Cancer Letters. 57 (1991) 103-108 Elsevier Scientific Publishers Ireland
103 Ltd.
Modulation of epidermal growth factor receptor phosphorylation by tumor necrosis factor L. Guazzoni,
P. Perego,
Division of Experimental Milan (Italy) (Received (Accepted
Oncology
tyrosine
P. Banfi and F. Zunino B, Istituto
Nazionale
per lo Studio
e la Cura
dei Tumori.
Via
Venezian
1. 20133
13 November 1990) 28 January 1991)
receptors of susceptible cells [ 191. Expression of membrane receptors for TNF appears necessary but not sufficient to determine its growth inhibitory or cytolytic action. Antitumor activity of TNF has been ascribed to a direct effect on tumor cells and an indirect effect on tumor vascularization. The early biochemical events occurring following TNF binding to specific receptors are not completely understood. Modulation of EGF receptor tyrosine kinase activity by TNF in sensitive human tumor cell lines has suggested a role of the EGF receptor in the cell response that results in growth inhibition [8]. The precise mechanism of the reported modulation remains unclear. It is known that PKC is involved in control of EGF receptor function [6, lo]. PKC itself has been implicated in the control of cell responsiveness to TNF. The possibility is supported by the following observations: (a) PKC activating agents induce down-regulation of the TNF receptor [20,11]; (b) PKC inhibitors were shown to block some TNFmediated effects [ 17,141. We examined, in a different TNF-sensitive cell line [7], a possible link between EGF receptor protein kinase activity and TNF action. In this approach, the specific tyrosine kinase activity of the EGF receptor was investigated with antiphosphotyrosine antibodies in intact cells.
Summary
The effects of tumor necrosis factor (TNF) on epidermal growth factor (EGF) receptor tyrosine phosphorylation were investigated in Swiss 3T3 cells, which are sensitive to TNF action. At cytotoxic levels, TNF produced an appreciable inhibition of EGF-induced autophosphorylarion of the receptor. A similar inhibition was detected even after prolonged treatment IL’-0-tetradecanoylphorbol-13-acetate with (TPA) which produces down-regulation of protein kinase C (PKC). According to this finding, TNF does not induce phosphorylation of the 80 kDa PKC-specific substrate. These results support the hypothesis that the inhibition of EGF receptor tyrosine phosphorylation is not mediated uia stimulation of PKC activity in intact Swiss 3T3 cells. Keywords:
epidermal
growth
factor;
tumor
necrosis factor Introduction
Like other cytokines, TNF exerts its biologic action through binding to specific cell surface Correspondence to: F. Zunino, Istituto Nazionale Tumori. Division of Experimental Oncology B. Via Venezian 1, 20133 Milan, Italy. 0304-3835/91/$03.50 Published and Printed
0 1991 Elsevier Scientific in Ireland
Publishers
Ireland
Ltd
104
Materials and Methods Reagents Recombinant human TNFa was purchased from Amersham Corp. (Amersham, Buckinghamshire, U.K.). EGF, platelet derived growth factor (PDGF), bombesin and TPA were purchased from Sigma Chemical Co. (St. Louis, MO). Cells and growth condition Swiss 3T3 fibroblasts were obtained from the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia (Brescia, Italy). They were routinely grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% calf serum (Colorado Serum Co., Denver, Colorado, USA) at 37°C in a humified atmosphere containing 5% CO*. Cytotoxic assay Cell survival was assayed by the calorimetric signal produced by cleavage of the tetrazolium salt 3-(4,5-dimethylthiazol-Z-yl)-2,5-diphenyltetrazolium bromide (MTT) [l]. Briefly, cells were harvested from exponential-phase maintenance cultures, dispensed into 96-well culture plates in 100 ~1 volume and treated with TNF. After incubation of the microtiter plates for 96 h, 10 ~1 of MTT working solution (5 mg/ml) was added to each well and cultures were incubated at 37°C for 4 h. The culture medium was removed from the wells and replaced with 100 ~1 of dimethylsulfoxide (DMSO). The absorbance of each well was measured using a microculture plate reader at 550 nm interfaced with an Apple computer. Tyrosine phosphorylation of EGF receptor Cells were seeded at 1. 