Nickel compounds induce phosphorylation of histone H3 at serine 10 by activating JNK–MAPK pathway

Abstract

Nickel (Ni) is a known carcinogen, although the mechanism of its carcinogenicity is not clear. Here, we provide evidence that Ni can induce phosphorylation of histone H3 at its serine 10 residue in a c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)-dependent manner. Ni induces the phosphorylation of JNK, with no effect on the phosphorylation states of the extracellular signal-regulated kinase (ERK) or p38 mitogen-activated protein kinases. An inhibitor of JNK eliminated the Ni-initiated JNK-mediated induction of histone H3 phosphorylation at serine 10, whereas inhibitors specific for ERK or p38 kinases had no effect on the phosphorylation levels of histone H3 at serine 10 (P-H3S10) in Ni-treated cells. A complete loss of Ni ion-induced phosphorylation of H3S10 was observed when JNK was specifically knocked down with RNAi. These results are the first to show the specific JNK-mediated phosphorylation of histone H3 at its serine 10 residue. We show that addition of Ni to an in vitro P-H3S10 dephosphorylation reaction does not change the loss of phosphorylation in the reaction, supporting the notion that Ni causes H3S10 phosphorylation via the JNK/SAPK pathway. It is likely that modification of H3S10 is one of a growing number of epigenetic changes believed to be involved in the carcinogenesis caused by Ni.

Introduction

The eukaryotic genome is packaged into chromatin, whose fundamental subunit is the nucleosome. Each nucleosome contains 146 bp of DNA wrapped around an octamer of histones (1). Two copies of each of histone H2A, H2B, H3 and H4 form the histone core octamer. Posttranslational modifications (i.e. acetylation, methylation, phosphorylation, ubiquitination, etc.) of the N- and C-terminal tails of these histones play an important role in regulating chromatin biology (2,3).

Studies have shown that histone phosphorylation disrupts histone–DNA interactions and destabilizes chromatin structure (4–6). Phosphorylation of histone H3 at serine 10 (S10) is crucial for chromosome condensation and cell-cycle progression and is regarded as a marker of mitosis (7). Conversely, it has been observed that histone H3 at serine 10 (H3S10) phosphorylation plays an important role in the induction of immediate-early (IE) genes, such as c-jun and c-fos, and that mitogen-activated protein kinase (MAPK) pathways are involved (8).

MAPK signaling pathways feature a series of phosphorylation cascades. Upon receiving the extracellular signals such as growth factors, hormones and stress stimuli, the MAPKs are activated by phosphorylation on threonine and tyrosine by the MAPK kinases, which are activated by serine/threonine phosphorylation by MAPK kinase kinases. Additional protein kinases and members of the Ras and Rho families of small GTPases may also participate at the upstream of MAPK kinase kinases. Once activated, MAPKs can alter the activity of target transcription factors and transcriptional coregulators by phosphorylation, resulting in changes in the expression of genes that mediate cell growth, proliferation, differentiation, apoptosis and transformation.

It has been reported that the level of phosphorylated H3S10 in cells increases dramatically in response to stimulation of the MAPK pathway (9). The MAPK cascades play an important role in eukaryotic gene regulation by orchestrating the phosphorylation of transcription factors and coactivators responsible for the induction of IE genes. The phosphorylation of H3S10 has also been shown to play a role in the MAPK cascade-mediated induction of IE genes (10). In fact, phosphorylation of histone H3 has thus far been seen as indissociable from IE gene induction (11).

There are four distinct MAPK cascades elucidated so far: (i) the extracellular signal-regulated kinase (ERK)/MAPK 1 and 2 pathway; (ii) the p38 pathway; (iii) the c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway and (iv) the big mitogen-activated protein kinase/ERK5 pathway.

Whether by exposure to mitogen or other stimuli of diverse nature, such as growth factors or protein synthesis inhibitors, the activation of MAPK cascades elicits the phosphorylation of certain chromosomal proteins, including histone H3. This has been referred to as ‘the nucleosomal response’ (12), and it correlates well with the induction of IE genes, suggesting a role for H3 phosphorylation in transcriptional activation. However, phosphorylation of H3 at S10 has long been established as a mark of mitosis and chromatin condensation (13). It may seem puzzling that the same modification is involved in the opposing functions of transcriptional activation and chromosome condensation. One possible explanation is that H3 phosphorylation opens the chromatin fiber, allowing access by nuclear factors to the underlying genome, and that these nuclear factors may then function in a repressive capacity to further condense chromatin (13).

