Induction of ERK1/2 and Histone H3 Phosphorylation within the Outer Stripe of the Outer Medulla of the Eker Rat by 2,3,5-Tris-(Glutathion-S-yl)hydroquinone

Jing Dong*,{dagger}, Jeffrey I. Everitt{ddagger}, Serrine S. Lau* and Terrence J. Monks*,1

* Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona Health Sciences Center, Tucson, Arizona 85721; {dagger} Department of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712; and {ddagger} GlaxoSmithKline, Research Triangle Park, North Carolina 27709

Received February 28, 2004; accepted April 20, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,5-tris-(glutathion-S-yl)-hydroquinone (TGHQ), a metabolite of hydroquinone (HQ), generates reactive oxygen species (ROS) in cultured renal epithelial cells and binds to tissue macromolecules within the rat kidney. The potential mechanisms by which TGHQ induces nephrotoxicity and nephrocarcinogenesis have been examined in cell culture models, but less is known concerning the molecular mechanisms of TGHQ-induced nephrotoxicity in vivo. In LLC-PK1 cells, TGHQ induces phosphorylation of both mitogen-activated protein kinase and histone H3, which likely promotes inappropriate chromatin condensation and mitotic catastrophe. Using the Eker (Tsc-2 mutant) rat as a model, we show by immunohistochemistry that TGHQ (7.5 µmol/kg) selectively induces ERK1/2 phosphorylation within the outer stripe of the outer medulla (OSOM) of the kidney. ERK1/2 phosphorylation is time-dependant, occurring as early as 1 h following treatment, and reaching maximal levels by 4 h. Subsequently, ERK1/2 phosphorylation returns to baseline levels by 24 h post treatment. ERK1/2 phosphorylation was confirmed by western blot analysis of OSOM tissue. Increases in histone H3 phosphorylation occurred subsequent to ERK1/2 phosphorylation (8 h), and reached a peak by 24 h, coincident with histological evidence of tissue necrosis. In contrast to studies in cell culture, neither JNK/SAPK nor p38 MAPK phosphorylation were significantly altered after TGHQ administration in vivo, as evidenced by western blot and immunohistochemical analyses. These data indicate that activation of the ERK1/2 pathway precedes overt cytotoxicity and that the signaling pathways activated by TGHQ in vivo and in vitro differ.

Key Words: TGHQ; MAPK; histone H3; Eker rats; ROS.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species (ROS) are associated with a variety of human diseases and toxicity (Bolton et al., 2000Go). Renal proximal tubule epithelial cells are particularly sensitive to oxidative stress-induced damage. However, the molecular mechanisms by which ROS cause injury in renal epithelial cells remain unclear. 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ) is a potent nephrotoxic metabolite of hydroquinone (HQ). TGHQ causes oncotic/necrotic cell death of renal proximal tubule epithelial cells, and likely mediates HQ-induced nephrotoxicity and nephrocarcinogenicity (Lau et al., 1988Go, 2001Go; Peters et al., 1997Go). TGHQ maintains the ability to redox-cycle and generate ROS (Towndrow et al., 2000Go; Weber et al., 2001Go) and covalently binds to cellular macromolecules (Kleiner et al., 1998aGo,bGo). The one-electron-reduced form of TGHQ may react with O2, yielding superoxide anion radical, which ultimately gives rise to hydroxyl radical and hydrogen peroxide (Monks and Lau, 1998Go). Quinone thioethers induce rapid ROS-dependent DNA damage, growth arrest, and cell death in a well-established in vitro model of porcine renal proximal tubule epithelial cells (LLC-PK1) (Jeong et al., 1996Go; 1997aGo,bGo; Mertens et al., 1995Go).

