Tumor Necrosis Factor Increases the Number of Epidermal Growth ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 5, Issue of February 15, pp. 1950-1954 1987 Printed in il..S.A.

Tumor Necrosis Factor Increases theNumber of Epidermal Growth Factor Receptors on Human Fibroblasts* (Received for publication, August 21, 1986)

From the $Department of Microbiology and Kaplan Cancer Center, the lDepartment of Pharmmology, New York University Medical Center, New York, New York 10016 and 11 The Rockefeller University, New York, New York 10021

Tumor necrosis factor (TNF) was shown previously to stimulate the growth of human FS-4 fibroblasts. Here we show that human recombinant TNF can increase the binding of epidermal growth factor (EGF) to these cells. Incubation with TNF resulted in a 4080%increase in the number ofEGF-bindingsites with no apparent change in receptor binding affinity. The increase in EGF binding was apparent 8-12 h after the addition of TNF. TNF also increased the amount of EGF receptor protein immunoprecipitated from cells labeled with [35S]methionine.Stimulation of EGF receptor protein synthesis was demonstrable 2-4 h following TNF treatment. TNF increased EGF binding with a dose-response relationship similar to that reported earlier for the mitogenic action. Increased expression of EGF receptors, due to enhanced synthesis of the EGF receptor protein, may be functionally related to the mitogenic action of TNF in human fibroblasts.

(51, the regulation of HLA-A and B antigen expression on vascular endothelial cells and dermal fibroblasts (6), the inhibition of virus replication, and the induction of &interferon in human fibroblasts (7). T N F also stimulates the growth of humanfibroblasts (8, 9);the mitogeniceffect of T N F in fibroblasts is similar to that seen with the classical growth factors such as epidermal growth factor (EGF). EGF can promote the growth of many types of cells (1012). On binding to its receptor, EGF stimulates the phosphorylation of the receptor and of other cellular proteins (12-17). The EGF-binding and the kinase activity both reside within the samemolecule (18,191. Many factorswere shown to affect the binding of EGF to certain cells by altering the receptor number and/or affinity. Platelet-derived growth factor (PDGF) (20-22) and tumor promoting phorbol esters (23-25) were shown t o decrease the binding of radiolabeled EGF to its receptors, while transforming growth factor-@(TGF-P) (26) and estrogen (27) were found to increase EGF binding by increasing theexpression of EGF receptors. Modulation of the EGF receptor, by these and other factors, may result in an altered response to EGF, as was shown with TGF-/3 (28). Instimulatingthe growth of humanfibroblasts TNF Tumor necrosis factor (TNF)' is a monocyte/macrophage derived protein, originally described as a mediator of hemor- showed a marked synergism with insulin, while the actions of TNF and EGF were less thanadditive(9).Thisfinding rhagic tumor necrosis in animals injected sequentially with response Bacillus Calmette-Guerin and endotoxin(1).T N F is identical suggested the activationof some common pathway in between t o the lipoprotein lipase suppressing hormone, cachectin (2). to EGF and TNF. To investigate the relationship The mostextensively studied activityof T N F is its cytostatic the mitogenic actions of TNF and EGF, we examined the or cytotoxic effect on some tumor cells in culture. Recently, effect of TNF on theexpression of EGF receptors in human T N F was shown to have a wide spectrum of biological activ- fibroblasts. We report that TNF canincrease the number of ities on normal cells in addition to its anti-tumor activity. EGF receptors. We also show that the increased receptor These activities include induction of interleukin-1 in human expression results from an increase in the synthesis of the endothelial cells (3), mononuclear cells (4), and diploid fibro- EGF receptor. blasts,' the production of collagenase and prostaglandin E' EXPERIMENTALPROCEDURES

* This work wassupported in part by National Institutes of Health Grants AI-12948 (to J. V.), AM-27083 (to F. R. M.), CA-37754 (to S. J. D.), and by a grant from Suntory Limited (to J. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. Supported by predoctoral fellowship Cancer Research Training Grant T32 CA-09161. ** To whom correspondence should be addressed Dept. of Microbiology, New York University School of Medicine, 550 First Ave., New York, NY 10016. The abbreviations used are: TNF, tumor necrosis factor; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; TGF, tranforming growth factor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FBS, fetal bovine serum; HEPES, 4(2-hydroxyethyl)-l-piperazineethanesulfonic;Tricine, N-Tris(hydroxymethy1)metbylglycine. J. Le, D. Weinstein, U. Gubler, and J . VilLek (1987) J. Zmmunol., in press.

