Low Concentrations Of Paraquat Induces Early Activation Of Extracellular Signal-Regulated Kinase 1/2, Protein Kinase B, And C-Jun N-terminal Kinase 1/2 Pathways: Role Of C-Jun N-Terminal Kinase in Paraquat-Induced Cell Death

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

Mireia Niso-Santano, Jose´ M. Mora´n, Lourdes Garcı´a-Rubio, Ana Go´mez-Martı´n, Rosa A. Gonza´lez-Polo,Germa´n Soler,§ and Jose´ M. Fuentes

Abstract:

Paraquat is a herbicide with a potential risk to induce parkinsonism due to its demonstrated neurotoxicity and its strong structural similarity to 1-methyl-4-phenylpyridinium (MPP+), a well-known neurotoxin that causes a clinical syndrome similar to Parkinson’s disease (PD). However, at present very little is known about the signaling pathways activated by paraquat in any cell system. In this study, we have investigated the effect of paraquat on extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and protein kinase B (PKB)activation in E18 cells. Low concentrations of paraquat stimulated very early increases in ERK1/2, JNK1/2, and PKB phosphorylation. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) inhibited early paraquat-induced increases in PKB phosphorylation. Furthermore, early paraquat-mediated increases in ERK1/2 activation were sensitive to the mitogen-activated protein kinase kinase 1 (MEK1) inhibitor PD 98059(2#-amino-3#-methoxyflflavone), whereas JNK1/2 responses were blocked by the JNK1/2 inhibitor SP 600125 (anthra[1-9-cd]pyrazol-6(2H)-one). Pretreatment with wortmannin, LY 294002, or PD 98059 did not affect paraquat cell death in E18 cells. In contrast, SP 600125 significantly decreased paraquat-induced cell death in E18 cells. In conclusion, we have shown that low concentrations of paraquat stimulate robust very early increases in ERK1/2, JNK1/2, and PKB phosphorylation in E18 cells. Furthermore, the data presented suggest that inhibition of the JNK1/2 pathway protects E18 cells from paraquat-induced cell death and supports the fact that inhibition of early activation of JNK1/2 can constitute a potential strategy in PD treatment. Key Words: paraquat; low concentrations; JNK; cell death; Parkinson.

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INTRODUCTION

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the death of dopaminergic neurons in the sub-Constantia nigra in conjunction with intracytoplasmic inclusions known as Lewy bodies (Olanow and Tatton, 1999). Only 5–10% of PD patients have a familial form of this disease with an autosomal dominant mode of inheritance (Gasser, 2001). Genetic factors are important in young-onset patients, but they aren't likely to play a major role in the more common sporadic PD. By contrast, epidemiological studies indicate several environmental factors that increase the risk of developing PD(Parkinson’s disease). These included pesticides, herbicides, industrial chemicals, farming, and living in a rural environment (Cory-Slechta et al.,2005; Gasser, 2001; Landrigan et al., 2005; Tanner and BenShlomo, 1999). The most convincing argument in support of an environmental factor in PD relates to the discovery of the biologic effects of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrarchy-dropyridine). This central nervous system toxin produces a syndrome that is both clinically and anatomically similar toPD (Kopin and Markey, 1988; Langston et al., 1983). In this sense, the widely used herbicide paraquat (1,1#-dimethyl-4,4#-bypiridinium) has been suggested as a putative risk factor based on both its structural homology to MPPþ, the active metabolite of MPTP (Tanner and Langston, 1990), and on reports of parkinsonism correlated with exposure to the agent(Hertzman et al., 1990; Hubble et al., 1993; Jimenez-Jimenezet al., 1992; Liou et al., 1997; Wang et al., 1992). However, while the signaling pathways involved in MPPþ cell death are well known (Halvorsen et al., 2002; Gomez-Santos et al., 2002; Gonzalez-Polo et al., 2003), very little is known about the signaling pathways activated by paraquat (Cheng et al., 2003; Chun et al., 2001; Peng et al., 2004) Anyway, there are no studies about early events observed after exposure to low concentrations of paraquat.

