A cross-talk between NFAT and NF-κB pathways is crucial for nickel-induced COX-2 expression in Beas-2B cells

Abstract

Cyclooxygenase-2 (COX-2) is a critical enzyme implicated in chronic inflammation-associated cancer development. Our studies have shown that the exposure of Beas-2B cells, a human bronchial epithelial cell line, to lung carcinogenic nickel compounds results in increased COX-2 expression. However, the signaling pathways leading to nickel-induced COX-2 expression are not well understood. In the current study, we found that the exposure of Beas-2B cells to nickel compounds resulted in the activation of both nuclear factor of activated T cell (NFAT) and nuclear factor-κB (NF-κB). The expression of COX-2 induced upon nickel exposure was inhibited by either a NFAT pharmacological inhibitor or the knockdown of NFAT3 by specific siRNA. We further found that the activation of NFAT and NF-κB was dependent on each other. Since our previous studies have shown that NF-κB activation is critical for nickel-induced COX-2 expression in Beas-2B cells exposed to nickel compounds under same experimental condition, we anticipate that there might be a cross-talk between the activation of NFAT and NF-κB for the COX-2 induction due to nickel exposure in Beas-2B cells. Furthermore, we showed that the scavenging of reactive oxygen species (ROS) by introduction of mitochondrial catalase inhibited the activation of both NFAT and NF-κB, and the induction of COX-2 due to nickel exposure. Taken together, our results defining the evidence showing a key role of the cross-talk between NFAT and NF-κB pathways in regulating nickel-induced COX-2 expression, further provide insight into the understanding of the molecular mechanisms linking nickel exposure to its lung carcinogenic effects.

Fig. (1). Induction of COX-2 by nickel in Beas-2B cells

A, 8×10³ Beas-2B COX-2-luc massl cells seeded into each well of a 96-well plate were treated with either 0.5 mM NiCl₂ or 2 µg/cm² NiS for various time points as indicated (A, left). For dose response studies, the cells were treated with various doses of nickel compounds for 48 h (A, right). The cells were then extracted with lysis buffer for the luciferase activity assay using the Promega luciferase assay kit. The results were expressed as the COX-2 induction relative to the medium control (relative COX-2 induction). Each bar indicates the mean and standard deviation of triplicate wells. The asterisk (*) indicates a significant increase from medium control cells (P<0.05). B, Beas-2B cells (2×10⁵) were seeded into each well of 6-well plates, and cultured in 10% FBS DMEM at 37°C. When the cell density reached 70–80%, the culture medium was replaced with 0.1% FBS DMEM. After being cultured overnight, the cells were exposed to nickel compounds at the indicated dosages for 24 h or 48 h. The cells were extracted for Western blot using specific antibodies against COX-2 and β-Actin, respectively.

Fig. (2). Essential role of NFAT pathway in COX-2 induction by nickel compounds in Beas-2B cells

A. Beas-2B-NFAT-luc massl cells (8×10³) were seeded into each well of a 96-well plate. After being cultured at 37°C overnight, the cells were treated with either 0.5 mM NiCl₂ or 2 µg/cm² NiS for various time points as indicated (left). For dose response studies, the cells were treated with various doses of nickel compounds for 24 h (right). Then the luciferase activity was determined. The results were expressed as the NFAT activation relative to the medium control (relative NFAT activity). Each bar indicates the mean and standard deviation of triplicate wells. The asterisk (*) indicates a significant increase from medium control cells (P<0.05). B. 8×10³ Beas-2B-NTAT-luc massl cells (left) or Beas-2B-COX-2-luc massl cells (right) seeded into each well of a 96-well plate were pretreated with CsA of various doses as indicated and then exposed to 0.5 mM NiCl₂. Then the luciferase activity was determined. The asterisk (*) indicates a significant decrease from the nickel-treated cells without CsA (P<0.05). C. 8×10³ Beas-2B COX-2-luc siNTAT NFAT massl cells or Beas-2B-COX-2-luc massl cells seeded into each well of a 96-well plate were exposed to 2 µg/cm² NiS for various time points (left) or various doses of NiS for 48 h (right). Then the luciferase activity was determined. The asterisk (*) indicates a significant decrease from Beas-2B-COX-2-luc massl cells (P<0.05). D. Both Beas-2B siNFAT and Beas-2B vector control cells (2×10⁵) were seeded into each well of 6-well plates, and cultured in 10% FBS DMEM at 37°C. When the cell density reached 70–80%, the culture medium was replaced with 0.1% FBS DMEM. After being cultured overnight, the cells were treated with either 0.5 mM NiCl₂ or 2 µg/cm² NiS for 48h. The expression of COX-2 protein was determined using Western blot.