Materials-Recombinant Escherichia coli-derived human TNF was produced and purified (specific activity 4.8 X lo7 units/mg) at the Suntory Institute for Biomedical Research, Osaka, Japan. The material appeared homogeneous when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (29). EGF was prepared from mouse salivary glands as described by Savage and Cohen (30). Rabbit polyclonal antiserum against the denatured human EGF receptor was used in all immunoprecipitation experiments (31). ~ - [ ~ ~ S ] M e t b i o nand i n eNalZ5Iwere purchased from New England Nuclear. EGF was iodinated to a specific activity of 6-7 X 10' cpm/ ng as described previously (32). Cell Culture-The human diploid FS-4 fibroblast line, isolated a t the New York University Medical Center from a foreskin (33), was used in all experiments. FS-4 cells were grown in minimum Eagle's medium supplemented with 6 mM HEPES, 3 m M Tricine, 50 pg/ml gentamicin, and 5% heat-inactivated (56 "C, 30 min) fetal bovine serum (Gibco). FS-4 cells were used at passage level 13-15. lZ5Z-EGFBinding Assays-FS-4 fibroblasts were seeded in 24-well plates (1 X IO5cells/well in 1 ml of medium) and incubated at 37 "C in ahumidified CO, incubator. TNF was added directly to thegrowth

1950

1951

TNF Stimulates EGF Receptor Synthesis medium of confluent cultures 24 h after cell seeding. Control cultures were incubated in the absence of TNF. After a specific incubation period, the FS-4 cells were rinsed twice with 1ml of minimum Eagle's medium/HEPES (20 mM, pH 7.4) containing 1mg/ml bovine serum albumin (Sigma) and incubated for 10 min at 37 "C. The medium was then removed and 0.5 ml of ice-cold minimum Eagle's medium/ HEPES containing 5 mg/ml bovine serum albumin and '"I-EGF was added. After 4 h at 4 "C, the cells were washed five times with icecold minimum Eagle's medium/HEPES. The cells were then solubilized in 1 ml of 0.1 N NaOH, 0.1% Triton X-100 and theradioactivity was counted. Protein concentration in the solubilized cells was determined with the aid of the Bradford reagent (34) purchased from BioRad. For all experiments, specific binding is the difference between total binding and nonspecific binding. Nonspecific binding was determined induplicate in the presence of a 500-foldexcess of unlabeled EGF. Specific binding is expressed per pg of cell protein. Saturation binding data were evaluated with the aid of the Equilibrium Binding Data Analysis Program (35,36). Isotopic Labelingof Cells-Approximately 4.5 X lo5 FS-4 cells were seeded per well (in 2 ml of medium) in 6-well plates. After a 24-48 h incubation, the mediumwas removed and replaced with 1 ml of methionine-free Dulbecco's modified Eagle's medium (Gibco) supplemented with 1 pg/ml L-methionine and 5% fetal bovine serum. TNF (10 ng/ml) was added for the times indicated. The cells were labeled with [35S]methionine(50 pCi/ml) for the last 6 h before the harvesting of cultures. To label the precursor (M, = 160,000) of the mature EGF receptor, the following procedure was used. TNF was added directly to theoriginal growth medium and, after aspecific incubation period, the medium was replaced with 1 ml of methionine-free Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and [35S] methionine (100 pCi/ml). Cultures were then incubated in the presence of [35S]methioninefor only 45 min prior to harvesting (37). TNF was also present throughout this 45-min labeling period. Immunoprecipitation and SDS-PAGE-Labeled cells were washed three times with CaZf/M%+-freephosphate-buffered saline and were scraped off the plates in an EDTA (50 mM) solution. Aliquots of the cell suspensions were lysed and theprotein concentration was determined. The labeled cells were then lysed and immunoprecipitated as previously described (37), with some modification. Briefly, the cells were centrifuged in an Eppendorf microfuge and the pellet was lysed in hot 2% sodium dodecyl sulfate (SDS; in 20 mM Tris hydrochloride, pH 7.4). The samples were heated for 5 min at 100 "C, diluted 1:20 with radioimmune precipitation buffer (38) containing 10 mM EDTA and no SDS, and centrifuged for 2 min. The pellet was discarded. Before immunoprecipitation, each sample was adjusted so that equal amounts of cell protein would be immunoprecipitated. Antiserum (2.5 pl) was mixed with the cell extracts at room temperature for 60 min. Thereafter, 40 pl of radioimmune precipitation buffer/protein ASepharose CL-4B (Pharmacia) was added for 30 min at 4 "C. The beads were washed three times with radioimmune precipitation buffer (1 ml) and one time with 50 mM Tris hydrochloride, pH 7.4. The immunoprecipitates were boiled in electrophoresis sample buffer and subjected to 7% SDS-PAGE as described by Laemmli (39). The gels were fixed, stained,treated for 30 min with Enlightening (New England Nuclear), dried, and exposed (Kodak XAR-5 film) for 6-12 days.

