tnf/egfr.042301-j of biological

Transactivation of EGFR by TNFHirota et al.

Redox-sensitive transactivation of epidermal growth

factor receptor by tumor necrosis factor confers the NF-kB

activation

Kiichi Hirota, ‡ § Miyahiko Murata, ¶ Tatsuya Itoh, ‡ Junji Yodoi , || and

Kazuhiko Fukuda ‡

From ‡ Department of Anesthesia, Kyoto University Hospital, Kyoto

University, 54 Shogoin-Kawaharacho, Sakyo-Ku, Kyoto, 606-8507, JAPAN, ¶

Department of Integrative Brain Science, Graduate School of Medicine, Kyoto

University, Yoshida, Sakyo-Ku, Kyoto, JAPAN and || Department of Biological

Responses, Institute of Virus Research, Kyoto University, 54 Shogoin-

Kawaharacho, Sakyo-Ku, Kyoto, 606-8507, JAPAN

------------------------------------------------------------------------------------

§ To whom correspondence should be addressed;

phone +81-75-751-3436, fax +81-75-752-3259, e-mail address:

[email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 3, 2001 as Manuscript M011021200 by guest on M

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Running Title

Transactivation of EGFR by TNF

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Summary

Cross-communication between different signaling systems allows the

integration of the great diversity of stimuli that a cell receives under varying

physiological situations. In this paper we have explored a possibility that

tumor necrosis factor (TNF) receptor signal crosstalks with epidermal growth

factor (EGF) receptor signal on the nuclear factor kappa B (NF-kB) activation

pathway. We have demonstrated that overexpression of EGFR in NIH3T3

cells significantly enhances TNF-induced NF-kB-dependent luciferase activity

even without EGF , that EGF treatment has a synergistic effect on the induction

of the reporter activity and that this enhancement is suppressed by AG1478,

EGFR-specific tyrosine kinase inhibitor. We also have shown that TNF

induces tyrosine phosphorylation and internalization of the overexpressed EGFR

in NIH3T3 cells and the endogenously expressed EGFR in A431 cells and that

the transactivation by TNF is suppressed by N -acetyl-L-cysteine or

overexpression of an endogenous reducing molecule, thioredoxin but not by

phosphatidylinositol 3-kinase inhibitors and PKC inhibitor. Taken together,

these evidences strongly suggests that EGFR transactivation by TNF, which is

regulated in a redox-dependent manner, is playing a pivotal role in TNF-induced

NF-kB activation.

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Introduction

A cardinal concept in signal transduction is that receptors are activated by

highly specific interactions with cognate ligands. Hence each receptor recognizes

one or a few structurally related ligands, and each ligand stereochemically

"fits" one or perhaps two structurally related receptors. The specificity of

biological responses generated to extracellular signals depends on adherence to

this rule. For some time, however, it has been clear that structurally unrelated

receptors may communicate with each other in what is termed receptor

transactivation or cross-talk. While receptor cross-talk is no doubt real, its

biological importance remains unclear. Understanding the mechanism(s) by

which inter-receptor communication occurs may offer the experimental means

to explore the underlying biology.

The initial steps in the major pathway utilized by TNF (tumor necrosis

factor) 1 to activate the NF-kB are fairly well understood (1,2). The NF-kB

activation is usually initiated by the interaction of TNF with TNFR1 (p55),

one of two distinct surface receptors, the other being TNFR2 (p75). Ligand-

induced clustering of TNFR1 leads to the recruitment of (TRADD) to the

intracellular portion of the receptor (3). Receptor-bound TRADD recruits

both the protein kinase designated receptor-interacting protein (RIP) and the

4

ring/zinc finger protein TNF receptor-associated factor 2 (TRAF2). These


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two adapter proteins lead to the activation of the NF-kB as well as the

activation of the stress-activated protein kinases (SAPKs) such as c-Jun N-terminal

