Introduction

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Lead is a widespread environmental pollutant, the major health concerns of which relate to its developmental neurotoxicity (Ballinger et al., 1987). Lead is also classified by the International Agency for Research on Cancer as a group 2B carcinogen (possible human carcinogen). In animals, there is evidence of lead inducing renal adenomas, lung adenomas, and cerebral gliomas (ATSDR, 1999). Excess of renal (Steenland et al., 1992), lung (Anttila et al., 1995), and brain (particularly astrocytomas) (Anttila et al., 1996) cancers, have also been found in epidemiological studies in lead-exposed workers. As lead-induced gene mutations in mammalian cells have been usually observed only at high toxic concentrations (Zelikoff et al., 1988), such genotoxicity may not be the result of direct damage to DNA but may occur by indirect mechanisms, such as inhibition of DNA repair (Hartwig, 1994). There is, however, evidence that lead can increase proliferation of rat and mouse kidney cells (Choie and Richter, 1974), rat liver cells (Liu et al., 1997), vascular smooth-muscle cells (Fujiwara et al., 1995), and spleen cells (Razani-Boroujerdi et al., 1999), suggesting that it may act as a tumor promoter.

The established receptors for potent tumor promoters, such as the phorbol esters, are the classical and novel isozymes of protein kinase C (PKC) (Mellor and Parker, 1998). A large body of evidence exists which indicates that lead can activate PKC in different cellular systems and under different experimental conditions (reviewed in Costa, 1998). We have recently found that lead can stimulate DNA synthesis and cell cycle progression in human astrocytoma cells and that this effect is due to a selective activation of PKC by lead (Lu et al., 2001). However, the signal transduction pathway leading from PKC activation to DNA synthesis remains to be elucidated.

In the present study, we investigated the ability of lead to activate the MAPK cascade and the role of PKC in this signal transduction pathway. MAPKs are a family of protein kinases playing a central role in signal transduction and thought to mediate diverse processes ranging from transcription of protooncogenes to programmed cell death (Derkinderen et al., 1999; Pearson et al., 2000). The best studied MAPK are ERK1 and ERK2 (p42 and p44 MAPK), which are activated by mitogens and play a central role in cell proliferation (Ferrell, 1996). ERK1/2 are regulated upstream by a MAPK kinase (MEK1/2), which in turn is activated by Raf kinases, particularly Raf-1 (Ferrell, 1996; Derkinderen et al., 1999). PKC has been shown to activate Raf-1 kinase, either by direct phosphorylation or by modulating its membrane association (Kolch et al., 1993; Schonwasser et al., 1998). Activated ERK1/2 translocate to the nucleus where they phosphorylate and activate other kinases, transcription factors, and other target proteins (Derkinderen et al., 1999; Pearson et al., 2000) including the family of p90 kDa ribosomal S6 kinases (p90^RSK), which seem to play a relevant role in cell proliferation (Frodin and Gammeltoft, 1999). The aim of the present study was, therefore, to investigate whether lead would stimulate this cascade of signal transduction events (PKC Raf-1 MEK1/2  ERK1/2  p90^RSK) leading to proliferation of human astrocytoma cells.

	Experimental Procedures

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Materials. Dulbecco's modified Eagle's medium, fetal bovine serum (FBS), penicillin/streptomycin, and trypsin were purchased from Invitrogen (Carlsbad, CA). [methyl-³H]Thymidine (6.7 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Anti-phosphoERK1/ERK2 antibody and horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody were obtained from Promega (Madison, WI). Anti-phospho-Elk-1, anti-phospho-MEK1/2, anti-phospho-p90^RSK (Ser381), and immobilized anti-phospho-MAPK antibodies were obtained from New England Biolabs (Beverly, MA). Anti-ERK1/ERK2 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The protease inhibitor cocktail tablets were obtained from Roche Molecular Biochemicals (Indianapolis, IN). The myristoylated PKC peptide inhibitor, based on the pseudosubstrate region for classical PKC (Myr-Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-Lys-Asn-Val), was purchased from Promega. Lead acetate and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Lead acetate was dissolved in deionized water and prepared as a 2 mM stock solution.

Cell Culture. The human astrocytoma cell line 1321N1 (kindly donated by Dr. J. H. Brown, University of California at San Diego) was maintained in low-glucose Dulbecco's modified Eagle's medium, supplemented with 5% FBS, 100 U/ml penicillin G, and 100 µg/ml streptomycin in 75-cm² flasks under a humidified atmosphere of 5% CO₂/95% air at 37°C. Cells were subcultured every 7 days, and the growth medium was changed every 3 or 4 days. Cells were seeded in 24-well plates at the density of 2.5 × 10⁴/ml for the proliferation experiments and in 100-mm dishes at the density of 1 × 10⁵/ml for Western blot experiments.

