Distinct Contributions of JNK and p38 to Chromium Cytotoxicity and Inhibition of Murine Embryonic Stem Cell Differentiation

Background Potassium dichromate [Cr(VI)] is a widespread environmental toxicant responsible for increased risk of several human diseases. Cr(VI) exposure leads to activation of mitogen-activated protein kinases (MAPKs), including c-Jun N-terminal kinase (JNK)1/2, p38, and extracellular-signal regulated kinase (ERK)1/2. Objectives We evaluated the contribution of MAPKs to Cr(VI) toxicity. Methods Phosphorylation of MAPKs and their downstream effectors was evaluated by Western immunoblotting; reactive oxygen species were measured by DCFDA (5′,6′-chloromethyl-2′-7′-dichlorofluorescin diacetate) labeling and flow cytometry, and glutathione and glutathione disulfide levels were determined by monochrome graphic spectroflurometer. Cytotoxicity was assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and colony formation. Embryoid body (EB) differentiation was evaluated by contracting cardiomyocyte formation, and real-time polymerase chain reaction (RT-PCR) was used for cardiomyocyte-specific and stem-cell-specific gene expression. Results Acute treatment of mouse embryonic stem (ES) cells with 50 μM Cr(VI) induced the rapid phosphorylation of JNK, p38, and ERK and their respective downstream transcription factors, c-JUN, activating transcription factor-2, and ELK1. MAPK activation and cytotoxicity induction were partially blocked by pretreatment with the antioxidant N-acetyl cysteine. Ablation of the upstream MAP kinase kinase (MAP2K7) in ES cells prevented JNK activation, whereas ablation of MAP2K4 prevented both JNK and p38 activation. Using specific MAPK inhibitors and MAP2K4- and MAP2K7-deficient ES cells, we showed that JNK reduced acute Cr(VI) cytotoxicity, p38 potentiated it, and ERK had no effect. At low submicromolar concentrations, Cr(VI) caused MAP2K4/7-dependent JNK activation and MAP2K4-dependent p38 activation and strongly inhibited contracting cardiomyocyte development in wild-type ES cells, but much less so in Map2k7(−/−) cells. Conclusion Each MAPK distinctly contributes to chromium toxicity. Whereas JNK prevents and p38 promotes acute cytotoxicity, JNK contributes to optimal inhibition of ES cell differentiation by chromium.

volume 117 | number 7 | July 2009 • Environmental Health Perspectives Research Potassium dichromate [Cr(VI)] is the most toxic form of chromium produced by industrial processes and is a common environmental contaminant (Langard 1990). Humans exposed to Cr(VI) under environmental or occupational settings show increased risk of asthma, nasal septum and skin ulcerations, allergic and contact dermatitis, and respiratory cancers (Salnikow and Zhitkovich 2008). Experimental mice exposed to chromate exhibit acute tissue damage, including testicular lesions, kidney tubular necrosis, and liver toxicity, whereas Cr(VI) ingestion via drinking water leads to increased DNA damage and ultraviolet-induced skin tumor formation in mice (Davidson et al. 2004;Ueno et al. 2001). In addition, Cr(VI) is a developmental toxicant that causes developmental defects, including postimplantation losses, resorptions, reduced fetal weight, and malformations (Junaid et al. 1996;Kanojia et al. 1998).
Particulate Cr(VI) undergoes dissolution on the cell surface, and the chromate oxyanion enters the cells through the nonspecific phosphate/sulfate ion transporters (O'Brien et al. 2003). Once inside the cell, chromate can be rapidly reduced by antioxidants, such as ascorbate, glutathione, and NAD(P)H, to generate reactive Cr(V) and Cr(IV) intermediates that are strong oxidants. The Cr intermediates are subsequently reduced to form the thermodynamically stable Cr(III), which is primarily responsible for DNA adducts formation and mutagenicity (Salnikow and Zhitkovich 2008). On the other hand, Cr(VI) has been shown to activate cellular signaling pathways that in turn lead to either promoting (Salnikow and Zhitkovich 2008;Wakeman et al. 2005;Wang and Shi 2001) or preventing Cr(VI) toxicity (Chen et al. 2002;He et al. 2007).