kinase (JNK) and p38 MAPK (3). IL-1 also activates NF-kB and SAPKs using

a similar pathway initiated by ligand binding to the type 1 IL-1 receptor

(IL-1R1) and involving adapter proteins MyD88, IL-1 receptor-associated

kinase (IRAK), and TRAF6 (4). The precise connection between RIP-TRAF2

or IRAK-TRAF6 complexes and NF-kB are less well established but are

thought to involve the MAPK kinase kinase family members MEKK-1 or

NF-kB-inducing kinase (NIK) (5). Both of these protein kinases can directly

phosphorylate and activate IkB kinase (IKK) which is a multiprotein enzyme

complex consisting of IKK-1, IKK-2, and NEMO (also known as IKKg)

subunits (2,6-9). IKK-a and IKK-b phosphorylate members of the IkB

family which share a common mechanism for regulation of NF-kB. In resting

cells, IkB proteins bind to and mask the nuclear localization sequence of

NF-kB resulting in the retention of the transcription factor in the cytosol.

Phosphorylation of IkB by IKKs targets this protein for rapid ubiquitination

and degradation by the proteasome (2,10). This process releases NF-kB which

then translocates to the nucleus and induces transcription. MEKK-1, but not

NIK, catalyzes the phosphorylation and activation of yet other MAPK kinases

5

and is probably responsible for the parallel activation of SAPKs (11-13).


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Recently, an alternative signaling pathway has been described in some

cells in which Akt directly phosphorylates IKKs leading to the activation of

NF-kB, independent of MEKK-1 and NIK (14). This pathway thus allows

growth factors such as PDGF to activate NF-kB (15). Moreover, TNF and

IL-1 have been found to activate the phosphatidylinositol 3-kinase (PI3K) /Akt

pathway, potentially providing an alternative pathway for TNF and IL-1 to

activate NF-kB that is independent of NIK or MEKK-1. The importance of

this alternative NF-kB pathway compared with the previously described

TRADD/RIP/TRAF2 or MyD88/IRAK/TRAF6 cytokine signaling pathways is

unknown and may vary with the cell type. TNF and IL-1 also activate MAPK,

but this pathway is even less well defined.

The epidermal growth factor (EGF) receptor is ubiquitously expressed in

various tissues and is thus positioned to influence a wide range of responses,

depending on the coordinate expression of the cognate ligand (16,17). A

number of reports have demonstrated that various extracellular stimuli, unrelated

to EGF-like ligands, can also activate the EGF receptor (18). These diverse

stimuli include numerous agonists such as carbachol, bombesin, thrombin, and

LPA for heptahelical G protein-coupled receptors, cytokine receptors such as

prolactin, growth hormone adhesion receptors such as integrins, membrane-

6

depolarizing agents such as KCl, and environmental stress factors such as


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ultraviolet irradiation, g irradiation, oxidants, heat shock and hyperosmotic

shock (19-21). Activation of EGF receptors by such seemingly unrelated and

indirect means is potentially biologically significant (18-20).

Together, these evidences prompted us to investigate a possibility of

crosstalk between TNFa-induced signals and EGFR-mediated signals on the

NF-kB activation. In this paper, we have demonstrated that TNFa transactivates

EGFR in a redox-sensitive manner and this activation enhances the NF-kB

7

activation.


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Experimental Procedures

Cell lines and reagents

A murine embryonic fibroblast cell line NIH3T3 was obtained from Dr.

H. Sabe (Osaka Biosicence Institute, Osaka, Japan). A human embryo kidney

cell-derived cell line HEK293 and human carcinoma A431 cells were from the

American Type Culture Collection (Rockville, MD). Cells were maintained in

DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (50

U/ml penicillin and 100 µg/ml streptomycin) at 37˚C under a humidified

atmosphere of 5% CO2. Recombinant human TNFa and recombinant human

EGF were purchased from Boehringer Mannheim GmbH. Monoclonal antibodies

raised against EGF receptor (E12020) and activated EGF receptor (E12120)

were from BD Transduction Laboratory. Tyrphostin AG1478, wortmannin,

LY294002, PD98059 and GF109203X were from Calbiochem. N -acetyl-L-

cysteine (NAC) was from Sigma.