Measurement of DNA Synthesis. Incorporation of [methyl-³H]thymidine into cell DNA was measured as described previously (Guizzetti et al., 1996). Briefly, cells were seeded at the density of 2.5 × 10⁴/ml in 24-well plates. After 4 days in medium supplemented with 5% FBS, cells were switched to serum-free medium supplemented with 0.1% bovine serum albumin for 48 h, before treatment. Treatment with lead or other compounds was for 24 h. One µCi/well of [methyl-³H]thymidine was included for the last 6 h of the incubation at 37°C under an atmosphere of 5% CO₂/95% air. At the end of the incubation, cells were washed twice with cold PBS and fixed in methanol. Unincorporated [³H]thymidine was removed by two washes with ice-cold 10% trichloroacetic acid and one wash of ice-cold 0.5% trichloroacetic acid. The monolayer was dissolved in 500 µl of 1 M NaOH, and 250 µl was transferred to scintillation fluid and counted for radioactivity in a Beckman LS5000 CE scintillation counter (Beckman Coulter, Inc., Fullerton, CA).

Western Blot Analyses. Cells were seeded on 100-mm dishes in medium containing 5% FBS and were switched to serum-free medium when confluent. Treatments were done after 48 h of serum starvation. After treatment, cells were harvested in buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerol phosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 15% final volume protease inhibitor cocktail. Proteins were quantified using the Bradford method, and a 5× sample buffer was added. After boiling for 5 to 10 min, samples containing 20 to 50 µg of proteins were loaded on a 10% SDS-polyacrylamide gel. After separation, proteins were transferred to Immobilon membranes (Millipore Corporation, Bedford, MA), which were incubated with the appropriate primary antibodies overnight at 4°C and then 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (dilution 1:2000). Bands were revealed by chemiluminescent detection using an enhanced chemiluminescence kit (Amersham Biosciences, Arlington Heights, IL), and densitometrically quantified using a Millipore Image System.

Immunoprecipitation and Kinase Assay. After chemical stimulation, cells were harvested in a lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM -glycerol phosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 15% final volume protease inhibitor cocktail. Cell extracts (100-200 µg) were incubated with immobilized phosphospecific (Thr²⁰²/Tyr²⁰⁴) MAPK antibody overnight at 4°C. The immune complexes were washed twice with lysis buffer and then twice with kinase buffer containing 25 mM Tris, pH 7.5, 5 mM glycerol phosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, and 10 mM MgCl₂. Each complex was then suspended in 25 µl of kinase buffer with 200 µM ATP and 1 µg of the substrate, the GST-Elk-1 fusion protein, and incubated at 30°C for 30 min. The reactions were terminated by addition of 3× SDS sample buffer. The samples were analyzed by SDS-polyacrylamide gel electrophoresis and Western blot as described above. MAPK activity was measured by using a phospho-Elk-1 antibody.

Statistical Analysis. Each experiment was performed at least three times. All statistical tests were carried out using the StatView 512 program (Abacus Concepts, Berkeley, CA) or Microsoft Excel (Microsoft, Redmond, WA) on a Macintosh personal computer (Cupertino, CA). One-way analysis of variance followed by Fisher's least significant difference test was used to determine significant difference between treatments.

	Results

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Lead Causes MAPK (ERK1/ERK2) Activation in 1321N1 Human Astrocytoma Cells. Time course experiments showed that lead caused a rapid activation of ERK1/2 with a maximum at 15 min and a decrease to control level after 4 h (Fig. 1A). Dose-response experiments showed that lead-induced MAPK activation was concentration-dependent (Fig. 1B). Levels of phosphorylated ERK1 and ERK2 were similar in control cells as well as in cells stimulated with lead or with the phorbol ester TPA (Fig. 1C). An immunocomplex kinase assay using GST-Elk-1 as a substrate indicated that lead also increased MAPK activity (Fig. 1D). Total MAPK protein levels, measured by an anti-ERK1/2 antibody, did not change upon lead exposure (Fig. 1E). In this range of concentrations (1-50 µM), lead did not have any cytotoxic effect and was able to stimulate DNA synthesis and cell cycle progression in astrocytoma cells (Lu et al., 2001).

