the proliferation of fetal rat hepatocytes in MX82 medium (Hoffmann, Piasecki and Paul, 1989). Canalis and Raisz (1979) showed that the effect of insulin in increasing the rate of collagen synthesis in cultured rat calvarial cells is inhibited by EGF. Our findings on the effects of these factors on [‘HI-thymidine incorporation, alkaline phosphatase activity and [2,3-‘HI-proline incorporation in rat pulp cells indicate that insulin inhibits the stimulatory action of EGF on the pulp cell proliferation and that insulin-mediated stimulation of cell differentiation is inhibited by EGF. TGF-fi seems to regulate insulin action on pulp cell differentiation. Insulin, TGF-8, and EGF are reported to act on various cells through membrane-bound receptors (Carpenter et al., 1975; Cuatrecasas ef al., 1975; Frolik et al., 1984). These interactions of factors may
be expressed at the receplor level. lo sume cell lines {intestinal epithelial cells; IEC-6), insulin produces enhanced binding of EGF to 11s receptor (Conteas. McMorrow artd Luk, I9891+buT in other cell lines like Swiss 3Tj, insulin reduces the binding of ECF(‘z51) TVI;he cells (Corps and Brawn. 1988). Our dara and those ol others (Kurakaw er al, i 987: Nakamura et ni., f983; Canalis el crf,. 1977) thus indicate that the rulr UTthese Factors may be differmt in each orpan or cell. The reSUf& SLI~@sl that the proiiferarion dilkrentiarlan of pip ccllr are regulated nor onls by each of these kmm but also ty their combina&m ric~nul~fert~enlmrs--ENrare grateful to Ilk s. Kasupai of the Tokyo Medical and Dental University. fz his generaus gift of the RPC-CZA cell line, and to Dr L. D. Frye for reviewing the manuscript. REFERENCES Bembenek ;M. E.. W11lrsD. H. Jr and Liberti 1. P. (1982) The
cffecr of insulin on collagen production in isolated chondrosarroma chondrocytes. Biorh~m. b~&~.c. &s. cm nrun. 106, 338-345. Ber R.. Kubota T., Sodek J. and Aubin J. E (1991) Efiecls of transforming growth factor-/I on normal clonaf bone cdl poptJlarions. Bio&nt. Ceil BinI 69, 1X-140, Bortell R.. Earone L. M.. Tassioari M. S., Lian J. B. and Stein G. S. { IYWf Gene expression dtiring endochondrai bone development: evidence for coordinatr expression of transforming growth factor PI and collagen type I. d. Celf. &ockeni. 44, 8 l-9 I Caaalis E. $1980) Effcc~ UT insuiiniiiie growth Factor I on DNA and protein syntbssis in c&u+ rat calvaria. J. c/m. Inresl. 66, 70%719. Canalis E. (1983) E&t of hormones and grabth factcrs on alkaline phosphatase activify and collagen synthesis in cultured rat calvariac. Mefu&%~~ 32, I4420. Canalis E. and Raisz L. G. (1979) Eflect of epidermal growth factor on bore formation m riiro. Endocrittoio,p 104, 862-869. Canalis E. M., Dietrich J, W,, M&a D. M-and Raisz L. G. <197Tj Hormonal control of bone collagen synthesis h &ro. effects of insulin and giucagon. Endorrinaio#y 100, 668-67J.
Carpenter C., Lembach K. J.. Morrisrm M. M. and Cohen S. (19Vj Characterization of the biading of “ii-l&elcd epidtrmai growth factor to human fibroblasrs, _f. biu!. Clrem Cheer 7.
250, 42974304.
L., Mallory 1. R. and Chang S. L (1989) Modulatjon of transforming grorvth raclor-8 acrions in rat osteoblast-like cells: the e!Tects of bFGF and EGF. Gr01t1h Factors I, 335-345, Cnnteas C N., McMorroa- B and tuk G. D, (1989) Moduiation of epidermal growth factor-induced ~41 proMeration and receptor binding by it?s&x III cultured Intestinal epithelial cells. Biorhent. &$I~s. Res. Comm:w. 161,414319, Corps A. N. and Brown K. D. (1988] Insulin-like growth factor I and insulin reduce epidcrma! growth factor binding to Swiss 3T3 cells by 3~ inClrtet mcchamsm that is apparently independent ofproTein kinase C FEES LPI:. 233, 303-306. Craig R. G.. Rowe D. W., Petersen D. Fr’.and Krcam B. E. (1989) lcsulin increases Ihe steady state lewd of ?-t(l) procollagen mRNA In the osteoblast-rich segment of fetal r#t Ch’ilria. &nduuckdug~ 125, 143&l&37. CuatrecasasP,, HolienbergM. D., ChangK. J.and BennettV. [I 975) Hormone receptor complexes and their modulation of membrane fraction, Rec. Prag. I%UX RQ~.31, 37-91.