Plasmids

An expression vector of GFP-fused human EGF receptor (EGFR), in

which the GFP motiety is fused to carboxyl-terminus of EGFR, pEGFR-EGFP

was kindly provided by Dr. Alexander Sorkins (University of Colorado Health

8

Science Center). Point mutation of EGFR, K721A, was performed by a


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PCR-based technique (22) based on pEGFR-EGFP and results in

pEGFR(K721A)-EGFP. pEGFP-N3 was obtained from Clontech. Expression

plasmids of TRAF2 and IKKa were kindly provide by Drs. Reiko Shinkura

and Kazuhiro Kitada, Kyoto University (23). An expression plasmid for

human thioredoxin, pcDNA3-TRX was described previously (24,25). pNF-

kB-Luc and pSV40-b-Galactosidase were from Stratagene and Promega,

respectively.

Transfection and Reporter gene assay

Cells were plated in 24-well plates at a density of 5x104 cells per well.

The Fugene 6 reagent (Roche) was used for transfection. In each transfection,

250 ng of pNF-kB-Luc and 100 ng of pSV40-b-galactosidase as an internal

control were pre-mixed and used. In the case of NIH3T3 cells, 250 ng of

pEGFR-EGFP or pEGFP-N3 plasmids were used additionally. At 12 h after

transfection, cells were serum-starved (0.1%) or not. After further 12 h

incubation, cells were treated with TNFa and/or EGF with or without kinase

inhibitors. After 6 h, the cells were harvested and the luciferase activity was

determined using an assay system (Promega) with a luminometer, Lumat

LB9507 (Berthold) (24-27). The relative fold induction of luciferase activity

was calculated after normalization dividing the luciferase activity by b-

9

galactosidase activity. Each experiment was done at least two times in triplicate


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and data shown is a representative of them.

Western blotting

After treatments, cells were lysed with a buffer (5mM Hepes; 250 mM

NaCl, 10%(v/v) glycerol, 0.5% Nonidet P-40, 100 µM Va3VO4 supplemented

by Complete protease inhibitor cocktail, Roche). Whole cell lysate (50 µg)

were separated on 7.5 % SDS-PAGE gels. Western blotting analysis was

performed following the protocol described previously (24,26). Briefly, after

electro-blotting, the PVDF membranes (Millipore, Bedford, MA) were treated

with 5% (w/v) skim milk in TBS-T (20mM Tris-HCl, pH7.6/137 mM NaCl/0.5

% Tween 20), and incubated with antigen-specific antibodies against EGF

receptor (1:2500) or activated form EGF receptor (1:1000), followed by

incubation with peroxidase-conjugated anti-mouse IgG (1:2000) (Amersham

Pharmacia Biotech AB, Uppsala, Sweden). The epitope was visualized with an

ECL Western blot detection kit (Amersham Pharmacia Biotech AB).

Confocal microscopy

NIH3T3 cells were plated onto chamber slides (LabTech) and transfected

by pEGFP-N3 or pEGFR-EGFP. After 12 h, cells were serum-starved (0.1

% serum) for 12 h and then stimulated for 15 min in the absence or presence

of TNF or EGF. Cells were fixed in 4 % paraformaldehyde and mounted in

10

90% glycerol with 1 mg/ml p-phenylenediamine and examined with a confocal


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microscope, MRC-1024 (Bio-Rad Lab., Hercules, CA) (24).

Statistical analysis

Statistical significance between 2 groups was tested using the unpaired

Student’s t test. Differences were considered statistically significant at a value

11

of P < 0.05.


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Results

Overexpression of EGF receptor enhances TNFaaaa-induced NF-

kkkkB transcriptional activation in NIH3T3 cells

Because NIH3T3 cells express EGFR only in low density, we can examined

effects of EGFR on the TNFa-induced NF-kB activation by overexpression of

EGFR. In figure 1A, pEGFR-EGFP enhanced the NF-kB-dependent luciferase

activity compared to pEGFP-N3 (lane 2). Concentration of serum did not

cause any significant difference (lane 2 and 5). EGF has been shown to

activate NF-kB in rat aortic smooth muscle cells (28) and A431 carcinoma

cells (29), but not in human microvascular endothelial cells (30). Therefore,

we examined the effect of EGF on the NF-kB activation in NIH3T3 cell.