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Activation of MAPK by lead acetate. A, time course of lead (5 µM Pb)-induced phosphorylation of ERK1/2. B, concentration-dependent phosphorylation of ERK1/2 by lead (Pb, 5 min). C, quantitation of the effect of lead (10 µM Pb) and TPA (200 ng/ml), both at 15 min, on ERK1/2 phosphorylation (mean ± S.E.; n = 3). D, lead (Pb; 15 min) increases MAPK activity, measured by an immunocomplex assay using GST-Elk-1 as substrate. E, ERK1/2 protein levels did not change after lead (Pb) treatment (15 min). Blots shown are from one experiment, which was repeated three times with similar results. *, significantly different from control, p < 0.05.

Role of PKC in Lead-Induced MAPK Activation. To determine the role of PKC in lead-induced MAPK activation, cells were pretreated with GF109203X, which acts as a competitive inhibitor for the ATP-binding site of PKC, for 30 min prior to lead treatment. Lead-induced MAPK activation was significantly decreased in the presence of GF109203X (Fig. 2A). Additionally, cells were treated with the phorbol ester TPA (200 ng/ml) for 24 h before lead treatment. Such prolonged treatment of TPA is known to down-regulate PKC and  in astrocytoma cells (Guizzetti et al., 1998). The ability of lead and TPA to activate MAPK was also inhibited under this condition (Fig. 2B). In contrast, platelet-derived growth factor (PDGF), which has been shown to activate MAPK independently of PKC (Baron et al., 2000), retained its ability to activate MAPK after PKC down-regulation.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Role of PKC in lead-induced phosphorylation of ERK1/2. A, the PKC inhibitor GF109203X (30 min before lead) inhibits activation of MAPK by lead (10 µM Pb, 15 min). B, lead (10 µM Pb), TPA (200 ng/ml), and PDGF (25 ng/ml) caused activation of MAPK (left). In cells pretreated with TPA (200 ng/ml, 24 h) to down-regulate PKC, the effects of lead and TPA were inhibited, whereas PDGF still caused MAPK activation. Blots shown are from one experiment, which was repeated three times with similar results.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Activation of MAPK by lead is mediated by PKC. A, the pseudosubstrate to PKC (25 µM, 30 min) inhibits MAPK activation by lead (10 µM Pb, 15 min) but not by PDGF (25 ng/ml). B, lead (10 µM Pb), TPA (200 ng/ml), and PDGF (25 ng/ml) activate MAPK (left). In cells pretreated with lead (100 µM Pb, 24 h) to selectively down-regulate PKC (Lu et al., 2001), the effect of lead is inhibited, whereas TPA and PDGF remained effective in activating MAPK. Blots shown are from one experiment, which was repeated three times with similar results.

MEK Inhibitors Inhibit Lead-Induced MAPK Activation and DNA Synthesis. MEK1/2, the kinase upstream of ERK1/2, was also activated by lead treatment (10 µM), as shown by an increase in its phosphorylated form (Fig. 4A). Activation of MEK1/2 by lead was inhibited by the PKC inhibitor GF109203X and by down-regulation of PKC through prolonged TPA treatment (200 ng/ml, 24 h) (Fig. 4A). Two MEK1/2 inhibitors, PD98059 and UO126, were able to inhibit MAPK activation induced by lead (Fig. 4B). Lead-induced DNA synthesis, as measured by [methyl-³H]thymidine incorporation into cell DNA, was also blocked by the two MEK1/2 inhibitors (Fig. 4C). PD98059 and UO126 have been recently found not to be specific for MEK1/2 but to also inhibit MEK5, the kinase that phosphorylates ERK5 (BMK1), a MAPK which may also be involved in the mitogenic response to growth factors (Kamakura et al., 1999; Kato et al., 2000). Lead, however, did not activate ERK5 in astrocytoma cells (Fig. 4D), suggesting that the effects of the two MEK inhibitors on lead-induced DNA synthesis can be ascribed to their action on MEK1/2.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effect of lead on MEK1/2. A, lead (10 µM Pb, 15 min) caused MEK1/2 phosphorylation. The effect of lead was inhibited by PKC down-regulation by TPA pretreatment (200 ng/ml, 24 h) and by pretreatment with the PKC inhibitor GF109203X (5 µM, 30 min). B, lead (10 µM Pb)-induced ERK1/2 phosphorylation is inhibited by the MEK inhibitors UO126 and PD98059 (30 min). C, lead-induced DNA synthesis (10 µM, 24 h) is inhibited by PD98059 and UO126 (mean ± S.E.; n = 3). D, lead (Pb) does not activate ERK5 in astrocytoma cells. Blots shown are from one experiment, which was repeated three times with similar results.