TNF Dose (pg)

FIG. 1. Effect of different concentrations of T N F on laa1E G F binding. FS-4 cells were incubated with various concentrations of TNF for 24 h at 37 "C. lz5I-EGF (10 ng/ml) binding at 4 "C was determined as described under "Experimental Procedures." Cellbound radioactivity is expressed as percent radioactivity of control cultures (0.47 pg of '"I-EGF/pg cell protein) incubated in parallel in the absence of TNF. Results are presented as the mean (& S.D.) of triplicate determinations and the figure is a representative example of three separate experiments.

5

m 120

t 0

J 4

8

12 16 Hours

20

24

FIG. 2. Increase in "'1-EGF binding after different times of incubation with TNF. Cells were cultured in the presence of 10 ng/ ml of TNF at 37 "C for the times indicated. '=I-EGF (10 ng/ml) binding at 4 "C was quantitated as described under "Experimental Procedures." Cell-bound radioactivity is expressed as percent radioactivity of control cultures (0.31 pgof '"I-EGF/pgcell protein) incubated without TNF for 24 h. The datapoints representthe means (*S.D.) of three determinations. The figure shown is representative of three separate experiments.

Nonspecific binding, measured in the presence of a 500-fold excess of unlabeled EGF, was approximately 5% of the total binding. In other experiments, treatment with 100 pg to 10 ng/ml of TNF resulted in a 40-80% increase in EGF binding (data not shown). Time Courseof TNF-stimulated Increase in EGF BindingThe increase in EGF binding first became apparent by 12 h RESULTS after the addition of TNF (10 ng/ml) (Fig. 2). An additional Increased Binding of EGF to Fibroblasts Incubated with increase occurred between 12 and 24 h. Other experiments TNF-Different concentrations of TNF were added to FS-4 showed that further incubation with TNF did not lead to a cultures andthe cells were incubated for 24 h. The amount of greater increase in EGF binding. Cells cultured in the presence lZ5I-EGFbinding was then determined (Fig. 1).The results of TNF for 2 or 5 days also bound 50-90% more 'T-EGF are expressed as percent lz5I-EGF bound to cells relative to than the controls (data not shown), indicating that the incontrol cultures incubated in the absence of TNF. A TNF creased capacity to bind EGFin cells cultured in the presence dose of 10 pg/ml ( 3 X10-13 M) slightly increased lZ5I-EGF of TNF is maintained for several days. binding, while an optimal stimulation of 67% was seen on TNF Increases the Number of EGF Binding Sites without incubation with TNF at 1 ng/ml ( 3 x lo-" M ) . This dose is Changing the Affinity of the Receptors-To determine whether similar to that determined to be optimal for the mitogenic increased EGF binding results from a change in receptor effect (9) and is about 10-fold less than the apparent Kd of number or affinity, saturation binding analysis was performed TNF binding to its receptor in FS-4 and other cell types (8, in control cultures and in cells treated with TNF (10 ng/ml) 29, 40). Although treatment with TNF for 24 h did not result for 24 h. When specific binding of T - E G F to FS-4 cells at in an increase in cell number, there was a slight increase in 4 "C was analyzed as a function of increasing concentrations the amount of cellular protein (data not shown). To correct of lZ5I-EGF,cells treated with TNF bound significantly more for this change, specific binding is expressed per pg of protein. EGF than the control cultures (Fig. 3, inset). Computer anal-