Treatment with EGF by itself did not have any significant effects (lane 3) but

made significant synergistic effect with TNF treatment (lane 4). This enhancing

effect of EGFR were in a dose-dependent manner of pEGFR-EGFP plasmid

(Fig. 1B).

Kinase Inhib i tors suppress TNFaaaa- induced NF-kkkkB

transcriptional activation in NIH3T3 cells and HEK293 cells.

The EGFR is a transmembrane protein and the ligation of EGF to the

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receptor results in an increase in the tyrosine kinase activity of the cytoplasmic


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domain and the autophosphorylation of tyrosine residues (31). Various protein

kinases such as PKC, MAPK, and EGFR itself are considered to be involved in

the tyrosine phosphorylation of EGFR indirectly or directly (16). In order to

explore the molecular mechanisms, we used a series of kinase inhibitors (Fig.

2A). The effect of expression of pEGFR-EGFP was suppressed dose-dependently

by treatment with Tyrphostin AG1478 (right columns of lane 2-4), which is a

rather selective EGFR tyrosine kinases inhibitors. AG1478 treatment did not

have significant effects TNFa-induced NF-kB activation in pEGFP-N3-

transfected NIH3T3 cells (left columns of lane 2-4). In contrast, both PI3K

inhibitors, LY294002 and wortmannin and a PKC inhibitor, GF109203X

suppressed the NF-kB activation in both pEGFP-N3- and pEGFR-EGFP-

transfected NIH3T3 cells (lane 5-8 and 11-12). Because a line of reports have

demonstrated that reactive oxygen intermediates (ROIs) play a pivotal role in

the TNF-induced NF-kB activation, we tried a potent antioxidant NAC in this

assay. Interestingly, NAC almost completely abolished EGFR-mediated

enhancement of the NF-kB activation induced by TNF (lane 13). In contrast,

the MEK1 inhibitor, PD98059, did not have any significant effects on the

NF-kB activation (lane 9 and 10). Treatment with AG1478 or NAC caused

similar suppressive effects the NF-kB activation in HEK293 cells (Fig 2B) as

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well as in pEGFR-EGFP-transfected NIH3T3 cells.


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As next, we examined the effect of overexpression of kinase dead EGFR

(K721A) on the TNF-induced NF-kB-dependent gene expression in HEK293

cells. As shown in figure 2C, expression of EGFR(K721A)-EGFP chimera

blocked the NF-kB-dependent gene expression in a plasmid dose-dependent

manner. Taken together with result using A1478, the tyrosine phosphorylation

of EGFR plays very critical roles in TNF-induced NF-kB activation process.

EGF transactivation by TNF is not suppressed by inhibitors of

PI3K and PKC but by NAC treatment and TRX expression.

As next, we examined if EGFR could be activated by TNFa or not using

an antibody which specifically recognizes the “activated” EGFR, of which

tyrosine residues were phosphorylated (32). We detected significant signals of

activated EGFR (upper row) in pEGFR-EGFP-transfected NIH3T3 cells treated

by EGF (Fig. 3A, lane 4) or TNF (lane 5). Neither 100 µM of LY294002

(lane 7) nor 10 µM of GF109203X (lane 8) affected TNF-induced EGFR

activation. In contrast, NAC dose-dependently suppressed the EGFR

phosphorylation by TNF (lane 9 and 10). EGFR endogenously expressed in

A431 cells were also phosphorylated by TNF treatment (Fig. 3B, lane 3) and

this phosphorylation was suppressed by AG1478 (lane 4) or NAC treatment

(lane 5) as well as exogenously expressed EGFR in NIH3T3 cells. Moreover,

14

expression of an endogenous protein with antioxidant property, TRX blocked


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phosphorylation of EGFR expressed in NIH3T3 cells (Fig. 3C).