Several stimuli, including MPPþ and paraquat, can activate an array of intracellular signaling cascades that are closely associated with both cell death and cell survival pathways. For example, MPPþ (Gomez-Santos et al., 2002; Halvorsen et al.,2002) activates members of the mitogen-activated protein kinase (MAPK) family and protein kinase B (PKB). The TheMAPK family members are activated by MPPþ or paraquat include extracellular signal-regulated kinases 1 and 2 (ERK1/2),c-Jun N-terminal kinases (JNK1 and JNK2), and p38 MAPKs.It is generally accepted that ERK1/2 and PKB activation promotes cell survival by activating anti-apoptotic signaling pathways, whereas activation of JNK1/2 and p38 MAPK is associated with neuronal cell death (Harper and LoGrasso,2001; Shin et al., 2001; Xia et al., 1995). Activation of the phosphatidylinositol 3-kinase (PI-3K)/PKB pathway is involved in neuronal survival (Shin et al., 2001). Particularly, activation of the JNK1/2 pathway can play a critical role in MPPþand paraquat toxicity (Cassarino et al., 2000; Halvorsen et al.,2002; Peng et al., 2004) and consequently in the cellular processes that are affected in PD(Parkinson’s disease).

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MATERIALS AND METHODS

Cell line and culture.

Spontaneously immortalized rat brain neuroblasts, E18 cells, were used in this study. The E18 cell line has been obtained by spontaneous immortalization from cultures of 18-day-old fetal rat cerebral cortices and was kindly provided by Dr. A. Mun˜oz (Instituto de InvestigacionesBiome´dicas, CSIC, Madrid, Spain). E18 cells represent primitive neuroblasts that express NF 68 and the primitive neuronal marker nestin but lack the astrocyte marker, glial fibrillary acidic protein. After partial differentiation induction with dibutyryl-cAMP, the cells express additional neuronal markers such as NF 145, NF 220, and neuron-specific enolase (Mun˜oz, personal communication). Cells were grown in Hanks’ F-12 (Hyclone, Brevieres, France) and supplemented with 10% FCS (Hyclone), streptomycin (100 mg/ml), and penicillin (100 U/ml). Cells were seeded at 5 3 10
5 in a 75-cm2 tissue culture flask (TPP, Trasadingen, Switzerland) and incubated at 37 C under a 5% CO2/95% air atmosphere. Cultures were passaged once a week by trypsinization using a trypsin-EDTA solution (Hyclone).

Cell treatments.

Confluent cells (~ 80%) in 75-cm2 tissue culture flasks were trypsinized and seeded in tissue culture dishes at a concentration of 5 3104 cells/cm2. Twenty-four hours later, the medium was aspirated and replaced with fresh medium alone or medium containing the indicated concentrations of paraquat. In further experiments, different concentrations of kinase inhibitors(LY 294002, wortmannin, SP 600125, and PD(Parkinson’s disease) 98059, all from Tocris, Bristol, United Kingdom) were added 30 min before the paraquat exposure. As kinase inhibitors were dissolved in dimethyl sulfoxide, controls were made with the highest concentration used (0.2% vol/vol). Dimethyl sulfoxide addition did not affect the viability values of control plates.

Cell viability assay.

Cell viability was determined by the colorimetricMTT assay (Mosmann, 1983). In viable cells, the mitochondrial enzyme succinate dehydrogenase can metabolize MTT into a formazan dye that absorbs light at 570 nm. For these experiments, 24-well test plates were used. At the end of, each treatment, 100 ll of MTT prepared at a concentration of 5 mg/ml in PBS was added to each plate. Following 3 h of incubation, the medium was decanted, and the formazan precipitates were solubilized with acidic isopropanol (0.04–0.1 N HCl in absolute isopropanol). The absorbance of converted dye was measured at a wavelength of 570 nm with background subtraction at 630–690 nm. The absorbance of the untreated cultures was set at 100%.