104/cm2 on 5-cm petri dishes, grown to confluency and then left for 24 h in DMEM containing 0.2% serum, 0.1% bovine serum albumin and 10 mM 4- (2-hydroxyethyl) - 1-piperazineethanesulfonic acid (HEPES) pH 7.2, to obtain the quiescent state. Confluent and growth-arrested monolayers were incubated with 10m6 M TPA or TNF at the indicated concentrations, at 37”C,
with shaking. After 15 min EGF (50 pg/ml) was added for 3 min. The plates were then chilled on ice and media immediately removed. Cells were rinsed three times with phosphate-buffered saline and scraped in sodium dodecylsulfate (SDS) sample buffer [ 131 containing 1 mM phenylmethyl-sulfonyf fluoride (PMSF), 125 pg/ml leupeptin, 0.1 mM Na2Mo04 and 1 mM NasV04. The extracts were then boiled and sonicated. Samples from each plate were fractionated by SDSPAGE (polyacrylamide gel electrophoresis) [13] and blotted on nitrocellulose sheets. Filters were immunodecorated with an antiphosphotyrosine serum prepared as originally described by Ross et al. [15] and purified by affinity chromatography [5]. To detect the immunocomplexes the nitrocellulose blots were incubated with ‘251-protein A (Amersham) at 400 000 cpm/ml and exposed to autoradio-
graphy. Phosphorylation of the 80-kDa protein Swiss 3T3 fibroblasts were grown to confluency in 24-well plates. The cultures were washed with DMEM without phosphate, and the cells were incubated with the medium containing carrier-free 32Pi at 0.2 mCi/ml at 37°C for 3 h [16]. The cells were then washed with DMEM without phosphate at 37°C and exposed to TNF or other agents for various times; the reaction was stopped by removing the medium. Cells were immediately extracted with 0.5% Triton X-100, 10 mM 1,4triperazinediethanesulfonic acid (PIPES) pH 6.9, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), 10 mM NaF and 1 mM PMSF at 0°C with shaking for 5 min. SDS sample buffer containing 1 mM PMSF, 125 pg/ml leupeptin, 0.1 mM Na2Mo04 and 1 mM Na3V04 was added to supernatants containing the 80 kDa heat-stable protein [4]. To determine the radioactivity incorporated, casein (8 mg/ml) was used as vehicle, and samples were precipitated with 10% trichloroacetic acid/ 10 mM sodium pyrophosphate on Whatman 3MM filters. Filters were washed twice with 5% trichloroacetic acid/l0 mM sodium pyrophos-
105
phate and with acetone at 0°C and dried, and the radioactivity was determined by liquid scintillation. Samples were resoluted by SDSPAGE (10% acrylamide). Gels were fixed, dried and exposed to autoradiography. Results
Effect of TNF on the growth of Swiss 3T3 cells The effect of TNF on the growth of Swiss 3T3 fibroblasts was examined following exposure to the agent for 96 h. The doseresponse curve for the effect of TNF is shown in Fig. 1. According to the MIT assay, the percentage of control absorbance reflects the surviving fraction of the cells. The TNF effect was found to be dose-dependent, and the concentration required to inhibit 50% cell growth was 1.55 nM. Thus, exponentially growing Swiss 3T3 cells showed a sensitivity to the antiproliferative action of TNF comparable to that of other sensitive cell systems [19]. Effect of TNF on EGF receptor autophosphorylation
To determine whether TNF affects EGF receptor tyrosine phosphorylation, Swiss 3T3 mouse fibroblast monolayers were incubated with 0.1, 1 and 10 nM TNF for 15 min and then exposed to EGF (50 ng/ml) for 3 min. The results of a representative experiment are shown in Fig. 2A. The densitometric analysis
04 0.01
I 0.1
1
10
TNF CONCENTRATION (nM)
activity of TNF on Swiss 3T3 Fig. 1. Cytotoxic fibroblasts. The cells were exposed to TNF for 96 h. Cell survival was measured by the MTT assay.