If the observed tyrosine phosphorylation reflects EGFR activation, it

should lead to multimerization and internalization of EGFR (17). To examine

whether stimulation of TNFR leads to internalization of transactivated EGFR,

we examined intracellular localization of overexpressed EGFR-EGFP in NIH3T3

cells with confocal immunofluorescence microscopy. Figure 4 shows that in

unstimulated cells transfected with pEGFR-EGFP, EGFR localizes primarily

to the cell surface (B). TNFa (500 ng/ml) treatment of these cells, however,

leads to an increase in EGFR localized inside the cells (C). EGF (500 ng/ml)

treatment also leads to the internalization (D). GFP homogeneously expressed

in cells and did not have any particular localization pattern (A).

TRAF2-induced NF-kkkkB activation is sensitive but IKKaaaa-induced

N F -kkkkB activation is not to AG1478 in HEK293 cells

To explore the AG1478 sensitive step in the NF-kB activation, we

overexpressed signaling intermediate molecules, TRAF2 and IKKa in HEK293

cells (Fig. 5). Overexpression of TRAF2 induced significantly NF-kB-dependent

gene expression and this was suppressed by treatment with AG1478 (250 nM)

(Fig. 5A). In contrast, AG1478 (250 nM) had not any significant effect on the

IKKa-induced NF-kB activation (Fig. 5B).

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TRAF2 induced EGFR phosphorylation in a redox-dependent


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manner in NIH3T3 cells

As next, we examined effect of TRAF2 on the EGFR phosphorylation.

HEK293 cells were transfected by TRAF2 expression vector with or without

NAC treatment. As shown in figure 6, EGFR phosphorylation was significantly

enhanced by TRAF2 overexpression (lane 3) and the enhancement was abolished

by NAC treatment (lane 4). H2O2 by itself enhanced EGFR phosphorylation

16

(lane 2).


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Discussion

In this paper, we have described the EGFR transactivation by TNF and its

involvement in TNF-induced NF-kB activation. We have demonstrated that

the transactivation is redox-sensitive but on activity neither PI3K nor PKC. In

contrast, NF-kB activation process is redox-sensitive and dependent on the

activity of PI3K and PKC but not MAPK.

The EGFR is a transmembrane glycoprotein and the ligation of EGF to

the receptor results in the dimer formation, an increase in the tyrosine kinase

activity of the cytoplasmic domain, and the autophosphorylation of several

tyrosine residues (33,34). Intracelluar tyrosine kinases such as Src and focal

adhesion kinase and EGFR itself are considered to be involved in the tyrosine

phosphorylation (18). Under certain circumstances, intracellualr tyrosine

kinase activity is dependent on other kinases such as PKC, PI3K, and MAPK.

Our results of EGFR transactivation assay using anti-activated EGFR antibody

demonstrate that treatment with a PI3K inhibitor, LY294002 and a PKC

inhibitor, GF109203X, do not influence the EGFR transactivation by TNF

(Fig. 3A). Because a MEK inhibitor, PD98059 does not have any effects on

TNF-induced NF-kB activity in NIH3T3 cells, MAPK may not be involved in

this transactivation. Interestingly and very importantly, NAC, a very potent

17

antioxidant, treatment almost completely abolished TNF-induced EGFR


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transactivation and suppressed NF-kB activation (Fig. 3A, lane 9 and 10). On

the other hand, treatment with pharmacological inhibitors of PI3K and PKC

significantly suppress TNF-induced EGFR-dependent NF-kB activation (Fig.

2A, lane 5, 6, 7, 8, 11, and 12). Taken together, it is highly suggested that

steps sensitive to PI3K inhibitor and PKC inhibitor are located at the downstream

of EGFR in the TNF-induced NF-kB activation pathway.

Reactive oxygen intermediate (ROI) plays an essential role in the TNF-

induced signaling pathways to NF-kB and stress-activated protein kinases (35).

