INTRODUCTION

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Arsenite, the trivalent arsenic compound, is a potent carcinogen to which there is significant worldwide exposure through natural contamination of food and drinking water (3). In humans, chronic exposure is associated with an increased incidence of skin and bladder cancers (3, 16). Although it appears to be nonmutagenic alone, in cultured cells arsenite potentiates the mutagenic effects of short-wavelength UV radiation (UV-C) (23, 34, 63). Hence, arsenite has been suggested to act as a tumor promoter in the carcinogenic process (6, 59). While the basis for such tumor-promoting activity is unclear, the ability of arsenite to interact with protein thiol groups and thereby alter the activities of key regulatory proteins is likely to contribute to its carcinogenic properties (6, 37).

The mitogen-activated protein (MAP) kinase cascade leading to the activation of ERK is crucial for regulating cell growth and differentiation. Initiation of this signaling pathway via growth factor receptors has been studied extensively (2, 11, 56). Ligand-mediated dimerization of growth factor receptors triggers the activation of receptor-type tyrosine kinases, resulting in autophosphorylation of tyrosine residues (2, 24, 53, 56). These residues then serve as docking sites for the recruitment of downstream signaling mediators necessary for the activation of membrane-localized Ras (15, 53). For example, the adapter protein Grb2 binds to the phosphotyrosine residues through its Src homology 2 (SH2) domain to bring the Ras guanine nucleotide exchange factor, Son of Sevenless (Sos), from the cytoplasm to the vicinity of Ras through its two Src homology 3 domains. In addition, the Grb2-Sos complexes can be recruited to the phosphotyrosine sites through another adaptor protein, Shc (4). Shc binds to certain receptor phosphotyrosine sites through its phosphotyrosine-binding domain; Shc then becomes tyrosine phosphorylated itself and thereby provides additional docking sites for Grb2 (3, 4, 15, 53). The recruitment of Grb2-Sos complexes to the phosphotyrosine sites leads to Ras activation, which then initiates the phosphorylation cascade starting with the activation of the proto-oncoprotein Raf, a serine kinase that phosphorylates and activates ERK kinase (MEK), which in turn activates ERK (2, 56). ERK is responsible for the phosphorylation of a variety of cellular proteins including downstream kinases, transcription factors, and components of the protein synthesis machinery (11).

In cultured cells, dysregulation of the ERK pathway through alterations in any one of several mediators involved in the cascade (e.g., mutation of growth factor receptors, Shc, Ras, Raf, and MEK) can lead to cellular transformation (7, 14, 15, 33, 40). Indeed, many tumors exhibit elevated ERK activity (32, 54, 57). It is not surprising, therefore, that a common feature of known tumor promoters is their ability to perturb the ERK signaling pathway. For example, although acting through different mechanisms, the tumor promoters phorbol ester, okadaic acid, and butylated hydroxytoluene hydroperoxide all lead to the activation of ERK. Phorbol esters act through activation of protein kinase C (leading to activation of Raf) (29), while okadaic acid acts through the inhibition of serine/threonine phosphatases (18). The mechanism responsible for ERK activation by butylated hydroxytoluene hydroperoxide is not fully understood but appears to involve steps upstream of Ras, as the activation of ERK by this agent is Ras dependent (20). In keeping with arsenite's putative role as a tumor promoter, we have recently observed that arsenite treatment leads to a significant elevation in ERK activity in Rat1 and PC12 cells (37). This activation is dependent on Ras and is sensitive to the presence of the growth factor receptor poison suramin (60). These findings suggested that arsenite may act early in the pathway, either at a step involving the growth factor receptors directly or during the events serving to activate the growth factor receptors (37). Better understanding of the signal transduction pathway leading to ERK activation by arsenite could provide important insight into its carcinogenic mechanisms. In the present report, we focus on the early events in the signaling pathway leading to ERK activation. We provide evidence that arsenite treatment results in tyrosine phosphorylation of the Shc adapter protein, leading to its enhanced interaction with Grb2. We further demonstrate that epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase is required for these interactions and the subsequent activation of ERK. The ability of arsenite to usurp this growth regulatory pathway is likely to contribute to its tumor-promoting properties.