Lead Activates p90^RSK in a PKC- and MAPK-Dependent Manner. Lead also induced a concentration-dependent phosphorylation of p90^RSK, a target of ERK1/2, which seems to play a role in cell proliferation (Frodin and Gammeltoft, 1999) (Fig. 5A). Pretreatments with TPA (200 ng/ml, 24 h) to down-regulate PKC, or with the PKC inhibitor GF109203X, attenuated the effect of lead on p90^RSK, whereas the MEK inhibitor UO126 completely abolished it (Fig. 5B).

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Activation of p90RSK by lead. A, lead (5-50 µM Pb) and TPA (200 ng/ml) stimulate p90RSK phosphorylation. B, lead (10 µM Pb)-induced phosphorylation of p90RSK is inhibited by down-regulation of PKC with TPA (200 ng/ml, 24 h), by the PKC inhibitor GF109203X (5 µM) and by the MEK inhibitor UO126 (5 µM). Blots shown are from one experiment, which was repeated three times with similar results.

Lead Does Not Activate PI3K and p70^S6K. The selective activation of PKC by lead may be due to an interaction of this metal with the C2 domain of the enzyme, which confers calcium/phosphatidylserine binding to classical PKC isozymes (Mellor and Parker, 1998). Such domain is not unique for PKC, as it is found in other calcium- and phospholipid-binding proteins, such as phosphatidylinositol 3-kinase (PI3K), which plays a central role in cell proliferation (Coulonval et al., 2000). We found that lead was unable to cause phosphorylation of Akt/PKB, a major substrate of PI3K (Fig. 6A). Phosphorylation of p70^S6K, which is a target for PI3K (Coulonval et al., 2000), was also not affected by lead (Fig. 6B). Furthermore, although in some systems activation of MAPK is regulated by PI3K (Toker, 2000), activation of ERK1/2 by lead was not affected by the PI3K inhibitor LY294002 (Fig. 6C).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effect of lead on PI3K and on p70S6K. A, lead (10 µM Pb, 15 min) and TPA (200 ng/ml, 15 min) had no effect on phosphorylation of the PI3K substrate Akt/PKB, whereas the positive control PDGF (25 ng/ml) did. B, lead (10 µM Pb, 15 min) did not induce phosphorylation of p70S6K, whereas PDGF (25 ng/ml) did. C, lead (10 µM Pb)-induced phosphorylation of ERK1/2 was not affected by the PI3K inhibitor LY294002 (1 µM). Blots shown are from one experiment, which was repeated three times with similar results.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Although the ability of lead to increase DNA synthesis in different cell types had been previously reported (Choie and Richter, 1974; Liu et al., 1997), the intracellular mechanisms involved in this effect had not been elucidated. We have recently shown that lead causes a concentration-dependent increase in DNA synthesis and cell cycle progression in human astrocytoma cells and that this effect was dependent upon stimulation of PKC (Lu et al., 2001). In the present study, we investigated whether lead would activate the MEK-MAPK-RSK cascade in a PKC-dependent manner, to gain a better understanding of the signal transduction pathway that may be activated by lead and may underlie the increased DNA synthesis. There is evidence that several different stimuli can activate the MEK/MAPK pathway in a PKC-dependent manner (Abe et al., 1998; Axmann et al., 1998). PKC can activate Raf-1 kinase, either by direct phosphorylation (Kolch et al., 1993) or by modulating its membrane association (Schonwasser et al., 1998); Raf-1, in turn, can phosphorylate MEK, which then activates the ERK1/2 MAPK (McDonald et al., 1993; Derkinderen et al., 1999). Among the substrates of ERK1/2, p90^RSK is considered to be relevant for the mitogenic response (Frodin and Gammeltoft, 1999). We found that lead was able to activate ERK1/2 in human astrocytoma cells, as evidenced by an increase in the levels of phosphorylated ERK1/2 and an increase in their activity, without changes in the level of proteins. Experiments with the PKC inhibitor GF109203X and with down-regulation of PKC by prolonged treatment with TPA indicated that activation of MAPK was dependent upon PKC. As human 1321N1 astrocytoma cells express only the PKC and  TPA-sensitive isozymes, further experiments were carried out, which indicated that PKC mediates the effect of lead on MAPK. PKC is also the only PKC isozyme activated by lead in human astrocytoma cells (Lu et al., 2001).

