Department of Environmental Toxicology, University of Occupational and Environmental Health, 11 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
Received May 18, 1999; accepted August 30, 1999
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ABSTRACT |
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Key Words: c-Jun N-terminal kinase; c-jun; c-Jun protein; mercury chloride; LLC-PK1 cells; nephrotoxicity.
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INTRODUCTION |
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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, 1996; Schaeffer and Weber, 1999
). In response to various environmental stresses, JNK is activated by dual phosphorylation on Thr183 and Tyr185 (Dérijard et al., 1994
; Kyriakis and Avruch, 1996
), and in turn phosphorylates Ser63 and Ser73 in the amino terminal activation domain of c-Jun protein (Hibi et al., 1993
). The resultant phosphorylation enhances the transcriptional activity of c-Jun and its heterodimer AP-1 (Karin, 1995
). Evidence indicates that JNK is involved in the induction of apoptosis in various types of cells (Chen et al., 1996
; Eilers et al., 1998
; Gajate et al., 1998
; Verheij et al., 1996
; Xia et al., 1995
). We therefore examined whether HgCl2 can activate JNK signaling pathways in LLC-PK1 cells.
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MATERIALS AND METHODS |
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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, 1990).
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, 1998). 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 (189) 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 (SDSPAGE) 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., 1997). The sequences of the forward and reverse c-jun primers were 5'-TATGAGGAACCGCATCGCTG-3' and 5'-TAGCATGAGTTGGCACCCACTG-3', respectively (Chung et al., 1996
). These amplified a 196-bp product corresponding to nucleotide numbers 9671162 on the porcine c-jun gene (Chung et al., 1996
). 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., 1997
).
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., 1998).
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.
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RESULTS |
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DISCUSSION |
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We have found previously that HgCl2 induced the expression of c-fos gene in LLC-PK1 cells (Matsuoka et al., 1997). 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., 1993
). 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., 1993
). Because JNK can phosphorylate and activate not only c-Jun but also ATF-2 (van Dam et al., 1995
) and Elk-1 (Cavigelli et al., 1995
) 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- and IL-Iß, and various cellular stresses such as UV radiation, ionizing radiation, heat shock, hydrogen peroxide, and chemical mutagens (Kyriakis and Avruch, 1996
). We have found previously that CdCl2 activated JNK in LLC-PK1 cells (Matsuoka and Igisu, 1998
). 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., 1998
; Ramesh et al., 1999
) and zinc sulfate activated JNK in human bronchial epithelial cells (Samet et al., 1998
). 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., 1997; Smith et al., 1987
), which is an important regulator of JNK activity (Mitchell et al., 1995
). Pretreatment with an intracellular Ca2+ chelator, BAPTA/AM, abolished the CdCl2-induced JNK phosphorylation completely in the present and previous studies (Matsuoka and Igisu, 1998
). 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, 1997), 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., 1996
). 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., 1997; Chen et al., 1996
; Eilers et al., 1998
; Gajate et al., 1998
; Guo et al., 1998
; Preston et al., 1996
; Xia et al., 1995
). 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, 1996
). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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