The nephrotoxicity of polyphenolic glutathione (GSH) conjugates is dependent on the high activity of {gamma}-glutamyl transpeptidase ({gamma}-GT) within the brush border membrane of the proximal tubule epithelial cells (Lau et al., 1988Go; Monks and Lau, 1998Go). The activity of {gamma}-GT is required for the generation of the corresponding cysteine conjugates, which are subsequently transported into cells via the L-amino acid transport system. ROS generation and covalent binding by the cysteine conjugates and the subsequent activation or inactivation of signaling pathways likely contribute to TGHQ-induced nephrotoxicity. A major signaling pathway that responds to various external stresses, including oxidative stress, is the mitogen-activated protein kinase (MAPK) pathway (Martindale and Holbrook, 2002). The MAPK family is comprised of three major subfamilies: the extracellular signal-regulated protein kinase (ERK), the c-Jun N-terminal kinases/stress-activated protein kinase (JNK/SAPK), and the p38 MAPK (Cobb, 1999Go). Upon activation, following phosphorylation of the tyrosine and threonine residues, MAPKs subsequently activate a variety of substrates, the majority of which are transcription factors. One indirect downstream substrate of MAPKs is histone H3, which is phosphorylated subsequent to ERK activation, probably through the activation of either 90 kDa ribosomal S6 kinase 2 (RSK2) or mitogen and stress activated protein kinase 1 (MSK1) (Tikoo et al., 2001Go). TGHQ induces the activation of all three major subfamilies of MAPK (Ramachandiran et al., 2002Go) and histone H3 phosphorylation (Tikoo et al., 2001Go) in LLC-PK1 cells. The inappropriate phosphorylation of histone H3 leads to premature chromatin condensation (PCC) and oncotic cell death in LLC-PK1 cells (Tikoo et al., 2001Go). Inhibition of ERK phosphorylation by PD098059, a MAPK kinase (MEK1/2) inhibitor, decreases TGHQ-induced histone H3 phosphorylation (Tikoo et al., 2001Go) and cell death (Ramachandiran et al., 2002Go) in LLC-PK1 cells. Whether similar signaling cascades contribute to the acute nephrotoxicity of TGHQ in vivo is not known and is the focus of the current studies.

We are utilizing the Eker rat (Tsc-2EK/+) as our animal model with the intent of coupling TGHQ-induced acute nephrotoxicity to the subsequent development of renal tumors by identifying the early molecular changes that support renal tumor formation. Eker rats carry a germ-line insertion in the tuberous sclerosis tumor suppressor gene (Tsc-2), which predisposes the animals to renal tumors (Lau et al., 2001Go; Walker et al., 1992Go; Yoon et al., 2001Go). Overexpression of ERK1/2 is often found in human neoplasia and tumors. For example, constitutive ERK activation was found in 48% of renal carcinomas examined (Oka et al., 1995Go). Moreover, PD184352, an inhibitor of ERK activation, suppressed tumor growth in vivo (Duesbery et al., 1999Go; Sebolt-Leopold et al., 1999Go). TGHQ induces an intensive increase of phospho-ERK1/2 expression in LLC-PK1 cells. In addition, both p38 MAPK and JNK/SAPK, which play important roles in inflammation, tumorigenensis and apoptosis (Tian et al., 2000Go), are also activated by TGHQ rapidly and intensively in LLC-PK1 cells (Ramachandiran et al., 2002Go). In the present study, we investigated the effects of TGHQ on MAPK activation and histone H3 phosphorylation in Eker rats to identify early molecular changes that may contribute to HQ and TGHQ-mediated carcinogenicity in vivo, and to determine the extent to which the in vitro model is predictive of molecular responses in vivo.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Caution. TGHQ is nephrotoxic and nephrocarcinogenic in rats and therefore must be handled with protective clothing and in a well-ventilated hood.

Chemicals and reagents. TGHQ was freshly synthesized and purified in our laboratory according to established protocols (Lau et al., 1988Go). Antibodies for phospho-p42/44 MAPK, p42/44 MAPK, phospho-histone H3 (Ser10), phospho-p38 MAPK, p38 MAPK, phospho-JNK/SAPK, and JNK/SAPK were all purchased from Cell Signaling Technology, Inc. (Beverly, MA). Secondary antibodies and the ABC immuno-staining kit were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Hematoxylin was acquired from Vector Laboratories, Inc. (Burlingame, CA). All other chemicals were from Sigma (St. Louis, MO), or from Fisher Scientific (Houston, TX) and of the highest grades available.