To confirm the results obtained by the MTT assay, cell death was also assessed by measuring the release of the cytosolic enzyme lactate dehydrogenase (LDH)to the culture medium using a colorimetric LDH assay kit (Roche, Indianapolis, IN) according to the manufacturer’s instructions. LDH leakage was defined as the ratio of LDH activity in the culture medium to the total activity per well(3100). Comparable data were obtained using both methods.

Preparation of cell extracts and Western blot analysis.

Following experimental treatments, the cells (cultured in 60-mm dishes) were rinsed twice with cold PBS and removed by scrapping and then centrifuged at 900 3 g for 5 min at 4 C. Cells were lysed in a buffer containing 50mM HEPES, pH 7.5, 300mMNaCl, 1% Triton X-100, 2mM EDTA, 5mM MgCl2, 25mM NaF, 1mMNa3VO4, and Protease Inhibitor Cocktail (Sigma, St Louis, MO). Cells were centrifuged at 13,000 3 g for 5 min at 4 C. Supernatants were stored at
80 C until analysis by Western blot. Protein concentration was measured according to Bradford (1976) using BSA as standard.

Equal amounts of proteins (10 LG per condition) were resolved in 12% SDSgel electrophoresis and transferred to PVDF membranes according to conventional partially modified methods (Fuentes et al., 2000). Briefly, proteins were transferred (250 mA for 60 min) to PVDF membranes using Mini Trans-Blot Cell apparatus (Bio-Rad, Hercules, CA). The procedure for immunodetection includes the transfer and blocking of the membrane (60 min at room temperature) with TTBS (10mM Tris/HCl, pH 7.5, 150mM NaCl, and 0.2%Tween-20) containing 10% nonfat dried milk. Membranes were then incubated for 60 min at room temperature with rabbit polyclonal primary antibodies (all from Cell Signalling, Beverly, MA, diluted 1:1000) in TTBS þ 10% non-fat dried milk or 5% BSA. After washing (for two 5-min periods with TTBS), membranes were incubated (60 min at room temperature) with peroxidase-conjugated anti-rabbit secondary antibodies (1:5000 in TTBS with 10% nonfat dried milk). After washing (for two 5-min periods and one 10-min period), the detection of bound antibodies was visualized by chemiluminescence using the ECL-plus reagent (Amersham Biosciences, Orsay, France) and analyzed by Quantity One software (Bio-Rad). Actin content was analyzed as a control using a rabbit polyclonal antibody (from Sigma) diluted 1:2500 in TTBSwith 10% nonfat dried milk).

Statistical analysis.

Each experiment was repeated at least three times with a satisfactory correlation between the results of individual experiments. The data shown are those of a representative experiment; each group was an average of three to four culture dishes. All data are expressed as the mean ±SEM. Results were analyzed by ANOVA. The p values less than 0.05 were considered significant.

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RESULTS

Cytotoxic Effects of Paraquat in E18 Cells

As previously reported (Cappelletti et al., 1998; Chun et al.,2001; Gonzalez-Polo et al., 2004), several cell lines are sensitive to the toxic properties of paraquat. Exposition to paraquat caused a dose-dependent reduction in cell viability. (Fig. 1.) In this study, E18 cells treated with 25lM paraquat for24 h exhibited a 50% loss of cell viability. Cell viability was evaluated by measuring the transformation to MTT into a formazan dye that absorbs light at 570 nm. This assay is extensively used to monitor cell death in several cell lines, including E18 cells (Donaire et al., 2005; Garcia-Roman et al.,2001), and different insults, including paraquat (Chun et al.,2001; Gonzalez-Polo et al., 2004).

