Cytokine induction of NO synthase II in human DLD‐1 cells: roles of the JAK‐STAT, AP‐1 and NF‐ κ B‐signaling pathways

1 In human epithelial‐like DLD‐1 cells, nitric oxide synthase (NOS) II expression was induced by interferon‐γ (100 u ml −1 ) alone and, to a larger extent, by a cytokine mixture (CM) consisting of interferon‐γ, interleukin‐1β (50 u ml −1 ) and tumor necrosis factor‐α (10 ng ml −1 ). 2 CM‐induced NOS II expression was inhibited by tyrphostin B42 (mRNA down to 1%; nitrite production down to 0.5% at 300 μ M ) and tyrphostin A25 (mRNA down to 24%, nitrite production down to 1% at 200 μ M ), suggesting the involvement of janus kinase 2 (JAK‐2). Tyrphostin B42 also blocked the CM‐induced JAK‐2 phosphorylation (kinase assay) and reduced the CM‐stimulated STAT1α binding activity (gel shift analysis). 3 CM reduced the nuclear binding activity of transcription factor AP‐1. A heterogenous group of compounds, that stimulated the expression of c‐fos/c‐jun, enhanced the nuclear binding activity of AP‐1. This group includes the protein phosphatase inhibitors calyculin A, okadaic acid, and phenylarsine oxide, as well as the inhibitor of translation anisomycin. All of these compounds reduced CM‐induced NOS II mRNA expression (to 9% at 50 n M calyculin A; to 28% at 500 n M okadaic acid; to 18% at 10 μ M phenylarsine oxide; and to 19% at 100 ng ml −1 anisomycin) without changing NOS II mRNA stability. In cotransfection experiments, overexpression of c‐Jun and c‐Fos reduced promoter activity of a 7 kb DNA fragment of the 5′‐flanking sequence of the human NOS II gene to 63%. 4 Nuclear extracts from resting DLD‐1 cells showed significant binding activity for transcription factor NF‐κB, which was only slightly enhanced by CM. The NF‐κB inhibitors dexamethasone (1 μ M ), 3,4‐dichloroisocoumarin (50 μ M ), panepoxydone (5 μg ml −1 ) and pyrrolidine dithiocarbamate (100 μ M ) produced no inhibition of CM‐induced NOS II induction. 5 We conclude that in human DLD‐1 cells, the interferon‐γ–JAK‐2‐STAT1α pathway is important for NOS II induction. AP‐1 (that is downregulated by CM) seems to be a negative regulator of NOS II expression. NF‐κB, which is probably important for basal activity of the human NOS II promoter, is unlikely to function as a major effector of CM in DLD‐1 cells.British Journal of Pharmacology (1998) 125 , 193–201; doi: 10.1038/sj.bjp.0702039