Position-specific histone modifications carry specific signals for functional outcomes, and combinations of modifications can provide a unique way to vary signals and shape different outcomes. In light of this, it is interesting to note that methylation at the adjacent K9 residue could not occur when the S10 was phosphorylated (14), whereas acetylation at the nearby K14 depends upon the preceding S10 phosphorylation, and is itself required for the subsequent methylation of K9 (15). Therefore, S10 must be first phosphorylated and then dephosphorylated in order for K9 methylation to occur. Further studies of this phenomenon determined that trimethylation of K9 is a constant component, whereas the S10 phosphorylation mark is the flexible component of a ‘binary methylation/phosphorylation switch’ that regulates heterochromatin protein-1 interaction with mitotic chromatin at H3K9me3 and dissociation from trimethylated, S10-phosphorylated histone H3- (H3K9me3S10ph-) containing chromatin (16,17).

Nickel (Ni) compounds exhibit carcinogenic properties based on epidemiological, experimental animal and cell culture studies (18–20). However, Ni compounds are found to be non-mutagenic as revealed by many conventional mutagenesis assays (21–23). Epigenetic mechanisms have been implicated in the actions of some mutagenic carcinogens, and it has been hypothesized that DNA methylation and chromatin condensation alter gene expression (24), thus providing a plausible non-genotoxic mechanism to heritable alterations in gene expression induced by Ni.

Exposure of cells to Ni compounds disrupts epigenetic homeostasis. Previous studies have shown that exposure of cells to Ni compounds [soluble nickel chloride (NiCl₂) and insoluble nickel subsulfide (Ni₃S₂)] results in intracellular accumulation of Ni ion, a loss of acetylation at all four core histones, and increased methylation of histone H3 lysine 9 at the global level (25,26). Research on the mechanisms by which Ni compounds alter histone modifications has revealed that Ni exposure decreases histone acetylation by inhibiting histone acetyltransferase activity, but has no effect on histone deacetylases (27). More recently, it has been shown that Ni increases methylation of histone H3 lysine 9 by inhibiting the activity of a histone demethylase with no or very slight inhibition of G9a methyltransferase (26).

Nickel sulfide was shown to induce activation of MAPK signaling (28) and to do so through its induction of oxidative stress. NiCl₂ was reported to induce the phosphorylation of the ERK, p38 kinase and JNK/SAPK in monocyte-derived dendritic cells (29). Moreover, nickel sulfate (NiSO₄) was shown to activate JNK, and target-gene expression upon Ni treatment was significantly affected by JNK inhibition (30).

Ni compounds generate intracellular oxidants after several hours of exposure in cells, as detected by the dichlorofluorescein fluorescent assay method or by electron spin resonance (31,32). It was suggested that Ni-induced reactive oxygen species play an important role in the activation of signaling pathways such as MAPKs.

In this paper, we report the specific and Ni-induced JNK-mediated phosphorylation of H3S10. Owing to the ambiguous nature of the extant data on MAPK-mediated phosphorylation of H3S10, we reasoned that the previous studies of Ni-mediated activation of MAPK (30) provided a key method with which to investigate the mechanism of the phosphorylation of H3S10 through the MAPK cascade signaling. Here, we show unambiguously that the JNK/SAPK, and not the ERK or p38, MAPK pathway is the means by which Ni compounds induce phosphorylation of H3S10.

Materials and methods

Cell culture

Human lung carcinoma A549 cells were cultured in Ham's F-12 K medium (Invitrogen, Fredericks, MD) and human bronchial epithelial Beas-2B cells were cultured in Dulbecco's Modified Eagle Medium (Invitrogen, Fredericks, MD) supplemented with 10% fetal bovine serum (Omega Scientific, Tarzana, CA) and 1% penicillin–streptomycin (Invitrogen, Fredericks, MD). Cells were maintained at 37°C as monolayers in a humidified atmosphere containing 5% CO₂. Cells were passaged at 70–80% confluence by trypsinization. All treatments were administered when cell density reached ∼70–80% confluence.