TNF Stimulates EGF Synthesis Receptor

1952

t

2-1

\

2 6 1014 18 22 26 x)

'251-EGF Bound. pM

FIG. 3. Scatchard analysis of '"I-EGF binding on control and TNF-treated cells. Cells were incubated in the presence (0) or absence ( 0 )of T N F (10 ng/ml) for 24 h a t 37 "C. "'1-EGF binding (4 "C) was determined as in the previous experiments. Inset shows the specific binding of '''I-EGF to the control and TNF-treated cultures. The cells were incubated a t 4 "C with increasing concentrations of '*'I-EGF. Specific binding (per pg of protein) was calculated as described under "Experimental Procedures" and is expressed as the mean (+ S.D.) of four values. The figure shown is representative of three experiments performed on separate days.

ysis of the data by the Equilibrium Binding Data Analysis program (35, 36) showed that TNF increased the number of specific EGFbindingsites by 76% withoutchangingthe apparent affinity (Fig. 3). The Kd values for control and TNFtreated cells were 1.8 and 1.7 nM, respectively. The correlation

a

coefficient for both values was -0.98. The Bmaxvalues were 15.8 (a 1.12) pM and 29.2 (f 2.42) PM for the control and TNF-treated cultures,respectively (or 0.179 PM and 0.315 PM when expressed per pg of protein for the control and TNFtreated cultures,respectively). The calculated number of bindingsites/cell was approximately 40,000 and 25,000 in the TNF-treated and control cultures,respectively. The Equilibrium Binding DataAnalysis programshowed a Hillcoefficient close to unity for both samples (TNF, 0.994 f 0.028; control, 0.997 & 0.036), demonstrating a single class of non-interacting Kd values rangedfrom binding sites. In other experiments the 1.6 to 2.3 nM with no significant difference between control and TNF-treated cells. TNF Stimulates Synthesisof the EGF Receptor-The EGF receptor protein was immunoprecipitated from TNF-treated or control cells with the aid of a rabbit antiserum prepared against the denatured human EGF receptor (31). This antiserum specifically recognizes both the 160-kDa precursor and the 170-kDa mature form of the EGF receptor. Before immunoprecipitation, allsamples were adjusted to the same protein concentration. Fig. 4u illustrates a typical immunoprecipitation experiment analyzed by 7% SDS-PAGE. Addition of T N F (10 ng/ml) for 6 h (lane C ) , 12 h (lane D),and 24 h (lane E ) resulted inan increase in theamount of immunoprecipitable EGF receptor protein during a 6-h pulse with ["S]methionine when compared to the control without T N F (lune B). Boththe 160- and 170-kDa forms of the receptor were increased indicating an increase in both synthesisandmaturation of the receptor during this period.

b

A

B

C

D

E

-160.000

FIG. 4. Immunoprecipitation of the EGF receptor from cells treated for various times with TNF.a, cells were treated with 10 ng/ml of T N F a 37 t "C for 6 h ( l a n e C), 12 h ( l a n e D),and 24 h ( l a n e s A and E ) . Controls were incubated in the absence of T N F ( l a n e B ) but were subjected to the same manipulations. Cultures were labeled with ['%]methionine (50 pCi/ml) for the last 6 h of culture. Cell lysates were immunoprecipitated with 2.5 pl of rabbit anti-EGF receptor serum ( l a n e s B-E) or 2.5 pl of normal rabbit serum ( l a n e A ) as described under "Experimental Procedures." b, cells were treated with T N F (10ng/ml) at 37 "C for 2 h ( l a n e C), 4 h ( l a n e D),5 h ( l a n e E ) , and 6 h ( l a n e s A and F). Control cells were incubated without T N F ( l a n e B). The cells were labeled for the last 45 min with 1 0 0 pCi/ml of ['5S]methionine. Cell extracts were immunoprecipitated with 2.5 pl of rabbit anti-EGF receptor serum ( l a n e s B-F) and 2.5 pl of normal rabbit serum ( l a n e A ) as above. Because incubation with T N F increased protein concentration in the cell lysate (see above) and ita effect on the transport of ["SI methionine has notbeen previously determined, care was taken that equal amounts of protein and equal counts of All samples [%]methionine be subjected to immunoprecipitation in each experimental group in both experiments. were separated on 7% SDS-polyacrylamide gels and theM,= 160,000and 170,000 regions of the gel are indicated.