Intracellularly generated ROI has been shown to act as a second messenger of

the signaling (36,37). It has been also shown that EGFR activation is modulated

by redox principle (38-40). In fact, ROIs enhance the EGFR phosphorylation

by a ligand-independent mechanism (38,40-42). Although it is unclear how

ROI is involved in this process, one of plausible mechanism is the reversible

inactivation of protein tyrosine phosphatases (PTP) (38). Because all PTPs

have catalytic sites containing reactive cysteine residues, which form a thiol-

phosphatase intermediate during catalysis, oxidation of these residues leads to

their inactivation. Thiol-mediated redox systems such as glutathione and

thioredoxin (TRX) systems have been demonstrated to be involved in these

process (22,24,26,43). As well as treatment with NAC , overexpression of

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TRX suppressed TNF-induced EGFR phosphorylation (Fig. 3C). We have


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shown that redox-sensitive steps in TNF-induced NF-kB signaling pathway are

down from TRAF2 and up to IKK in HEK293 cells (27). In addition, in this

study, we have shown that TRAF2-overexpression induces EGFR

phosphorylation in a redox-dependent manner and TRAF2-induced NF-kB

activation is AG1478-dependent (Fig. 5 and 6). Consistent with this observation,

recently Liu et al. showed that not only TNF but also just overexpression of

TRAF2 fosters the production of ROI in transfected cells and that overexpression

of TRX or treatment with antioxidants suppressed part of downstream signaling

cascades (44). Rac1 is an essential subunits of NADPH oxidase system in even

non-phagocytotic cells and overexpression of constitutively active mutant of

Rac1 confers generation of ROI in certain cells and overexpression of dominant

negative mutant suppresses ROI generation by growth factor such as EGF and

PDGF as well as by TNF (45-49). Together with our results (Fig. 4 and 5),

ROI of downstream of TNFR and TRAF2 may play a role in the EGFR

transactivation process. In this study we have clearly demonstrated that treatment

with AG1478 significantly suppresses TRAF2-induced NF-kB-dependent gene

expression but not IKKa-induced gene expression (Fig. 5). Moreover, TRAF2

induces EGFR phosphorylation in a redox-dependent manner (Fig. 6). In

addition to signaling by protein-to-protein interaction, diffusible second

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messenger, ROI, mainly H2O2 downstream of TNFR-TRAF2 complex may


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contribute certainly to this signaling pathway.

Considering ubiquitous expression of EGFR in various types of cells,

particularly in cancer cells, transactivation of EGFR by TNF may play very

important roles in the activation of NF-kB in physiological and pathophysiogical

scenarios. Further studies on molecular mechanism of this phenomenon may

lead to better understanding of TNF signaling pathway. In particular, it may

be very interesting to determine whether TNF-induced transactivation of EGFR

involves proteolytic cleavage of membrane bound EGFR ligands such as HB-EGF

in the case of G protein-coupled receptor ligands including carbachol, bombesin,

and thrombin (50). Moreover, it also seems interesting and significant to

determine whether the transactivation potentiates other signaling pathways

20

downstream of TNFR such as MAPKs activation.


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Acknowledgments

We thank Drs. Alexander Sorkin, Reiko Shinkura, and Hajime Kitada for

providing plasmids, Drs. Takumi Kawabe, Toshifumi Tetsuka, Katsushi Miura,

Shin Kurosawa and Kaikobad Irani for critical reading of the manuscript, Dr.

Kenjiro Mori for his encouragement Ms. Kanako Murata for technical help

and Ms. Yoko Kanekiyo and Etsuko Kobayashi for secretarial help.

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Footnotes

This work was supported in part by a Grant-in-Aid for Scientific Research

from the Ministry of Education, Science and Culture of Japan

1. The abbreviations used are: TNF, tumor necrosis factor; EGF, epidermal

growth factor; NF-kB, nuclear factor kappa B; EGFR, EGF receptor;

TRX, thioredoxin; PAGE, polyacrylamide gel electrophoresis, ROI,

reactive oxygen intermediate; MAPK, mitogen activate protein kinase;

NAC, N -acetyl-L-cysteine; PI3K, phosphatidylinositol 3-kinase

28


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Legend for Figures

Figure1 Enhancement of the TNFa-induced NF-kB transcriptional

activation by overexpression of EGF receptor in NIH3T3 cells

NIH3T3 cells (5x104 cells/well) were transfected by pEGFR-EGFP or

pEGFP-N3 plasmids. 12 h after transfection, cells were treated with 100

ng/ml of TNF (lane 2, 4, and 5) and/or 500 ng/ml of EGF (lane 3 and 4).

