Transient Suppression of Ligand-mediated Activation of Epidermal Growth Factor Receptor by Tumor Necrosis Factor-α through the TAK1-p38 Signaling Pathway
2007; Elsevier BV; Volume: 282; Issue: 17 Linguagem: Inglês
10.1074/jbc.m608723200
ISSN1083-351X
AutoresPattama Singhirunnusorn, Yoko Ueno, Mitsuhiro Matsuo, Shunsuke Suzuki, Ikuo Saiki, Hiroaki Sakurai,
Tópico(s)HER2/EGFR in Cancer Research
ResumoEpidermal growth factor receptor (EGFR) has been shown to be activated by specific ligands as well as other cellular stimuli including tumor necrosis factor-α (TNF-α). In the present study, we found that cellular stress suppressed ligand-mediated EGFR activity. Both TNF-α and osmotic stress rapidly induced phosphorylation of EGFR. This phosphorylation of EGFR and the activation of mitogen-activated protein kinases and NF-κB occurred independently of the shedding of extracellular membrane-bound EGFR ligands and intracellular EGFR tyrosine kinase activity. Transforming growth factor-β-activated kinase 1 (TAK1) was involved in the TNF-α-induced signaling pathway to EGFR. In addition, experiments using chemical inhibitors and small interfering RNA demonstrated that p38α is a common mediator for the cellular stress-induced phosphorylation of EGFR. Surprisingly, the modified EGFR was not able to respond to its extracellular ligand due to transient internalization through the clathrin-mediated mechanism. Furthermore, turnover of p38 activation led to dephosphorylation and recycling back to the cell surface of EGFR. These results demonstrated that TNF-α has opposite bifunctional activities in modulating the function of the EGFR. Epidermal growth factor receptor (EGFR) has been shown to be activated by specific ligands as well as other cellular stimuli including tumor necrosis factor-α (TNF-α). In the present study, we found that cellular stress suppressed ligand-mediated EGFR activity. Both TNF-α and osmotic stress rapidly induced phosphorylation of EGFR. This phosphorylation of EGFR and the activation of mitogen-activated protein kinases and NF-κB occurred independently of the shedding of extracellular membrane-bound EGFR ligands and intracellular EGFR tyrosine kinase activity. Transforming growth factor-β-activated kinase 1 (TAK1) was involved in the TNF-α-induced signaling pathway to EGFR. In addition, experiments using chemical inhibitors and small interfering RNA demonstrated that p38α is a common mediator for the cellular stress-induced phosphorylation of EGFR. Surprisingly, the modified EGFR was not able to respond to its extracellular ligand due to transient internalization through the clathrin-mediated mechanism. Furthermore, turnover of p38 activation led to dephosphorylation and recycling back to the cell surface of EGFR. These results demonstrated that TNF-α has opposite bifunctional activities in modulating the function of the EGFR. Epidermal growth factor receptor (EGFR) 2The abbreviations used are: EGFR, epidermal growth factor receptor; NF-κB, nuclear factor-κB; IKK, IκB kinase; TNF, tumor necrosis factor; TAK1, transforming growth factor-β-activated kinase 1; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; ADAM, A disintegrin and metalloprotease; FACS, fluorescence-activated cell sorter; E3, ubiquitin-protein isopeptide ligase. 2The abbreviations used are: EGFR, epidermal growth factor receptor; NF-κB, nuclear factor-κB; IKK, IκB kinase; TNF, tumor necrosis factor; TAK1, transforming growth factor-β-activated kinase 1; MAPK, mitogen-activated protein kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; ADAM, A disintegrin and metalloprotease; FACS, fluorescence-activated cell sorter; E3, ubiquitin-protein isopeptide ligase. is a member of the receptor tyrosine kinase family and plays a critical role in a wide variety of cellular functions, including proliferation, differentiation, and apoptosis (1Ullrich A. 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Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (740) Google Scholar, 43Shinohara H. Yasuda T. Aiba Y. Sanjo H. Hamadate M. Watarai H. Sakurai H. Kurosaki T. J. Exp. Med. 2005; 202: 1423-1431Crossref PubMed Scopus (130) Google Scholar, 44Choo M.K. Sakurai H. Koizumi K. Saiki I. Int. J. Cancer. 