Paraquat-Induced Activation of ERK1/2 in E18 Cells

Increases in ERK1/2 activation in E18 cells treated with paraquat were monitored by Western blotting using a phosphospecific ERK1/2 (Thr202/Tyr204) antibody. The exposition of E18neuroblasts cells with 25lM paraquat produced a marked increase in the phosphorylation (Thr202/Tyr204) status of ERK1/2 (44/42 kDa) (Fig. 2A). Maximal increases in the phosphorylation occurred after 5 min, after which phosphorylation levels slowly decline to near basal levels. Furthermore, the increases in ERK1/2 phosphorylation induced by paraquat were not concentration-dependent (Fig. 2B). Pretreatment with the theMEK1 inhibitor, PD(Parkinson’s disease) 98059 (50lM; Dudley et al., 1995)completely inhibited paraquat-induced increases in ERK1/2phosphorylation (Fig. 3).

Paraquat-Induced Activation of PKB in E18 Cells

PKB activation in E18 cells was detected by Western blotting using a phospho-specific (Ser473) antibody. Stimulation of E18 cells with 25lM paraquat produced a marked increase in PKB phosphorylation (Fig. 4A). This increase occurs after 20 min, comparable to that of ERK1/2 phosphorylation after phosphorylation levels slowly declined to near basal levels (Fig. 4A). Furthermore, paraquat-mediated increases in PKB phosphorylation were concentration-dependent with a maximum at 25lM of paraquat (Fig. 4A). As PI-3K is required for PKB activation, the role of PI-3K in paraquat-induced PKB activation in E18 cells was, therefore, explored using the PI-3K inhibitors wortmannin and LY 294002.

Paraquat-mediated increases in PKB phosphorylation were completely blocked following pretreatment (30 min) of E18cells with 100nM wortmannin and 30lM LY 294002 (Fig. 5). These observations demonstrate that a PI-3K–dependent pathway mediates paraquat-induced increases in PKB phosphorylation in E18 cells.

Paraquat-Induced Activation of JNK1/2 in E18 Cells

Previous studies have shown that paraquat activates JNK1/2in neurons (Chun et al., 2001; Peng et al., 2004) and nonneuronal cells (Bennett et al., 2001; Cheng et al., 2003). However, concentrations used are largely higher than those employed in this work. Additionally, the times selected to visualize JNK1/2 activation are also longer. JNK1/2 activation in E18 cells was detected by Western blotting using a phosphospecific JNK (Thr183/Tyr185) antibody. Stimulation of E18 cells with 25lM paraquat produced a marked and early increase inJNK (46/54 kDa) phosphorylation (Fig. 6A). These increases were maximal after 5 min after which phosphorylation levels decreased toward basal levels (Fig. 6A). However, paraquatmediated increases in JNK1/2 phosphorylation were not concentration-dependent (Fig. 6B). Paraquat-mediated increases in JNK1/2 phosphorylation were sensitive to the theJNK1/2 inhibitor SP 600125 (10lM; Fig. 7; Bennett et al.,2001). Finally, paraquat (25lM) did not stimulate measurable increases in p38 MAPK phosphorylation in E18 cells in the same time-course experiments (data not shown). These experiments were performed using a phosphospecific p38 MAPK(Thr180/Tyr182) antibody.

Measurement of Cell Viability After Treatment of E18 Cells with Paraquat and Kinase Inhibitors

Having shown that paraquat produces in all cases an early activation ERK1/2, PKB, and JNK1/2 in E18 cells, we subsequently determined the role of the fast activation of these kinase pathways in cell death induced by low concentrations of paraquat using pharmacological specific inhibitors. As shown in Figure 1, exposure of E18 cells to 25lM paraquat-induced a significant reduction in cell viability (around 50%). To investigate the role of ERK1/2, PKB, and JNK1/2 in paraquat-induced cell death, E18 cells were preincubated for 30 min with the following kinase inhibitors: PD(Parkinson’s disease) 98059 (50lM; MEK1/2inhibitor), LY 294002 (30lM; PI-3K inhibitor), wortmannin(100nM; PI-3K inhibitor), and SP 600125 (10lM; JNK1/2 inhibitor). As shown in Figure 8 (and it is summarized in Table 1), wortmannin, LY 294002, and PD 98059 had no significant effect on the loss of cell viability induced by 25lM paraquat.

