Epidermal Growth Factor Receptor Is a Common Mediator of Quinone-induced Signaling Leading to Phosphorylation of Connexin-43

Rat liver epithelial cells were exposed to three quinones with different properties: menadione (2-methyl-1,4-naphthoquinone, vitamin K3), an alkylating as well as redox-cycling quinone, the strongly alkylating p-benzoquinone (BQ), and the non-arylating redox-cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). All three quinones induced the activation of extracellular signal-regulated kinase (ERK) 1 and ERK 2 via the activation of epidermal growth factor receptor (EGFR) and MAPK/ERK kinases (MEK) 1/2. ERK activation resulted in phosphorylation at Ser-279 and Ser-282 of the gap junctional protein, connexin-43, known to result in the loss of gap junctional intercellular communication. Another EGFR-dependent pathway was stimulated, leading to the activation of the antiapoptotic kinase Akt via phosphoinositide 3-kinase. The activation of EGFR-dependent signaling by these quinones was by different mechanisms: (i) menadione, but not BQ or DMNQ, inhibited a protein-tyrosine phosphatase regulating the EGFR, as concluded from an EGFR dephosphorylation assay; (ii) although menadione-induced activation of ERK was unimpaired by pretreatment of cells with N-acetyl cysteine, activation by BQ and DMNQ was prevented; (iii) cellular glutathione (GSH) levels were strongly depleted by BQ. The mere depletion of GSH by application of diethyl maleate EGFR-dependently activated ERK and Akt, thus mimicking BQ effects. GSH levels were only moderately decreased by menadione and not affected by DMNQ. In summary, EGFR-dependent signaling was mediated by protein-tyrosine phosphatase inactivation (menadione), GSH depletion (BQ), and redox-cycling (DMNQ), funneling into the same signaling pathway.

boxylation of blood coagulation factors involving the K vitamins, or redox cycling as a result of quinone reduction followed by reoxidation with molecular oxygen, concomitantly generating the superoxide anion (2,3). Further, a variety of alkylation reactions are observed, including Michael-type additions of sulfhydryl groups to quinones (4) or DNA alkylation (5). This biochemistry of quinones is exploited in cancer chemotherapy, such as with mitomycin c or certain anthraquinone derivatives including doxorubicin, yet the exact contributions of both redox and alkylation reactions to either the desired or adverse effects of these compounds are not fully elucidated.
Cells respond to stimuli such as those imposed by xenobiotic quinones by activating stress-responsive signaling cascades regulating cellular proliferation and survival. These include pathways activated by DNA damage, such as p53-related events (6), and general stress-responsive cascades, such as mitogen-activated protein kinase (MAPK) 1 cascades, the phosphoinositide 3-kinase (PI3K)/Akt cascade, and others (7,8).
Not only are intracellular signaling pathways affected that regulate the survival and proliferation of the respective cell harboring these signaling cascades, but a stress response may also consist of disconnecting a cell from its environment, e.g. by down-regulation of gap junctional intercellular communication (GJC) to prevent diffusion of xenobiotics or toxic metabolites thereof within a tissue. Gap junctions allow the diffusion of compounds of low molecular mass (up to about 1 kDa) between the cytoplasms of adjacent cells (9), a process known to be regulated by phosphorylation of the gap junctional proteins, the connexins (Cx) (10,11).
Recently, the exposure of rat liver epithelial cells to menadione (2-methyl-1,4-naphthoquinone, vitamin K 3 ), which is both a redox-cycling and an alkylating quinone, was shown to lead to the activation of extracellular signal-regulated kinase (ERK) 1 and ERK 2, which are well known for their prominent role in the regulation of cellular proliferation (12). ERK activation resulted in phosphorylation of the gap junctional channel protein Cx43 and down-regulation of GJC (8). The activation of ERK was blocked by inhibitors of the direct upstream kinases of ERK 1/2, MAPK/ERK kinase (MEK) 1, and MEK 2 and by inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase, leading to the hypothesis that ligandindependent activation of the EGFR by menadione was responsible for the above described effects (8).
