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.

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.

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.

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.