S T A N L E Y COHEN,* G R A H A M CARPENTER,* and K E N N E T H J. LEMBACH*
Interaction of Epidermal Growth Factor (EGF) with Cultured Fibroblasts I. II. III. IV. V.
Introduction Chemical and Physical Properties of E G F Effects of E G F in Vivo and in Organ Culture Systems Stimulation of Fibroblast Proliferation b y E G F 1 2 5 Interactions of I - E G F with Human Fibroblasts 1 2 5 A. Kinetics of I - E G F Binding 1 2 5 B. Fate of Cell-Bound I-EGF 1 2 5 C . Specificity of I - E G F Binding V I . Conclusions References
265 266 267 268 274 275 279 281 283 284
I. Introduction The presence of a factor in extracts of the submaxillary gland of the male mouse which causes precocious opening of the eyelids and eruption of the incisors in newborn mice was first noted in 1962 (Cohen, 1962). A polypeptide ( M W 6045) responsible for this effect has been isolated and termed epidermal growth factor ( E G F ) . Its biological activity is due to a direct stimulation of the proliferation and keratinization of epidermal tissue. Recent investigations have shown that the growth-stimulating effects of E G F are not limited to epidermal cells; fibroblasts in culture also respond to this polypeptide. The chemical and biological properties of E G F have been reviewed in detail (Cohen and Taylor, 1974; Cohen and Savage, 1974). In this report we are presenting a partial sum* Department of Biochemistry, Vanderbilt ITniversity, School of Medicine, Nashville, Tennessee. 265
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mary of these reviews (Sections II and I I I ) , together with some of our more recent results on the interaction of E G F with human fibroblasts in culture (Sections IV and V ) .
II. Chemical and Physical Properties of EGF E G F is a single-chain polypeptide containing a total of 53 amino acid residues (Taylor et al., 1972). All the common amino acids are present except lysine, alanine, and phenylalanine. The molecule contains 6 halfcystine residues in disulfide bridges. E G F is antigenic and is sensitive to proteolytic digestion. It is heat stable, nondialyzable, and has an isoelectric point at pH 4.6. The complete amino acid sequence of E G F and the location of the three disulfide bridges have been reported (Savage and Cohen, 1972; Savage et al, 1972, 1973) and are shown in Fig. 1. In crude homogenates of the submaxillary gland of the adult male mouse, E G F is a component of a high molecular weight complex. This biologically active complex has been isolated and characterized (Taylor et al., 1970, 1974a). The complex has a molecular weight of approximately 72,000 and can be reversibly dissociated, under a variety of conditions, into two molecules of E G F and two molecules of an EGF-binding protein ( M W 29,000). The EGF-binding protein is an arginine esterase that exhibits a hydrolytic specificity for arginine esters (Taylor et al., 1974b). The additional observation that E G F possesses a carboxy-terminal arginine residue suggests that E G F may be generated from a precursor protein by the possible proteolytic action of the EGF-binding esterase. The EGF-binding esterase is antigenically related, but not identical, to the arginine esterase associated with the nerve growth factor.
FIG. 1. The amino acid sequence and disulfide linkages of epidermal growth factor.
EGF Interaction with Cultured Fibroblasts
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III. Effects of EGF in Vivo and in Organ Culture Systems The precocious opening of the eyelids following the daily subcutaneous injection of microgram quantities of E G F into newborn animals is ascribed mainly to an enhancement of epidermal growth and keratinization, as ascertained from histological studies (Cohen and Elliott, 1963). E G F also stimulates the proliferation of corneal epithelium when applied T topically to the eyes of rabbits w ith corneal wounds (Frati et al, 1972; Savage and Cohen, 1973). A direct stimulatory effect of E G F on epithelial cell proliferation in vitro has been demonstrated in a number of organ culture systems (Cohen, 1965; Turkington, 1969; Savage and Cohen, 1973). These include embryonic skin, embryonic cornea, and mammary gland expiants. The effects of E G F are not species specific; expiants derived from the chick, mouse, and human are responsive. The stimulation of epidermal proliferation in organ cultures of chick embryo skin is dependent upon a number of conditions, among which are the age of the embryo and the presence of dermal cells. A number of biochemical effects of E G F have been studied employing an organ culture system in which sheets of pure epidermis, derived from the back skin of 9-day chick embryos, are cultured on Millipore filters in a variety r of media w ith or without microgram quantities of E G F (Hoober and Cohen, 1967a,b; Cohen and Stastny, 1968; Stastny and Cohen, 1970). The observations reported are summarized below: E G F appeared to rapidly stimulate the transport of certain metabolites. Within 15 minutes after the addition of EGF, there was an approximate 2-fold stimulation of the uptake of radioactive aminoisobutyric acid and uridine into the trichloroacetic acid-soluble fraction of the cells. This uptake was not prevented by inhibitors of protein synthesis such as cycloheximide. E G F stimulated the synthesis and net accumulation of protein and R N A . Under these conditions (epidermal sheets on Millipore filters) no stimulation of D N A synthesis was noted. After a 90-minute incubation period with labeled uridine in the presence and in the absence of EGF, sucrose sedimentation analysis of the postmitochondrial supernatants revealed that the synthesis of all types of cytoplasmic R N A discernible on the gradient (4 S, 18 S, 28 S, and heterogeneous R N A ) were stimulated (approximately 4-8-fold). Soon after the addition of E G F there was a conversion of preexisting
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ribosomal monomers into functional polysomal structures. This ribosomal monomer-polysome conversion was detected on sucrose gradients 30 minutes after the addition of E G F to the culture medium, and was observed even in the presence of cycloheximide or in a simple salt medium. These results suggest, therefore, that the initial transfer of monosomes to polysomes does not require the synthesis of new protein or increased transport of amino acids or glucose. As expected from the increase in the ratio of polysomes to monosomes in the epidermal cells under the influence of E G F , the total ribosomal population from the EGF-treated cells was more active in cell-free protein synthesis than that of control cells. Finally, in a manner similar to that observed in a number of cell types when presented with a growth stimulus, E G F induced a marked (40fold), but transient, increase of ornithine decarboxylase activity in cultures of chick embryo epidermis, resulting in the intracellular accumulation of putrescine. Thus, we have described a series of metabolic alterations which accompany the growth-stimulating effects of E G F on epidermal cells. Many of these changes appear to take place in a variety of cells when a growth stimulus is applied.
IV. Stimulation of Fibroblast Proliferation by EGF Recently we initiated an investigation of animal cells cultured in vitro as a potential experimental system in which to examine the mechanism by which E G F stimulates growth. These studies were prompted by the observation of Armelin (1973) that EGF, at a concentration of 100 ng/ml, could stimulate the proliferation of mouse 3T3 cells, and by our own finding that the growth factor induces a marked increase in ornithine decarboxylase activity in these cells and their SV40 virus transformants. After confirming the results of Armelin, we initiated a study of the interaction of E G F with other cell types in vitro, including various epithelial cells and human skin fibroblasts. In a limited series of studies, we were unable to obtain quiescent, nondividing populations of cultured epithelial cells which could clearly be stimulated to proliferate by EGF. A good growth response of human fibroblasts to E G F was observed, however, and a study of the interaction of the factor with these cells was initiated. Human fibroblasts were selected for further study since it was considered that these cells, which remain diploid and exhibit a finite lifetime in culture (Hayflick and Moorhead, 1961), might retain more normal characteristics than established cell lines, such as 3T3.