2006; 118: 2758-2764Crossref PubMed Scopus (40) Google Scholar).We have recently reported that gefitinib, an EGFR tyrosine kinase inhibitor, abrogated the intrahepatic metastasis of murine hepatocellular carcinoma (27Ueno Y. Sakurai H. Matsuo M. Choo M.K. Koizumi K. Saiki I. Br. J. Cancer. 2005; 92: 1690-1695Crossref PubMed Scopus (28) Google Scholar). TNF-α-induced cellular responses, such as the activation of MAPK, expression of integrin, and invasion are also regulated by the ADAM-mediated transactivation of EGFR. In the present study, we found that TNF-α actually suppressed the ligand-mediated activation of EGFR via TAK1-p38-mediated signaling in HeLa cells.EXPERIMENTAL PROCEDURESAntibodies and Reagents—An anti-phospho-TAK1 (Thr-187) antibody was generated as described previously (40Singhirunnusorn P. Suzuki S. Kawasaki N. Saiki I. Sakurai H. J. Biol. Chem. 2005; 280: 7359-7368Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Other phosphospecific antibodies against p38, JNK, ERK, p65, S6K, and EGFR (Tyr-845, -1045, -1068, and -1173) were purchased from Cell Signaling Technology. Antibodies against TAK1 (M-579), p38 (C-20-G), JNK (FL), ERK1 (C-16), ERK2 (C-14), p65 (C-20-G), EGFR (1005), c-Cbl (C-15), clathrin heavy chain (C-20), Cbl-b (C-20) and Actin (C-11) were obtained from Santa Cruz Biotechnology. A neutralizing anti-EGFR monoclonal antibody (clone LA1, mouse IgG1) and isotype control IgG1 were purchased from Upstate Biotechnology and R & D Systems, respectively. Recombinant human TNF-α and EGF were obtained from R & D Systems, SB203580, SP600125, U0126, SC-514, GM6001, PD153035, and AG825 from Merck Biosciences, and recombinant human p38α, a TAK1-TAB1 fusion protein, and λ-phosphatase from Upstate Biotechnology. 5Z-7-oxozeaenol, a selective TAK1 inhibitor, was a gift from Chugai Pharmaceutical Co., Ltd. All of the chemical inhibitors were dissolved in Me2SO, and the final concentration of Me2SO was <0.1%.Cell Cultures—HeLa and A549 cells were maintained in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2.Transfection of Small Interfering RNAs—Duplex small interfering RNAs (siRNAs) with two nucleotides overhanging at the 3′ end of the sequence were designed at iGENE Therapeutics and synthesized at Hokkaido System Science Co., Ltd. The target sequences for TAK1, p38α, clathrin heavy chain, c-Cbl, Cbl-b, and firefly luciferase (GL2) were reported previously (14Huang F. Kirkpatrick D. Jiang X. Gygi S. Sorkin A. Mol. Cell. 2006; 21: 737-748Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 40Singhirunnusorn P. Suzuki S. Kawasaki N. Saiki I. Sakurai H. J. Biol. Chem. 2005; 280: 7359-7368Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 46Motley A. Bright N.A. Seaman M.N. Robinson M.S. J. Cell Biol. 2003; 162: 909-918Crossref PubMed Scopus (550) Google Scholar). HeLa cells were transfected with the siRNAs in a final concentration of 25–50 nm using Lipofectamine reagents. At 72 h post-transfection, the cells were stimulated.Phosphatase and Kinase Reactions—EGFR immunoprecipitated from untreated or TNF-α-stimulated HeLa cells were incubated with λ-phosphatase at 30 °C for 30 min or with recombinant p38α and TAK1-TAB1 fusion protein in the presence of [32P]ATP. Phosphatase activity was analyzed as a shift in mobility on immunoblotting. The kinase activity was visualized by autoradiography.Immunoblotting—After the stimulation, whole cell lysates were prepared as described previously. Cell lysates were resolved by 7.5, 10, or 12.5% SDS-PAGE and transferred to an Immobilon-P nylon membrane (Millipore). The membrane was treated with BlockAce (Dainippon Pharmaceutical Co., Ltd., Suita, Japan) and probed with primary antibodies. The antibodies were detected using horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat IgG (DAKO) and visualized with the ECL system (Amersham Biosciences). Some antibody reactions were carried out in the Can Get Signal solution (TOYOBO).FACS Analysis—After the stimulation, HeLa cells were harvested in phosphate-buffered saline. Cells were fixed with 2% paraformaldehyde for 20 min at room temperature. Where indicated, cells were permeabilized in phosphate-buffered saline containing 0.1% Triton