Bone and Mint-ml, 15 (1991) 125-136
Elsevier
125
BAM 00397
Epidermal growthfactor attenuates cell proliferationby down-regulatingthe transformin growth factor-p receptor in the osteoblasticcell line MC3T3-El
Hiroaki Jikihara’, Hiromasa Ikegami’, Masahiro Sakata’, Ken-ichiro Morishige’, Yoshiko Fujita’, Hirohisa Kurachi’, Akira Miyake’, Osamu Tanizawa’ and Naoki Terakawa* ‘Department of Obstetricsand Gynecology, Osaka UniversityMedical School, Osaka, and 2Department of Obstetricsand Gynecology, Tottori Universio Medical School, Tottori, Japan
(Received 5 November 1990) (Accepted 2 July 1991)
summary We investigated the role of transforming growth factor /?I (TGF-8,) in growth regulation of the murine osteoblastic cell line, MC3T3-El. We detected TGF-/3 activity in the conditioned medium of MC3T3-El cells and its activity was increased by acid treatment of the bioassay system using CC164 cells. Northern blot analysis revealed the expression of TGF-fi, precursor messenger ribonucleic acid (mRNA). MC3T3-El cells possessed a single class of high affinity TGF-/3, receptor (2.8 x 104sites per cell, Kd = 196 PM). Cross-linking studies revealed three specific TGF-B receptors with molecular masses of 65,95 and 280 kDa. Both TGF-/I and epidermal growth factor (EGF) stimulated rH]-thymidine incorporation into cells. EGF decreased the number of TGF-#? receptor dose-dependently, and pretreatment of cells with 10 nM EGF attenuated cell growth by TGF-@. All these data suggested that TGF-@might be an autocrine growth factor in murine osteoblastic MC3T3-El cells and that EGF might modulate the growth of cells by down-regulating the TGF-/I receptor level.
Key words: Transforming growth factor-@; Transforming growth factor-p receptor; Epidermal growth factor; Osteoblastic MC3T3-El cell line
Introduction
Transforming growth factor-@,(TGF-PI), a potent modulator of cell growth, is a polypeptide with a molecular mass of 25 000, consisting of two subunits [1,2]. Correspondence to: H. Jikihara, Department of Obstetrics and Gynecology, Osaka University Medical School, l-l-50, Fukushima, Fukushimaku, Osaka 553, Japan. 0169~6009i91/$03.50@)1991 Elsevier Science Publishers B.V.
126 TGF-#lcan promote the growth of cells derived from mesenchymal tissues, such as normal rat kidney (NRK) fibroblasts [1,2]. TGF-/I, has also been suggested to have important functions in bone metabolism because it is synthesized and expressed abundantly in bone cells [3-51. There have been several controversial reports about the differentiation and proliferation by TGF-13,in various osteoblast-like cells [6-131. The effects of TGF-/I, on MC3T3-El cells appeared to be biphasic, possibly reflecting the complex mechanisms of action: the short term exposure of cells to TGF-/I, inhibits the proliferation, whereas chronic exposure of these cells to TGF-& stimulates cellular proliferation and inhibits the differentiation into osteoblastic phenotype [7,9]. Robey et al. [5] examined the binding of TGF-j?in various types of bone-derived cells and suggested a possible action of TGFq3 in an autocrine manner in osteoblast cells. TGF-fi receptor modulation is not well understood. So far, only epidermal growth factor (EGF) has been reported to down-regulate TGF-/3receptor in NRK cells [14]. EGF is a polypeptide growth factor with a wide range of effects on the growth and differentiation of a variety of cells. But its effects on TGF-/3receptors are still unknown. In this study, we examined the autocrine effects of TGF-/3on the cell growth and also examined the modulation of TGF-fi receptor levels by EGF in mouse clonal osteoblast-like cell line MC3T3-El.
Materials and Methods Materials and cells
MC3T3-El cells were established in 1981 as a cell line from a newborn C57BW6 mouse calvaria [15]. The cells, in 10 cm diameter plastic culture dishes (Corning Glass Works, Corning, NY) were cultured at 37 “C in a humidified atmosphere of 5% CO2in air. The culture medium was a-MEM (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS, Irvine Scientific, Santa Ana, CA). Cells were subcultured every 3 days after dispersion with 0.003% pronase E (Kaken Chemical, Tokyo, Japan) and 0.002% disodium ethylenediaminetetraacetic acid in calcium- and magnesium-free PBS. CC164 cells (kindly provided by Dr. Ishii, Laboratory of Mitsubishikasei, Yokohama, Japan) were maintained in RPM1 (Gibco) with 10% FBS. TGF-@,, [lz51]TGF-/3, and anti-TGF-p, immunoglobulin were purchased from R&D Systems, (Minneapolis, MN). EGF was purchased from Wakunaga Pharmaceutical (Hiroshima, Japan). TGF-/?, cDNA probe was a kind gift from Dr Ric Derynck (Genentech, CA). Incorporation of PHI-thymidine
MC3T3-El cells were plated in 6-wellculture dishes in a-MEM with 10% FBS, and incubated for 48 h and were then washed with serum-free a-MEM and cultured in fresh medium containing 10% FBS. Cells were incubated with TGF-/I, (1 @ml) or EGF (lo’* M) for 12-48 h, 37 kBq [3H]-thymidinewas added to each well for the last 3 h of culture. Then trichloroacetir:acid precipitable material was solubilized in