Animal treatment and dose selection rationale. Frozen kidney tissues derived from male Eker rats (Tsc-2EK/+) were from previous in vivo studies (Habib et al., 2003Go). Briefly, male Eker rats (Tsc-2EK/+) were obtained from the University of Texas MD Anderson Cancer Center, Smithville, TX. Animals were treated with a single dose of TGHQ (7.5 µmol/kg in PBS, pH 7.4, iv). The dose of TGHQ (7.5 µmol/kg) used for the present study causes necrosis of renal proximal tubule epithelial cells evident both histologically and by examination of biochemical markers. We have utilized lower doses of TGHQ (2.5 µmol/kg) to study its ability to produce tumors in a chronic exposure model (Yoon et al. 2002Go). Due to the special breeding required to produce sufficient numbers of the mutant Eker rats, and the high costs of such breeding, we did not feel justified in using either lower nontoxic doses, or higher doses that cause severe renal injury and death (the dose-response to TGHQ is particularly steep). The selected dose therefore reflects a mildly toxic dose that permits the tissue to mount a coordinated survival/repair response. Control rats were administered phosphate-buffered saline (PBS) only. Animals were euthanized at 0, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h following TGHQ treatment. Kidneys were removed and dissected longitudinally. Half of the kidney was preserved in 10% formalin in PBS, 0.01 M, pH 7.4. The outer stripe of the outer medulla (OSOM) was excised from the remaining kidney sections and immediately snap frozen in liquid nitrogen.

Western-blot analysis. Frozen OSOM tissues were homogenized and lysed in RIPA buffer (1XPBS, 1% Nonidet P-40 [NP-40], 0.5% sodium deoxycholate, 0.1% SDS) containing phosphatase inhibitors (1 mM NaVO4, 10 mM ß-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM NaF) and protease inhibitors (one tablet of cocktail protease inhibitors, Roche Molecular Biochemicals, Indianapolis, IN for 10 ml lysis buffer, and 0.1 mg/ml PMSF). Aliquots (50 µg) of the lysates were separated by 10% SDS–PAGE and transferred to nitrocellulose membranes. The membranes were blocked in blocking solution containing 5% nonfat dry milk in TBS buffer with 0.1% Tween-20 for 1 h, and then incubated with primary antibodies (phospho-p42/44 MAPK [polyclonal, 1:1000], p42/44 MAPK [polyclonal, 1:1000], phospho-p38 [monoclonal, 1:2000] and phospho-JNK/SAPK [polyclonal, 1:1000]) overnight at 4°C in blocking solution or 5% BSA in TBS containing 0.1% Tween-20. The membranes were then washed and incubated with secondary antibodies (goat anti-rabbit IgG-HRP, or goat anti-mouse IgG-HRP, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h at room temperature in blocking solution. Membranes were finally developed with the ECL western-blotting detection reagents, and exposed to Hyperfilm (Amersham Pharmacia Biotech, UK).

Immunohistochemistry. Paraffin sections (4 µm) were incubated at 65°C overnight, and then deparaffinized and rehydrated in xylene, 100% ethanol, and 95% ethanol, separately. The sections were then incubated in 0.5% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. The slides were then heated at 100°C for 10 min in 10 mM sodium citrate buffer (pH 6.0) to unmask antigens. Subsequently, the slides were incubated with primary antibodies (phospho-p42/44 MAPK [polyclonal, 1:50], phospho-p38 MAPK [monoclonal, 1:50], phospho-JNK/SAPK [polyclonal, 1:50], and phospho-histone H3 [Ser10] [monoclonal, 1:50]) at 4°C overnight. The slides were then incubated with secondary antibodies (goat anti-rabbit IgG, biotin-conjugated; or goat anti-mouse, biotin-conjugated) at room temperature for 30 min. An ABC staining kit was used to probe the proteins of interest. Briefly, the slides were incubated with AB enzyme reagents provided by the ABC staining kit, for 30 min at room temperature, washed in PBS 3 times at 5 min each, and then incubated with peroxide substrate solution (DAB, substrate, in substrate buffer) for 5 to 10 min. Finally, the slides were washed in double distilled water, counterstained with hematoxylin, and observed by light microscopy. Images were taken with a Kodak DC120 digital camera and processed with Adobe Photoshop 6.0 software.

Statistical analysis. Data are shown as mean ± STDEV. Mean values were compared by analysis of variance with a post hoc Student's Newman Kuels test; p values less than 0.05 were considered to be statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nephrotoxicity of TGHQ
TGHQ (7.5 µmol/kg, iv)-induced nephrotoxicity in the kidneys of Eker rats was confirmed by microscopic examination. By 4 h after TGHQ administration, morphological damage within proximal tubules located in the OSOM was observed. The brush border became diffuse and the tubular lumen became filled with cellular debris (Fig. 1). Kidneys of untreated Eker rats appeared unaffected.