Then, cells underwent the luciferase assay following a protocol in “materials

and methods”. In lane 5, the culture media was replaced with the media

with 0.5 % serum. The results are the means ± SD and presented as

fold-increases in luciferase activity over the baseline seen with the mock

transfectant without treatment. This is a representative of two independent

experiment which were done in triplicate. #; P< 0.05

Figure 2 Effects of kinase inhibitors on the TNFa-induced NF-kB

transcriptional activation.

NIH3T3 cells were transfected by reporter plasmid and pEGFR-EGFP-

or pEGFP-N3 (A). 12 h after transfection, cells were pretreated with

AG1478, LY294002, wortmannin, PD98059, GF109203X, or NAC for 1

h and then treated with TNF. HEK293 cells (B, C) were transfected by

29

reporter plasmid and pEGFR-EGFP, pEGFR-EGFP(K721A) or pEGFP-N3


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plasmids as indicated (C) and pretreated with AG1478 or NAC for 1 h

(A). 6 h after incubation, cells underwent the luciferase assay following

the protocol in “materials and methods”. The results are the means ± SD

and presented as fold-increases in luciferase activity over the baseline

seen with the column without TNF treatment. This is a representative of

two independent experiment which were done in triplicate. (A); %; P<

0.05 compared with pEGFP-N3 treated with TNF, #; P< 0.05 compared

with pEGFR-EGFP treated with TNF. (B and C); #; P< 0.05

Figure 3 TNF transactivates EGFR

NIH3T3 cells (4 x 106) were transfected by pEGFP-N3 or pEGFR-EGFP

and incubated for 12 h. The transfected NIH3T3 cells (A, C) and A431

cells (B) were serum-starved for 12 h and then stimulated for 15 min in

the absence or presence of TNF (500 ng/ml), EGF (500 ng/ml) , NAC

(A, lane 9; 20 mM, and A, lane 10 and B, lane 5 ; 40 mM) or AG1478

(250 nM). In (C), cells were co-transfected with pEGFR-EGFP and

pcDNA3-TRX or pcDNA3 before treatment. Cells were harvested and

then subjected to western blotting analysis using antibodies raised against

EGF receptor and activated EGF receptor as described in “Materials and

30

Methods”.


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Figure 4 Internalization of EGFR by TNF

NIH3T3 cells on chamber slides were transfected with pEGFP-N3 or

pEGFR-EGFP. 12 h after, cells were serum-starved (0.1 % serum) for

12 h and then stimulated for 15 min in the absence (A and B) or presence

of 500 ng/ml of TNF (C) or 500 ng/ml of EGF (D). Cells were washed

by PBS and fixed with 4% paraformaldehyde in PBS and subjected to

analysis with confocal microscopy.

Figure 5 TRAF2-induced NF-kB activation is sensitive but IKKa-induced

NF-kB activation is not to AG1478 in HEK293 cells

HEK293 cells were transfected with mock, TRAF2 (A), or IKKa (B)

expression plasmids. 12 h after transfection, cells were treated with or

without AG1478 (250 nM). After 8 h incubation, cells were harvested

and underwent the luciferase assay. The results are the means ± SD and

presented as fold-increases in luciferase activity over the baseline seen

with the mock transfectant without treatment. This is a representative of

two independent experiment which were done in triplicate.

31

Figure 6 TRAF2-induced EGFR transactivation is sensitive to NAC


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NIH3T3 cells (4 x 106) were transfected by pcDNA3 (lane 1 and 2),

TRAF2 (lane 3 and 4) with pEGFP-EGFR. 6 h after transfection, cells

were serum-starved for 12 h. In lane 2, cells were treated with H202 (200

µM). Cells were harvested and then subjected to western blotting analysis

using antibodies raised against EGF receptor and activated EGF receptor

as described in “Materials and Methods”.

32


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- -- + --- + + +

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Kiichi Hirota, Miyahiko Murata, Tatsuya Itoh, Junji Yodoi and Kazuhiko FukudaB activationκfactor confers the NF-

Redox-sensitive transactivation of epidermal growth factor receptor by tumor necrosis

published online May 3, 2001J. Biol. Chem.

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