Frhch J.. Grow B., tirbrherr hi. sod Balsw S. (19851 1.25 DihydroxyviLammD, is required for growthindeper&nt expression of alkaline phasphatase in cullurtti rat osteoblasts. Calc. Zs5. ffu. 3?, 639-645. Froiik C. A., Wakefield t. .M., Smith D. M. and Sporn M. 8. (1933)Characterization of a membrarle receptor for transforming growth factor-b in normal rat kidney fibroblasts. J. &of. C!tm. 259, 10,995-l I,UOU. Wata R., Rcssem C , Brittga~P. Jr, Hru 51.Y. and Y&t-kin N. C. 11990) Epidennal growth factor regulates gene expression of both epithellal and mesenchymal cells in mou5e molar tooth organs in culture. CeNBroLlrtrt(,Rep, 14, 539-519. Hoffmann B., &w&i A. and Paul D. (1989) Praliferatioa of fetal rat hepatocytes in response to growth factors and hormones in primary culture. J. Ce0 Physiol. 139, 654-662. KasPerk C. K , Wergedal J. E.. Mahan S.. Long D. L.. Lau K. H. W. and Baylink D. 5. 0990) Jnteractions ofgrowth factors present in bone matrix with bone cells: effects on DNA synthesis and a&line phosphatase. Growrh Fucrors 3, [47-I% Kasupai S.,
Adachi M. and Uvura H. (39833 Establishment and cbaraclcriralion of aclonal &II I& (RPC-CZA) from dental pulp of the rat incisor. /3&s cvaf &al. 33, 887-891. %&am 3. E,, Smith M. D.. CanaIis E. and Raisr L. G. (1985) Characterization of the effect of insulin on collagen synthesis in f&f rat bone. Etdmid~~~ 116, 296-332. Kream B., Harrison J., Bailq R., Petersen D., Rowe 5, an& Lichxler A. (19&9) Hormonal regulation 3f collagen gene
expression in osteablastic cells-overview and new findings. Con!l. T&s. Res. 20, 187-192. Kurokowa M., Lynch K. and Podolsky D. K. (1987) Effects of gmwh fa;Wrs on arr intestinal epithelial cell linetransfoxnirrg growth factor #I inhibits proliferation and stimulates dlfferentlation. Blochem. bioph_w Rex Cornmrm. 142, 775-782. Liang R.-F., Nishimura S . Kawabata .4., Ohhara Y.. Tajima hf., ?ilaru~ama S.. Hirose K., Hanazawa S., Kitano S. and Sato S. [1988) Etfecrs of transforming growth factor-p and epidermal growth factor on pulp cells from rat incisor. 5. Meikai f_Xrl. Sch.Dent. 17, 433-438 Liang R.-f, NisRimura S., ,Maruyama S., Xanazawa S., Kitano S. and Sa~o S. {1990f Effects of transforming growth factor-b and epidermal growth factor on clonal lar pulp cells. 4r& ora! Biol. 35, 7-1 I. Lowry 0. H., Roberts N. R., Wu M. L.. Hixon W. S. and Crawford E, j. jf953) The quantitative hi,tochemistry of bram. Il. Enzyme measurement. $. bid. Chem. 207,19-37. NagatsT.. lshrda H., Kido J.. Hamasaki A. and Wakano Y. (1987) EKec~s of insulin and parathyroid hormone on glycosaminaglycan syathesir of developing bovine denta! pulp in culture. J. &m Mhwf Merah 4, 192-198. ?Jiakamura T., Tomita Y., Hirai R., Yamaoka K.. Kaji K and lchihara A. (1985) Inhibitory eKecr of transforming growth factor+ cm DNA synthesis of adult rat heparooytesin +-nary cultnz. Hinr&w~ bioph:._r.RG. Commun. 133, IWZ-lOSO.
