Inhibition of small G proteins of the rho family by statins or clostridium difficile toxin B enhances cytokine-mediated induction of NO synthase II.
In order to investigate the involvement of Ras and/or Rho proteins in the induction of the inducible isoform of nitric oxide synthase (NOS II) we used HMG-CoA reductase inhibitors (statins) and Clostridium difficile toxin B (TcdB) as pharmacological tools. Statins indirectly inhibit small G proteins by preventing their essential farnesylation (Ras) and/or geranylgeranylation (Rho). In contrast, TcdB is a glucosyltransferase and inactivates Rho-proteins directly. Human A549/8- and DLD-1 cells as well as murine 3T3 fibroblasts were preincubated for 18 h with statins (1 - 100 microM) or TcdB (0.01-10 ng ml(-1)). Then NOS II expression was induced by cytokines. NOS II mRNA was measured after 4 - 8 h by RNase protection assay, and NO production were measured by the Griess assay after 24 h. Statins and TcdB markedly increased cytokine-induced NOS II mRNA expression and NO production. Statin-mediated enhancement of NOS II mRNA expression was reversed almost completely by cotreatment with mevalonate or geranylgeranylpyrophosphate. It was only slightly reduced by farnesylpyrophosphate. Therefore, small G proteins of the Rho family are likely to be involved in NOS II induction. In A549/8 cells stably transfected with a luciferase reporter gene under the control of a 16 kb fragment of the human NOS II promoter (pNOS2(16)Luc), statins produced only a small increase in cytokine-induced NOS II promoter activity. In contrast, statins had a considerable superinducing effect in DLD-1 cells stably transfected with pNOS2(16)Luc. In conclusion, our studies provide evidence that statins and TcdB potentiate cytokine-induced NOS II expression via inhibition of small G proteins of the Rho family. This in turn results in an enhanced NOS II promoter activity and/or a prolonged NOS II mRNA stability.
Hausding
M
M
Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55101 Mainz, Germany.
Witteck
A
A
Rodriguez-Pascual
F
F
von Eichel-Streiber
C
C
Förstermann
U
U
Kleinert
H
H
D016475:3T3 Cells; D000818:Animals; D000069059:Atorvastatin; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology*; D016207:Cytokines / Q000502:physiology; D004347:Drug Interactions; D004790:Enzyme Induction; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors*; D015971:Gene Expression Regulation, Enzymologic; D006538:Heptanoic Acids / Q000494:pharmacology; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D008148:Lovastatin / Q000494:pharmacology; D008798:Mevalonic Acid / Q000494:pharmacology; D051379:Mice; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011758:Pyrroles / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D016207:Cytokines; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D011106:Polyisoprenyl Phosphates; D011758:Pyrroles; C031815:toxB protein, Clostridium difficile; D008148:Lovastatin; D000069059:Atorvastatin; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; C002963:geranylgeranyl pyrophosphate; D008798:Mevalonic Acid