Mercury Chloride Activates c-Jun N-Terminal Kinase and Induces c-jun Expression in LLC-PK1 Cells

Masato Matsuoka1, Bambang Wispriyono, Yoshihisa Iryo and Hideki Igisu

Department of Environmental Toxicology, University of Occupational and Environmental Health, 1–1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan

Received May 18, 1999; accepted August 30, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In response to various environmental stresses including heavy metals, the c-Jun N-terminal kinase (JNK) is phosphorylated and then it phosphorylates c-Jun protein. In the present study, effects of mercury chloride (HgCl2) on JNK signalling pathway were examined in LLC-PK1 cells. When exposed to 10 or 20 µM HgCl2, the level of phosphorylated JNK and the activity of JNK assayed in vitro using glutathione-S-transferase-c-Jun as substrate increased markedly. The level of phosphorylated JNK increased 30 min after HgCl2 exposure and remained elevated even at 8 h. On the other hand, no changes were found in the total amount of JNK protein. Consistent with the activation of JNK, c-Jun proteins phosphorylated on Ser63 and Ser73 were accumulated in cells exposed to HgCl2. Concomitantly, the levels of c-jun mRNA and c-Jun protein were elevated. When compared to other heavy metal compounds such as manganese chloride, zinc chloride, cadmium chloride, and lead chloride, HgCl2 could phosphorylate JNK more markedly. Neither intracellular Ca2+ nor sulfhydryl groups appeared to play a major role in the activation of JNK by HgCl2 exposure in this porcine renal epithelial cell line.

Key Words: c-Jun N-terminal kinase; c-jun; c-Jun protein; mercury chloride; LLC-PK1 cells; nephrotoxicity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inorganic mercury has been used widely in industry and known to cause renal proximal tubular damage (Clarkson, 1997Go). Among the inorganic compounds, the soluble forms such as mercury chloride (HgCl2) are highly toxic (Clarkson, 1997Go). It has also been reported that the treatment with 35 µM HgCl2 for 7 h causes apoptosis in LLC-PK1 cells (Duncan-Achanzar et al., 1996Go), an established renal epithelial line with characteristics of the proximal tubule (Gstraunthaler, 1988Go). We have previously found that the treatment with 20 µM HgCl2 increased c-fos mRNA level and resulted in the accumulation of c-Fos protein, a component of the heterodimeric transcriptional factor AP-1, before the development of DNA fragmentation in LLC-PK1 cells (Matsuoka et al., 1997Go). However, effects of HgCl2 exposure on the signal transduction pathway leading to cell death or apoptosis in the proximal tubular cells have not been clarified.

The c-Jun N-terminal kinase (JNK), also known as stress-activated protein kinase (SAPK), belongs to the mitogen-activated protein kinase (MAPK) superfamily, which transmits extracellular signals into the nucleus (Kyriakis and Avruch, 1996Go; Schaeffer and Weber, 1999Go). In response to various environmental stresses, JNK is activated by dual phosphorylation on Thr183 and Tyr185 (Dérijard et al., 1994Go; Kyriakis and Avruch, 1996Go), and in turn phosphorylates Ser63 and Ser73 in the amino terminal activation domain of c-Jun protein (Hibi et al., 1993Go). The resultant phosphorylation enhances the transcriptional activity of c-Jun and its heterodimer AP-1 (Karin, 1995Go). Evidence indicates that JNK is involved in the induction of apoptosis in various types of cells (Chen et al., 1996Go; Eilers et al., 1998Go; Gajate et al., 1998Go; Verheij et al., 1996Go; Xia et al., 1995Go). We therefore examined whether HgCl2 can activate JNK signaling pathways in LLC-PK1 cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture.
LLC-PK1 cells, a porcine renal epithelial cell line, were obtained from Health Science Research Resources Bank (Osaka, Japan), and grown in medium 199 supplemented with 3% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (GIBCO BRL, Life Technologies, Inc., Rockville, MD) in a humidified atmosphere of 5% CO2, 95% air at 37°C. For each experiment, exponentially growing LLC-PK1 cells were plated at 5 x 105 cells/well in 6-well culture plates, cultured for 1 day, and maintained in serum-free medium for another day. Medium 199 contained 0.05 mg/l glutathione (reduced form) and 0.1 mg/l L-cysteine HCl • H2O.