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FIG. 1. Increased ERK1/2 phosphorylation within the OSOM of TGHQ-treated Eker rat kidney: Cells expressing phospho-ERK1/2 stain brown, phospho-ERK1/2 negative cells stain blue with hematoxylin counterstained. Representative treatment times (0, 4, 8, 24 h) are shown under the photomicrographs, taken under 400 x (Bar = 20 µm) or 40 x (Bar = 200 µm) total magnification. CS, cytosolic staining; NS, nuclear staining; SHD, shedding.

 
ERK1/2 Phosphorylation
ERK1/2 phosphorylation was absent in the proximal tubule of control rat kidneys (Fig. 1). TGHQ-induced ERK1/2 phosphorylation was time-dependent, appearing as early as 1 h following TGHQ treatment and reaching maximal levels between 4 and 8 h after TGHQ treatment. Levels of phospho-ERK1/2 returned to control levels by 24 h. We found the staining of phospho-ERK1/2 to be specifically localized within the OSOM. Phospho-ERK1/2 was primarily located within the cytoplasm of the proximal tubule epithelial cells at earlier time points after TGHQ administration, although some nuclear staining was also present. Nuclear staining of phospho-ERK1/2 became more intense and prominent at later time points following TGHQ treatment. Additional immunoreactivity was also found in the lumen of the proximal tubules in lesional regions, probably due to the release of the cytoplasmic contents after cell membrane damage. Constitutive staining for phospho-p42/44 MAPK was present in the distal tubules, in both control and treated animal kidneys, but did not increase upon TGHQ treatment. The findings suggest that phospho-ERK1/2 translocates from cytoplasm to nucleus, but this initial interpretation will require confirmation of immunolocalization using confocal microscopy. Western-blot analysis, specifically on dissected OSOM tissue, revealed that ERK1/2 phosphorylation changed in a similar pattern to that found with immunohistochemical staining (Fig. 2).



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FIG. 2. Western-blot analysis confirms increases in ERK1/2 phosphorylation in OSOM tissue of Eker rat kidneys after TGHQ treatment: Frozen OSOM tissues of untreated or treated Eker rats were homogenized, lysed, and analyzed by western blot analysis using a phospho-specific antibody for p-ERK1/2. (A) ERK1/2 phosphorylation upon TGHQ treatment. p-ERK1/2 (upper two bands), and total ERK1/2 (lower two bands) are shown, and the illustrated blot is typical of at least three independent experiments. (B). Statistical analysis on the changes of p-ERK1/2. p-ERK1/2 are compared and normalized to total ERK1/2, at each TGHQ treatment time point, and data are shown as fold-increase compared to untreated controls. Statistical significance is at *p < 0.05.

 
p38 MAPK and JNK1/2 Phosphorylation
The phosphorylation status of p38 MAPK (Fig. 3) and JNK1/2 (Fig. 4) within the OSOM changed little following TGHQ treatment, compared with the phosphorylation of ERK1/2, as determined by immunohistochemistry. Constitutive staining was observed throughout the kidney, mainly in glomeruli, the outer medulla, the inner medulla, and endothelial cells. We observed a slight increase in phospho-p38 MAPK (Fig. 3) and phospho-JNK1/2 (Fig. 4) within the OSOM 12 h after TGHQ administration, but this was observed only within limited loci with much less intensity than that observed for phospho-ERK1/2. Western-blot analysis on the OSOM tissues, using phospho-specific antibodies, confirmed the constitutive phosphorylation of p38 MAPK (Fig. 5) and JNK1/2 (Fig. 6) in both untreated and treated animal kidneys, and failed to detect any changes following TGHQ treatment. Preferential phosphorylation of JNK2 compared to JNK1 within OSOM tissue of both untreated and TGHQ-treated Eker rats was observed with a phospho-specific antibody for phospho-JNK1/2. But the mechanism of the preferential phosphorylation of JNK2 is unclear (Fig. 6).