Further experiments were then carried out to elucidate the signaling steps upstream and downstream of MAPK. Lead was able to activate MEK1/2, the kinases that directly phosphorylate ERK1/2, and this effect was also dependent upon PKC. Two inhibitors of MEK, PD98059 and UO126, blocked the ability of lead to activate MAPK, as expected, and, at the same concentrations, they inhibited lead-induced DNA synthesis, thus supporting the main involvement of this signaling pathway in the mitogenic effect of this metal. These two MEK1/2 inhibitors have been recently shown to also inhibit MEK5, the kinase that phosphorylates ERK5 (Kamakura et al., 1999), a MAPK that is involved in mitogenic signaling (Kato et al., 2000; Pearson et al., 2000). Lead, however, was unable to induce phosphorylation of ERK5 in astrocytoma cells, suggesting that the inhibitory effect of PD98059 and UO126 on lead-induced DNA synthesis may be ascribed to inhibition of MEK1/2.

The additional link between lead-activated PKC and MEK1/2 may be represented by Raf-1 kinase (Abe et al., 1998), as PKC has been shown to activate Raf-1 kinase, either by direct phosphorylation or by modulating its membrane association (Kolch et al., 1993; Schonwasser et al., 1998). In a number of experiments, we attempted measuring activation of Raf-1 kinase by lead. MEK1 was used as a substrate to carry out an immunocomplex kinase assay after immunoprecipitation with an anti-Raf-1 antibody. Although we consistently found an increase in Raf-1 activity upon exposure to lead (10 µM), the effect of lead was small (30% above basal), so that experiments with PKC inhibitors were not carried out (data not shown). The finding suggests that Raf-1 may link PKC to MEK1/2 in the pathway leading to MAPK activation by lead. However, an alternative interpretation of this result (in addition to the possibility of a low sensitivity of the method used to measure Raf-1 activation) is that lead-activated PKC may in turn activate MEK1/2 through another MEK kinase (Pearson et al., 2000). Downstream of MAPK, we found that lead could activate p90^RSK, one of the substrates of ERK1/2, which can activate various transcription factors and seems to play a role in cell proliferation (Zhao et al., 1996; Frodin and Gammeltoft, 1999). Activation of p90^RSK also depended on activation of PKC and MEK1/2, as expected.

Selective activation of PKC by lead in human astrocytoma cells (Lu et al., 2001) may be due to an interaction of this metal with the C2 domain of the enzyme, which confers calcium/phosphatidylserine binding to classical PKC isozymes (Nalefski and Falke, 1996; Mellor and Parker, 1998). Such C2 domain is not unique for PKC, as it is found in several other calcium- and phospholipid-binding proteins, such as synaptotagmin, phospholipase, or PI3K (Nalefski and Falke, 1996; Rizo and Sudhof, 1998). The latter is of interest in the context of an effect of lead on DNA synthesis, as PI3K plays a central role in cell proliferation (Leevers et al., 1999; Coulonval et al., 2000). We found that lead did not stimulate phosphorylation of Akt/PKB, a major substrate of PI3K (Coffer et al., 1998). Lead also did not activate another member of the S6 kinase family, the p70^S6K, which is activated by PI3K (Coulonval et al., 2000). Furthermore, although in some systems activation of MAPK is regulated by PI3K (Toker, 2000), this is not the case with lead, as the PI3K inhibitor LY294002 did not affect lead-induced ERK1/2 activation. However, in a preliminary experiment, we have found that LY294002 (1 µM) inhibited lead-induced DNA synthesis (by 40%), suggesting that activation of other PI3K substrates may be also involved in the mitogenic effect of lead (H. Lu, unpublished).

In summary, the results of this study indicate that the ability of lead to induce DNA synthesis and cell proliferation in human 1321N1 astrocytoma cells is mediated by activation of the MEK1/2 and ERK1/2, signal transduction pathway, via a PKC-dependent manner. Activation of MEK and MAPK by lead had been previously found in PC12 cells (Ramesh et al., 1999) but was not studied in the context of cell proliferation. Our results suggest that by activating the ERK1/2 pathway, lead may act as a tumor promoter in transformed glial cells, although such findings may not extend to other cell types. In this context, however, our present results are of interest in light of the observed increased risk of lead exposure for brain tumors, most notably astrocytomas (Anttila et al., 1996).