EGF Interaction with Cultured Fibroblasts
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Monolayer cultures of human fibroblasts were initiated from expiants of newborn foreskin and were maintained by standard cell culture techniques. Cells were routinely grown in Dulbecco's medium supplemented with 10% fetal calf serum and were utilized between the 6th and 20th passages in culture (split ratio, 1:4). Quiescent, nondividing cells were obtained either by growth of the cultures to confluence (density-dependent growth inhibition) or by nutritional shift-down to a serum concentration limiting cell proliferation. These conditions are generally considered to result in an inhibition of growth in the Gl phase of the cell cycle, possibly at a common regulatory site (Pardee, 1974). In the experimental protocol used in the present studies, a 48-hour interval at confluence or in serum-deficient medium generally preceded the application of a growth stimulus (EGF or serum). Figure 2 shows the time course of D N A replication elicited by the addition to serum-limited cultures of either E G F (5 ng/ml) or serum (10% v/v, 6-7 mg of protein per milliliter). In each case the rate of D N A replication was found to increase after a lag period of approximately 12 hours, reaching a maximum value about 20-24 hours after the stimulus. These data are consistent with the anticipated release from a Gl blockade of cell multiplication, and indicate in addition that the fibroblasts respond to physiologically low concentrations of the growth factor. The latter point is emphasized by the data presented in Fig. 3, which shows the dependence of D N A replication on the concentration of E G F added to the culture medium. EGF was found to elicit a maximal growth response at a concentration of approximately 5 ng/ml (0.8 nilf), while a half-maximal effect, obtained from a double reciprocal plot of the data, T w as observed at about 0.8 ng/ml. At concentrations of E G F above 5 ng/ml, the effect on D N A replication was decreased. These data agree reasonably well with the results of Hollenberg and Cuatrecasas (1973). It should also be noted that a concentration of E G F of as little as 0.1 ng/ml was found to induce a significant (4-5-fold) increase in the rate of D N A replication. On the basis of these results, EGF is as effective in the stimulation of fibroblast proliferation in vitro as the fibroblast growth factor (FGF) purified from pituitary extracts by Gospodarowicz (1974) or the multiplication-stimulating activity ( M S A ) isolated from transformed cells by Dulak and Temin (1973). The stimulation of D N A replication by E G F varied in different experiments from 25 to 100% of that elicited by a serum shift-up. As shown below, the response of the cells to EGF is dependent upon the culture conditions previously used to obtain quiescent cells and, in particular, upon the serum content of the extracellular medium. The initiation of D N A replication in response to E G F was followed
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HOURS FIG. 2 . Induction of D N A replication in human fibroblasts b y epidermal growth 2 factor ( E G F ) . Experimental cultures (Falcon flasks, 2 5 c m ) were initiated from trypsinized cell suspensions and were incubated for 2 4 hours in growth medium containing 1 0 % calf serum. T h e cultures were then washed with balanced salt solution, and a nutritional shift-down to medium containing 1 % calf serum was imposed. After an interval of 4 8 hours to effect growth inhibition, cultures were stimulated b y the addition of either E G F ( 5 n g / m l ) or calf serum ( 7 m g / m l ) . T h e incorporation of Ή - l a b e l e d thymidine ( 1 8 . 4 C i / m m o l e , 1 ^ C i / m l ) into D N A was measured during 1-hour labeling intervals commencing at the indicated times. All points represent the average value of duplicate cultures, which varied b y less than 1 0 % . T h e protein content ranged from 3 0 0 to 4 0 0 μg per culture. Cultures stimulated b y E G F , # unstimulated cultures, χ
φ ; cultures stimulated χ.
b y serum shift-up,
Ο
Ο;
by an increase in cell number. As shown in Table I, E G F was found to induce an overgrowth of confluent cultures, as well as to elicit proliferation of subconfluent, serum-limited cells. Derivatives of E G F lacking the carboxy-terminal two amino acids, Leu-Arg-COOH
( E G F - 2 ) , or lacking the carboxy-terminal five amino
acids, Trp-Trp-Glu-Leu-Arg-COOH
( E G F - 5 ) , were also tested for their
ability to stimulate D N A replication. As shown in Table II, E G F - 2 was as effective
as E G F , while E G F - 5
exhibited only about 1-5% of the
growth-stimulating activity of the native factor. The carboxy-terminal
271
EGF Interaction with Cultured Fibroblasts
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FIG. 3. Stimulation of D N A replication as a function of the epidermal growth factor ( E G F ) concentration. Cultures were incubated for 48 hours following a nutritional shift-down to medium containing 0.5% calf serum. E G F at the indicated 3 concentration was then added at zero time, and the incorporation of H-labeled thymidine (100 m C i / m m o l e , 1 yuCi/ml) was measured during a 4-hour labeling interval beginning at 20 hours. T h e data represent the average of duplicate cultures, which varied b y less than 10%, and have been corrected for the incorporation o b served in unstimulated controls (0.35 ± 0.02 cpm per microgram of protein). T h e cultures contained 300-400 μg of cell protein. T h e inset represents a double reciprocal 3 plot of these data, where V represents the incorporation of H-labeled thymidine in cpm per microgram of cell protein.
pentapeptide was totally inactive in stimulating D N A replication, and no synergism was observed in combination with EGF-5. Thus, the Trp-TrpGlu sequence appears to be critical for full activity of EGF, and as shown in Section V, this sequence was also found to influence significantly the binding of the E G F molecule to human fibroblasts. It is of interest that both E G F - 2 and E G F - 5 possess growth factor activity equivalent to that of E G F when assayed in vivo (Savage et al., 1972). These observations and the present findings thus suggest that the carboxy-terminal pentapeptide may be readily removed in vivo by a trypsinlike protease, reducing the activity of E G F and E G F - 2 to that exhibited by EGF-5.
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I
G R O W T H STIMULATION BY E G F
A
Total cell number X 1 0 "
6
Initial serum concentration (%)
Unstimulated
EGF
Calf Serum
0.5 1.0 10.0
0.39 ± 0.03 0.69 ± 0.03 1.02 ± 0.01
0.71 ± 0 . 0 5 1.06 ± 0.01 1.65 ± 0.01
1.27 ± 0 . 0 4 1.57 ± 0 . 0 8 1.55+0.03
α
Replicate experimental cultures were initiated as described in Fig. 2. After 24 hours, the cells were washed with balanced salt solution and the culture fluids were replaced with growth medium containing various initial concentrations of calf serum. After a growth period of 72 hours, the cultures were supplemented with either E G F (10 n g / m l ) , calf serum ( 1 0 % ) , or no addition. After an additional 72 hours of incubation, cell suspensions were prepared by trypsinization, and the cells were enumerated with the use of a Coulter counter. Duplicate or triplicate flasks were analyzed for each point, and the average cell number and deviation are presented. T A B L E II STIMULATION
OF D N A
Stimulus EGF EGF-2 EGF-5 Pentapeptide Pentapeptide EGF-5
plus
REPLICATION BY DERIVATIVES
OF
EGF
a
Concentration (ng/ml)
Relative stimulation
5 0.5 5 0.5 50 5 50 50 + 50
100 36 95 48 44 15 3 50
(%)
a
Experimental cultures were initiated as described in Fig. 2. Following a 48-hour incubation in growth medium containing 0 . 5 % calf serum, E G F or its derivatives were added at the indicated con3 centrations, and the incorporation of H-thymidine (100 m C i / mmole, 1 ^ C i / m l ) into D N A was measured between 20 and 24 hours after the addition of the stimulus. The results were corrected for the incorporation observed in control flasks without any additions, and are expressed as a percentage of the value obtained with native E G F at a concentration of 5 n g / m l .
The stimulation of D N A replication and cell multiplication elicited by EGF in cultures of human fibroblasts was found to be dependent upon the presence of serum in the culture medium. As shown in Fig. 4, the
EGF
Interaction with Cultured
273
Fibroblasts
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SERUM PROTEIN CONCENTRATION ( MG/ML) FIG. 4 . Effect of the extracellular concentration of serum protein on the stimulation of D N A replication b y epidermal growth factor ( E G F ) . Cultures of human fibroblasts were incubated for 4 8 hours following nutritional shift-down to a growth medium containing 0 . 5 % calf serum as described in Fig. 2 . T h e cells were then washed twice with balanced salt solution and the cultures were incubated for an additional 4 8 hours in media containing the indicated concentration of calf serum protein. After this interval, the cultures received at zero time either E G F ( 5 n g / m l ) or no addition. T h e incorporation of Ή - l a b e l e d thymidine ( 1 0 0 m C i / m m o l e , 1 /uCi/ml) into D N A was measured during the interval from 2 0 to 2 4 hours following the addition of E G F . All points represent the average value of duplicate cultures, which generally varied b y less than 10%. Control cultures stimulated b y the addition of calf serum ( 7 m g / m l ) incorporated 13.7 cpm/Vg per microgram of cell protein. Cultures stimulated with E G F , Ο Ο; unstimulated cultures, φ φ.
addition of E G F to resting cultures preincubated in serum-free medium induced a negligible increase in D N A replication. A marked synergistic effect on D N A replication was observed when the growth factor was added to cells cultured in the presence of low concentrations of serum, which alone did not significantly enhance D N A synthesis. This requirement for serum could not be replaced by comparable levels of bovine serum albumin (data not shown) and therefore probably is not due to the trivial need for extracellular protein. W e have also been unable to eliminate the serum requirement with the use of hydrocortisone, which has been shown by Armelin (1973) and Gospodarowicz (1974) to complement a pituitary factor in stimulating the growth of mouse fibroblasts
Stanley Cohen et al.