Chemicals and antibodies

All histone antibodies were from Upstate (Lake Placid, NY). β-Actin antibody was from Sigma (St Louis, MO), and JNK antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibody-bound proteins were detected by using appropriate alkaline phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary antibody (obtained from Cell Signaling Technology, Danvers, MA) and an enhanced chemifluorescent (for AP) or enhanced chemiluminescent (for HRP) western blotting system (from Amersham, Piscataway, NJ). All other reagents were obtained from Sigma unless otherwise specified.

siRNA transfection

siRNA target human JNK was purchased from Dharmacon (Lafayette, CO) and its gene accession number is NM_139049 (Catalog number: L-003514-00). Non-coding siRNA (siControl) was also purchased from Dharmacon; the sequence was not provided (Catalog number: D-001210-03). The A549 cells were seeded in a six-well plate (2.5 × 10⁵ cells per well) and incubated at 37^oC for 24 h. Next, cells were transfected with 100 pmol siRNA and 5 μl Lipofectamine 2000 reagent (Invitrogen; Catalog number: 11668-019) and incubated for 48 h, at which time they were treated with 1.0 mM NiCl₂ and incubated for another 24 h.

Immunofluorescence staining

Cells cultured on slides (BD Falcon) were washed with 1× phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were then permeabilized with 0.2% Triton X-100 at room temperature for 5 min. After three washes with Tris-buffered saline (TBS), cells were quenched in fresh 0.1% borohydride in TBS for 5 min. Cells were then blocked by incubation with blocking buffer (10% horse serum, 1% bovine serum albumin in TBS) for 45 min at room temperature. The primary antibodies that were diluted in 1% bovine serum albumin using TBS were incubated with the coverslips overnight in a cold room. After three washes with TBS, cells were incubated with the fluorescently labeled secondary antibodies for 45 min at room temperature in the dark, followed by another three washes. The coverslips were then mounted using the ProLong Gold Antifade Reagent with 4′,6-diamidino-2-phenylindole and stored at room temperature overnight before being visualized using a fluorescent microscope (Model AX 70; Olympus, Melville, NY).

Histone extraction

Cells cultured in 150 mm dishes were washed with ice-cold 1× PBS twice and lysed in 1 ml NIB buffer (10 mM Tris–HCl, pH 7.4, 2 mM MgCl₂, 3 mM CaCl₂, 1% Nonidet P-40) for 15 min. The pellet was collected by centrifugation at 10 000g for 10 min and washed in 1 ml high salt NIB buffer (500 mM NaCl in NIB) for 15 min. The pellet was collected by centrifugation at 14 000g for 10 min and resuspended in 300 μl 0.4 N H₂SO₄. After 90 min of incubation on ice, the supernatant was collected by centrifugation at 14 000g for 15 min and mixed with 1.2 ml cold acetone overnight at −20°C. The histones were collected by centrifugation at 14 000g for 15 min and air-dried and then were suspended in 4 M urea to make a final histone concentration of 1 mg/ml.

Whole-cell protein extraction

Cells cultured in six-well plates were washed with ice-cold 1× PBS twice and lysed with 100 μl lysis buffer [10 mM Tris–HCl, pH 7.4, 1% sodium dodecyl sulfate (SDS), 1.0 mM sodium orthovanadate] for 15 min. The cells were transferred to an Eppendorf tube and sonicated to reduce viscosity by applying 10 one second pulses with a Branson Sonifier 450. The samples were stored at −20°C until use.

Western blot

Protein concentrations were measured using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA), and the proteins were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene difluoride membrane. To assess protein loading, gels were stained with Bio-Safe Coomassie Stain (Bio-Rad) after transfer. After blocking for 1 h with a freshly prepared 3% dry milk–TBS (0.1 M Tris and 150 mM NaCl) solution (TBS-MLK), membranes were typically incubated overnight at 4°C with gentle agitation in a solution of specific antibodies diluted in TBS-MLK. Membranes were washed by gentle agitation in 0.1 M Tris, 150 mM NaCl and 0.1% Tween-20 three times (5 min per wash) and then incubated with appropriate AP- or HRP-conjugated secondary antibodies for 3 h at 4°C with gentle agitation. After three more washes with 0.1 M Tris, 150 mM NaCl and 0.1% Tween-20, the presence of secondary antibody was detected by chemical fluorescence following an enhanced chemifluorescent (for AP) or enhanced chemiluminescent (for HRP) Western blotting protocol.