	MATERIALS AND METHODS

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Cell culture and treatments. Wild-type and Ras N17 PC12 cells, kindly provided by G. Cooper (61), were grown in a 37°C humidified atmosphere containing 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc., Gaithersburg, Md.) supplemented with 10% fetal bovine serum (HyClone, Logan, Utah) and 5% horse serum (Life Technologies, Inc.). Cells were serum starved by placement in serum-free medium for at least 16 h prior to treatment with arsenite or EGF. Sodium arsenite (Sigma Chemical Co., St. Louis, Mo.) was added directly into the medium to a final concentration of 400 µM. EGF stimulation was carried out by directly adding EGF (Life Technologies) into the medium to a final concentration of 100 ng/ml. Suramin (300 µM) and tyrphostin AG1478 (30 or 40 nM) (Calbiochem, San Diego, Calif.) were added to the medium immediately before addition of sodium arsenite. All reagents were left in the medium until cells were harvested.

Down-modulation of EGFR. Down-modulation of EGFR was carried out by incubating PC12 cells with EGF essentially as previously described by Carpenter and Cohen (5). PC12 cells cultured in serum-free medium overnight were stimulated with EGF (1 µg/ml) for 1 h. The EGF-containing medium was then removed, and the cells were washed twice with phosphate-buffered saline (PBS). The cells were then kept in serum-free medium for another 2 h at 37°C to allow the internalization of EGF-EGFR complexes before a second treatment with EGF, nerve growth factor (NGF), or arsenite. Replenishment of EGFR was accomplished by incubation of PC12 cells with down-regulated EGFR in DMEM containing 10% fetal bovine serum for 10 h. After replenishment of EGFR, cells were serum starved for another 14 h and subsequently treated with arsenite, EGF, or NGF.

In vitro binding assays. The glutathione S-transferase (GST)-Grb2 SH2 domain-expressing plasmid was generously provided by H. Gram (43). The GST-Grb2 SH2 domain fusion protein was produced in Escherichia coli and purified by glutathione-Sepharose affinity chromatography followed by fast protein liquid chromatography (FPLC) purification procedures. Twenty micrograms of the glutathione-free GST-Grb2 SH2 domain fusion protein was incubated with 400 µg of soluble cell lysates at 4°C for 2 h. Glutathione-Sepharose beads (Pharmacia Biotech Inc., Piscataway, N.J.) were then added to the samples, and the samples were incubated for an additional hour with gentle rotation. The Sepharose beads were recovered through gentle centrifugation and washed extensively with the lysis buffer. Proteins recovered with the GST-Grb2 SH2 domain fusion protein were separated through a 10% NuPAGE Bis-Tris gel (Novex, San Diego, Calif.) and subjected to Western blot analysis.

Antibodies, immunoprecipitation, and immunoblotting. Polyclonal anti-Shc, monoclonal anti-Grb2, and anti-pan ERK antibodies were purchased from Transduction Laboratories (Lexington, Ky.). Polyclonal antibodies against EGFR and ERK2 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Polyclonal anti-phospho-p42/p44 ERK antibody was purchased from Promega Co. (Madison, Wis.). Antiphosphotyrosine monoclonal antibody 4G10 was purchased from Upstate Biotechnology Inc. (Lake Placid, N.Y.). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Amersham Co. (Arlington Heights, Ill.).

Immune complex kinase assay. The kinase activity of ERK was assessed by an immune complex kinase assay as previously described, using myelin basic protein (MBP) as a substrate (35-37). Briefly, soluble cell lysates containing 400 µg of protein were incubated with 1 µg of rabbit polyclonal anti-ERK2 antibody and 20 µl of protein A-Sepharose beads at 4°C for 16 h with gentle rotation. The immunoprecipitates were then washed three times with lysis buffer, three times with wash buffer (500 mM LiCl, 100 mM Tris-HCl [pH 7.6], 0.1% Triton X-100, 1 mM dithiothreitol), and three times with kinase assay buffer (20 mM morpholinepropanesulfonic acid [pH 7.2], 2 mM EGTA, 10 mM MgCl₂, 1 mM dithiothreitol, 0.1% Triton X-100). The kinase reactions were carried out at 30°C for 20 min in 55 µl of kinase assay buffer containing 10 µM ATP, 10 µCi of [-³²P]ATP, 20 mM MgCl₂, and 6 µg of MBP (Sigma).