127 0.2 ml of 0.2 N NaOH and the radioactivity of the material was determined in an Isocap 300 liquid scintillation counter (Hewlett Packard). [‘251]-TGF-~,binding studies MC3T3-El cells (5 X 104)were plated in 24-well Falcon plastic dishes and cultured in a-MEM with 10% FBS for 3 days. When the cells were subconfluent, whole cell monolayer binding assays were performed as previously described [ 14,161. In binding saturation studies, cells were incubated with 4-320 pM [1251]-TGF-~,with or without loo-fold excess of unlabeled TGF-#l, in 0.1% BSA/Dulbecco’s modified Eagle’s medium for 3 h at 15 “C. Then the cells were washed four times with icecold binding buffer containing 0.1% BSA and solubilized in 1% Triton X-100, 20 mM Hepes pH 7.4, and 10% glycerol buffer for 30 min at 37 “C. Radioactivity in aliquots of cell lysates was determined in a gamma counter (efficiency for **‘I,75%). Scatchard analysis [17] was performed to determine the Kd and maximum number of binding sites. Cell numbers were determined in replicate wells treated in the same way, The effects of preincubations with 10 nM EGF for 18 h on [‘251]-TGF-#?, specific binding were examined using 2-250 pM [‘251]-TGF-#l,with or without lOOfold excess of unlabeled TGF-/l,. The values of [ 1251]-TGF-/3,specific bindings were normalized by cell numbers. The effects of preincubation with EGF (0.1-100 nM) for 5-45 h on [*251]-TGF-#?, specific binding were also examined. Affinity labeling studies The procedure used was as described previously [ 18,191. Confluent monolayers of MC3T3-El cells were incubated in 10 cm* tissue culture plates with 125 pM [1251]TGF$$ with or without loo-fold excess of unlabeled TGF-#l,. After incubating for 4 h at 15 “C, the monolayer cells were washed with cold binding buffer, and the crosslinking agent disuccinimidyl suberate (DSS), 0.25 mM, (Pierce, Rockford, IL) was added for 15 min. The cross-linking reaction was quenched by adding 0.25 M sucrose, 10 mM Tris pH 7.4, 1 mM EDTA, 0.3 mM phenylmethylsulfonyl fluoride, 1 pglml pepstatin and 1 &ml leupeptin. Then the cells were scraped off the plates, collected by centrifugation at 12 000 x g at 4 “C for 10 min and extracted with Triton X-100 as described by Masague and Like [20]. The detergent-soluble fraction was subjected to SDS-polyacrylamide gel electrophoresis (5-19% SDS gradient) as described previously [21]. The gels were stained with 0.25% Coomassie Brilliant Blue, destained with 7% acetic acid, dried, and exposed to Kodak XARJ X-ray film at -70 “C for 7 days. Quantitative analysis of TGF-/3activity in conditioned medium TGF-p activity in the conditio:ied medium was assessed by measuring the inhibition of [3H]-thymidine incorporation into CC1 64 mink lung epithelial cells as described previously [16,22,23]. The CC1 64 cells (1 ;’ 104) were incubated with unconcentrated conditioned medium or a-MEM containing various concentrations of TGF@i in 96-well plates (Corning). After 24 h incubation, 37 kBq/well [3H]-thymidine was added and incubation continued for 4 h. Then radioactivity incorporated into
128 CC164 cells was determined. TGF-j3activity was determined from a standard curve for TGF$, prepared in parallel. For neutralization studies, rabbit anti-TGF$ immunoglobulin (IgG) or non-immune IgG were used. RNA preparationand northernblotanalysis
cellular RNA was isolated from MC3T3-El cells by the guanidine cesium chloride method as described by Chirgwin et al. [24]. Northern blotting was performed by the method of Davis et al. [24]. Twenty yg of total RNA was denatured, fractionated in 1% agarosel0.66 M formaldehyde gel and transferred to a nitrocellulose filter. The filter was baked at 80 “C for 2 h, prehybridized and hybridized at 42 “C with a [j2P]-labeledTGF-/l, cDNA probe in 50% formamide, 5x SSC, 5 x Denhardt’s solution, 50 mM sodium phosphate (pH 7.0), 250 ,ug/ml denatured salmon sperm DNA and 0.1% SDS. The filter was washed under highly stringent conditions and autoradiographed. Total
Statistical analysis
Values were expressed as means and standard errors. Homoscedasticity of data was analyzed by Bartlett test. The significanceof differences was assessed by analysis of variance, followed by multiple comparisons of Scheffe or Turkey. P values of less than 0.01 or 0.05 were considered significant. All experiments were repeated two or three times with similar results and representative experiments are shown.
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anti-TGF-0 Ab
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+
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-
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Fig. 1. TGF-fl activity in conditioned mediumof MC3DEl cells. Twenty-five~1 of conditioned media from MC3’TSEl cells cultured for 48 h were added to CC164 cell cultures and incubated for 24 h after which [3H]-thymidineincorporationinto CC164 cells was assessed. Results are expressedas percentages of [3H]-thymidineincorporated into CCI 64 cells vs that in the absence of the conditioned media. Control, RPM1medium;Ab, antibody @g/ml). 'Pc 0.05; **PC 0.01.
129 Results TGF-/3 activityin MC3T3- El conditioned medium
DNA synthesis of CC1 64 cells was inhibited by serum-free conditioned medium of MC3T3-El cells. The TGF-#l activity in their culture medium was calculated to be 2040 + 157 pg/ml/x lo6 cells148 h by the interpolation of values to a standard curve for TGF-/I,. TGF-/3 is reported to be mainly produced as a latent form that can be activated by acid or heat treatment [16,22]. As shown in Fig. 1, acid-treated conditioned medium inhibited DNA synthesis of CC1 64 cells more than untreated one in 40%. Moreover, TGF-/I, activity in acid-treated conditioned medium from MC3T3-El cells was neutralized by anti-TGF$l IgG but not by non-immune IgG. These results suggested that MC3T3-El cells secreted TGF-/I,. TGF$, mRNA (2.5 kb) was detected in MC3T3-El cells by Northern blot analysis using TGF-b, cDNA as a probe (Fig. 2). These results show that these osteoblast-like cells produce and secrete TGF-p,. Effects of TGFj3, on DNA synthesis
TGF-#l, added to the culture medium with 10% FBS stimulated the [3H]-thymidine
,
-
2.5 kb
Fig. 2. RNA blot analysis of TGF-/I mRNA MC3T3-El cells. Total cellular RNA was isolated from MC3T3-El cells and 20 pg was electrophoresed in 1% agarosel0.56 M formaldehyde gel. Fractionated RNA transferred to a nitrocellulose filter and hybridized with [“*PI-TGF-B, cDNA and autoradiographed.