Noda M. and Rodan G. A. (1986) Type-p transforming growth factor inhibits proliferation and expression of alkaline phosphatase jn mu&e osteoblasi-like cells. &achcm. bioplty.5. Rex. Commun. 140, X&5. oidd S., Suzuki M. and Sasaki S. (1379) Nature and biwzhcmical characteristics of alkaline phosphatase. J, &IF Minerof kferab. 12, 25-33. Orlowski W. A. and Doyle J. L. (1976) Collagen metabolism in the pulps of rat tecrh. Archr arul Rio!. 21, 391-392. Petcrkofsky B. and Diegeelmxnn R. (1971) Use of a mixture 01 prsteinase-free collagenases for the specific as~dy of
radioactive collagen in the presence of other protrins. Aiacknrblyr IQ, 988-993.
Insulin-induced differentiation in pulp cells Pfeilschifter J., D’Souza S. M. and Mundy G. R. (1987) Effects of transforming growth factor-b on osteoblastic osteosarcoma cells. Endocrinology 121, 212-218. Schwartz P. L.. Wettenhall R. E. H.. Troedel M. A. and Bomstein J.‘(l970) A long-term ‘effect of insulin on collagen synthesis by newborn rat bone in vitro. Diabetes 19, 465-466.
Shuttleworth C. A., Ward J. L. and Hirschmann P. N. (1979) In cico incorporation of [‘HI-proline into rabbit dental pulp collagen. Archs oral Biol. 24, 613-615. Stevens R. L., Nissley S. P.. Kimura J. H., Rechler M. M., Caplan A. I. and Hascall V. C. (1981) Effects of insulin and multiplication-stimulating activity on proteoglycan biosynthesis in chondrocytes from the swarm rat chondrosarcoma. J. biol. Chem. 256, 2045-2052. Strauss D. S. (1984) Growth stimulation actions of insulin in ritro and in cico. Endow. Rev. 5, 356369.
795
Tashjian A. H. Jr and Levine L. (1978) Epidermal growth factor stimulates prostaglandin production and bone resorption in cultured mouse calvaria. Biochem. biophp. Res. Commun. 85, 966-975.
Weiss R. E. and Reddi A. H. (1980) Influence of experimental diabetes and insulin on matrix-induced cartilage and bone differentiation. Am. J. Physiol. 238, EZOO-E207. Wlodarski K. H. and Reddi A. H. (1986) Alkaline phosphatase as a marker of osteo-inductive cells. Cult. Tiss. Inf. 39, 382-385.
Yamamura T. (1985) Differentiation of pulpal cells and inductive influences of various matrices with reference to pulpal wound healing. J. dent. Res. 64, 530-540. Yee J. A. (1985) Stimulation of alkaline phosphatase activity in cultured neo-natal mouse calvarial bone cells by parathyroid hormone. Cult. Tiss. Inr. 37, 530-538.
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
I
I
Control 0.1
I
I
I
I
1
5
10
50
I
I
I
100 500 1000
Wml)
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
**
6
*** *** (I
I 5-
Insulin Control l
f
** \ MP
**
/
Control
Insulin
I;_
I 0
I
I
I
I
I
I
2
3
4
2
3
4
Incubation Fig. 2. Time incorporation confluence (in incorporation).
g B -z 5 z
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).
1312Ulo-
; i .z : 9 $ .-c ? .; 5x
32-
L I m -
Io-
9876-
x_
54-
-
Control
Ins
EGF
TGF+
Ins EtF
Ins
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
792
R.-F. LIANG
et al
50-
F 'g
40-
& E" ;; 30.= C 2
0
Control
Ins
EGF
TGF-13
IflS
Ins
EiF
T&3
EGF TG;-13
I!p E!F Y/-C ii)! -13
Fig. 4. Effect of insulin in combination with EGF and,or TGF-P on alkaline phosphatase (ALPase) activity in clonal rat pulp cells. The cells were exposed to the growth factor(s) for 48 h after they had become nearly confluent. Ins, 500 ng/ml, EGF, 0.5 ng ml; TGF-8, 0.5 ngjml. Each column and bar represent the mean + SE from five dishes. +**p < 0.001compared with control. l**p < 0.001compared with TGFq3. Op < 0.05, oop c 0.001 compared with EGF. ArAp < 0.001 compared with EGF + TGF-8. dAAp < 0.001 compared with Ins (Student’s t-test):
EGF
TGF-/3
TGF-fi
Ins t
EtjF TGF-fi
Fig. 5. ER’ectsof insulin in combination with EGF and/or TGF-8 on collagen synthesis in clonal rat pulp ceils. Once they had reached confluence the cells were exposed to the growth factor(s) for 72 h. Ins, 500 ngjml; EGF, 0.5 ng/ml; TGF-p, 0.5 ng/ml. Each column and bar represent the mean f SE from four dishes. **p < 0.01, ***p < 0.001 compared with control. 00~ < 0.01,000~ < 0.001 compared with Ins. 'p < 0.05,+ + +p < 0.001 compared with EGF. 000~ < 0.001 compared with Ins + TGF-fi. "p < 0.05 compared with TGF-fl (Student’s l-test).