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FIG. 3. TGHQ has little effect on p38 MAPK phosphorylation within the OSOM of Eker rat kidney: p-p38 MAPK expressing cells are stained brown with DAB, whereas p-p38 MAPK negative cells are counter-stained blue with hematoxylin. Selected treatment times are shown below the pictures. Bar = 200 µm for lower magnification (40 x); bar = 20 µm for higher magnification (400 x).

 


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FIG. 4. TGHQ does not induce significant JNK1/2 phosphorylation within the OSOM of Eker rat kidneys: In the photomicrographs, p-JNK1/2-expressing cells are stained brown, whereas p-JNK1/2 negative cells are counter-stained blue with hematoxylin. Treatment times are shown below the pictures. Bar = 200 µm for lower magnification (40 x); bar = 20 µm for higher magnification (400 x).

 


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FIG. 5. Western-blot analysis confirms the lack of induction of p38 MAPK phosphorylation after TGHQ treatment: Frozen OSOM tissue from untreated and TGHQ-treated Eker rats were homogenized, lysed, and analyzed by Western-blot using a phospho-specific antibody for p-p38 MAPK. The upper band shows unchanged p-p38 MAPK, and the lower band shows unchanged total p38 MAPK. The blot is a representative of at least 3 independent experiments.

 


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FIG. 6. Western-blot analysis confirms the lack of induction of JNK1/2 phosphorylation after TGHQ treatment: Frozen OSOM tissue from untreated or TGHQ-treated Eker rat were homogenized, lysed and analyzed by western-blot using a phospho-specific antibody for p-JNK1/2. The upper two bands represent the p-JNK1/2, and the lower two bands represent the total JNK1/2. The blot is a representative of at least 3 independent results.

 
Histone H3 Phosphorylation
Immunohistochemistry revealed increased phospho-histone H3 (Ser10) 8 h following TGHQ treatment, reaching maximal levels between 12 and 24 h (Fig. 7), and returning to control levels by 72 h. Western-blot analysis was performed on pooled samples from two to four animals at each time point, and results confirmed the immunohistochemical findings (Fig. 8). The presence of phospho-histone H3 within glomeruli likely contributes to the high expression levels in the control western-blot analyses, consistent with the immunohistochemical findings. However, the significant increases in phospho-histone H3 levels revealed by western-blot analyses, which exhibit the same kinetics as observed by immunohistochemistry, reflects changes occurring specifically within the OSOM, the target of TGHQ-induced toxicity. Thus, increases in phospho-histone H3 (Ser10) were found mostly within the OSOM and were associated with tubules exhibiting pathologic features. We also observed that phospho-histone H3 appeared in both the nuclei and cytoplasm of the proximal tubule epithelial cells, but most prominently in the nuclei at earlier time points (8 h), with increased intensity of cytoplasmic immunoactivity at later time points (12 h), probably due to the loss of membrane integrity.



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FIG. 7. TGHQ increases histone H3 phosphorylation at Ser10 within The OSOM of TGHQ-treated Eker rat kidneys: p-Histone H3 (Ser10)-expressing cells are stained brown, whereas cells staining negative for p-histone H3 (Ser10) are counter-stained blue with hematoxylin. Time of treatment is shown below the photomicrographs, with meter bar of 200 µm or 20 µm for lower or higher total magnification (40 x or 400 x). CS, cytosolic staining; NS, nuclear staining; SHD, shedding.

 