274
cultured in the absence of added serum. Similarly, the use of insulin, which has been reported to replace certain requirements of cultured cells for serum, did not allow an E G F response in the absence of serum. The requirement of serum for the proliferative stimulus induced by E G F is in apparent conflict with the results of Hollenberg and Cuatrecasas (1973), who observed an E G F effect on human fibroblasts incubated in medium containing albumin as the only added protein. W e have been able to confirm these results, but in this experimental protocol no attempt is made to eliminate carefully the serum proteins remaining from the previous growth in complete medium. In view of the limited concentration of serum necessary for the response to EGF, it seems likely that sufficient residual serum remains in the experimental cultures under these conditions to obtain an E G F effect on D N A replication in the absence of any added serum. The reasons for the requirement of serum for the E G F response remain unclear. It should be noted that human fibroblasts may be maintained in a viable state for extended intervals in the absence of serum, which argues against the need for the cell survival factor shown to be required by mouse fibroblasts (Lipton et al., 1971). In this regard it should also be stressed that cells preincubated in serum-free medium remain fully responsive to growth stimulation elicited by a serum shift-up. W e have also found, as shown in Section V, that the low concentrations of serum necessary to observe the proliferative response to E G F do not affect the binding of the growth factor to human fibroblasts. The large synergistic effect observed between E G F and serum suggests that the growth factor and a serum component are each providing a complementary activity necessary for cell proliferation.
V . Interactions of
125
I'EGF with Human Fibroblasts
Studies of cell proliferation suggest that the primary event which occurs when mammalian cells are stimulated by exogenous mitogens is the interaction of the mitogen with the plasma membrane of the target cell. However, the significance of mitogen-membrane interactions to the growth control of mammalian cells is not well understood. It is not known whether these membrane interactions have direct or indirect effects on cell growth. In order to investigate the mitogenic activity of EGF, we have studied the binding characteristics of this molecule to human foreskin fibroblasts (Carpenter et al., 1975). To measure
1 2 5
I - E G F binding, replicate cultures in 60-mm Falcon plas-
EGF Interaction with Cultured Fibroblasts
275
tic petri dishes were inoculated with 0.25 X 10° cells in 5 ml of modified Dulbecco's medium supplemented with 10% calf serum. Cultures were incubated at 37° in a humidified atmosphere of 2.5% C 0 2 in air for 4 - 5 e days. Cell densities of 0.5 to 1.0 X 10 cells per dish were attained. In each experiment, duplicate culture:, were harvested by trypsinization and the cell number was determined with a Coulter counter. The human fibroblasts for these studies were used between the 6th and the 18th passage in culture (split ratio, 1:4). Although the number of cells per dish varied e between 0.5 to 1.2 X 10 in different experiments, there was only a 10% variance in cell number per dish in any one experiment. The cells were washed twice with 4-ml portions of Hanks' solution, and 1.4 ml of the binding medium (modified Dulbecco's medium plus 0.1% bovine serum albumin) were added to each dish. Labeled E G F or other components were added to a final volume of 1.5 ml. After incubation at 37°C, un1 2 5 bound I - E G F was removed by washing the cells eight times with a total wash volume of 13 ml of cold Hanks' solution containing 0.1% boT vine serum albumin. To solubilize the cells, 1 ml of 0.5 Ν N a O H w as added to each culture dish. After standing for 1-2 hours at 37°C, the contents of each dish was transferred to counting vials and the radioactivity measured. All experiments were performed in duplicate or triplicate. "Specific" binding was determined by measuring the difference in cellbound radioactivity in the presence and absence of 20 /xg of unlabeled EGF. The cell-bound radioactivity in the presence of this excess amount of E G F was considered to be "nonspecific." In the experiments reported here, the "nonspecific" binding amounted to less than 2% of the total binding. All the data have been corrected for this low background.
A . Kinetics of I-EGF Binding 125
The binding of E G F to fibroblasts has been studied by utilizing prepa12r, 1 rations of E G F labeled with I by a lactoperoxidase technique. The 12r, details of this procedure and the isolation of I-labeled monoiodo-EGF by DEAE-cellulose chromatography are described elsewhere (Carpenter et al., 1975). This iodinated derivative is as biologically active as native EGF, judged by the in vivo assay described by Cohen (1962). Also, ex12r periments described below demonstrate that E G F and T - E G F bind to fibroblasts with equal affinities. 12r
The binding of T - E G F to human fibroblasts is both time and temperature dependent as shown in Fig. 5. The data in this experiment indi1
The iodination of E G F was carried out b y Dr. Martin Morrison, St. Jude Children's Research Hospital, Memphis, Tennessee.
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Stanley Cohen et al. CNi
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FIG. 5. Time course of I-labeled epidermal growth hormone ( E G F ) binding to human fibroblasts at 37° and 0 ° C . T o each culture dish containing 1.5 ml of standard 1 2 5 binding medium were added 16 ng of I - E G F , 1438 cpm/ng. The culture dishes were incubated at the indicated temperatures. A t each of the time intervals, duplicate dishes were selected, and the binding reaction was terminated b y washing.
cate that the binding was maximal after 40 minutes' incubation at either 37° or 0 ° C . Interestingly, the amount of labeled E G F bound at 3 7 ° C was 2.5 times the maximal amount bound at 0 ° C . In this regard, Inoue (1974) has reported that Ehrlich ascites tumor cells bind twice as many molecules of
131
I-concanavalin A at 3 7 ° C compared to 0 ° C . The data
in Fig. 5 also show that after 60 minutes' incubation at 3 7 ° C the amount of cell-bound radioactivity decreases. After 5 hours' incubation at this temperature, approximately 33% of the maximal level of bound radioactivity remains bound to the cells. An identical pattern of binding was noted when the 0.1% albumin in the binding medium was replaced by 0.5% (final concentration) calf serum. Additional experiments have demonstrated that continued incubation (up to 24 hours) under these conditions does not result in a further net loss of cell-bound radioactivity. We have found that the inclusion either of the protease inhibitors, benzyl guanidobenzoate or tosyl-L-lysine chloromethyl ketone, or of calf serum (10%) in the initial binding medium prevented the decrease in cell-bound radioactivity during a 6-hour incubation period under the conditions described in Fig. 5. The inhibitory effect of serum may be due to the activity of protease inhibitors normally present in serum. The effect of monoiodo-EGF concentration on the binding reaction is presented in Fig. 6. This experiment shows that the binding of monoiodoEGF to human fibroblasts is a saturable process. At the lowest concentra-
277
EGF Interaction with Cultured Fibroblasts
6
0
100
200
300
400
500
600
'I-EGF (MxlO") FIG. 6. Effect of monoiodo-epidermal growth factor ( E G F ) concentration on binding to human fibroblasts. Indicated concentrations of labeled E G F ( 9 7 , 4 4 8 c p m / n g ) were added to culture dishes and the specific binding was determined after a 1-hour 125 incubation at 3 7 ° C . Inset shows results obtained at lowest I - E G F concentrations. - 12
tion of the growth factor employed, 5.6 Χ Ι Ο M, 15.3% of the labeled 9 E G F was bound to the cells and at the highest concentration, 5.6 X 10~ My 1.9% was bound. It was determined from a Scatchard plot, Fig. 7, of the binding data that the apparent dissociation constant of the binding - 10 reaction under these conditions is 4.3 Χ Ι Ο M and that each cell is 125 capable of binding approximately 97,000 molecules of I - E G F . The low bound/free ratios that were measured at monoiodo-EGF concentrations - 11 less than 5.6 Χ Ι Ο M are believed to be an artifact since a 2-hour incubation time is required to reach maximal binding at such low concentrations. In the experiment illustrated in Figs. 6 and 7, however, a 1-hour incubation period was used to minimize the time-dependent release of cell-bound radioactivity observed at higher E G F concentrations. To obviate these difficulties and to assess their possible influences upon the calculated values for the binding reaction, the following experiment was 1 2 5 carried out. The binding of I - E G F was measured after a 2-hour incubation period in the presence of tosyl-L-lysine chloromethyl ketone to inhibit the release of cell-bound radioactivity. Under these conditions, at n concentrations below 5.6 X 1 0 ~ M, approximately 30% of the labeled EGF remained bound to the cells. Therefore, the increased incubation time and the presence of the protease inhibitor have a significant effect upon the binding reaction at low E G F concentrations. In contrast to the Scatchard plot previously presented in Fig. 7, the bound/free ratios ob-
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Stanley Cohen et al. 0.24r
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0
20
40
60
80
100
120
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FIG. 7. Scatchard plot of binding data presented in Fig. 6.