A
B
Effect of TNF on EGF-induced tyrosine Fig. 2. phosphorylation of the EGF receptor in Swiss 3T3 cells. (A) Quiescent monolayers were incubated with 10m6 M TPA or TNF at the indicated concentrations for 15 min, then exposed to 50 ng/ml EGF for 3 min. Total cell proteins were fractionated by SDS-PAGE, and tyrosine phosphorylation of the EGF receptor was visualized by Western blotting using anti-phosphotyrosine antibodies as described in Materials and Methods. (B) Cells were pre-incubated with 10e6 M TPA for 24 h before treatment as described in (A).
of the results, summarized in Table I, indicates that tyrosine phosphorylation of the EGF receptor was reduced of about 40% in cells treated with 1 and 10 nM TNF, whereas a lower concentration (0.1 nM) had no appreciable inhibitory effect. To determine whether PKC was involved in the observed reduction of EGF receptor autophosphorylation following treatment with TNF, a similar experiment was performed with cells chronically exposed to TPA. Such a treatment is known to down-regulate PKC through an increased rate of degradation [ 181. As expected, following incubation with lo6 M TPA for 24 h, treatment with TPA for 15 min no longer had an inhibitory effect on EGF receptor tyrosin
106
Table 1. Effect of TNF on EGF-induced tyrosine phosphorylation of the EGF receptor in Swiss 3T3 cells. Control refers to phosphorylation of EGF receptor in cells exposed to EGF. See legend to Fig. 2 for experimental details.
12345678910
KDa -80
TNF concentration
0.1 nM 1 nM 10 nM
% Control Without TPA preincubation
After 24 h preincubation with TPA
98 f 12’ 61 zt 5** 60 zt 4*’
112 f 13’ 60 j, 0.5’ 57 f 1.5’
*Average value of 2 experiments *Average value of 3 experiments l
phosphorylation. In the PKC-deficient cells, 1 and 10 nM TNF still inhibited tyrosine phosphorylation of the EGF receptor to the same extent (Fig. 2B; Table I). These data suggest that PKC activation is not necessary for transmodulation of the EGF receptor by TNF. Effect of TNF on phosphorylation specific 80-kDa substrate of PKC
of
the
In a different approach to test whether PKC was involved in transmodulation of the EGF receptor by TNF, the ability of TNF to induce phosphorylation of the specific substrate of PKC (80 K) was examined in quiescent cultures of Swiss 3T3 fibroblasts. Serumdeprived fibroblasts were exposed to 0.1, 1 and 10 nM TNF for 5 and 15 min. To control the function of the experimental system, PDGF and bombesin, which are known to affect the activity of PKC with stimulation of the phosphorylation of the 80 K protein, were added to the cells for 5 min. In contrast, 0.1, 1 and 10 nM TNF did not activate PKC (Fig. 3). These data suggest that the effect of TNF on the EGF receptor is not mediated via stimulation of PKC activity in intact fibroblastic cells.
Fig. 3. Effect of TNF on 80 KDa protein phosphorylation. Quiescent cultures of Swiss 3T3 cells were labeled with 32P for 3 h and were exposed to 4 ng/ml PDGF, 10 nM Bombesin for 5 min (lanes 2 and 3), and to 0.1, 1 and 10 nM TNF for 5 min (lanes 4, 5, 6) or 15 min (lanes 8, 9, 10) Untreated cells were extracted at 5 min (lane 1) and at 15 min (lane 7). The samples were analyzed for “P incorporation into 80.kDa protein as described in Materials and Methods.