Insulin-induced differentiation in pulp cells
increase in alkaline phosphatase
activity effected by of EGF. TGF-/? had a similar inhibitory effect, but weaker than that of EGF. The strongest inhibition of the insulininduced activity was found when the three factors were added simultaneously. On the contrary, the inhibition of this enzyme activity by EGF or TGF-/3 used alone was blocked by insulin (Fig. 4). Collagen synthesis assessed by the measurement of the incorporation of [2,3-‘HI-proline into collagenasedigestible protein was increased by insulin. This increase was reduced by the combination of insulin with EGF, but was potentiated by the addition of TGF-/I. EGF alone decreased the collagen synthesis, but no effect was seen when TGF-B was used alone. The combination of the three factors showed a similar effect to that of EGF alone (Fig. 5). All experiments were repeated at least three times, and the results followed the same pattern. insulin was inhibited
by the addition
DISCUSSION
Insulin has a mitogenic effect on a variety of cells in aitro, including osteoblasts, chondrocytes and fibroblasts (Strauss, 1984). In fetal rat bone, insulin at 10-9-10-8 M increases the rate of collagen synthesis in the osteoblast-rich layer (Canalis et al., 1977; Canalis. 1980). Insulin also has a stimulatory effect on the proliferation and differentiation of chondrocytes or mesenchymal cells of bone in vivo (Weiss and Reddi, 1980). More recently, insulin was reported to be concerned with the regulation of DNA synthesis, alkaline phosphatase activity, and collagen synthesis of bone cells and tissues (Schwartz et al., 1970; Stevens et al., 1981; Canalis, 1983; Bembenek, Willis and Liberti, 1982; Kream et al., 1985, 1989; Craig et al., 1989). Nagata et al. (1987) also showed that insulin enhanced glycosaminoglycan synthesis in developing bovine dental pulp cells. These reports suggest that insulin may play a part in hard tissue formation and dentinogenesis. Collagen is the major organic component of dental pulp. In citro incorporation of [“HI-proline into rat (Orlowski and Doyle, 1976) and rabbit (Shuttleworth, Ward and Hirschmann, 1979) pulp suggested a high rate of metabolic activity in this collagen. Collagen is also indispensable in the initial stage of dentine formation. Alkaline phosphatase plays an important part in hard tissue development, and is used as a marker enzyme of osteoblastic differentiation (Yee, 1985; Fritsch ef al., 1985; Wlodarski and Reddi, 1986). In dental pulp, Oida, Suzuki and Sasaki (1979) found that alkaline phosphatase activity is present in the odontoblastic, subodontoblastic, and pulp cell layer, with the activity being highest in the subodontoblastic layer. This indicates that alkaline phosphatase activity may be important for the differentiation of undifferentiated mesenchymal pulp cells to differentiated odontoblast cells. We found that insulin decreased [)H]-thymidine incorporation, an indicator of proliferation, in clonal rat pulp cells whereas it increased the alkaline phosphatase activity and collagen synthesis in these cells, which have a relatively high alkaline phosphatase activity even in the absence of the hormone. There is
793
a possibility that these results might have been due to an increase in cell number, but the fact that [‘HIthymidine incorporation by insulin-treated cells was decreased compared with that by control cells at all times suggests that this was not the case. The reason why [3HI-thymidine incorporation was decreased by insulin in the case of RPC-C2A cells but increased in other types of cells is not understood, but it does show that insulin inhibits the proliferation and accelerates the differentiation of these cells. On the other hand, TGF-/I and EGF are also involved in bone metabolism (Tashjian and Levine, 1978; Noda and Rodan, 1986; Pfeilschifter, D’Souza and Mundy, 1987), and they may play a part as modulators of regulatory mechanisms of differentiation and/or proliferation of pulp cells (Liang et al., 1988). We found that EGF increased the [‘HIthymidine incorporation and inhibited the alkaline phosphatase activity and the (2,3-‘HI-proline incorporation, which suggests that EGF stimulates cell proliferation and inhibits differentiation. Hata ef al. (1990) showed that EGF inhibits synthesis of type