tained at low hormone concentrations in this experiment (Fig. 8) correlate more closely with the ratios at the higher concentrations. An analysis of the Scatchard plot in Fig. 8 indicates that each cell is capable of bind1 2 5 ing approximately 115,000 molecules of I - E G F and that the binding 10 reaction has an apparent dissociation constant of 2.7 X 10~ M. This value for the apparent dissociation constant is lower than the value of - 10 4.3 Χ Ι Ο M obtained from the binding experiment conducted in the absence of a protease inhibitor and limited to a 1-hour incubation period. However, there is very little difference in the calculated average number 1 2 5 of I - E G F binding sites per cell under the two sets of conditions. Hollenberg and Cuatrecasas (1974) investigated the binding of an uncharacter1 2 5 ized preparation of I - E G F to human fibroblasts and reported that the 10 binding reaction has an apparent dissociation constant of 3 Χ 10~ M, which is in agreement with the value we have reported. However, they 1 2 5 indicated that each cell has an average of 40,000 binding sites for I EGF which is significantly less than the value of approximately 100,000
EGF Interaction with Cultured Fibroblasts
279
0.5 r
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12
plot of binding data determined
chloromethyl ketone (20 /xg/ml).
1 2 5
80
60
BOUND ( Μ χ ΙΟ )
I - E G F , 5.6 χ 10
12
in the
to 5.6 χ Ι Ο
presence of ts-Lys10
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with fibroblasts for 2 hours at 3 7 ° C .
sites that we have reported. This difference may be due to different experimental conditions employed in the two experiments. Recently, Revoltella et al. (1974) demonstrated that the binding of nerve growth factor to neuroblastoma cells is greatly influenced by the stage of the cell cycle. It is not known how the cell cycle affects the binding of E G F to human fibroblasts.
B. Fate of Cell-Bound
TEGF
125
In order to determine the rate at which cell-bound radioactivity is released from cells following binding, we have carried out the following 1 2 5 experiment. Fibroblasts were incubated with I - E G F for 1 hour at 37°C. Then the cultures were washed with warm Hanks' medium to remove all unbound radioactivity, the standard binding medium was added to each culture dish, and the cultures were reincubated at 37°C. At various time intervals thereafter, two cultures were removed and the amount of cell-bound radioactivity was determined. The data (Fig. 9) indicated that the cell-bound radioactivity decreases with a half-life of approximately 60 minutes. In a similar experiment an excess of unlabeled E G F was added after the unbound radioactive E G F was removed to determine whether the presence of the growth factor affected the rate of release.
Stanley Cohen et al.
280
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FIG. 9. Dissociation of cell-bound I-labeled epidermal growth factor ( E G F ) in 125 presence and in the absence of E G F . I - E G F , 6.6 ng/ml, was incubated with fibroblasts for 1 hour at 37°C. The medium was removed and the cells were washed with Hanks' medium containing 0.1% bovine serum albumin. Finally, 1.5 ml of standard binding medium with E G F , 20 Atg/ml (O O), and without E G F (# φ) were added. The cultures were incubated at 37°C, and at the indicated time intervals, duplicate dishes were selected and washed, and the cell-bound radioactivity was determined.
De Meyts et al. (1973) have reported that the rate of release of cellbound insulin is increased by the presence of insulin in the medium. As shown in Fig. 9 the presence of E G F in the medium has no effect on the rate of release of cell-bound radioactivity. 1 2 5 The fact that protease inhibitors prevent the release of bound I - E G F from cells has suggested to us that subsequent to the binding of the growth factor to the plasma membrane the molecule is subject to the action of cellular proteases. Therefore, we have analyzed the radioactivity that is released from cells into the medium. Gel filtration of the radio1 2 active product and of a sample of T - E G F showed that the radioactivity 125 released into the medium was not intact I - E G F , but rather was associ-
EGF Interaction with Cultured Fibroblasts
281
ated with a much smaller molecule. The radioactive material was identified as monoiodotyrosine on the basis of paper electrophoresis at three T different pH values and paper chromatography in tw o different solvents. These results demonstrate that proteolytic degradation of bound 1 2 5 I - E G F occurs with the release of monoiodotyrosine into the medium. However, two important questions in this regard have not been answered. It is not known whether the proteolysis occurs at the cell surface or whether the bound E G F is subject to endocytosis and degradation by intracellular proteases. Also, it is not known wdiether the entire E G F molecule is degraded or whether a limited proteolysis sufficient to liberate one labeled tyrosine residue occurs. This second question may be answered once the location of the iodinated tyrosine residue (s) is known.
C. Specificity of I-EGF Binding 125
1 2 5
W e have determined the specificity of I - E G F binding with respect to the following: (1) native E G F ; (2) derivatives of E G F ; (3) other polypeptide hormones; and (4) various cell types. To determine whether the monoiodinated form of E G F and native E G F bind to fibroblasts with similar affinities, the following experiment was 125 performed. Mixtures of I-labeled monoiodo-EGF and native EGF, containing a saturating quantity of the iodinated derivative and varying amounts of native EGF, were permitted to bind to cells and the amount of cell-bound radioactivity was determined after 1 hour of incubation at 37°C. The results of this experiment, presented in Fig. 10, demonstrate ο
UJ I M m
Si
100
u_ Ο
75
It
50
tu >
25
ζ ω
< _ι or
' UNLABELED EGF ADDED (ng)
FIG. 10. Competition of native epidermal growth hormone ( E G F ) and m o n o i o d o E G F for binding to human fibroblasts. Indicated amounts of E G F and 75 ng of m o n o i o d o - E G F (1784 c p m / n g ) were added simultaneously to culture dishes containing 1.5 ml of standard binding medium. Specific binding of the labeled E G F to fibroblasts was determined after 1 hour of incubation at 37°C. The solid line represents a theoretical dilution curve of 75 ng of m o n o i o d o - E G F by varying amounts of native E G F . The points are experimental values.
282
Stanley Cohen et al.
that the amount of labeled E G F bound to the cells was directly dependent on the relative quantities of native E G F added. Therefore, both iodinated and native E G F appear to bind to the cells with equal affinities. Also, the inclusion of 10% rabbit antiserum to E G F inhibited the binding of 1 2 5 I - E G F to the cells by 99%. In view of the data presented on the relative growth stimulating effects of derivatives of EGF, the binding affinities of E G F - 2 and EGF-5 to 1 2 5 the fibroblasts were estimated by their abilities to compete with I1 2 5 EGF. Although E G F - 2 competed with I - E G F as well as native EGF, EGF-5 was only approximately 5% as effective as the native molecule. The isolated carboxy-terminal pentapeptide had no effect on the binding. The results indicate that of the five carboxy-terminal amino acids only the Trp-Trp-Glu sequence influences the binding of the native molecule. Thus, the binding affinities of these derivatives parallel their growthstimulating effects on human fibroblasts. TABLE BINDING OF
I - E G F TO CULTURED
6
Y-l
xc NC-37 CHO K-NRK MA-184 HPME 3T3 SV101 NRK AR-963 ΕΡΟ-Η8
CELLS
0
T o t a l I - E G F bound 6 ( c p m / 1 0 cells)
Specific binding 1 2 5 of I-EGF 6 ( c p m / 1 0 cells)
90 187 220 167 83 13,500 7,120 3,525 4,224 4,060 803 9,798
Interaction of Epidermal Growth Factor (EGF) with Cultured Fibroblasts I. II. III. IV. V.