Discussion
As a direct result of TNF-receptor interaction, different effects may be triggered at the cellular level. These include growth inhibition and cytolytic action but growth stimulatory effects have also been described [19]. The mechanism of such biologic effects is likely to be quite different. However, the nature of the signal evoked upon TNF-receptor binding remains unclear. The expression of the TNF receptor alone is not sufficient to determine inhibitory or stimulatory effects of TNF [3]. In an attempt to investigate the post-receptor message that leads to inhibitory events, we selected a cell line responsive to the cytotoxic action of TNF. This TNF-sensitive cell line is also characterized by the expression of EGF receptors. The results presented in this study indicate that TNF caused a reduction of about 40% of EGF-induced tyrosine phosphorylation of the receptor itself. This effect, evidenced with antiphosphotyrosine antibodies, is in agreement with observations reported by
107
other authors, in a similar cell system (i.e. of fibroblasts), using immunoprecipitation [32P]EGF receptor [2]. In contrast, stimulation of EGF receptor tyrosine protein kinase activity by TNF has been described by Donato et al. [8] in human carcinoma cells. It is well known that the EGF receptor is modulated by PKC, as indicated by specific enzyme activators [6,10]. To investigate the molecular basis of the reduction of EGF receptor phosphorylation, TNF effects on Swiss 3T3 fibroblasts were examined following downregulation of PKC. Under these conditions, a 40% reduction of EGF receptor phosphorylation was still detected. This finding is consistent with a PKC independent mechanism of transmodulation of the EGF receptor by TNF. Relevant to this point is the observation that TNF did not induce phosphorylation of the 80 kDa specific substrate for PKC. In addition, a lack of phosphatidic acid production following TNF exposure provides indirect evidence that the mechanism of EGF receptor modulation by TNF was independent of PKC activation (not shown). Indeed, it has been reported that TNF treatment did not cause phosphorylation of Thr 654 of the EGF receptor (i.e., a site of PKCphosphorylation) [2]. However, an increased phosphorylation of EGF receptor protein in serine residues (as well as in other proteins) in TNF-treated cells has been reported [3,12]. This could be the result of activation of serine kinase or other protein kinases. Thus, a tentative explanation for the TNF effects on Swiss 3T3 cells observed in our study is that reduction of the tyrosine kinase activity of the EGF receptor occurs through phosphorylation of a site other than Thr 654. The possibility of phosphorylation of the EGF receptor at different sites has been already described following cell treatment with thapsigargin [9]. This multiple phosphorylation presumably involves different protein kinases.
and
serum in the
References 1
2
3
4
5
6
7
8
9
10
11
Acknowledgements The authors thank Drs. R.A. Gambetta
C. Lanzi for the antiphosphotyrosine and Ms. L. Zanesi for her assistance preparation of the manuscript.
Alley, M.C., Sendiers, D.A., Monks, A., Hursey, M.L.. Czerwinski, M.J., Fine. D.L., Abbott, B.J., Mayo, J.G., Schoemaker, R.H. and Boyd, M.R. (1988) Feasibility of drug screening with panels of human tumor cell lines using microculture tetrazolium assay. Cancer Res., 48, 589-601. Bird, T.A. and Saklatvala. J. (1989) IL-l and TNF transmodulate epidermal growth factor receptors by a protein kinase C-independent mechanism. J. Immunol., 142, 126-133. Bird. T.A. and Saklatvala, J. (1990) Down-modulation of epidermal growth factor receptor affinity in fibroblasts treated with interleukin 1 or tumor necrosis factor is associated with phosphorylation at a site other than threonine 654. J. Biol. Chem., 265, 235-240. Blackshear, P.J., Wen. L., Glynn, B.P. and Witters. L.A. (1986) Protein kinase C-stimulated phosphorylation in vitro of a M, 80,000 protein phosphorylated in response to phorbol esteri: and growth factors in intact fibroblasts. J. Bi01. Chem., 261, 1459-1469. Cirillo, D.M., Gaudino, G., Naldini, C., et al. (1986) Receptor for bombesin with associated tyrosine kinase activity. Mol. Cell. Biol.. 6, 4641-4649. Cachet, C., Gill, G.N., Meisenhelder, J., Cooper, J.A. and Hunter, T. (1984) C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity. J. Biol. Chem., 259, 2553-2558. Collins, M.K.L. and Rozengurt. E. (1984) Homologous and heterologous mitogenic desensitization Swiss 3T3 cells to phorbol ester and vasopressin: role of receptor and postreceptor steps. J. Cell. Physiol., 118. 133-142. Donate, N.J., Gallick. G.E., Steck, P.A. and Rosenblum. M.G. (1989) Tumor necrosis factor modulates epidermal growth factor receptor phosphorylation and kinase activity in human tumor cells. J. Biol. Chem., 264. 20474-20481. Friedman, B.. van Amsterdam, J.. Fujiki, H. and Rosner. M.R. (1989) Phosphorylation at threonjne-654 is not required for negative regulation of the epidermal growth factor receptor by non-phorbol tumor promoters. Proc. Natl. Acad. Sci. U.S.A., 86, 812-816. Ivashita, S. and Fox, C.F. (1984) Epidermal growth factor and potent phorbol tumor promoters induce epidermal growth factor receptor phosphorylation in a similar but distinctively different manner in human epidermoid carcinoma A431 cells. J. Biol. Chem., 259. 2559-2567. Johnson, S.E. and Baglioni C. (1988). Tumor necrosis factor receptors and cytocidal activity are down-regulated by activators of protein kinase C. J. Biol. Chem., 263, 5686-5692.
