Regulation by ceramide of epidermal growth factor signal transduction and mitogenesis in cell lines overexpressing the growth factor receptor

Abstract

Ceramide has emerged as a pleiotropic signal mediator of cellular responses including differentiation, proliferation, cell cycle arrest and apoptosis. In the present study we evaluated the effect of cell permeant ceramide analogues on ligand-induced tyrosine phosphorylation of the EGF receptor (EGFR), phospholipase C gamma (PLC gamma) activity and cell proliferation. Treatment with N-acetylsphingosine (C2-cer) and N-hexanoylceramide (C6-cer) prevented EGF-induced tyrosine trans-phosphorylation of the receptor in two different cell lines overexpressing the human EGFR (A431 and EGF-T17 cells). In contrast, treatment of A431 and EGFR-T17 cells with C2-cer or C6-cer did not affect the ligand binding capacity of the receptor, an effect that was however: observed after TPA-induced activation of PKC. In addition EGF-stimulated PLC gamma activity was transiently decreased in A431 cells treated with C6-cer and only a modest, albeit significant reduction on ligand-induced H-3-InsP(3) generation was observed in EGFR-T17 cells pretreated with ceramide. We also examined the effect of C2-cer on serum (A431)- or EGF (EGFR-T17)-induced cell proliferation Treatment of EGFR-T17 cells with C2-cer (0.1-10 muM) did not affect cell viability, but prevented EGF-induced H-3-thymidine incorporation in a dose-dependent manner. In contrast, H-3-thymidine incorporation in serum-stimulated A431 cells decreased only at the higher doses of C2-cer used (1-10 muM), being this effect accompanied by a slight, albeit significant (20-25%), reduction in cell viability.