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Lead-Stimulated p38MAPK-Dependent Hsp27 Phosphorylation
Toxicology and Applied Pharmacology 178, 44 –51 (2002) doi:10.1006/taap.2001.9320, available online at http://www.idealibrary.com on
Lead-Stimulated p38 MAPK-Dependent Hsp27 Phosphorylation Rodrigo B. Leal,* ,1 Fabiano M. Cordova,* Lynn Herd,† Larisa Bobrovskaya,† and Peter R. Dunkley† *Departamento de Bioquı´mica, Centro de Cieˆncias Biolo´gicas (CCB), Universidade Federal de Santa Catarina, Floriano´polis, Santa Catarina, Brazil 88040-900; and †The School of Biomedical Sciences, Faculty of Medicine and Health Sciences, The University of Newcastle, Callaghan, New South Wales, Australia 2308 Received July 30, 2001; accepted September 28, 2001
as an important target for Pb 2⫹, with both enzyme activation and inhibition occurring in a concentration-dependent manner (Markovac and Goldstein, 1988; Murakami et al., 1993; Bressler et al., 1999; Sun et al., 1999). In addition, modulation of Ca 2⫹/calmodulin-dependent protein kinase II (CaM-KII) activity through a mechanism dependent on calmodulin activation by Pb 2⫹ has been observed (Kern and Audesirk, 1995). In these examples Pb 2⫹ can directly substitute for Ca 2⫹ in the activation of Ca 2⫹-dependent events. Pb 2⫹ can also affect other signaling pathways and neurological processes. It can modulate neuronal plastic events such as long-term potentiation (Gilbert et al., 1999), long-term depression (Zhao et al., 1999), neurite outgrowth (Kern and Audesirk, 1995), and learning and memory (Johnston and Goldstein, 1998). Pb 2⫹ can also stimulate neurotransmitter release from adrenal chromaffin cells (Tomsig and Suszkiw, 1993), neurons (Cory-Slechta, 1995), and PC12 cells (Bressler et al., 1996). It can increase protein expression of IEGs in PC12 cells (Kim et al., 2000) as well as stress response proteins in astroglial cells (Opanashuk and Finkelstein, 1995). The mechanism(s) whereby Pb 2⫹ modulates these events is not established and may, or may not, involve direct substitution for Ca 2⫹. Mammals express at least four distinctly regulated groups of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases (ERK)-1/2, Jun NH 2-terminal kinases (JNKs), p38 MAPK, and ERK5 (Chang and Karin, 2001). It has been shown that a variety of metal ions can cause activation of some members of the MAPK family as well as provoke downstream consequences, such as activation of transcription factors and apoptosis (Samet et al., 1998; Hung et al., 1998; Gala´n et al., 2000). It is possible that some of the actions of Pb 2⫹ in the nervous system could be due to activation of these kinases. However, there have been no systematic studies on the effects
Lead-Stimulated p38 MAPK-Dependent Hsp27 Phosphorylation. Leal, R. B., Cordova, F. M., Herd, L., Bobrovskaya, L., and Dunkley, P. R. (2002). Toxicol. Appl. Pharmacol. 178, 44 –51. Lead (Pb 2ⴙ) is a cytotoxic metal ion whose mechanism of action is not established. However, Pb 2ⴙ is known to interact with a wide variety of molecules involved in signal transduction. In this study the effect of Pb 2ⴙ on protein phosphorylation in bovine adrenal chromaffin cells and human SH SY5Y cells was examined. Cells were incubated with 32P i for 1 h in the presence of Pb 2ⴙ (1–10 M) and the proteins were separated by two-dimensional PAGE. An increase in the phosphorylation of a number of proteins was observed in response to Pb 2ⴙ, including three spots, MW 25 kDa, and pI’s in the range 4.0 – 4.5. These proteins were immunoidentified as three isoforms of the heat-shock protein 27 kDa (Hsp27), and the identity of the most basic spot was confirmed by amino acid sequencing. Phosphorylation of p38 MAPK was increased by Pb 2ⴙ and the effect of Pb 2ⴙ on Hsp27 phosphorylation was blocked by the p38 MAPK inhibitor SB203580 (1 M). The results were similar for bovine chromaffin cells and human SH SY5Y cells. This is the first report showing that Pb 2ⴙ can modulate the phosphorylation state of Hsp27 via activation of the p38 MAPK pathway. © 2002 Elsevier Science Key Words: lead; p38 MAPK; Hsp27; bovine adrenal chromaffin cells; human SH SY5Y cells; protein phosphorylation.
Heavy metals are environmental contaminants that may disturb cellular signaling and cause irreversible damage to biological systems (DeMoor and Koropatnick, 2000). Lead (Pb 2⫹) is a neurotoxic agent that can cause permanent cognitive deficits in children (Cory-Slechta, 1995; Bressler et al., 1999) and it is recognized as a significant problem in many countries (Okada et al., 1997; Needleman, 2000). It has been well documented that Pb 2⫹ interacts with a variety of molecules involved in signal transduction, such as Ca 2⫹ channels, Ca 2⫹ binding proteins, and Ca 2⫹-dependent protein kinases (Bressler et al., 1999). Classically, protein kinase C (PKC) has been seen
Ca 2⫹/calmodulin-dependent protein kinase II; ERK, extracellular signal-regulated kinase; Hsp, heat-shock protein; IgG–HRP, horseradish peroxidase linked to immunoglobulin G; JNK, c-Jun amino-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAPK2, MAPK-activated protein kinase 2; NBT, nitroblue tetrazolium; PDB, phorbol 12,13-dibutyrate; 32P i, [ 32P]orthophosphate; PKA, protein kinase A; PKC, protein kinase C; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TH, tyrosine hydroxylase.
To whom correspondence should be addressed. Fax: ⫹55 48 3319672. E-mail: [email protected]. 2 Abbreviations used: BACC, bovine adrenal chromaffin cells; BCIP, 5bromo-4-chloro-3-indolyl phosphate; BSA, bovine serum albumin; CaM-KII, 1
0041-008X/02 $35.00 © 2002 Elsevier Science All rights reserved.
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LEAD STIMULATES Hsp27 PHOSPHORYLATION VIA P38 MAPK
of Pb 2⫹ on MAPKs or their downstream effectors. Stimulation of ERK and JNK phosphorylation by 1 M Pb 2⫹ and the activation of NF- and AP-1 were demonstrated in PC12 cells over a 1-h period (Ramesh et al., 1999). Additionally, exposure of PC12 cells to toxic levels of lead amplifies NGFinduced neurite outgrowth and ERK phosphorylation (Williams et al., 2000). To our knowledge no studies on the effects of Pb 2⫹ on p38 MAPK have been published. The overall aim of this study was to determine the effects of Pb 2⫹ on protein phosphorylation in bovine adrenal chromaffin cells (BACCs) and human neuroblastoma SH SY5Y. We were especially interested in the effects of Pb 2⫹ on the MAPKs and their downstream effectors. METHODS Chemicals. Lead acetate was obtained from BDH. SB203580, anisomycin, bovine serum albumin (BSA), Hepes, EGTA, and nicotine were purchased from Sigma. PD98059 was obtained from Biomol Research Laboratories. The protein phosphatase inhibitor okadaic acid and the kinase inhibitors Ro-31– 8220 (PKC), H-89 (PKA), and KN-93 (CaMKII) were obtained from Sapphire Bioscience. Anti-phospho-p38 MAPK antibody was purchased from Santa Cruz. Anti-phospho-Hsp27 antibody and secondary anti-sheep antibody HRP-conjugated were obtained from Upstate Biotechnology. Anti-Hsp27 antibody, anti-phospho-ERK1/2 antibody, forskolin, and phorbol 12,13-dibutyrate (PDB) were from Calbiochem. [ 32P]orthophosphate ( 32P i; carrier free; 10 mCi/ml) was from Du Pont NEM. Acrylamide/bisacrylamide and NBT/BCIP were from Bio-Rad. Hybond nitrocellulose (ECL), secondary antibodies (rabbit and mouse) HRP-conjugated, and ECL detection reagents were obtained from Amersham–Pharmacia. Sequence grade trypsin was from Promega. All other reagents were of analytical grade or tissue culture grade for cell culture. Cell cultures. BACCs were isolated from adrenal glands by pronase and collagenase digestion followed by Percoll density gradient centrifugation, as described by Bunn et al. (1995). Cells were then plated onto six-well culture plates in a supplemented DMEM medium (Bunn et al., 1995) at a density of 2 ⫻ 10 6 cells per well and maintained at 37°C. Cultured cells were used for experimentation between the 3rd and 12th days. SH-SY5Y cells are a catecholaminergic neuroblastoma cell line derived from humans (Encinas et al., 2000). SH-SY5Y cells were plated onto rat tail collagen-coated six-well plastic culture plates at a density 1 ⫻ 10 6 cells/well and maintained at 37°C. Cultured cells were used for experimentation 16 –24 h after plating. Cell treatment and 32P i labeling. BACC were removed from the incubator and placed on a warming plate held at 37°C. Cells were washed twice with 1 ml of Hepes-buffered saline containing 10 mM Hepes, 154 mM NaCl, 5.2 mM KCl, 1.2 mM MgCl 2, 1.2 mM CaCl 2, and 6 mM glucose, pH 7.4. Incubation was then performed for 1 h with a further 1 ml of the same medium containing 32 P i 50 Ci/ml. Lead acetate was dissolved in double-deionized water before use. The metal was added to the incubation medium at different times and in different concentrations (1–10 M). Nicotine (10 M; 5 min), forskolin (10 M; 10 min), PDB (400 nM; 15 min), protein kinase inhibitors such as SB203580 (1–5 M; 15-min preincubation plus all of the incubation period) and PD98059 (20 M; 15-min preincubation plus all of the incubation period), and the protein phosphatase (PP1/PP2A) inhibitor okadaic acid (1 M; 15-min preincubation plus all of the incubation period) were each used in order to characterize the phosphorylation system modulated by Pb 2⫹. SH-SY5Y cells were rinsed twice with 1 ml of Hepes-buffered saline and then treated with Pb 2⫹ (1–10 M, 1 h) or anisomycin (10 g/ml, 0.5 h) in the presence or absence of 1 M SB203580. One-dimensional gel electrophoresis. The reactions were stopped with 300 l per well of SDS-stopping solution (4% SDS, 2 mM EDTA, 8%