108
12
Kavr, P. and Saklatvala, J. (1988) lnterleukin 1 and tumor necrosis factor increase phosphorylation of fibroblast proteins. FEBS Letters, 241, 6-10.
13
Laemmli. U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227. 680-685 Raz, A., Wyche, A. and Needleman, P. (1989) Temporal and pharmacological division of fibroblast cyclooxygenase expression into transcriptional and translation phases. Proc. Natl. Acad. Sci. U.S.A., 86, 1657- 1661. Ross, A.M.. Baltimore, D. and Eisen, H.N. (1981) Phosphotyrosine-containing proteins isolated by affinity chromatography with antibodies to a synthetic hapten. Nature, 294, 654-656. Rozengurt, E.. Rodriguez-Pena. M. and Smith, K.A. (1983) Phorbol esters, phospholipase C, and growth factors rapidly stimulate the phosphorylation of a Mr 80,000 protein in intact quiescent 3T3 cells. Proc. Natl. Acad. Sci. U.S.A., 80. 7244-7248. Sehgal, P.B.. Walther, 2. and Tamm. I. (1987). Rapid enhancement of &interferon/B-cell differentiation factor
14
15
16
17
18
19
20
21
in human fibroblasts by BSF-2 gene expression diacylglycerols and the calcium ionophore A23187. Proc. Natl. Acad. Sci. U.S.A., 84, 3663-3667. Stauber, G.B.. Aiyer, R.A. and Aggarwal, B.B. (1988) Human tumor necrosis factor-o receptor. Purification by immunoaffinity chromatography and initial characterization. J. Biol. Chem., 263, 19098-19014. Sugarman. B. J., Aggarwal, B.B., Hass, P.E., Figari, I.S., Palladino. M.A. and Shepard. H.M. (1985) Recombinant human tumor necrosis factor-o: effects on proliferation of normal and transformed cells in vitro. Science, 230, 943-945. Unglaub, R.. Maxeiner, B., Thoma, B.. Pfizenmaier. K., Scheurich, K. and Scheurich, P. (1987). Downregulation of tumor necrosis factor (TNF) sensitivity via modulation of TNF binding capacity by protein kinase C activators. J. Exp. Med., 166, 1788-1797. Young, S.. Parker, P.J., Ullrich. A. and Stabel, S. (1987) Down-regulation of protein kinase C is due to an increased rate of degradation.Biochem. J.. 244. 775-779.
103 Ltd.
Modulation of epidermal growth factor receptor phosphorylation by tumor necrosis factor L. Guazzoni,
P. Perego,
Division of Experimental Milan (Italy) (Received (Accepted
Oncology
tyrosine
P. Banfi and F. Zunino B, Istituto
Nazionale
per lo Studio
e la Cura
dei Tumori.
Via
Venezian
1. 20133
13 November 1990) 28 January 1991)
receptors of susceptible cells [ 191. Expression of membrane receptors for TNF appears necessary but not sufficient to determine its growth inhibitory or cytolytic action. Antitumor activity of TNF has been ascribed to a direct effect on tumor cells and an indirect effect on tumor vascularization. The early biochemical events occurring following TNF binding to specific receptors are not completely understood. Modulation of EGF receptor tyrosine kinase activity by TNF in sensitive human tumor cell lines has suggested a role of the EGF receptor in the cell response that results in growth inhibition [8]. The precise mechanism of the reported modulation remains unclear. It is known that PKC is involved in control of EGF receptor function [6, lo]. PKC itself has been implicated in the control of cell responsiveness to TNF. The possibility is supported by the following observations: (a) PKC activating agents induce down-regulation of the TNF receptor [20,11]; (b) PKC inhibitors were shown to block some TNFmediated effects [ 17,141. We examined, in a different TNF-sensitive cell line [7], a possible link between EGF receptor protein kinase activity and TNF action. In this approach, the specific tyrosine kinase activity of the EGF receptor was investigated with antiphosphotyrosine antibodies in intact cells.