TNF Stimulates EGF Receptor Synthesis There appeared to be less 160-kDa receptor protein immunoprecipitated from cells treated for 24 h with T N F when compared with the 6- and 12-hsamples. However, the amount of mature EGF receptor (170 kDa) remained high after 24 h with TNF. No EGF receptor bands could be detected in the lysate of TNF-treated cells immunoprecipitated with normal rabbit serum (lune A ) . A kinetic analysis of the stimulation of EGF receptor synthesis by TNF is shown in Fig. 4b. In each group the cells were incubated with 100 pCi/ml of [%]methionine for the last 45 min of culture. During this time, only the 160-kDa precursor should be labeled (37). The results indicate that stimulation of EGF receptor synthesis was apparent between 2 and 4 h after the addition of TNF. The 160-kDa precursor was, in fact, theonly form of the EGFreceptor detected. Cells treated with T N F (10 ng/ml) for 24 h and labeled for 45 min with [%]methionine also showed a detectable increase in the amount of the 160-kDa protein immunoprecipitated, but this increase was less than that seen at 6 h after T N F treatment (data notshown). DISCUSSION

1953

binding affinity of the high affinity sites (28). The ability of TGF-P to stimulate EGFreceptor expression may be related to the actions of this agent and EGF on normal rat kidney cell growth (28). However, Massagui. (52) observed only a decrease in EGFbinding in normal rat kidney cells in response to TGF-P. It is interesting that EGF itself was found to increase the synthesisof its own receptor (53,541. The precise physiological role for any of theseevents remains to be established. Recently, TNF has been shown to induce the synthesis of two unknown proteins (36 and 42 kDa) ( 5 5 ) and of interleukin-1' in human fibroblasts. In this reportwe show that TNF can also stimulate the synthesis of another protein, namely, the EGFreceptor. Together these findings indicate that TNF is a potent regulator of cellular protein synthesis. Since the dose-response relationship for the increase in EGF binding by T N F is similar to that seen for the mitogenic stimulation (9), increased expression of EGF receptors may be related to the mitogenic action of TNF in human fibroblasts. Acknowledgments-We thank theSuntory Institute for Biomedical Research for the generous supply of TNF, Dr. T. Noguchi and his colleagues for encouragement and support, and Cristy Minton and Ilene Toder for preparation of the manuscript.