Protein extraction for in vitro phosphorylation assays

Cells cultured in 150 mm dishes were washed with ice-cold 1× PBS twice and lysed with 1 ml ice-cold Pagano buffer [20 mM Tris–HCl (pH 7.4), 2 mM dithiothreitol, 0.25 mM ethylenediaminetetraacetic acid, 10 μg/ml leupeptin and 10 μg/ml pepstatin]. The suspension was kept on ice and sonicated as described above. After centrifugation at 14 000g for 10 min, the supernatants were divided into aliquots and stored at −80°C.

In vitro phosphatase assay

Five micrograms of extracted histones dissolved in H₂O were incubated for 1 h with 10 μl of a phosphorylation mix containing 40 mM Tris (pH 7.4), 5 mM MgCl₂, 10% glycerol, 1 mM dithiothreitol and 20 μg of protein extract. The reaction was terminated by addition of 10% volume of SDS–PAGE loading buffer and boiling (100°C) for 5 min. Finally, the samples were resolved by 15% SDS–PAGE, transferred to a polyvinylidene difluoride membrane and Western blotted with antibody against phosphorylated H3S10.

Results

Increased histone H3S10 phosphorylation induced by Ni compounds

Based on their water solubility, Ni compounds can be categorized as soluble (e.g. NiCl₂ and NiSO₄) and insoluble (e.g. nickel sulfide, Ni₃S₂ and nickel monoxide). Occupational exposure to mixed soluble and insoluble Ni compounds has been associated with an increased risk of respiratory cancer (33). Since inhalation is the primary route for human exposure to Ni compounds, the human lung carcinoma A549 cells and human bronchial epithelial Beas-2B cells were utilized as the cell model for the present study. We studied the effect of both soluble and insoluble Ni on histone H3S10 phosphorylation and on the induction of MAPK cascades.

The cytotoxicity of Ni compounds in A549 cells has been previously measured using 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (34), cell growth (35) and colony formation assays (26), demonstrating conclusively that NiCl₂ at ≤1 mM does not significantly impact cell viability. Treatment for 24 or 48 h with either NiCl₂ at 0.5 and 1 mM or Ni₃S₂ at 0.5 and 1.0 μg/cm² show comparable toxicities with one another. Colony formation assay was conducted in Beas-2B cells exposed to various concentrations of Ni ions for 24 h, and the results showed that 0.25 mM NiSO₄ did not have a significant effect on colony formation. So, we used this as well as lower doses of NiS0₄ (0.125 and 0.25 mM) to treat Beas-2B cells for 24 h.

Environmental stimuli, such as arsenite (36), 12-O-tetradecanoylphorbol-13-acetate (12) and ultraviolet radiation (37), have been shown to induce phosphorylation of H3S10 through various signal transduction pathways. Here, A549 cells were treated with soluble NiCl₂ (1 mM) or insoluble Ni₃S₂ (0.5 and 1 μg/cm²) for 24 h. After exposure, the histones were isolated as described in Materials and Methods, and western blots were performed using antibody against P-H3S10. Exposure of A549 cells to Ni compounds resulted in increased phosphorylated H3S10 (Figure 1A). In Beas-2B, a more normal cell than the transformed A549 cell line, Ni ion exposure also increased phosphorylation of histone H3S10 (Figure 1C).

Fig. 1.

Increased phosphorylation of histone H3S10 by Ni compounds. (A) A549 cells were treated with 1.0 mM NiCl₂ or 0.5 or 1.0 μg/cm² Ni₃S₂ for 24 h. Isolated histones were separated by 15% SDS–PAGE and subjected to western blotting with antibody against phosphorylated H3S10 (P-H3S10). Lower panel shows the gel stained with Coomassie Blue after transfer to monitor for histone loading. (B) Increased phosphorylation of histone H3S10 by Ni compounds, as measured by immunofluorescence staining. The A549 cells were treated with 1.0 mM NiCl₂ for 24 h. Cells were fixed and the presence of phosphorylated H3S10 was detected by immunofluorescence as described in Materials and Methods. (C) Beas-2B cells were treated with 0.125 or 0.25 mM of NiCl₂ for 24 h. Five micrograms of histone was used to detect the levels of H3S10 phosphorylation by western blot analysis. Coomassie Blue staining was used to verify that the loading of histones was similar in all lanes.