Once activated, the MAPKs can translocate to the nucleus, where they phosphorylate transcription factors causing gene expression changes. The activated MAPKs exhibit distinct specificity in nuclear transcription factor phosphorylation. For instance, JNKs are the upstream kinases for c-JUN, whereas both JNK and p38 can phosphorylate ATF2; the ERKs, on the other hand, specifically catalyze the phosphorylation of ELK1. Through the distinct connections to upstream regulators and downstream effectors, each MAPK exhibits unique roles in converting transient biochemical signals to permanent changes of gene expression in the control of cell functions.
The existing literature describes divergent effects of Cr(VI) on MAPK signaling, as activation patterns vary depending on different experimental systems, treatment concentrations, time of exposure, and target cell type used for the studies (Chuang and Yang 2001;O'Hara et al. 2003;Samet et al. 1998;Tessier and Pascal 2006). Several studies show that high concentrations of Cr(VI) can activate all three MAP kinases and that the activation may be mediated via reactive oxygen species (ROS)-dependent mechanisms. Conversely, lower concentrations of Cr(VI) may activate selective MAPK subgroups in a highly celltype-specific fashion, whereas activation might be ROS independent. Induction of MAP kinase pathways was shown to contribute to specific toxic responses to Cr(VI) (Wakeman et al. 2005;Wang and Shi 2001), although in other cases could not be linked to a defined end point of Cr(VI) toxicity (Ceryak et al. 2004;Chuang and Yang 2001). Collectively, these observations suggest that MAPK activation is due to the combinatorial effects of Cr(VI) bioprocessing and dynamic interactions of Cr species with other cellular components within the local environment. As a consequence, the contribution of each MAPK pathway to chromium toxicity will be better evaluated individually in the specific cellular context and exposure condition. In this paper, we characterized the effects of high and low concentrations of Cr(VI) on MAPK activation profiles in wild-type (WT) and Map2k4 (-/-) and Map2k7 (-/-) embryonic stem (ES) cells. Using these cells, we show that Cr(VI) activates all three MAPKs, but each of them leads to specific transcription factor modifications and makes unique contributions to Cr(VI) cytotoxicity and differentiation inhibition.
Cell culture and adenoviral infection. We precoated culture dishes used for mouse ES cells with 0.1% gelatin for 20 min. The ES cells of WT, Map2k4 (-/-) , and Map2k7 (-/-) were maintained in ES complete medium, containing DMEM, 15% FBS knockout serum replacement, 2 mM l-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin, 1% nonessential amino acids, 0.1mM β-mercaptoethanol, and 10 3 units/µL LIF. We carried out infection of the ES cells with adenovirus as described previously (Deng et al. 2006). At 48 hr after infection, we subjected the cells to subsequent treatments with Cr(VI) or harvested them for Western blot analyses.
Chronic toxicity assay was performed as described (Franken et al. 2006). Briefly, we plated 5,000 ES cells onto gelatin pretreated 6-mm plates in ES complete medium in the absence and presence of various doses of Cr(VI). Cells were allowed to grow for 7 days, and the ES complete medium was replenished every 3 days. We washed the colonies with phosphate-buffered saline, fixed, and stained them with 0.3% crystal violet mixed in 25% ethanol for 2 min and counted them under the microscope. We used three plates for each treatment condition.
Cell lysate preparation and immunoblotting. ES cells were challenged with Cr(VI) at various conditions described above. Cell lysates were prepared and subjected to SDS-PAGE and transferred to nitrocellulose membranes for Western blotting analyses as described elsewhere (Peng et al. 2007).
Cellular ROS and glutathione measurement. We measured the ROS levels in the cells by CM-H 2 DCFDA labeling, and identified the fluorescence-positive cells by FACS analyses as described previously (Peng et al. 2007). Glutathione and glutathione disulfide (GSSG) levels were determined as detailed previously (Senft et al. 2002), with samples prepared in triplicate for statistical analysis.