Northern blot analysis. Northern blot analysis was carried out as previously described (36). Briefly, serum-starved PC12 cells were pretreated with or without tyrphostin AG1478 (40 nM) for 30 min prior to arsenite treatment. Arsenite was directly added to the medium to a final concentration of 400 µM. Cells were harvested 30 min after the addition of arsenite, and total RNA was extracted by using Stat 60 (Teltest "B," Friendswood, Tex.). Northern blot analysis was performed with rat c-fos and c-jun cDNAs and 18S oligonucleotides as probes.

Cell cycle distribution analysis. PC12 cells were plated into six-well plates and cultured until reaching 50 to 60% confluence. Cells were serum starved by being placed in serum-free medium for 48 h and were then treated with 400 µM arsenite for another 24 h. Cells were harvested before and after the treatment. Cell cycle distribution was analyzed by flow cytometry as previously described (17). Briefly, 10⁶ to 2 × 10⁶ cells were collected and fixed in 70% ethanol for 30 min. Fixed cells were washed with PBS once, incubated with 1 µg of RNase A per ml for 30 min at 37°C, and then stained with propidium iodide (Boehringer Mannheim, Indianapolis, Ind.). The stained cells were analyzed on a FACscan flow cytometer for relative DNA content based on red fluorescence. The percentages of the cells in the various cell cycle compartments were determined by using the MULTICYCLE software program (Phoenix Flow Systems, San Diego, Calif.).

	RESULTS

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Arsenite activates ERK through a signaling pathway mediated by Ras and MEK. We have previously reported that arsenite can activate ERK MAP kinase through a Ras-dependent and suramin-sensitive signaling pathway (37). Results presented in Fig. 1 confirmed these earlier observations and provided additional information regarding the kinetics of ERK activation by arsenite and its dependency on MEK in PC12 cells. The kinase activity of ERK was assessed by an immune complex kinase assay using MBP as a substrate. An increase in ERK activity was apparent within 10 min of exposure to 400 µM arsenite (Fig. 1A). Maximum activity was observed between 20 and 30 min of treatment, with a return to near baseline levels by 60 min. This time course is similar to that seen following treatment with EGF, but the magnitude of activation by arsenite is somewhat lower than that seen with the growth factor. Since ERK activation requires dual phosphorylation of both threonine and tyrosine residues in the kinase subdomain VIII (2, 11, 56), active ERK can be detected by the antibody specific for dually phosphorylated ERK. Consistent with the time course for ERK activation detected by immunocomplex kinase assay, Western blot analysis of cell lysates using the anti-active ERK (phospho-ERK) antibody also showed a transient increase in ERK phosphorylation (Fig. 1B, upper panel). In contrast to the time-dependent increase in the level of active ERK, Western blot analysis with a pan-ERK antibody which recognizes both the phosphorylated and nonphosphorylated forms of ERK1 and ERK2 demonstrated that total ERK protein levels did not change in response to arsenite treatment (Fig. 1B, lower panel).

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Arsenite-stimulated ERK activation depends on upstream signals. Serum-starved PC12 cells were treated with either 400 µM arsenite or 100 ng of EGF per ml for the indicated times (in all figures, ' denotes minutes). (A) Time course of ERK activation following arsenite treatment of PC12 cells. Kinase activity of ERK was measured by an immune complex kinase assay using MBP as a substrate. (B) Western blot analysis of phosphorylated and total ERK protein in arsenite-treated cells. Upper panel, phospho-ERK detected with an anti-phospho-ERK antibody; lower panel, total ERK levels detected with an anti-pan-ERK antibody. (C) Effect of MEK inhibitor PD98059 on phosphorylation of ERK in response to arsenite. Cells were preincubated with 50 µM PD98059 for 30 min before addition of arsenite to the medium. Phospho-ERK was detected by Western blot analysis using the phospho-ERK-specific antibody. (D) Comparison of ERK kinase activities in wild-type (WT) and Ras N17 mutant PC12 cells. ERK activity was assessed by an immune complex kinase assay using MBP as a substrate and quantitated by using ImageQuant software from Molecular Dynamics (Sunnyvale, Calif.). Results are representative of three separate experiments.