To confirm that Ni exposure causes increased phosphorylation of H3S10, immunofluorescence analysis was performed. As shown in Figure 1B, there was a clear elevation in the intensity of the fluorescent signal corresponding to phosphorylated H3S10 in cells exposed to NiCl₂. In addition, it was found that the percentage of cells in G₂/M phase (indicated by exceptionally strong fluorescence visible to the naked eye) in exposed and non-exposed cells does not significantly differ. An analysis of the cell-cycle effect of a 24 h exposure to 1 mM NiCl₂ in A549 cells indicated a loss in the amount of cells in S phase (21–12%) and a corresponding increase in the percentage of cells in G₁ (63–73%) but no significant change in cells in G₂/M. Thus, the observed increased levels of phosphorylated H3S10 are not simply due to a higher number of Ni-exposed cells undergoing mitosis as compared with their control counterparts (i.e. those without Ni exposure).

Ni induces phosphorylation of histone H3S10 through JNK–MAPK pathway

Ni-induced phosphorylation of H3S10 may be a consequence of increased H3S10 kinase activity or decreased phosphatase activity. It has been reported that the level of phosphorylated H3S10 in cells increases dramatically in response to stimulation of the MAPK pathway (9). Since this pathway is activated by Ni exposure (30), the role of MAPK in Ni-induced phosphorylation of H3S10 was studied by pretreatment of cells with specific inhibitors of the MAPK pathways. As shown in Figure 2A, exposure of A549 cells to NiCl₂ (1.0 mM) resulted in increased phosphorylation of JNK, whereas the phosphorylation levels of neither ERK nor p38 were affected under the same conditions. Furthermore, pretreatment of the cells with the JNK inhibitor, SP600125 (50 μM), not only abrogated Ni-induced phosphorylation of JNK but also eliminated the Ni-induced increased levels of H3S10 phosphorylation. To confirm the precise role of JNK in mediating the Ni ion-induced phosphorylation of H3S10, JNK was knocked down using RNAi, and this also resulted in a loss of H3S10 phosphorylation in Ni-exposed cells (Figure 2C). In contrast, pretreatment of A549 cells with the ERK or p38 inhibitors, PD 98059 (50 μM) or SB 202190 (2 μM), respectively, had an effect neither on the control levels of phosphorylated JNK nor on the Ni-induced levels of phosphorylated H3S10 (Figure 2B). Collectively, these results suggested that Ni compounds induced H3S10 phosphorylation through the JNK–MAPK pathway.

Fig. 2.

Ni induces phosphorylation of histone H3S10 through the JNK–MAPK pathway. (A) A549 cells treated with 1.0 mM NiCl₂ for 24 h. (B) A549 cells were treated with the JNK inhibitor (50 μM), the ERK inhibitor PD 98059 (50 μM) or p38 inhibitor SB 203580 (2 μM) for 1 h and then with 1.0 mM NiCl₂ for 24 h. (C) A549 cells were transfected with siRNA targeting human JNK and non-coding control siRNA for 48 h and then were treated with 1.0 mM NiCl₂ for another 24 h. Cells were then lysed and Western blots were conducted using antibodies against phosphorylated JNK (P-JNK), JNK, phosphorylated ERK (P-ERK), phosphorylated-p38 (P-p38), phosphorylated H3S10 (P-H3S10), α-tubulin and actin. The membrane was blotted with anti-P-JNK and was reblotted with antibodies against JNK and actin following a stripping procedure. Actin serves as the loading control in (A) and α-tubulin serves as the loading control in (C). The lower panel in (B) shows the core histones stained with Coomassie Blue in the posttransfer gel as a loading control. In (C), phosphorylation of H3S10 signal was quantified using software Image J from National Institutes of Health and normalized with loading control, and the numbers below the figure indicate the quantitative numerical assessment of H3S10 phosphorylation.

Ni does not inhibit H3 phosphatase activity

To further investigate the mechanism of Ni-induced phosphorylation of histone H3S10, we developed an in vitro phosphatase assay to probe any possible dephosphorylation that might occur as a result of Ni treatment. Total histones purified from A549 cells containing basal levels of phosphorylated H3S10 were incubated with cellular extracts from untreated control cells at 37°C. The products were then electrophoretically resolved by SDS–PAGE, and Western blot analysis with the antiphosphorylated H3S10 antibody allowed us to measure the amount of phosphorylated H3S10 remaining after the reaction. A schematic of the in vitro phosphatase assay is shown in Figure 3A.