Differentiation of ES cells into contracting myocardial cells.
We conducted cardiac differentiation of ES cells as described previously (Gribaldo et al. 2002). On day 0, we aggregated ES cells (5 × 10 4 cells/mL) by culturing in 20-µL hanging drops in cell culture medium without LIF. On day 3, the cell aggregates formed embryoid bodies (EBs), and the EBs were transferred to 24-well plates at 1 EB/well. On day 14, we counted the number of EBs with contracting myocardial areas under light microscopy. In some experiments, Cr(VI) was included in the growth medium for the entire time of differentiation. We replenished the culture medium with or without chemicals every 3 days.
Real-time polymerase chain reaction. We isolated total cellular RNA from WT and Map2k7 (-/-) ES cells and EBs, which were allowed to differentiate for 14 days in the absence and presence of 0.1 µM Cr(VI). We performed cDNA preparation and real-time PCR as described previously (Peng et al. 2007). Primers for mouse stem-cell-specific genes, Notch-1: 5´-AAT CTT GTC CAG ACA GGT GCC AGA and 5´-TCT GCT TAT GCC TCA AGG GAA CCA, Sox-2: 5´-AAA GGA GAG AAG TTT GGA GCC CGA and 5´-GGG CGA AGT GCA ATT GGG ATG AAA, Oct-4: 5´-GGC GTT CTC TTT GGA AAG GTG TTC and 5´-CTC GAA CCA CAT CCT TCT CT and Nanog: 5´-AGG GTC TGC TAC TGA GAT GCT CTG and 5´-CAA CCA CTG GTT TTT CTG CCA CCG; for cardiomyocyte-specific genes, Mhca: 5´-CTG CTG GAG AGG TTA TTC CTC G and 5´-GGA AGA GTG AGC GGC GCA TCA AGG and Mhcb: 5´-TGC AAA GGC TCC AGG TCT GAG GGC and 5´-GCC AAC ACC AAC CTG TCC AAG TTC; and for housekeeping genes, Actinb: 5´-TCT TGG GTA TGG AAT CCT GTG GCA and 5´-TCT CCT TCT GCA TCC TGT CAG CAA and Gapdh: 5´-TCA ACA GCA ACT CCC ACT CTT CCA and 5´-ACC CTG TTG CTG TAG CCG TAT TCA were used for RT-PCR.
Statistical analyses. We conducted statistical comparisons using Student two-tailed paired t-test and analysis of variance. We considered values of p < 0.05 statistically significant.

High-concentration Cr(VI) activates MAPKs and downstream transcription factors in mouse ES cells.
We examined MAPK activation in mouse ES cells treated with Cr(VI) at 50 µM for various lengths of time. Cr(VI) caused early and delayed ERK activation, as indicated by phospho-ERK2 (p42) induction, and delayed JNK and p38 activation, as indicated by the appearance of phospho-JNK (p54 and p46) and phospho-p38 ( Figure 1A). Correspondingly, Cr(VI) induced the MAPK downstream events, including phosphorylation of c-JUN, ATF2, and ELK1 in WT ES cells ( Figure 1B). In addition, there was an apparent elevation of c-JUN and c-FOS proteins at the delayed phase of exposure ( Figure 1D).