We here demonstrate that quinones of different structures and reactivities all activate the same signaling pathway, resulting in Cx43 phosphorylation. Both a strongly alkylating quinone, p-benzoquinone (BQ), as well as an exclusive redoxcycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), cause an EGFR-dependent activation of ERK and the PI3K/Akt cascade, as does menadione, an alkylating and redox-cycling compound. Regarding the mechanism of activation, protein-tyrosine phosphatase (PTPase) inhibition, glutathione depletion, and redox cycling are proposed to be responsible for the activation of the pathway by menadione, BQ, and DMNQ, respectively. It thus appears that different modes of action of the various quinones are similarly interpreted and integrated by the cell and funneled into the same signaling pathway that regulates cellular proliferation and intercellular communication.

EXPERIMENTAL PROCEDURES
Cell Culture-WB-F344 rat liver epithelial cells (13) with stem celllike properties (14) were a kind gift of Dr. James E. Trosko, East Lansing, MI. They were held in Dulbecco's modified Eagle's medium (Sigma) supplemented with (final concentrations) 10% (v/v) fetal calf serum (BioWest, Frickenhausen, Germany), 2 mM L-glutamine, and penicillin/streptomycin. HeLa cells (European Collection of Cell Cultures, Salisbury, UK) were grown under identical conditions. Quinones were from Sigma (menadione), Merck (BQ), and Calbiochem (DMNQ), and diethyl maleate (DEM) was from Sigma. Stocks of these compounds were in Me 2 SO and stored frozen in the dark. Cells grown to confluence in 7-cm 2 dishes were exposed to quinones diluted in serum-free medium for the times indicated. In experiments with inhibitors of signaling cascades (U0126, compound 56, AG1478, wortmannin, LY294002, all from Alexis, Lausen, Switzerland, or Calbiochem) culture medium was removed, and cells were briefly washed with PBS prior to preincubation for 30 min with either Me 2 SO (control) or the respective inhibitor (diluted from stock solutions in Me 2 SO) diluted in serum-free medium. Inhibitors were also present in the medium during exposure to the respective quinone tested.

FIG. 1. ERK activation by quinones.
WB-F344 rat liver epithelial cells were exposed to menadione (MQ, 50 M), BQ (100 M), or DMNQ (100 M) for the times indicated (A) or for 15 min (B) followed by determination of ERK phosphorylation and total ERK levels by Western blotting. The MEK inhibitor U0126 was used at 10 M. Me 2 SO was taken as vehicle control "C" for the quinones and for U0126. Data are representative of at least three independent experiments.

FIG. 2. Induction of connexin phosphorylation by quinones.
WB-F344 rat liver epithelial cells were exposed to Me 2 SO (vehicle control, C), MQ (50 M), BQ (50 M), or DMNQ (100 M) for 30 min. Phosphorylation of Cx43 at Ser-279 and Ser-282 was then analyzed by dot blotting (A) and immunohistochemistry (B). In A, loading control was by detection of total Cx43. In B, nuclei were stained with DAPI for orientation. Data are representative of at least three independent experiments. was a kind gift from Dr. Kerstin Leykauf and Dr. Angel Alonso from the German Cancer Research Center, Heidelberg, Germany, was taken for dot blotting analysis. For dot blotting, 2 l of the lysates in SDS-PAGE sample buffer were applied to a nitrocellulose membrane and air-dried. Immunodetection was as described above for Western blotting.
For immunohistochemistry, WB-F344 cells were grown to confluence on coverslips in 3-cm-diameter plastic dishes, briefly washed in PBS, and kept in serum-free medium overnight before exposure to the respective agent or Me 2 SO (vehicle control). After treatment, cells were washed twice with cold PBS and fixed with 5 ml/dish of methanol for 15 min at Ϫ20°C. Fixed cells were then thoroughly washed with ice-cold PBS followed by blocking with 5% (v/v) normal goat serum (Invitrogen) in PBS containing 0.3% (v/v) Triton X-100 for 90 min at room temperature. For detection of phosphorylated connexin-43, cells were incubated at 4°C overnight under slight agitation with rabbit polyclonal anti-phospho-connexin-43 antibody (sc-12900-R) diluted 1:1,500 in PBS containing 1% (v/v) goat serum. Cells were then washed with PBS and incubated with an Alexa 488-coupled goat anti-rabbit IgG (HϩL) antibody (Molecular Probes, Eugene, OR) for 1 h at 37°C. Nuclear staining was performed after intense washing with PBS by addition of 4Ј,6diamidino-2-phenylindole (DAPI, 0.2 g/ml final concentration) in PBS for 15 min followed again by intense washing and embedding. Images were taken with a Zeiss Axiovert fluorescent microscope coupled to a CCD camera (ORCA II, Hamamatsu, Japan).
