Exposure to B a P is an epidemiologically
Exposure to B[a]P is an epidemiologically proven cause of lung cancer (Hecht, 2003; Rojas et al., 2004; Alexandrov et al., 2010), and the formation of B[a]PDE-N2-dG adducts is considered to be the critical event in lung tumorigenesis by B[a]P. On the other hand, there is evidence suggesting that the AhR and MAPK signaling pathways interact during the initiation and promotion of human tumors, producing disturbances in the cell cycle, proliferation and apoptosis, along with the toxic effects caused by the metabolic transformation of B[a]P into carcinogenic metabolic products (Perez et al., 2008; Chramostová et al., 2004; Andrysík et al., 2006; Hoffer et al., 1996; Tan et al., 2002, Tan et al., 2004; Ding et al., 2009; Occhi et al., 2015; Puga et al., 2009). However, the involvement of MAPK in the biotransformation of AhR ligands, such as B[a]P, is not clear. Additionally, the molecular cross talk between AhR and the MAPK cascade has not been analyzed in non-tumorigenic bronchial epithelium. Thus, the present study was designed to answer the question whether the kinases, ERK 1/2, are involved in the AhR-dependant metabolic activation of B[a]P and B[a]PDE-N2-dG adduct formation in the immortalized human bronchial epithelial cell line, BEAS-2B. We first observed that B[a]P was not cytotoxic for BEAS–2B cells. A modest but not significant reduction in viability was detected after 36 h incubation, suggesting that toxic effects might only be seen at longer exposure times. This is in agreement with a recent report showing that exposure of VH10 fibroblasts to the B[a]P metabolic product, BPDE, for up to 72 h increased the apoptosis frequency, and after 14 days of exposure the metabolic competence was dramatically reduced (Christmann et al., 2016). A well-documented effect of exposure to B[a]P is the AhR-associated induction of CYP1A1 (Nebert et al., 2004; Courcot et al., 2012; Bersten et al., 2013; Uppstad et al., 2010; Kim et al., 2004). Accordingly, a significant increase of CYP1A1 transcription and protein levels were observed in BEAS–2B 360A with all concentrations of B[a]P tested. Previously, published data indicated that B[a]P or its metabolites can modulate the MAPK pathway, preceding or coinciding with AhR activation (Andrysík et al., 2006; Hoffer et al., 1996; Tan et al., 2002, Tan et al., 2004; Ding et al., 2009; Occhi et al., 2015) and, as a consequence, disturbs cellular processes. In spite of this knowledge, there is no evidence of the participation of ERK 1/2 in the AhR-dependant metabolic activation of B[a]P and its related genotoxic effects in lung cells. Here, we have shown that exposure to B[a]P rapidly leads to activation of the ERK 1/2 kinases. This means that B[a]P can induce both, transcriptional and non-transcriptional effects in lung cells. Reports using different cell models and various AhR ligands indicate that ERK kinases might be involved in the AhR pathway (Tan et al., 2002, Tan et al., 2004; Ding et al., 2009). Here, by using the chemical MEK 1/2 inhibitor, U0126, we found that inhibition of ERK 1/2 altered the AhR pathway by decreasing the CYP1A1 protein induction. These data suggest that ERK 1/2 may participate in the CYP1A1 induction in lung cells. It should be noted that the chemical inhibitor, U0126, has been related to AhR-mediated transcriptional activation in non-lung cellular models (Andrieux et al., 2004). Although our results showed that incubating cells with U0126 alone did not induce an increment in CYP1A1 expression, we consider that a more appropriate approach to demonstrate the participation of ERK 1/2 in the transformation of bronchium epithelial cells would be the inhibition of the kinase using specific knock down systems such as the introduction of a dominant-negative mutant of ERK 1/2 to prevent kinase activation, or by using specific shRNA. Production of BPDE-N2-dG adducts is a central step leading to lung carcinogenesis, and it has always been associated with the canonical B[a]P/AhR/CYP1A1 pathway. Interestingly, we demonstrated that blocking ERK 1/2 phosphorylation inhibited induction of CYP1A1 and consequently reduced the formation of BPDE-N2-dG