X-100. The cells were resuspended in 100 μl of FACS buffer (phosphate-buffered saline containing 0.5% bovine serum albumin and 0.05% NaN3) containing 1 μg of anti-EGFR monoclonal antibody (LA1) and incubated on ice for 30 min. After washing with FACS buffer, the cells were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG antibody (DAKO) on ice for 30 min and analyzed by the FACSCalibur system (BD Biosciences). The ratio of internalization of EGFR was calculated using the median values of fluorescence.Statistical Analysis—The significance of differences between groups was determined by applying Student's two-tailed t test. Values of p < 0.05 were considered significant.RESULTSStress-induced Modifications of EGFR—HeLa cells have commonly been used for characterization of the TNF-α- and EGF-induced signaling pathways. We first confirmed activation of the downstream signaling pathways. Fig. 1A shows that both TNF-α and EGF rapidly activated MAPKs (ERK, JNK, and p38) at similar time points within 10 min. In contrast, TAK1 and NF-κB p65 were only activated by TNF-α. The total expression level of these proteins was comparable (data not shown).We next investigated the effects of TNF-α on the phosphorylation of EGFR. Interestingly, TNF-α rapidly induced a shift in the mobility of EGFR on SDS-PAGE within 10 min (Fig. 1A). In contrast, the mobility of other EGFR family members (ErbB2–4) was not changed (data not shown) in HeLa cells. EGF also caused a mobility shift within 10 min (Fig. 1A). It should be noted that, although EGF induced strong phosphorylation at Tyr-845, -1045, -1068, and -1173 in the intracellular tyrosine kinase domain of EGFR, only a faint tyrosine phosphorylation was detected in response to TNF-α (Fig. 1A). These results indicated that the TNF-α-induced mobility shift of EGFR is independent of the tyrosine phosphorylation.Once EGFR is activated by a specific ligand, it has been shown to rapidly enter a program of degradation through the phosphorylation of Tyr-1045 and subsequent c-Cbl-mediated ubiquitination. In fact, the form shifted by EGF largely disappeared within 2 h (Fig. 1B). In contrast, the TNF-α-induced mobility shift was transient, and there was a return to the control level at 60 min (Fig. 1B). A similar mobility shift was observed in A549 human lung adenocarcinoma cells (Fig. 1B). High osmotic stress with additional 300 mm NaCl also caused a rapid shift in the mobility of EGFR without the tyrosine phosphorylation (Fig. 1C); however, it was sustained for at least 2 h. Osmotic stress also induced prolonged activation of MAPKs but not TAK1 and NF-κB (Fig. 1C). Interestingly, no obvious degradation of EGFR was observed in cells treated with TNF-α and osmotic stress.Stress-induced Modification of EGFR Is Independent of ADAM-mediated Shedding of EGFR Ligand—G-protein-coupled receptor-mediated transactivation of EGFR has been shown to be mediated by the ADAM-dependent release of membrane-bound extracellular EGFR ligands. To explore the involvement of this mechanism in stress-induced modification of EGFR, we first examined the effects of PD153035, a potent EGFR tyrosine kinase inhibitor. Fig. 2A shows that PD153035 at 0.1 μm completely inhibited the EGF-induced mobility shift as well as the tyrosine phosphorylation of EGFR, whereas AG825, a selective ErbB2 tyrosine kinase inhibitor, had no inhibitory effect. In contrast, although a higher concentration (10 μm) of PD153035 blocked the TNF-α-induced mobility shift of EGFR, the TNF-α-induced modification was not influenced by PD153035 at the concentration having an effect on the EGFR tyrosine kinase (1 μm). These results indicate that the tyrosine kinase activity of EGFR was not involved in the mobility shift (Fig. 2B).FIGURE 2Effects of anti-EGFR agents on stress-induced modification of EGFR. HeLa cells were pretreated with the indicated concentrations (μm) of PD153035 (PD)(A and B), AG825 (AG)(A and B), or GM6001 (GM)(C and D) for 30 min and then stimulated with 10 ng/ml EGF (A), 20 ng/ml TNF-α (B and C), or 300 mm NaCl (Osmo)(D) for 10 min. E, cells were pretreated with an anti-EGFR monoclonal antibody (1 μg/ml) or an isotype-matched control IgG1 (1 μg/ml) for 2 h and then stimulated