Introduction Chemical and Physical Properties of E G F Effects of E G F in Vivo and in Organ Culture Systems Stimulation of Fibroblast Proliferation b y E G F 1 2 5 Interactions of I - E G F with Human Fibroblasts 1 2 5 A. Kinetics of I - E G F Binding 1 2 5 B. Fate of Cell-Bound I-EGF 1 2 5 C . Specificity of I - E G F Binding V I . Conclusions References
265 266 267 268 274 275 279 281 283 284
I. Introduction The presence of a factor in extracts of the submaxillary gland of the male mouse which causes precocious opening of the eyelids and eruption of the incisors in newborn mice was first noted in 1962 (Cohen, 1962). A polypeptide ( M W 6045) responsible for this effect has been isolated and termed epidermal growth factor ( E G F ) . Its biological activity is due to a direct stimulation of the proliferation and keratinization of epidermal tissue. Recent investigations have shown that the growth-stimulating effects of E G F are not limited to epidermal cells; fibroblasts in culture also respond to this polypeptide. The chemical and biological properties of E G F have been reviewed in detail (Cohen and Taylor, 1974; Cohen and Savage, 1974). In this report we are presenting a partial sum* Department of Biochemistry, Vanderbilt ITniversity, School of Medicine, Nashville, Tennessee. 265
266
Stanley Cohen et al.
mary of these reviews (Sections II and I I I ) , together with some of our more recent results on the interaction of E G F with human fibroblasts in culture (Sections IV and V ) .
II. Chemical and Physical Properties of EGF E G F is a single-chain polypeptide containing a total of 53 amino acid residues (Taylor et al., 1972). All the common amino acids are present except lysine, alanine, and phenylalanine. The molecule contains 6 halfcystine residues in disulfide bridges. E G F is antigenic and is sensitive to proteolytic digestion. It is heat stable, nondialyzable, and has an isoelectric point at pH 4.6. The complete amino acid sequence of E G F and the location of the three disulfide bridges have been reported (Savage and Cohen, 1972; Savage et al, 1972, 1973) and are shown in Fig. 1. In crude homogenates of the submaxillary gland of the adult male mouse, E G F is a component of a high molecular weight complex. This biologically active complex has been isolated and characterized (Taylor et al., 1970, 1974a). The complex has a molecular weight of approximately 72,000 and can be reversibly dissociated, under a variety of conditions, into two molecules of E G F and two molecules of an EGF-binding protein ( M W 29,000). The EGF-binding protein is an arginine esterase that exhibits a hydrolytic specificity for arginine esters (Taylor et al., 1974b). The additional observation that E G F possesses a carboxy-terminal arginine residue suggests that E G F may be generated from a precursor protein by the possible proteolytic action of the EGF-binding esterase. The EGF-binding esterase is antigenically related, but not identical, to the arginine esterase associated with the nerve growth factor.
FIG. 1. The amino acid sequence and disulfide linkages of epidermal growth factor.
EGF Interaction with Cultured Fibroblasts
267
III. Effects of EGF in Vivo and in Organ Culture Systems The precocious opening of the eyelids following the daily subcutaneous injection of microgram quantities of E G F into newborn animals is ascribed mainly to an enhancement of epidermal growth and keratinization, as ascertained from histological studies (Cohen and Elliott, 1963). E G F also stimulates the proliferation of corneal epithelium when applied T topically to the eyes of rabbits w ith corneal wounds (Frati et al, 1972; Savage and Cohen, 1973). A direct stimulatory effect of E G F on epithelial cell proliferation in vitro has been demonstrated in a number of organ culture systems (Cohen, 1965; Turkington, 1969; Savage and Cohen, 1973). These include embryonic skin, embryonic cornea, and mammary gland expiants. The effects of E G F are not species specific; expiants derived from the chick, mouse, and human are responsive. The stimulation of epidermal proliferation in organ cultures of chick embryo skin is dependent upon a number of conditions, among which are the age of the embryo and the presence of dermal cells. A number of biochemical effects of E G F have been studied employing an organ culture system in which sheets of pure epidermis, derived from the back skin of 9-day chick embryos, are cultured on Millipore filters in a variety r of media w ith or without microgram quantities of E G F (Hoober and Cohen, 1967a,b; Cohen and Stastny, 1968; Stastny and Cohen, 1970). The observations reported are summarized below: E G F appeared to rapidly stimulate the transport of certain metabolites. Within 15 minutes after the addition of EGF, there was an approximate 2-fold stimulation of the uptake of radioactive aminoisobutyric acid and uridine into the trichloroacetic acid-soluble fraction of the cells. This uptake was not prevented by inhibitors of protein synthesis such as cycloheximide. E G F stimulated the synthesis and net accumulation of protein and R N A . Under these conditions (epidermal sheets on Millipore filters) no stimulation of D N A synthesis was noted. After a 90-minute incubation period with labeled uridine in the presence and in the absence of EGF, sucrose sedimentation analysis of the postmitochondrial supernatants revealed that the synthesis of all types of cytoplasmic R N A discernible on the gradient (4 S, 18 S, 28 S, and heterogeneous R N A ) were stimulated (approximately 4-8-fold). Soon after the addition of E G F there was a conversion of preexisting
268
Stanley Cohen et al.
ribosomal monomers into functional polysomal structures. This ribosomal monomer-polysome conversion was detected on sucrose gradients 30 minutes after the addition of E G F to the culture medium, and was observed even in the presence of cycloheximide or in a simple salt medium. These results suggest, therefore, that the initial transfer of monosomes to polysomes does not require the synthesis of new protein or increased transport of amino acids or glucose. As expected from the increase in the ratio of polysomes to monosomes in the epidermal cells under the influence of E G F , the total ribosomal population from the EGF-treated cells was more active in cell-free protein synthesis than that of control cells. Finally, in a manner similar to that observed in a number of cell types when presented with a growth stimulus, E G F induced a marked (40fold), but transient, increase of ornithine decarboxylase activity in cultures of chick embryo epidermis, resulting in the intracellular accumulation of putrescine. Thus, we have described a series of metabolic alterations which accompany the growth-stimulating effects of E G F on epidermal cells. Many of these changes appear to take place in a variety of cells when a growth stimulus is applied.
IV. Stimulation of Fibroblast Proliferation by EGF Recently we initiated an investigation of animal cells cultured in vitro as a potential experimental system in which to examine the mechanism by which E G F stimulates growth. These studies were prompted by the observation of Armelin (1973) that EGF, at a concentration of 100 ng/ml, could stimulate the proliferation of mouse 3T3 cells, and by our own finding that the growth factor induces a marked increase in ornithine decarboxylase activity in these cells and their SV40 virus transformants. After confirming the results of Armelin, we initiated a study of the interaction of E G F with other cell types in vitro, including various epithelial cells and human skin fibroblasts. In a limited series of studies, we were unable to obtain quiescent, nondividing populations of cultured epithelial cells which could clearly be stimulated to proliferate by EGF. A good growth response of human fibroblasts to E G F was observed, however, and a study of the interaction of the factor with these cells was initiated. Human fibroblasts were selected for further study since it was considered that these cells, which remain diploid and exhibit a finite lifetime in culture (Hayflick and Moorhead, 1961), might retain more normal characteristics than established cell lines, such as 3T3.