Arsenite induces tyrosine phosphorylation in PC12 cells. Tyrosine phosphorylation plays an essential role in ERK activation by growth factors and by G-protein-mediated events (10, 15, 39, 53, 64). To investigate the role of tyrosine-phosphorylated proteins in the response to arsenite, a protein tyrosine phosphorylation profile was generated. PC12 cells were treated with 400 µM arsenite and then harvested at different times. The lysates were examined by Western blot analysis using the antiphosphotyrosine antibody 4G10. As shown in Fig. 2A, several proteins (marked by arrows) were found to undergo rapid tyrosine phosphorylation upon arsenite stimulation, similar to that seen following EGF treatment. The apparent molecular masses of the affected proteins on the 10% NuPAGE blot were 170, 54, 45, 39, and 36 kDa, respectively. EGF stimulation did not increase the tyrosine phosphorylation of the 39-kDa protein but did result in tyrosine phosphorylation of a 41-kDa protein that was not affected by arsenite treatment. Interestingly, tyrosine phosphorylation of the 45-kDa (hereafter referred to as p45) and 39-kDa (hereafter referred to as p39) proteins by arsenite was drastically reduced in the Ras N17 cells, while tyrosine phosphorylation of the 170-, 54-, and 36-kDa proteins was unchanged relative to parental cells. The identity of the p39 protein is unclear, but its tyrosine phosphorylation pattern suggests that it may represent a component of a stress-specific signaling pathway that is partially mediated through Ras.

View larger version (40K): [in this window] [in a new window]   FIG. 2.   Protein tyrosine phosphorylation profile of arsenite-treated PC12 cells. (A) Wild-type and Ras N17 PC12 cells were treated with 400 µM arsenite or 100 ng of EGF per ml for the indicated times. The cell extracts were separated on 10% NuPAGE Bis-Tris gel. Tyrosine-phosphorylated proteins were detected by immunoblotting with the antiphosphotyrosine antibody 4G10. The major tyrosine-phosphorylated bands induced by arsenite or EGF are indicated by arrows. (B) Protein tyrosine phosphorylation profile in arsenite-treated PC12 cells was generated on a 4 to 12% gradient NuPAGE Bis-Tris gel. Tyrosine-phosphorylated proteins were detected by immunoblotting with the antiphosphotyrosine antibody 4G10. Arrows indicate the major tyrosine-phosphorylated bands induced by arsenite or EGF. (C) Shc isoforms comigrate with tyrosine-phosphorylated proteins induced by arsenite or EGF. The blot shown in panel B was stripped and reprobed with an Shc-specific rabbit polyclonal antibody. Shown is a single lane from this blot (wild-type, arsenite-treated cells), as all lanes showed identical patterns.

Arsenite induces tyrosine phosphorylation of Shc and enhances its association with Grb2. Shc serves as an adapter protein in growth factor signaling through its interaction with Grb2 (4). To determine if it provides a similar function during stress signaling, we examined whether Shc indeed associated with Grb2 following arsenite treatment. Shc was immunoprecipitated from the cell lysates by using the rabbit anti-Shc antibody that recognizes all three Shc isoforms. The immunoprecipitates were then separated by NuPAGE Bis-Tris gel electrophoresis, and tyrosine-phosphorylated proteins in the immune complexes were detected by Western blot analysis using the mouse monoclonal antiphosphotyrosine antibody 4G10. As shown in the upper panel of Fig. 3A, arsenite treatment led to an enrichment in tyrosine-phosphorylated Shc proteins (p52^Shc and p46^Shc) which were absent in control cells. Interestingly, a 170-kDa tyrosine-phosphorylated protein (p170) was also detected in the Shc immune complexes isolated from the arsenite-treated cells but not from the control cells (Fig. 3A, upper panel). This result suggested that arsenite treatment led to an interaction between the tyrosine-phosphorylated protein p170 and Shc. Western blot analysis of the anti-Shc immunoprecipitates with an anti-Grb2 antibody indicated that Grb2 did coimmunoprecipitate with Shc in the arsenite-treated samples (Fig. 3A, lower panel). Although a small amount of Grb2 in the Shc immune complexes could be detected in the Shc immune complexes as early as 10 min following arsenite exposure, the amount of Grb2 in the Shc immunocomplexes increased markedly after 20 min (Fig. 3A, lower panel; compare the first three lanes). Again, little Grb2 protein was detectable in the negative controls (time zero). Importantly, the enrichment of Grb2 protein in the Shc immune complexes correlated precisely with the kinetics of Shc tyrosine phosphorylation (compare the upper and lower panels in Fig. 3A), while the amounts of Shc and Grb2 detected by Western blot analysis in the total cell lysates remained unchanged throughout the entire treatment period (Fig. 3B).