I collagen in mouse molar organ culture, which agrees with our results on [2,3-‘HI-proline incorporation. The effects of TGF-/I on the alkaline phosphatase activity of these cells were the same as its effects on bone cells (Kasperk et al., 1990; Ber ef al., 1991). It is reported that TGF-/I stimulates the synthesis of type I collagen in bone cells (Bortell ef al., 1990; Ber et al., 1991). However, we did not find such an effect of TGF-P on (2,3-‘HI-proline incorporation. Our finding is similar to that of Chen, Mallory and Chang (1989) who, using osteoblastic cells from fetal rat calvariae, showed that TGF-/l inhibited alkaline phosphatase activity in a dose-dependent manner but failed to increase collagen synthesis, even at a dose as high as 20mg/ml. This finding, as well as ours, is also in sharp contrast with the response of primary cultures of rat skin fibroblasts (Chen er al., 1989). The effects of various growth factors used singly or in combination are quite variable, depending on the target cells examined. For example, the combination of TGF-/I and’or EGF with insulin inhibits the stimulatory effect of insulin on [-‘HI-thymidine incorporation in epithelium of the rat intestinal crypt (IEC-6) (Kurokowa, Lynch and Podolsky, 1987). It is also reported that the stimulatory effect of insulin and EGF on DNA synthesis in adult rat hepatocytes was inhibited by TGF-/? (Nakamura et al., 1985) but insulin plus EGF stimulated the proliferation of fetal rat hepatocytes in MX82 medium (Hoffmann, Piasecki and Paul, 1989). Canalis and Raisz (1979) showed that the effect of insulin in increasing the rate of collagen synthesis in cultured rat calvarial cells is inhibited by EGF. Our findings on the effects of these factors on [‘HI-thymidine incorporation, alkaline phosphatase activity and [2,3-‘HI-proline incorporation in rat pulp cells indicate that insulin inhibits the stimulatory action of EGF on the pulp cell proliferation and that insulin-mediated stimulation of cell differentiation is inhibited by EGF. TGF-fi seems to regulate insulin action on pulp cell differentiation. Insulin, TGF-8, and EGF are reported to act on various cells through membrane-bound receptors (Carpenter et al., 1975; Cuatrecasas ef al., 1975; Frolik et al., 1984). These interactions of factors may
be expressed at the receplor level. lo sume cell lines {intestinal epithelial cells; IEC-6), insulin produces enhanced binding of EGF to 11s receptor (Conteas. McMorrow artd Luk, I9891+buT in other cell lines like Swiss 3Tj, insulin reduces the binding of ECF(‘z51) TVI;he cells (Corps and Brawn. 1988). Our dara and those ol others (Kurakaw er al, i 987: Nakamura et ni., f983; Canalis el crf,. 1977) thus indicate that the rulr UTthese Factors may be differmt in each orpan or cell. The reSUf& SLI~@sl that the proiiferarion dilkrentiarlan of pip ccllr are regulated nor onls by each of these kmm but also ty their combina&m ric~nul~fert~enlmrs--ENrare grateful to Ilk s. Kasupai of the Tokyo Medical and Dental University. fz his generaus gift of the RPC-CZA cell line, and to Dr L. D. Frye for reviewing the manuscript. REFERENCES Bembenek ;M. E.. W11lrsD. H. Jr and Liberti 1. P. (1982) The
cffecr of insulin on collagen production in isolated chondrosarroma chondrocytes. Biorh~m. b~&~.c. &s. cm nrun. 106, 338-345. Ber R.. Kubota T., Sodek J. and Aubin J. E (1991) Efiecls of transforming growth factor-/I on normal clonaf bone cdl poptJlarions. Bio&nt. Ceil BinI 69, 1X-140, Bortell R.. Earone L. M.. Tassioari M. S., Lian J. B. and Stein G. S. { IYWf Gene expression dtiring endochondrai bone development: evidence for coordinatr expression of transforming growth factor PI and collagen type I. d. Celf. &ockeni. 44, 8 l-9 I Caaalis E. $1980) Effcc~ UT insuiiniiiie growth Factor I on DNA and protein syntbssis in c&u+ rat calvaria. J. c/m. Inresl. 66, 70%719. Canalis E. (1983) E&t of hormones and grabth factcrs on alkaline phosphatase activify and collagen synthesis in cultured rat calvariac. Mefu&%~~ 32, I4420. Canalis E. and Raisz L. G. (1979) Eflect of epidermal growth factor on bore formation m riiro. Endocrittoio,p 104, 862-869. Canalis E. M., Dietrich J, W,, M&a D. M-and Raisz L. G. <197Tj Hormonal control of bone collagen synthesis h &ro. effects of insulin and giucagon. Endorrinaio#y 100, 668-67J.