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FIG. 8. Western-blot analysis confirms an increase in histone H3 phosphorylation after TGHQ treatment: Frozen OSOM tissue from untreated or TGHQ-treated Eker rats were homogenized, lysed, sonicated, and analyzed by western blot analysis using a phospho-specific antibody for phospho-histone H3 (Ser10). Lysates from 2–4 animals from each time point (0–72 h) were pooled for analysis. The upper band represents the phopho-histone H3 (S10), and the lower band represents the total histone H3.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study we have shown that TGHQ induces the time-dependent phosphorylation of ERK1/2, followed by the subsequent phosphorylation of histone H3, within the OSOM of Eker rats. More importantly, increases in ERK1/2 phosphorylation precede the morphological findings of acute toxicity, consistent with a role for these phosphorylation events in toxicity. TGHQ-induced DNA damage precedes nephrotoxicity and was determined with immunostaining of 8-oxo-dG, which was at maximal levels 8 h after TGHQ administration in Eker rats (Habib et al., 2003Go). The acute toxicity of TGHQ is mainly localized to proximal tubule epithelial cells within the OSOM, due to the high concentrations of {gamma}-GT in these cells. Urinary {gamma}-GT levels increase rapidly after administration of renal toxicants, followed by increases in the urinary excretion of cytosolic components such as glutathione S-transferase (GST). Thus, urinary {gamma}-GT and GST activity serve as predictive indices of renal toxicity in vivo. After TGHQ treatment, urinary {gamma}-GT activity in Eker rats increases as early as 4 h, and rapidly reaches maximum levels at 8 h, which are sustained for at least 48 h (Habib et al., 2003Go). In contrast, urinary GST activity, an indicator of renal cell shedding into the tubular lumen, increases progressively and reaches peak levels 24 h after TGHQ treatment (Habib et al., 2003Go). Thus, initial increases in GST occur coincidentally with increases in histone H3 phosphorylation.

In contrast, we performed immunohistochemical experiments comparing Eker rats (possessing a mutation in one allele of the Tsc-2 tumor-suppressor gene) and their "wild-type" equivalents (Long-Evans), and did not observe any differences in the expression of phospho-ERK1/2 and phospho-histone H3 between the wild-type and the mutant Eker rats (data not shown). Consistent with these findings, TGHQ induces markedly increased cell proliferation and increased ERK activity within the OSOM of the kidneys in both wild-type and mutant Eker rats (Yoon et al., 2002Go). Loss of the remaining wild-type allele of the Tsc-2 gene is required for TGHQ-induced nephrocarcinogenicity. The Eker rat therefore represents an excellent model with which to examine chemical-induced nephrotoxicity and nephrocarcinogenicity.

Two downstream substrates of ERK1/2 are MSK1 and RSK2, both of which exhibit histone H3 kinase activity (Thomson et al., 1999Go). Consistent with the in vitro findings, ERK1/2 phosphorylation preceded histone H3 phosphorylation in vivo, suggesting that histone H3 may also be a downstream substrate of phospho-ERK1/2 in vivo. However, in contrast to TGHQ-mediated activation of p38 MAPK and JNK1/2 in LLC-PK1 cells, neither of these kinases was significantly altered within the OSOM of Eker rats. Thus, the contribution of p38 MAPK and JNK1/2 to TGHQ-induced nephrotoxicity in vivo remains unclear. Western-blot analysis of OSOM tissue from TGHQ-treated Eker rats confirmed the immunohistochemical findings, and revealed little change in phospho-p38 MAPK and phospho- JNK/1/2 throughout the observed time points. Our data are consistent with the constitutive expression of JNK1/2 and of corresponding JNK1/2 phosphorylation reported in proximal tubules of adult rats (Omori et al., 2000Go).

The roles of the MAPKs in hypertrophy, ischemia/ reperfusion-induced injury, chronic renal disease, angiotensinogen gene expression, and endoplasmic reticulum stress response have been the subject of many investigations (Hannken et al., 2000Go; Hsieh et al., 2002Go; Hung et al., 2003Go; Khan et al., 2001Go; Park et al., 2002Go). However, relatively less is known concerning the pattern of activation of the MAPK pathways in kidneys in response to ROS-generating chemical insult in vivo. The effects of ERK1/2 activation and the subsequent changes in the phosphorylation status of their substrates is controversial. Thus, ERK1/2 activation may be responsible for both cell proliferation and differentiation. The duration of ERK1/2 activation is also critical for cell signaling decisions in PC12 cells (Marshall, 1995Go); short-term ERK activation leads to proliferation whereas sustained ERK activation leads to differentiation. In the present model, ERK1/2 phosphorylation was maximal 4 h after TGHQ treatment, and returned to control levels by 24 h. The phosphorylation of ERK1/2 and subsequent histone H3 phosphorylation may constitute signals to the proximal tubule epithelial cells to activate the necessary machinery for cell division and proliferation. However, these same cells are simultaneously experiencing extensive DNA damage, with the concomitant activation of growth arrest and DNA damage inducible signals (gadd153 mRNA upregulation, and downregulation of histone mRNA (Jeong et al., 1996Go)). These two conflicting signaling pathways may contribute to premature chromatin condensation and mitotic catastrophe. Moreover, cells that survive the TGHQ-induced ROS-dependent stress by activating specific characteristics/signaling pathways, or by excessive proliferation after tissue damage, may acquire the potential to develop into tumors. The relationship between TGHQ-induced cell proliferation and TGHQ-induced nephrotoxicity and nephrocarcinogenicity has been previously established (Lau et al., 2001Go; Peters et al., 1997Go; Yoon et al., 2002Go). The mechanism likely involves cytotoxicity and compensatory cell proliferation followed by, in Eker rats, loss of tuberin expression. ERK activation is associated with mitogenesis, and histone H3 phosphorylation is a marker of cell proliferation. We hypothesize that TGHQ-induced DNA damage (Habib et al., 2003Go) increases the frequency of mutations, and perhaps loss of heterozygosity at the Tsc-2 locus, in the highly proliferative environment that exists in renal proximal tubules in response to tissue injury. In addition, the constitutive phosphorylation of JNK1/2 and p38 MAPK (Figs. 3GoGo6) may indicate that Eker rats, even in the absence of stress, exhibit a unique spectrum of signaling pathways that predispose these animals to the development of spontaneous renal tumors.