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Arsenite stimulates Shc tyrosine phosphorylation and its interaction with Grb2. (A) Upper panel, arsenite-induced tyrosine phosphorylation of Shc. Cellular extracts from arsenite-treated PC12 cells were immunoprecipitated with anti-Shc polyclonal antibody (pAb) and protein (Prot or Pro) A-Sepharose beads. Shc immunoprecipitates were resolved by NuPAGE Bis-Tris gel and Western blotted (WB) with the antiphosphotyrosine (anti-PY) antibody 4G10. Cell lysates (60-min arsenite treatment) and immunoprecipitates obtained in the absence of either Shc antibody or lysates were run as controls. Arrows indicate the major tyrosine-phosphorylated bands detected. Lower panel, arsenite-enhanced interaction between Grb2 and Shc. The immunoblot used in the upper panel was stripped and then blotted with a monoclonal anti-Grb2 antibody. The position of Grb2 is indicated. (B) Western blot analysis of total Shc and Grb2 levels in control and arsenite-treated cells. (C) Coomassie blue staining of the GST-Grb2 SH2 domain fusion protein. Recombinant GST-Grb2 SH2 domain fusion protein was produced in E. coli and purified by glutathione-Sepharose affinity column followed by FPLC. The fusion protein was then separated by SDS-polyacrylamide gel electrophoresis and stained. (D) Arsenite stimulates interaction between Grb2 SH2 domain and Shc in vitro. Cell lysates prepared from arsenite- or EGF-treated cells were incubated with the glutathione-free GST-Grb2 SH2 fusion protein for 2 h at 4°C. GST-Grb2 SH2 domain fusion protein and proteins associated with it were recovered by glutathione-Sepharose affinity chromatography and subjected to immunoblotting with the antiphosphotyrosine 4G10 or the anti-Shc antibodies.

Activation of EGFR is required for arsenite-induced Shc tyrosine phosphorylation and ERK activation. Tyrosine phosphorylation of Shc can be regulated by both receptor-type and nonreceptor-type protein tyrosine kinases (4). It has been shown that the EGFR tyrosine kinase can directly phosphorylate Shc (55). The observations that the tyrosine-phosphorylated protein p170 was found to coimmunoprecipitate with tyrosine-phosphorylated Shc in arsenite-treated cells (Fig. 3A) and also to copurify with tyrosine-phosphorylated Shc by using the Grb2 SH2 domain fusion protein (Fig. 3D) raised the possibility that the 170-kDa tyrosine-phosphorylated protein p170 might be the tyrosine kinase responsible for Shc phosphorylation. Because EGF treatment also enhanced the tyrosine phosphorylation of this protein and its size was similar to that of the EGFR, we investigated whether EGFR was involved in the arsenite-triggered signaling cascade. Western blot analysis using a polyclonal anti-EGFR antibody indicated that EGFR did indeed comigrate with the 170-kDa tyrosine-phosphorylated protein seen in both arsenite- and EGF-treated cells (Fig. 4A, right panel).

View larger version (61K): [in this window] [in a new window]   FIG. 4.   Role of EGFR in mediating arsenite-induced protein tyrosine phosphorylation. (A) Control and arsenite-treated PC12 cells were subjected to Western blot (WB) analysis using the antiphosphotyrosine (anti-PY) monoclonal antibody 4G10 and an anti-EGFR polyclonal antibody. The same blot was sequentially probed with the two antibodies. The band recognized by the EGFR antibody is indicated by an arrow. EGFR detected in the right panel comigrates with the 170-kDa tyrosine-phosphorylated protein seen in the left panel. (B) Effects of suramin and tyrphostin AG1478 on the tyrosine phosphorylation of EGFR. Suramin (300 µM) or tyrphostin AG1478 (40 nM) was added at the same time as arsenite (400 µM). Cell lysates were immunoprecipitated (IP) with the anti-EGFR polyclonal antibody, and the immunoprecipitates were subjected to Western blot analysis using the antiphosphotyrosine antibody 4G10. (C) Effect of suramin and tyrphostin AG1478 on overall arsenite-induced protein tyrosine phosphorylation. PC12 cells were treated with arsenite in the presence or absence of suramin or tyrphostin AG1478 under the conditions described above. Cell lysates were analyzed by Western blotting with the antiphosphotyrosine antibody 4G10. Arrows denote proteins whose tyrosine phosphorylation was affected by suramin or tyrphostin AG1478.

