Tyrosine kinase inhibitors and antioxidants modulate NF-kappaB and NOS-II induction in retinal epithelial cells.
Bovine retinal pigmented epithelial (RPE) cells express an inducible nitric 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 nitrite release was inhibited in a concentration-dependent manner by these compounds. Analysis by Northern blot showed that this inhibitory effect correlated with a decrease in NOS-II mRNA accumulation. Analysis by electrophoretic mobility shift assay of the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) involved in NOS-II induction demonstrated that LPS alone or combined with IFN-gamma induced NF-kappaB binding. NF-kappaB activation was not changed by the presence of tyrosine kinase inhibitors but was totally prevented by PDTC pretreatment. Immunocytochemistry experiments confirmed the reduction of the nuclear translocation of NF-kappaB only by PDTC. Our results 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 inhibiting NF-kappaB activation. Furthermore, the LPS-IFN-gamma-induced NOS-II mRNA accumulation was sensitive to cycloheximide, suggesting that, in addition to NF-kappaB, transcriptional factors that require new protein synthesis are involved in NOS-II induction.
Développement, Vieillissement, et Pathologie de la Rétine, Unité 450 Institut National de la Santé et de la Recherche Médicale, Association Claude Bernard, 75016 Paris, France.
V
V
Faure
Y
Y
Courtois
O
O
Goureau
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D016227:Benzoquinones; D002083:Butylated Hydroxyanisole / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D019833:Genistein / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D047029:Lactams, Macrocyclic; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011759:Pyrrolidines / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D017828:Rifabutin / Q000031:analogs & derivatives; D013859:Thiocarbamates / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D000975:Antioxidants; D016227:Benzoquinones; D004791:Enzyme Inhibitors; D047029:Lactams, Macrocyclic; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011759:Pyrrolidines; D011809:Quinones; D012333:RNA, Messenger; D013859:Thiocarbamates; D017828:Rifabutin; D002083:Butylated Hydroxyanisole; C020972:pyrrolidine dithiocarbamic acid; C020754:herbimycin; D007371:Interferon-gamma; D003513:Cycloheximide; D019833:Genistein; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D011505:Protein-Tyrosine Kinases