Cytokine induction of NO synthase II in human DLD-1 cells: roles of the JAK-STAT, AP-1 and NF-kappaB-signaling pathways.
1. In human epithelial-like DLD-I cells, nitric oxide synthase (NOS) II expression was induced by interferon-gamma (100 u ml(-1)) alone and, to a larger extent, by a cytokine mixture (CM) consisting of interferon-gamma, interleukin-1beta (50 u ml(-1)) and tumor necrosis factor-alpha (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 microM) and tyrphostin A25 (mRNA down to 24%, nitrite production down to 1% at 200 microM), 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 STAT1alpha 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 microM 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-kappaB, which was only slightly enhanced by CM. The NF-kappaB inhibitors dexamethasone (1 microM), 3,4-dichloroisocoumarin (50 microM), panepoxydone (5 microg ml(-1)) and pyrrolidine dithiocarbamate (100 microM) produced no inhibition of CM-induced NOS II induction. 5. We conclude that in human DLD-1 cells, the interferon-gamma-JAK-2-STAT1alpha 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-kappaB, 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.
Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany.
H
H
Kleinert
T
T
Wallerath
G
G
Fritz
I
I
Ihrig-Biedert
F
F
Rodriguez-Pascual
D A
DA
Geller
U
U
Förstermann
D016207:Cytokines / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D050832:Interferon-Stimulated Gene Factor 3; D007371:Interferon-gamma / Q000378:metabolism; D007375:Interleukin-1 / Q000378:metabolism; D053614:Janus Kinase 2; D008387:Marine Toxins; D016328:NF-kappa B / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D016207:Cytokines; D004791:Enzyme Inhibitors; D050832:Interferon-Stimulated Gene Factor 3; D007375:Interleukin-1; D008387:Marine Toxins; D016328:NF-kappa B; D010080:Oxazoles; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide; C079868:gamma interferon activation factor; D019319:Okadaic Acid; C059041:calyculin A; D007371:Interferon-gamma; D004247:DNA; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D010749:Phosphoprotein Phosphatases