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-mercaptoethanol, and 50 mM Tris, pH 6.8). The cells were scraped from each well and solubilized. Samples were fractionated by SDS–PAGE using gradient gels 7.5–15% or 12% gels (Bunn et al., 1995). When the phosphoproteins were 32P i radiolabeled, 40 g protein per track was applied and they were detected by autoradiography (Dunkley et al., 1997). For phosphorylation measurement through Western blot, 60 g protein per track was applied for phospho-ERK1/2 immunodetection, and 120 g protein per track was applied for phospho-p38 MAPK and phospho-Hsp27 immunodetection. Two-dimensional electrophoresis and microsequencing. In order to analyze the proteins by two-dimensional electrophoresis (2D), the reactions were stopped with 10% TCA (4°C). Cells were scraped and centrifuged at 15,000g for 5 min in a refrigerated microfuge. The pellet was washed twice with 4% TCA and once with ethanol 80%. The final pellet was resuspended in 300 l of rehydration solution (7 M urea, 2 M thiourea, 4% Chaps, 2% ampholines, 40 mM Tris, 0.3% dithiothreitol, and bromophenol blue). The sample (3 mg protein) was applied to IPG strips by in-gel hydration. IPG strips (Pharmacia, pH 4 –7, 13 cm) were rehydrated for at least 6 h in the rehydration solution to their original thickness of 0.5 mm. The strips were then focused in the first dimension on a Pharmacia LKB Multiphor system. The gel strips were run at 300 V for 5 h and then were increased to 3500 V over 5 h, before continuing at 3500 V for 6 h. Before the second dimension was run strips were equilibrated in equilibration solution (6 M urea, 20% glycerol, 2% SDS, 375 mM Tris, pH 8.8, plus 1% DTT or 4% iodoacetamide). IPG strips were placed in the top of 1-mm-thick, 12% SDS–PAGE gels and held in place by 1% agarose. The gels were then run at 25 mA/gel until the blue dye front had reached the bottom of the gel (approximately 5 h). Proteins were electrotransferred to PVDF membrane using 10 mM Caps buffer with 10% methanol at 600 mA overnight and stained with Coomassie blue. The membranes were exposed to X-ray films and the phosphoproteins were identified on the autoradiograms. One of the phosphoproteins modulated by Pb 2⫹ [spot 3 in Fig. 4 (25 kDa, pI 4.5)], known as pp25, was then immunoblotted using a specific anti-Hsp27 (rabbit) antibody. For microsequencing the electrotransfer to PVDF was omitted. Instead the gel was stained with Coomassie blue and dried before the gel was exposed to X-ray film. The pp25 protein was identified and the stained spot was cut from the gel. The protein was then digested with 0.5% sequencing grade trypsin in 200 mM NH 4HCO 3 for 16 h and the resulting peptides were separated by microbore HPLC using a (10 – 60%) acetonitrile gradient. Two peptides were selected and applied to Biobrene-treated glass filters. Pulsed liquid phase N-terminal sequencing was then carried out on a PE-ABI Procise HT automated protein sequencer. Immunoblotting. BACC proteins separated by one-dimensional (1D) SDS–PAGE were transferred to nitrocellulose (Jarvie and Dunkley, 1995). The membranes were blocked with 2% BSA in TBS-T (0.1% Tween 20 v/v, 25 mM Tris, 150 mM NaCl, pH 7.5). In BACC samples phospho-p38 MAPK was detected with anti-phospho-p38 MAPK (1:1000; incubated overnight); phosphoERK1/2 was detected with anti-phospho-ERK1/2 (1:10,000; incubated 2 h); all antibodies were diluted in TBS-T (150 mM NaCl, 10 mM Tris, pH 7.6, 0.05% Tween-20) containing 2% BSA. After extensive washing using TBS-T, the membranes were incubated 1.5 h with secondary antibody. Either anti-rabbit IgG–HRP (horseradish peroxidase) (1:4000 in 1% BSA/TBS-T) or anti-mouse IgG–HRP (1:10,000) was used as a secondary antibody. The reactions were developed by ECL. Hsp27 was identified on PVDF, after separation of proteins by 2D electrophoresis, using the anti-Hsp27 (rabbit) antibody (1:2000 in 2% BSA/TBS-T). The reaction was determined by a colorimetric assay using anti-rabbit IgG conjugated to alkaline phosphatase and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrate. For SH-SY5Y cells the immunodetection of proteins was performed as described for BACCs, except that the phosphorylation of Hsp27 was measured by immunoblotting using an anti-phospho-Hsp27 antibody (1:500 dilution in TBST, 4°C, overnight) and anti-sheep IgG-HPR (1:2000 in TBS-T, 1.5 h) as a secondary antibody. The color reactions were developed by ECL as described above.
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LEAL ET AL.
Quantification and statistical analysis. Protein phosphorylation was measured by scanning the autoradiograms and the bands were quantified using the Analyst program from Bio-Rad. Data are presented as means ⫾ SD. Statistical significance was assessed by one-way ANOVA followed by Duncan’s test, when appropriate. A value of p ⬍ 0.05 was considered to be significant.
RESULTS
Incubation of BACC for 1 h with 32P i leads to phosphorylation of many proteins, including tyrosine hydroxylase (TH) (Fig. 1A, track C). Addition of 1 M Pb 2⫹ increased the phosphorylation of a number of these proteins. The most obvious effect was a significant increase in the 25-kDa band (pp25; Fig. 1A). This effect was dose dependent up to 10 M (Fig. 1B). We found that the effect of Pb 2⫹ on pp25 was also time dependent and could not be detected for incubations of less than 15 min but was maximal at 60 min using 10 M Pb 2⫹. However, not all proteins showed increased phosphorylation in response to Pb 2⫹ (Fig. 1A). Detailed analysis of the activity and the phosphorylation of individual sites on TH (Cheah et al., 1999) indicated that there was no significant effect of 10 M Pb 2⫹ on this protein (data not shown). The mechanism of Pb 2⫹ action on pp25 phosphorylation was investigated. In control experiments we confirmed that the effects were not due to the counter ion acetate. It was determined that the effect of Pb 2⫹ was not due to an increase in the uptake rate of 32P i, as preincubation of BACC with 32P i for various periods, prior to Pb 2⫹ addition, did not alter the effects of Pb 2⫹ on pp25 phosphorylation. The effect of Pb 2⫹ on pp25 was essentially the same in the presence or absence of extracellular Ca 2⫹. The kinase inhibitors KN-93 (CaM-KII), PD98059 (ERK1/2), Ro-31– 8220 (PKC), and H89 (PKA) were not able to block basal or Pb 2⫹-stimulated pp25 phosphorylation, although these inhibitors had previously been shown to be effective in BACC at the concentrations used here (Bobrovskaya et al., 1998, 2001; Cheah et al., 1999). Other studies indicated that Ca 2⫹ influx into BACC in response to nicotine or K ⫹ depolarization significantly increased pp25 phosphorylation (data not shown). Additionally, the PP1/PP2A inhibitor okadaic acid also caused a marked increase of pp25 phosphorylation under nonstimulated conditions (data not shown). The p38 MAPK inhibitor, SB203580, blocked both basal and Pb 2⫹-stimulated pp25 phosphorylation (Fig. 2) and this occurred over a concentration range between 1 and 5 M. The inactive inhibitor of p38 MAPK, SB202474, did not change basal or Pb 2⫹-stimulated pp25 phosphorylation (Fig. 2). In order to characterize the effect of Pb 2⫹ on MAPKs the phosphorylation states of p38 MAPK, ERK1/2, and JNK were evaluated. Pb 2⫹ significantly increased the phosphorylation of p38 MAPK from 1 to 10 M in a dose-dependent manner in BACCs (Fig. 3). On the other hand, Pb 2⫹ did not increase the phosphorylation of ERK1/2 or JNK even at 10 M (data not shown). The proteins present in the 25-kDa band were examined using a 2D gel separation procedure. Figure 4 shows autora-
FIG. 1. Pb 2⫹ increases the phosphorylation of BACC proteins including a 25-kDa band. BACCs were incubated with 32 P i for 1 h in the presence or absence of Pb 2⫹ . Proteins were solubilized and separated by 1D SDS– PAGE. (A) Representative autoradiograph showing the effect of Pb 2⫹ on the overall phosphorylation pattern of proteins. (B) Effects of Pb 2⫹ on pp25 phosphorylation. The quantification of the pp25 band was performed by densitometric analysis and the data are expressed as percentages of the control (considered 100%). The values are means of three to five experiments ⫾ SD. Statistical significance: *p ⬍ 0.05; **p ⬍ 0.001 compared to control. #p ⬍ 0.05 vs 1 M.