Immunoprecipitation-Cells were grown in 70-cm 2 cell culture dishes. After treatment, cells were washed twice with ice-cold PBS and lysed in 500 l of IP buffer (30 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 5 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, 1% Triton X-100) per dish by incubating on ice for up to 30 min. Lysed cells were collected with a cell lifter and transferred to Eppendorf cups. Lysates were frozen at Ϫ80°C, thawed, and centrifuged to pellet non-soluble fractions. The resulting supernatants were transferred to fresh Eppendorf cups, and protein was determined according to Bradford. 700 g of protein were immunoprecipitated using 5 g of rabbit polyclonal anti-EGFR antibody (Upstate Biotechnology, Lake Placid, NY) and 50 l of a 50% (w/v) slurry of protein A-agarose (Upstate Biotechnology; preequilibrated in IP buffer) under end-over-end rotation at 4°C overnight. The samples were centrifuged and washed twice with fresh IP buffer and once with IP wash buffer (25 mM Tris, pH 6.8; 1 mM EDTA). After addition of 40

FIG. 3. Role of EGFR in menadione-induced ERK activation.
In A, WB-F344 cells were exposed to menadione (50 M) for 15 min with or without pre-and coincubation with inhibitors of the EGFR tyrosine kinase (AG1478 and compound 56, c56; 10 M each) followed by analysis of ERK phosphorylation by Western blotting. Exposure to inhibitors alone (not shown) did not yield results different from control (Ϫ). Me 2 SO served as vehicle control for menadione and for the inhibitors. In B, cells were treated with EGF at 400 ng/ml (ϩ) or control treated (Ϫ) for 1 h followed by washing and incubation in serum-free medium for 2 h. Cells were then exposed to menadione at the indicated concentrations or EGF (100 ng/ml) for 15 min followed by lysis and Western blotting. In C, cells were exposed to menadione at the concentrations given for 15 min followed by lysis and IP of the EGFR and determination of tyrosine phosphorylation by Western blotting. Detection of EGFR in the precipitates served as control for successful IP and equal loading; EGF (100 ng/ml for 15 min) was used as positive control for tyrosine phosphorylation. Data are representative of at least three independent experiments.

FIG. 4. Quinone-induced activation of EGFR-dependent signaling.
In A, WB-F344 cells were exposed to Me 2 SO (vehicle control, C), BQ (50 M), or DMNQ (100 M) for 30 min with or without pre-and coincubation with inhibitors of the EGFR tyrosine kinase (AG1478 and compound 56, c56; 10 M each) followed by analysis of ERK phosphorylation by Western blotting. Exposure to inhibitors alone (not shown) did not yield results different from control (Ϫ). Me 2 SO served as vehicle control both for menadione and for the inhibitors. In B, cells were exposed to MQ (50 M), BQ, or DMNQ (100 M each) in the absence or presence of inhibitors of phosphoinositide 3-kinase, wortmannin (100 nM), and LY294002 (20 M). Phosphorylation of Akt at Ser-473 was detected by Western blotting. Data are representative of at least three independent experiments. l of 4ϫ SDS-PAGE sample buffer to the washed protein A pellets, samples were boiled, and the proteins were resolved on 8% Tris-glycine gels followed by blotting onto polyvinylidene difluoride membranes. Detection of phosphorylated tyrosines was with monoclonal anti-phosphotyrosine antibody (4G10; Upstate Biotechnology). Membranes were blocked for 1 h in 1% (w/v) bovine serum albumin in TBST, briefly washed with TBST, and incubated with the primary antibody diluted 1:1,000 in 1% bovine serum albumin in TBST at 4°C overnight. The secondary antibody was diluted in TBST. Human recombinant EGF (R&D Systems, Minneapolis, MN) was taken as positive control.
Assay of EGF Receptor Dephosphorylation-The assay was essentially performed as described by Knebel et al. (17). Briefly, HeLa cells were grown to 80 -100% confluency and serum-starved overnight. EGF receptor tyrosine phosphorylation was stimulated by incubation in the presence of EGF (100 ng/ml) for 5 min. The cells were washed with PBS, treated with the respective quinone (100 M) or Me 2 SO (as vehicle control) for 15 min. The quinone was removed, and fresh serum-free medium containing the EGFR tyrosine kinase inhibitor compound 56 (Calbiochem; 10 M) was added to prevent any further autophosphorylation of the receptor. After 30 s, medium was quickly removed, and cells were lysed in 2ϫ SDS-PAGE sample buffer followed by SDS-PAGE on a gel of 8% (w/v) acrylamide and Western blotting with detection of phosphorylated tyrosine residues (4G10 monoclonal antibody, Upstate Biotechnology).