In recent years, genetic inactivation of MAP2Ks has been shown to specifically block MAP kinase activation, and the MAP2Kdeficient cells are used to sort out the roles of the MAPKs in variety of cell functions (Wada et al. 2008). We examined MAPK activation by Cr(VI) in the MAP2K4-deficient and MAP2K7-deficient ES cells ( Figure 1C). Cr(VI) exposure did not alter MAP2K4/ MAP2K7 expression. Compared with the induction in the WT cells, p-JNK and p-p38 induction by Cr(VI) in the Map2k4 (-/-) cells was almost completely abolished [ Figure 1A; volume 117 | number 7 | July 2009 • Environmental Health Perspectives see Supplemental Material, Figure 1 (available online at http://www.ehponline.org/ members/2009/0800157/suppl.pdf)]. Correspondingly, there were no detectable p-c-JUN and activating transcription factor-2 (p-ATF-2) in the Map2k4 (-/-) cells [ Figure 1B; see Supplemental Material, Figure 1 (http://www.ehponline.org/members/2009/0800157/suppl.pdf)]. The phosphorylation of JNK and c-JUN was markedly suppressed in the Map2k7 (-/-) cells, whereas the phosphorylation of p38 and ATF-2 was unaffected. The phosphorylation of ERK and ELK1 were not overtly affected by MAP2K4ablation and MAP2K7-ablation, indicating that these MAP2Ks were not essential for ERK pathway activation by Cr(VI) ( Figures  1A and 1B). Based on these observations, we conclude that the Cr(VI) signals that activate JNK are mediated by both MAP2K4 and MAP2K7 and those that activate p38 by MAP2K4; however, neither MAP2K4 nor MAP2K7 are required for ERK activation and the induction of c-JUN and c-FOS expression ( Figure 1D). To confirm that MAP2K7 conferred resistance to Cr(VI) cytotoxicity, we overexpressed MAP2K7 in Map2k7 (-/-) cells using an adenovirus-mediated gene delivery system. At 48 hr of infection, the levels of MAP2K7 in Ad-MAP2K7-infected, but not Ad-GFP infected, Map2k7 (-/-) cells were similar to or even higher than those in the WT cells ( Figure 2D). When exposed to Cr(VI), the Ad-MAP2K7-infected Map2k7 (-/-) cells reached a survival rate similar to that of the WT cells, much higher than the parental uninfected or Ad-GFP-infected cells ( Figure 2E).

MAP2K7 and JNK reduce, but p38 promotes, cytotoxicity induced by high concentration of Cr(VI). Cr(VI) cytotoxicity in WT ES cells depended on concentration
To test whether MAPK activation was responsible for Cr(VI) cytotoxicity, we treated the WT ES cells with specific MAPK inhibitors prior to Cr(VI) exposure. Our previous work had established that at 5 µM concentration short-term treatment, SP600125 was specific for JNK inhibition, and similarly, SB202190 was specific for p38 and PD98059 for ERK (Deng et al. 2006). Whereas inhibition of JNK sensitized the cells to Cr(VI) cytotoxicity, inhibition of p38 reduced it, and inhibition of ERK had no effect ( Figure 2F). Furthermore, pretreatment of cells with CHX to inhibit protein synthesis significantly reduced, but did not completely prevent, Cr(VI) cytotoxicity in WT ES cells ( Figure 2F), suggesting that gene transcription and de novo protein synthesis contribute partially to Cr(VI) cytotoxicity. Considering that Map2k7 (-/-) cells are defective in JNK activation and are more sensitive to Cr(VI) cytotoxicity, a condition similar to that of WT cells treated with the JNK inhibitors, we suggest that JNK activation is required for reducing Cr(VI) cytotoxicity. On the other hand, cells treated with the p38 inhibitor and Map2k4 (-/-) cells, defective in p38 activity, are both somewhat Cr-resistant, suggesting that p38 activation may augment Cr(VI) cytotoxicity.