diograms from a series of 2D gels after transference of the proteins from BACCs to PVDF. Three radioactive spots were present in the 25-kDa range (Fig. 4; spots 1, 2, and 3). Each of these spots showed an increase in phosphorylation in response to Pb 2⫹. Both basal and Pb 2⫹-stimulated phosphorylation of all three spots were substantially blocked by SB203580 (1 M).
LEAD STIMULATES Hsp27 PHOSPHORYLATION VIA P38 MAPK
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substantially increased protein and phosphate incorporation, spot 1 was also identified as Hsp27 using the anti-Hsp27 antibody. The phenomenon observed in BACCs was then studied in human neuroblastoma cell line SH-SY5Y. Cells were treated with different concentrations of Pb 2⫹ in the presence or absence of SB203580 and the phosphorylation of p38 MAPK and Hsp27 was determined. A protein synthesis inhibitor, anisomycin (10 g/ml, 0.5 h), was used as a positive control as we have previously shown that anisomycin effectively increased both p38 MAPK and Hsp27 phosphorylation in BACCs (Bobrovskaya et al., 2001). It was found that SH-SY5Y cells Pb 2⫹ (1–10 M) produced a dose-dependent phosphorylation of both p38 MAPK (Fig. 6A) and Hsp27 (Fig. 6B). The phosphorylation of Hsp27 was completely inhibited by 1 M SB203580 in response to 5 M Pb 2⫹ and was partially inhibited in response to 10 M Pb 2⫹. Also, SB203580 significantly inhibited the anisomycin-stimulated Hsp27 phosphorylation. Thus, the phenomenon of p38 MAPK-dependent Hsp27 phosphorylation is not restricted to only bovine chromaffin cells but is also observed in human neuroblastoma cells. FIG. 2. The specific p38 MAPK inhibitor SB203580 blocked the Pb 2⫹-induced phosphorylation of pp25. In order to determine the involvement of p38 MAPK, BACCs were preincubated 15 min with 1 M SB203580, the active p38 MAPK inhibitor (SBa) or 1 M of its inactive analogue SB202474 (SBi) and they were maintained throughout the incubation in the continuing presence of Pb 2⫹ and 32P i. Control cells were 32P i labeled in the presence or absence of Pb 2⫹ (5 and 10 M) but without addition of inhibitor or analogue.
Pb 2⫹ also increased the phosphorylation of many other proteins, but their phosphorylation was not inhibited by SB203580. The spots shown in Fig. 4 (Pb 2⫹) are shown in Fig. 5A at higher resolution. Spot 3 corresponded to the major Coomassie blue-stained protein at the position of the pp25 band (Fig. 5B). Spot 2 was Coomassie stained but only lightly. Spot 1 was between other major Coomasssie blue-stained spots but was not itself stained. Spot 3 was cut out, digested with trypsin, and the peptides were fractionated on a microbore HPLC column. The identity of the pp25 protein was confirmed by N-terminal sequencing. Two peptides from a tryptic digestion of spot 3 were analyzed and the sequences were determined to be VSLDVNHFAPEE and SATQSAEITIPV. A BLASTP search of the SWISSPROT database determined that these sequences uniquely matched with the 27-kDa heat-shock protein (Hsp27). To confirm the identity of spot 3 as Hsp27, an antibody against Hsp27 was used (Fig. 5C). Both spots 2 and 3 were shown to be isoforms of Hsp27. In addition, spots 4 – 6 were also stained with the same antibody. Spot 6 corresponded to a major Coomassie blue-stained spot that was unphosphorylated. Spots 4 and 5 represent minor Hsp27 isoforms. Spot 1 was not stained with Coomassie blue or with anti-Hsp27 antibody in these experiments. However, in other experiments performed with
FIG. 3. Modulation of p38 MAPK phosphorylation by Pb 2⫹. BACC were incubated 1 h in presence of various concentrations of Pb 2⫹. Proteins were separated by 1D electrophoresis and transferred to a nitrocellulose membrane. Phosphorylation of p38 MAPK was detected by specific antibodies against the phosphorylated region of the kinase and reactions were developed by ECL. Phosphorylation was quantified by densitometric analysis. The data are expressed as percentages of the control (considered 100%). The values are means of three to five experiments ⫾ SD. Statistic significance: *p ⬍ 0.05; **p ⬍ 0.001 compared to control. Phospho-p38 MAPK (P-p38 MAPK) shows difference between treatments (p ⬍ 0.05).
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LEAL ET AL.
FIG. 4. Modulation of protein phosphorylation by Pb 2⫹ analyzed by 2D electrophoresis. BACC were labeled in the same conditions described in the legend to Fig. 2. However, the proteins were separated by 2D electrophoresis. Pb 2⫹ caused a consistent increase on the level of phosphorylation of three phosphoproteins (spots 1, 2, and 3) and the effect was blocked by the specific p38 MAPK inhibitor SB203580 (1 M).
DISCUSSION
The classical studies of Pb 2⫹ action on signal transduction pathways point to PKC and CaM kinase as the main targets (Markovac and Goldstein, 1988; Kern and Audesirk 1995; Belloni-Olivi et al., 1996; Bressler et al., 1999; Kim et al., 2000), and in these cases Pb 2⫹ can directly substitute for calcium. We found here that Pb 2⫹ increases the phosphorylation of a large number of proteins and many of these effects are likely to be via PKC and/or CaM kinase mechanisms. The most obvious effect of Pb 2⫹ was on the phosphorylation of pp25. However, the effect of Pb 2⫹ on this protein was unlike the effects seen on other proteins in this study, in that it was not mediated by PKC or CaMKII and it was blocked by the p38 MAPK inhibitor SB203580. There was also other specificity in the effect of Pb 2⫹ on protein phosphorylation, as the major BACC phosphoprotein tyrosine hydroxylase was apparently unaffected. We do not know which of the effects of Pb 2⫹ on protein phosphorylation constitute its primary toxic effect. The data presented in this paper indicate for the first time that Pb 2⫹ can increase the phosphorylation of p38 MAPK and Hsp27 in BACCs over 1 h of incubation at doses as low as 1 M. In addition we have established that essentially the same results occur in SH SY5Y cells, which is a catecholaminergic neuroblastoma cell line derived from humans. This suggests that the results obtained are likely to be applicable to human
FIG. 5. Analysis of Hsp27 isoforms after exposition of BACCs to Pb 2⫹. Cells were incubated for 1 h with Pb 2⫹. Proteins were separated by 2D PAGE and transferred to PVDF. The figure shows part of the membrane containing the 25-kDa region. (A) Autoradiogram of the spots 1–3 shown in Fig. 4 Pb 2⫹. (B) Coomassie blue-stained membrane showing the main proteins detected. Spots 2 and 3 indicate the major proteins stained by Coomassie blue. a, b, and c indicate proteins used for reference of spot 1, which ran below spot c. Spot b was determined to be the ubiquitin C terminal hydrolase by protein sequencing. (C) Immunodetection of Hsp27 performed with anti-Hsp27 antibody. The numbers 2, 3, 4, 5, and 6 indicate the spots that reacted with the antibody.
neurons. Despite the difficulty in comparing in vitro and in vivo models of metal exposure, it is important to highlight that our results show an effect of Pb 2⫹ at concentrations attained in tissues in some in vivo regimes. Goulart et al. (2001) showed that, in 21-day-old rats exposed to Pb 2⫹ acetate (8 mg/kg),
FIG. 6. Modulation of p38 MAPK and Hsp27 phosphorylation by Pb 2⫹ in SH-SY5Y cells. SH-SY5Y cells were treated with Pb 2⫹ (1, 5, or 10 M for 1 h) or anisomycin (Anis, 10 g/ml, 0.5 h) in the presence or absence of 1 M SB203580 (SB) as described under Methods. SH-SY5Y cell proteins were separated by SDS–PAGE. The phosphorylation of p38 MAPK (p-p38 MAPK) (A) and Hsp27 (p-Hsp27) (B) were measured by immunoblotting SH-SY5Y cell proteins with anti-phospho-p38 MAPK or anti-phospho-Hsp27 antibody, respectively.