Treatment with metals.
HgCl2 and lead chloride (PbCl2) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and manganese chloride (MnCl2), zinc chloride (ZnCl2), and cadmium chloride (CdCl2) were from Sigma Chemical Co. (St. Louis, MO). Stock solutions of each metal compound were dissolved in water and sterilized by filtration. LLC-PK1 cells were incubated with serum-free medium containing the appropriate concentration of HgCl2 or other metals for 1 h at 37°C. In the time course study, cells were incubated with 10 µM HgCl2 for 30 min to 8 h. Untreated control cells were incubated with serum-free medium without HgCl2 and treated identically to the cells exposed to HgCl2.

An intracellular Ca2+ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM, Calbiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide (DMSO). Cells were preincubated with serum-free medium containing either 10 µM BAPTA/AM or 0.03% DMSO for 30 min. After washing with phosphate-buffered saline (PBS), cells were incubated with 10 µM HgCl2 or 20 µM CdCl2 for 1 h at 37°C.

To prevent the possible interaction between Hg2+ and sulfhydryl groups within and on the membrane, cells were preincubated with serum-free medium containing 5, 10, or 20 µM of the nonpolar maleimide N-ethylmaleimide (NEM) and the polar maleimide N-hydroxymaleimide (NHM, Sigma Chemical Co.) for 1 h, respectively. After washing with PBS, cells were incubated with 10 µM HgCl2 for another hour. In LLC-PK1 cells, 20 µM NEM did not show a significant effect on the protein content of the cell layer (Templeton, 1990Go).

Western blots.
Levels of JNK phosphorylated on Thr183 and Tyr185, total (phosphorylation state-independent) JNK, c-Jun phosphorylated on Ser63, c-Jun phosphorylated on Ser73, and total (phosphorylation state-independent) c-Jun were determined by the Western blot analysis as described previously (Matsuoka and Igisu, 1998Go). The antibodies used were phospho-specific SAPK/JNK (Thr183/Tyr185) antibody, SAPK/JNK antibody, phospho-specific c-Jun (Ser63) II antibody, phospho-specific c-Jun (Ser73) antibody, and c-Jun antibody (New England Biolabs, Inc., Beverly, MA). Bands on the developed film (Hyperfilm, Amersham Pharmacia Biotech, Tokyo, Japan) were quantified with NIH Image Version 1.61.

JNK activity assay.
Activity of JNK was measured using SAPK/JNK assay kit (New England Biolabs) according to the instruction from the manufacturer. Briefly, cell lysates were incubated with glutathione-S-transferase (GST)-c-Jun (1–89) coupled to glutathione-sepharose beads, and the precipitated JNK was subjected to in vitro kinase assay using GST-c-Jun as substrate. Phosphorylation of GST-c-Jun on Ser63 was analyzed after sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting with phospho-specific c-Jun (Ser63) antibody.

RT-PCR analysis.
Isolation of total RNA and the reverse transcription-polymerase chain reaction (RT-PCR) analysis were carried out as described previously (Matsuoka et al., 1997Go). The sequences of the forward and reverse c-jun primers were 5'-TATGAGGAACCGCATCGCTG-3' and 5'-TAGCATGAGTTGGCACCCACTG-3', respectively (Chung et al., 1996Go). These amplified a 196-bp product corresponding to nucleotide numbers 967–1162 on the porcine c-jun gene (Chung et al., 1996Go). Primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Clontech (Palo Alto, CA) and their amplified product was used to normalize that of c-jun as reported previously (Matsuoka et al., 1997Go).

Determination of intracellular glutathione.
The concentration of total glutathione (reduced and disulfide forms) was determined according to the method of Tietze (1969), with slight modifications as described previously (Wispriyono et al., 1998Go).

Cell viability assay.
LLC-PK1 cells were plated at 5.0 x 104 cells/well in 96-well culture plates, cultured for 1 day, and maintained in serum-free medium for another day. At the end of the incubation with metals, cell viability was assayed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; Boehringer Mannheim, Tokyo, Japan) conversion according to instructions from the manufacturer.

