Ni is a major component of ambient air particulate matter (1
) that is associated with inappropriate immune responses (35
), airway hyperresponsiveness (7
), and increased cardiopulmonary disease (1
). Mechanisms of action for inhaled Ni that are independent of its antigenicity remain poorly defined. It is evident, however, that without induction of protective or adaptive genes, such as increasing MT in airway epithelium, the pathogenic responses to inhaled Ni are greatly enhanced (20
). These studies used the BEAS-2B cells to model the terminal bronchiolar epithelial responses to Ni. While these cells are SV40 immortalized, we have demonstrated that their responses to metals are nearly identical to primary normal human bronchiolar epithelial cells cultured on an air/liquid interface (27
) and mouse bronchiolar epithelium in vivo
). Induction of MT after Ni exposure in these cells closely matches responses observed in mouse lungs (33
), and thus the mechanisms for this induction in BEAS-2B cells are likely to be broadly applicable. As indicated, MT induction by Ni and other toxic metals is a protective response. However, since only Zn has been shown to directly induce the gene, it remains unclear how Ni and other metals signal for indirect gene activation. The current studies indicate that Ni transcriptionally activates MT2A by first mobilizing intracellular Zn to bind with MTF-1. Ni does not elicit ROS generation to mobilize labile Zn pools, but may displace Zn from thiols or interact with regulatory reduced thiols to stimulate MTF-1 transactivation.
MT regulates zinc homeostasis and is induced by various stressors, including metals (13
). We demonstrated that Ni exacerbated acute lung injury in MT null mice (20
), acknowledging its protective function in the lung. MT is basally expressed in the airway epithelium (38
) and inducible by exogenous chemicals (39
). Our data in corroborate previous findings that Ni induces MT2A mRNA (13
). This induction was rapid and time-dependent, with the highest expression after 4 hours of Ni exposure. Ni also increased MT protein levels after 4 hours of Ni exposure (). There are multiple transcription factors that drive MT2A gene expression, but the activation of MTF-1 has an essential role by binding to MREs present in the promoter region of MT2A (24
). Ni increased transactivation of MRE (), which occurred through the increased binding activity of MTF-1. Exposure to Ni facilitated the translocation of MTF-1 to the nucleus (). Also, Ni did not induce MT in cells deficient in MTF-1 (). However, since only Zn can directly activate and increase DNA binding of MTF-1 (24
), the data support the hypothesis that Ni-induced MT2A is caused by redistributed Zn stimulating MTF-1 DNA binding.
Soluble Ni increased intracellular ROS (), which is consistent with our previous report using particulate Ni3
). Likewise, NAC and AA prevented Ni-increased intracellular ROS levels. However, in the previous report, neither antioxidant affected transcriptional induction of SERPINE1 after Ni exposures (34
). In contrast, in the current study, only NAC prevented Ni-induced MT2A transcript levels (). Thus, production of ROS may not be involved in Ni-induced MT2A, but free thiols may regulate the ability of Ni to induce certain genes. NAC acts as an antioxidant by increasing the intracellular free thiol pool and increasing synthesis of glutathione (GSH) (41
). GSH and oxidized gluthothione (GSSG) are key regulators of the release of Zn from MT (42
). GSSG interacts with MT to facilitate the transfer of Zn from MT (43
). However, in reducing conditions, when GSH concentrations are high, Zn remains bound to MT (42
). The data from suggest that adding NAC pushed the thiol balance toward enhanced Zn binding to MT and prevented its mobilization to interact with MTF-1. Exposure to high levels of Ni has been shown to reduce the intracellular GSH content and increase the GSSG/GSH ratio (44
). Hence, it is possible that in our system Ni decreased the GSH content of the cell to facilitate Zn mobilization. However, this is unlikely given the low level of oxidant generation in response to Ni and the overwhelming amount of intracellular GSH relative to the amount of Ni added.
Transcriptional activation of MT2A by Ni required Zn mobilization, since pretreatment with TPEN, a Zn chelator, abrogated Ni-induced MRE transactivation and increased MT2A transcripts (). Although TPEN is often used as a Zn-specific chelator, it has affinities for other divalent metals including Ni (15
). However, the stochiometry of TPEN metal chelation is such that the low concentration used in the present study was sufficient to bind only 5% of the added Ni. Furthermore, the ability of Zn, but not Ni, to rescue the response observed () indicates that the added TPEN predominantly bound the intracellular Zn released by Ni to inhibit Ni-stimulated MT induction. This inhibition did not result from toxicity, since TPEN was added at a concentration that did not cause cell death (data not shown). Thus, the observed results consistently support a role for mobilization of endogenous Zn in Ni-stimulated signaling. Moreover, increased FluoZin-3 fluorescence after Ni exposure () established that Ni increases free intracellular Zn levels, as was previously demonstrated in hepatocytes (46
In summary, we demonstrated that Ni increases free intracellular Zn levels to induce MT2A transcript levels. This was not at the level of MT itself, since Ni has a low affinity for MT, relative to Zn, and cannot displace Zn from MT (15
). The precise mechanism of how Ni is redistributing Zn has not been investigated fully, but remains an area of interest. However, this study elucidates the mechanism of Ni-induced MT2A and provides a better understanding of how metals other than Zn produce this adaptive response. This understanding may offer insight for developing protective strategies to reduce pathogenic airway responses to inhaled metals.