Glutathione Determination-Total glutathione and glutathione disulfide were determined enzymatically according to Ref. 18 with minor modifications. Briefly, cells on 7-cm 2 cell culture dishes were lysed by scraping them in 250 l of ice-cold HCl (10 mM) followed by one freeze/ thaw cycle, brief sonication on ice, and centrifugation at 20,000 ϫ g for 10 min to remove cell debris. Aliquots of the supernatants were kept for protein determination according to Bradford. For glutathione determination, protein was precipitated from the supernatant with 5% (w/v; final concentration) 5-sulfosalicylic acid on ice. Samples were vortexed and centrifuged at 20,000 ϫ g for 10 min. Total glutathione (GSH plus GSSG) and, after blocking thiols with 2-vinylpyridine, GSSG were determined from the supernatant using 5,5Ј-dithionitrobenzoic acid in the presence of NADPH and glutathione reductase (18).

Activation of ERK 1/2 and Connexin-43
Phosphorylation after Exposure to Quinones-Exposure of cells to menadione is known to result in the activation of ERK 1/2 and ERK-dependent phosphorylation of connexin-43 (Cx43), resulting in a decreased intercellular communication (8). Two other quinones of different reactivities were tested for ERK activation and Cx43 phosphorylation: BQ, a strong alkylator, and DMNQ, an exclusive redox-cycler, were compared with menadione, which is known both to undergo redox cycling and to alkylate. Exposure of cells to any of the three quinones resulted in the strong dual phosphorylation of ERK 1/2 (Fig. 1A), which is mediated by MEK 1 and MEK 2, the kinases directly upstream of ERK 1/2, as U0126, a specific inhibitor of MEK 1/2 activation, completely (BQ, DMNQ) or largely (menadione) blocked ERK phosphorylation (Fig. 1B). When cells were exposed to any of the quinones for 15 min followed by aspiration of quinone-containing media and postincubation in serum-free medium, the activation of ERK 1/2 remained equally strong with slight decreases observable starting after 2 h of postincubation (data not shown).
Role of the Epidermal Growth Factor Receptor in Quinoneinduced Signaling-Based on inhibitor studies, it was proposed that menadione-induced activation of ERK 1/2 is mediated by the activation of the EGFR (8). As demonstrated in Fig. 3A, AG1478 and compound 56, two specific inhibitors of the EGFR tyrosine kinase, largely blocked menadione-induced ERK activation. Another strategy used to demonstrate the dependence of ERK activation by menadione on EGFR was based upon the fact that permanent exposure of cells to high concentrations of growth factors may result in an enhanced internalization of the respective receptors (19 -21). Exposure of WB-F344 cells to high concentrations of EGF (400 ng/ml) for 1 h followed by washing and 2 h of incubation with serum-free medium rendered cells refractory for further activation by EGF (100 ng/ml) or menadione (10, 25 M), as seen in Fig. 3B. Taken together, menadione-induced ERK activation is dependent on EGFR stimulation. The activation of the EGFR upon exposure to menadione was demonstrated by immunoprecipitation and detection of tyrosine-phosphorylated receptor molecules (Fig. 3C). Like menadione, BQ-and DMNQ-induced ERK activation relies on EGFR activation, as demonstrated using AG1478 and compound 56 (Fig. 4A), both of which abrogated ERK phosphorylation induced by exposure of cells to these quinones. If the EGFR is activated in cells exposed to menadione, BQ, or DMNQ, downstream signaling pathways other than the MEK/ ERK pathway should also be stimulated. In fact, the PI3K/Akt cascade was activated in WB-F344 cells as serine 473 phosphorylation of Akt was strongly enhanced after treatment with menadione, BQ, or DMNQ and abrogated in the presence of either of two structurally unrelated inhibitors of PI3K, wortmannin or LY294002 (Fig. 4B). Taken together, menadione, BQ, and DMNQ all activate the EGFR as well as downstream signaling pathways.