Statistical analysis.
Results were expressed as mean ± SD. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Bonferroni multiple comparison test. When mRNA levels of two groups were compared, Welch's t-test was used. p < 0.05 was considered as statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular Glutathione Level
Treatment with HgCl2 for 1 h reduced the intracellular glutathione level in a dose-dependent manner (Fig. 1Go). At this time, no change of cell viability was observed when MTT-based cytotoxicity assay was used (data not shown).



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FIG. 1. Dose effects of HgCl2 on the level of intracellular glutathione. LLC-PK1 cells were incubated with 0, 1, 5, or 10 µM HgCl2 for 1 h, and intracellular glutathione level was determined. Each column and bar represents mean ± SD of five experiments. * p < 0.01, ** p < 0.001 compared to control (0 µM HgCl2).

 
Phosphorylated and Total JNK Proteins
Two isoforms, 54-kDa (p54) and 46-kDa (p46), which arise from alternative mRNA splicing at the extreme C terminus (Kyriakis and Avruch, 1996Go; Kyriakis et al., 1994Go), were detected with phospho-specific SAPK/JNK (Thr183/Tyr185) antibody or SAPK/JNK antibody (Figs. 2A and 3GoGo). In LLC-PK1 cells treated with 10 µM HgCl2 for 1 h, the level of JNK phosphorylated on Thr183 and Tyr185 increased (Figs. 2A and 2BGo). Exposure to 20 µM HgCl2 for 1 h also induced the accumulation of phosphorylated JNK with almost the same potency as 10 µM (data not shown). In contrast, the level of total (phosphorylation state-independent) JNK protein was not changed by HgCl2 exposure (Fig. 2AGo). In the time course study, the level of phosphorylated JNK increased after 30 min and remained elevated at 8 h (Fig. 3Go), whereas total JNK level was not changed during this period (Fig. 3Go).



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FIG. 2. Dose effects of HgCl2 on the accumulation of phosphorylated JNK. LLC-PK1 cells were incubated with 0, 1, 5, or 10 µM HgCl2 for 1 h, and cell lysates were subjected to Western immunoblotting. (A) Representative immunoblots obtained with phospho-specific JNK antibody (upper panel) or phosphorylation state-independent JNK antibody (lower panel). (B) Densitometric analysis of phosphorylated JNK. Each value was expressed as the ratio of phosphorylated JNK level to total JNK level, and the value of control (0 µM HgCl2) was set to 1. Each column and bar represents mean ± SD of three experiments. * p < 0.05 compared to control.

 


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FIG. 3. Time course of the HgCl2-induced accumulation of phosphorylated JNK. LLC-PK1 cells were incubated with 10 µM HgCl2 for 15 min to 8 h, and cell lysates were subjected to Western immunoblotting using phospho-specific JNK antibody (upper panel) or phosphorylation state-independent JNK antibody (lower panel). The untreated control is 0 min. Result shown is representative of two experiments.

 
JNK Activity
Consistent with the phosphorylation of JNK by HgCl2, the activity of JNK assayed in vitro using GST-c-Jun as substrate increased in a dose-dependent manner (Figs. 4A and 4BGo).



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FIG. 4. Dose effects of HgCl2 on JNK activity. LLC-PK1 cells were incubated with 0, 1, 5, or 10 µM HgCl2 for 1 h, and cell lysates were used for in vitro kinase reaction with GST-c-Jun (1–89) as substrate. Phosphorylation of GST-c-Jun (1–89) was analyzed after Western immunoblotting using phospho-specific c-Jun (Ser63) antibody. (A) A representative immunoblot. (B) Densitometric analysis of GST-c-Jun (1–89) phosphorylated on Ser63. Each value was expressed as the fold increase with respect to control (0 µM HgCl2). Each column and bar represents mean ± SD of three experiments. * p < 0.001 compared to control.