130
**
I I
C
T
C
E
T
C
E
T 48
12 ~ncubati&4tirne
(hr)
E
Fig. 3. Effects of TGF-fit and EGF on [3H]-thymidine incorporation into MC3T3-El cells. Cells were plated into 4well culture dishes in a-MEM with 10% FBS, and after incubation for 48 h, TGFj3 (1 nglml) or EGF (lo’* M) was added for 12-48 h. Cells were pulsed with 37 kBq of {5H].thymidine for the last 3 h of the culture period. C, control; T, TGF-/3, (1 @ml); E, EGF (10-s M). **P c 0.01.
incorporation into cells. Although the effects of TGF-b were not significantat 12 h, significant stimulation was observed after 24 h and 48 h incubation of cells with TGF-JS(Fig. 3). EGF stimulated DNA synthesis after incubation for 24 h and 48 h. TGF$, also stimulated [3H]-thymidineincorporation to the cells in serum-free medium but the increase was not significant (data not shown).
TGFP
+
-
Fig. 4. Affinity cross linking of TGF-/I receptors in MC3T3-El cells. Autoradiograms of [1251]-TGF-b, (125 PM) crosslinked to cell surface proteins in the presence (left) or absence (right) of lOO-foldexcess of unlabeled TGF-#I,.
131
B/F
TGFBl (PM)
Fig. 5. Scatchard analysis of [tzsI].TGF$?, bindings in MC3T3-El cells treated with 10 nM EGF for 18 h (A) or without EGF ( ). MC3T3mEl cells (5 x 104) were used for binding studies. Details are described in Materials and Methods. Inset: specific binding of [‘251]-TGF-#I in cells treated with 10 nM EGF for 18 h (A) or control (0). 2-250 pM [‘251]-TGF-/?were added for the last 3 h of the incubation period with or without lOO-foldexcess of unlabeled TGF-j?,.
Expression of TGF-/?receptorin MC3T3-El cells Cross-linking studies in MC3T.3-El cells revealed three specific TGF-b receptors with molecular masses of 280,95 and 65 kDa (Fig. 4). Figure 5 (inset) shows the saturation curve in [1251]-TGF-j3,binding studies. Scatchard analysis indicated that MC3T3-El cells possessed a single class of high affinity TGF-B receptor (2.8 X 104 sites per cell, Kd = 196 PM).
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10
20
30
EGF treatment time
40
I 50
(hr)
Fig. 6. Time-dependent inhibition of [ 1251]-TGF-/? specific binding by EGF (10 nM). 74 pM [ 1251]-TGF-/I was added for the last 3 h of the incubation period and all receptor binding assays were performed at the same time under identical conditions to avoid interassay deviation. **P C 0.01.
132
.s 2,ooc g g ._ % 6 1,000 b zi N
gfg. 7. Dose-dependent effect of EGF on the reduction in TGF+I bindings in MC3T3-El cells. Cells were incubated with various doses of EGF (10-lo-lo-’ M) for 18 h and [lzsI]-TGF-@binding assays were performed as in Fig. 6. +P < 0.05. **P < 0.01.
Effect of EGFon cell growthand itseffect on TGF-fi receptorlevel After the incubation with 10 nM EGF for 18 h, [12SI]-TGF-/3, specific binding to
MC3T3-El cells was assessed. The treatment by EGF did not alter the binding affinity of the TGF-Breceptors, but reduced their number (1.7 x lo4 sites per cell, Kd = 207 PM). Figure 6 shows the time course of the reduction in TGF-/3bindings in
I-*-lF=F=il 3 20( f ii! 6 ._
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Ffg. 8. Effect of EGF on the stimulation of [3H]-thymidine incorporation into MC3T3-El cells by [email protected] cells were exposed to TGF-/I (1 ng/ml) and/or EGF (10 nM) for 18 h in a medium containing 10% FBS and then washed with serum-free a-MEM and incubated with TGF-/3 (1 @ml) in medium containing 10% FBS. After a 24-h period, incorporation of [3H]-thymidme was determined as described in Materials and Methods. *P< 0.05. l*P< 0.01.
133 the cells by 10 nM EGF; incubation with EGF for 20 h reduced the [1251]-TGF-pspecific binding by about 40% and this level remained reduced up to 48 h. When MC3T3-El cells were incubated with various concentrations of EGF for 18 h, EGF decreased the level of TGF-/I receptors dose-dependently, and 10 nM EGF caused a significant decrease in TGF-/! receptors (Fig. 7). Although TGF-@ stimulated the [3H]-thymidine incorporation into MC3T3-El cells, TGF-#I failed to stimulate it after the EGF treatment for 18 h (Fig. 8). This result also supports the fact that EGF down-regulated the TGF-p receptor level in these cells.