with TNF-α, osmotic stress, or EGF for 10 min. Whole cell lysates were immunoblotted with anti-EGFR, anti-EGFR (pY845), and anti-EGFR (pY1068) antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)GM6001, a broad spectrum inhibitor for metalloproteases, has widely been used as an ADAM inhibitor that blocks the transactivation of EGFR. However, GM6001 was not able to block the TNF-α and osmotic stress-induced mobility shift (Fig. 2, C and D). Furthermore, a neutralizing monoclonal anti-EGFR antibody did not inhibit the TNF-α- and osmotic stress-induced modifications of EGFR, although it effectively abrogated the EGF-induced activation of EGFR (Fig. 2E). Collectively, these results clearly demonstrated that the stress-induced modification of EGFR occurs independent of the ADAM-mediated shedding of the ligands.Effects on the Downstream Signaling Pathways—We and others have demonstrated that TNF-α-induced signaling pathways are partly dependent on the tyrosine kinase activity of EGFR in several types of cells (27Ueno Y. Sakurai H. Matsuo M. Choo M.K. Koizumi K. Saiki I. Br. J. Cancer. 2005; 92: 1690-1695Crossref PubMed Scopus (28) Google Scholar, 34Chen W.N. Woodbury R.L. Kathmann L.E. Opresko L.K. Zangar R.C. Wiley H.S. Thrall B.D. J. Biol. Chem. 2004; 279: 18488-18496Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 35Argast G.M. Campbell J.S. Brooling J.T. Fausto N. J. Biol. Chem. 2004; 279: 34530-34536Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Therefore, we next confirmed the effects of EGFR blockers on stress-induced signaling pathways in HeLa cells. Although PD153035 clearly blocked EGF-induced signaling pathways in a concentration-dependent manner (Fig. 3C), TNF-α-induced activation of p38, JNK, ERK, TAK1, and NF-κB was resistant at up to 10 μm (Fig. 3A). Similarly, osmotic stress-induced signaling pathways were not affected (Fig. 3B). In addition, neither GM6001 nor the EGFR neutralizing antibody blocked the TNF-α signaling pathways (Fig. 3, D and E). These results clearly demonstrated that the modification of EGFR in response to TNF-α and osmotic stress is not a process of EGFR transactivation.FIGURE 3Effects of anti-EGFR agents on the downstream signaling pathways. HeLa cells were pretreated with the indicated concentrations (μm) of PD153035 (PD) for 30 min (D) and an anti-EGFR monoclonal antibody (E) as described in the legend to Fig. 2 and then stimulated with TNF-α (A), NaCl (high asmolarity, Osmo)(B), or EGF (C) for 5 min (for TAK1 and p65) or 10 min (for the other kinases). Whole cell lysates were immunoblotted with phosphospecific and control (Cont) antibodies against p38, JNK, ERK, TAK1, and p65.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Signaling Pathways Leading to EGFR—As shown in Fig. 1, TNF-α and osmotic stress rapidly induces intracellular signaling pathways. To identify the signaling pathway leading to the modification of EGFR, we first tried to examine the effects of chemical inhibitors for downstream kinases. The TNF-α-induced modification of EGFR was completely inhibited by pretreatment with 5Z-7-oxozeaenol and SB203580, inhibitors for TAK1 and p38, respectively (Fig. 4A). SB203580 (but not 5Z-7-oxozeaenol) also abrogated the osmotic stress-induced modification (Fig. 4A). In contrast, inhibitors for IKK, JNK, and MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) (SC-514, SP600125 and U0126, respectively) did not block modification of EGFR (Fig. 4A). Similar results were obtained in experiments using siRNA against TAK1 and p38α (Fig. 4, B and C). In contrast, these siRNAs were not effective in blocking the shift in the mobility of EGFR caused by EGF (Fig. 4, B and C).FIGURE 4Role of TAK1 and p38α in stress-induced phosphorylation of EGFR. A, HeLa cells were pretreated with 5Z-7-oxozeaenol (0.3 μm), SC-514 (30 μm), SB203580 (20 μm), or SP600125 (20 μm) for 30 min and then stimulated with TNF-α or osmotic stress by 300 mm NaCl (high asmolarity, Osmo) for 10 min. Cell lysates were immunoblotted with anti-EGFR antibody. Cells were transfected with siRNA (50 nm) against TAK1 (si-TAK1)(B), p38α (si-p38α)(C), and luciferase (si-luc)(B and C). At 72 h post-transfection, the cells were stimulated with