Role of Protein-tyrosine Phosphatase Inactivation in EGFR Activation by Quinones-One possible way of activating a receptor tyrosine kinase such as the EGFR in the absence of ligand would be to disrupt negative regulation of the receptor. It was demonstrated that the activation of receptor tyrosine kinases by ultraviolet irradiation and reactive oxygen species relies on the inactivation of a PTPase regulating the respective receptor (22). To test for the involvement of a PTPase in the activation of the EGFR upon exposure to quinones, cells were treated with EGF followed by addition of the EGFR tyrosine kinase inhibitor, compound 56. HeLa cells were used for these

FIG. 5. Protein-tyrosine phosphatase inhibition by quinones.
In A, HeLa cells were stimulated with EGF (100 ng/ml) for 5 min. The cells were washed with PBS, treated with 100 M of MQ, BQ, or DMNQ or Me 2 SO (as vehicle control) for 15 min. The quinone was removed, and fresh serum-free medium containing the EGFR tyrosine kinase inhibitor compound 56 (c56, 10 M) was added to prevent any further autophosphorylation of the receptor. After 30 s, medium was quickly removed, and cells were lysed followed by Western blotting with detection of phosphorylated tyrosine residues and of total EGFR. B, schematic interpretation of A. MQ inhibits PTPase dephosphorylating the EGFR, resulting in a net phosphorylation of the receptor. experiments because they responded better to EGF treatment, yielding more intense tyrosine phosphorylation than WB-F344 cells, which is crucial for the assay. Stimulation of the cells with EGF resulted in tyrosine phosphorylation of the receptor (Fig. 5A, lane 2), which disappeared after addition of compound 56 (Fig. 5A, lane 3). This can be explained only by the existence of a tyrosine phosphatase that dephosphorylates the activated receptor (Fig. 5B). In the presence of menadione, however, dephosphorylation was blocked, resulting both in an enhanced responsiveness of the cells to EGF (Fig. 5A, lane 4 versus lane  2) and in the maintenance of tyrosine phosphorylation of the EGFR even in the presence of the EGFR tyrosine kinase inhibitor (Fig. 5A, lane 5). On the contrary, neither BQ nor DMNQ had the same effect (Fig. 5A, lanes 7 and 9), pointing to mechanisms other than PTPase inactivation being responsible for EGFR activation by these quinones. On the Role of Glutathione in Quinone-induced EGFR and ERK Activation-The results obtained from experiments on PTPase inhibition render it highly likely that menadione-induced EGFR activation is due to inactivation of a PTPase negatively regulating the receptor. Menadione both is an alkylator and undergoes redox cycling in the cell. Both the alkylation of a PTPase and its oxidation by reactive oxygen species generated upon redox cycling are feasible mechanisms of inactivating the enzyme. All PTPases known so far harbor an essential cysteine thiolate at their active site that is prone to oxidation (23) and alkylation. The fact that menadione is capable of inactivating isolated PTPases in the absence of cell lysate (8) and that DMNQ, an exclusive redox cycler, did not inactivate a PTPase regulating the EGFR (Fig. 5A) rule out redox cycling as the major mechanism for PTPase inactivation. In line with the alkylating properties of menadione, it interacts with GSH, as can be seen from the difference in UV-visible spectra of menadione before and after reaction with GSH: the calculated sum spectrum of GSH plus menadione significantly differs from the spectrum measured after mixing the two compounds (Fig. 6A, top). Even more dramatic spectral changes were observed with BQ, whereas no differences between calculated and measured spectra were seen for DMNQ (Fig. 6A,  middle and bottom). This is in line (i) with BQ being a strong alkylator that is known to easily react with GSH, not only forming monoglutathionylated hydroquinone but also di-, tri-, and tetra-(glutathionyl)-hydroquinone in cells (4,24); and (ii) with DMNQ being a non-alkylating quinone: both the C2 and the C3 positions are occupied. In accordance with the spectra in Fig. 6A, about 35% of the cellular GSH was depleted in cells exposed to menadione already after 15 min, whereas about 90% was lost after exposure to BQ, and no more than a tendency toward GSH depletion was observed with DMNQ ( Fig. 6B, top). Significant accumulation of glutathione disulfide was found only in cells exposed to menadione; a tendency to accumulate GSSG was seen with DMNQ (Fig. 6B, bottom). The extensive accumulation of GSSG in menadione-treated cells may be explained by redox cycling of menadione in combination with its known inhibitory effect on glutathione reductase (25): superoxide derived from redox cycling would yield hydrogen peroxide that is reduced by glutathione peroxidase at the expense of GSH which, in turn, ends up as GSSG that cannot be reduced by glutathione reductase if menadione is present.