LEAD STIMULATES Hsp27 PHOSPHORYLATION VIA P38 MAPK
during the second stage of rapid postnatal brain growth, the animals achieved Pb 2⫹ concentrations of approximately 0.5, 4.0, and 0.8 M for brain, kidney, and blood, respectively. Therefore, different cells clearly have the capacity to accumulate Pb 2⫹ to different extents (Sierra et al., 1989). The activation of p38 MAPK by Pb 2⫹ is supported, first, by the evidence that its phosphorylation was increased in response to the metal ion. Second, the effect of Pb 2⫹ on Hsp27 phosphorylation was blocked by the established inhibitor of p38 MAPK, SB203580, but not by the inactive analogue, SB202474. How then is p38 MAPK activated by Pb 2⫹ and what pathway is used to increase Hsp27 phosphorylation? p38 MAPK represents a family of four homologous kinases, p38␣, p38, p38␥, and p38␦, usually involved in stress responses (Meilke and Herdegen, 2000; Nebreda and Porras, 2000). SB203580 is an inhibitor particularly of p38␣ and p38 (Kumar et al., 1997) and these isoforms are therefore the most likely to be the ones activated by Pb 2⫹. p38 MAPKs are activated by dual phosphorylation on threonine 180 and tyrosine 182. This phosphorylation is mediated by a signaling cascade ending in MAP kinase kinase (MKK3/MKK6). It is possible that these, or other upstream enzymes, are directly activated by Pb 2⫹ or are activated indirectly by reactive oxygen species formed in response to Pb 2⫹ (Sandhir et al., 1994; Clerk et al., 1998). Alternatively, Pb 2⫹ might inhibit tyrosine phosphatase activity. A novel dual protein phosphatase (MKP-7) that is specific to p38␣, p38, and JNK was characterized. MKP-7 has a critical cysteine at the active site (Tanoue et al., 2001), which might be potentially a target for metals such as Pb 2⫹. Despite all of these possibilities, it is noteworthy that the effect of Pb 2⫹ was specific to the p38 MAPK pathway, as the metal did not increase the phosphorylation of either ERK1/2 or JNK. It is established that p38 MAPK activates MAPK-activated protein kinase-2 (MAPKAPK2) in BACCs (Thomas et al., 1997; Bobrovskaya et al., 2001) and Hsp27 is a well-established substrate of MAPKAPK2 (Stokoe et al., 1992; Mielke and Herdegen, 2000). It is therefore assumed that p38 MAPK activation by Pb 2⫹ results in activation of MAPKAPK2, which phosphorylates Hsp27. The inhibition of Hsp27 phosphorylation by SB203580 is consistent with this conclusion. The possible involvement of PKC, CaMKII, or PKA in Hsp27 phosphorylation is ruled out by the fact that inhibitors of these kinases had no effect on the phosphorylation of Hsp27. On the other hand, the PP2A/PP1 inhibitor okadaic acid caused a major increase in Hsp27 phosphorylation. It has been demonstrated that Hsp27 can be directly dephosphorylated by PP2A (Cairns et al., 1994). In addition, it has been shown that okadaic acid can also activate the signaling pathways involving p38 MAPK (Chen et al., 2000). Therefore, the PP inhibitor could modulate the Hsp27 phosphorylating system in BACCs by both mechanisms, activating p38 MAPK and inhibiting Hsp27 dephosphorylation. It is not known whether Pb 2⫹ can inhibit PP2A activity directly. The further question about these data is: What are the
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pathophysiological consequences of Hsp27 phosphorylation? Heat-shock proteins are a family of stress-inducible proteins that show a functional heat-shock element in their promoter. Within the cell they can form large oligomers, which serve as chaperones that bind other proteins and regulate their conformation and activity (Sharp et al., 1999). Mammalian Hsp27 is rapidly phosphorylated by MAPKAP kinase 2/3 at two or three residues (Ser15, Ser78, and Ser82) in response to various extracellular stresses (Landry et al., 1992). When BACCs were exposed to Pb 2⫹, three acidic spots showed increased phosphorylation on the 2D separations (Fig. 4, spots 1–3) and these spots corresponded to phosphorylated isoforms of Hsp27 (Fig. 5). Pb 2⫹ could induce phosphorylation on additional sites in Hsp27, which decreased the pI of the protein and caused a mobility shift toward the acidic electrode. Similar findings were described with Hsp27 in macrophages exposed to Cd 2⫹ (Radloff et al., 1998). Unphosphorylated Hsp27 is an oligomeric protein that works as chaperone and confers resistance against oxidative stress (Rogalla et al., 1999). After phosphorylation Hsp27 decreases its oligomeric size, resulting in a loss of both its chaperone activity and its ability to protect against oxidative stress (Rogalla et al., 1999). On the other hand, Hsp27 is a component of a signal transduction pathway that can regulate microfilament dynamics (Lavoie et al., 1993; Benndorf et al., 1994; Landry and Huot, 1999). It is supposed that Hsp27 phosphorylation confers stabilization of actin filaments, which might result in cell protection (Lavoie et al., 1993; Benndorf et al., 1994). Therefore, the activation of the p38 MAPK pathway in response to Pb 2⫹, followed by phosphorylation of Hsp27, may lead to a decrease in its chaperone action and/or a change in its capacity to maintain cell shape. The present work is the first report showing that Pb 2⫹ can stimulate the p38 MAPK pathway and modulate Hsp27 phosphorylation, perhaps contributing to the toxicity of Pb 2⫹. ACKNOWLEDGMENTS This work was performed primarily at the University of Newcastle while Dr. R. B. Leal was a visiting scientist funded by CNPq, Brazil. The NH&MRC of Australia, the research management of the University of Newcastle, and the Hunter Medical Research Institute are thanked for supporting this research.
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DeMoor, J. M., and Koropatnick, D. J. (2000). Metals and cellular signaling in mammalian cells. Cell. Mol. Biol. 46, 367–381.
Nebreda, A. R., and Porras, A. (2000). p38 MAP kinases: Beyond the stress response. Trends Biochem. Sci. 25, 257–260.
Dunkley, P. R., Jarvie, P. E., and Sim, A. T. R. (1997). Protein phosphorylation and dephosphorylation in the nervous system. In Neurochemistry: A Practical Approach (A. J. Turner and H. Bachelard, Eds.), pp. 201–228. Oxford Univ. Press, New York.
Needleman, H. L. (2000). The removal of lead from gasoline: Historical and personal reflections. Environ. Res. 84, 20 –35.
Encinas, M., Iglesias, M., Liu, Y., Wang, H., Muhaisen, A., Cena, V., Gallego, C., and Comella, J. (2000) Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent human neuron-like cells. J. Neurochem. 75, 991–1003. Gala´n, A., Garcı´a-Bermejo, M. L, Troyano, A., Vilaboa, N. E., Blas, E., Kazanietz, M. G., and Aller, P. (2000). Stimulation of p38 mitogen-activated protein kinase is an early regulatory event for cadmium-induced apoptosis in human promonocytic cells. J. Biol. Chem. 275, 11418 –11424.
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Ramesh, G. T., Manna, S. K., Aggarwal, B. B., and Jadhav, A. L. (1999). Lead activates nuclear transcription factor-, activator protein-1, and aminoterminal c-Jun kinase in pheochromocytoma cells. Toxicol. Appl. Pharmacol. 155, 280 –286.
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Rogalla, T., Ehrnsperger, M., Preville, X., Kotlyarov, A., Lutsch, G., Ducasse, C., Paul, C., Wieske, M., Arrigo, A-P., Buchner, J., and Gaestel, M. (1999). Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor ␣ by phosphorylation. J. Biol. Chem. 274, 18947–18956.