TNF-α, osmotic stress, or EGF for 10 min. Cell lysates were immunoblotted with antibodies against EGFR, phospho-p38, p38, and TAK1. D, EGFR was immunoprecipitated (IP) from untreated or TNF-α-stimulated HeLa cells and then incubated with λ-phosphatase (λ-PPase) in vitro. The shift in mobility was analyzed by immunoblotting with anti-EGFR antibody. E, EGFR was immunoprecipitated from untreated HeLa cells and then incubated with recombinant p38α and TAK1-TAB1 fusion protein in the presence of [32P]ATP. Incorporation of [32P]phosphate was visualized by autoradiography after SDS-PAGE. Cont, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TNF-α-induced Phosphorylation of EGFR—To identify the kinds of modifications that occurred on EGFR, we first investigated the possibility of phosphorylation. EGFR was immunoprecipitated from cells untreated or treated with TNF-α for 10 min, and the immunoprecipitates were incubated with λ-phosphatase in vitro. Fig. 4D shows that the reduced mobility was completely restored, indicating that phosphorylation at unknown Ser/Thr residues is involved, at least in part, in the TNF-α-induced modification of EGFR.As shown above, p38α and TAK1 are possible candidates for Ser/Thr kinases. We therefore performed in vitro kinase assays using recombinant kinases. Whole cell lysates prepared from unstimulated HeLa cells were immunoprecipitated with normal IgG or anti-EGFR antibody. The immunoprecipitates were then incubated with recombinant p38α or TAK1-TAB1 fusion protein (an active TAK1 protein) in the presence of 32P-labeled ATP. In the absence of recombinant kinases, EGFR had an autophosphorylation activity. Both p38α and TAK1 induced the incorporation of radioactivity into the EGFR (Fig. 4E); however, the activity was not as strong as that against the known substrates ATF2 and MKK6, respectively (data not shown).Stress-induced Phosphorylation Suppressed Ligand-mediated Activation of EGFR—We investigated the role of the transient modification of EGFR in ligand-mediated activation. HeLa cells were pretreated with TNF-α for 10 or 60 min and then stimulated with EGF for another 2 or 10 min in the presence of TNF-α. The 10-min pretreatment induced a complete shift in the mobility of EGFR (Fig. 5A). The stimulation with EGF did not induce any additional mobility shift. Surprisingly, the EGF-induced tyrosine phosphorylation of EGFR was impaired by the pretreatment with TNF-α for 10 min (Fig. 5A). Interestingly, with the disappearance of the mobility shift at 60 min after pre-TNF-α stimulation, the ability of EGFR to respond to its ligand was largely restored (Fig. 5A). The reduced tyrosine phosphorylation of EGFR is correlated with the suppression of EGF-induced activation of the downstream ribosomal p70 S6 kinase (p70S6K) (Fig. 5B). These results clearly demonstrated that the TNF-α-induced modification of EGFR suppressed the ability of the receptor to respond to its extracellular ligands.FIGURE 5Transient suppression of EGFR by TNF-α. A, HeLa cells were pretreated with TNF-α for 10 or 60 min and then stimulated with EGF for another 2 or 10 min. Whole cell lysates were immunoblotted with anti-EGFR, anti-EGFR (pY845), and anti-EGFR (pY1068) antibodies. B, HeLa cells were pretreated with TNF-α for 10 min and then stimulated with EGF for another 10 min. Whole cell lysates were immunoblotted with anti-phospho-S6K and anti-S6K antibodies. C and D, after pretreatment with 5Z-7-oxozeaenol (0.3 μm) for 30 min, cells were treated with TNF-α for 10 min and then further stimulated with EGF for another 10 min. Whole cell lysates were immunoblotted with anti-EGFR, anti-EGFR (pY845), anti-EGFR (pY1068), anti-phospho-p38, anti-p38, and anti-TAK1 antibodies. The ratios of phosphorylated EGFR (pY845 and pY1068) and total EGFR are shown in D. *, p < 0.01 from three independent dishes. DMSO, dimethylsulfoxide (Me2SO).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate whether the suppression of EGFR is mediated through the TAK1-p38 pathway, we tried to test the effect of a TAK1 inhibitor. HeLa cells were pretreated with the inhibitor before being treated with TNF-α and EGF. In the absence of TNF-α pr
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