To delineate the role of GSH depletion in the activation of ERK 1/2 by quinones, cells were exposed to N-acetyl cysteine (NAC), a cell-permeant thiol employed to deliver antioxidant capacity to cells that serves as a precursor of cysteine which is utilized for GSH synthesis. ERK activation by menadione and BQ, but not by DMNQ, was prevented by the concomitant presence of NAC with the quinones (Fig. 7A). This was probably due to the direct interaction of NAC, which was applied in a 300 -600-fold molar excess, with menadione or BQ, respectively. No such interaction with the non-alkylating DMNQ is to be expected. Indeed, spectral changes of the quinones in the presence of NAC were very similar to those observed with GSH in Fig. 6A (not shown). If, however, cells were treated with NAC for 1-3 h and then washed before exposure to the quinones, both BQ and DMNQ did not activate ERK 1/2 anymore, whereas menadione-induced ERK activation was unimpaired (Fig. 7B). Consistent with these data, no ERK activation was seen with any of the quinones, if preincubation of NAC was followed by coincubation of the quinones with NAC (not shown). The mechanism of menadione-induced ERK activation thus obviously differs from the path taken by BQ and DMNQ, in accordance with Fig. 5A, which demonstrates that inhibition of a PTPase regulating the EGFR was exclusively brought about by menadione.
Glutathione Depletion Leads to Activation of EGFR-dependent Signaling-To test whether the depletion of GSH might cause the activation of EGFR-dependent signaling, cells were exposed to DEM, a glutathione S-transferase substrate that is coupled to GSH, thus depleting the thiol. Exposure of WB-F344 cellstovariousconcentrationsofDEMresultedinaconcentrationdependent activation of ERK 1/2 and of Akt (Fig. 8A). Eightythree percent (83 Ϯ 3%, means Ϯ S.D., n ϭ 3) of cellular FIG. 8. ERK activation induced by GSH depletion. Rat liver epithelial cells were exposed to diethyl maleate at the indicated concentrations for 60 min followed by lysis and analysis of ERK and Akt phosphorylation and total ERK/Akt levels by Western blotting. Inhibitors of platelet-derived growth factor receptor (AG1295, 10 M) or EGFR (AG1478, compound 56 (c56), both 10 M) were added to the cells 30 min prior to DEM. Me 2 SO served as vehicle control (0 or Ϫ) both for DEM and for the inhibitors. Exposure to inhibitors alone (not shown) did not yield results different from control (Ϫ). Data are representative of three independent experiments. glutathione was depleted with 1 mM DEM under these conditions. Interestingly, GSH depletion had to be rapid, i.e. within minutes (as with BQ), to result in the activation of ERK: exposure of cells to buthionine sulfoximine (4 mM), an inhibitor of GSH biosynthesis, for 24 h decreased total glutathione by 71 Ϯ 9% (means Ϯ S.D., n ϭ 3) of control, but no ERK activation was seen (data not shown).
ERK and Akt activation was blocked in the presence of inhibitors of the EGFR, AG1478, and compound 56. Interestingly, this activation was also partly prevented by AG1295, an inhibitor of the platelet-derived growth factor receptor tyrosine kinase (Fig. 8B), pointing to a minor role of another receptor tyrosine kinase in addition to EGFR. As with menadione or BQ, the depletion of GSH by the application of DEM entails phosphorylation of the ERK 1/2-specific sites of Cx43, Ser-279 and Ser-282, as demonstrated by dot blotting (Fig. 9A) and immunocytochemistry (Fig. 9B) using two different antibodies.

Significance of ERK Activation by Quinones-
The activation of ERK 1/2 is usually connected with cell proliferation, which is due to the substrates of the kinases, including transcription factors as well as key enzymes involved in nucleotide and protein synthesis (see Ref. 12 for review). Connexin-43 is also phosphorylated by ERK 1/2, resulting in an attenuation of GJC (10,11,26). Three Cx43 sites were demonstrated to be phosphorylated by ERK 1/2, Ser-255 as well as Ser-279/Ser-282, all of which are located in the C-terminal cytoplasmic domain of the protein (10,11). Recently, ERK 5 was implicated in Cx43 phosphorylation at Ser-255 and impairment of GJC (27).