EGF Interaction with Cultured Fibroblasts
269
Monolayer cultures of human fibroblasts were initiated from expiants of newborn foreskin and were maintained by standard cell culture techniques. Cells were routinely grown in Dulbecco's medium supplemented with 10% fetal calf serum and were utilized between the 6th and 20th passages in culture (split ratio, 1:4). Quiescent, nondividing cells were obtained either by growth of the cultures to confluence (density-dependent growth inhibition) or by nutritional shift-down to a serum concentration limiting cell proliferation. These conditions are generally considered to result in an inhibition of growth in the Gl phase of the cell cycle, possibly at a common regulatory site (Pardee, 1974). In the experimental protocol used in the present studies, a 48-hour interval at confluence or in serum-deficient medium generally preceded the application of a growth stimulus (EGF or serum). Figure 2 shows the time course of D N A replication elicited by the addition to serum-limited cultures of either E G F (5 ng/ml) or serum (10% v/v, 6-7 mg of protein per milliliter). In each case the rate of D N A replication was found to increase after a lag period of approximately 12 hours, reaching a maximum value about 20-24 hours after the stimulus. These data are consistent with the anticipated release from a Gl blockade of cell multiplication, and indicate in addition that the fibroblasts respond to physiologically low concentrations of the growth factor. The latter point is emphasized by the data presented in Fig. 3, which shows the dependence of D N A replication on the concentration of E G F added to the culture medium. EGF was found to elicit a maximal growth response at a concentration of approximately 5 ng/ml (0.8 nilf), while a half-maximal effect, obtained from a double reciprocal plot of the data, T w as observed at about 0.8 ng/ml. At concentrations of E G F above 5 ng/ml, the effect on D N A replication was decreased. These data agree reasonably well with the results of Hollenberg and Cuatrecasas (1973). It should also be noted that a concentration of E G F of as little as 0.1 ng/ml was found to induce a significant (4-5-fold) increase in the rate of D N A replication. On the basis of these results, EGF is as effective in the stimulation of fibroblast proliferation in vitro as the fibroblast growth factor (FGF) purified from pituitary extracts by Gospodarowicz (1974) or the multiplication-stimulating activity ( M S A ) isolated from transformed cells by Dulak and Temin (1973). The stimulation of D N A replication by E G F varied in different experiments from 25 to 100% of that elicited by a serum shift-up. As shown below, the response of the cells to EGF is dependent upon the culture conditions previously used to obtain quiescent cells and, in particular, upon the serum content of the extracellular medium. The initiation of D N A replication in response to E G F was followed
270
Stanley Cohen et al.
Lu
\O
oc CL
HOURS FIG. 2 . Induction of D N A replication in human fibroblasts b y epidermal growth 2 factor ( E G F ) . Experimental cultures (Falcon flasks, 2 5 c m ) were initiated from trypsinized cell suspensions and were incubated for 2 4 hours in growth medium containing 1 0 % calf serum. T h e cultures were then washed with balanced salt solution, and a nutritional shift-down to medium containing 1 % calf serum was imposed. After an interval of 4 8 hours to effect growth inhibition, cultures were stimulated b y the addition of either E G F ( 5 n g / m l ) or calf serum ( 7 m g / m l ) . T h e incorporation of Ή - l a b e l e d thymidine ( 1 8 . 4 C i / m m o l e , 1 ^ C i / m l ) into D N A was measured during 1-hour labeling intervals commencing at the indicated times. All points represent the average value of duplicate cultures, which varied b y less than 1 0 % . T h e protein content ranged from 3 0 0 to 4 0 0 μg per culture. Cultures stimulated b y E G F , # unstimulated cultures, χ
φ ; cultures stimulated χ.
b y serum shift-up,
Ο
Ο;
by an increase in cell number. As shown in Table I, E G F was found to induce an overgrowth of confluent cultures, as well as to elicit proliferation of subconfluent, serum-limited cells. Derivatives of E G F lacking the carboxy-terminal two amino acids, Leu-Arg-COOH
( E G F - 2 ) , or lacking the carboxy-terminal five amino
acids, Trp-Trp-Glu-Leu-Arg-COOH
( E G F - 5 ) , were also tested for their
ability to stimulate D N A replication. As shown in Table II, E G F - 2 was as effective
as E G F , while E G F - 5
exhibited only about 1-5% of the
growth-stimulating activity of the native factor. The carboxy-terminal
271
EGF Interaction with Cultured Fibroblasts
ο er ο-
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o CL
tr
o o
o
5
10 EGF (NG/ML)
FIG. 3. Stimulation of D N A replication as a function of the epidermal growth factor ( E G F ) concentration. Cultures were incubated for 48 hours following a nutritional shift-down to medium containing 0.5% calf serum. E G F at the indicated 3 concentration was then added at zero time, and the incorporation of H-labeled thymidine (100 m C i / m m o l e , 1 yuCi/ml) was measured during a 4-hour labeling interval beginning at 20 hours. T h e data represent the average of duplicate cultures, which varied b y less than 10%, and have been corrected for the incorporation o b served in unstimulated controls (0.35 ± 0.02 cpm per microgram of protein). T h e cultures contained 300-400 μg of cell protein. T h e inset represents a double reciprocal 3 plot of these data, where V represents the incorporation of H-labeled thymidine in cpm per microgram of cell protein.
pentapeptide was totally inactive in stimulating D N A replication, and no synergism was observed in combination with EGF-5. Thus, the Trp-TrpGlu sequence appears to be critical for full activity of EGF, and as shown in Section V, this sequence was also found to influence significantly the binding of the E G F molecule to human fibroblasts. It is of interest that both E G F - 2 and E G F - 5 possess growth factor activity equivalent to that of E G F when assayed in vivo (Savage et al., 1972). These observations and the present findings thus suggest that the carboxy-terminal pentapeptide may be readily removed in vivo by a trypsinlike protease, reducing the activity of E G F and E G F - 2 to that exhibited by EGF-5.
272
Stanley Cohen et al. TABLE
I
G R O W T H STIMULATION BY E G F
A
Total cell number X 1 0 "
6
Initial serum concentration (%)
Unstimulated
EGF
Calf Serum
0.5 1.0 10.0
0.39 ± 0.03 0.69 ± 0.03 1.02 ± 0.01
0.71 ± 0 . 0 5 1.06 ± 0.01 1.65 ± 0.01
1.27 ± 0 . 0 4 1.57 ± 0 . 0 8 1.55+0.03
α
Replicate experimental cultures were initiated as described in Fig. 2. After 24 hours, the cells were washed with balanced salt solution and the culture fluids were replaced with growth medium containing various initial concentrations of calf serum. After a growth period of 72 hours, the cultures were supplemented with either E G F (10 n g / m l ) , calf serum ( 1 0 % ) , or no addition. After an additional 72 hours of incubation, cell suspensions were prepared by trypsinization, and the cells were enumerated with the use of a Coulter counter. Duplicate or triplicate flasks were analyzed for each point, and the average cell number and deviation are presented. T A B L E II STIMULATION
OF D N A
Stimulus EGF EGF-2 EGF-5 Pentapeptide Pentapeptide EGF-5
plus
REPLICATION BY DERIVATIVES
OF
EGF
a
Concentration (ng/ml)
Relative stimulation
5 0.5 5 0.5 50 5 50 50 + 50
100 36 95 48 44 15 3 50
(%)
a
Experimental cultures were initiated as described in Fig. 2. Following a 48-hour incubation in growth medium containing 0 . 5 % calf serum, E G F or its derivatives were added at the indicated con3 centrations, and the incorporation of H-thymidine (100 m C i / mmole, 1 ^ C i / m l ) into D N A was measured between 20 and 24 hours after the addition of the stimulus. The results were corrected for the incorporation observed in control flasks without any additions, and are expressed as a percentage of the value obtained with native E G F at a concentration of 5 n g / m l .
The stimulation of D N A replication and cell multiplication elicited by EGF in cultures of human fibroblasts was found to be dependent upon the presence of serum in the culture medium. As shown in Fig. 4, the
EGF
Interaction with Cultured
273
Fibroblasts
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0.2
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SERUM PROTEIN CONCENTRATION ( MG/ML) FIG. 4 . Effect of the extracellular concentration of serum protein on the stimulation of D N A replication b y epidermal growth factor ( E G F ) . Cultures of human fibroblasts were incubated for 4 8 hours following nutritional shift-down to a growth medium containing 0 . 5 % calf serum as described in Fig. 2 . T h e cells were then washed twice with balanced salt solution and the cultures were incubated for an additional 4 8 hours in media containing the indicated concentration of calf serum protein. After this interval, the cultures received at zero time either E G F ( 5 n g / m l ) or no addition. T h e incorporation of Ή - l a b e l e d thymidine ( 1 0 0 m C i / m m o l e , 1 /uCi/ml) into D N A was measured during the interval from 2 0 to 2 4 hours following the addition of E G F . All points represent the average value of duplicate cultures, which generally varied b y less than 10%. Control cultures stimulated b y the addition of calf serum ( 7 m g / m l ) incorporated 13.7 cpm/Vg per microgram of cell protein. Cultures stimulated with E G F , Ο Ο; unstimulated cultures, φ φ.
addition of E G F to resting cultures preincubated in serum-free medium induced a negligible increase in D N A replication. A marked synergistic effect on D N A replication was observed when the growth factor was added to cells cultured in the presence of low concentrations of serum, which alone did not significantly enhance D N A synthesis. This requirement for serum could not be replaced by comparable levels of bovine serum albumin (data not shown) and therefore probably is not due to the trivial need for extracellular protein. W e have also been unable to eliminate the serum requirement with the use of hydrocortisone, which has been shown by Armelin (1973) and Gospodarowicz (1974) to complement a pituitary factor in stimulating the growth of mouse fibroblasts
Stanley Cohen et al.