View larger version (33K): [in this window] [in a new window]   FIG. 5.   Central role of EGFR in mediating arsenite-induced ERK activation. (A) Effects of suramin and tyrphostin AG1478 on arsenite-induced ERK activation. PC12 cells were treated with arsenite (400 µM) in the presence or absence of 300 µM suramin or 40 nM tyrphostin AG1478 for 30 min. ERK activity was analyzed by an immune complex kinase assay using MBP as a substrate and quantitated by using ImageQuant software (Molecular Dynamics). (B) Effects of suramin and tyrphostin AG1478 on arsenite-induced ERK phosphorylation. Cell lysates from cells treated with arsenite in the absence or presence of suramin or tyrphostin AG1478 were analyzed by Western blotting using the phospho-ERK-specific antibody.

View larger version (49K): [in this window] [in a new window]   FIG. 6.   Pretreatment of cells with EGF inhibits a subsequent arsenite response. (A) PC12 cells were pretreated with EGF (1 µg/ml) for 1 h (+) or not pretreated (), as indicated at the bottom. The cells were then washed and incubated in EGF-free medium for the time intervals shown at the bottom. The cells were further treated with EGF (100 ng/ml) for 10 min or NGF (100 ng/ml) for 5 min (second treatment, shown at the top). Cell lysates following these different treatments were subjected to Western blot analysis using anti-EGFR polyclonal antibody (upper panel). The same blot was sequentially probed with anti-phospho-ERK-specific antibody (lower panel). (B) PC12 cells were pretreated with EGF (1 µg/ml) for 1 h (+) or not pretreated (), as indicated at the bottom. The cells were then washed and incubated in EGF-free medium for the time intervals shown at the bottom. The cells were further treated (second treatment) with 400 µM arsenite for an additional 30 min. Total cellular proteins were subjected to Western blot (WB) analysis using anti-phospho-ERK-specific antibody (upper panel) or the antiphosphotyrosine (anti-PY) monoclonal antibody 4G10 (lower panel). EGFR-mediated protein tyrosine phosphorylation stimulated by arsenite is indicated by arrows.

View larger version (34K): [in this window] [in a new window]   FIG. 7.   Treatment of PC12 cells with the EGFR-selective inhibitor tyrphostin AG1478 prevents arsenite-induced Shc tyrosine phosphorylation and its interaction with EGFR and Grb2. PC12 cells were treated with EGF or arsenite in the presence or absence of 30 nM tyrphostin AG1478. Cell lysates containing 4.5 mg of protein were subjected to immunoprecipitation (IP) by using 1 µg of anti-Shc polyclonal antibody. Immunoprecipitates were subjected to Western blot (WB) analysis using the antiphosphotyrosine (anti-PY) antibody 4G10 (A), anti-EGFR antibody (B), and anti-Grb2 antibody (C). The same blot was sequentially probed with the three antibodies.

Arsenite induces expression of c-fos and c-jun and stimulates DNA synthesis. Growth factors and other mitogens have been shown to stimulate the expression of a number of immediate-early genes that play a critical role in mediating proliferation (1). Among these are the c-fos and c-jun genes, whose inductions are largely dependent on MAP kinase pathways (1, 11, 27). Since arsenite treatment is also known to induce the expression of c-fos and c-jun (6), we investigated the potential role of EGFR in mediating this response. Arsenite treatment of PC12 cells resulted in the rapid induction of both c-fos and c-jun mRNA levels (4.8- and 3.8-fold, respectively [Fig. 8A]). Pretreatment of cells with tyrphostin AG1478 almost completely abolished c-fos induction and partially inhibited c-jun expression, supporting a role for the EGFR in mediating these effects (Fig. 8A). To gain further evidence for the ability of arsenite to influence cell proliferation, cells were examined by fluorescence-activated cell sorting analysis for changes in cell cycle distribution following treatment with the agent. Cells were first subjected to 48 h of serum starvation to enrich for cells in the G₁ phase. At this point only a small number of cells were present in S phase (14%). Cells were then either left untreated or treated with arsenite and examined 24 h later. As shown in Fig. 8B, in the absence of arsenite treatment there was a further reduction in the number of cells in S phase. In contrast, arsenite treatment led to a significant increase in the number of cells in S phase (31%), indicative of increased DNA synthesis. Thus, arsenite does appear to enhance cell proliferation, consistent with its role as a tumor promoter.

