OXIDATIVE STRESS INDUCES CROSS-REGULATION BETWEEN EGFR AND BER PATHWAYS IN HUMAN THYROCYTES

Introduction. The main clinical concern about thyroid nodules (TNs) is their possible hidden malignancy. To date mechanisms involved in TNs malignant transition are unresolved. In thyroid H2O2 is produced in large quantities and may play a role in the pathogenesis of TNs. Increased levels of 8-oxo-guanidine DNA adducts were recently detected in TNs and contribute to mutagenesis, which, in turn may result in cell proliferation and survival. DNA base lesions, generated by an excess of reactive oxygen species, are physiologically removed by Base Excision Repair (BER) pathway. A link between Epidermal Growth Factor Receptor (EGFR) signaling and DNA repair mechanisms has been documented, however, their cross-regulation is poorly understood in thyroid tissue pathophysiology. This study investigated the mechanisms involved in cross-regulation between EGFR and BER molecular pathways in normal human thyrocytes after H2O2 exposure and EGF treatment. Materials and Methods. Normal human thyroid epithelial cells, Nthy-ori3-1, were treated with H2O2, alone or combined with EGF and inhibitors PD98059, and LY294002 to specifically block the MAPK (Erk) kinases and PI3-kinase pathways. EGFR and BER pathways expression and activation were tested by Western Blot and quantitative gene expression (qRT-PCR). Results. In response to H2O2, alone or combined with EGF, we observed a time-dependent increase of OGG1 and MUTYH gene expression that was reversed by EGF treatment and by LY alone or combined with H2O2. EGF alone induces EGFR over-expression as well as LY, alone and H2O2 combined. Noteworthy H2O2, alone and combined with EGF, induced a fast kinetic of ErbB2 gene expression. In contrast LY, alone and combined with H2O2, reduced its expression. These results indicated that ErbB2 is modulated in response to oxidative stress through an Akt-dependent mechanism. Surprisingly, PD enhanced the ErbB2 expression compared with untreated and H2O2-treated cells. Therefore, H2O2 treatment, alone or combined with EGF, LY, and PD resulted in EGFR activation at tyrosine residue 1068. Similar results were obtained analyzing MAPK phosphorylation except after co-treatment with EGF and H2O2. Finally, the analysis of PARP1 expression level showed a moderate reduction after treatments with H2O2 combined with EGF and inhibitors. Conclusion. Our observations present the first evidence that a pronounced stress with H2O2 may induce a cross-regulation between EGF and BER pathways in thyroid epithelial cells.