LEAD STIMULATES Hsp27 PHOSPHORYLATION VIA P38 MAPK Samet, J. M., Graves, L. M., Quay, J., Dailey, L. A., Devlin, R. B., Ghio, A. J., Wu, W., Bromberg, P. A., and Reed, W. (1998). Activation of MAPKs in human bronchial epithelial cells exposed to metals. Am. J. Physiol. 275, L551–L558. Sandhir, R., Julka, D., and Gill, K. D. (1994). Lipoperoxidative damage on lead exposure in rat brain and its implications on membrane bound enzymes. Pharmacol. Toxicol. 74, 66 –71. Sharp, F. R., Massa, S. M., and Swanson, R. A. (1999). Heat-shock protein protection. TINS 22, 97–99. Sierra, E. M., Rowles, T. K., Martin, J., Bratton, G. R., Womac, C., and Tiffany-Castiglioni, E. (1989). Low level lead neurotoxicity in a pregnant guinea pig model: Neuroglial enzyme activities and brain trace metal concentrations. Toxicology 59, 81–96. Stokoe, D., Engel, K., Campbell, D. G., Cohen, P., and Gaestel, M. (1992). Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett. 313, 307–313. Sun, X., Tian, X., Tomsig, J. L., and Suszkiw, J. B. (1999). Analysis of
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differential effects of lead on protein kinase C isozymes. Toxicol. Appl. Pharmacol. 156, 40 – 45. Tanoue, T., Yamamoto, T., Maeda, R., and Nishida, E. (2001). A novel MAPK phosphatase MKP-7 acts preferentially on JNK/SAPK and p38␣ and  MAPKs. J. Biol. Chem. 276, 26629 –26639. Thomas, G., Haavik, J., and Cohen, P. (1997). Participation of stress activated protein kinase cascade in the activation of tyrosine hydroxylase in chromaffin cells. Eur. J. Biochem. 217, 715–722. Tomsig, J. L., and Suszkiw, J. B. (1993). Intracellular mechanism of leadinduced norepinephrine release from bovine chromaffin cells. Am. J. Physiol. 265, C1630 –C1636. Williams, T. M., Ndifor, A. M., Near, J. T., and Reams-Brown, R. R. (2000). Lead enhances NGF-induced neurite outgrowth in PC12 cells by potentiating ERK/MAPK activation. Neurotoxicology 21, 1081–1089. Zhao, W-F., Ruan, D-Y., Xu, Y-Z., Chen, J-T., Wang, M., and Ge, S-Y. (1999). The effects of chronic lead exposure on long-term depression in area CA1 and dentate gyrus of rat hippocampus in vitro. Brain Res. 818, 153–159.
Lead-Stimulated p38 MAPK-Dependent Hsp27 Phosphorylation Rodrigo B. Leal,* ,1 Fabiano M. Cordova,* Lynn Herd,† Larisa Bobrovskaya,† and Peter R. Dunkley† *Departamento de Bioquı´mica, Centro de Cieˆncias Biolo´gicas (CCB), Universidade Federal de Santa Catarina, Floriano´polis, Santa Catarina, Brazil 88040-900; and †The School of Biomedical Sciences, Faculty of Medicine and Health Sciences, The University of Newcastle, Callaghan, New South Wales, Australia 2308 Received July 30, 2001; accepted September 28, 2001
as an important target for Pb 2⫹, with both enzyme activation and inhibition occurring in a concentration-dependent manner (Markovac and Goldstein, 1988; Murakami et al., 1993; Bressler et al., 1999; Sun et al., 1999). In addition, modulation of Ca 2⫹/calmodulin-dependent protein kinase II (CaM-KII) activity through a mechanism dependent on calmodulin activation by Pb 2⫹ has been observed (Kern and Audesirk, 1995). In these examples Pb 2⫹ can directly substitute for Ca 2⫹ in the activation of Ca 2⫹-dependent events. Pb 2⫹ can also affect other signaling pathways and neurological processes. It can modulate neuronal plastic events such as long-term potentiation (Gilbert et al., 1999), long-term depression (Zhao et al., 1999), neurite outgrowth (Kern and Audesirk, 1995), and learning and memory (Johnston and Goldstein, 1998). Pb 2⫹ can also stimulate neurotransmitter release from adrenal chromaffin cells (Tomsig and Suszkiw, 1993), neurons (Cory-Slechta, 1995), and PC12 cells (Bressler et al., 1996). It can increase protein expression of IEGs in PC12 cells (Kim et al., 2000) as well as stress response proteins in astroglial cells (Opanashuk and Finkelstein, 1995). The mechanism(s) whereby Pb 2⫹ modulates these events is not established and may, or may not, involve direct substitution for Ca 2⫹. Mammals express at least four distinctly regulated groups of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinases (ERK)-1/2, Jun NH 2-terminal kinases (JNKs), p38 MAPK, and ERK5 (Chang and Karin, 2001). It has been shown that a variety of metal ions can cause activation of some members of the MAPK family as well as provoke downstream consequences, such as activation of transcription factors and apoptosis (Samet et al., 1998; Hung et al., 1998; Gala´n et al., 2000). It is possible that some of the actions of Pb 2⫹ in the nervous system could be due to activation of these kinases. However, there have been no systematic studies on the effects
Lead-Stimulated p38 MAPK-Dependent Hsp27 Phosphorylation. Leal, R. B., Cordova, F. M., Herd, L., Bobrovskaya, L., and Dunkley, P. R. (2002). Toxicol. Appl. Pharmacol. 178, 44 –51. Lead (Pb 2ⴙ) is a cytotoxic metal ion whose mechanism of action is not established. However, Pb 2ⴙ is known to interact with a wide variety of molecules involved in signal transduction. In this study the effect of Pb 2ⴙ on protein phosphorylation in bovine adrenal chromaffin cells and human SH SY5Y cells was examined. Cells were incubated with 32P i for 1 h in the presence of Pb 2ⴙ (1–10 M) and the proteins were separated by two-dimensional PAGE. An increase in the phosphorylation of a number of proteins was observed in response to Pb 2ⴙ, including three spots, MW 25 kDa, and pI’s in the range 4.0 – 4.5. These proteins were immunoidentified as three isoforms of the heat-shock protein 27 kDa (Hsp27), and the identity of the most basic spot was confirmed by amino acid sequencing. Phosphorylation of p38 MAPK was increased by Pb 2ⴙ and the effect of Pb 2ⴙ on Hsp27 phosphorylation was blocked by the p38 MAPK inhibitor SB203580 (1 M). The results were similar for bovine chromaffin cells and human SH SY5Y cells. This is the first report showing that Pb 2ⴙ can modulate the phosphorylation state of Hsp27 via activation of the p38 MAPK pathway. © 2002 Elsevier Science Key Words: lead; p38 MAPK; Hsp27; bovine adrenal chromaffin cells; human SH SY5Y cells; protein phosphorylation.
Heavy metals are environmental contaminants that may disturb cellular signaling and cause irreversible damage to biological systems (DeMoor and Koropatnick, 2000). Lead (Pb 2⫹) is a neurotoxic agent that can cause permanent cognitive deficits in children (Cory-Slechta, 1995; Bressler et al., 1999) and it is recognized as a significant problem in many countries (Okada et al., 1997; Needleman, 2000). It has been well documented that Pb 2⫹ interacts with a variety of molecules involved in signal transduction, such as Ca 2⫹ channels, Ca 2⫹ binding proteins, and Ca 2⫹-dependent protein kinases (Bressler et al., 1999). Classically, protein kinase C (PKC) has been seen
Ca 2⫹/calmodulin-dependent protein kinase II; ERK, extracellular signal-regulated kinase; Hsp, heat-shock protein; IgG–HRP, horseradish peroxidase linked to immunoglobulin G; JNK, c-Jun amino-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKAPK2, MAPK-activated protein kinase 2; NBT, nitroblue tetrazolium; PDB, phorbol 12,13-dibutyrate; 32P i, [ 32P]orthophosphate; PKA, protein kinase A; PKC, protein kinase C; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TH, tyrosine hydroxylase.
To whom correspondence should be addressed. Fax: ⫹55 48 3319672. E-mail: [email protected]. 2 Abbreviations used: BACC, bovine adrenal chromaffin cells; BCIP, 5bromo-4-chloro-3-indolyl phosphate; BSA, bovine serum albumin; CaM-KII, 1
0041-008X/02 $35.00 © 2002 Elsevier Science All rights reserved.