274
cultured in the absence of added serum. Similarly, the use of insulin, which has been reported to replace certain requirements of cultured cells for serum, did not allow an E G F response in the absence of serum. The requirement of serum for the proliferative stimulus induced by E G F is in apparent conflict with the results of Hollenberg and Cuatrecasas (1973), who observed an E G F effect on human fibroblasts incubated in medium containing albumin as the only added protein. W e have been able to confirm these results, but in this experimental protocol no attempt is made to eliminate carefully the serum proteins remaining from the previous growth in complete medium. In view of the limited concentration of serum necessary for the response to EGF, it seems likely that sufficient residual serum remains in the experimental cultures under these conditions to obtain an E G F effect on D N A replication in the absence of any added serum. The reasons for the requirement of serum for the E G F response remain unclear. It should be noted that human fibroblasts may be maintained in a viable state for extended intervals in the absence of serum, which argues against the need for the cell survival factor shown to be required by mouse fibroblasts (Lipton et al., 1971). In this regard it should also be stressed that cells preincubated in serum-free medium remain fully responsive to growth stimulation elicited by a serum shift-up. W e have also found, as shown in Section V, that the low concentrations of serum necessary to observe the proliferative response to E G F do not affect the binding of the growth factor to human fibroblasts. The large synergistic effect observed between E G F and serum suggests that the growth factor and a serum component are each providing a complementary activity necessary for cell proliferation.
V . Interactions of
125
I'EGF with Human Fibroblasts
Studies of cell proliferation suggest that the primary event which occurs when mammalian cells are stimulated by exogenous mitogens is the interaction of the mitogen with the plasma membrane of the target cell. However, the significance of mitogen-membrane interactions to the growth control of mammalian cells is not well understood. It is not known whether these membrane interactions have direct or indirect effects on cell growth. In order to investigate the mitogenic activity of EGF, we have studied the binding characteristics of this molecule to human foreskin fibroblasts (Carpenter et al., 1975). To measure
1 2 5
I - E G F binding, replicate cultures in 60-mm Falcon plas-
EGF Interaction with Cultured Fibroblasts
275
tic petri dishes were inoculated with 0.25 X 10° cells in 5 ml of modified Dulbecco's medium supplemented with 10% calf serum. Cultures were incubated at 37° in a humidified atmosphere of 2.5% C 0 2 in air for 4 - 5 e days. Cell densities of 0.5 to 1.0 X 10 cells per dish were attained. In each experiment, duplicate culture:, were harvested by trypsinization and the cell number was determined with a Coulter counter. The human fibroblasts for these studies were used between the 6th and the 18th passage in culture (split ratio, 1:4). Although the number of cells per dish varied e between 0.5 to 1.2 X 10 in different experiments, there was only a 10% variance in cell number per dish in any one experiment. The cells were washed twice with 4-ml portions of Hanks' solution, and 1.4 ml of the binding medium (modified Dulbecco's medium plus 0.1% bovine serum albumin) were added to each dish. Labeled E G F or other components were added to a final volume of 1.5 ml. After incubation at 37°C, un1 2 5 bound I - E G F was removed by washing the cells eight times with a total wash volume of 13 ml of cold Hanks' solution containing 0.1% boT vine serum albumin. To solubilize the cells, 1 ml of 0.5 Ν N a O H w as added to each culture dish. After standing for 1-2 hours at 37°C, the contents of each dish was transferred to counting vials and the radioactivity measured. All experiments were performed in duplicate or triplicate. "Specific" binding was determined by measuring the difference in cellbound radioactivity in the presence and absence of 20 /xg of unlabeled EGF. The cell-bound radioactivity in the presence of this excess amount of E G F was considered to be "nonspecific." In the experiments reported here, the "nonspecific" binding amounted to less than 2% of the total binding. All the data have been corrected for this low background.
A . Kinetics of I-EGF Binding 125
The binding of E G F to fibroblasts has been studied by utilizing prepa12r, 1 rations of E G F labeled with I by a lactoperoxidase technique. The 12r, details of this procedure and the isolation of I-labeled monoiodo-EGF by DEAE-cellulose chromatography are described elsewhere (Carpenter et al., 1975). This iodinated derivative is as biologically active as native EGF, judged by the in vivo assay described by Cohen (1962). Also, ex12r periments described below demonstrate that E G F and T - E G F bind to fibroblasts with equal affinities. 12r
The binding of T - E G F to human fibroblasts is both time and temperature dependent as shown in Fig. 5. The data in this experiment indi1
The iodination of E G F was carried out b y Dr. Martin Morrison, St. Jude Children's Research Hospital, Memphis, Tennessee.
276
Stanley Cohen et al. CNi
O
I4r
X
LU H
0
2
3 HOURS
4
5
6
125
FIG. 5. Time course of I-labeled epidermal growth hormone ( E G F ) binding to human fibroblasts at 37° and 0 ° C . T o each culture dish containing 1.5 ml of standard 1 2 5 binding medium were added 16 ng of I - E G F , 1438 cpm/ng. The culture dishes were incubated at the indicated temperatures. A t each of the time intervals, duplicate dishes were selected, and the binding reaction was terminated b y washing.
cate that the binding was maximal after 40 minutes' incubation at either 37° or 0 ° C . Interestingly, the amount of labeled E G F bound at 3 7 ° C was 2.5 times the maximal amount bound at 0 ° C . In this regard, Inoue (1974) has reported that Ehrlich ascites tumor cells bind twice as many molecules of
131
I-concanavalin A at 3 7 ° C compared to 0 ° C . The data
in Fig. 5 also show that after 60 minutes' incubation at 3 7 ° C the amount of cell-bound radioactivity decreases. After 5 hours' incubation at this temperature, approximately 33% of the maximal level of bound radioactivity remains bound to the cells. An identical pattern of binding was noted when the 0.1% albumin in the binding medium was replaced by 0.5% (final concentration) calf serum. Additional experiments have demonstrated that continued incubation (up to 24 hours) under these conditions does not result in a further net loss of cell-bound radioactivity. We have found that the inclusion either of the protease inhibitors, benzyl guanidobenzoate or tosyl-L-lysine chloromethyl ketone, or of calf serum (10%) in the initial binding medium prevented the decrease in cell-bound radioactivity during a 6-hour incubation period under the conditions described in Fig. 5. The inhibitory effect of serum may be due to the activity of protease inhibitors normally present in serum. The effect of monoiodo-EGF concentration on the binding reaction is presented in Fig. 6. This experiment shows that the binding of monoiodoEGF to human fibroblasts is a saturable process. At the lowest concentra-
277
EGF Interaction with Cultured Fibroblasts
6
0
100
200
300
400
500
600
'I-EGF (MxlO") FIG. 6. Effect of monoiodo-epidermal growth factor ( E G F ) concentration on binding to human fibroblasts. Indicated concentrations of labeled E G F ( 9 7 , 4 4 8 c p m / n g ) were added to culture dishes and the specific binding was determined after a 1-hour 125 incubation at 3 7 ° C . Inset shows results obtained at lowest I - E G F concentrations. - 12
tion of the growth factor employed, 5.6 Χ Ι Ο M, 15.3% of the labeled 9 E G F was bound to the cells and at the highest concentration, 5.6 X 10~ My 1.9% was bound. It was determined from a Scatchard plot, Fig. 7, of the binding data that the apparent dissociation constant of the binding - 10 reaction under these conditions is 4.3 Χ Ι Ο M and that each cell is 125 capable of binding approximately 97,000 molecules of I - E G F . The low bound/free ratios that were measured at monoiodo-EGF concentrations - 11 less than 5.6 Χ Ι Ο M are believed to be an artifact since a 2-hour incubation time is required to reach maximal binding at such low concentrations. In the experiment illustrated in Figs. 6 and 7, however, a 1-hour incubation period was used to minimize the time-dependent release of cell-bound radioactivity observed at higher E G F concentrations. To obviate these difficulties and to assess their possible influences upon the calculated values for the binding reaction, the following experiment was 1 2 5 carried out. The binding of I - E G F was measured after a 2-hour incubation period in the presence of tosyl-L-lysine chloromethyl ketone to inhibit the release of cell-bound radioactivity. Under these conditions, at n concentrations below 5.6 X 1 0 ~ M, approximately 30% of the labeled EGF remained bound to the cells. Therefore, the increased incubation time and the presence of the protease inhibitor have a significant effect upon the binding reaction at low E G F concentrations. In contrast to the Scatchard plot previously presented in Fig. 7, the bound/free ratios ob-
278
Stanley Cohen et al. 0.24r
I
ι
ι
ι
ι
ι
ι
0
20
40
60
80
100
120
l 2 5
I-E6F
12
BOUND (MxlO )
FIG. 7. Scatchard plot of binding data presented in Fig. 6.