Fig. 3.

Ni does not inhibit any H3 phosphatase activity. (A) Schematic of the in vitro phosphatase assay. (B) Total histones (5 μg) purified from A549 cells were incubated with 20 μg of cellular extracts for 1 h at 37°C before the reaction was stopped by addition of SDS–PAGE loading buffer. Products were then separated by SDS–PAGE and subjected to western blotting. Remaining P-H3S10 was detected using antibody against P-H3S10. (C) A549 cells were treated with OA (10 and 100 nM) or NiCl₂ (1.0 mM) for 24 h. The cells were lysed and western blots were conducted using an antibody against P-H3S10. The lower panels show the gel stained with Coomassie Blue after transfer to assess whether the loading of histones was similar.

Since okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, has been shown to prevent dephosphorylation of phosphorylated H3S10 in vitro (5), we included it as a positive control in our in vitro phosphatase assay. As shown in Figure 3B, there was a dramatic loss of phosphorylated H3S10 when histones were incubated with control cell extracts in comparison with the levels in the absence of cell extract (i.e. H₂O used instead). Addition of NiCl₂ to the reaction mixture did not prevent this loss, whereas OA prevented the dephosphorylation of phosphorylated H3S10. Additionally, in order to confirm the effect of OA on phosphorylation of H3S10 in living cells, A549 cells were exposed to OA for 24 h. The results showed that OA led to a striking increase in the levels of H3S10 phosphorylation as compared with control cells (Figure 3C). These results indicated that, unlike OA, Ni-induced phosphorylation of H3S10 was not caused by inhibition of any dephosphorylating activity.

Taken together, the above data demonstrate that Ni ion exposure led to the activation of the JNK–MAPK pathway in A549 cells, which resulted in phosphorylation of H3S10. The results from the in vitro phosphatase assay showed that phosphatase activity was not affected by Ni ions and cannot account for the induction of H3S10 phosphorylation. Therefore, it is clear that Ni-induced phosphorylation of H3S10 is the consequence of an increased H3S10 kinase activity resulting from the activation of the JNK–MAPK pathway and is not the result of a decreased, i.e. Ni-inhibited, phosphatase activity.

Discussion

Phosphorylation of H3S10 has been linked to chromosome condensation as well as gene expression, although the mechanisms remain elusive. In this study, we used A549 cells as a model for MAPK cascade-driven phosphorylation of H3S10 induced by Ni ions, because A549 cells are human lung tissue-derived, and this exposure scenario is relevant to the airborne nature of the most common, and the most prevalent, exposures of particulate Ni in humans (33,38). Using kinase inhibitors, we found that Ni, in soluble or insoluble form, induced phosphorylation of H3S10 (Figure 1) through activation of the JNK/SAPK pathway by the upstream phosphorylation of JNK (Figure 2). Ni's effect was quite specifically mediated by this MAPK cascade, as inhibition of the ERK1/2 or p38 kinase cascade pathways by inhibitors specific for these MAPKs (PD 98059 and SB 203580, respectively) had no effect on the Ni-induced phosphorylation of H3S10 (Figure 2B). Additionally, knock down of JNK with RNAi inhibited the Ni ion-induced phosphorylation of H3S10. We believe that this is the first report showing that Ni's induction of histone H3 phosphorylation at serine 10 was mediated specifically by JNK and did not involve either the ERK or p38 MAPKs.

Our investigation of the underlying mechanism revealed that Ni exposure led to phosphorylation of JNK/MAPK, while phosphorylation of ERK and p38 remained unaffected. This Ni-activated JNK was required for phosphorylation of H3S10, as inhibition of JNK by both the inhibitor and RNAi significantly abolished Ni-induced H3S10 phosphorylation. Moreover, inhibition of phosphatase activity by Ni has been ruled out as a possible mechanism of the observed increase in H3S10 phosphorylation in Ni-treated cells because, in comparison with a known phosphatase inhibitor, OA, Ni ions did not attenuate the dephosphorylation of H3S10 (Figure 3B and C).