View larger version (32K): [in this window] [in a new window]   FIG. 8.   Arsenite induces c-fos and c-jun expression and enhances DNA synthesis. (A) Northern blot analysis of RNA extracted from arsenite-stimulated PC12 cells in the presence or absence of tyrphostin AG1478. PC12 cells were treated (Arsenite) or untreated (Control) with 400 µM arsenite for 30 min in the presence (Arsenite + AG1478) or absence of tyrphostin AG1478. The cells were harvested and analyzed for expression of c-fos mRNA, c-jun mRNA, and 18S rRNA by Northern blot analysis. The signals were quantitated with a PhosphorImager, and following normalization to 18S rRNA, relative fold mRNA induction for c-fos and c-jun mRNA expression was determined (bottom panel). (B) Arsenite increases cell population in S phase. PC12 cells were starved in serum-free medium for 48 h. Starved cells were either treated with 400 µM arsenite for 24 h or continual kept in serum-free medium for another 24 h. For cell cycle distribution analysis (as described in Materials and Methods), the cells were collected before arsenite treatment [Serum free (48 h)], after arsenite treatment [Serum free (48 h) + Ars (24 h)], or without treatment [serum free (72 h)]. The percentages of the cells in various cell cycle phases are indicated as means ± standard deviations. Results were from three different experiments.

	DISCUSSION

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We have previously demonstrated that arsenite treatment results in the activation of ERK MAP kinase through a Ras-dependent, suramin-sensitive pathway (37). Based on this observation, we hypothesized that the arsenite signal was transmitted through a pathway mediated by growth factor receptor-type tyrosine kinase(s). In this report, we have provided direct evidence to support this view. In particular, we have shown that EGFR undergoes tyrosine phosphorylation following arsenite treatment and that this event is associated with enhanced tyrosine phosphorylation of the Shc adapter protein, allowing for its increased interaction with Grb2.

The EGFR has been implicated in ERK activation by other extracellular stress signals including UV-C, H₂O₂, and asbestos (22, 25, 28, 47, 50, 52, 65). However, in none of the previous studies was the link between EGFR phosphorylation and activation of the ERK pathway established. Phosphorylation of Shc has been demonstrated to be a crucial step in the activation of the Ras/MEK/ERK pathway in response to growth factor stimulation (4). Our findings indicate that Shc is also an important mediator in arsenite-induced ERK activation, serving as an adapter for the recruitment of Grb2 and Sos to the membrane. Shc can be phosphorylated by both receptor-type and nonreceptor-type tyrosine kinases (4). Since a variety of stresses are known to activate nonreceptor-type tyrosine kinases (e.g., Src), it remains possible that these, rather than the intrinsic EGFR kinase activity, are responsible for Shc phosphorylation following arsenite treatment (12, 52). It is also clear that EGFR can be phosphorylated by nonreceptor-type tyrosine kinases, and in certain instances, EGFR does not rely on its intrinsic kinase activity for its signaling functions (39, 64). For example, Src family tyrosine kinases have been shown to be responsible for tyrosine phosphorylation of EGFR regulated by G-protein-coupled receptors (39). Similarly, JAK2, another nonreceptor tyrosine kinase, was found to phosphorylate EGFR in response to growth hormone treatment (64). In both instances, the phosphorylated EGFR was shown to be important for mediating downstream events, including Shc tyrosine phosphorylation and activation of the ERK pathway. In our case, abrogation of EGFR autophosphorylation by treatment with the highly specific EGFR inhibitor tyrphostin AG1478 also prevented the arsenite-induced Shc tyrosine phosphorylation and ERK activation. These findings argue strongly that EGFR phosphorylation is crucial for activation of the ERK signaling cascade in response to arsenite and also that intrinsic EGFR tyrosine kinase activity is required.

In addition to EGFR, other growth factor receptors including the T-cell receptor complex, interleukin-1 receptor, and the NGF receptor TrkA have been implicated in the transduction of stress signals by other toxic agents (21, 28, 30, 48, 50, 51). In particular, we have found that overexpression of TrkA in PC12 cells leads to enhanced activation of ERK in response to hydrogen peroxide treatment (21). Hence, it was somewhat surprising in this study to find that the EGFR tyrosine kinase-selective inhibitor tyrphostin AG1478 completely abolished the ERK activation in response to arsenite. This observation suggests that at least for PC12 cells, the response to arsenite is highly dependent on EGFR. Further support for this notion was obtained in experiments in which down-regulation of EGFR led to an attenuation of Shc tyrosine phosphorylation and ERK activation.