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of Pb 2⫹ on MAPKs or their downstream effectors. Stimulation of ERK and JNK phosphorylation by 1 M Pb 2⫹ and the activation of NF- and AP-1 were demonstrated in PC12 cells over a 1-h period (Ramesh et al., 1999). Additionally, exposure of PC12 cells to toxic levels of lead amplifies NGFinduced neurite outgrowth and ERK phosphorylation (Williams et al., 2000). To our knowledge no studies on the effects of Pb 2⫹ on p38 MAPK have been published. The overall aim of this study was to determine the effects of Pb 2⫹ on protein phosphorylation in bovine adrenal chromaffin cells (BACCs) and human neuroblastoma SH SY5Y. We were especially interested in the effects of Pb 2⫹ on the MAPKs and their downstream effectors. METHODS Chemicals. Lead acetate was obtained from BDH. SB203580, anisomycin, bovine serum albumin (BSA), Hepes, EGTA, and nicotine were purchased from Sigma. PD98059 was obtained from Biomol Research Laboratories. The protein phosphatase inhibitor okadaic acid and the kinase inhibitors Ro-31– 8220 (PKC), H-89 (PKA), and KN-93 (CaMKII) were obtained from Sapphire Bioscience. Anti-phospho-p38 MAPK antibody was purchased from Santa Cruz. Anti-phospho-Hsp27 antibody and secondary anti-sheep antibody HRP-conjugated were obtained from Upstate Biotechnology. Anti-Hsp27 antibody, anti-phospho-ERK1/2 antibody, forskolin, and phorbol 12,13-dibutyrate (PDB) were from Calbiochem. [ 32P]orthophosphate ( 32P i; carrier free; 10 mCi/ml) was from Du Pont NEM. Acrylamide/bisacrylamide and NBT/BCIP were from Bio-Rad. Hybond nitrocellulose (ECL), secondary antibodies (rabbit and mouse) HRP-conjugated, and ECL detection reagents were obtained from Amersham–Pharmacia. Sequence grade trypsin was from Promega. All other reagents were of analytical grade or tissue culture grade for cell culture. Cell cultures. BACCs were isolated from adrenal glands by pronase and collagenase digestion followed by Percoll density gradient centrifugation, as described by Bunn et al. (1995). Cells were then plated onto six-well culture plates in a supplemented DMEM medium (Bunn et al., 1995) at a density of 2 ⫻ 10 6 cells per well and maintained at 37°C. Cultured cells were used for experimentation between the 3rd and 12th days. SH-SY5Y cells are a catecholaminergic neuroblastoma cell line derived from humans (Encinas et al., 2000). SH-SY5Y cells were plated onto rat tail collagen-coated six-well plastic culture plates at a density 1 ⫻ 10 6 cells/well and maintained at 37°C. Cultured cells were used for experimentation 16 –24 h after plating. Cell treatment and 32P i labeling. BACC were removed from the incubator and placed on a warming plate held at 37°C. Cells were washed twice with 1 ml of Hepes-buffered saline containing 10 mM Hepes, 154 mM NaCl, 5.2 mM KCl, 1.2 mM MgCl 2, 1.2 mM CaCl 2, and 6 mM glucose, pH 7.4. Incubation was then performed for 1 h with a further 1 ml of the same medium containing 32 P i 50 Ci/ml. Lead acetate was dissolved in double-deionized water before use. The metal was added to the incubation medium at different times and in different concentrations (1–10 M). Nicotine (10 M; 5 min), forskolin (10 M; 10 min), PDB (400 nM; 15 min), protein kinase inhibitors such as SB203580 (1–5 M; 15-min preincubation plus all of the incubation period) and PD98059 (20 M; 15-min preincubation plus all of the incubation period), and the protein phosphatase (PP1/PP2A) inhibitor okadaic acid (1 M; 15-min preincubation plus all of the incubation period) were each used in order to characterize the phosphorylation system modulated by Pb 2⫹. SH-SY5Y cells were rinsed twice with 1 ml of Hepes-buffered saline and then treated with Pb 2⫹ (1–10 M, 1 h) or anisomycin (10 g/ml, 0.5 h) in the presence or absence of 1 M SB203580. One-dimensional gel electrophoresis. The reactions were stopped with 300 l per well of SDS-stopping solution (4% SDS, 2 mM EDTA, 8%
45
-mercaptoethanol, and 50 mM Tris, pH 6.8). The cells were scraped from each well and solubilized. Samples were fractionated by SDS–PAGE using gradient gels 7.5–15% or 12% gels (Bunn et al., 1995). When the phosphoproteins were 32P i radiolabeled, 40 g protein per track was applied and they were detected by autoradiography (Dunkley et al., 1997). For phosphorylation measurement through Western blot, 60 g protein per track was applied for phospho-ERK1/2 immunodetection, and 120 g protein per track was applied for phospho-p38 MAPK and phospho-Hsp27 immunodetection. Two-dimensional electrophoresis and microsequencing. In order to analyze the proteins by two-dimensional electrophoresis (2D), the reactions were stopped with 10% TCA (4°C). Cells were scraped and centrifuged at 15,000g for 5 min in a refrigerated microfuge. The pellet was washed twice with 4% TCA and once with ethanol 80%. The final pellet was resuspended in 300 l of rehydration solution (7 M urea, 2 M thiourea, 4% Chaps, 2% ampholines, 40 mM Tris, 0.3% dithiothreitol, and bromophenol blue). The sample (3 mg protein) was applied to IPG strips by in-gel hydration. IPG strips (Pharmacia, pH 4 –7, 13 cm) were rehydrated for at least 6 h in the rehydration solution to their original thickness of 0.5 mm. The strips were then focused in the first dimension on a Pharmacia LKB Multiphor system. The gel strips were run at 300 V for 5 h and then were increased to 3500 V over 5 h, before continuing at 3500 V for 6 h. Before the second dimension was run strips were equilibrated in equilibration solution (6 M urea, 20% glycerol, 2% SDS, 375 mM Tris, pH 8.8, plus 1% DTT or 4% iodoacetamide). IPG strips were placed in the top of 1-mm-thick, 12% SDS–PAGE gels and held in place by 1% agarose. The gels were then run at 25 mA/gel until the blue dye front had reached the bottom of the gel (approximately 5 h). Proteins were electrotransferred to PVDF membrane using 10 mM Caps buffer with 10% methanol at 600 mA overnight and stained with Coomassie blue. The membranes were exposed to X-ray films and the phosphoproteins were identified on the autoradiograms. One of the phosphoproteins modulated by Pb 2⫹ [spot 3 in Fig. 4 (25 kDa, pI 4.5)], known as pp25, was then immunoblotted using a specific anti-Hsp27 (rabbit) antibody. For microsequencing the electrotransfer to PVDF was omitted. Instead the gel was stained with Coomassie blue and dried before the gel was exposed to X-ray film. The pp25 protein was identified and the stained spot was cut from the gel. The protein was then digested with 0.5% sequencing grade trypsin in 200 mM NH 4HCO 3 for 16 h and the resulting peptides were separated by microbore HPLC using a (10 – 60%) acetonitrile gradient. Two peptides were selected and applied to Biobrene-treated glass filters. Pulsed liquid phase N-terminal sequencing was then carried out on a PE-ABI Procise HT automated protein sequencer. Immunoblotting. BACC proteins separated by one-dimensional (1D) SDS–PAGE were transferred to nitrocellulose (Jarvie and Dunkley, 1995). The membranes were blocked with 2% BSA in TBS-T (0.1% Tween 20 v/v, 25 mM Tris, 150 mM NaCl, pH 7.5). In BACC samples phospho-p38 MAPK was detected with anti-phospho-p38 MAPK (1:1000; incubated overnight); phosphoERK1/2 was detected with anti-phospho-ERK1/2 (1:10,000; incubated 2 h); all antibodies were diluted in TBS-T (150 mM NaCl, 10 mM Tris, pH 7.6, 0.05% Tween-20) containing 2% BSA. After extensive washing using TBS-T, the membranes were incubated 1.5 h with secondary antibody. Either anti-rabbit IgG–HRP (horseradish peroxidase) (1:4000 in 1% BSA/TBS-T) or anti-mouse IgG–HRP (1:10,000) was used as a secondary antibody. The reactions were developed by ECL. Hsp27 was identified on PVDF, after separation of proteins by 2D electrophoresis, using the anti-Hsp27 (rabbit) antibody (1:2000 in 2% BSA/TBS-T). The reaction was determined by a colorimetric assay using anti-rabbit IgG conjugated to alkaline phosphatase and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrate. For SH-SY5Y cells the immunodetection of proteins was performed as described for BACCs, except that the phosphorylation of Hsp27 was measured by immunoblotting using an anti-phospho-Hsp27 antibody (1:500 dilution in TBST, 4°C, overnight) and anti-sheep IgG-HPR (1:2000 in TBS-T, 1.5 h) as a secondary antibody. The color reactions were developed by ECL as described above.
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Quantification and statistical analysis. Protein phosphorylation was measured by scanning the autoradiograms and the bands were quantified using the Analyst program from Bio-Rad. Data are presented as means ⫾ SD. Statistical significance was assessed by one-way ANOVA followed by Duncan’s test, when appropriate. A value of p ⬍ 0.05 was considered to be significant.