Discussion MC3T3-El cells, a clonal line of osteoblastic cells derived from a mouse calvaria, have the phenotypic characteristics of osteoblasts: they synthesize type I collagen and have high alkaline phosphatase activity and high responsiveness to PTH and 1,25dihydroxyvitamin D, [26,27] and in prolonged culture they form bone in vitro ]281. Although there are several reports about the effects of TGF-/l on isolated osteoblast-like cells [6-131, results are not consistent. We found that treatment with TGF$, for 24 and 48 h stimulated proliferation of MC3T3-El cells, whereas Noda and Rodan reported that treatment with TGF-/I, for 6 days inhibited the growth of MC3T3-El cells [6]. These contradictory results were probably due to the differences in the incubation time because, consistent with our findings, Ibbotson et al. [7] reported that treatment of MC3T3-El cells with TGFq!?, for 24 h increased the cell numbers and DNA content. Ueno et al. [9] reported that the effects of TGF-/l on MC3T3-El cells appeared to be biphasic. The short-term exposure of cells to TGF-/3 may be during the cellular differentiation into osteoblastic phenotype with decreased cellular proliferation, whereas prolonged treatment causes cellular proliferation and inhibition of differentiation into osteoblastic phenotype. The prolonged treatment of TGF-#I seems to be consistent with our data. Robey et al. [S] found that osteoblasts synthesize TGF-/3 and have high affinity receptors for TGF-fi, and they suggested that the growth of these cells might be stimulated by TGF-/3 in an autocrine fashion. Noda and Vogel [29] also reported that bovine basic fibroblast growth factor increased the steady-state level of 2.5 kb TGF-p, mRNA in several osteoblast-like cells such as MC3T3-El cells. We have shown that MC3T3-El cells expressed TGF-/l on the bioassay system using CC1 64 cells and we have detected the 2.5 kb TGF-pi mRNA which is consistent with that reported by Noda and Vogel. Moreover, we have shown that these cells have a single class of high affinity receptor for TGF-/l. Wakefield et al. [ 141reported that the TGF$ binding was not altered by various agents that affected the cellular response to TGF-P, but that only EGF caused 20% decrease in TGFj3 binding in NRK cells. In the present study we found that EGF decreased the binding of TGF-8 to MC3T3El cells dose-dependently and that treatment of the cells with 10 nM EGF for 20 h
134 resulted in about 40% decrease in TGF$ receptor levels. Ueno et al. [9] reported that the exposure of MC3T3-El cells to TGF-/I initially increased the cell surface EGF receptor level and that the prolonged exposure to TGF-@resulted in a decrease in EGF receptors. They also found that TGF-/I did not enhance the stimulatory effect of EGF on cellular proliferation. Thus, they concluded that TGF-pi stimulated the cellular proliferation of osteoblastic MC3T3-El cells independently from its effect on modulating the EGF receptor levels. In this work we found that TGF-/l stimulated cellular proliferation, and that this effect was inhibited when the TGF-/3receptor level was suppressed by EGF. These findings suggest that the effect of TGF-@on cellular proliferation is modulated by the TGF-fi receptor level. The precise role of TGF-fl and its receptors remains unclear, but they must be important in osteoblast cells because osteoblasts synthesize and secrete large amounts of TGF-b. TGF-b is reported to decrease the alkaline phosphatase activity in MC3T3-El cells [6,7,9]. Thus TGF-#imay have roles not only in cell growth but also in differentiation in osteoblastic cells. If so, our finding of down-regulation of the TGF-J? receptor levels by EGF might represent a cellular protection mechanism which prevents excessive cellular proliferation by TGF-/3 by modulating cellular differentiation.
Acknowledgements
We thank Dr. H. Kurose and Dr. Y. Seino (Department of Pediatrics) for their generous supply of MC3T3-El cells and kind suggestionsin our work.
References 1 Roberts AB, Anzano MA, Lamb LC, Smith JM, Spom MB. New class of transforminggrowth factors potentiated by epidermal growth factor: isolation from non-neoplastic tissues. Proc Nat1Acad Sci USA 1981;78:5339-5343. 2 Tucker RF, Volkenant ME, Branum EL, Moses EL. Comparisonof intra- and extracellulartransforminggrowth factorsfrom non-transformedand chemicallytransformedmouse embryo cells. Cancer Res 1983;43:1581-1586. 3 Spom MB, Roberts AB, Wakefield LM, Assoian RK. Transforminggrowth factor-@ biological function and chemicalstructure. Science 1986;233:532-534. 4 Centrella M, Canalis E. Transformingand non-transforminggrowth factors are present in medium conditioned by fetal rat calvariae. Pmc Nat1Acad Sci USA 1985;82:7335-7339. 5 Robey PG. Young MF, Flanders KC, Roche NS, Kondaiah P, Reddi AH, Termine JD, Sporn MB, Roberts AB. Gsteoblasts synthesize and respond to transforminggrowth factor-type-/l (TGF-/3)in vitro. J Cell Biol1987;105:457-463. 6 Noda M, Rodan GA. Type-/Jtransforminggrowth factor inhibits proliferation and expression of alkaline phosphatasein murine osteoblast-likecells. Biochem Biophys Res Commun 1986;140:56-65. 7 Ibbotson K, Orcutt CM, Anghn AM, D’Souxa SM. Effects of transforminggrowth factorj3, and /t2 on a mouse clonal, osteoblast-like cell line MC3T3-El. J Bone Min Res 1989;4:37-45. 8 Eiford PR, Guenther HL, Felix R, Cecchini MG, Fleisch H. Transforminggrowth factor-@reduces