 
Phosphorylated and Total c-Jun Proteins
The phosphorylation status of c-Jun protein in cells exposed to HgCl2 was examined. Marked increases in the levels of c-Jun phosphorylated on Ser63 (Figs. 5A and 5BGo) and that on Ser73 (Figs. 5A and 5BGo) were found in cells treated with 10 µM HgCl2 for 1 h. In addition, the level of total (phosphorylation state-independent) c-Jun protein increased dose dependently (Figs. 5A and 5BGo). Treatment with 20 µM HgCl2 for 1 h also increased the levels of phosphorylated and total c-Jun proteins markedly (data not shown).



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FIG. 5. Dose effects of HgCl2 on the accumulation of phosphorylated and total c-Jun proteins. LLC-PK1 cells were incubated with 0, 1, 5, or 10 µM HgCl2 for 1 h, and cell lysates were subjected to Western immunoblotting. (A) Representative immunoblots obtained with phospho-specific c-Jun (Ser63) antibody (top panel), phospho-specific c-Jun (Ser73) antibody (middle panel), or phosphorylation state-independent c-Jun antibody (bottom panel). (B) Densitometric analysis of c-Jun phosphorylated on Ser63 (top panel), c-Jun phosphorylated on Ser73 (middle panel), and total c-Jun (bottom panel). Each value was expressed as the fold increase with respect to control (0 µM HgCl2). Each column and bar represents mean ± SD of three experiments. * p < 0.01 compared to control.

 
c-jun mRNA
We have found previously that the treatment with 20 µM HgCl2 for 1 h increased c-fos mRNA level in LLC-PK1 cells using the semiquantitative RT-PCR analysis (Matsuoka et al., 1997Go). In the present study, the level of c-jun mRNA was also determined. Treatment with 20 µM HgCl2 for 1 h increased the c-jun mRNA level markedly, whereas no significant change was found in the expression of GAPDH (Figs. 6A and 6BGo). Treatment with 5 µg/ml actinomycin-D, an inhibitor of transcription, abolished the HgCl2-induced c-jun expression (data not shown). These findings indicate that HgCl2 exposure induced the transcriptional activation of c-jun as well as c-fos gene in LLC-PK1 cells.



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FIG. 6. Induction of c-jun expression by HgCl2. (A) LLC-PK1 cells were incubated with medium (untreated control, lane b) or 20 µM HgCl2 (lane c) for 1 h, and then total RNA was isolated and subjected to RT-PCR analysis. Mixtures of RT-PCR product of c-jun (196-bp, 20 cycles) and GAPDH (983-bp, 22 cycles) on a 2% agarose gel stained with ethidium bromide. DNA marker ({phi}X174/Hae III digest) was run in lane a. Marker sizes were 1353, 1078, 872, 603, 310, 281, 271, 234, 194, and 118-bp from top to bottom. (B) Densitometric analysis of RT-PCR products. Each value of c-jun mRNA level was normalized with GAPDH mRNA level and the value of 0 µM HgCl2 (control) was set to 1. Each column and bar represents mean ± SD of five experiments. * p < 0.05 compared to control.

 
Comparison of Effects of Heavy Metals on JNK Phosphorylation
HgCl2 and CdCl2, both of which are nephrotoxic heavy metal compounds, increased the level of phosphorylated JNK in LLC-PK1 cells (p < 0.001, Figs. 7A and 7BGo). Phosphorylation was higher in cells treated with HgCl2 than with CdCl2 (p < 0.01). On the other hand, no significant increases were found in cells treated with the same dose (10 µM) of MnCl2, ZnCl2, or PbCl2 for 1 h (Figs. 7A and 7BGo). The change of cell viability as assayed with MTT conversion was not observed after 1 h exposure to any metal (data not shown).



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FIG. 7. Comparison of the effects of heavy metals on the accumulation of phosphorylated JNK. LLC-PK1 cells were incubated with 10 µM of each heavy metal compound for 1 h, and cell lysates were subjected to Western immunoblotting. (A) Representative immunoblots obtained with phospho-specific JNK antibody (upper panel) or phosphorylation state-independent JNK antibody (lower panel). Lane a, control (without metals); lane b, MnCl2; lane c, ZnCl2; lane d, CdCl2; lane e, HgCl2; lane f, PbCl2. (B) Densitometric analysis of phosphorylated JNK. Each value was expressed as the ratio of phosphorylated JNK level to total JNK level, and the value of control was set to 1. Each column and bar represents mean ± SD of six experiments. * p < 0.001 compared to control.