Based on both the studies reported here, and some of our other recent findings (25,39,40), we propose a model of the epigenetic effects of Ni compounds. To this model we can now add the phosphorylation of H3S10 mediated by MAPK cascades, bearing in mind that our studies show the specific participation of JNK, even to the exclusion of other MAPK family members. JNK has been implicated in H3S10 phosphorylation in other studies (12,30,36), but only in an apparently auxiliary role, and H3S10 phosphorylation has been thought to depend on other kinases, such as ERK, rather than JNK (12,36). There are many kinases that have been shown to phosphorylate H3S10, and it is beyond the scope of this paper to consider them all: further details of these kinases are reviewed in ref. (9). Although the JNK pathway delivers a signal that results in H3S10 phosphorylation, JNK does not phosphorylate it directly (9,37): its phosphorylation is presumed to be effected by kinases downstream of JNK (37).

Animal tissues exposed to insoluble Ni compounds (e.g. nickel sulfide and Ni₃S₂) retain particulate Ni within those tissues and the Ni is gradually assimilated by the cells through phagocytosis, resulting in Ni ion accumulation within the cell and the nucleus. In contrast, soluble Ni compounds (e.g. NiCl₂ and NiSO₄) are easily cleared away from the exposed tissue by biological fluids, but Ni ions derived from soluble Ni compounds may bind tightly to proteins and amino acids that they encounter within the cell. Ni ions structurally and/or chemically resemble essential metal ions such as zinc, copper, iron and manganese and thereby compete with them for binding to essential enzymes, with subsequent disruption of enzymatic processes. Effects on histone-modifying enzymes, which would contribute to Ni-induced epigenetic changes, could be expected to be particularly pronounced. These effects would include: inactivation of histone acetyltransferase that may lead to histone deacetylation; inactivation of histone demethylase that may increase histone methylation; and, inactivation of a histone deubiquitinase (39) causing increases in histone ubiquitination.

Histone modifications can be considered a program of response to environmental stimuli that gives the cell a distinctly greater responsiveness to stresses, which translates into greater survivability. This program provides an epigenetic code through which chromatin can initiate and preserve heritable patterns of gene expression throughout, and even beyond, the life of the organism. The central theme to our current understanding of the role of chromatin in cellular function is that both the combinatorial nature of the interpretation of histone marks by the cell and the chromatin structure induced by the histone modifications play a role in the epigenetic process (2). Together with DNA methylation, RNA associations and other molecular interactions, the histones play out this epigenetic code locally, in specific gene activation and silencing events, and globally, in developmental cycles.

Ni-induced specific and global alterations of histone modifications may work in combination to bring about an efficient repression of specific genes (e.g. tumor suppressor genes). In particular, these epigenetic silencing mechanisms may target specific genes, thereby marking chromatin in such a way that this silencing state is inherited over many cell generations. In this study, we have shown an example of Ni's ability to specifically and markedly alter the phosphorylation state of H3S10. JNK is a specific mediator of this Ni-induced alteration, and this may correspond to a collateral alteration of a second histone modification induced by Ni that somehow signals to, or is signaled by, P-H3S10 (13,41).

In summary, this study provides novel evidence that Ni induces phosphorylation of H3S10 through the activation of JNK, and not ERK or p38, MAPK. We illustrate the probable kinase cascade by use of type-specific kinase inhibitors as well as knock down of JNK with RNAi, and we further delineate the probable mechanism of this Ni-induced phosphorylation by discounting the possibility of inhibition of phosphatases through our comparison of Ni with a known phosphatase inhibitor, OA. In the future, we hope to find the precise kinase responsible for histone H3’s phosphorylation. We would eventually like to uncover other histone modifications, also induced by Ni, that specifically cross talk with the P-H3S10 mark, thus elucidating a Ni-specific epigenetic program response or deregulation.

Funding

National Institutes of Environmental Health Sciences (ES00260, ES014454, ES005512, ES010344 and T32-ES07324); National Cancer Institute (CA16087).

Glossary

Abbreviations

AP

alkaline phosphatase

ERK

extracellular signal-regulated kinase

HRP

horseradish peroxidase

H3S10

histone H3 at serine 10

IE

immediate-early

JNK

c-jun N-terminal kinase

MAPK

mitogen-activated protein kinase

Ni

nickel

NiCl₂

nickel chloride

Ni₃S₂

nickel subsulfide

Niso₄

nickel sulfate

OA

okadaic acid

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

SAPK

stress-activated protein kinase

SDS

sodium dodecyl sulfate

TBS

Tris-buffered saline