Summary
The effects of tumor necrosis factor (TNF) on epidermal growth factor (EGF) receptor tyrosine phosphorylation were investigated in Swiss 3T3 cells, which are sensitive to TNF action. At cytotoxic levels, TNF produced an appreciable inhibition of EGF-induced autophosphorylarion of the receptor. A similar inhibition was detected even after prolonged treatment IL’-0-tetradecanoylphorbol-13-acetate with (TPA) which produces down-regulation of protein kinase C (PKC). According to this finding, TNF does not induce phosphorylation of the 80 kDa PKC-specific substrate. These results support the hypothesis that the inhibition of EGF receptor tyrosine phosphorylation is not mediated uia stimulation of PKC activity in intact Swiss 3T3 cells. Keywords:
epidermal
growth
factor;
tumor
necrosis factor Introduction
Like other cytokines, TNF exerts its biologic action through binding to specific cell surface Correspondence to: F. Zunino, Istituto Nazionale Tumori. Division of Experimental Oncology B. Via Venezian 1, 20133 Milan, Italy. 0304-3835/91/$03.50 Published and Printed
0 1991 Elsevier Scientific in Ireland
Publishers
Ireland
Ltd
104
Materials and Methods Reagents Recombinant human TNFa was purchased from Amersham Corp. (Amersham, Buckinghamshire, U.K.). EGF, platelet derived growth factor (PDGF), bombesin and TPA were purchased from Sigma Chemical Co. (St. Louis, MO). Cells and growth condition Swiss 3T3 fibroblasts were obtained from the Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia (Brescia, Italy). They were routinely grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% calf serum (Colorado Serum Co., Denver, Colorado, USA) at 37°C in a humified atmosphere containing 5% CO*. Cytotoxic assay Cell survival was assayed by the calorimetric signal produced by cleavage of the tetrazolium salt 3-(4,5-dimethylthiazol-Z-yl)-2,5-diphenyltetrazolium bromide (MTT) [l]. Briefly, cells were harvested from exponential-phase maintenance cultures, dispensed into 96-well culture plates in 100 ~1 volume and treated with TNF. After incubation of the microtiter plates for 96 h, 10 ~1 of MTT working solution (5 mg/ml) was added to each well and cultures were incubated at 37°C for 4 h. The culture medium was removed from the wells and replaced with 100 ~1 of dimethylsulfoxide (DMSO). The absorbance of each well was measured using a microculture plate reader at 550 nm interfaced with an Apple computer. Tyrosine phosphorylation of EGF receptor Cells were seeded at 1. 104/cm2 on 5-cm petri dishes, grown to confluency and then left for 24 h in DMEM containing 0.2% serum, 0.1% bovine serum albumin and 10 mM 4- (2-hydroxyethyl) - 1-piperazineethanesulfonic acid (HEPES) pH 7.2, to obtain the quiescent state. Confluent and growth-arrested monolayers were incubated with 10m6 M TPA or TNF at the indicated concentrations, at 37”C,
with shaking. After 15 min EGF (50 pg/ml) was added for 3 min. The plates were then chilled on ice and media immediately removed. Cells were rinsed three times with phosphate-buffered saline and scraped in sodium dodecylsulfate (SDS) sample buffer [ 131 containing 1 mM phenylmethyl-sulfonyf fluoride (PMSF), 125 pg/ml leupeptin, 0.1 mM Na2Mo04 and 1 mM NasV04. The extracts were then boiled and sonicated. Samples from each plate were fractionated by SDSPAGE (polyacrylamide gel electrophoresis) [13] and blotted on nitrocellulose sheets. Filters were immunodecorated with an antiphosphotyrosine serum prepared as originally described by Ross et al. [15] and purified by affinity chromatography [5]. To detect the immunocomplexes the nitrocellulose blots were incubated with ‘251-protein A (Amersham) at 400 000 cpm/ml and exposed to autoradio-
