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

Br J Pharmacol. 1998 Sep;125(1):193-201. doi: 10.1038/sj.bjp.0702039.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cytokines / metabolism*
  • DNA / metabolism
  • Enzyme Induction
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation, Enzymologic
  • Humans
  • Interferon-Stimulated Gene Factor 3
  • Interferon-gamma / metabolism
  • Interleukin-1 / metabolism
  • Janus Kinase 2
  • NF-kappa B / metabolism*
  • Nitric Oxide Synthase / biosynthesis*
  • Nitric Oxide Synthase / genetics
  • Nitric Oxide Synthase Type II
  • Okadaic Acid / pharmacology
  • Oxazoles / pharmacology
  • Phosphoprotein Phosphatases / antagonists & inhibitors
  • Phosphorylation
  • Plasmids / genetics
  • Promoter Regions, Genetic
  • Protein-Tyrosine Kinases / antagonists & inhibitors
  • Protein-Tyrosine Kinases / metabolism*
  • Proto-Oncogene Proteins c-fos / biosynthesis
  • Proto-Oncogene Proteins c-fos / genetics
  • Proto-Oncogene Proteins c-jun / biosynthesis
  • Proto-Oncogene Proteins c-jun / genetics
  • Proto-Oncogene Proteins*
  • RNA, Messenger / metabolism
  • Signal Transduction
  • Transcription Factor AP-1 / metabolism*
  • Transcription Factors / metabolism*
  • Transfection
  • Tumor Cells, Cultured
  • Tumor Necrosis Factor-alpha / metabolism
  • Tyrphostins / pharmacology


  • Cytokines
  • Enzyme Inhibitors
  • Interferon-Stimulated Gene Factor 3
  • Interleukin-1
  • NF-kappa B
  • Oxazoles
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-fos
  • Proto-Oncogene Proteins c-jun
  • RNA, Messenger
  • Transcription Factor AP-1
  • Transcription Factors
  • Tumor Necrosis Factor-alpha
  • Tyrphostins
  • alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide
  • gamma interferon activation factor
  • Okadaic Acid
  • calyculin A
  • Interferon-gamma
  • DNA
  • NOS2 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Protein-Tyrosine Kinases
  • JAK2 protein, human
  • Janus Kinase 2
  • Phosphoprotein Phosphatases