In murine 3T3 fibroblasts, different second messenger pathways resulting in the induction of NO synthase II (iNOS) converge in the activation of transcription factor NF-kappaB.
Transcription factor NF-kappaB is essential for the induction of nitric oxide synthase (NOS) II (iNOS) by bacterial lipopolysaccharide in murine macrophages (Xie, Q. W., Kashiwabara, Y., and Nathan, C. (1994) J. Biol. Chem. 269, 4705-4708). In 3T3 fibroblasts, agents other than cytokines are efficacious inducers of NOS II expression. In addition to cytokines such as interferon-gamma or tumor necrosis factor-alpha, protein kinase C-stimulating agents such as tetradecanoylphorbol-13-acetate, or cyclic AMP-elevating agents such as forskolin and 8-bromo-cAMP markedly increased NOS II mRNA (measured by Sl nuclease and RNase protection analyses), NOS II protein (determined by Western blotting), and NOS activity (measured by chemiluminescence detection of NO2-). Transforming growth factor-beta1 (which is an inhibitor of NOS II induction in other cell types) potentiated NOS II mRNA expression produced by all inducing agents listed, whereas dexamethasone, pyrrolidine dithiocarbamate and 3,4-dichloroisocoumarin (inhibitors of NF-kappaB activation) suppressed NOS II mRNA induction in response to all stimulants. In electrophoretic mobility shift assays, nuclear protein extracts from 3T3 cells stimulated with any of the inducing agents significantly slowed the migration of an NF-kappaB-binding oligonucleotide, whereas nuclear extracts from untreated control cells did not. These experiments indicate that NF-kappaB is the key control element for the induction of NOS II in response to at least three different second messenger pathways in 3T3 cells.
Kleinert
H
H
Department of Pharmacology, Johannes Gutenberg University, Mainz, Federal Republic of Germany.
Euchenhofer
C
C
Ihrig-Biedert
I
I
Förstermann
U
U
D016475:3T3 Cells; D000199:Actins / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D015290:Second Messenger Systems / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D000199:Actins; D017931:DNA Primers; D016328:NF-kappa B; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D003907:Dexamethasone; D019001:Nitric Oxide Synthase; D020794:Receptor Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C