RESULTS
Incubation of BACC for 1 h with 32P i leads to phosphorylation of many proteins, including tyrosine hydroxylase (TH) (Fig. 1A, track C). Addition of 1 M Pb 2⫹ increased the phosphorylation of a number of these proteins. The most obvious effect was a significant increase in the 25-kDa band (pp25; Fig. 1A). This effect was dose dependent up to 10 M (Fig. 1B). We found that the effect of Pb 2⫹ on pp25 was also time dependent and could not be detected for incubations of less than 15 min but was maximal at 60 min using 10 M Pb 2⫹. However, not all proteins showed increased phosphorylation in response to Pb 2⫹ (Fig. 1A). Detailed analysis of the activity and the phosphorylation of individual sites on TH (Cheah et al., 1999) indicated that there was no significant effect of 10 M Pb 2⫹ on this protein (data not shown). The mechanism of Pb 2⫹ action on pp25 phosphorylation was investigated. In control experiments we confirmed that the effects were not due to the counter ion acetate. It was determined that the effect of Pb 2⫹ was not due to an increase in the uptake rate of 32P i, as preincubation of BACC with 32P i for various periods, prior to Pb 2⫹ addition, did not alter the effects of Pb 2⫹ on pp25 phosphorylation. The effect of Pb 2⫹ on pp25 was essentially the same in the presence or absence of extracellular Ca 2⫹. The kinase inhibitors KN-93 (CaM-KII), PD98059 (ERK1/2), Ro-31– 8220 (PKC), and H89 (PKA) were not able to block basal or Pb 2⫹-stimulated pp25 phosphorylation, although these inhibitors had previously been shown to be effective in BACC at the concentrations used here (Bobrovskaya et al., 1998, 2001; Cheah et al., 1999). Other studies indicated that Ca 2⫹ influx into BACC in response to nicotine or K ⫹ depolarization significantly increased pp25 phosphorylation (data not shown). Additionally, the PP1/PP2A inhibitor okadaic acid also caused a marked increase of pp25 phosphorylation under nonstimulated conditions (data not shown). The p38 MAPK inhibitor, SB203580, blocked both basal and Pb 2⫹-stimulated pp25 phosphorylation (Fig. 2) and this occurred over a concentration range between 1 and 5 M. The inactive inhibitor of p38 MAPK, SB202474, did not change basal or Pb 2⫹-stimulated pp25 phosphorylation (Fig. 2). In order to characterize the effect of Pb 2⫹ on MAPKs the phosphorylation states of p38 MAPK, ERK1/2, and JNK were evaluated. Pb 2⫹ significantly increased the phosphorylation of p38 MAPK from 1 to 10 M in a dose-dependent manner in BACCs (Fig. 3). On the other hand, Pb 2⫹ did not increase the phosphorylation of ERK1/2 or JNK even at 10 M (data not shown). The proteins present in the 25-kDa band were examined using a 2D gel separation procedure. Figure 4 shows autora-
FIG. 1. Pb 2⫹ increases the phosphorylation of BACC proteins including a 25-kDa band. BACCs were incubated with 32 P i for 1 h in the presence or absence of Pb 2⫹ . Proteins were solubilized and separated by 1D SDS– PAGE. (A) Representative autoradiograph showing the effect of Pb 2⫹ on the overall phosphorylation pattern of proteins. (B) Effects of Pb 2⫹ on pp25 phosphorylation. The quantification of the pp25 band was performed by densitometric analysis and the data are expressed as percentages of the control (considered 100%). The values are means of three to five experiments ⫾ SD. Statistical significance: *p ⬍ 0.05; **p ⬍ 0.001 compared to control. #p ⬍ 0.05 vs 1 M.
diograms from a series of 2D gels after transference of the proteins from BACCs to PVDF. Three radioactive spots were present in the 25-kDa range (Fig. 4; spots 1, 2, and 3). Each of these spots showed an increase in phosphorylation in response to Pb 2⫹. Both basal and Pb 2⫹-stimulated phosphorylation of all three spots were substantially blocked by SB203580 (1 M).
LEAD STIMULATES Hsp27 PHOSPHORYLATION VIA P38 MAPK
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substantially increased protein and phosphate incorporation, spot 1 was also identified as Hsp27 using the anti-Hsp27 antibody. The phenomenon observed in BACCs was then studied in human neuroblastoma cell line SH-SY5Y. Cells were treated with different concentrations of Pb 2⫹ in the presence or absence of SB203580 and the phosphorylation of p38 MAPK and Hsp27 was determined. A protein synthesis inhibitor, anisomycin (10 g/ml, 0.5 h), was used as a positive control as we have previously shown that anisomycin effectively increased both p38 MAPK and Hsp27 phosphorylation in BACCs (Bobrovskaya et al., 2001). It was found that SH-SY5Y cells Pb 2⫹ (1–10 M) produced a dose-dependent phosphorylation of both p38 MAPK (Fig. 6A) and Hsp27 (Fig. 6B). The phosphorylation of Hsp27 was completely inhibited by 1 M SB203580 in response to 5 M Pb 2⫹ and was partially inhibited in response to 10 M Pb 2⫹. Also, SB203580 significantly inhibited the anisomycin-stimulated Hsp27 phosphorylation. Thus, the phenomenon of p38 MAPK-dependent Hsp27 phosphorylation is not restricted to only bovine chromaffin cells but is also observed in human neuroblastoma cells. FIG. 2. The specific p38 MAPK inhibitor SB203580 blocked the Pb 2⫹-induced phosphorylation of pp25. In order to determine the involvement of p38 MAPK, BACCs were preincubated 15 min with 1 M SB203580, the active p38 MAPK inhibitor (SBa) or 1 M of its inactive analogue SB202474 (SBi) and they were maintained throughout the incubation in the continuing presence of Pb 2⫹ and 32P i. Control cells were 32P i labeled in the presence or absence of Pb 2⫹ (5 and 10 M) but without addition of inhibitor or analogue.
Pb 2⫹ also increased the phosphorylation of many other proteins, but their phosphorylation was not inhibited by SB203580. The spots shown in Fig. 4 (Pb 2⫹) are shown in Fig. 5A at higher resolution. Spot 3 corresponded to the major Coomassie blue-stained protein at the position of the pp25 band (Fig. 5B). Spot 2 was Coomassie stained but only lightly. Spot 1 was between other major Coomasssie blue-stained spots but was not itself stained. Spot 3 was cut out, digested with trypsin, and the peptides were fractionated on a microbore HPLC column. The identity of the pp25 protein was confirmed by N-terminal sequencing. Two peptides from a tryptic digestion of spot 3 were analyzed and the sequences were determined to be VSLDVNHFAPEE and SATQSAEITIPV. A BLASTP search of the SWISSPROT database determined that these sequences uniquely matched with the 27-kDa heat-shock protein (Hsp27). To confirm the identity of spot 3 as Hsp27, an antibody against Hsp27 was used (Fig. 5C). Both spots 2 and 3 were shown to be isoforms of Hsp27. In addition, spots 4 – 6 were also stained with the same antibody. Spot 6 corresponded to a major Coomassie blue-stained spot that was unphosphorylated. Spots 4 and 5 represent minor Hsp27 isoforms. Spot 1 was not stained with Coomassie blue or with anti-Hsp27 antibody in these experiments. However, in other experiments performed with
FIG. 3. Modulation of p38 MAPK phosphorylation by Pb 2⫹. BACC were incubated 1 h in presence of various concentrations of Pb 2⫹. Proteins were separated by 1D electrophoresis and transferred to a nitrocellulose membrane. Phosphorylation of p38 MAPK was detected by specific antibodies against the phosphorylated region of the kinase and reactions were developed by ECL. Phosphorylation was quantified by densitometric analysis. The data are expressed as percentages of the control (considered 100%). The values are means of three to five experiments ⫾ SD. Statistic significance: *p ⬍ 0.05; **p ⬍ 0.001 compared to control. Phospho-p38 MAPK (P-p38 MAPK) shows difference between treatments (p ⬍ 0.05).
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FIG. 4. Modulation of protein phosphorylation by Pb 2⫹ analyzed by 2D electrophoresis. BACC were labeled in the same conditions described in the legend to Fig. 2. However, the proteins were separated by 2D electrophoresis. Pb 2⫹ caused a consistent increase on the level of phosphorylation of three phosphoproteins (spots 1, 2, and 3) and the effect was blocked by the specific p38 MAPK inhibitor SB203580 (1 M).