In an earlier study, highly purified recombinant TNF was shown to be a potent mitogen for human diploid fibroblasts REFERENCES (9). Maximal stimulation of cell growth was observed at TNF concentrations between 1 and 10 ng/ml ( 3 X 10"'-3 x lo-" 1. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., and Williamson, B. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 3666M ) . Recently T N F was shown to induce p2 interferonin 3670 human fibroblasts and it was suggested that this interferon 2. Beutler, B., Greenwald, D., Hulmes, J. D., Chang, M., Pan, Y.-C. could act as an autocrine inhibitorof cell proliferation (7). It E., Mathison, J., Ulevitch, R., and Cerami, A. (1985) Nature appears that TNF, perhaps in conjunction with other factors, 316, 552-554 may play a role in the regulation of the growth of some cells. 3. Nawroth, P. P., Bank, I., Handley, D., Cassimeris, J., Chess, L., TNF, like EGF, acted synergistically with insulin in promotand Stern, D. (1986) J. Exp. Med. 163, 1363-1375 4. Dinarello, C. A., Cannon, J. G., Wolff, S. M., Bernheim, H.A., ing the growth of FS-4 cells (9). On the other hand, the Beutler, B., Cerami, A., Figari, I. S., Palladino, M. A., Jr., and combined mitogenic action of TNF and EGF,when added to O'Connor, J. V. (1986) J. Exp. Med. 163,1433-1450 cells simultaneously, was less than additive. This result sug5. Dayer, J.-M. Beutler, B., and Cerami, A. (1985) J. Exp. Med. gested that TNF and EGFmay activate similar pathways in 162,2163-2168 cell cycle progression. 6. Collins, T., Lapierre, L. A., Fiers, W., Strominger, J. L., and To explore the possible involvement of EGF or an EGFPober, J. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,446-450 7. Kohase, M., Henriksen-DeStefano, D., May, L. T., VilEek, J., and like molecule in the mitogenic action of TNF, we examined Sehgal, P. B. (1986) Cell 4 5 , 659-666 the effect of TNF on the expression of EGF receptors. Our 8. Sugarman, B. J., Aggarwal, B. B., Hass, P. E., Figari, I. S., results show clearly that TNF can increase the number of Palladino, M. A., Jr., and Shepard, H. M. (1985) Science 230, EGF-binding sites without changing receptor binding affinity. 943-945 Immunoprecipitationstudies revealed thatTNF also in9. VilEek, J., Palombella, V. J., Henriksen-DeStefano, D., Swenson, C., Feinman, R., Hirai, M., and Tsujimoto, M. (1986) J. Exp. creased EGF receptor protein synthesis. In addition to FS-4 Med. 163,632-643 cells we examined the effect of human TNF on EGF binding in confluent BALB/c 3T3 cells. Treatment of 3T3 cells with 10. Carpenter, G., Lembach, K. J., Morrison, M. M., and Cohen, S. (1975) J. Bid. Chem. 2 5 0 , 4297-4304 TNF resulted in a 100-200% increase in the number of EGF 11. Carpenter, G., and Cohen, S. (1979) Annu. Reu. Biochern. 48, binding sites (data not shown). These results suggest that 193-216 enhancement of EGF receptorexpression by T N F isnot 12. Carpenter, G. (1984) Cell 37,357-358 13. Cohen, S., Carpenter, G., and King, L., Jr. (1980) J. Biol. Chem. confined to human fibroblasts. 255,4834-4842 Many other factors have been shown to modulate the EGF (23-25), diacylglycerol (41, 42), 14. Ushiro, H., and Cohen, S. (1980) J. Bwl. Chem. 255,8363-8365 receptor. Phorbolesters 15. Hunter, T., and Cooper, J. A. (1981) Cell 24, 741-752 PDGF (20-22), and fibroblast growth factor (43) were shown 16. Gill, G. N., and Lazar, C. S. (1981) Nature 293, 305-307 to decrease the bindingof radiolabeled EGF to culturedcells. 17. Downward, J., Parker, P., and Waterfield, M.D. (1984) Nature Phorbol esters anddiacylglycerol appear to phosphorylate the 311,483-485 EGF receptor as a result of an activation of protein kinase C 18. Cohen, S., Ushiro, H., Stoscheck, C., and Chinkers, M. (1982) J. Bi01. Chern. 257, 1523-1531 (41, 44-47). The kinase phosphorylates threonine 654 in the S. A., Cohen, S., and Staros, J. V. (1982) J. Biol. Chem. human EGF receptor and this eventseems to regulate ligand 19. Buhrow, 257,4019-4022 binding andinternalization (48-50). PDGF, on theother 20. Wrann, M., Fox, C. F., and Ross, R. (1980) Science 210, 1363hand, appears todecrease EGF binding by a different mech1365 (ie. independent of protein 21. Wharton, W., Leof, E., Pledger, W. J., and O'Keefe, E. J. (1982) anismthantumorpromoters Proc. Natl. Acad. Sci. U. S. A. 79, 5567-5571 kinase C) (51). In contrast tophorbol esters and PDGF,estrogen (27) and 22. Collins, M. K. L., Sinnett-Smith,J. W., and Rozengurt, E. (1983) J. Biol. Chem. 258,11689-11693 TGF-P (26)were shown to increase EGF receptor levels. TGF- 23. Lee, L.-S., and Weinstein, I. B. (1978) Science 2 0 2 , 313-315 /3 increased the numberof lowand high affinity sites in normal 24. Shoyab, M., DeLarco, J. E., and Todaro, G. J. (1979) Nature rat kidney fibroblasts only after a transient decrease in the 279,387-391