Fig. (3). A cross-talk between the activation of NFAT and NF-κB due to nickel exposure

A. Beas-2B-NF- B-luc DN-NFAT massl cells or Beas-2B-NF-B-luc massl cells (8 10³) seeded into each well of a 96-well plate were treated with various doses of nickel compounds. Then the luciferase activity was determined as described in “Materials and Methods” section. The asterisk (*) indicates a significant decrease from Beas-2B-NF-B-luc massl cells (P < 0.05). B. Both Beas-2B siNFAT and Beas-2B vector control cells (2 10⁵) were seeded into each well of 6-well plates, and cultured in 10% FBS DMEM at 37ûC. When the cell density reached 70–80%, the culture medium was replaced with 0.1% FBS DMEM. After being cultured overnight, the cells were treated with 3 g/cm² NiS for either 30min or 180min. The expression of p-IKK, IKK , and IKK was determined using Western blot as previously described. C. 8 10³ Beas-2B NFAT-luc IKK-KM massl cells or Beas-2B NFAT-luc massl cells seeded into each well of a 96-well plate were exposed to various doses of nickel compounds. Then the luciferase activity was determined. The asterisk (*) indicates a significant decrease from Beas-2B NFAT-luc massl cells (P < 0.05).

Fig. (4). The essential role of ROS in the activation of NFAT and NF-κB, and the induction of COX-2 upon nickel exposure

A. Beas-2B-NFAT-luc massl cells or Beas-2B-NF-κB-luc massl cells (8×10³) seeded into each well of a 96-well plate were pretreated with 250U/ml SOD, 20mM NAC, 50000U/ml catalase, or 600mM sodium formate for 1 h. Then, the cells were treated with lmM NiCl₂ or 2µg/cm² NiS for additional 24 h in the presence of the same reagents. And the luciferase activity was determined and the asterisk (*) indicates a significant decrease from nickel-treated cells (P<0.05). B. 8×10³ Beas-2B-NFAT-luc massl cells (left) or Beas-2B-COX-2-luc massl cells (right) seeded into each well of a 96-well plate were pretreated with 20000U/ml (20KU/ml) catalase for 1 h. Then, the cells were treated by 2µg/cm² NiS for additional 24 h in the presence of catalase. The luciferase activity was determined and the asterisk (*) indicates a significant decrease from the cells treated by nickel alone (P<0.05). C. Identification of the Cat H₂O₂ scavenging activity was performed as described in “Materials and Methods.” The asterisk (*) indicates a significant decrease from vector control cells (P<0.05). D. Beas-2B CAT massl cells, Beas-2B mCAT mass2 cells, or Beas-2B vector control cells (2×10⁵) were seeded into each well of 6-well plates, and cultured in 10% FBS DMEM at 37°C. When the cell density reached 70–80%, the culture medium was replaced with 0.1% FBS DMEM. After being cultured overnight, the cells were treated with various doses of NiS for 24 h. Then the expression of COX-2 was determined using Western blot as previously described. E. Schematic illustration of cell signaling pathways involved in nickel-induced COX-2 expression.

Fig. (5)

The proposed mechanisms for the mediation of chronic lung inflammation and carcinogenesis upon nickel exposure.