 
Effects of Pretreatment with BAPTA/AM on JNK Phosphorylation
The effects of intracellular Ca2+ chelator BAPTA/AM on the HgCl2- or CdCl2-induced JNK phosphorylation were examined. Treatment with BAPTA/AM alone did not affect the phosphorylation status of JNK (Fig. 8Go). In cells pretreated with 10 µM BAPTA/AM for 30 min, CdCl2-induced JNK phosphorylation was suppressed almost completely without alteration of total JNK level (Fig. 8Go). In contrast, pretreatment with BAPTA/AM did not affect the HgCl2-induced JNK phosphorylation clearly (Fig. 8Go).



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FIG. 8. Effects of pretreatment with BAPTA/AM on the HgCl2- or CdCl2-induced JNK phosphorylation. LLC-PK1 cells were preincubated with 0.03% DMSO or 10 µM BAPTA/AM for 30 min, and then washed with phosphate-buffered saline. After incubation with 10 µM HgCl2 or 20 µM CdCl2 for 1 h, cell lysates were subjected to Western immunoblotting using phospho-specific JNK antibody (upper panel) or phosphorylation state-independent JNK antibody (lower panel). Result shown is representative of four experiments.

 
Effects of Pretreatment with Maleimides on JNK Phosphorylation
To prevent the possible interaction between Hg2+ and sulfhydryl groups within the cells, the nonpolar maleimide NEM, which alkylates sulfhydryl groups, was used. Treatment with 20 µM NEM alone did not affect the phosphorylation status of JNK (Fig. 9Go).. Pretreatment with varying doses (5, 10, and 20 µM) of NEM for 1 h failed to suppress the HgCl2-induced JNK phosphorylation (Fig. 9Go). The polar maleimide NHM, which interacts only with proteins on the external leaflet of the membrane (Rosenspire et al., 1998Go), also did not affect the phosphorylation of JNK by HgCl2 (data not shown).



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FIG. 9. Effects of pretreatment with NEM on the HgCl2-induced JNK phosphorylation. LLC-PK1 cells were preincubated with NEM for 1 h, and then washed with phosphate-buffered saline. After incubation with or without 10 µM HgCl2 for another hour, cell lysates were subjected to Western immunoblotting using phospho-specific JNK antibody (upper panel) or phosphorylation state-independent JNK antibody (lower panel). Result shown is representative of three experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In response to the exposure of 10 or 20 µM HgCl2, the level of JNK phosphorylated on Thr183 and Tyr185 and the activity of JNK assayed in vitro using GST-c-Jun as substrate increased markedly in LLC-PK1 cells. In the time course study, the level of phosphorylated JNK increased 30 min after HgCl2 exposure and remained elevated even at 8 h. Consistent with these findings, c-Jun proteins phosphorylated on Ser63 and Ser73 were accumulated in cells exposed to HgCl2. On the other hand, no changes were found in the total amount of JNK protein. These results clearly show that HgCl2 activated JNK through the phosphorylation of JNK, but not the elevation of its protein level, in this renal epithelial cell line.

We have found previously that HgCl2 induced the expression of c-fos gene in LLC-PK1 cells (Matsuoka et al., 1997Go). In the present study, exposure to HgCl2 increased the levels of c-jun mRNA and c-Jun protein, another member of AP-1. Thus, the activation of JNK by HgCl2 was accompanied by the increased expression of immediate early genes including c-jun and c-fos in LLC-PK1 cells. The major regulators of c-jun promoters are prebound heterodimers of c-Jun and ATF-2 on two promoter elements (van Dam et al., 1993Go). On the other hand, the serum response element, one of c-fos promoters, is activated by Elk-1 through the stimulation of ternary complex formation (Marais et al., 1993Go). Because JNK can phosphorylate and activate not only c-Jun but also ATF-2 (van Dam et al., 1995Go) and Elk-1 (Cavigelli et al., 1995Go) on these promoters, HgCl2 might induce the expression of c-jun gene and in part c-fos through the activation of JNK in LLC-PK1 cells.