1954

TNF Stimulates EGF Receptor Synthesis

25. King, A.C., and Cuatrecasas, P. (1982)J. Biol. Chem. 257,30533060 26. Assoian, R. K., Frolik, C.A., Roberts, A. B., Miller, D. M., and Sporn, M. B. (1984) Cell 36, 35-41 27. Mukku, V. R., and Stancel, G. M. (1985) J. Biol. Chem. 260, 9820-9824 28. Assoian, R.K. (1985) J. Biol. Chem. 260, 9613-961745. 29. Tsujimoto, M., Yip, Y. K., and VilEek, J. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,7626-7630 30. Savage, C. R., Jr., and Cohen, S. (1972) J. Biol. Chem. 247, 7609-7611 48. 31. Decker, S. (1984)Arch. Biochem. Biophys. 228, 621-626 32. Carpenter, G., and Cohen, S. (1976) J. Cell Biol. 71, 159-171 33. VilEek, J., and Havell, E. A. (1973) Proc. Natl. Acad. Sci.U. S. A. 70,3909-3913 34. Bradford, M. M. (1976)Anal. Biochem. 72, 248-254 35. McPherson, G.A. (1983) Comput. ProgramsBiomed. 17, 107114 36. McPherson, G. A. (1985) J. Pharmacal. Methods 1 4 , 213-228 37. Decker, S. J. (1984) Mol. Cell. Bwl. 4,571-575 38. Karess, R. E., Hayward, W. S., and Hanafusa, H. (1979) Proc. Natl. Acad. Sci. U. S. A . 76, 3154-3158 39. Laemmli, U. K. (1970) Nature 227,680-685 40. Tsujimoto, M., Yip, Y. K., and VilEek, J. (1986) J. Zmmunol. 136,2441-2444 41. McCaffrey, P. G., Friedman, B., and Rosner, M. R. (1984)J.Bwl. 9567 Chem. 259, 12502-12507

42. Sinnett-Smith, J. W., and Rozengurt, E. (1985) J. Cell. Physwl. 124,81-86 43. Rozengurt, E., Collins, M., Brown, K. D., and Pettican, P. (1982) J. Bwl. Chem. 257,3680-3686 44. Cochet, C., Gill, G. N., Meisenhelder, J., Cooper, J. A., and Hunter, T. (1984) J. Bwl. Chem. 259, 2553-2558 Davis, R. J., and Czech,M. P. (1984) J. Biol. Chem. 259, 85458549 46. Decker, S. J. (1984) Mol. Cell. Biol. 4, 1718-1724 47. Fearn, J. C., and King, A. C. (1985) Cell 40,991-1000 Hunter, T., Ling, N., and Cooper, J. A. (1984) Nature 311,480483 49. Davis, R. J., and Czech, M. P. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,1974-1978 50. Lin, C. R., Chen, W. S., Lazar, C. S., Carpenter, D., C. G.Gill, N., Evans, R. M., and Rosenfeld, M. G. (1986) Cell 44, 839848 51. Olashaw, N. E., O'Keefe, E. J., and Pledger, W. J. (1986) Proc. Natl. Acad. Sci. U. S. A . 83, 3834-3838 52. Massaguir, J. (1985) J. Cell Bwl. 100, 1508-1514 53. Clark, A. J. L., Ishii, S., Richert, N., Merlino, G. T., and Pastan, I. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,8374-8378 54. Earp, H. S., Austin, K. S., Blaisdell, J., Rubin, R. A., Nelson, K. G., Lee, L. W., and Grisham, J. W. (1986) J. Biol. Chem. 261, 4777-4780 55. Kirstein, M., and Baglioni, C. (1986) J. Bwl. C k m . 261, 9565-