DISCUSSION

Recently, various studies have increased interest in the possibility that environmental neurotoxins such as pesticides may be related to the development of nongenetic PD(Parkinson’s disease) (CorySlechta et al., 2005; Landrigan et al., 2005; Norris et al., 2004; Ritz and Yu, 2000; Sherer et al., 2001). PQ is one of the possible herbicides that are involved in PD, because of a similar chemical structure with MPPþ and the strong correlation between the incidence of the disease and the amount of PQused (Lanska, 1997; Liou et al., 1997; Ritz and Yu, 2000). However, the mechanism of the neuronal toxicity occurring underexposure to low levels of paraquat has not been determined. In any case, previous works are not interested in the early process evidenced after the exposition to paraquat. The major finding of the present study is that we have shown that E18neuroblast cells exposed to low concentrations of paraquatrapidly increased the activated phosphorylation of the generally antiapoptotic signaling pathways PI-3K/PKB and MEKERK and also the generally proapoptotic JNK1/2 MAPK. In contrast, paraquat did not stimulate time- or concentration-dependent measurable increases in p38 MAPK phosphorylation. As indicated above, we used a low concentration of paraquat. The relevance of the fact whether paraquat can get into the brain or not because it is a charged molecule has been debated. However, recent findings (Shimizu et al., 2001)revealed that paraquat can use the neutral amino acid pump to get into the brain. The findings presented in this work indicate that only a low concentration of paraquat is needed in the cell to rapidly stimulate several signal pathways including NK, implicated in cell death processes. These early activations due to the low concentration of paraquat are proposed, for the first time, in the present work.

Little attention has been dedicated to studying the changes in phosphorylation levels of PKB or ERK1/2 induced by paraquat(Cheng et al., 2003; Peng et al., 2004). In any case, previous reports (Peng et al., 2004) use relatively high concentrations of paraquat (400lM), measuring the phosphorylation degree ofERK after 12–18 h of exposure. At these times, they did not observe changes in the p-ERK levels. In our work, we detected an early (5–10 min) and important activation of ERK1/2 (Fig.2A). This activation declines slowly to the basal levels. In our conditions, the levels of phosphorylated ERK1/2 (after 12–18h) are the same as in the control, indicating that this pathway is not active at these times (data not shown) as described by Peng et al. (2004). This activation is not concentration-dependent, indicating the result that the robust stimulation of ERK is obtained and maintained with very low concentrations of paraquat. Changes in phosphorylated PKB levels have still not been described after paraquat exposure. We show in Figure4 that the results are time and concentration-dependent, showing a maximal effect after a 20-min exposition with25lM paraquat. That is, early paraquat-mediated increases in PKB phosphorylation are comparable with those observed forERK1/2. These data reveal an early increase in activating the phosphorylation of both PKB and ERK survival pathways. There are no data about early activation of PKB or ERK in paraquat-induced cell death. However, previous studies (Halvorsen et al., 2002) describe a similar early increase in both pathways in neuroblastoma SH-SY5Y cells exposed to MPPþ.In this case, phosphorylation levels do not decay toward the basal level. These data are particularly interesting due to the previously related similar structure between paraquat andMPPþ, indicating a different beginning of the cellular machinery implicated in the effect of both molecules.