N-acetyl cysteine partially blocks highconcentration Cr(VI)-induced MAPK activation and cytotoxicity. It has been suggested that Cr(VI) can activate MAPKs via ROSdependent and ROS-independent mechanisms (Kim and Yurkow 1996;O'Hara et al. 2003). We labeled the WT ES cells, either untreated or treated with Cr(VI) and H 2 O 2 , with ROS-activated fluorescent dye CM-H 2 DCFDA and measured intracellular ROS levels by flow cytometry. Compared with the untreated cells, ROS accumulation was higher in Cr(VI)-treated cells, but the levels induced by Cr(VI) were lower than those induced by H 2 O 2 ( Figure 3A). Map2k4 (-/-) and Map2k7   and 3C). Cellular redox homeostasis is largely dependent on GSH, which when oxidized by ROS, forms GSSG. We found higher GSSG in ES cells treated by Cr(VI) than in untreated cells, further supporting the idea that Cr(VI) induced oxidative stress and caused GSH depletion ( Figure 3D) To test whether NAC can prevent MAPK activation and cytotoxicity in our system, we treated WT cells with NAC prior to Cr(VI) exposure. In NAC pretreated cells, there was a significant decrease in the induction of p-JNK, p-p38, and p-ERK by Cr(VI) at the late exposure times (6 hr), but induction of p-ERK at early times (10 min) did not seem to be affected ( Figure 3E). Induction of the nuclear factors p-JUN, p-ATF2, and c-FOS was also significantly reduced by NAC pretreatment ( Figure 3F). Moreover, NAC significantly prevented Cr(VI) cytotoxicity as measured by both the acute and chronic toxicity ( Figures  3G and 3H). Protection by NAC was much more effective than by MAPK inhibitors or by MAP2K gene ablation, indicating that NAC offers a broader protection against Cr(VI) cytotoxicity and that one of the downstream events of NAC may be to reduce the delayed MAPK activation.

The role of MAPKs in low-concentration Cr(VI) effects.
Having established the MAPK activation patterns in response to high Cr(VI) concentrations, we asked whether the MAPKs, particularly JNK and p38, were similarly affected by MAP2K4/MAP2K7 ablation in response to low concentrations of Cr(VI), more comparable with potential environmental or occupational human exposures. We treated the ES cells with 0.1 µM Cr(VI) for various times ranging from 1 to 10 days and examined the phospho-JNK and total-JNK and p38 ( Figure 4A). Lowconcentration Cr(VI) caused a clear induction of p-JNK in WT ES cells. In contrast to high-concentration Cr(VI), which strongly induced phosphorylation of both p46 and p54 JNK isoforms ( Figure 1A), low-concentration Cr(VI) only weakly activated the p46 JNK isoform. Similarly, low-concentration Cr(VI) induced weaker and delayed p38 phosphorylation. JNK activation by low-concentration Cr(VI) was completely abolished in Map2k4 (-/-) and Map2k7 (-/-) cells, whereas p38 activation was unaffected in Map2k7 (-/-) but blocked in Map2k4 (-/-) cells, similar to the activation patterns in response to high concentration ( Figure 1A).
We next examined the contributions of MAPKs to chronic toxicity of Cr(VI) using colony formation assays ( Figure 4B). In comparison with the WT ES cells, Map2k4 (-/-) cells showed significantly higher chronic survival rate in response to 1 µM Cr(VI) treatment for 7 days, whereas Map2k7 (-/-) cells had markedly decrease survival rate. Based on these observations, we conclude that, like high-concentration Cr(VI), low concentration activates JNK through MAP2K4/7 and p38 through MAP2K4 in ES cells and that MAP2K4 and MAP2K7 have similar effect on the cytotoxicity of low and high concentrations of Cr(VI).

Suppression of ES cell differentiation by chromium requires MAP2K7.
Developing rhythmically contractile cardiomyocytes from EBs were used to evaluate the developmental effects of MAPKs and Cr(VI). Although 30% of WT EBs and 60% of MAP2K7-deficient EBs developed rhythmic contractile cardiomyocytes, none of the MAP2K4-deficient EBs formed them ( Figure 5A and data not shown). Because p38 α-deficient ES cells have failed cardiomyocyte differentiation, it is possible that the MAP2K4-deficient cells lack p38 activation and therefore have a defective cardiac differentiation (Aouadi et al. 2006). Cr(VI) at 1 µM completely abolished WT EB to form contractile cardiomyocytes, but at 0.01 µM had no effect at all (data not shown). At 0.1 µM, Cr(VI) caused a 95% suppression of cardiac differentiation in WT EBs, but less than 50% suppression in MAP2K7-deficient EBs. Based on these evaluations, we conclude that MAP2K7 inactivation might offer protection against Cr(VI) toxicity in the inhibition of ES cell differentiation (Figures 5A and 5B).