DISCUSSION
The classical studies of Pb 2⫹ action on signal transduction pathways point to PKC and CaM kinase as the main targets (Markovac and Goldstein, 1988; Kern and Audesirk 1995; Belloni-Olivi et al., 1996; Bressler et al., 1999; Kim et al., 2000), and in these cases Pb 2⫹ can directly substitute for calcium. We found here that Pb 2⫹ increases the phosphorylation of a large number of proteins and many of these effects are likely to be via PKC and/or CaM kinase mechanisms. The most obvious effect of Pb 2⫹ was on the phosphorylation of pp25. However, the effect of Pb 2⫹ on this protein was unlike the effects seen on other proteins in this study, in that it was not mediated by PKC or CaMKII and it was blocked by the p38 MAPK inhibitor SB203580. There was also other specificity in the effect of Pb 2⫹ on protein phosphorylation, as the major BACC phosphoprotein tyrosine hydroxylase was apparently unaffected. We do not know which of the effects of Pb 2⫹ on protein phosphorylation constitute its primary toxic effect. The data presented in this paper indicate for the first time that Pb 2⫹ can increase the phosphorylation of p38 MAPK and Hsp27 in BACCs over 1 h of incubation at doses as low as 1 M. In addition we have established that essentially the same results occur in SH SY5Y cells, which is a catecholaminergic neuroblastoma cell line derived from humans. This suggests that the results obtained are likely to be applicable to human
FIG. 5. Analysis of Hsp27 isoforms after exposition of BACCs to Pb 2⫹. Cells were incubated for 1 h with Pb 2⫹. Proteins were separated by 2D PAGE and transferred to PVDF. The figure shows part of the membrane containing the 25-kDa region. (A) Autoradiogram of the spots 1–3 shown in Fig. 4 Pb 2⫹. (B) Coomassie blue-stained membrane showing the main proteins detected. Spots 2 and 3 indicate the major proteins stained by Coomassie blue. a, b, and c indicate proteins used for reference of spot 1, which ran below spot c. Spot b was determined to be the ubiquitin C terminal hydrolase by protein sequencing. (C) Immunodetection of Hsp27 performed with anti-Hsp27 antibody. The numbers 2, 3, 4, 5, and 6 indicate the spots that reacted with the antibody.
neurons. Despite the difficulty in comparing in vitro and in vivo models of metal exposure, it is important to highlight that our results show an effect of Pb 2⫹ at concentrations attained in tissues in some in vivo regimes. Goulart et al. (2001) showed that, in 21-day-old rats exposed to Pb 2⫹ acetate (8 mg/kg),
FIG. 6. Modulation of p38 MAPK and Hsp27 phosphorylation by Pb 2⫹ in SH-SY5Y cells. SH-SY5Y cells were treated with Pb 2⫹ (1, 5, or 10 M for 1 h) or anisomycin (Anis, 10 g/ml, 0.5 h) in the presence or absence of 1 M SB203580 (SB) as described under Methods. SH-SY5Y cell proteins were separated by SDS–PAGE. The phosphorylation of p38 MAPK (p-p38 MAPK) (A) and Hsp27 (p-Hsp27) (B) were measured by immunoblotting SH-SY5Y cell proteins with anti-phospho-p38 MAPK or anti-phospho-Hsp27 antibody, respectively.
LEAD STIMULATES Hsp27 PHOSPHORYLATION VIA P38 MAPK
during the second stage of rapid postnatal brain growth, the animals achieved Pb 2⫹ concentrations of approximately 0.5, 4.0, and 0.8 M for brain, kidney, and blood, respectively. Therefore, different cells clearly have the capacity to accumulate Pb 2⫹ to different extents (Sierra et al., 1989). The activation of p38 MAPK by Pb 2⫹ is supported, first, by the evidence that its phosphorylation was increased in response to the metal ion. Second, the effect of Pb 2⫹ on Hsp27 phosphorylation was blocked by the established inhibitor of p38 MAPK, SB203580, but not by the inactive analogue, SB202474. How then is p38 MAPK activated by Pb 2⫹ and what pathway is used to increase Hsp27 phosphorylation? p38 MAPK represents a family of four homologous kinases, p38␣, p38, p38␥, and p38␦, usually involved in stress responses (Meilke and Herdegen, 2000; Nebreda and Porras, 2000). SB203580 is an inhibitor particularly of p38␣ and p38 (Kumar et al., 1997) and these isoforms are therefore the most likely to be the ones activated by Pb 2⫹. p38 MAPKs are activated by dual phosphorylation on threonine 180 and tyrosine 182. This phosphorylation is mediated by a signaling cascade ending in MAP kinase kinase (MKK3/MKK6). It is possible that these, or other upstream enzymes, are directly activated by Pb 2⫹ or are activated indirectly by reactive oxygen species formed in response to Pb 2⫹ (Sandhir et al., 1994; Clerk et al., 1998). Alternatively, Pb 2⫹ might inhibit tyrosine phosphatase activity. A novel dual protein phosphatase (MKP-7) that is specific to p38␣, p38, and JNK was characterized. MKP-7 has a critical cysteine at the active site (Tanoue et al., 2001), which might be potentially a target for metals such as Pb 2⫹. Despite all of these possibilities, it is noteworthy that the effect of Pb 2⫹ was specific to the p38 MAPK pathway, as the metal did not increase the phosphorylation of either ERK1/2 or JNK. It is established that p38 MAPK activates MAPK-activated protein kinase-2 (MAPKAPK2) in BACCs (Thomas et al., 1997; Bobrovskaya et al., 2001) and Hsp27 is a well-established substrate of MAPKAPK2 (Stokoe et al., 1992; Mielke and Herdegen, 2000). It is therefore assumed that p38 MAPK activation by Pb 2⫹ results in activation of MAPKAPK2, which phosphorylates Hsp27. The inhibition of Hsp27 phosphorylation by SB203580 is consistent with this conclusion. The possible involvement of PKC, CaMKII, or PKA in Hsp27 phosphorylation is ruled out by the fact that inhibitors of these kinases had no effect on the phosphorylation of Hsp27. On the other hand, the PP2A/PP1 inhibitor okadaic acid caused a major increase in Hsp27 phosphorylation. It has been demonstrated that Hsp27 can be directly dephosphorylated by PP2A (Cairns et al., 1994). In addition, it has been shown that okadaic acid can also activate the signaling pathways involving p38 MAPK (Chen et al., 2000). Therefore, the PP inhibitor could modulate the Hsp27 phosphorylating system in BACCs by both mechanisms, activating p38 MAPK and inhibiting Hsp27 dephosphorylation. It is not known whether Pb 2⫹ can inhibit PP2A activity directly. The further question about these data is: What are the
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pathophysiological consequences of Hsp27 phosphorylation? Heat-shock proteins are a family of stress-inducible proteins that show a functional heat-shock element in their promoter. Within the cell they can form large oligomers, which serve as chaperones that bind other proteins and regulate their conformation and activity (Sharp et al., 1999). Mammalian Hsp27 is rapidly phosphorylated by MAPKAP kinase 2/3 at two or three residues (Ser15, Ser78, and Ser82) in response to various extracellular stresses (Landry et al., 1992). When BACCs were exposed to Pb 2⫹, three acidic spots showed increased phosphorylation on the 2D separations (Fig. 4, spots 1–3) and these spots corresponded to phosphorylated isoforms of Hsp27 (Fig. 5). Pb 2⫹ could induce phosphorylation on additional sites in Hsp27, which decreased the pI of the protein and caused a mobility shift toward the acidic electrode. Similar findings were described with Hsp27 in macrophages exposed to Cd 2⫹ (Radloff et al., 1998). Unphosphorylated Hsp27 is an oligomeric protein that works as chaperone and confers resistance against oxidative stress (Rogalla et al., 1999). After phosphorylation Hsp27 decreases its oligomeric size, resulting in a loss of both its chaperone activity and its ability to protect against oxidative stress (Rogalla et al., 1999). On the other hand, Hsp27 is a component of a signal transduction pathway that can regulate microfilament dynamics (Lavoie et al., 1993; Benndorf et al., 1994; Landry and Huot, 1999). It is supposed that Hsp27 phosphorylation confers stabilization of actin filaments, which might result in cell protection (Lavoie et al., 1993; Benndorf et al., 1994). Therefore, the activation of the p38 MAPK pathway in response to Pb 2⫹, followed by phosphorylation of Hsp27, may lead to a decrease in its chaperone action and/or a change in its capacity to maintain cell shape. The present work is the first report showing that Pb 2⫹ can stimulate the p38 MAPK pathway and modulate Hsp27 phosphorylation, perhaps contributing to the toxicity of Pb 2⫹. ACKNOWLEDGMENTS This work was performed primarily at the University of Newcastle while Dr. R. B. Leal was a visiting scientist funded by CNPq, Brazil. The NH&MRC of Australia, the research management of the University of Newcastle, and the Hunter Medical Research Institute are thanked for supporting this research.
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