Carpenter C., Lembach K. J.. Morrisrm M. M. and Cohen S. (19Vj Characterization of the biading of “ii-l&elcd epidtrmai growth factor to human fibroblasrs, _f. biu!. Clrem Cheer 7.
250, 42974304.
L., Mallory 1. R. and Chang S. L (1989) Modulatjon of transforming grorvth raclor-8 acrions in rat osteoblast-like cells: the e!Tects of bFGF and EGF. Gr01t1h Factors I, 335-345, Cnnteas C N., McMorroa- B and tuk G. D, (1989) Moduiation of epidermal growth factor-induced ~41 proMeration and receptor binding by it?s&x III cultured Intestinal epithelial cells. Biorhent. &$I~s. Res. Comm:w. 161,414319, Corps A. N. and Brown K. D. (1988] Insulin-like growth factor I and insulin reduce epidcrma! growth factor binding to Swiss 3T3 cells by 3~ inClrtet mcchamsm that is apparently independent ofproTein kinase C FEES LPI:. 233, 303-306. Craig R. G.. Rowe D. W., Petersen D. Fr’.and Krcam B. E. (1989) lcsulin increases Ihe steady state lewd of ?-t(l) procollagen mRNA In the osteoblast-rich segment of fetal r#t Ch’ilria. &nduuckdug~ 125, 143&l&37. CuatrecasasP,, HolienbergM. D., ChangK. J.and BennettV. [I 975) Hormone receptor complexes and their modulation of membrane fraction, Rec. Prag. I%UX RQ~.31, 37-91.
Frhch J.. Grow B., tirbrherr hi. sod Balsw S. (19851 1.25 DihydroxyviLammD, is required for growthindeper&nt expression of alkaline phasphatase in cullurtti rat osteoblasts. Calc. Zs5. ffu. 3?, 639-645. Froiik C. A., Wakefield t. .M., Smith D. M. and Sporn M. 8. (1933)Characterization of a membrarle receptor for transforming growth factor-b in normal rat kidney fibroblasts. J. &of. C!tm. 259, 10,995-l I,UOU. Wata R., Rcssem C , Brittga~P. Jr, Hru 51.Y. and Y&t-kin N. C. 11990) Epidennal growth factor regulates gene expression of both epithellal and mesenchymal cells in mou5e molar tooth organs in culture. CeNBroLlrtrt(,Rep, 14, 539-519. Hoffmann B., &w&i A. and Paul D. (1989) Praliferatioa of fetal rat hepatocytes in response to growth factors and hormones in primary culture. J. Ce0 Physiol. 139, 654-662. KasPerk C. K , Wergedal J. E.. Mahan S.. Long D. L.. Lau K. H. W. and Baylink D. 5. 0990) Jnteractions ofgrowth factors present in bone matrix with bone cells: effects on DNA synthesis and a&line phosphatase. Growrh Fucrors 3, [47-I% Kasupai S.,
Adachi M. and Uvura H. (39833 Establishment and cbaraclcriralion of aclonal &II I& (RPC-CZA) from dental pulp of the rat incisor. /3&s cvaf &al. 33, 887-891. %&am 3. E,, Smith M. D.. CanaIis E. and Raisr L. G. (1985) Characterization of the effect of insulin on collagen synthesis in f&f rat bone. Etdmid~~~ 116, 296-332. Kream B., Harrison J., Bailq R., Petersen D., Rowe 5, an& Lichxler A. (19&9) Hormonal regulation 3f collagen gene
expression in osteablastic cells-overview and new findings. Con!l. T&s. Res. 20, 187-192. Kurokowa M., Lynch K. and Podolsky D. K. (1987) Effects of gmwh fa;Wrs on arr intestinal epithelial cell linetransfoxnirrg growth factor #I inhibits proliferation and stimulates dlfferentlation. Blochem. bioph_w Rex Cornmrm. 142, 775-782. Liang R.-F., Nishimura S . Kawabata .4., Ohhara Y.. Tajima hf., ?ilaru~ama S.. Hirose K., Hanazawa S., Kitano S. and Sato S. [1988) Etfecrs of transforming growth factor-p and epidermal growth factor on pulp cells from rat incisor. 