To study EB differentiation at the molecular level, we examined the expression of stem-cell-specific and cardiomyocyte-specific markers by RT-PCR. Compared with undifferentiated ES cells, the differentiated WT and Map2k7 (-/-) EBs had decreased expression of stem-cell-specific genes, such as Notch, Sox-2, Nanog, and Oct-4, but elevated expression of cardiomyocyte genes, such as MhcA and MhcB, indicative of cardiomyocyte differentiation ( Figures 5C and 5D). Such changes in gene expression were clearly suppressed by Cr(VI) in WT but not in Map2k7 (-/-) EBs. Thus, this  (-/-) , and Map2k7 (-/-) adenoviral-infected cells were treated with 50 µM Cr(VI) for 10 hr. Cell survival was determined by MTS assays, and each value was presented as mean ± SD from four replicates. (F) WT ES cells were pretreated with chemical inhibitor for JNK (SP60015, 5 µM), p38 (SB202190, 5 µM), ERK (PD98059, 5 µM), and protein synthesis (CHX, 1 µg/mL) for 1 hr. The cells were incubated with Cr(VI) at 50 µM for 8 hr. Cell survival rates were analyzed by MTS assay, and each value was presented as mean ± SD from four replicates. **p < 0.01. # p < 0.001.

Discussion
Cr(VI) exposure is associated with broad classes of adverse health effects in humans and in animal models, including respiratory cancer, impaired organ function, immune system defects, birth defects, and decreased fertility (Junaid et al. 1996;Kanojia et al. 1998;Salnikow and Zhitkovich 2008), but the molecular mechanisms underlying disease development are not well understood. In the present studies, we show that one consequence of Cr(VI) exposure of mouse ES cells is the early and delayed activation of MAPKs. There is a good correlation between the MAPKs and their corresponding downstream transcription factor modifications. These results, together with the findings that Cr(VI) cytotoxicity requires de novo protein synthesis, indicate that the MAPKs may activate transcriptional programs for gene expression, which in turn influences cell fate determination in response to Cr(VI) (Figure 6). Similar MAPK induction by Cr(VI) has been observed in various cells types (Ceryak et al. 2004;Chuang and Yang 2001;O'Hara et al. 2003;Samet et al. 1998;Tessier and Pascal 2006;Wakeman et al. 2005), but the specific contributions of the different MAPKs to Cr(VI) toxicity are poorly defined. Studies based solely on pharmaceutical MAPK inhibitors used at high concentrations that have been shown to cause nonspecific effects (Tan et al. 2002) often generate diametrically opposite results (Chuang and Yang 2001;Wakeman et al. 2005). We used instead the combined approach of MAP2K-deficient cells and lowdose MAPK inhibitors for short-term treatment to reveal the distinct contributions each MAPK makes to acute Cr(VI) cytotoxicity. We show that JNK alleviates, p38 promotes, and ERK has no obvious effect on cytotoxicity of Cr(VI) at high and low concentrations ( Figure 6). Antagonistic roles of JNK and p38 have been described previously, in that JNK positively regulates proliferation and oncogenic transformation but negatively regulates cellular senescence, whereas p38 suppresses proliferation and transformation but potentiates cellular senescence (Wada et al. 2008). Our studies for the first time report opposing effects of JNK and p38 exist for Cr(VI) cytotoxicity.
In addition to its acute cytotoxicity, Cr(VI) is a known developmental toxicant, causing postimplantation losses, resorptions, reduced fetal weight, and malformations including reduced ossification (Junaid et al. 1996;Kanojia et al. 1998). We have found that Cr(VI) is a strong developmental toxicant that, at concentrations as low as 0.1 µM, causes 95% inhibition of cardiac differentiation of EBs, corresponding to the repression of cardiomyocyte-specific gene induction. This observation is in general agreement with the findings by Stummann et. al. showing that 1.8 µM Cr(VI) causes 50% inhibition of the cardiac differentiation of D3 ES cells (Stummann et al. 2007). However, the ES cell lines used in our studies appear to be even more sensitive to Cr(VI), perhaps because they originated from the Sv129 mouse strain.

