Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

Abstract

Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-l-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H2O2, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H2O2. Furthermore, both arsenic and H2O2 were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation.

Keywords: Akt; Arsenic; Cytoplasmic translocation; EZH2; Oxidative stress; Phosphorylation.

Fig. 1

Involvement of oxidative stress in As³⁺-induced kinase activation and EZH2 phosphorylation in BEAS-2B cells. (A) BEAS-2B cells were treated with 20 µM As³⁺ for 0, 1, 2, or 4 h with or without NAC pretreatment for 2 h. S21 phosphorylation of the EZH2 (pEZH2^S21) protein was determined by Western blotting. The most right lanes (0 h As³⁺) are NAC only treatment. (B–D) The activation of Akt (B), STAT3 and JNK (C), and Erk and p38 (D) were determined in the BEAS-2B cells treated with 20 or 80 µM As³⁺ for 2 h in the presence or absence of NAC pretreatment for 2 h. In each panel, the most right lanes (without As³⁺, —) are NAC only groups. (E) H₂O₂ induces pEZH2^S21 and Akt activation in a time-dependent manner. (F) Dose-dependent activation of Akt kinase in the BEAS-2B cells treated with the indicated concentrations of H₂O₂ for 5 min.

Fig. 2

Oxidative stress contributes to As³⁺-induced EZH2 phosphorylation and kinase activation in A549 cells. (A) A549 cells were treated with 20 µM As³⁺ for the indicated time with or without NAC pretreatment for 2 h. The levels of pEZH2^S21 and Akt activation were determined by Western blotting. The NAC only groups were indicated as 0 h As³⁺ treatment. (B) Dose- and time-dependent EZH2 phosphorylation and kinases activation in A549 cells treated with H₂O₂. (C) Lower concentrations of As³⁺ induce JNK but not Akt activation in the A549 cells treated with As³⁺ for 72 h. (D) NAC was unable to inhibit lower concentration As³⁺-induced EZH2 phosphorylation in the cells cultured for 72 h.

Fig. 3

Both As³⁺ and H₂O₂ induce the interaction of Akt and EZH2 and Akt-dependent phosphorylation of the exogenous EZH2 overexpressed in HEK-293 cells. (A) HEK-293 cells were transfected with 3myc-tagged EZH2 expression vector for 24 h followed by the treatment of the cells with 20 µM As³⁺ for 2 h in the presence or absence of 10 mM NAC, followed by Western blotting using the indicated antibodies. (B) The transfected HEK-293 cells were treated with As³⁺ or H₂O₂, followed by Western blotting as in (A). (C) Anti-myc tag or control IgG was used in immunoprecipitation (IP) to pull down the exogenousmyc-tagged EZH2. The IP was then subjected to Western blotting using antibodies recognizing the phosphorylated Akt substrate motif (RXRXXS*/T*), myc tag, pAkt, and Akt.

Fig. 4

Both As³⁺ and H₂O₂ induce cytoplasmic localization of the EZH2 protein in BEAS-2B cells. (A) BEAS-2B cells expressing GFP-EZH2 were treated with 20 µM As³⁺ for 2 h. The intracellular distribution of EZH2 was determined by immunofluorescent microscopy. (B) NAC prevented cytoplasmic localization of the EZH2 protein induced by As³⁺ in BEAS-2B cells transfected with GFP-EZH2. H₂O₂ was used as a control of ROS. (C) Statistical analysis of the cytoplasmic translocation ratio of the EZH2 protein in the cells treated with As³⁺ in the presence or absence of NAC. Data are expressed as the mean ± SD, n=3, ** p < 0.05.

Fig. 5

Increased cytoplasmic localization of the phosphorylated and total EZH2 in BEAS-2B cells treated with As³⁺ or H₂O₂. (A) BEAS-2B cells were treated with 20 or 80 µM As³⁺ for 2 h with or without NAC pretreatment. Cellular fractions were made to extract the cytoplasmic and nuclear proteins. Lamin A/C and GAPDH were used as indications of the nuclear and cytoplasmic fractions. Lanes 4 and 10 are NAC only groups. Right panel shows semi-quantification of the ratio between pEZH and EZH2 in the cytoplasm. (B) BEAS-2B cells were treated with 0.2 mM H₂O₂ for the indicated times. The levels of phosphorylated EZH2, total EZH2, lamin A/C, and GAPDH were determined in the cytoplasmic and nuclear fractions, respectively. Right panel shows semi-quantification of the ratio between pEZH and EZH2 in the cytoplasm.