graphy. Phosphorylation of the 80-kDa protein Swiss 3T3 fibroblasts were grown to confluency in 24-well plates. The cultures were washed with DMEM without phosphate, and the cells were incubated with the medium containing carrier-free 32Pi at 0.2 mCi/ml at 37°C for 3 h [16]. The cells were then washed with DMEM without phosphate at 37°C and exposed to TNF or other agents for various times; the reaction was stopped by removing the medium. Cells were immediately extracted with 0.5% Triton X-100, 10 mM 1,4triperazinediethanesulfonic acid (PIPES) pH 6.9, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), 10 mM NaF and 1 mM PMSF at 0°C with shaking for 5 min. SDS sample buffer containing 1 mM PMSF, 125 pg/ml leupeptin, 0.1 mM Na2Mo04 and 1 mM Na3V04 was added to supernatants containing the 80 kDa heat-stable protein [4]. To determine the radioactivity incorporated, casein (8 mg/ml) was used as vehicle, and samples were precipitated with 10% trichloroacetic acid/ 10 mM sodium pyrophosphate on Whatman 3MM filters. Filters were washed twice with 5% trichloroacetic acid/l0 mM sodium pyrophos-
105
phate and with acetone at 0°C and dried, and the radioactivity was determined by liquid scintillation. Samples were resoluted by SDSPAGE (10% acrylamide). Gels were fixed, dried and exposed to autoradiography. Results
Effect of TNF on the growth of Swiss 3T3 cells The effect of TNF on the growth of Swiss 3T3 fibroblasts was examined following exposure to the agent for 96 h. The doseresponse curve for the effect of TNF is shown in Fig. 1. According to the MIT assay, the percentage of control absorbance reflects the surviving fraction of the cells. The TNF effect was found to be dose-dependent, and the concentration required to inhibit 50% cell growth was 1.55 nM. Thus, exponentially growing Swiss 3T3 cells showed a sensitivity to the antiproliferative action of TNF comparable to that of other sensitive cell systems [19]. Effect of TNF on EGF receptor autophosphorylation
To determine whether TNF affects EGF receptor tyrosine phosphorylation, Swiss 3T3 mouse fibroblast monolayers were incubated with 0.1, 1 and 10 nM TNF for 15 min and then exposed to EGF (50 ng/ml) for 3 min. The results of a representative experiment are shown in Fig. 2A. The densitometric analysis
04 0.01
I 0.1
1
10
TNF CONCENTRATION (nM)
activity of TNF on Swiss 3T3 Fig. 1. Cytotoxic fibroblasts. The cells were exposed to TNF for 96 h. Cell survival was measured by the MTT assay.
A
B
Effect of TNF on EGF-induced tyrosine Fig. 2. phosphorylation of the EGF receptor in Swiss 3T3 cells. (A) Quiescent monolayers were incubated with 10m6 M TPA or TNF at the indicated concentrations for 15 min, then exposed to 50 ng/ml EGF for 3 min. Total cell proteins were fractionated by SDS-PAGE, and tyrosine phosphorylation of the EGF receptor was visualized by Western blotting using anti-phosphotyrosine antibodies as described in Materials and Methods. (B) Cells were pre-incubated with 10e6 M TPA for 24 h before treatment as described in (A).
of the results, summarized in Table I, indicates that tyrosine phosphorylation of the EGF receptor was reduced of about 40% in cells treated with 1 and 10 nM TNF, whereas a lower concentration (0.1 nM) had no appreciable inhibitory effect. To determine whether PKC was involved in the observed reduction of EGF receptor autophosphorylation following treatment with TNF, a similar experiment was performed with cells chronically exposed to TPA. Such a treatment is known to down-regulate PKC through an increased rate of degradation [ 181. As expected, following incubation with lo6 M TPA for 24 h, treatment with TPA for 15 min no longer had an inhibitory effect on EGF receptor tyrosin
106
Table 1. Effect of TNF on EGF-induced tyrosine phosphorylation of the EGF receptor in Swiss 3T3 cells. Control refers to phosphorylation of EGF receptor in cells exposed to EGF. See legend to Fig. 2 for experimental details.