5. Meikai f_Xrl. Sch.Dent. 17, 433-438 Liang R.-f, NisRimura S., ,Maruyama S., Xanazawa S., Kitano S. and Sa~o S. {1990f Effects of transforming growth factor-b and epidermal growth factor on clonal lar pulp cells. 4r& ora! Biol. 35, 7-1 I. Lowry 0. H., Roberts N. R., Wu M. L.. Hixon W. S. and Crawford E, j. jf953) The quantitative hi,tochemistry of bram. Il. Enzyme measurement. $. bid. Chem. 207,19-37. NagatsT.. lshrda H., Kido J.. Hamasaki A. and Wakano Y. (1987) EKec~s of insulin and parathyroid hormone on glycosaminaglycan syathesir of developing bovine denta! pulp in culture. J. &m Mhwf Merah 4, 192-198. ?Jiakamura T., Tomita Y., Hirai R., Yamaoka K.. Kaji K and lchihara A. (1985) Inhibitory eKecr of transforming growth factor+ cm DNA synthesis of adult rat heparooytesin +-nary cultnz. Hinr&w~ bioph:._r.RG. Commun. 133, IWZ-lOSO.
Noda M. and Rodan G. A. (1986) Type-p transforming growth factor inhibits proliferation and expression of alkaline phosphatase jn mu&e osteoblasi-like cells. &achcm. bioplty.5. Rex. Commun. 140, X&5. oidd S., Suzuki M. and Sasaki S. (1379) Nature and biwzhcmical characteristics of alkaline phosphatase. J, &IF Minerof kferab. 12, 25-33. Orlowski W. A. and Doyle J. L. (1976) Collagen metabolism in the pulps of rat tecrh. Archr arul Rio!. 21, 391-392. Petcrkofsky B. and Diegeelmxnn R. (1971) Use of a mixture 01 prsteinase-free collagenases for the specific as~dy of
radioactive collagen in the presence of other protrins. Aiacknrblyr IQ, 988-993.
Insulin-induced differentiation in pulp cells Pfeilschifter J., D’Souza S. M. and Mundy G. R. (1987) Effects of transforming growth factor-b on osteoblastic osteosarcoma cells. Endocrinology 121, 212-218. Schwartz P. L.. Wettenhall R. E. H.. Troedel M. A. and Bomstein J.‘(l970) A long-term ‘effect of insulin on collagen synthesis by newborn rat bone in vitro. Diabetes 19, 465-466.
Shuttleworth C. A., Ward J. L. and Hirschmann P. N. (1979) In cico incorporation of [‘HI-proline into rabbit dental pulp collagen. Archs oral Biol. 24, 613-615. Stevens R. L., Nissley S. P.. Kimura J. H., Rechler M. M., Caplan A. I. and Hascall V. C. (1981) Effects of insulin and multiplication-stimulating activity on proteoglycan biosynthesis in chondrocytes from the swarm rat chondrosarcoma. J. biol. Chem. 256, 2045-2052. Strauss D. S. (1984) Growth stimulation actions of insulin in ritro and in cico. Endow. Rev. 5, 356369.
795
Tashjian A. H. Jr and Levine L. (1978) Epidermal growth factor stimulates prostaglandin production and bone resorption in cultured mouse calvaria. Biochem. biophp. Res. Commun. 85, 966-975.
Weiss R. E. and Reddi A. H. (1980) Influence of experimental diabetes and insulin on matrix-induced cartilage and bone differentiation. Am. J. Physiol. 238, EZOO-E207. Wlodarski K. H. and Reddi A. H. (1986) Alkaline phosphatase as a marker of osteo-inductive cells. Cult. Tiss. Inf. 39, 382-385.
Yamamura T. (1985) Differentiation of pulpal cells and inductive influences of various matrices with reference to pulpal wound healing. J. dent. Res. 64, 530-540. Yee J. A. (1985) Stimulation of alkaline phosphatase activity in cultured neo-natal mouse calvarial bone cells by parathyroid hormone. Cult. Tiss. Inr. 37, 530-538.