More attention has been dedicated to the role of JNK1/2pathway in mediating signals contributing to the paraquat-induced cell death. In this study, we have shown that low concentrations (25lM) of paraquat triggered significant increases in phosphorylated JNK1/2 (Fig. 6). As occurs in PKBor ERK1/2, this activation is time-dependent with a very early beginning (5 min) and with a slow return to the basal levels. Previous works (Cheng et al., 2003; Chun et al., 2001; Peng et al., 2004) show late (12–18 h) activations of JNK1/2pathway produced in any case by relatively high concentrations of paraquat (from 400lM [Peng et al., 2004] to 800lM [Chunet al., 2001]). In our conditions, the phosphorylation degree ofJNK1/2 at the indicated times is comparable to the control. Overall, these results indicate for the first time that cell exposure to a very low concentration of paraquat produces an early activation of PKB, ERK1/2, and JNK1/2 pathways.

Having established that paraquat triggers early PKB, ERK1/2, and JNK1/2 activation in E18 cells, we then investigated the role of these protein kinase pathways in paraquat-induced cell death using several pharmacological inhibitors. Cell viability was monitored by measuring the transformation to MTT into a formazan dye that absorbs light at 570 nm. MTT assay has been used to accurately measure neuronal injury following a variety of insults including MPPþ or paraquat (GonzalezPolo et al., 2003, 2004; Schmuck et al., 2002; Sheng et al.,2002; Storch et al., 2004), and it is the most used method to determine cell death. As shown in Figure 8, pretreatment with the selective JNK1/2 inhibitor SP 600125 significantly reduced paraquat-induced cell death. This concentration of SP 600125completely blocked paraquat-induced activation of JNK1/2(Fig. 7). These data suggest that inhibition of the early activation of the JNK1/2 pathway protects cells from paraquat-induced cell death. These data are in agreement with previous studies (Chun et al., 2001; Peng et al., 2004), which have shown that JNK1/2 is involved in neuronal cell death triggered by paraquat. However, these studies use between 15 and 30times more concentration of paraquat than that employed in this study. We also demonstrate that low concentrations of paraquat can produce not only activation but also an early activation of this pathway. The protection observed with SP600125 also indicates that the JNK1/2 pathway is involved in cell death induced by paraquat. Additionally, JNK1/2 inhibition has been designed as a potential strategy in treating PD(Parkinson’s disease) in several models both in vivo and in vitro (Wang et al., 2004).

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Previous studies have shown that both PKB and ERKpathways are involved in neuronal survival (Guyton et al.,1996; Shin et al., 2001). In this study, we have shown that low concentrations of paraquat produce and early stimulate both PKB and ERK pathways. These observations suggest that paraquat-induced PKB and ERK activation may be involved in protecting cells against paraquat-induced cell death. To investigate this possibility, we used the structurally unrelated PI-3K inhibitors wortmannin and LY 294002 and the ERK1/2inhibitor PD(Parkinson’s disease) 98059. Interestingly, both PKB and ERK inhibition did not alter cell viability in presence of paraquat, suggesting that, in our conditions, increased PKB and ERKactivation is not essential for survival in paraquat-induced cell death in E18 cells.

It is well known that the mechanism of paraquat neurotoxicity is, among other factors, mediated via oxidative stress(Gonzalez-Polo et al., 2004; Mollace et al., 2003). In this sense, recently a very fast cytosolic oxidative stress in early apoptotic events implicated in paraquat-induced cell death has been described (Gonzalez-Polo et al., 2004). As oxidative stress activates members of the MAPK family (such as ERK1/2and JNK1/2) and PKB (Kamata and Hirata, 1999), this early activation of ERK1/2, JNK1/2, and PKB can be attributed to the rapid oxidative stress-induced after paraquat exposition.

In summary, our results show for the first time that low concentrations of paraquat stimulate very early and robust increases in PKB, ERK1/2, and JNK1/2 phosphorylation inE18 cells. In addition, we have shown that inhibition of the early activation of the JNK1/2 pathway protects E18 cells from paraquat-induced cell death. This result points toward the existence of some early events (as rapid JNK1/2 activation) that play an essential role in the cell death induced by low concentrations of paraquat. This result also opens a new and exciting line in the study of paraquat toxicity and its possible implication in the development of PD(Parkinson’s disease).

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