Efeito dos ligantes do Receptor do Fator de Crescimento Epidermal (EGFR) para sua translocação nuclear

Abstract

The epidermal growth factor receptor (EGFR) once in touch with their ligands is activated and becomes able to generate a range of signals such as proliferation, differentiation, migration and cell survival. Recent studies demonstrated that the activated EGFR can generate intranuclear effects, giving rise a new signaling mode. The receptor overexpression in the nucleus is related with tumor treatment resistance and it agressiveness. Since recent data show the importance of nuclear EGFR for tumor signaling, this study aimed to evaluated the nuclear translocation of EGFR stimulated by the ligands EGF, HB-EGF, TGF-, -cellulin, Amphiregulin and Epiregulin. For that, we used the hepatic human endothelial tumor cells - SKHep-1, which express high levels of EGFR. We observed that translocation to the nucleus happens after stimulation with EGF, HB-EGF, TGF- and -cellulin and the higher the concentration of stimulus, the greater the rate of nuclear translocation. The ligands Amphiregulin and Epiregulin did not generate nuclear translocation of EGFR. The activation of tyrosines, serines and threonines in the carboxiterminus of EGFR were evaluated by western blot after stimulus with 100 ng/mL of which ligand and we observed a different profile of phosphorylation of specific residues. In general, the binding sites that are less phosphorylated are the ones stimulated by the ligands Amphiregulin and Epiregulin. The cellular migrations experiment was performed to evaluate whether the EGFR ligand were efficient in the wound healing in the cell monolayer. The ligands EGF, HB-EGF, TGF-, -cellulin and Epiregulin showed a higher wound closure rate when compared with the groups Amphiregulin and serum free. Finally the growth curve and the clonogenic assay indicated that the ligands were not able to generate cellular division and colony formation when used alone in the serum free medium. All together, these data indicated that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF- and -cellulin and the same ligands generate more phosphorylation of tyrosines in the carboxiterminus of EGFR.