12345678910
KDa -80
TNF concentration
0.1 nM 1 nM 10 nM
% Control Without TPA preincubation
After 24 h preincubation with TPA
98 f 12’ 61 zt 5** 60 zt 4*’
112 f 13’ 60 j, 0.5’ 57 f 1.5’
*Average value of 2 experiments *Average value of 3 experiments l
phosphorylation. In the PKC-deficient cells, 1 and 10 nM TNF still inhibited tyrosine phosphorylation of the EGF receptor to the same extent (Fig. 2B; Table I). These data suggest that PKC activation is not necessary for transmodulation of the EGF receptor by TNF. Effect of TNF on phosphorylation specific 80-kDa substrate of PKC
of
the
In a different approach to test whether PKC was involved in transmodulation of the EGF receptor by TNF, the ability of TNF to induce phosphorylation of the specific substrate of PKC (80 K) was examined in quiescent cultures of Swiss 3T3 fibroblasts. Serumdeprived fibroblasts were exposed to 0.1, 1 and 10 nM TNF for 5 and 15 min. To control the function of the experimental system, PDGF and bombesin, which are known to affect the activity of PKC with stimulation of the phosphorylation of the 80 K protein, were added to the cells for 5 min. In contrast, 0.1, 1 and 10 nM TNF did not activate PKC (Fig. 3). These data suggest that the effect of TNF on the EGF receptor is not mediated via stimulation of PKC activity in intact fibroblastic cells.
Fig. 3. Effect of TNF on 80 KDa protein phosphorylation. Quiescent cultures of Swiss 3T3 cells were labeled with 32P for 3 h and were exposed to 4 ng/ml PDGF, 10 nM Bombesin for 5 min (lanes 2 and 3), and to 0.1, 1 and 10 nM TNF for 5 min (lanes 4, 5, 6) or 15 min (lanes 8, 9, 10) Untreated cells were extracted at 5 min (lane 1) and at 15 min (lane 7). The samples were analyzed for “P incorporation into 80.kDa protein as described in Materials and Methods.
Discussion
As a direct result of TNF-receptor interaction, different effects may be triggered at the cellular level. These include growth inhibition and cytolytic action but growth stimulatory effects have also been described [19]. The mechanism of such biologic effects is likely to be quite different. However, the nature of the signal evoked upon TNF-receptor binding remains unclear. The expression of the TNF receptor alone is not sufficient to determine inhibitory or stimulatory effects of TNF [3]. In an attempt to investigate the post-receptor message that leads to inhibitory events, we selected a cell line responsive to the cytotoxic action of TNF. This TNF-sensitive cell line is also characterized by the expression of EGF receptors. The results presented in this study indicate that TNF caused a reduction of about 40% of EGF-induced tyrosine phosphorylation of the receptor itself. This effect, evidenced with antiphosphotyrosine antibodies, is in agreement with observations reported by
107
other authors, in a similar cell system (i.e. of fibroblasts), using immunoprecipitation [32P]EGF receptor [2]. In contrast, stimulation of EGF receptor tyrosine protein kinase activity by TNF has been described by Donato et al. [8] in human carcinoma cells. It is well known that the EGF receptor is modulated by PKC, as indicated by specific enzyme activators [6,10]. To investigate the molecular basis of the reduction of EGF receptor phosphorylation, TNF effects on Swiss 3T3 fibroblasts were examined following downregulation of PKC. Under these conditions, a 40% reduction of EGF receptor phosphorylation was still detected. This finding is consistent with a PKC independent mechanism of transmodulation of the EGF receptor by TNF. Relevant to this point is the observation that TNF did not induce phosphorylation of the 80 kDa specific substrate for PKC. In addition, a lack of phosphatidic acid production following TNF exposure provides indirect evidence that the mechanism of EGF receptor modulation by TNF was independent of PKC activation (not shown). Indeed, it has been reported that TNF treatment did not cause phosphorylation of Thr 654 of the EGF receptor (i.e., a site of PKCphosphorylation) [2]. However, an increased phosphorylation of EGF receptor protein in serine residues (as well as in other proteins) in TNF-treated cells has been reported [3,12]. This could be the result of activation of serine kinase or other protein kinases. Thus, a tentative explanation for the TNF effects on Swiss 3T3 cells observed in our study is that reduction of the tyrosine kinase activity of the EGF receptor occurs through phosphorylation of a site other than Thr 654. The possibility of phosphorylation of the EGF receptor at different sites has been already described following cell treatment with thapsigargin [9]. This multiple phosphorylation presumably involves different protein kinases.
and
serum in the
References 1
2
3
4
5
6
7
8
9
10
11
Acknowledgements The authors thank Drs. R.A. Gambetta
C. Lanzi for the antiphosphotyrosine and Ms. L. Zanesi for her assistance preparation of the manuscript.
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