adducts, suggesting that ERK 1/2 activity may participate, at least in part, in the genotoxic damage produced by B[a]P. In concordance with our observations, there are experimental data suggesting that induction of CYP1A1 can occur through non-canonical pathways; furthermore, induction of CYP1A1 can take place independently of AhR (Delescluse et al., 2000). Experimental data in cancer-derived cell models suggest that the AhR-free tyrosine kinase, Src, can mediate induction of the MAPK cascade by activating membrane receptors in the absence of specific ligands (Xie et al., 2012; Pontillo et al., 2011). Based on this evidence, we hypothesized that Src could provide the link between the B[a]P-activated AhR and ERK 1/2 pathway in BEAS–2B cells. We showed that the inhibition of Src leads to reduction of both phosphorylation of ERK 1/2 kinases and the amount of CYP1A1 protein. Our results suggest that Src is an upstream mediator of ERK 1/2 activation, and the consequent induction of CYP1A1 in non-tumor bronchium epithelial cells exposed to B[a]P. Some reports suggest that the progression of the AhR pathway depends on phosphorylation of various components of the AhR-associated complex. For instance, it has been shown that phosphorylation of the AhR at tyrosine residues regulate its ability to bind to DNA (Minsavage et al., 2003). In addition, phosphorylation of HSP90 at serine/threonine residues modulates the formation of a functional cytosolic AhR multiprotein complex in vitro (Ogiso et al., 2004). Protein kinases, such as PKC (protein kinase C) and PTKs (protein tyrosine kinases), have been implicated in the regulation of AhR activity (Backlund and Ingelman-Sundberg, 2005). However, the specific kinases involved in AhR signaling have not been characterized yet. We have observed that active ERK 1/2 participates in the AhR-dependant CYP1A1 induction, which may be explained by previous observations showing that MAPKs are able to enhance transcription of ARNT; this enhancement in turn may increase transcription of AhR-target genes (Backlund and Ingelman-Sundberg, 2005). Normally, ERK 1/2 is activated by upstream kinases belonging to a cell receptor/ligand-associated signaling cascade. However, in our system no specific receptors were stimulated. Thus, we hypothesized that an intermediate kinase might be responsible for the phosphorylation of ERK 1/2. Former studies have demonstrated that Src mediates phosphorylation of ERK 1/2 in different types of cells, including lung fibroblasts (Tanaka et al., 2004; Li et al., 2013; Lee et al., 2014; Ikuta et al., 2009). In agreement with preceding reports, we observed that inhibition of Src completely abolished B[a]P–induced ERK 1/2 phosphorylation, suggesting that Src might participate in a receptor-independent mechanism leading to the activation of the MAPK signaling pathway in BEAS–2B cells. On the other hand, our results showed that Src is also a regulator of AhR nuclear translocation in BEAS–2B cells, since inhibition of Src with dasatinib hampered AhR nuclear translocation promoted by B[a]P. Src has already been acknowledged as a regulator of both ligand-dependent and ligand-independent AhR signaling (Backlund and Ingelman-Sundberg, 2005), but direct participation on nuclear translocation in lung cells had not been reported, the only evidence showing that Src regulates nuclear translocation of AhR was found in a model of prostate cancer (Ghotbaddini et al., 2017). It has been proposed that intracellular localization of AhR is regulated by phosphorylation of residues in the proximity of its N-terminal domain (Ikuta et al., 2009); our observations suggest that Src may be the kinase producing the phosphorylation required for AhR translocation. However, experimental procedures will be needed, as a ChIP assay, in order to confirm the Src participation in AhR translocation and correct DNA binding. Our results showed that the canonical AhR pathway is the principal route involved in the B[a]P-adducts formation. However, Src-mediated phosphorylation of both AhR and ERK 1/2 is required for an adequate AhR signaling pathway, B[a]P metabolic processes and their related genotoxic damage production.