The arsenite-induced activation of ERK is not restricted to PC12 cells, as we have observed that arsenite produces similar effects in other cell types, including Rat1 fibroblasts (37), human epidermoid carcinoma A431 cells, and transformed human embryonal kidney 293 cells (data not shown). However, our findings do contrast with several reports by others. For example, Rouse et al. (49) failed to detect ERK activation in arsenite-treated PC12 cells. Similarly, Cavigelli et al. (6) did not observe activation of ERK in arsenite-treated HeLa cells, although JNK was highly activated. While the reasons for the differences between our studies and these remain unclear, they may reflect differences in the EGFR content of the different cell lines or strains used. In support of this notion, we have observed that compared to our PC12 cells, HeLa cells contain very low levels of EGFR (data not shown). Like Cavigelli et al. (6), we too have reported that JNK is highly activated in response to arsenite treatment in HeLa cells, as is p38 (37). However, the activation of both JNK and p38 occurs through mechanisms which do not involve EGFR, as the activation cannot be blocked by treatment with either suramin (37) or AG1478 (unpublished observations), nor is it dependent on Ras (37). This is consistent with the suggestion by Cavigelli et al. that arsenite-induced JNK activation occurs primarily through inhibition of a specific JNK phosphatase (6).

What is the mechanism through which arsenite activates EGFR? At least two distinct possibilities exist: (i) inhibition of tyrosine phosphatases that are involved in the inactivation of the EGFR tyrosine kinase, and (ii) up-regulation of intrinsic EGFR tyrosine kinase activity through direct interaction with the receptor. The first scenario assumes that cells exhibit significant spontaneous tyrosine kinase activity that is normally kept in check by protein tyrosine phosphatases which serve to inactivate the kinase. Studies of Knebel et al. (28) have provided strong evidence that certain treatments such as radiation, oxidants, and alkylating agents act through such a mechanism. Essential thiol groups on the tyrosine phosphatases are the presumed targets of the adverse agents, but the specific phosphatases involved have not yet been identified. Given the high reactivity of arsenite for vicinal dithiols, it is possible that arsenite also produces such effects, leading to elevated EGFR tyrosine kinase activity. The second possibility is that arsenite can actually mimic the action of ligands to activate the receptor tyrosine kinase. EGFR contains extracellular cysteine-rich domains that are important for ligand-triggered dimerization (24, 53). By reacting with vicinal dithiols in these domains, arsenite could alter the conformation of EGFR, resulting in an increase of its intrinsic tyrosine kinase activity. In support of this model, we observed that cotreatment of cells with arsenite and orthovanadate, a general tyrosine phosphatase inhibitor, synergistically enhanced the protein tyrosine phosphorylation (data not shown).

Arsenite is a potent carcinogen (3) that is believed to play an important role in the development of certain cancers such as skin and bladder tumors (3, 8, 9, 26). For example, arsenite ingested through contaminated water supplies was found to be associated with a high rate of bladder and skin cancer in regions of developing countries. In addition, a high rate of these cancers has also been reported in patients receiving Fowler's solution (potassium arsenite) for the treatment of psoriasis in the 1940s to 1970s (8, 31). The potential role of EFGR in mediating arsenite's carcinogenic effects is intriguing. Consistent with its tumor-promoting properties, arsenite treatment led to enhanced DNA synthesis and induction of the proliferation-associated genes c-fos and c-jun (Fig. 8). That the induction of c-fos was almost completely abolished in the presence of tyrphostin AG1478 highlights the essential role of the EGFR in mediating this response. Abnormal activation of EGFR and related tyrosine kinases such as Neu, ErbB-3, and ErbB-4 has been reported for a variety of human cancers (19, 33, 38, 42, 58). In many cases, elevated Shc tyrosine phosphorylation has also been observed (45). Finally, suramin as well as antibodies targeting members of the EGFR tyrosine kinase family have proven effective in the treatment of certain tumors (33, 60). Our findings indicating that arsenite usurps the EGF/EGFR signaling pathway to activate ERK provides a mechanism for its tumor-promoting properties and offers a potential target for therapeutic strategies aimed at preventing or inhibiting arsenite-induced tumor growth.