tained at low hormone concentrations in this experiment (Fig. 8) correlate more closely with the ratios at the higher concentrations. An analysis of the Scatchard plot in Fig. 8 indicates that each cell is capable of bind1 2 5 ing approximately 115,000 molecules of I - E G F and that the binding 10 reaction has an apparent dissociation constant of 2.7 X 10~ M. This value for the apparent dissociation constant is lower than the value of - 10 4.3 Χ Ι Ο M obtained from the binding experiment conducted in the absence of a protease inhibitor and limited to a 1-hour incubation period. However, there is very little difference in the calculated average number 1 2 5 of I - E G F binding sites per cell under the two sets of conditions. Hollenberg and Cuatrecasas (1974) investigated the binding of an uncharacter1 2 5 ized preparation of I - E G F to human fibroblasts and reported that the 10 binding reaction has an apparent dissociation constant of 3 Χ 10~ M, which is in agreement with the value we have reported. However, they 1 2 5 indicated that each cell has an average of 40,000 binding sites for I EGF which is significantly less than the value of approximately 100,000
EGF Interaction with Cultured Fibroblasts
279
0.5 r
0.4 LlI
LU
0.3 3 Ο 0Û
0.2
0.1
20
0
40 ,25
I-EGF
FIG. 8. Scatchard
12
plot of binding data determined
chloromethyl ketone (20 /xg/ml).
1 2 5
80
60
BOUND ( Μ χ ΙΟ )
I - E G F , 5.6 χ 10
12
in the
to 5.6 χ Ι Ο
presence of ts-Lys10
M was incubated
with fibroblasts for 2 hours at 3 7 ° C .
sites that we have reported. This difference may be due to different experimental conditions employed in the two experiments. Recently, Revoltella et al. (1974) demonstrated that the binding of nerve growth factor to neuroblastoma cells is greatly influenced by the stage of the cell cycle. It is not known how the cell cycle affects the binding of E G F to human fibroblasts.
B. Fate of Cell-Bound
TEGF
125
In order to determine the rate at which cell-bound radioactivity is released from cells following binding, we have carried out the following 1 2 5 experiment. Fibroblasts were incubated with I - E G F for 1 hour at 37°C. Then the cultures were washed with warm Hanks' medium to remove all unbound radioactivity, the standard binding medium was added to each culture dish, and the cultures were reincubated at 37°C. At various time intervals thereafter, two cultures were removed and the amount of cell-bound radioactivity was determined. The data (Fig. 9) indicated that the cell-bound radioactivity decreases with a half-life of approximately 60 minutes. In a similar experiment an excess of unlabeled E G F was added after the unbound radioactive E G F was removed to determine whether the presence of the growth factor affected the rate of release.
Stanley Cohen et al.
280
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FIG. 9. Dissociation of cell-bound I-labeled epidermal growth factor ( E G F ) in 125 presence and in the absence of E G F . I - E G F , 6.6 ng/ml, was incubated with fibroblasts for 1 hour at 37°C. The medium was removed and the cells were washed with Hanks' medium containing 0.1% bovine serum albumin. Finally, 1.5 ml of standard binding medium with E G F , 20 Atg/ml (O O), and without E G F (# φ) were added. The cultures were incubated at 37°C, and at the indicated time intervals, duplicate dishes were selected and washed, and the cell-bound radioactivity was determined.
De Meyts et al. (1973) have reported that the rate of release of cellbound insulin is increased by the presence of insulin in the medium. As shown in Fig. 9 the presence of E G F in the medium has no effect on the rate of release of cell-bound radioactivity. 1 2 5 The fact that protease inhibitors prevent the release of bound I - E G F from cells has suggested to us that subsequent to the binding of the growth factor to the plasma membrane the molecule is subject to the action of cellular proteases. Therefore, we have analyzed the radioactivity that is released from cells into the medium. Gel filtration of the radio1 2 active product and of a sample of T - E G F showed that the radioactivity 125 released into the medium was not intact I - E G F , but rather was associ-
EGF Interaction with Cultured Fibroblasts
281
ated with a much smaller molecule. The radioactive material was identified as monoiodotyrosine on the basis of paper electrophoresis at three T different pH values and paper chromatography in tw o different solvents. These results demonstrate that proteolytic degradation of bound 1 2 5 I - E G F occurs with the release of monoiodotyrosine into the medium. However, two important questions in this regard have not been answered. It is not known whether the proteolysis occurs at the cell surface or whether the bound E G F is subject to endocytosis and degradation by intracellular proteases. Also, it is not known wdiether the entire E G F molecule is degraded or whether a limited proteolysis sufficient to liberate one labeled tyrosine residue occurs. This second question may be answered once the location of the iodinated tyrosine residue (s) is known.
C. Specificity of I-EGF Binding 125
1 2 5
W e have determined the specificity of I - E G F binding with respect to the following: (1) native E G F ; (2) derivatives of E G F ; (3) other polypeptide hormones; and (4) various cell types. To determine whether the monoiodinated form of E G F and native E G F bind to fibroblasts with similar affinities, the following experiment was 125 performed. Mixtures of I-labeled monoiodo-EGF and native EGF, containing a saturating quantity of the iodinated derivative and varying amounts of native EGF, were permitted to bind to cells and the amount of cell-bound radioactivity was determined after 1 hour of incubation at 37°C. The results of this experiment, presented in Fig. 10, demonstrate ο
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FIG. 10. Competition of native epidermal growth hormone ( E G F ) and m o n o i o d o E G F for binding to human fibroblasts. Indicated amounts of E G F and 75 ng of m o n o i o d o - E G F (1784 c p m / n g ) were added simultaneously to culture dishes containing 1.5 ml of standard binding medium. Specific binding of the labeled E G F to fibroblasts was determined after 1 hour of incubation at 37°C. The solid line represents a theoretical dilution curve of 75 ng of m o n o i o d o - E G F by varying amounts of native E G F . The points are experimental values.
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Stanley Cohen et al.
that the amount of labeled E G F bound to the cells was directly dependent on the relative quantities of native E G F added. Therefore, both iodinated and native E G F appear to bind to the cells with equal affinities. Also, the inclusion of 10% rabbit antiserum to E G F inhibited the binding of 1 2 5 I - E G F to the cells by 99%. In view of the data presented on the relative growth stimulating effects of derivatives of EGF, the binding affinities of E G F - 2 and EGF-5 to 1 2 5 the fibroblasts were estimated by their abilities to compete with I1 2 5 EGF. Although E G F - 2 competed with I - E G F as well as native EGF, EGF-5 was only approximately 5% as effective as the native molecule. The isolated carboxy-terminal pentapeptide had no effect on the binding. The results indicate that of the five carboxy-terminal amino acids only the Trp-Trp-Glu sequence influences the binding of the native molecule. Thus, the binding affinities of these derivatives parallel their growthstimulating effects on human fibroblasts. TABLE BINDING OF
I - E G F TO CULTURED
6
Y-l
xc NC-37 CHO K-NRK MA-184 HPME 3T3 SV101 NRK AR-963 ΕΡΟ-Η8
CELLS
0
T o t a l I - E G F bound 6 ( c p m / 1 0 cells)
Specific binding 1 2 5 of I-EGF 6 ( c p m / 1 0 cells)
90 187 220 167 83 13,500 7,120 3,525 4,224 4,060 803 9,798