Figure 3. NAC suppresses MAPK activation and cytotoxicity induced by high-concentration Cr(VI). (A) WT
ES cells were treated with 50 µM Cr(VI) for 6 hr or 50 µM H 2 O 2 for 2 hr. The cells were labeled with 10 µM CM-H 2 DCFDA for 30 min and were subjected to flow cytometric analysis. DCF-positive cells were identified by CellQuest analysis. Cells were either pretreated with 5 mM NAC for 4 hr or left untreated prior to exposure to 50 µM Cr(VI) for various times. (B) Western blotting was used to measure the expression of HO-1 and actin in WT ES cells treated with Cr(VI) 50 µM for various times. (C) The relative levels of HO-1 protein expression were quantified by gel imaging in ES cells with or without NAC and Cr(VI) treatment. (D) Cells exposed to Cr(VI) at 50 µM for various times were analyzed for the cellular level of GSSG. Western blot was done to measure (E) phosphorylated and total JNK, p38, and ERK and (F) p-JUN, p-ATF2, c-FOS, and actin. The results were quantified by chemiluminescence imaging and the fold induction of p-JNK over JNK, p-38 over P38, p-ERK over ERK, and p-c-Jun, p-ATF2, and c-Fos over actin was calculated. (G) WT, Map2k4 (-/-) , and Map2k7 (-/-) ES cells were either pretreated with 5 mM NAC for 4 hr or left untreated prior to exposure to 50 µM Cr(VI) for 8 hr. Cell survival was measured by the MTS assay. (H) WT cells were exposed to 1 µM or 10 µM Cr(VI) in the presence or absence of 5 mM NAC. Colonies were counted on day 8 of treatment. The values in B, D, and E are shown as mean ± SD from at least three experiments. The ratio of phosphoprotein versus total protein or actin was calculated in each sample, and the fold induction was calculated in comparison with the untreated control of the same cell type. *p < 0.05. **p < 0.01. # p < 0.001. Although the chemical inhibitors for shortterm treatment can effectively block MAPK activity and cytotoxicity of chromium, the JNK inhibitor SP600125 completely abrogated the EB formation when used for a longer treatment period (data not shown). These observations suggest that long-term application of these chemicals may exhibit nonspecific toxicity irrelevant to JNK inhibition, because in vivo JNK1/ JNK2 double ablation does not cause global differentiation defects (Kuan et al. 1999).
Because JNK is the only known downstream target of MAP2K7, the Map2k7 (-/-) cells have been conveniently used to evaluate physiologic consequences of JNK loss of function (Wada et al. 2008). Indeed, we show that JNK is the sole MAPK whose activation by Cr(VI) is effectively abrogated in Map2k7 (-/-) ES cells. Intriguingly, Cr(VI) causes only a slight suppression of contractile cardiomyocyte formation in the Map2k7 (-/-) EBs and does not cause reduction of cardiomyocyte-specific gene expression in these cells. Because Cr(VI) fails to activate JNK in Map2k7 (-/-) cells, it is possible that blocking JNK reduces Cr(VI) toxicity on the inhibition of cardiomyocyte differentiation. The seemingly contrasting role that the JNK pathway appears to have by inhibiting cardiomyocyte differentiation on the one hand and preventing acute and chronic cytotoxicity on the other is not entirely surprising, because the JNK pathway is known to regulate diverse transcriptional programs, participating in a broad spectrum of cell responses.
