TYROSINE KINASE INHIBITORS AND ANTIOXIDANTS MODULATE NF-KAPPA-B AND NOS-II INDUCTION IN RETINAL EPITHELIAL-CELLS

Bovine retinal pigmented epithelial (RPE) cells express an inducible n itric oxide synthase (NOS-II) after activation with interferon-gamma ( IFN-gamma) and lipopolysaccharide (LPS). Experiments were performed to investigate the effects of tyrosine kinase inhibitors (genistein and herbimycin A) and antioxidants [pyrrolidine dithiocarbamate (PDTC) and butyl hydroxyanisol] on NOS-II induction. The LPS-IFN-gamma-induced n itrite release was inhibited in a concentration-dependent manner by th ese compounds. Analysis by Northern blot showed that this inhibitory e ffect correlated with a decrease in NOS-II mRNA accumulation. Analysis by electrophoretic mobility shift assay of the activation of the tran scription factor nuclear factor-kappa B (NF-kappa B) involved in NOS-I I induction demonstrated that LPS alone or combined with IFN-gamma ind uced NF-kappa B binding. NF-kappa B activation was not changed by the presence of tyrosine kinase inhibitors but was totally prevented by PD TC pretreatment. Immunocytochemistry experiments confirmed the reducti on of the nuclear translocation of NF-kappa B only by PDTC. Our result s demonstrated the existence in retinal pigmented epithelial cells of different intracellular signaling pathways in NOS-II induction, since tyrosine kinase inhibitors blocked NOS-II mRNA accumulation without in hibiting NF-kappa B activation. Furthermore, the LPS-IFN-gamma-induced NOS-II mRNA accumulation was sensitive to cycloheximide, suggesting t hat, in addition to NF-kappa B, transcriptional factors that require n ew protein synthesis are involved in NOS-II induction.