Menadione as well as BQ and DMNQ all activate ERK 1/2 ( Fig. 1), resulting in the phosphorylation of Cx43 (Fig. 2), which is known to result in an attenuation of GJC (8). This is of significance for cancer chemotherapy approaches exploiting the so-called bystander effect that is based upon the direct diffu- sion of active drug between the cytoplasms of adjacent cells. Many chemotherapeutics are quinones, such as mitomycin c or the anthraquinone derivatives doxorubicin and daunorubicin. Exposure of cancerous tissue to these drugs is aimed at killing the respective cells. If, however, the very agent employed induces Cx phosphorylation, thus impairing GJC, the outcome of chemotherapy might be suboptimal because diffusion between cytoplasms is impaired. We here devise a way of sustaining GJC in the presence of quinones: the concomitant addition of inhibitors of the EGFR-MEK-ERK pathway would prevent Cx phosphorylation and reestablish GJC.
Different Modes of Activation of EGFR-dependent Signaling by Quinones, Role of PTPases and GSH-Receptor tyrosine kinases such as the EGFR or the platelet-derived growth factor receptor forms appear to be activated by a variety of stressful stimuli, mediating the activation of downstream signaling events in the absence of the respective ligands. Such stimuli include several different reactive oxygen and nitrogen species, such as hydrogen peroxide (21) or peroxynitrite (28), as well as heavy metal ions (29) or anticancer agents such as cisplatin (30).
How should menadione and other quinones lead to the activation of the EGFR and of downstream signaling? A hypothesis widely accepted for the activation of EGFR signaling by reactive oxygen species is that of the inactivation of a phosphotyrosine phosphatase (or PTPase) negatively regulating the EGFR (31), and indeed, isolated PTPases are inhibited by menadione (8) as well as other naphthoquinone derivatives (32). It is demonstrated in Fig. 5 that in a cellular environment, menadione (but not BQ or DMNQ) also blocks PTPase activity. The identity of the PTPase inhibited is yet unknown. However, the same pathways activated by menadione, leading to EGFR, MEK, and ERK activation as well as Cx phosphorylation and GJC down-regulation, are also activated by an inhibitor of Cdc25A, 2 a dual specificity PTPase involved in cell cycle regulation that is known to interact with the EGFR (33). In the case of menadione, we propose that direct alkylation of a PTPase is the major mechanism for PTPase inactivation and EGFR stimulation. First, menadione-induced ERK activation was demonstrated to be independent of NAD(P)H:quinone oxidoreductase-1 (8), which reduces menadione to the corresponding hydroquinone that may then undergo redox cycling. Secondly, menadione may directly interact with isolated PTPases (see above). Thirdly, incubation of cells with NAC prior to exposure to menadione did not impair ERK activation by the quinone (different from BQ and DMNQ; Fig. 7).
PTPase inactivation does not necessarily need to be by direct interaction of the enzyme with the quinone. Rather, an indirect mechanism might also be considered that, at the expense of GSH, relies on the regeneration of PTPases that are oxidized during cellular metabolism. A depletion of cellular GSH such as by BQ or DEM might therefore render PTPases and other oxidant-sensitive signaling proteins prone to oxidative inactivation. As PTPase inactivation was not seen for BQ, either other target proteins have to be considered or only a slight PTPase inactivation occurred that escaped detection. The latter, however, is questionable because of the strong effects on kinase activation of BQ. Thioredoxins or peroxiredoxins are examples for proteins known for their regulatory role in redox signaling. Like PTPases, they harbor thiolates at their reactive sites and can be oxidized to sulfenic acids by metabolically generated H 2 O 2 (34). The sulfenic acids can be reduced by GSH, leading to the reactivation of the proteins. If, however, cellular GSH levels are strongly diminished, such as with BQ and DEM (Fig. 6B), reduction and reactivation are impaired. DMNQ, undergoing redox cycling, does neither inactivate a PTPase (Fig. 5) nor significantly deplete GSH levels in the cell within 15 min (Fig. 6), the time required to see the activation of the signaling pathways investigated. It may be speculated that superoxide and hydrogen peroxide formation oxidize signaling proteins such as the aforementioned, entailing effects identical to those seen under conditions with depleted GSH levels. In summary, three different quinones, the alkylating and redox cycling menadione, the strong alkylator BQ, and the exclusive redox cycler DMNQ, activate distinct signaling mechanisms converging at the level of EGFR activation and leading to the phosphorylation of Cx43 (Fig. 10).