The MAP2Ks are immediately upstream kinases responsible for MAPK phosphorylation and activation. It has been shown that MAP2K4 and MAP2K7 distinctly phosphorylate JNK at tyrosine and threonine, respectively (Lawler et al. 1998). We find that Cr(VI) reduces JNK phosphorylation and completely abolished c-JUN phosphorylation in Map2k4 (-/-) and Map2k7 (-/-) cells. This observation seems to support the notion that MAP2K4 and MAP2K7 phosphorylate distinct sites at the Thr-X-Tyr motif of JNK and that phosphorylation of both tyrosine and threonine residues is required for optimal JNK activity in c-JUN phosphorylation. Besides JNK activation, MAP2K4 is also known to activate p38. In ES cells, MAP2K4 appears to Cell lysates were used for Western blot analyses for the phospho-JNK and total JNK and p38. Arrowheads point at different JNK isoforms. The ratio of phosphoprotein versus total protein was calculated in each sample, and the fold induction was calculated in comparison with the untreated control of the same cell type. (B) Cells were treated with 0.1 µM or 1 µM Cr(VI) for 7 days, and colonies were counted on day 8 of treatment. Each value represents the mean ± SD from six replicates. *p < 0.05.  Cr(VI) undergoes intracellular reduction, resulting in the generation of toxic Cr intermediates and thermodynamically stable Cr(III), which are ultimately responsible for DNA damage (Salnikow and Zhitkovich 2008), and of ROS, which contribute to cytotoxicity. Cr(VI) reduction is carried out by cellular antioxidant systems such as glutathione and ascorbate. Ascorbate has been shown to increase Cr(VI) reductive activation, enhance toxicity, and potentiate mutagenicity, suggesting that ascorbate, commonly used as a redox scavenger to prevent toxicity of other environmental agents, is not suitable for counteracting Cr(VI) toxicity (Salnikow and Zhitkovich 2008). We find that incubation of cells in ascorbate at 1 mM for a shorter time (< 12 hr) offers no protection against Cr(VI) cytotoxicity, but for a long time (> 20 hr) is by itself toxic to ES cells even in the absence of Cr(VI) (data not shown). This observation precludes the possibility of further evaluating the role of ascorbate in Cr(VI) cytotoxicity in our experimental systems. On the other hand, NAC exhibits a remarkable protection against Cr(VI) cytotoxicity. Furthermore, inclusion of 5 mM NAC in the growth medium does not completely abrogate, but significantly reduces, the delayed MAPK activation by Cr(VI). There are multiple possibilities for NAC to exert protection, including potentiation of Cr(VI) extracellular bioreduction, elevation of intracellular GSH to promote the removal of ROS, and promotion of electron transfer for direct Cr(VI) intracellular reduction (Guttmann et al. 2008;Salnikow and Zhitkovich 2008), all of which could be the reason for NAC to decrease Cr(VI)-induced MAPK activity. It is worth noting that EB and contractile cardiomyocyte formation are completely abrogated in the presence of 0.5 mM NAC in the medium (data not shown), indicating that excess cysteine can also disrupt normal ES cell differentiation programs.
Our understanding of the molecular events mediating cellular responses to Cr(VI) is rapidly expanding. In addition to causing DNA damage and modifying chromatin, Cr(VI) can induce MAPKs to affect the wellbeing of the organism. Moreover, we find that each MAPK member makes a distinct contribution to Cr(VI) cytotoxicity and inhibition of differentiation ( Figure 6). The experimental systems described in our studies may provide useful tools to evaluate the contributions of the MAPK members to other Cr(VI) toxic end points, such as cell cycle progression, differentiation, development, and movement. Figure 6. Distinct contributions of JNK and p38 to Cr(VI) toxicity. Cr(VI) induced the activation of the MAPKs via multiple mechanisms that can be ROS dependent and independent. The activation of JNK and p38, but not ERK, is mediated through MAP2K4 and MAP2K7. Specifically, MAP2K4 and MAP2K7 both are required for optimal JNK activation, but only MAP2K4 is essential for p38 activation. Using cells deficient in MAP2K4 and MAP2K7, we were able to delineate the distinct roles JNK and p38 play in the cytotoxicity and developmental toxicity of chromium.