135 the phenotypic expression of osteoblastic MC3T3-El cells in monolayer cdture. Bone 1987;8:259-262. 9 Ueno S, Yamamoto I, Yamamuro T, Okumura H, Ohta S, Lee K, Kasai R, Konishi J. Transforming growth factor fi modulates proliferation of osteoblastic cells: relation to its effect on receptor levels for epidermal growth factor. J Bone Min Res 1989;4:165-171. 10 Centrella M, McCarthy TL, Canalis E. Skeletal tissue and transforming growth factor ,3. FASEB 1988;2:3066-3073. 11 Pfeilschifter J, Seyedin SM, Mundy GR. Transforming growth factor beta inhibits bone resorption in fetal rat long bone cultures. J Clin Invest 1988;82:680-685. 12 Wrana JL, Maeno M, Hawrylyshyn B, Yao K, Domenicucci C, Sodek J. Differential effects oftransforming growth factor-/l on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations. J Cell Biol1988;106:915-924. 13 Antosz ME, Bellows CG, Aubin JE. Effects of transforming growth factor @and epidermal growth factor on cell proliferation and formation of bone nodules in isolated fetal rat calvaria cells. J Cell Physiol1989;140:386-395. 14 Wakefield LM, Smith DM, Masui T, Harris CC, Sporn MB. Distribution and modulation of the cellular receptor for transforming growth factor-beta. J Cell Biol1987;105:965-975. 15 Kodama H, Amagi Y, Sudo H, Kasai S, Yamamoto S. Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Jpn J Oral Biol1981;23:899-901. 16 Valverius EM, Walker-Jones D, Bates SE, Stampfer MR, Clark R, McCormick F, Dickson RB, Lippman ME. Production of and responsiveness to transforming growth factor+ in normal and oncogene-transformed human mammary epithelial cells. Cancer Res 1989;49:6269-6274. 17 Scatchard G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949;51:660-672. 18 Segarini PR, Rosen DM, Seyedin SM. Binding of transforming growth factor-/l to cell surface proteins varies with cell type, Molec Endocrinol1989;3:261-272. 19 Arteaga CL, Tandon AK, von Hoff DD, Osborne CK. Transforming growth factorb: potential autocrine growth inhibitor of estrogen receptor-negative human breast cancer cells. Cancer Res 1988;48:3898-3904. 20 Masague J, Like B. Cellular receptors for type p transforming growth factor: ligand binding and affinity labeling in human rodent cell lines. 3 Biol Chem 1985;260:2636-2645. 21 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteri0phage.T. Nature 1970;227:680-685. 22 Kim IC, Schomberg DM. The production of transforming growth factor-/l activity by rat granulosa cell cultures. Endocrinology 1989;124:1345-1351. 23 Knabbe C, Lippman ME, Wakefield LM, Flanders KC, Kasid A, Derynck R, Dickson RB. Evidence that transforming grawth facto@ is a hormonally regulated negative growth factor in human breast cancer cells. Cell 198?,48:417-428. 24 Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochem 1979;18:5294-5299. 25 Davis LG, Dibner MD, Battey JF. Formaldehyde gel for electrophoretic separation of RNA and northern blot. In: Basic Methods in Molecular Biology. New York: Elsevier, 1986;143-146. 26 Ghta S, Shigeno C, Yamamoto I, Okumura H, Lee K, Ueno S, Konishi J, YamamuroT. Parathyroid hormone down-regulates the epidermal growth factor receptor in clonal osteoblastic mouse Calvarial cells, MC3T3-El: possible mediation by adenosine 3’,5’-cyciic monophosphate. Endocrinology 1989;124:2419-2426. 27 Kurihara N, Ikeda K, Hakeda Y, Tsunoi M, Maeda N, Kumegawa M. Effect of 1,25dihydroxyvitamin D3 on alkaline phosphatase activity and collagen synthesis in osteoblastic cells, clone MC3T3El. Biochem Biophys Res Commun 1984;119:767-771. 28 S&o H, Ko&+ma H, Amagai Y, Yamamoto S, Kasai S. In vitro differentiation and Calcification in a new cional osteogenic cell line derived from newborn mouse calvaria. J Cell Biol1983;96:191-198. 29 Noda M, Vogel R. Fibroblast growth factor enhances type /?i transforming growth factor gene expression in osteoblast-iike cells. J Cell Biol1989;109:2529-2535.
Elsevier
125
BAM 00397
Epidermal growthfactor attenuates cell proliferationby down-regulatingthe transformin growth factor-p receptor in the osteoblasticcell line MC3T3-El
Hiroaki Jikihara’, Hiromasa Ikegami’, Masahiro Sakata’, Ken-ichiro Morishige’, Yoshiko Fujita’, Hirohisa Kurachi’, Akira Miyake’, Osamu Tanizawa’ and Naoki Terakawa* ‘Department of Obstetricsand Gynecology, Osaka UniversityMedical School, Osaka, and 2Department of Obstetricsand Gynecology, Tottori Universio Medical School, Tottori, Japan
(Received 5 November 1990) (Accepted 2 July 1991)
summary We investigated the role of transforming growth factor /?I (TGF-8,) in growth regulation of the murine osteoblastic cell line, MC3T3-El. We detected TGF-/3 activity in the conditioned medium of MC3T3-El cells and its activity was increased by acid treatment of the bioassay system using CC164 cells. Northern blot analysis revealed the expression of TGF-fi, precursor messenger ribonucleic acid (mRNA). MC3T3-El cells possessed a single class of high affinity TGF-/3, receptor (2.8 x 104sites per cell, Kd = 196 PM). Cross-linking studies revealed three specific TGF-B receptors with molecular masses of 65,95 and 280 kDa. Both TGF-/I and epidermal growth factor (EGF) stimulated rH]-thymidine incorporation into cells. EGF decreased the number of TGF-#? receptor dose-dependently, and pretreatment of cells with 10 nM EGF attenuated cell growth by TGF-@. All these data suggested that TGF-@might be an autocrine growth factor in murine osteoblastic MC3T3-El cells and that EGF might modulate the growth of cells by down-regulating the TGF-/I receptor level.
Key words: Transforming growth factor-@; Transforming growth factor-p receptor; Epidermal growth factor; Osteoblastic MC3T3-El cell line
Introduction
Transforming growth factor-@,(TGF-PI), a potent modulator of cell growth, is a polypeptide with a molecular mass of 25 000, consisting of two subunits [1,2]. Correspondence to: H. Jikihara, Department of Obstetrics and Gynecology, Osaka University Medical School, l-l-50, Fukushima, Fukushimaku, Osaka 553, Japan. 0169~6009i91/$03.50@)1991 Elsevier Science Publishers B.V.