JNK has been known to be activated in response to inflammatory cytokines such as TNF-{alpha} and IL-Iß, and various cellular stresses such as UV radiation, ionizing radiation, heat shock, hydrogen peroxide, and chemical mutagens (Kyriakis and Avruch, 1996Go). We have found previously that CdCl2 activated JNK in LLC-PK1 cells (Matsuoka and Igisu, 1998Go). In the present study, HgCl2 exposure induced more marked phosphorylation of JNK than CdCl2. In contrast, other heavy metal compounds such as MnCl2, ZnCl2, and PbCl2 did not phosphorylate JNK in LLC-PK1 cells. It has been reported that MnCl2 and lead acetate activated JNK in rat pheochromocytoma PC12 cells (Hirata et al., 1998Go; Ramesh et al., 1999Go) and zinc sulfate activated JNK in human bronchial epithelial cells (Samet et al., 1998Go). These findings suggest that effects of heavy metals on the JNK pathway might be different among cell types, and the activation of JNK in LLC-PK1 cells by HgCl2 and CdCl2 might be related to their nephrotoxic potential.

It has been shown that both HgCl2 and CdCl2 exposure elevate the concentration of intracellular Ca2+ (Benters et al., 1997Go; Smith et al., 1987Go), which is an important regulator of JNK activity (Mitchell et al., 1995Go). Pretreatment with an intracellular Ca2+ chelator, BAPTA/AM, abolished the CdCl2-induced JNK phosphorylation completely in the present and previous studies (Matsuoka and Igisu, 1998Go). In contrast, pretreatment with BAPTA/AM did not suppress the HgCl2-induced JNK phosphorylation clearly. Therefore, unlike the mechanism of JNK phosphorylation by CdCl2, HgCl2 did not depend on the intracellular Ca2+ to activate JNK in LLC-PK1 cells.

Inorganic mercury has high affinity for sulfhydryl groups (Clarkson, 1997Go), and we found that the level of intracellular glutathione was reduced in response to HgCl2 exposure. We therefore examined whether the prevention of interaction between Hg2+ and sulfhydryl groups could suppress the phosphorylation of JNK. However, HgCl2-induced JNK phosphorylation was not suppressed by pretreatment with either the nonpolar maleimide NEM or the polar maleimide NHM. These results indicate that the sulfhydryl groups within or on the membrane did not play a major role in the HgCl2-induced JNK phosphorylation, at least in LLC-PK1 cells. On the other hand, exposure to HgCl2 has been reported to stimulate the generation of hydrogen peroxide in LLC-PK1 cells (Nath et al., 1996Go). Therefore, the involvement of reactive oxygen species in the HgCl2-induced JNK activation remains to be examined.

Exposure to HgCl2 increased the level of phosphorylated JNK and induced the expression of c-jun and c-fos genes at the same time in LLC-PK1 cells. The persistent activation of JNK and the subsequent accumulation of c-Jun and c-Fos proteins have been reported to be responsible for the induction of apoptosis in various types of cells (Bossy-Wetzel et al., 1997Go; Chen et al., 1996Go; Eilers et al., 1998Go; Gajate et al., 1998Go; Guo et al., 1998Go; Preston et al., 1996Go; Xia et al., 1995Go). In the renal tubular epithelial cells, apoptosis seems to function for the rapid clearance of dying cells and for the protection of surrounding tissues from ischemic and toxic injuries (Lieberthal and Levine, 1996Go). Therefore, JNK may be one of the important cellular signal transduction pathways in the proximal tubular cells, which are damaged by nephrotoxic heavy metals including HgCl2.


    ACKNOWLEDGMENTS
 
This work was supported in part by a Grant-in-Aid for Science Research from the Ministry of Education, Science, Sports, and Culture, and a Special Research Grant from the University of Occupational and Environmental Health, Japan.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +81 (93) 692-4790. E-mail: masatomm{at}med.uoeh-u.ac.jp. Back


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