Cytokine induction of NO synthase II in human DLD-1 cells: Roles of the JAK-STAT, AP-1 and NF-κB-signaling pathways

Abstract

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 μM) and tyrphostin A25 (mRNA down to 24%, nitrite production down to 1% at 200 μM), 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 nM calyculin A; to 28% at 500 nM okadaic acid; to 18% at 10 μM 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 μM), 3,4-dichloroisocoumarin (50 μM), panepoxydone (5 μg ml-1) and pyrrolidine dithiocarbamate (100 μM) 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.