126 TGF-#lcan promote the growth of cells derived from mesenchymal tissues, such as normal rat kidney (NRK) fibroblasts [1,2]. TGF-/I, has also been suggested to have important functions in bone metabolism because it is synthesized and expressed abundantly in bone cells [3-51. There have been several controversial reports about the differentiation and proliferation by TGF-13,in various osteoblast-like cells [6-131. The effects of TGF-/I, on MC3T3-El cells appeared to be biphasic, possibly reflecting the complex mechanisms of action: the short term exposure of cells to TGF-/I, inhibits the proliferation, whereas chronic exposure of these cells to TGF-& stimulates cellular proliferation and inhibits the differentiation into osteoblastic phenotype [7,9]. Robey et al. [5] examined the binding of TGF-j?in various types of bone-derived cells and suggested a possible action of TGFq3 in an autocrine manner in osteoblast cells. TGF-fi receptor modulation is not well understood. So far, only epidermal growth factor (EGF) has been reported to down-regulate TGF-/3receptor in NRK cells [14]. EGF is a polypeptide growth factor with a wide range of effects on the growth and differentiation of a variety of cells. But its effects on TGF-/3receptors are still unknown. In this study, we examined the autocrine effects of TGF-/3on the cell growth and also examined the modulation of TGF-fi receptor levels by EGF in mouse clonal osteoblast-like cell line MC3T3-El.
Materials and Methods Materials and cells
MC3T3-El cells were established in 1981 as a cell line from a newborn C57BW6 mouse calvaria [15]. The cells, in 10 cm diameter plastic culture dishes (Corning Glass Works, Corning, NY) were cultured at 37 “C in a humidified atmosphere of 5% CO2in air. The culture medium was a-MEM (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS, Irvine Scientific, Santa Ana, CA). Cells were subcultured every 3 days after dispersion with 0.003% pronase E (Kaken Chemical, Tokyo, Japan) and 0.002% disodium ethylenediaminetetraacetic acid in calcium- and magnesium-free PBS. CC164 cells (kindly provided by Dr. Ishii, Laboratory of Mitsubishikasei, Yokohama, Japan) were maintained in RPM1 (Gibco) with 10% FBS. TGF-@,, [lz51]TGF-/3, and anti-TGF-p, immunoglobulin were purchased from R&D Systems, (Minneapolis, MN). EGF was purchased from Wakunaga Pharmaceutical (Hiroshima, Japan). TGF-/?, cDNA probe was a kind gift from Dr Ric Derynck (Genentech, CA). Incorporation of PHI-thymidine
MC3T3-El cells were plated in 6-wellculture dishes in a-MEM with 10% FBS, and incubated for 48 h and were then washed with serum-free a-MEM and cultured in fresh medium containing 10% FBS. Cells were incubated with TGF-/I, (1 @ml) or EGF (lo’* M) for 12-48 h, 37 kBq [3H]-thymidinewas added to each well for the last 3 h of culture. Then trichloroacetir:acid precipitable material was solubilized in
127 0.2 ml of 0.2 N NaOH and the radioactivity of the material was determined in an Isocap 300 liquid scintillation counter (Hewlett Packard). [‘251]-TGF-~,binding studies MC3T3-El cells (5 X 104)were plated in 24-well Falcon plastic dishes and cultured in a-MEM with 10% FBS for 3 days. When the cells were subconfluent, whole cell monolayer binding assays were performed as previously described [ 14,161. In binding saturation studies, cells were incubated with 4-320 pM [1251]-TGF-~,with or without loo-fold excess of unlabeled TGF-#l, in 0.1% BSA/Dulbecco’s modified Eagle’s medium for 3 h at 15 “C. Then the cells were washed four times with icecold binding buffer containing 0.1% BSA and solubilized in 1% Triton X-100, 20 mM Hepes pH 7.4, and 10% glycerol buffer for 30 min at 37 “C. Radioactivity in aliquots of cell lysates was determined in a gamma counter (efficiency for **‘I,75%). Scatchard analysis [17] was performed to determine the Kd and maximum number of binding sites. Cell numbers were determined in replicate wells treated in the same way, The effects of preincubations with 10 nM EGF for 18 h on [‘251]-TGF-#?, specific binding were examined using 2-250 pM [‘251]-TGF-#l,with or without lOOfold excess of unlabeled TGF-/l,. The values of [ 1251]-TGF-/3,specific bindings were normalized by cell numbers. The effects of preincubation with EGF (0.1-100 nM) for 5-45 h on [*251]-TGF-#?, specific binding were also examined. Affinity labeling studies The procedure used was as described previously [ 18,191. Confluent monolayers of MC3T3-El cells were incubated in 10 cm* tissue culture plates with 125 pM [1251]TGF$$ with or without loo-fold excess of unlabeled TGF-#l,. After incubating for 4 h at 15 “C, the monolayer cells were washed with cold binding buffer, and the crosslinking agent disuccinimidyl suberate (DSS), 0.25 mM, (Pierce, Rockford, IL) was added for 15 min. The cross-linking reaction was quenched by adding 0.25 M sucrose, 10 mM Tris pH 7.4, 1 mM EDTA, 0.3 mM phenylmethylsulfonyl fluoride, 1 pglml pepstatin and 1 &ml leupeptin. Then the cells were scraped off the plates, collected by centrifugation at 12 000 x g at 4 “C for 10 min and extracted with Triton X-100 as described by Masague and Like [20]. The detergent-soluble fraction was subjected to SDS-polyacrylamide gel electrophoresis (5-19% SDS gradient) as described previously [21]. The gels were stained with 0.25% Coomassie Brilliant Blue, destained with 7% acetic acid, dried, and exposed to Kodak XARJ X-ray film at -70 “C for 7 days. Quantitative analysis of TGF-/3activity in conditioned medium TGF-p activity in the conditio:ied medium was assessed by measuring the inhibition of [3H]-thymidine incorporation into CC1 64 mink lung epithelial cells as described previously [16,22,23]. The CC1 64 cells (1 ;’ 104) were incubated with unconcentrated conditioned medium or a-MEM containing various concentrations of TGF@i in 96-well plates (Corning). After 24 h incubation, 37 kBq/well [3H]-thymidine was added and incubation continued for 4 h. Then radioactivity incorporated into
128 CC164 cells was determined. TGF-j3activity was determined from a standard curve for TGF$, prepared in parallel. For neutralization studies, rabbit anti-TGF$ immunoglobulin (IgG) or non-immune IgG were used. RNA preparationand northernblotanalysis
cellular RNA was isolated from MC3T3-El cells by the guanidine cesium chloride method as described by Chirgwin et al. [24]. Northern blotting was performed by the method of Davis et al. [24]. Twenty yg of total RNA was denatured, fractionated in 1% agarosel0.66 M formaldehyde gel and transferred to a nitrocellulose filter. The filter was baked at 80 “C for 2 h, prehybridized and hybridized at 42 “C with a [j2P]-labeledTGF-/l, cDNA probe in 50% formamide, 5x SSC, 5 x Denhardt’s solution, 50 mM sodium phosphate (pH 7.0), 250 ,ug/ml denatured salmon sperm DNA and 0.1% SDS. The filter was washed under highly stringent conditions and autoradiographed. Total
Statistical analysis
Values were expressed as means and standard errors. Homoscedasticity of data was analyzed by Bartlett test. The significanceof differences was assessed by analysis of variance, followed by multiple comparisons of Scheffe or Turkey. P values of less than 0.01 or 0.05 were considered significant. All experiments were repeated two or three times with similar results and representative experiments are shown.