The shuttling of MAPKs between the cytoplasm and nucleus plays an important role in regulating MAPK function (Adachi et al., 2000Go). Activated ERK1/2 usually translocates from the cytoplasm to the nucleus, and inactivated ERK1/2 binds to MEK1/2 and relocalizes to the cytoplasm, assisted by the nuclear export signal on MEK1/2 (Adachi et al., 2000Go). The nuclear export of ERK1/2 is inhibited by leptomycin B, which binds to a component of the nuclear export complex, CRM1, and blocks nuclear export (Adachi et al., 2000Go). Nuclear translocation of activated ERK1/2 may participate in the activation of several targets, mainly transcription factors, and culminate in nuclear histone H3 phosphorylation. Following nuclear export of ERK1/2 to the cytoplasm, or breakdown of the nuclear and/or plasma membranes, phospho-ERK1/2 and phospho-histone H3 are detectable in the cytoplasm and/or within the lumen of the proximal tubules (Fig. 1), indicating necrotic cell death. With the development of new biological tools such as leptomycin B, importin antisense, and confocal microscopy, it will be possible to monitor the localization of MAPKs following TGHQ treatment.

In summary, we have shown that TGHQ induces time- dependent phosphorylation of both ERK1/2 and histone H3 within the OSOM of Eker rats. Phosphorylation of ERK1/2 and histone H3 are associated with oncotic/necrotic cells. In addition, the nuclear shuttling of ERK1/2 H3 was observed in response to TGHQ treatment. In contrast, phosphorylation of p38 MAPK and JNK1/2 remained unchanged after TGHQ treatment.


    ACKNOWLEDGMENTS
 
The authors would like to acknowledge the Center for Research on Environmental Diseases, Histology Core Facility, MD Anderson Cancer Research Center, Science Park, Smithville, Texas for the preparation of the paraffin slides and assistance with immunohistological techniques. This work was supported in part by NIEHS Center Grants P30 ES-07784 and P30 ES-06694, DK59491 to TJM, and GM39338 to SSL. Portions of this work were presented in abstract form at the 2002 Annual Meeting of the Society of Toxicology (Toxicol. Sci. 72(Suppl.), 64).


    NOTES
 

1 To whom correspondence should be addressed. Tel: (520) 626-9906. Fax: (520) 626-2466. E-mail: scouser{at}pharmacy.arizona.edu.


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 RESULTS
 DISCUSSION
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Lau, S. S., Monks, T. J., Everitt, J. I., Kleymenova, E., and Walker, C. L. (2001). Carcinogenicity of a nephrotoxic metabolite of the "nongenotoxic" carcinogen hydroquinone. Chem. Res. Toxicol. 14, 25–33.[CrossRef][ISI][Medline]

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Yoon, H. S., Monks, T. J., Everitt, J. I., Walker, C. L., and Lau, S. S. (2002). Cell proliferation is insufficient, but loss of tuberin is necessary, for chemically induced nephrocarcinogenicity. Am J Physiol. Renal. Physiol. 283, F262–270.[Abstract/Free Full Text]