t
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2
I
acid
treated
anti-TGF-0 Ab
. -
+
+
-
-
5
control .
Fig. 1. TGF-fl activity in conditioned mediumof MC3DEl cells. Twenty-five~1 of conditioned media from MC3’TSEl cells cultured for 48 h were added to CC164 cell cultures and incubated for 24 h after which [3H]-thymidineincorporationinto CC164 cells was assessed. Results are expressedas percentages of [3H]-thymidineincorporated into CCI 64 cells vs that in the absence of the conditioned media. Control, RPM1medium;Ab, antibody @g/ml). 'Pc 0.05; **PC 0.01.
129 Results TGF-/3 activityin MC3T3- El conditioned medium
DNA synthesis of CC1 64 cells was inhibited by serum-free conditioned medium of MC3T3-El cells. The TGF-#l activity in their culture medium was calculated to be 2040 + 157 pg/ml/x lo6 cells148 h by the interpolation of values to a standard curve for TGF-/I,. TGF-/3 is reported to be mainly produced as a latent form that can be activated by acid or heat treatment [16,22]. As shown in Fig. 1, acid-treated conditioned medium inhibited DNA synthesis of CC1 64 cells more than untreated one in 40%. Moreover, TGF-/I, activity in acid-treated conditioned medium from MC3T3-El cells was neutralized by anti-TGF$l IgG but not by non-immune IgG. These results suggested that MC3T3-El cells secreted TGF-/I,. TGF$, mRNA (2.5 kb) was detected in MC3T3-El cells by Northern blot analysis using TGF-b, cDNA as a probe (Fig. 2). These results show that these osteoblast-like cells produce and secrete TGF-p,. Effects of TGFj3, on DNA synthesis
TGF-#l, added to the culture medium with 10% FBS stimulated the [3H]-thymidine
,
-
2.5 kb
Fig. 2. RNA blot analysis of TGF-/I mRNA MC3T3-El cells. Total cellular RNA was isolated from MC3T3-El cells and 20 pg was electrophoresed in 1% agarosel0.56 M formaldehyde gel. Fractionated RNA transferred to a nitrocellulose filter and hybridized with [“*PI-TGF-B, cDNA and autoradiographed.
130
**
I I
C
T
C
E
T
C
E
T 48
12 ~ncubati&4tirne
(hr)
E
Fig. 3. Effects of TGF-fit and EGF on [3H]-thymidine incorporation into MC3T3-El cells. Cells were plated into 4well culture dishes in a-MEM with 10% FBS, and after incubation for 48 h, TGFj3 (1 nglml) or EGF (lo’* M) was added for 12-48 h. Cells were pulsed with 37 kBq of {5H].thymidine for the last 3 h of the culture period. C, control; T, TGF-/3, (1 @ml); E, EGF (10-s M). **P c 0.01.
incorporation into cells. Although the effects of TGF-b were not significantat 12 h, significant stimulation was observed after 24 h and 48 h incubation of cells with TGF-JS(Fig. 3). EGF stimulated DNA synthesis after incubation for 24 h and 48 h. TGF$, also stimulated [3H]-thymidineincorporation to the cells in serum-free medium but the increase was not significant (data not shown).
TGFP
+
-
Fig. 4. Affinity cross linking of TGF-/I receptors in MC3T3-El cells. Autoradiograms of [1251]-TGF-b, (125 PM) crosslinked to cell surface proteins in the presence (left) or absence (right) of lOO-foldexcess of unlabeled TGF-#I,.
131
B/F
TGFBl (PM)
Fig. 5. Scatchard analysis of [tzsI].TGF$?, bindings in MC3T3-El cells treated with 10 nM EGF for 18 h (A) or without EGF ( ). MC3T3mEl cells (5 x 104) were used for binding studies. Details are described in Materials and Methods. Inset: specific binding of [‘251]-TGF-#I in cells treated with 10 nM EGF for 18 h (A) or control (0). 2-250 pM [‘251]-TGF-/?were added for the last 3 h of the incubation period with or without lOO-foldexcess of unlabeled TGF-j?,.
Expression of TGF-/?receptorin MC3T3-El cells Cross-linking studies in MC3T.3-El cells revealed three specific TGF-b receptors with molecular masses of 280,95 and 65 kDa (Fig. 4). Figure 5 (inset) shows the saturation curve in [1251]-TGF-j3,binding studies. Scatchard analysis indicated that MC3T3-El cells possessed a single class of high affinity TGF-B receptor (2.8 X 104 sites per cell, Kd = 196 PM).
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10
20
30
EGF treatment time
40
I 50
(hr)
Fig. 6. Time-dependent inhibition of [ 1251]-TGF-/? specific binding by EGF (10 nM). 74 pM [ 1251]-TGF-/I was added for the last 3 h of the incubation period and all receptor binding assays were performed at the same time under identical conditions to avoid interassay deviation. **P C 0.01.
132
.s 2,ooc g g ._ % 6 1,000 b zi N
gfg. 7. Dose-dependent effect of EGF on the reduction in TGF+I bindings in MC3T3-El cells. Cells were incubated with various doses of EGF (10-lo-lo-’ M) for 18 h and [lzsI]-TGF-@binding assays were performed as in Fig. 6. +P < 0.05. **P < 0.01.
Effect of EGFon cell growthand itseffect on TGF-fi receptorlevel After the incubation with 10 nM EGF for 18 h, [12SI]-TGF-/3, specific binding to
MC3T3-El cells was assessed. The treatment by EGF did not alter the binding affinity of the TGF-Breceptors, but reduced their number (1.7 x lo4 sites per cell, Kd = 207 PM). Figure 6 shows the time course of the reduction in TGF-/3bindings in
I-*-lF=F=il 3 20( f ii! 6 ._
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Ffg. 8. Effect of EGF on the stimulation of [3H]-thymidine incorporation into MC3T3-El cells by [email protected] cells were exposed to TGF-/I (1 ng/ml) and/or EGF (10 nM) for 18 h in a medium containing 10% FBS and then washed with serum-free a-MEM and incubated with TGF-/3 (1 @ml) in medium containing 10% FBS. After a 24-h period, incorporation of [3H]-thymidme was determined as described in Materials and Methods. *P< 0.05. l*P< 0.01.
133 the cells by 10 nM EGF; incubation with EGF for 20 h reduced the [1251]-TGF-pspecific binding by about 40% and this level remained reduced up to 48 h. When MC3T3-El cells were incubated with various concentrations of EGF for 18 h, EGF decreased the level of TGF-/I receptors dose-dependently, and 10 nM EGF caused a significant decrease in TGF-/! receptors (Fig. 7). Although TGF-@ stimulated the [3H]-thymidine incorporation into MC3T3-El cells, TGF-#I failed to stimulate it after the EGF treatment for 18 h (Fig. 8). This result also supports the fact that EGF down-regulated the TGF-p receptor level in these cells.
Discussion MC3T3-El cells, a clonal line of osteoblastic cells derived from a mouse calvaria, have the phenotypic characteristics of osteoblasts: they synthesize type I collagen and have high alkaline phosphatase activity and high responsiveness to PTH and 1,25dihydroxyvitamin D, [26,27] and in prolonged culture they form bone in vitro ]281. Although there are several reports about the effects of TGF-/l on isolated osteoblast-like cells [6-131, results are not consistent. We found that treatment with TGF$, for 24 and 48 h stimulated proliferation of MC3T3-El cells, whereas Noda and Rodan reported that treatment with TGF-/I, for 6 days inhibited the growth of MC3T3-El cells [6]. These contradictory results were probably due to the differences in the incubation time because, consistent with our findings, Ibbotson et al. [7] reported that treatment of MC3T3-El cells with TGFq!?, for 24 h increased the cell numbers and DNA content. Ueno et al. [9] reported that the effects of TGF-/l on MC3T3-El cells appeared to be biphasic. The short-term exposure of cells to TGF-/3 may be during the cellular differentiation into osteoblastic phenotype with decreased cellular proliferation, whereas prolonged treatment causes cellular proliferation and inhibition of differentiation into osteoblastic phenotype. The prolonged treatment of TGF-#I seems to be consistent with our data. Robey et al. [S] found that osteoblasts synthesize TGF-/3 and have high affinity receptors for TGF-fi, and they suggested that the growth of these cells might be stimulated by TGF-/3 in an autocrine fashion. Noda and Vogel [29] also reported that bovine basic fibroblast growth factor increased the steady-state level of 2.5 kb TGF-p, mRNA in several osteoblast-like cells such as MC3T3-El cells. We have shown that MC3T3-El cells expressed TGF-/l on the bioassay system using CC1 64 cells and we have detected the 2.5 kb TGF-pi mRNA which is consistent with that reported by Noda and Vogel. Moreover, we have shown that these cells have a single class of high affinity receptor for TGF-/l. Wakefield et al. [ 141reported that the TGF$ binding was not altered by various agents that affected the cellular response to TGF-P, but that only EGF caused 20% decrease in TGFj3 binding in NRK cells. In the present study we found that EGF decreased the binding of TGF-8 to MC3T3El cells dose-dependently and that treatment of the cells with 10 nM EGF for 20 h
134 resulted in about 40% decrease in TGF$ receptor levels. Ueno et al. [9] reported that the exposure of MC3T3-El cells to TGF-/I initially increased the cell surface EGF receptor level and that the prolonged exposure to TGF-@resulted in a decrease in EGF receptors. They also found that TGF-/I did not enhance the stimulatory effect of EGF on cellular proliferation. Thus, they concluded that TGF-pi stimulated the cellular proliferation of osteoblastic MC3T3-El cells independently from its effect on modulating the EGF receptor levels. In this work we found that TGF-/l stimulated cellular proliferation, and that this effect was inhibited when the TGF-/3receptor level was suppressed by EGF. These findings suggest that the effect of TGF-@on cellular proliferation is modulated by the TGF-fi receptor level. The precise role of TGF-fl and its receptors remains unclear, but they must be important in osteoblast cells because osteoblasts synthesize and secrete large amounts of TGF-b. TGF-b is reported to decrease the alkaline phosphatase activity in MC3T3-El cells [6,7,9]. Thus TGF-#imay have roles not only in cell growth but also in differentiation in osteoblastic cells. If so, our finding of down-regulation of the TGF-J? receptor levels by EGF might represent a cellular protection mechanism which prevents excessive cellular proliferation by TGF-/3 by modulating cellular differentiation.
Acknowledgements
We thank Dr. H. Kurose and Dr. Y. Seino (Department of Pediatrics) for their generous supply of MC3T3-El cells and kind suggestionsin our work.
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